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Carmen Molina-París
Grant Lythe Editors
Mathematical,
Computational
and Experimental
T Cell Immunology
Mathematical, Computational and Experimental
T Cell Immunology
Carmen Molina-París • Grant Lythe
Editors
Mathematical,
Computational and
Experimental T Cell
Immunology
Editors
Carmen Molina-París Grant Lythe
Department of Applied Mathematics, Department of Applied Mathematics,
School of Mathematics School of Mathematics
University of Leeds University of Leeds
Leeds, UK Leeds, UK
ISBN 978-3-030-57203-7 ISBN 978-3-030-57204-4 (eBook)

https://doi.org/10.1007/978-3-030-57204-4
© Springer Nature Switzerland AG 2021

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of
the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information
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The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
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protective laws and regulations and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this book
are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or
the editors give a warranty, expressed or implied, with respect to the material contained herein or for any
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This Springer imprint is published by the registered company Springer Nature Switzerland AG
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
to Federico-ico and Sofía-pía,
may they always be good, happy and free.
Stimulation of naive CD4+ T cells with PMA and Ionomycin.
Naive CD4+ T cells from a BALB/c mouse were isolated and approximately 50,000
cells were seeded in a U-bottomed 96 well plate. The cells were activated with
10 ng/mL of Phorbol 12-myristate 13-acetate and 0.1 μM Ionomycin. This picture
was taken 36 hours post-activation.
vii
Contents
1 Cytokine Receptor Signaling and CD4/CD8 Lineage Choice

during T Cell Development in the Thymus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Megan A. Luckey and Jung Hyun Park
2 An Agent-Based Model of T Helper Cell Fate Decisions in the
Thymus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Sahamoddin Khailaie, Philippe A. Robert, and Michael
Meyer-Hermann
3 Modelling Naive T Cell Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Benedict Seddon, Sanket Rane, and Andrew J. Yates
4 Mechanistic Models of CD4 T Cell Homeostasis and
Reconstitution in Health and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Joanna Lewis and Joseph F. Standing
5 Modeling the Dynamics of CD4+ T Cells in HIV-1 Infection . . . . . . . . . 81
Ruy M. Ribeiro
6 Modelling the Response to Interleukin-7 Therapy in
HIV-Infected Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Rodolphe Thiébaut, Laura Villain, Chloé Pasin,
and Daniel Commenges
7 Modeling Immunopathology During Persistent Viral Infections. . . . . . 109
Veronika I. Zarnitsyna, Philip L. F. Johnson, Joseph N. Blattman,
and Rustom Antia
8 Delayed Differentiation Makes Many Models Compatible with
Data for CD8+ T Cell Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Aridaman Pandit and Rob J. de Boer
9 Inferring Differentiation Order in Adaptive Immune
Responses from Population-Level Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Alexander S. Miles, Philip D. Hodgkin, and Ken R. Duffy
ix
x Contents
10 Experimental and Mathematical Approaches to Quantify

Recirculation Kinetics of Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Vitaly V. Ganusov and Michio Tomura
11 The Public Face and Private Lives of T Cell Receptor Repertoires . . . 171
Pradyot Dash and Paul G. Thomas
12 Population Dynamics of Immune Repertoires. . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Jonathan Desponds, Andreas Mayer, Thierry Mora,
and Aleksandra M. Walczak
13 Mathematical Modelling of T Cell Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Nicola C. Trendel and Omer Dushek
14 Agent-Based Model of Heterogeneous T-Cell Activation in Vitro . . . . . 241
Shamik Majumdar, Carmen Molina-París, Dipankar Nandi, and
Grant Lythe
15 CTLA-4-Mediated Ligand Trans-Endocytosis:
A Stochastic Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Luis de la Higuera, Martín López-García, Grant Lythe,
and Carmen Molina-París
16 Automated Gating and Dimension Reduction of
High-Dimensional Cytometry Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Sharon X. Lee, Geoffrey J. McLachlan, and Saumyadipta Pyne
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Chapter 1
Cytokine Receptor Signaling
and CD4/CD8 Lineage Choice during
T Cell Development in the Thymus
Megan A. Luckey and Jung Hyun Park
1.1 Introduction
T cell immunity is controlled by two major subsets of T cells that can be identified
by their distinct expression of the coreceptor molecules CD4 and CD8. T cells that
express the CD4 coreceptor recognize peptide antigens in the context of MHC class
II molecules and provide helper functions. T cells that express the CD8 coreceptor,
in contrast, react with peptide antigens in the context of MHC class I and exhibit
cytolytic activities [50, 82]. Notably, there is a remarkable correlation between
coreceptor expression and T cell function, such that CD4 coreceptor expression
marks helper lineage T cells, whereas CD8 coreceptor expression is associated
with cytolytic activity. The association between coreceptor phenotype and T effector
function is thought to be imposed during T cell development in the thymus, where
immature thymocytes that express both CD4 and CD8 coreceptor molecules, i.e.,
CD4+ CD8+ double-positive (DP) cells, make lineage decision into either CD4
single-positive (CD4SP) or CD8 single-positive (CD8SP) T cells [8, 28, 88]. The
process of CD4 versus CD8 cell fate decision has fascinated immunologists for a
long time. However, deciphering the molecular basis of this process turned out to
be a daunting task. A major obstacle is the multiplicity of events that occur at the
same time when CD4 versus CD8 lineage commitment is made. Most prominently,
CD4/CD8 lineage choice coincides with the T cell receptor (TCR)-induced positive
selection, which upregulates TCR expression. However, positive selection also
induces changes in coreceptor expression, so that the exact cellular phenotype at
lineage decision had remained obscure and confusing [88].
M. A. Luckey · J. H. Park ()

Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, MD, USA
e-mail: Parkhy@mail.nih.gov
© Springer Nature Switzerland AG 2021 1

C. Molina-París, G. Lythe (eds.), Mathematical, Computational and Experimental T Cell
Immunology, https://doi.org/10.1007/978-3-030-57204-4_1
2 M. A. Luckey and J. H. Park
Because TCR specificity imposes MHC restriction that is further matched with
the appropriate CD4 or CD8 coreceptor expression, classically, CD4/CD8 lineage
choice has been considered a developmental event that is primarily controlled by
TCR signaling [5, 28, 36, 113]. However, TCR signaling alone cannot satisfactorily
explain the entire process of CD4 versus CD8 lineage commitment. In fact, it
became evident that cell fate decision in the thymus requires additional regulatory
pathways, working in concert with TCR signaling to determine the lineage choice.
A series of experimental results followed by further genetic evidence revealed this
requirement to be the signaling by intra-thymic cytokines. Thus, cytokine signaling
activity specifies CD4/CD8 lineage fate during positive selection and further
imposes T cell function and promotes maturation of post-selection thymocytes
[10, 67, 74, 117]. In this article, we aimed to review our current understanding of
CD4/CD8 lineage choice during T cell development and to highlight recent findings
on the role of cytokine receptor signaling in this process.
1.2 T Cell Development and CD4/CD8 Lineage Choice

in the Thymus
Because both CD4SP and CD8SP cells originate from DP thymocytes, early
attempts to explain CD4 versus CD8 lineage choice were proposed around the
idea that lineage decision was made by selectively extinguishing the expression of
one or the other coreceptor [23, 103]. The molecular identity of the signals that
would instruct the termination of either CD4 versus CD8 coreceptor expression,
however, remained disputed. Different hypotheses and their variations of stochastic
or instructional mechanisms were put forward to explain the progression of DP into
SP cells [4, 5, 7, 28, 103]. Because forced expression of an MHC class I (MHC-I)
restricted TCR results in the generation of CD8 T cells, but MHC class II (MHC-II)-
restricted TCR transgenes produce CD4 T cells, evidently, lineage fate is encoded
in the specificity of the TCR. However, it was not known how this information was
extracted by the immune system and then translated into lineage specification. An
attractive model that has been favored for a long time proposed that MHC-I- versus
MHC-II-restricted TCRs differ in their signaling strength, such that strong TCR
signals terminate CD8 expression and drive CD4 lineage choice, whereas weak TCR
signals terminate CD4 expression and impose CD8 lineage fate [5, 28, 36, 103].
With regard to the molecular basis of distinct TCR signaling strength, it was noted
that CD4 coreceptors, which preferentially associate with MHC-II-restricted TCR,
binds significantly larger amounts of the tyrosine kinase Lck than CD8 coreceptors,
which associates with MHC-I-specific TCR [97, 99, 100]. Thus, the “strength-of-
signal” model posits that CD4/CD8 lineage choice is made at the DP stage, where
quantitative differences in TCR signals instruct the expression of an MHC-matched
coreceptor that associates with the signal-transducing TCR [5, 36, 37, 56, 108].
1 Cytokine Receptor Signaling and CD4/CD8 Lineage Choice during T Cell. . . 3
It was thus surprising when it was discovered that the downregulation of

coreceptor expression in TCR-signaled DP cells was not a binary decision of
terminating CD4 versus CD8 expression. Instead, DP cells are pre-programmed
to terminate only Cd8, and not Cd4 gene expression upon TCR signaling [16].
Consequently, positive selection of immature thymocytes, which precedes their
lineage commitment [4, 42], induces the differentiation of DP cells into tran-
scriptionally Cd4+ Cd8− but phenotypically CD4+ CD8lo cells. And, it is at this
CD4+ CD8lo intermediate stage that the CD4 versus CD8 lineage decision occurs
[9, 10, 16]. These results required a conceptual change in our view of CD4 versus
CD8 lineage choice, because they demonstrated that lineage commitment could
not be simply explained by the loss of the coreceptor that is not involved in
positive selection. Instead, these findings demonstrated that CD8SP differentiation
first needs to terminate CD8 expression at the CD4+ CD8lo intermediate stage
before re-expressing CD8 during their further maturation into CD8 T cells [10,
16]. The process of terminating CD8 coreceptor expression followed by CD8 re-
expression is referred to as “coreceptor reversal” [10], and it is a core principle
of the “kinetic signaling” model, which is currently considered to supply the best
molecular explanation for CD4/CD8 lineage choice in the thymus [61, 88] (Fig. 1.1).
According to this model, positive selection in DP cells results in the selective
termination of CD8, but not CD4 coreceptor expression. Notably, the loss of CD8
expression does not affect MHC-II-restricted TCR signaling that depends on CD4.
Fig. 1.1 The kinetic signaling model of CD4/CD8 lineage decision. Positive selection of immature
DP thymocytes induces the termination of Cd8 coreceptor gene expression that converts DP
cells into transcriptionally Cd4+ Cd8− and phenotyptically CD4+ CD8lo intermediate cells. If the
positive selecting TCR signal persists, such as MHC-II-restricted TCRs that do not depend on
CD8, persistent TCR signals upregulate the expression of the CD4-specifying transcription factor
ThPOK and desensitize cytokine receptors to impose CD4 lineage fate. However, if TCR signaling
ceases, such as for MHC-I-restricted TCRs that require CD8 coreceptor expression, the cessation
of TCR signals permits cytokine receptor signaling. It is then the intra-thymic cytokines that induce
the expression of the CD8 lineage-specifying transcription factor Runx3 which downregulates CD4
transcription, upregulates CD8 expression, and antagonizes ThPOK to seal CD8 lineage fate
Thus, MHC-II-specific TCR signals persist during CD4/CD8 lineage decision, and
persistent TCR signals upregulate the expression of the transcription factor ThPOK
which specifies CD4 lineage choice [32, 90]. TCR signaling further desensitizes
cytokine receptors in positively selected cells that could otherwise signal to induce
the expression of the CD8 lineage-specifying factor Runx3 [24, 67, 74]. By contrast,
the downregulation of CD8 terminates the signaling of MHC-I-specific TCRs, such
that ThPOK expression cannot be induced, and the possibility of CD4 lineage
specification is aborted. Consequently, the kinetic signaling model posits that it is
the kinetics – not the strength – of TCR signaling that determines the CD4 versus
CD8 lineage fate during T cell development in the thymus.
While cessation of TCR signaling abolishes ThPOK upregulation, it was difficult
to explain how the absence of TCR signal would induce Runx3 expression. It is now
known that the cessation of TCR signals is necessary to sensitize cytokine receptors,
and it is cytokine receptor signaling that upregulates Runx3 expression and imposes
CD8 lineage fate [24, 67, 74] (Fig. 1.1). Thus, two distinct signals govern T cell fate
in the thymus; TCR signals drive CD4 helper lineage differentiation, and cytokine
signaling specifies CD8 cytolytic lineage choice.
1.3 Cytokine Requirement in T Cell Development
Cytokine receptor signaling plays a critical role throughout the generation and
maintenance of T cells, and it is especially important for the earliest stages of T cell
development in the thymus. In particular, cytokines of the common γ-chain family
(γc) play a non-redundant role in thymopoiesis, as illustrated in the dramatically
reduced thymocyte numbers and developmental block in γc-deficient mice [11,
20] (Fig. 1.2). The γc cytokine family has six members, namely, IL-2, IL-4, IL-
7, IL-9, IL-15, and IL-21, which all share γc for ligand binding and signaling
[106]. However, the absence of IL-7 or its proprietary receptor IL-7R α-chain
(IL-7Rα) phenocopies γc deficiency, indicating that IL-7 signaling through the IL-
7Rα/γc receptor complex is the molecular basis for the γc requirement during T
cell development [6, 89, 105]. Due to the prominent role of IL-7 in providing
pro-survival signals to lymphocytes [27, 80, 102], early studies to understand the
IL-7 requirement in T cell development heavily focused on the anti-apoptotic role
of IL-7 signals [6, 89, 105]. Such views were further supported by experimental
data reporting significantly improved thymopoiesis upon the introduction of anti-
apoptotic Bcl-2 transgenes into IL-7Rα- or γc-deficient mice [3, 51, 69, 104].
However, careful analysis of immature CD4, CD8 double-negative (DN) thymo-
cytes revealed that the developmental and phenotypic block in γc-deficient mice was
incompletely rescued by forced Bcl-2 expression. While transgenic expression of
Bcl-2 substantially improved the survival and numbers of thymocytes in γc-deficient
Bcl-2-transgenic mice, there was still a dramatic developmental arrest at the DN2
(CD44+ CD25+ ) to DN3 (CD44− CD25+ ) stage of thymocyte differentiation. These
results and other studies indicated that pro-survival signals alone are insufficient to
Fig. 1.2 Cytokine receptor expression during T cell differentiation in the thymus. Cytokine recep-
tor signaling plays critical roles during the early thymocyte development and during CD4/CD8
lineage choice upon positive selection. While most thymocyte subpopulations express copious
amounts of IL-7Rα and γc, immature DP thymocytes are unique as they have terminated IL-7Rα
and have downregulated γc expression, which renders them non-responsive to IL-7 and other pro-
survival cytokines. Understanding the role and contribution of individual γc family members in
CD4/CD8 lineage choice remains an active area of research
restore proper thymocyte differentiation in the absence of γc signaling [60, 81].

Thus, cytokine signals in early T cell development are required for reasons beyond
their anti-apoptotic effects. Contrary to its requirement in the earliest immature
thymocytes, the role of cytokine signaling in the late stages of T cell development,
specifically during or after positive selection, has not been much appreciated. The
chief reasons for such neglect included the observations that genetic ablation of
most cytokine or cytokine receptor expression, other than IL-7/IL-7Rα, did not
have significant effects on T cell development or lineage differentiation [106]. For
example, a deficiency in IL-2, which is a major T cell proliferation cytokine, did
not affect the overall thymopoiesis [86]. Nor did the deficiencies in other major
cytokines, including IL-4, IL-10, IL-15, and IL-21, show any noticeable defects in
T cell development [40, 43, 52, 53]. Thus, in contrast to its role in effector T cell
polarization or proliferation, cytokine signaling in the thymus was considered to
play only a minor role in T cell development. Accordingly, the unique requirement
of IL-7/IL-7Rα signaling had been explained by the non-redundant anti-apoptotic
effects of IL-7. Pro-survival signals are necessary to maintain viability and drive
proliferation of pre-selection immature thymocytes until they express a functional
TCR receptor. Once functional TCRs are expressed, TCR signals and not cytokine
signals would drive lineage decision of immature DP thymocytes. Consequently,
in conventional models of CD4/CD8 lineage commitment, there is no mechanistic
requirement for cytokine signaling in T cell differentiation [7, 28, 36]. In this
regard, the kinetic signaling model is unique, because it predicts the requirement
of a cellular signal other than TCR signals to induce CD8 and downregulate CD4
expression in uncommitted CD4+ CD8lo cells [10, 61]. Such an idea is inherent
to the kinetic signaling model, because the model posits that TCR signaling is
necessarily terminated in MHC-I engaged cells as the loss of CD8 expression
destabilizes CD8 coreceptor-dependent TCR engagement (Fig. 1.1). Therefore,
TCR signals are unlikely to drive CD8 lineage specification. Instead, CD8 lineage
specification signal was initially proposed to be provided by intra-thymic cytokines,
specifically by IL-7 [10]. This idea was later confirmed in a series of in vivo
animal studies, where IL-7 signaling was specifically terminated in pre-selection
DP thymocytes by the conditional deletion of γc, IL-7Rα, or STAT5a/b expression
[67, 74]. STAT5 is a transcriptional activator that acts downstream of IL-7Rα/γc
and induces the expression of Runx3 [35, 74]. Notably, the absence of STAT5
or IL-7Rα/γc expression did not affect CD4 lineage choice but had profound
effects on CD8 lineage commitment, resulting in a substantial loss (~70%) of
CD8SP thymocyte number and frequency [24, 67, 74]. Differentiation into CD4SP
thymocytes, on the other hand, remained unaffected, illustrating a fundamental
difference in lineage specification signals between CD4 and CD8 T cells.
1.4 Cytokine Signaling in the CD4/CD8 Lineage Choice

of Developing Thymocytes
Pre-selection DP thymocytes are precluded from intra-thymic cytokine signaling

by multiple mechanisms, including their loss of cytokine receptor expression, their
anatomical position in the cytokine-poor thymic cortex, and abundant expression
of Suppressor Of Cytokine Signaling-1 (SOCS1), a potent inhibitor of cytokine
signaling [14, 116]. TCR-induced positive selection releases DP thymocytes from
these constraints by upregulating the expression of cytokine receptors, including
γc, IL-7Rα, and IL-21Rα [76, 79], and by inducing chemokine receptor expression,
such as CCR7, which drives their migration into the cytokine-rich medullary region
for further selection and maturation [17, 54, 98, 114]. Finally, the downregulation
of SOCS1 expression restores cytokine responsiveness in post-selection thymocytes
[14, 118]. Accordingly, a major role of positive selecting TCR signals is the
conversion of cytokine-unresponsive thymocytes into cytokine-responsive cells that
also have access to the appropriate cytokines. However, cytokine responsiveness
is not restored in all positively selected cells. Instead, it remains suppressed during
CD4 lineage specification, presumably by persistent TCR signaling that desensitizes
cytokine receptor signaling. The mechanistic details of cytokine receptor desensiti-
zation are not fully understood. However, it is known that TCR signaling induces the
destabilization and loss of JAK1 proteins, which interferes with the signaling of γc
family cytokines [41]. TCR signaling was also proposed to cleave the cytosolic tail
of γc by activation of the calcium-activated cysteine protease calpain, which would
debilitate γc cytokine signaling [71].
During thymic lineage differentiation, persistent TCR signals also induce the
expression of the CD4-specifying transcription factor ThPOK [32, 90]. Notably,
ThPOK acts as a transcriptional activator to upregulate the expression of SOCS
molecules, such as SOCS1, SOCS3, and Cish [64]. Because members of the
SOCS family molecules are potent inhibitors of cytokine signaling [115], their
increased abundance by ThPOK curtails cytokine signaling during CD4 lineage
differentiation. In fact, suppression of cytokine signaling is a critical step in CD4
lineage differentiation that was found to be sufficient to restore CD4 lineage
specification in the absence of ThPOK [64].
Upregulation of ThPOK expression is a critical event in CD4/CD8 lineage
choice because ThPOK is both necessary and sufficient to impose CD4 lineage
fate during positive selection [32, 34, 68, 90]. Consequently, ThPOK expression
is tightly controlled during T cell development. ThPOK expression is suppressed in
pre-selection DP thymocytes by the concerted action of the transcription factors
Runx1 and MAZR [83] and requires GATA-3 for its induction during CD4
lineage differentiation [107]. A naturally occurring ThPOK-mutant mouse strain
that expresses non-functional ThPOK proteins is unable to generate CD4 helper T
cells and thus has been referred to as helper deficient (HD) [42]. MHC-II-restricted
thymocytes are positively selected in HD mice, but they fail to commit into CD4
cells and instead undergo lineage redirection into CD8 T cells. Strikingly, enforced
expression of SOCS1 effectively rescued the CD4 T cell-deficiency in HD mice
[64]. Thus, suppression of cytokine signaling could replace the ThPOK requirement
in CD4 lineage specification, resulting in the generation of ThPOK-deficient CD4 T
cells [64]. Such ThPOK-deficient CD4 T cells still exerted helper function, because
they upregulated CD40L expression upon TCR stimulation. These results indicated
that cytokine signaling, or the absence thereof, underpins the basis of CD4/CD8
lineage decision.
In the same vein, while enforced expression of ThPOK redirected MHC class
I-selected thymocytes into CD4 lineage T cells [32, 90], ablation of SOCS1 could
correct lineage choice in ThPOK-transgenic mice, resulting in the re-appearance
of CD8SP thymocytes and mature CD8 T cells [64]. Collectively, these results
revealed that the persistence of positively selected TCR signals is primarily required
to disable cytokine signaling during CD4 lineage differentiation. These findings
agree that it is cytokine signaling which directs CD4/CD8 lineage choice during
T cell development in the thymus.
1.5 Cytokine-Driven Molecular Circuitry of CD4/CD8

Lineage Choice
The molecular basis for a cytokine requirement in CD8 lineage differentiation can be
explained by the fact that CD8 specification is driven by Runx3, whose expression
in turn is induced by cytokine signaling [2, 67, 74]. Runx3 is selectively induced
upon CD8, but not CD4, lineage differentiation and is required to activate the Cd4
silencer and to upregulate CD8 coreceptor expression. These are precisely the two
core events of coreceptor reversal [31, 74, 96], and thus, they place Runx3 as a
central regulator of CD8 T cell lineage fate. Runx3 gene expression is controlled
by two distinct promoter elements, i.e. proximal and distal, and they regulate
Runx3 transcription in a tissue-specific manner. The distal promoter-driven Runx3
(Runx3d) initiates protein translation at the distal exon 1, and it encodes for a Runx3
protein with 19 extra amino acids at the N-terminal end compared to the Runx3
protein produced from the proximal exon 1 [21]. Functional differences between
the Runx3 molecules that are expressed either from the distal versus the proximal
promoter are not known. But, their tissue-specific expression is quite distinct.
Runx3d is specifically expressed in CD8 lineage T cells, whereas the proximal
promoter-driven Runx3 is mostly found in cells outside of the lymphoid system [21,
63]. The precise molecular pathway involved in regulating distal versus proximal
promoter-driven Runx3 expression is not mapped.
CD8 lineage differentiation has been associated with the cessation of TCR
signaling [88]. However, it has not been straightforward to understand how the
loss of TCR signals would induce the upregulation of Runx3 to specify CD8
lineage choice. New insights into this process were recently provided in a study
where the timing and duration of MHC class I-mediated signals were examined
in great detail [47]. The analyses revealed the surprising finding that CD8 lineage
differentiation was achieved in two sequential phases. Phase 1 is triggered by TCR
signaling and results in the gradual loss of CD8 coreceptor expression, concurrent
to the acquisition of cytokine responsiveness. Phase 2, in contrast, is triggered and
driven by cytokine receptor signaling, and it is associated with an incremental
gain of CD8 coreceptor expression, concomitant to a decrease in CD4 coreceptor
abundance. Analysis of Rag2-deficient OT-I TCR transgenic thymocytes further
revealed that Runx3d reporter expression was not induced in MHC-I-signaled
CD4+ CD8lo intermediate cells, unless positively selected TCR signals persisted
long enough to induce surface CCR7 expression [47]. Expression of the chemokine
receptor CCR7 is induced by TCR engagement of cortical thymocytes, and in vitro
studies found it to be upregulated within 48 hours of TCR stimulation [98]. The
duration of phase 1 normally does not exceed 53 hours. and it can be significantly
shortened by weaker TCR signals. Interestingly, the duration of phase 2 inversely
correlated with the length of phase 1, and it ranged between 32 and 79 hours. Thus,
the accelerated progression in phase 2 compensates for any prolonged residency in
phase 1. This mechanism is referred to as “signaling compensation,” and it provides
the molecular basis for the unexpected observation that the duration of positively
selected TCR signals (phase 1) inversely correlates with the time span to undergo
coreceptor reversal (phase 2) [47]. Collectively, these data documented that post-
selection CD4+ CD8lo thymocytes comprise two subsets, where CCR7-negative
CD4+ CD8lo cells are in phase 1 and do not express Runx3d, whereas CCR7-positive
CD4+ CD8lo cells are in phase 2, express Runx3d, and have committed to CD8
lineage fate.
Understanding the molecular mechanism that drives Runx3 expression during
coreceptor reversal is a core issue in CD4/CD8 cell fate decision. Experimental
results strongly advocate for a role of cytokines, and not of TCR signaling, in
inducing Runx3 expression [24, 64, 67, 74]. Specifically, the γc family cytokines
IL-7 and IL-15 are known to be the major players in Runx3 upregulation and
CD8 cell differentiation in the thymus [10, 67, 74]. However, the lack of IL-7
or γc signaling did not completely abolish CD8 cell generation in the thymus
[11, 20, 24, 67, 78, 94, 105], which questioned if all CD8 lineage differentiation
is driven by cytokine signaling. Potential redundancy among cytokines could
provide a potential explanation, but this had not been formally demonstrated.
A recent report addressed this issue [24], and the results provided a definitive
answer to this long-standing question. Accordingly, there is significant redundancy
among γc and non-γc cytokines in inducing Runx3d expression, and the lineage
commitment of all CD8 T cells is dependent on cytokine receptor signaling [24].
Because γc-deficient mice still contain residual CD8SP cells, in that study, a
new genetic model of combined cytokine deficiency was developed that not only
lacked γc but also IL-6, IFNγ, and TSLP signaling and was thus referred to
as “CytoQuad” (γccKO IL-6KO IFNγRKO TSLPRKO ) mice [24]. CytoQuad mice are
severely impaired in generating CD8SP cells, and it is the first in vivo model that
directly demonstrates redundancies among γc and non-γc cytokines in upregulating
Runx3d and imposing CD8 lineage fate. Notably, CytoQuad mice still produced
some CD8 T cells, but they were less than 8% in their frequency compared with
wild-type mice. These residual CD8 cells were presumably generated independently
of JAK-STAT signaling as they appeared in SOCS1-transgenic mice [24]. In-
depth analysis of these few CD8 T cells revealed that these cells were generated
by JAK-STAT-independent TGFβ signaling. Consequently, the genetic deletion of
TGFβR1 in combination with the removal of all γc and non-γc lineage-specifying
cytokines resulted in the complete loss of CD8SP cells. These results unequivocally
established that the development of CD8 T cells is exclusively driven by in vivo
cytokine signaling.
Notably, the CytoQuad mouse also demonstrated that the identity of individual
lineage-specifying cytokines does not matter much, as it is the total amount of
cytokine signals that is critical to confer CD8 lineage fate [24]. All lineage-
specifying cytokines induce Runx3d, and it is likely that MHC-I-selected thymo-
cytes are signaled by multiple intra-thymic cytokines during CD8 cell differen-
tiation. In agreement, all CD8SP thymocytes in wild-type mice express CD103
which is a signature of TGFβ signaling [22], and they also have upregulated Bcl-2,
which is induced by γc cytokines [24, 67], indicating that CD8SP cells normally
receive both cytokine signals. Currently, it remains unclear to which extent each
lineage-specifying cytokine would contribute to CD8 cell differentiation. However,

genetic ablation of IL-7Rα in DP thymocytes, which results in the most dramatic
loss of CD8SP thymocytes among all lineage-specifying cytokines [67], indicated
that IL-7 signaling would be the primary driver of CD8 lineage specification in the
thymus [67, 74].
1.6 IL-7 Receptor Expression during T Cell Development
IL-7 signaling is critical for T cell development in the thymus, but the receptor
for IL-7 is not found on all thymocytes [76], and its expression is developmen-
tally regulated during thymic maturation (Fig. 1.2). The signaling-competent IL-7
receptor is composed of the IL-7-proprietary IL-7Rα chain and the γc, which is
shared with other members of the γc cytokine family [66, 106]. IL-7Rα expression
is required to generate all lymphocytes, including some innate lymphoid cells (ILC),
specifically ILC2 and ILC3 [19]. All lymphocytes express IL-7Rα at some point
during their development, as demonstrated with IL-7Rα fate-mapping mice [84].
However, not all lymphocytes express and maintain IL-7Rα expression throughout
their life. Mechanistically, it is known that the developmental requirement for IL-
7Rα expression differs among cell types. γδ T cells and B cells depend on IL-7R
signaling for their antigen receptor recombination, whereas conventional αβ T
cells require IL-7R signaling for survival and proliferation during early thymic
development [38, 66, 89]. Notably, upon maturation and export into peripheral
tissues, B cells terminate IL-7Rα expression and become independent of IL-7
signaling for their survival. However, T cells remain dependent on IL-7, and they
require intermittent IL-7R signaling for long-term survival and maintenance in the
periphery [46, 85, 93]. Because of its importance in both T cell generation and
maintenance, the molecular mechanisms of IL-7Rα gene expression and regulation
have been extensively investigated. The current literature indicates that IL-7Rα
gene expression is primarily regulated by transcriptional mechanisms. Several
transcription factors positively regulate IL-7Rα expression by acting on either the
promoter or a conserved enhancer region (CNS-1) that is located 3.5-kb upstream of
the transcription initiation site [44, 55]. In addition, transcriptional activators, such
as GABPα, Runx1, FoxO1, and glucocorticoid receptors, induce the expression of
IL-7Rα [12, 21, 44, 73, 111], whereas transcriptional repressors, such as Foxp3,
Foxp1, and Gfi1, suppress IL-7Rα expression [26, 59, 62, 75, 87]. Whether the
CNS-1 enhancer alone would be sufficient to control IL-7Rα gene expression or if
other regulatory elements are involved in these processes has not been clear.
To address this question, IL-7Rα CNS-1-deficient mice were generated and
examined for IL-7Rα expression [1]. Surprisingly, CNS-1-deficient thymocytes
were virtually indistinguishable with respect to their IL-7Rα expression compared
to WT controls, indicating that the CNS-1 activity is not required for IL-7Rα
expression in thymocytes [1]. The developmental stage-specific regulation of IL-
7Rα expression was also unaffected by the absence of CNS-1 because IL-7Rα
was correctly expressed on DN and SP thymocytes and absent on DP cells of
CNS-1-deficient thymocytes. On the other hand, CNS-1 deficiency substantially
diminished IL-7Rα expression and impaired both the IL-7-dependent survival and
proliferation of mature peripheral T cells. Consequently, both CD4 and CD8 naive
T cell numbers were significantly reduced in CNS-1-deficient mice. These results
revealed a developmentally controlled requirement for CNS-1 to upregulate IL-7Rα
expression, such that the CNS-1 enhancer plays a limited role in thymocytes but
is required for IL-7Rα expression in mature T cells [1, 44, 73]. These data further
indicated that regulatory elements other than CNS-1 are involved in controlling IL-
7R expression in thymocytes, but this remains to be tested. Previously, an intronic
site in the Il7r gene was shown to contain a binding site for the transcriptional
repressor Gfi1 that suppresses IL-7Rα expression in CD8 lineage cells [59, 75]. But,
whether this element participates in the thymocyte-specific regulation of IL-7Rα
remains unclear. Additionally, a long-coding RNA known as lnc-IL7R was recently
identified within the sense strand of the 3 untranslated region (3’UTR) of the human
IL-7Rα gene [18]. Lnc-IL7R encodes a 1,427-nucleotide transcript that shares the
poly-A tail with the IL-7Rα gene. It would be interesting to examine if Lnc-IL7R
interferes with IL-7Rα mRNA expression. Collectively, these results demonstrate
the utilization of distinct molecular mechanisms to control IL-7Rα expression in
immature versus mature T cells, and they further illustrate that our understanding of
IL-7Rα regulation is still far from complete.
A major question that remains unanswered is the signaling mechanism that
drives the re-expression of IL-7Rα on post-selection thymocytes. During thymocyte
development, IL-7Rα is highly expressed on immature DN cells but is curiously
terminated upon differentiation into DP cells. In mature T cells, IL-7 signaling
downregulates the transcription and expression of its own receptor [59, 75], so
that the termination of IL-7Rα expression in DP cells might be the consequence
of IL-7 signaling in DN2/3 thymocytes. However, unlike in mature T cells, IL-
7Rα expression on DP thymocytes does not rebound in the absence of IL-7;
thus, the suppressive mechanisms differ between mature T cells and immature DP
thymocytes. Moreover, persistent IL-7Rα expression on DP thymocytes could be
detrimental to T cell differentiation because it would interfere with the selection of a
TCR-dependent immunocompetent repertoire by providing indiscriminate survival
signals to immature thymocytes, regardless of their TCR specificity. Thus, precisely
timed and controlled IL-7Rα expression provides the molecular basis for a variety
of aspects in T cell development and, most prominently, contributes to lineage fate
decision of CD4 versus CD8 T cells.
1.7 Cytokine Signaling in the Post-Selection Maturation

of Thymocytes
Fixing CD4/CD8 lineage fate is not the final step in thymic differentiation, and
post-selection CD4SP and CD8SP thymocytes undergo further maturation before
they are exported to peripheral tissues. Indeed, freshly selected SP thymocytes
exhibit a semi-mature phenotype that is characterized by residual expression of
CD24 and having not yet induced expression of the maturation marker Qa-2
(CD24hi Qa-2lo ) [48]. The non-classical MHC-I molecule Qa-2 is commonly used as
a surface marker to identify the most mature thymocytes [101]. Qa-2 acquisition was
previously found to be mediated by Aire+ medullary thymic epithelial cells [57],
and recent studies further refined the underlying mechanism to be driven by intra-
thymic type I IFN signaling [109]. While the physiological role of Qa-2 on mature
thymocytes is yet to be defined, cytokines play a role in the terminal maturation of
thymocytes, as demonstrated by their requirement to upregulate Qa-2.
Maturation of post-selection thymocytes is also accompanied by increased
expression of the chemokine receptor CCR7 and the downregulation of CD69.
Employing these markers, late-stage maturation of post-selection thymocytes,
which are identified by TCRβ+ CCR7+ [98], can be further divided into three
distinct stages. Based on CD69 and MHC-I expression, CD69+ MHCI-I− thymo-
cytes are referred to as semi-mature (SM) cells, CD69+ MHCI-I+ cells as mature
1 (M1) cells, and CD69− MHC-I+ cells as mature 2 (M2) cells [109]. Rag2-
GFP reporter mice revealed a linear relationship among these populations, where
newly selected cells are SM that become M1 and then mature into M2 cells
[109]. Interestingly, maturation of post-selection thymocytes was accompanied by
an increase in IFN-signaling gene signature, such that STAT1 and IRF7 were found
in greater abundance in M1 and M2 cells [109]. Because medullary thymic epithelial
cells constitutively produce IFNβ [58, 72], these results indicated that medullary
thymocytes are exposed to and signaled by type I IFNs and that IFN signaling
would further drive the maturation and differentiation of post-selection thymocytes,
including the acquisition of Qa-2 expression [109].
Along these lines, the migration of positively selected thymocytes into the
medulla is a critical event in thymocyte maturation [25, 70, 92]. Both the negative
selection of auto-reactive thymocytes and generation of Foxp3+ Treg cells require
the thymus medulla [17, 91]. Moreover, the medulla, and particularly the cortico-
medullary junction, is thought to be an important source of intra-thymic cytokines,
such as IL-7 and IL-15 [45, 77]. Importantly, these γc cytokines not only specify
lineage fate but also act as potent pro-survival factors for post-selection thymocytes.
Upon positive selection, the pro-survival pathway of thymocytes is switched
from an RORγt-mediated mechanism, which drives anti-apoptotic Bcl-xL protein
expression, to TCR or cytokine receptor signaling-dependent mechanisms that
upregulate Bcl-2 protein expression [29, 49, 65]. In particular, Bcl-2 is induced in
MHC-II-selected thymocytes by TCR signals, whereas Bcl-2 in MHC-I-selected
thymocytes is upregulated by signaling of cytokines, specifically by cytokines
of the γc family [24]. Notably, it was recently demonstrated that CD8 lineage
specification can be uncoupled from pro-survival signals, so that two distinct classes
of “lineage-specifying cytokines” exist. The non-γc cytokines IL-6, IFNγ, TSLP,
and TGFβ specified CD8 lineage differentiation but lacked pro-survival function.
The γc cytokines IL-7 and IL-15, on the other hand, imposed CD8 cell fate and also
provided pro-survival signals by upregulating Bcl-2 [24, 67]. Thus, intra-thymic γc
cytokines, such as IL-7 and IL-15, are uniquely equipped to maximize CD8 cell
generation, as they not only direct CD8 cell fate but also maintain survival of the
newly generated CD8SP cells. Additionally, cytokines of the γc family can drive
the proliferation of post-selection SP thymocytes, for example, by IL-15, whose
absence results in a significant loss of BrdU incorporation in CD8SP thymocytes
[15], or by IL-21, whose in vivo administration results in a significant expansion
of mature SP thymocytes [79]. Additionally, IL-7 is reported to be required for the
intra-thymic expansion of post-selection thymocytes in neonates [30]. The extent to
which such cytokine-driven expansion of post-selection thymocytes plays a role in
determining the ratio of CD4 versus CD8 thymocytes is an interesting issue that still
needs to be resolved.
1.8 Conclusion and Perspectives
The generation of effector T cells in peripheral tissues is an actively investigated

area of research. In contrast, the mechanisms that drive the development and
differentiation of T cells in the thymus are less clear and remain controversial.
Nonetheless, the identification of lineage-specific “master regulators” that impose
either the CD4 or the CD8 lineage fate has greatly advanced our understanding
of the molecular circuitry in thymic cell fate decision. In this regard, we now
understand that the zinc finger protein ThPOK imposes CD4 lineage choice,
whereas the transcription factor Runx3 directs CD8 lineage differentiation [33,
95, 110]. However, the cellular signals that regulate the expression of the master
regulators themselves have remained unclear.
In T helper subsets, the expression of lineage-specifying transcriptional regula-
tors is primarily induced by cytokine receptor signaling [112, 119]. It seems that
the same principle also applies to CD4/CD8 lineage choice in the thymus where
cytokine receptor signaling induces the expression of the CD8-specifying factor
Runx3, but the absence thereof maintains the expression of the CD4-specifying
factor ThPOK [33, 95]. Thus, during T cell development in the thymus, TCR
signaling drives the selection, but cytokine signaling determines the lineage fate of
immature thymocytes. This concept is fully compatible with the original observation
that positive selection and lineage choice are temporally distinct events [42], and it
fully aligns with the prediction of the “kinetic signaling model” that posits cellular
signals other than TCR stimulation would drive the lineage choice and co-receptor
reversal of CD8 T cells [61, 88]. The intra-thymic signals that drive lineage choice
were initially proposed to be γc family cytokines, specifically IL-7 [10, 117]. Since
the original report, however, it has become clear that IL-7 is not unique in its ability
to direct CD8 lineage fate decision. Instead, certain non-γc cytokines can also direct
CD8 lineage choice, and there is not only some redundancy among γc cytokines but
also a compensatory effect of non γc-cytokines in CD8 cell differentiation [67, 74,
94]. These findings are notable because they can explain the appearance of CD8SP
cells in IL-7Rα- or γc-deficient mice, which still generate Runx3+ CD8SP cells
despite the lack of IL-7 and/or IL-15 signaling [67, 74, 94]. Nonetheless, whether
it is the signaling by other cytokines that can replace IL-7 or IL-15 signaling had
not been formally established. The definitive evidence demonstrating that cytokine
signaling is absolutely required to generate CD8 T cells was only provided with the
availability of CytoQuad mice that additionally lacked TGFβ receptor expression
[24]. In these mice, all lineage-specifying cytokines are absent. Consequently, CD8
T cell generation was completely abolished. These results unequivocally establish
that CD8 lineage commitment is a process that is dependent on cytokine signaling
and thus document an essential role for in vivo cytokines in directing thymocyte
lineage choice.
It remains to be assessed how cytokine signaling is controlled, so that CD4
lineage cells are excluded from lineage-specifying cytokine signals. Moreover, it
is also unclear whether quantitative and qualitative differences in cytokine signaling
produce CD8 cells with distinct function. In this regard, post-commitment IL-4
signaling induces the generation of IFNγ-producing innate-like CD8 T cells [39],
and aberrant IFNγ signaling was previously shown to redirect MHC-II-restricted
thymocytes into CD8 lineage cells [13]. Thus, there is certainly plasticity and some
lineage errors during CD4/CD8 lineage commitment, but these pathways also seem
to involve cytokine signaling. Because the developmental history of T cells in the
thymus is so much entangled with cytokine receptor signaling, it is not surprising to
find the same mechanism to operate T cell differentiation in mature T cells. In fact,
effector T cell differentiation in the periphery mirrors the developmental process in
the thymus which re-emphasizes the importance of understanding the molecular
mechanisms in thymic development to truly understand the differentiation and
effector functions of mature T cells.
Acknowledgments We thank Drs. Changwan Hong (Pusan University), Alfred Singer (NCI,
NIH), and Yousuke Takahama (NCI/NIH) for the critical review and comments. This work was
supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for
Cancer Research.
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Chapter 2
An Agent-Based Model of T Helper Cell
Fate Decisions in the Thymus
Sahamoddin Khailaie, Philippe A. Robert, and Michael Meyer-Hermann
2.1 Introduction
The recognition of conserved features among pathogens is not sufficient to face

all different types of threats that might occur throughout an individual’s lifetime.
In particular, evolving vertebrates are exposed to highly mutating pathogens that
can easily escape the detection by innate immune receptors. Despite providing
an immediate recognition and defense against pathogens, the innate immune
system cannot provide long-lasting immunity to a host, because it is limited by
germline encoded receptors. Unlike the innate immune system, the adaptive immune
system is made of receptors that are acquired throughout life by random somatic
recombination, such as the T cell receptor (TCR), expressed on T cells, and the B
cell receptor (BCR), expressed on B cells. This process allows a limited number
of genes to generate a repertoire of antigen receptors with enormous diversity
S. Khailaie
Department of Systems Immunology and Braunschweig Integrated Centre of Systems Biology,
Helmholtz Centre for Infection Research, Braunschweig, Germany
Centre for Individualised Infection Medicine, Hannover, Germany
P. A. Robert
M. Meyer-Hermann ()
Centre for Individualised Infection Medicine, Hannover, Germany
Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität
Braunschweig, Braunschweig, Germany
e-mail: mmh@theoretical-biology.de
© Springer Nature Switzerland AG 2021 21

C. Molina-París, G. Lythe (eds.), Mathematical, Computational and Experimental T Cell
Immunology, https://doi.org/10.1007/978-3-030-57204-4_2
22 S. Khailaie et al.
which could potentially recognize all pathogens present in nature. Consequently, the
acquisition of such new and random receptors allows the adaptive immune system
to be prepared for future and unknown challenges.
The initial repertoire carried by T cell progenitors has the harmful potential
to induce auto-immunity by the later recognition of protein fragments from the
host (self-peptides). It is therefore necessary that the T cell repertoire is shaped to
efficiently react to harmful pathogens (bacteria, viruses, parasites, and altered-self
such as cancer cells) while tolerating healthy tissues at the same time. This tolerance
is achieved in the thymus through a process called thymic selection.
After they express a properly recombined TCR, thymocytes sequentially
encounter various antigen presenting cells (APCs) each expressing diverse self-
peptides on their plasma membrane. Thanks to specific antigen presentation
mechanisms in the thymus, APCs present a large set of self-peptides (but not all) on
their MHC molecules. These self-peptides correspond to a subset of the proteins’
fragments of the individual healthy tissues. It is believed that thymocyte-APC
encounters and the quality of self-antigen recognition is the primary determinant of
cell fate decision in the thymus, namely, cell maturation and differentiation or cell
death [1, 2]. Thymocytes that complete the maturation steps leave the thymus as
major cell types of CD8+ cytotoxic T lymphocyte (CTLs) and CD4+ Tconvs which
contribute to the pro-inflammatory immune response or CD4+ CD25+ Foxp3+ Tregs
that suppress the immune response.
Here, we present the designing steps of an agent-based model for thymic selec-
tion, previously published in [3], which is suitable enough to result in the complex
emerging properties of thymic selection from simple mechanistic hypotheses. The
model provides a straightforward representation of signal processing by thymocytes
into their fate decision, in the thymus.
2.1.1 Affinity Model of Thymic Selection
The strength of binding between two proteins (affinity) is commonly represented

by a dissociation constant (Kd ). The Kd is the ratio of how easy it is to separate a
protein complex into its constituents to how easy it is to form a complex: a smaller
Kd indicates a stronger or higher affinity interaction. At the interface between two
interacting cells, the combined affinity of simultaneously interacting proteins is
referred to as avidity.
When we consider sequential cell interactions, we define the abundance of a
peptide as the fraction of APCs that express the peptide (Fig. 2.1). If a peptide is
highly abundant, thymocytes will interact with the peptide more frequently [4].
The classical affinity model of thymic selection considers that the cell fate of
a thymocyte is only determined by the affinity/avidity of its TCR toward the set
of self-peptide-MHC complexes (spMHC) encountered during thymic selection
2 An Agent-Based Model of T Helper Cell Fate Decisions in the Thymus 23
Fig. 2.1 Affinity, avidity and abundance of ligands. Affinity is a measure for the strength of
interaction between a TCR and a single peptide-MHC complex. The collective strength of
simultaneous binding of the identical TCR molecules on a single T cell to multiple peptide-MHC
complexes on the interacting APC is called avidity. Abundance of a self-peptide indicates the
fraction of thymic APCs that express the self-peptide
Fig. 2.2 Affinity/avidity model of thymic selection. (a) Most thymocytes (≈90% of pre-selection
cells) express TCRs that bind spMHCs too weakly, do not receive a survival signal and further
die by neglect (grey cells). Low affinity interactions of TCRs with spMHCs rescue cells from
death (blue cells), whereas high-affinity interactions induces cell death (red cells). Positively
selected thymocytes undergo cell maturation and migrate out of the thymus to enter the peripheral
T cell pool. (b) Modified affinity model of thymic selection. nTreg selection occurs within an
affinity-window between positive selection and negative selection thresholds. The boundaries of
this window can be subjected to stochastic influences. For example, in TCR-transgenic systems,
expression of a high affinity ligand may induce either nTreg or Tconv selection, or nTreg selection
and clonal deletion. Therefore, a broad range of affinities seems to be permissive for nTreg
differentiation, and the same TCRs can raise both nTreg and Tconv decisions [6]
(see Fig. 2.2a). According to this model, minimum affinity/avidity interactions are
required during positive selection for maturation of thymocytes and protection
against cell death. In contrast, during negative selection (also known as clonal
deletion), thymocytes that encounter high affinity/avidity interactions undergo
apoptosis (cell death) [5, 6]. The affinity model in its classical form does not state
how nTregs are selected.
2.1.2 Selection of Regulatory T Cells
TCR-transgenic mouse models were extensively used for studying thymic selection.
In these genetically engineered mice, all thymocytes express copies of an identical
αβ TCR, with the known specificity of the peptides inducing a high affinity. By
manipulating the antigen presentation in the thymus and by the expression of a
ligand with known TCR affinity via a second transgene, the role of affinity/avidity
in thymic selection has been investigated from different angles.
These studies showed that high-affinity interactions deviate clonal deletion to
nTreg differentiation, suggesting that nTregs would arise from thymocytes with
autoreactive (self-reactive) TCRs. It favors an improved version of the affinity
model in which nTreg selection occurs within an affinity-window between positive
selection and negative selection (Fig. 2.2b) [6].
Using an agent-based model of thymic selection [3], we show the outcome of
cell fate determination based on the integrated TCR signal over a sequence of
thymocyte-APC interactions. In the proposed model, not only the affinity/avidity of
interactions contribute to the fate decision, but the single cell history of self-peptide
encounters, and therefore the abundance of presented self-peptides.
2.2 Model
The model consists of agents (cells) each carrying a different, randomly generated
TCR, and aims at investigating which TCR sequences lead to which cell fate
decision. It requires to choose a representation of TCR, peptides, and MHC
sequences and to further derive the affinity between each TCR-pMHC interactions.
In this section, we try to make biologically relevant assumptions to incorporate such
properties in the model.
2.2.1 Representation of Proteins and Their Binding Affinity
Structural models for protein interactions are still far away from predicting the
affinity of any two interacting proteins in a reasonable time, that could be used at the
scale of several millions of cells going through thymic selection. Several simplified
representations of affinities of interactions have already been used in literature, such
as [7] in the context of B cells and antibodies. Probabilistic models take abstraction
of the sequences and derive affinities randomly from a distribution function [8, 9].
Each cell would get an average affinity for the pool of pMHC sequences. These
representations are quite limited for thymic selection because they do not take into
account sequence similarities between self-peptides or between TCRs and limit
the analysis of the output cells from thymic selection: which self-peptides do they
recognize? which self-peptides drove their selection or death? Here, we use binary
sequences as simplified structural representations of proteins [10–13]. They allow
representing a large diversity of TCR sequences and can be linked with an affinity
function that incorporates the critical properties of protein-protein interaction, such
as the synergy between neighboring interacting amino acids (domains) and the
capacity of similar sequences to have a similar affinity property.
Assume that the interaction interface of each of two interacting proteins (binding
regions) are represented by binary sequences X and Y , both with length L,
X = {xi }L
i=1 , Y = {yi }i=1 with xi , yi ∈ {0, 1}.
L
(2.1)
The following approach is used to calculate the affinity between two proteins.
The “matching sites” of two proteins are the sub-sequences of X and Y which are
complementing each other (opposite value). A binary sequence of matching sites,
called “Complementarity sequence” C, is defined as

1 xi = yi
C(X, Y ) = X ⊕ Y = {ci }L
i=1 with ci = . (2.2)
0 xi = yi
If such single point sites are adjacent to each other, they form matching sites and
therefore contribute more to the global affinity than other sparse and small matching
points. We list the lengths of consecutive 1s that exist in the complementarity
sequence C in another sequence named “Adjacency Matching Sites (AMS)”,
denoted by M(C), in the following way (shown by example in Fig. 2.3):
M(C)={mj } with mj =pj −qj +1 s.t.

{pj , qj ∈ [1, L] | (∀i ∈ [pj , qj ] : ci =1) ∧ (cpj −1 ⊆ 0) ∧ (cqj +1 ⊆ 0)}.
(2.3)
Here, pj and qj are the locations of the first and last 1s in j -th AMS. The global
affinity (A) of the interaction is defined as the sum of the sizes of the AMS raised to
power r, normalized to the maximum possible value,
r
j (mj )
A(X, Y ) = , (2.4)
Lr
The specificity parameter (r) allows tuning the relative contribution of the largest
AMS into the global affinity. This dependency is illustrated in Fig. 2.3. By increasing
the value of r, the dependency of the affinity to the largest AMS increases, which
results in a high sensitivity of affinity to a single bit alteration in this region of the
sequence and a dramatic decrease in the interaction affinity. Such a dependency
to single point mutations (key mutations) has also been observed experimentally
[14]. Note that the affinity A is not linked to the physiochemical properties of
protein-protein interactions (such as on-rate, off-rate, or dissociation constant) and
Fig. 2.3 Interaction affinity and signal integration [3]. The affinity between peptide-MHC
complexes and TCRs, both represented as binary sequences, is calculated by obtaining the
complementarity sequence of the two sequences (Ci (TCR, pMHCi ), see Eq. (2.2))/Algorithm (1),
and the adjacency matching sites (Mi (Ci ) – see Eq. (2.3)). The affinity depends on the grouping
effect of adjacency matches (Ai (TCR, pMHCi ) – see Eq. (2.4)). In the illustrated example,
according to Eq. (2.4), the contribution of the largest adjacency match to the affinity is 37.5%
with r = 1. This contribution increases to 71% with r = 3. A bit mutation in the center of the
largest adjacency match region decreases the affinity approximately by 12.5% and 66% with r = 1
and r = 3, respectively. See Table 2.1 for the simulation values
is only a measure of how well the binary sequence representations of proteins are
complementing each other. Therefore, the dimension of affinity A is arbitrary. The
algorithm to calculate the affinity of two binary sequences is given in Algorithm 1.
Algorithm 1 Affinity calculation of two binary sequences
1: procedure AFFINITY(Sequence X, Sequence Y, specificity r)
2: s ←− 0 Length of the current matching
3: sum ←− 0 Will be the accumulated affinity
4: L ←− length of X (and Y)
5: for i from 0 to L-1 do
6: if X[i] = Y [i] then
7: s ←− s + 1
8: else
9: sum ←− sum + s r Matching contributions added at their ending
10: s ←− 0
11: end if
12: end for
13: sum ←− sum + s r
14: Return sumLr
15: end procedure
2.2.2 TCR-pMHC Interaction Surface
The interaction between a TCR and a peptide-MHC complex occur through well-
defined domains. On the TCR side, these domains are called complementarity
regions (CDR1/2/3). The middle of the binding interface (CDR3) makes contact
with the peptide and the MHC. CDR1/2 loops surround the central CDR3-peptide
region and are in contact with the top of MHC helices [15–17]. While CDR1 and
CDR2 are mostly conserved, the CDR3 loop is a hypervariable element of the TCR
which mediates a major part of the interaction between MHC and TCR. The variable
part of the TCR binds to both the peptide and the borders of MHC surrounding the
peptide. Since the conserved part of TCR interacting with MHC would contribute
equally to each TCR-pMHC interaction, it is not considered in the model.
We assume that the binary sequences representing TCR and pMHC proteins
are aligned and the middle part of the TCR sequence with length Lp contacts the
peptide sequence, and the sides of the TCR sequence (each with length LMHC /2)
contact the MHC sequence. This is illustrated in Fig. 2.3. The affinity of TCR-
pMHC interaction is computed using Eq. (2.4),
ApMHC = A(TCR, pMHC). (2.5)
We obtain the TCR affinity to a MHC by considering the MHC binary sequence
without the loaded peptide and the corresponding part of the TCR binary sequence,
AMHC = A(TCR, MHC). (2.6)
In a similar fashion, the affinity of a TCR to a single self-peptide (sp) is computed

by using the binary sequence of the self-peptide and the corresponding part of the
TCR sequence
Asp = A(TCR, sp). (2.7)
The separation of TCR-pMHC interaction affinity to MHC and peptide affinities

is a read-out that cells can actually not perceive during only one interaction, because
the signaling is always induced by the combined affinity of the self-peptide and the
MHC to the TCR; it provides us with an index to characterize TCRs according to
their specificities to a particular MHC and peptide (higher affinities indicate higher
specificities). An example of computing the affinity of a TCR to a self-peptide and
MHC is shown in Fig. 2.3.
2.2.3 Signal Integration
Two main questions are ignored by the affinity model. First, how can a T cell
perceive information about the affinity of its TCR toward the MHC molecules
versus the self-peptides it encounters? Second, how is the abundance of self-
peptides shaping the repertoire of selected cells of each subset (Tconv and Treg),
knowing that a T cell cannot scan all APCs in the thymus? To accurately explain
the biological properties of thymic selection, namely, the elimination of thymocytes
with low affinity to MHC or self-reactive thymocytes, a temporal integration of the
perceived signal by thymocytes has to be incorporated.
2.2.3.1 Fate Decision Requires Multiple Interactions
Each T cell carries a unique TCR protein sequence, expressed as thousands of

copies [18]. Upon migration in the thymus, T cells continuously sample their
microenvironment. In a the course of few hours, T cells interact with a considerable
number of APCs [19]. In the context of a single TCR-pMHC interaction, the affinity
is low and the resulting intracellular signals such as ERK or calcium flux [20] are
insufficient for selecting or activating T cell. Signaling events need to be prolonged
for several hours in order to overcome an activation threshold [21]. In order to
represent the aforementioned time-dependent properties of signaling, we assume
that thymocytes accumulate TCR signals and quantify them by activation and
accumulation of downstream intracellular signals. This assumption is irrespective
of the nature of the presented antigens (self and foreign pMHCs).
2.2.3.2 Integrated TCR Signal
Here, a randomly generated TCR is assigned to each agent (a thymocyte) at birth

and will interact with a series of APCs over time. There is no representation of
space, and in silico APC scanning is made by randomly assigning an APC to a
thymocyte at each time point. The random process of generating TCR sequences
and cell interactions are explained later. If the considered number of APCs is lower
than the number of thymocytes, a thymocyte may stay without interaction until the
next time point. For each thymocyte, and at each time-point, the TCR signaling
level increases with the summed affinity (avidity) of all simultaneous TCR-pMHC
bindings at the current thymocyte-APC interaction, and decays with a constant rate
over time (see Fig. 2.4).
At any thymocyte-APC interaction, the single affinity-dependent TCR ligation
signal pi (t), induced via ligation of TCR with the i-th presented pMHC on the
interacting APC, is:
Fig. 2.4 Serial encounter. Thymocytes continuously scan the thymic environment and interact
with thymic APCs each expressing a different subset of the diverse spMHCs pool. Depending
on the number of APCs, thymocytes may compete for interaction with APCs. Depending on the
affinity/avidity of TCR to presented spMHCs and the number of available APCs, integrated signal
fluctuates over time. Integrated signals contain the TCR stimulation history. The extent of this
history depends on how fast integrated signals decay over time

Ai during thymocyte − APC interaction
pi (t) = (2.8)
0 no thymocyte − APC interaction
where Ai is the TCR-pMHCi interaction affinity computed by Eq. (2.4).

The avidity v of a thymocyte-APC interaction is defined as the summation of
affinities for all presented pMHCs on APC (nspMHC )
nspMHC

v(t) = pi (t) (2.9)
i=1
The integrated TCR signal S(t) (ITS) is then updated according to all TCR-
pMHC ligation events following the formula:
dS(t)
= αv(t) − δS(t) (2.10)
dt
where α is the integration rate (with dimension 1/time) and δ is the degradation
rate (with dimension 1/time) of TCR-mediated intracellular signals. The value of α
sets the scale of the signaling and does not influence the qualitative interpretation
of our results. The value of δ sets the extent of memory of the ITS, i.e., how many
past encounters are integrated in the value of ITS at any given time. To investigate
the information encoded into ITS from the limited past encounters, we assumed a
sufficiently high value for δ. The qualitative interpretation of the results are not valid
for a very low value of δ.
Since foreign peptides are not normally presented by thymic resident APCs, the
ITS of thymocytes will implicitly contain information about the level of TCR self-
reactivity. Now, the question is to understand how the ITS encodes self-reactivity
of the carried TCR toward the self-peptides and MHC molecules presented in the
thymus.
2.2.4 Simulation Settings: Thymocyte-APC Interactions

and Signal Integration
We consider a set of Nsp random non-identical sequences as the self-peptide pool,

which is only a subset of all possible presentable peptides (2Lp for binary sequences
with length Lp ); the remaining ones are considered as the set of foreign-peptides.
In this simplified model, we consider only a single type of MHC in the thymus,
called the selecting-MHC, and which is presented by all APCs. Peptide sequences
are combined with MHC sequences as shown in Fig. 2.3.
An initial population of nT thymocytes is generated and assigned to a subset of
TCR binary sequences (among the 2L possible ones) chosen uniformly at random
without replacement. As an illustration, the distributions of self-peptide affinities
with the parameters from Table 2.1 are shown in Fig. 2.8.
We consider nAPC number of APCs, each carrying nspMHC spMHCs randomly
from the pool of spMHCs. It has been experimentally observed that self-peptides
in the thymic cortical region are diverse and of low-abundance [22, 23]. To be
compatible with this observation, we make sure that the self-peptide diversity
is sufficiently larger than the carrying capacity of an APC. For mathematical
convenience, a low value for self-peptide diversity (Nsp ) and carrying capacity of
APC (nspMHC ) is considered. In order to keep sufficient self-peptide diversity per
APC when Nsp > nspMHC , we distribute self-peptides among APCs in such a way
that spMHCs are not presented twice on a single APC. In this case, each self-peptide
is presented, on average, by a fraction of APCs equal to
nspMHC
F = (2.11)
Nsp
which shall become sufficiently small. It has been estimated that each APC may
present ≈1% of all self-peptides (see [3]). Using the parameter values in Table 2.1,
we assumed that each APC is expressing 2% of all different self-peptides.
According to Eq. (2.11), when we decrease the diversity of self-peptides while
keeping the total number of ligands (spMHCs) presented per APC in the thymus
(nAPC and nspMHC and consequently nAPC × nspMHC ), the abundance of each self-
peptide increases (see Fig. 2.1 for definition of abundance of a ligand).
2.2.4.1 T Cells and In Silico Scanning of APCs
At any given time-point, we assign a random interaction event between thymocytes

and APCs. Then, for each thymocyte, we calculate the affinity of TCRs to the
presented spMHCs, and we update the integrated TCR signal (ITS) S(t). We assume
that the duration of all thymocyte-APC interactions t is identical. To ensure a
uniform distribution of self-peptides, a sufficiently large number of APCs has to be
taken (nAPC nspMHC ). When the number of thymocytes is equal to or smaller
Table 2.1 Thymic selection model: parameter descriptions and values

Parameter Value Description
Nsp 500 Diversity of self-peptides
nT 106 Number of different thymocytes/TCR sequences
nAPC 106 Number of APCs
α .01 Activation/accumulation rate of TCR signaling
δ 0.1 Degradation rate of TCR signaling
r 7 Specificity parameter
L 24 Sequence length of TCR and self/foreign-peptide-MHC
LMHC 8 Sequence length of MHC
Lp 16 Sequence length of self/foreign-peptides
nspMHC 10 Number of distinct spMHCs presented by each APC
S(0) 0 Initial condition of integrated TCR signals
t 0.5 h Thymocyte-APC interaction duration
Ts 500t Thymic selection time-window
than the number of APCs (nT ≤ nAPC ), thymocytes do not compete for interacting
with APCs. Therefore, the number of thymocytes (nT ) does not need to represent
a physiological number but rather is chosen according to the desired statistical
significance. In contrast, when the number of APCs is smaller than the number
of thymocytes (nT > nAPC ), thymocytes compete for interacting with APCs at
any given time and with a uniform probability, and a subset of thymocytes remains
without interaction at each time-step. For simplicity and focusing on the impact of
TCR specificity to self-peptides on the ITS, the results are given in the absence of
competition for interacting APCs. The impact of APC availability on the results is
given and discussed in [3]. The parameters used in the presented results are given in
Table 2.1.
2.2.5 Dynamics of Integrated TCR Signal and Fate

Determination
Herein, after a simulation with parameter values given in Table 2.1, we checked
the temporal properties of TCR signaling on the scale of single cell. In Fig. 2.5,
some typical ITS curves are shown. As it can be seen in this figure, ITS generally
shows fluctuations. However, due to repeated interactions with APCs and affinity
contribution from the MHC which presents a mixture of self-peptides, the signal
never goes to zero. In addition to steady fluctuations, strong signaling peaks in
ITS can be observed. By recalling the fact that all the APCs carry identical MHCs
in the simulation, one possible reason for strong peaks could be the encounter of
cell with high affinity peptides; another possibility would be the encounter of cells
with multiple peptides with moderate affinities presented by a single APC, which
Fig. 2.5 Integrated TCR signal (ITS) and fate mapping. (a) Dynamic changes of integrated
TCR signal of two different thymocytes are shown schematically. Sequential interactions of a
thymocyte with APCs which are presenting diverse spMHCs results in fluctuations of the integrated
TCR signal. Within a window of sequential interactions, the sustained signaling level (SSL) is
defined as the minimum value of the ITS. The maximum ITS value in the same window of
interactions is defined as the transient signaling level (TSL). The values of SSL and TSL for each
thymocyte depend on the specificity of their TCRs to encountered spMHCs. (b) SSL and TSL of
all thymocytes are plotted against each other as a scatter diagram. SSL- and TSL-based rules for
thymic selections are estimated and shown
creates a high avidity thymocyte-APC interaction; or, consecutive moderate affinity

interactions with APCs could lead to an increase of the TCR signaling.
In order to investigate the information contained in ITS dynamics, we propose to
decompose the signal of each cell into two distinct components: the basal value of
ITS after a stabilization time-window, called “Sustained Signaling Level” (SSL)
which is determined by the minimum value of the ITS; the peak value, hereby
called “Transient Signaling Level” (TSL), obtained as the maximum value of the
ITS during the APC scanning period. These signaling components will be used for
thymocyte fate determination.
2.2.5.1 SSL-Based Positive Selection
A sustained TCR-mediated signaling necessary for cell survival requires repeated

engagements of TCRs with spMHCs [24]. The sustained levels of TCR-mediated
signals (e.g., ERK, calcium flux) are associated with positive selection [25–31].
These observations speak for a positive selection that relies on sustained TCR-
mediated signals in the thymic selection process. In the context of our model, we
incorporate this notion of positive selection as the elimination (death by neglect) of
thymocytes that have low SSL. We neglect thymocytes that are unable to sustain
their ITS at a higher level than the positive selection SSL threshold (Fig. 2.5b).
Approximately 5% of all preselection thymocytes are able to mature in the thymus
and enter the periphery [32]. By considering that 50% of the positively selected
thymocytes are clonally deleted by negative selection [33], we assume that 90% of
the cells die by neglect during positive selection.
2.2.5.2 TSL-Based Negative Selection
Strong transient increase in TCR-mediated signals observed experimentally are

associated with negative selection and clonal deletion events [25–27, 31]. These
observations speak for a negative selection that relies on a transient signaling events.
In the model, we assume that all cells with a TSL above a certain threshold are
negatively selected and clonally deleted from the repertoire (Fig. 2.5b). The TSL
threshold of negative selection is estimated from the experimental observation that
50% of the positively selected cells are clonally deleted during negative selection.
2.2.5.3 SSL-Based nTreg Selection
In the presented study, we focus on the selection of CD4+ , and our aim is to
incorporate a signal-based nTreg fate commitment; however, the previous defini-
tions apply for both contexts of CD4+ and CD8+ T cell development. Various
experimental evidence has shown that selection of nTregs in the thymus depends
on interaction of MHC class II-restricted TCRs with high affinity/avidity ligands
[34–38]. Therefore, it is believed that nTreg commitment happens to cells that
express self-reactive TCRs [39] and, therefore, perceive stronger TCR signals than
their Tconv progenitor counterpart [40]. Further, it has been suggested that nTregs
originate from thymocytes expressing TCRs with self-peptide affinities/avidities
that lie between the affinities/avidities thresholds that drive positive and negative
selection [4, 41, 42].
The ability of nTregs to sustain high strength TCR signals is not only required
for nTreg differentiation but also for their homeostasis and functionality in the
periphery [43]. In the context of our model, we assume that the selection of nTregs
requires a sufficiently high SSL. We consider an SSL-threshold for nTreg selection
after which only 5% of the selected repertoire can be selected as nTreg phenotype
(physiological value: 5–10% of total peripheral CD4+ T cells [44] or 4–6% of total
CD4+ thymocytes in mice [45]) (Fig. 2.5b). The percentage values assumed for
thymic output are used to determine the SSL- and TSL-thresholds of positive and
negative selections in the model retrospectively.
2.3 Simulation Results
2.3.1 TCR Specificity to MHC and Cognate Peptide Are

Encoded into Different Components of the Integrated
TCR Signal
We performed our numerical simulations according to the flow chart shown in

Fig. 2.6 and Algorithm 2. SSL and TSL, the two components of the integrated
Fig. 2.6 Simulation flow chart
TCR signal, are obtained for all thymocytes after an initial stabilizing time. The
distribution of SSL for thymocytes with identical MHC affinity is shown for all
possible MHC affinities in Fig. 2.7a. It is evident that MHC affinity and SSL
are positively correlated, meaning that thymocytes with higher affinity to MHC
sequence
√ have higher SSL (Pearson correlation coefficient p between SSL and
r
A(TCR, MHC) is ≈0.79). This correlation does not exist in a similar extent
between TSL and MHC affinity (data not shown, p ≈ 0.55). A similar analysis
between TSL and the maximum affinity to thymic ligands for all thymocytes reveals
a positive correlation (see Fig. 2.7b, p ≈ 0.75), which is not present at similar
extent between TSL and MHC affinity (data not shown, p ≈ 0.39). These results
suggest that the information about the affinity of TCRs to the selecting MHC and
their cognate spMHC are encoded into different dynamical components of the
integrated TCR signal. This is mainly possible due to the existence of diverse and
low-abundance self-peptides. Decreasing the self-peptide diversity as well as the
number of available APCs destroy the correlation between TCR affinities and the
signaling components (data not shown, see [3]).
Algorithm 2 Main simulation steps for thymic selection
1: procedure THYMIC SELECTION(All parameters)
2: mhc ←− a random binary sequence (size LMH C )
3: peptideSet ←− [] the pool of self-peptides
4: while size(peptideSet) < NSP
5: newP ep ←− random binary sequence (size LP )
6: if newP ep ∈
/ peptideSet then
7: peptideSet ←− peptideSet ∪ newP ep
8: end if
9: end while
10: Initiation of peptide presentation
11: AP C− list ←− []
12: for i from 1 to nAP C do

13: v ←− shuffle [1, 2, 3, . . . , nS P ]
14: Shuffling avoids to pick the same peptide twice (if nSP > nAP C )
15: If nSP < nAP C .nspMH C , shuffle [1, 2, . . . , nSP , 1, 2, . . . , , nSP , . . .]
16: newAP C ←− empty APC
17: for j from 1 to nspMH C do
18: newAP C.presentedP ool ←−
19: newAP C.presentedP ool ∪ (mhc + v[j − 1])
20: ‘+’ inserts the peptide on the MHC, as shown in Fig. 2.3
21: end for
22: AP C− list ←− AP C− list ∪ newAP C
23: end for
24: T− list ←− []
25: v ←− shuffle [1, 2, 3, . . . , 2L ] avoids to pick the same TCR twice
26: for i from 1 to nT do
27: newT ←− cell with TCR sequence = binary(v[i-1])
28: newT .signal ←− S(0)
29: T− list ←− T− list∪ newT
30: end for
31: Now running a thymic selection
32: for t from 0 to TS by steps of t do
33: shuffle AP C− list and T− list
34: for i from 1 to min(size(AP C− list), size(T− list)) do
35: UpdateInteraction(T− list[i − 1], AP C− list[i − 1], t, t)
36: end for
37: end for
38: A posteriori selection of cells
39: Sort T− list according to their SSL
40: T hreshold− P os ←− 10% best SSL value
41: P osSel− list ←− T cells with SSL ≥ T hreshold− P os
42: T hreshold− Neg ←− 50% best TSL value among P osSel− list
43: N egSel− list ←− T cells with T SL ≤ T hreshold− Neg
44: Sel− list ←− P osSel− list ∩ NegSel− list
45: end procedure
46: procedure UPDATEINTERACTION(T cell T, APC cell A, t, dt)
47: for each peptide-MHC pm on the APC do
48: A ←− aff inity(pm, T .T CR)
49: T .Signal ←− T .Signal + α.A.dt − δ.T .Signal
50: if t >strabilization time then
51: T .SSL = min(T .SSL, T .Signal)
52: T .T SL = max(T .T SL, T .Signal)
53: end if
54: end for
55: end procedure
The data structure represent agents properties following the scheme:

each APC cell a ’presentedPool’ of peptides
each T cell a ’TCR’ sequence
a ’signal’ value (ITC)
a ’SSL’ and ’TSL’ value
2.3.2 Distribution of Affinity to Self-Peptides in Preselection

Repertoire Is Preserved in Tconv and Treg Repertoires
It has been suggested that regulatory T cells might be selected due to high specificity
to self-peptides. To check this, we derived the distribution of affinities to self-
peptides for each TCR and for all the TCR sequences in the preselection, Tconv,
and Treg repertoires (Fig. 2.8a). It is evident that the initial distribution of affinities
is preserved in both Tconv and Treg repertoires.
Another way to measure the effect of thymic selections on the affinity of
thymocytes to self-peptides is to obtain the fraction of thymocytes that recognize
a self-peptide with a certain affinity. Figure 2.8b shows the average of such measure
for all self-peptides. It is evident that the initial distribution is preserved in Treg and
Tconv repertoires.
(A) (B)
Fig. 2.7 Distributions of SSL and TSL; previously published in [3]. (a) Distributions of SSL for
thymocytes with similar MHC affinity shown for all MHC affinity spectrum. (b) The maximum
affinity of each thymocyte to the spMHCs pool is obtained. The distributions of TSL for
thymocytes with identical maximum spMHC affinity is shown for all affinity spectrum. The SSL
is positively correlated with MHC affinity while TSL is positively correlated with the maximum
spMHC affinity. Since proteins are represented by binary sequences, only a finite set of MHC and
spMHC affinities are possible. The thymocytes with close affinity values are grouped. This applies
to all subsequent figures
(A) (B)
(C) (D)
Fig. 2.8 Distributions of self-peptide and spMHC affinity. (a) For each thymocyte, the affinity
to all self-peptides is calculated and the affinity distribution is obtained over all thymocytes. (b)
For each self-peptide, the number of thymocytes with different binding affinity is obtained and
averaged over all self-peptides in the pool. (c, d) Similar analysis as of (a, b) for spMHCs in the
pool
A similar analysis for spMHCs (but not self-peptides alone) shows that the initial
distribution is not preserved after thymic selection (see Fig. 2.8c, d). Treg and Tconv
repertoires contain TCRs that recognize thymic ligands with higher affinity, or
in other words, thymic selection eliminates TCRs with low affinity to thymically
expressed spMHCs.
2.3.3 Thymocytes with Low MHC Affinity Are Neglected

in Positive Selection
Positive selection neglects a vast majority of preselection cells. It is believed that

the aim of positive selection is to select TCRs with sufficient affinity to selecting
MHC, such that they are functional to recognize foreign peptides in the context
of self-MHC molecules. Therefore, we check how the initial distribution of MHC
affinities of thymocytes is shaped by thymic selections. Figure 2.9a shows that
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the continuity of life; that suffering is inseparable from life; that all life
is suffering; and that suffering is to be gotten rid of by the
suppression of desires, and by the extinction of personal existence.”
Here he makes his first great contrast in the teachings of Christ
and Gautama, and says in part: “It is noteworthy that both
Christianity and Buddhism agree in asserting that all creation
groaneth and travaileth in pain, in suffering, in tribulation. But mark
the vast, the vital distinction in the teaching of each. The one taught
men to be patient under affliction, and to aim at the utter annihilation
of the suffering body.” Further: “But, say the admirers of Buddhism,
at least you admit that the Buddha taught men to avoid sin, and to
aim at purity and holiness of life! Nothing of the kind. The Buddha
had no idea of sin as an offense against God; no idea of true
holiness. What he said was, Get rid of the demerit of evil actions,
and accumulate a stock of merit by good actions. And let me remark
here, that the determination to store up merit, like capital at a bank,
is one of those inveterate propensities of human nature, one of those
deep-seated tendencies in humanity which nothing but the divine
force imparted by Christianity can ever eradicate. It is forever
cropping up in the heart of man, as much in the West as in the East,
as much in the North as in the South; forever reasserting itself like a
pestilential weed, or like tares amidst wheat, forever blighting the
fruit of those good instincts which underlie man’s nature
everywhere.”
He shows the contrast of Gautama and Christ; the former
claiming to be self-sent, having no divine commission and no
external revelation, while the latter claimed to be sent from God; to
be the Son of God, whose every word has divine authority; that the
gospel is to be proclaimed to every man in all generations, and that
Christ himself was the Way, the Truth, and the Life. He proceeds to
contrast the Christian Bible with the Buddhist: “The characteristics of
the Christian’s Bible are that it claims to be a supernatural revelation,
yet it attaches no mystic, talismanic virtue to the mere sound of
words. On the other hand, Buddhism utterly repudiates all claim to
be a supernatural revelation; yet the very sound of its words is
believed to possess a meritorious efficacy capable of elevating any
one who hears it to heavenly abodes in future existences. In
illustration, I may advert to a legend current in Ceylon, that once on a
time five hundred bats lived in a cave where two monks daily recited
the Buddha’s law. These bats gained such merit by simply hearing
the sound of the words, that when they died they were all reborn as
men, and ultimately as gods.”
We are given another contrast in the kinds of self-sacrifice taught
by the two systems. “But again I hear the admirers of Buddhism say:
Is it not the case that the doctrine, like the doctrine of Christ, has
self-sacrifice as its keynote? Well, be it so. I admit that he related of
himself that, on a particular occasion in one of his previous births, he
plucked out his own eyes, and that on another he cut off his own
head as a sacrifice for the good of others; and that again, on a third
occasion, he cut his own body to pieces to redeem a dove from a
hawk. Yet note the vast difference of the sacrifice taught by the two
systems. Christianity demands the suppression of selfishness;
Buddhism demands the suppression of self, with the one object of
extinguishing all consciousness of self. In the one the true self is
elevated and intensified. In the other the true self is annihilated by a
false form of non-selfishness, which has for its real object not the
good of others, but the annihilation of the Ego, the utter extinction of
the illusion of personal individuality.”
The doctrines which Christ and Gautama bequeathed to their
followers present an equally great contrast. From the vast difference
between them it is comparatively easy to believe the statement from
Christ that he brought a divine revelation, and from Gautama that he
was self-sent and had no revelation to make to his followers. The
contrast between the two systems has been arranged by the same
author.
“According to Christianity: Fight, and overcome the world.
According to Buddhism: Shun the world, and overcome it.
“According to Christianity: Expect a new earth when the present
earth is destroyed; a world renewed and perfected; a purified world
in which righteousness is to dwell forever. According to Buddhism:
Expect a never-ending succession of evil worlds coming into
existence, developing, decaying, perishing, and reviving, and all
equally full of everlasting misery, disappointment, illusion, change,
and transmutations.
“According to Christianity, bodily existence is subject to only one
transformation. According to Buddhism, bodily existence is continued
in six conditions, through countless bodies of men, animals, demons,
ghosts, and dwellers in various hells and heavens; and that, too,
without any progressive development, but in a constant jumble of
metamorphoses and transmutations.
“Christianity teaches that life in heaven can never be followed by
a fall to a lower state. Buddhism teaches that life in a higher heaven
may be succeeded by a life in a lower heaven, or even by a life on
earth or in one of the hells.
“According to Christianity, the body of a man may be the abode
of the Spirit of God. According to Buddhism, the body, whether of
men or higher beings, can never be the abode of anything but evil.
“According to Christianity: Present your bodies as living
sacrifices, holy, acceptable to God, and expect a change to a
glorified body hereafter. According to Buddhism: Look to final
deliverance from all bodily life, present and to come, as the greatest
of all blessings, highest of all boons, and loftiest of all aims.
“According to Christianity, a man’s body can never be changed
into the body of a beast, or bird, or insect, or loathsome vermin.
According to Buddhism, a man, and even a god, may become an
animal of any kind, and even the most loathsome vermin may again
become a man or a god.
“According to Christianity: Stray not from God’s ways; offend not
against his holy laws. According to Buddhism: Stray not from the
eight-fold path of the perfect man, and offend not against yourself
and the perfect man.
“According to Christianity: Work the works of God while it is day.
According to Buddhism: Beware of action, as causing rebirth, and
aim at inaction, indifference, and apathy.
“According to the Christian Bible: Regulate and sanctify the
heart, desires, and affections. According to the Buddhist: Suppress
and destroy them utterly, if you wish for true sanctification.
“Christianity teaches that in the highest form of life, love is
intensified. Buddhism teaches that in the highest state of existence,
all love is extinguished.
“According to Christianity: Go and earn your own bread and
support your family. Marriage, it says, is honorable and the bed
undefiled, and married life is a field on which holiness may grow and
develop. Nay, more: Christ himself honored a wedding with his
presence, and took up little children in his arms and blessed them.
Buddhism, on the other hand, says: Avoid married life; shun it as if it
were a burning of live coals; or, having entered on it, abandon wife,
children, and home, and go about as celibate monks, engaging in
nothing but in meditation and recitation of the Buddha’s Law—that is
to say, if you aim at the highest degree of sanctification.”
Then comes greatest of all distinctions, which separates
Christianity and Buddhism.
“Christianity regards personal life as the most sacred of all
possessions. Life, it seems to say, is no dream, no illusion. ‘Life is
real. Life is earnest.’ Life is the most precious of all God’s gifts. Nay,
it affirms of God himself that he is the highest example of intense life,
of intense personality, the great ‘I Am That I Am,’ and teaches us
that we are to thirst for a continuance of personal life as a gift from
him, nay, more, that we are to thirst for the living God himself and for
conformity to his likeness; while Buddhism sets forth as the highest
of all aims the utter extinction of the illusion of personal identity—the
utter annihilation of the Ego—of all existence in any form whatever,
and proclaims as the true creed the ultimate resolution of everything
into nothing.”
“What shall I do to inherit eternal life?” says the Christian. “What
shall I do to inherit the eternal extinction of life?” says the Buddhist.
Surely in this comprehensive list of contrasts the great scholar
has shown that there is an immeasurable height of moral and
spiritual philosophy, and revealed truth concerning God and man in
the Christian religion that Buddhism never conceived. It has no
excellence in moral precept that is not better stated by Christianity.
Christianity sheds a broad, clear light on the way to find salvation
from sin. Buddhism has no light, and no consolation. The human
heart finds rest in the one, but the other can not bring a moment’s
peace to any anxious or agonizing soul. Buddhism is a pessimistic,
dark, and desolate system of philosophy, mistaken for religion.
CHAPTER XI
Ripened Fruit of Non-Christian Faiths
I N setting before the reader the following account of painful

scenes, most of which I have witnessed, in connection with
religious rites, I am aware that some may say that these facts,
though admitted to have taken place, are not characteristic of the
religion in which they are found. Having given much attention to this
pertinent question, I am convinced that they are some of the
legitimate fruit of religious systems without Christ. This book is not
written to theorize about religion, so much as to give an account of
what a missionary sees living in Burma in direct contact with its
varied people.
The theoretical teaching of Buddhism, Mohammedanism, or
Hinduism may be one thing, while the practical religious usages may
bear quite another character. A people may naturally be very
agreeable and have many lovable traits, and yet their religious rites
may degrade and not uplift the natural man. The saddest part of the
following account is found in its degradation of the ordinary healthful
sentiments of the people, in the name of religion.
Then, again, what is done openly in the name of any religion, is
done that it may be seen and recognized as of that religion.
Therefore, it is certainly characteristic. If the observance of this is
repeated, or is related to that which is of frequent occurrence, it is
certainly characteristic. That which is here recorded is the natural
fruitage of the religions which cheat the natural hunger of the human
heart for the favor of God, whom all have sinned against, but whose
loving mercy is not known among these Christless millions.
Before giving an account of the cruelties still observed by
devotees and fanatics, it is well to remember some terrible practices
which have been abolished in recent years. These include suttee, or
widow-burning, hook-swinging, the Juggernaut, and marriage of little
girls. These were all religious practices, but they were abolished by
the Government. Theoretically and practically, the English
Government in India is neutral in religion. Only a Christian
Government could be strictly neutral in religious matters, though
other Governments at times have been tolerant of other faiths to
some extent. By proclamation, the English rule in India is neutral in
religion. This proclamation is adhered to literally, so that a
Mohammedan, Hindu, or Buddhist has just as much protection under
the law as a Christian, and neither has any powers above the other.
How, then, does it come to pass that the Government has
interfered in religious rites? This was done only when such rites
actually took life, or endangered life. The Government’s first duty is
to protect the lives of its subjects, even against self-destruction,
where that is possible. Many questionable deeds are yet done by
devotees in which the Government has not interfered, though some
of them are exceedingly cruel, because they have not actually
endangered life.
But suttee, or widow-burning, has been prohibited by the
Government, though still practiced among the Hindus beyond the
English border. Bishop Thoburn gives an account of the burning of
four widows in Nepal, a little more than a quarter of a century ago.
The dead husband had been high in the service of the Indian
Government, and had been honored with a title by Queen Victoria.
He was a Hindu, and he died over the border in Nepal, and four of
his widows were burned with his body. This shows the spirit of
Hinduism where it is not restrained by a Christian law. You can not
burn people on any pretext under the British flag.
Hook-swinging was another horrible practice, which was put
down in the same way. Devotees were placed on hooks suspended
on long ropes fastened high above, and the hooks fastened deep in
the flesh of the devotees. The body was then swung from side to
side till its momentum tore the hooks from the flesh, and the torn and
bleeding body fell to the ground, perhaps to die, certainly to be
permanently maimed. The law suppressed this practice, and it is no
longer perpetuated except in remote regions and under great
secrecy.
The car Juggernaut, with its great idol, when drawn along the
roads at the Juggernaut festival evoked such fanaticism that men
threw themselves under its great wheels, and were crushed. This
practice has been prohibited by law. The festival is still observed, but
men do not throw themselves under the wheels. I have seen the
great car, with its hideous idol, drawn past our mission-house in
Rangoon, attended by thousands of Hindus, with all the noise and
confusion of an Oriental procession; but there is no blood on its
wheels now, and no crushed bodies are left in the street.
There was another even more terrible custom prevalent in India
for ages, in which the Government had to interfere. It has for
centuries been the custom of Hindus to give their little girls in
marriage when tender children. They were married at ten or eleven
years of age, or even at nine years. To appreciate this monstrous
cruelty it should be remembered that a child of the same age in
Western lands is much stronger than a child in India. There were
many mothers in India at twelve years of age. The cruelties of this
practice of child marriage were such that they can not be written. Let
it be remembered that this practice existed for hundreds of years,
and that practically all the Hindus approved it, although the sufferers
were their own children. And when the terribleness of the practice
was so pressed on the Government that it could not avoid taking
action, and consequently framed a bill to raise the age of consent to
twelve years, before which it would be unlawful to consummate
marriage, the whole Hindu world rose up in protest. They charged
the bill to the oppression of the Government. Fifty thousand Hindus
met in public protest in the city of Calcutta. This protest was from
Hindus directed against a righteous law for the protection of their
children from this age-old cruelty!
To the writer it has been an experience of a painful kind to find a
few Americans crying out against the “Oppression of England in
India,” when they are only echoing the cry of the Hindus against the
righteous law. But the law was enacted, and has had a wholesome
effect so far as it is not evaded by false statements of the age of the
girls, which are often made.
But if England had no other justification for her Government in
India than these four enactments, the Government would have much
to its credit. But these are legal protections thrown around her
people to prevent them taking life, or perpetuating cruelty in the
name of religion. If monstrous things are still done, it is a comfort to
know that these named have been abolished.
Once each year a sect of Mohammedans torture themselves by
running through the fire. This torture occurs during the feast which
follows the Mohammedan fast, or lent. During this fast the
Mohammedan community eat nothing from sunrise to sunset. They
may eat a great deal after sunset and before sunrise. Having fasted
in this manner for forty days, they feast for three days. But this does
not include all of the community. There is a division of the
Mohammedans dating back to the death of Mohammed. A quarrel
then arose over his successor as leader of the Mohammedans. One
party held to Hassan and Hoosan, the sons of the prophet; and the
opposing party stood for another leader. This division led to war, in
which Hassan and Hoosan were slain and their party defeated. This
contention has been maintained until the present, and those of the
conquering party are known as Sunni Mohammedans, and the
followers of Hoosan and Hassan are called Shia Mohammedans.
The latter will not feast with the other party, and take this occasion to
torture themselves by running through the fire, as a protest against
the opposing sect.
The preparations for this torture by fire are made deliberately,
and it is carried out on a large scale. First of all, they must secure the
permission of the Rangoon magistrate to this ordeal. Then they
publish the places where it will occur, for it is celebrated in several
localities on the same night. Ditches are dug deep enough to hold a
great mass of coals, and two or more rods in length. On the day
appointed, a large supply of dry wood being provided, a fire is
kindled in the ditches about noon, and is kept burning until long after
dark. Meantime the ditch has been filled with coals smoldering, and
kept alive, but not allowed to burn to ashes. As the earlier hours of
the night have been passing, multitudes of all nationalities have
gathered at the scene of fire-running. They have to be kept at a
distance by a cordon of rope stretched about a considerable circle.
As midnight passes, a spirit of expectancy takes possession of the
waiting multitude, which is increased by shouts and excitement in a
side street not far away. These are all according to the Oriental’s
idea of working up a climax of interest in his spectacular display. A
little after midnight the fanatics who have been selected to undergo
this torture come rushing from a side street, carrying banners and
shouting in increasing excitement as they enter the arena and
approach the fire on the run. Crowding close together at the end of
the ditch of fire, they wave their banners, chant, shout, and shriek
until a frenzy possesses them, and then they plunge into the fire with
their bared feet and legs! The first man to leap into the fire sinks
more than half way to his knees in the fiery pit, and the next step
also, and so through the ditch. As might be supposed, he plunges
through as fast as possible with his greatest strides. He is followed in
turn by every other of the twenty or more fanatics of his company.
They immediately collect at the other end of the ditch, and with
even greater frenzy than before scream, stamp the coals from feet,
and plunge again through the ditch. So from end to end they rush till
the coals are dragged out of the ditch clinging to their feet and legs,
or are kicked out on the surrounding ground. Then the fanatics
disappear, and the multitude of curious onlookers disperse.
This cruel practice is carried out every year among these
Mohammedan immigrants to the province of Burma. The idea seems
to be a frenzied appeal to God that their contention, settled by the
sword in the defeat of their party twelve centuries ago, was right.
This particular revolting exhibition may be a modern development;
but if so, it is but another evidence of the growth of fanatical
extravagance of an imperfect faith. But viewed in any light, it is a sad
commentary on the Christless Mohammedan world in this twentieth
century of the Christian era. It is a fact to be lamented that Christian
missions are not generally directed to the adherents of the
Mohammedan faith.
Lest it be thought that such fanaticism is only representative of
the lower class of people, let this circumstance be noted. On one
occasion my wife saw this “fire-running.” Just before the expected
approach of the company designated to run through the fire, a finely-
dressed Mohammedan merchant came within the ropes, with the air
of a man who intended to act as a self-appointed master of
ceremonies over these fanatics, lest they act too outrageously. But
when the excitement of the occasion was on, and these common
people were rushing through the fire, he began to show every
indication of rising excitement. He sat down, rose up, sat down
again. He took off one shoe, jumped up, and sat down again; then
off came both stockings, and he plunged into the fire like any other of
these frenzied people. This shows the terrible power of such
enormities over even the self-poised Mohammedan merchant.
Another scene we witnessed among the Hindus, even more
revolting than this annual exhibition among the Mohammedans. It
was on a Sabbath-day, and Bishop Thoburn was with us on his
biennial visit to Burma. The early morning service at the English
Church had been concluded, and we were going to the mission-
house in the cantonments. The day was growing almost intolerably
hot, especially under the direct rays of the sun, as it was in the latter
part of April, the hottest time of the year. As we rode along under the
protection of our covered tum-tum, we saw just ahead of us in the
middle of the Signal Pagoda Road, the main street between the city
and the suburbs, an excited company of Hindu pilgrims and their
attendants. It is a striking fact, too, that the scene we beheld was
very near a Christian church located on that road—a Christian
church dedicated to the worship of Him who died for all men—and
here by the side of that edifice on that Lord’s-day, nearly twenty
centuries after the gospel was proclaimed to a sin-darkened world,
was enacted one of the most distressing cruelties of heathenism,
and it is doubtful if the devotees, or their attendants, even dreamed
of the salvation which every Christian temple should suggest! That
Church does nothing for missions, being content to preach only to
those who bear the Christian name.
There was a company of about twenty men, eight of whom were
devotees, while the others urged them on their terrible way. Around
each devotee’s neck was an iron ring supporting twenty-four small
chains about two feet in length. On the end of each chain was a
large hook made of wire, and these two dozen hooks were buried
deep in the chest, sides, and back of each poor man. The flesh was
raised in great welts over the buried hooks! But to add to the horrors
of this torture, each man had an iron rod about the size of a slate-
pencil thrust through both cheeks, passing through the mouth, of
course. Another rod of equal size pierced the tongue, which was
drawn out of the mouth as far as could be done without plucking it
from its roots, the rod holding it in that drawn condition, as it was
held against the face by the strained muscles. These hooks and iron
rods piercing the flesh of body and face must have produced all the
agony that the human frame could endure. Yet the cruelties of the
heathen could add to even this. Most of the poorer natives of India
go barefooted. But these devotees wore wooden sandals, not to
protect the feet, but to torture them. Through these sandals from
below nails were driven and sharpened above, so that every step
each poor man took the weight of his body pressed upon the
upturned nails, and must have produced a refinement of agony. To
the natural weight of the body was added a wooden arch often used
among this class of Hindus in Rangoon as a symbol, this arch being
carried on the shoulder and adding possibly twenty pounds to press
down his tortured body upon those upturned nails! These poor
deluded sons of our unhappy race, these devotees of a Christless
faith, were agonizing along this highway under a pitiless tropical sun,
making their way to a Hindu shrine eight miles away! Their condition
was indescribable. Their attendants were urging them forward with
shouts, and were dashing water on their protruding tongues,
seemingly untouched with pity at their agony.
The very sight of this torture made the heart sick. I doubt if any
Christian man could have endured the sight for any length of time.
An indescribable faintness began to sweep over me; and the bishop,
who has a heart of great tenderness, could hardly speak; but as he
turned to me I noted an expression of anguish on his face, as he
said in broken tones: “That is the worst sight I have witnessed in
thirty-five years in India; but that is the ripened fruit of idolatry.”
Let the reader again recall that this occurred in the closing years
of the nineteenth century of our gospel era. Let him also be informed
that among all these thousands of Hindu immigrants to India there is
not one missionary giving his time to preaching Christ. The only
reason there is not such a missionary is because there is no money
in any mission treasury to send him. There is plenty of money in
Christian hands. If the Christian men and women of those lands that
are the heirs of all temporal blessings, and of the Christian joys of
the gospel centuries, could realize the blackness of the night that
has settled down on the Christless nations, who are heirs of
thousands of years of increasing idolatry, they would hasten the
messengers of life and light to these poor people.
If we turn to Buddhism and ask for correspondingly desperate
conditions, we are at once assured that they are not found. Its
friends would assure us of its elevated character as a religion. But I
am sure we find a sad enough condition among some of the most
faithful Buddhists, and a refinement of cruelty in all classes of the
adherents of the teachings of Gautama. The building of a pagoda is
regarded as the most meritorious deed, and even its repair or partial
regilding gives a man honor and merit. But the serving of a pagoda
renders a man an outcast. The only real outcast ever recognized in
Burmese Buddhism, which is free from the Indian caste system, is
the “pagoda slave.” Perhaps we ought to speak of this in the past
tense, as the English rule has made it possible for these slaves to
find their freedom, which was impossible under Burmese rule,
though even this legal liberty is not recognized by the Buddhists.
Under Buddhist, or Burmese, order, whole families were set
apart for the pagoda service. Once in that service they were
despised by their self-respecting co-religionists, and their children
after them forever suffered their disabilities. Sometimes a certain
number of families in a village were arbitrarily picked up and set
down at the pagoda for this purpose, henceforth to be banished from
the circle of respectability. Never could any man get out of this
degraded service. The heaviest penalties were laid upon any who
tried to aid him or his family to escape to any other calling.
Why this strange degradation of men and women who serve the
pagoda, when the building of a pagoda exalts its builder here and
hereafter, does not seem to be explained. Personally, I think it one of
the stony-hearted cruelties natural to this faith. The priests were fed
daily out of the household’s store; but the pagoda caretaker had to
fight with the ownerless dogs, with which Burma abounds, for some
of the food offered to the images of Gautama! When the slave died
he could not have respectable burial. Sometimes his body was
thrown out with the refuse for the dogs to eat. This settled policy of
Buddhism may be truly said to be one of its perfected fruits, not
mentioned by the friends of the faith from Western lands.
Under British rule these people were allowed to become sellers
of supplies for the pagoda service, or to go away where, unknown
and carefully disguising their former life, they might work into some
respectable occupation, but never with the consent of Buddhist
priests or self-respecting laymen, who had always despised the
servant of the temple. But the sight which always met the observers
when visiting the great Sway Dagon Pagoda in Rangoon until recent
years, was the line of lepers always piteously begging along its
ascending steps. They were classed with the pagoda slave, and
were despised. It can not be said that they were really pitied, even
though some corn or rice and an occasional copper coin were
thrown them by the Buddhist climbing the stair to Gautama’s shrine.
These lepers were born on these steps, diseased, rotted, and died
there! A more pitiable sight was never witnessed than these poor,
suffering creatures, practically expelled outside the bounds of
Buddhist sympathy for no fault of character or conduct. Of course,
the Buddhist would say that the leper’s foul disease was the demerit
of some past existence, and therefore he suffers justly in his
loathsome condition, and his ostracism from human sympathy! But
this is only another heartless invention of a much overrated religion.
The reader will have made the contrast. Centuries of labor and
millions in gifts to raise and perfect the great pagoda and to gild it
and to bejewel its tinkling bells, all in honor of eight human hairs;
while its own faithful adherents suffer and die without so much as a
shed being built to shelter them! Millions for gilding brick and mortar,
and not the least coin to build a hospital for suffering and despairing
men! This is Buddhism’s fruitage of the centuries. Let him praise the
tender sympathies of this faith who will. To me it is one of the most
heartless systems taught among men. The leper was an outcast
here, and taught to believe millions of years of existence in hell were
awaiting him hereafter. This was his portion in Buddhism.
Turn now and witness what Christianity has done for him. Within
the decade of my writing, the Christian missions of Burma became
strong enough to put their sympathies underneath the long suffering
lepers. A Scotch leper mission is aiding the Wesleyans at Mandalay.
Later the Baptists in Moulmein, and the Catholics on their own
account in Rangoon, built leper asylums, the Government aiding also
in their support; and the lepers in every municipality in Burma have
been gathered into these Christian institutions, their sores bound up,
medicines to alleviate their sufferings given, abundant food and
suitable clothing provided, the first time to most of them during their
agonized lives. Best of all, the gospel, with its help, love, and hope,
is preached to them who had been bound to suffering for ages to
come by their own religious system! If to the question, “Do missions
to the Burmese Buddhists pay?” there could be offered only these
three leper asylums, they would warrant the answer, “Yes.”
One very hot afternoon I went with an assistant of the Wesleyan
Leper Asylum, and took twenty-five of those lepers off the steps of
the Sway Dagon Pagoda, and securing their passage money from
the Rangoon municipality, I sent them off to Mandalay. Later I visited
that city and the asylum, and there beheld one hundred and forty of
the lepers gathered from many places in Burma. They were so well
cared for as to seem almost content in spite of their physical
suffering. They were fed, clothed, and had the gospel preached to
them in the true Christian spirit. Some of them were filled with peace
and joy by conscious communion with Him who is so mightily
preached by this institution of mercy. There was one leper, with
hands and feet fallen off, an eye gone, and the tongue nearly eaten
away; still on the piece of a face that remained there shone a light
seen only on the face of the man who communes with God. In this
glad vision I had my reward in having aided in a small way to send
so many afflicted ones to this Christian haven. A thousand times
since I have been made glad with the memory of what seemed that
hot afternoon a very commonplace piece of work. But as it retreats
into the past, and I know every poor sufferer will have had Christian
care all his days, it came to be recognized as one of my greatest
privileges as a missionary to have had some part in this work.
When in the future the traveler comes to Burma and visits the
pagoda, and when the resident passes through Rangoon’s streets,
and is not pained with the vision of lepers begging at his feet, let
them remember that for centuries these Buddhist lepers were
spurned by their own race and countrymen, and that it was the
Christian missionaries who gathered them into homelike asylums,
there to receive loving Christian care. Let them reflect that this
contrast is one of principle in the two faiths. The leper, agonizing in
hopeless despair on the pagoda steps, was the perfected fruit of
centuries of the teaching of the purest Buddhism to be found.
One more illustration of the practical teaching of Buddhism
presents itself. At Kemmendine, near Rangoon, is a Buddhist burial-
ground. There is a large pavilion near the entrance to the graveyard,
on the ceiling of which there have been various pictorial
representations of the teachings of Buddhism. Much of this is a
portrayal of the many Buddhist hells. But there was for years a
succession of pictures along one border representing the Buddhist
priest in the process of crushing out all sentiment and sympathy with
even the greatest human distress. The candidate for neikban must
destroy all desire; the last desire to give way is that for existence.
This pictorial representation was evidently made to show the process
of this suppression in progress. The yellow-robed priest, who should
represent the system, is seated in perfect composure looking on the
distress of a sick man. There is none to attend the sick, and the
priest, of course, gives no aid. The next picture shows the man
approaching the crisis of death; in the next he is actually dying. Then
follows a succession of pictures showing other stages of dissolution,
until only the scattered bones remain. Through all the series of
representations the priest sits with a face as expressionless as
marble, and has not moved a muscle. Complete indifference to all
experiences of human life is the virtue aimed at.
To show that this crushing out of all natural sensibility is a difficult
process, the artist has made another picture with a little humor in it.
The scattered bones suddenly become articulated, and the skeleton
makes a wild leap upon the priest. This unexpected jump of the
skeleton would be calculated to affright ordinary mortals to a degree;
but not so the priest. He only slightly turns his head. He has nearly
conquered all natural sentiment. The last picture shows a skull and
crossbone, and the motionless priest sitting in perfect composure of
features and of person. He has conquered all desire!
This is the picture on the pavilion; but now with the writer look on
the living reality. On one occasion I attended a cremation in that
burial-ground. Sometimes bodies are buried, and sometimes
cremated if the person was rich or much respected in life. The
funeral pyre was crudely made, and the burning presented a
revolting sight that need not be given in detail. When the body was
nearly consumed some of the people returned to the pavilion, and
with them the widow, a grown son and daughter, and some smaller
children of the deceased. Meantime five priests had come in from
the monastery, and sat in a row upon a platform at one end of the
structure. They had not been present at the place of burning. They
had rendered no service of consolation at all, though they may have
preached at the home the usual pronouncement of Buddhism, that
all existence leads to misery, and therefore the way out of misery is
to strive to get into neikban and cease to exist. More than this, the
funeral is the occasion over all others in which costly presents far
above the ability of the family are given to the poungyis, or priests.
But real consoling service to the sorrowing they give none. There is
nothing springing from sympathy, pity, or hope in this religion.
As these five well-fed priests sat on that platform, the broken-
hearted widow, son, and daughters came forward, and bowed down
and worshiped them. The bruised and broken human heart cried in
anguish and must cry for help, and Buddhism offers only the worship
of a yellow-robed priest! Buddhism has no God. It tries to crush all
human pity. But where shall the broken-hearted find rest? Worship
these yellow-robed priests! That is all. What about the priests? There
they sat, and chewed betel-nut and tobacco, spitting lazily at the
cracks in the platform, looking about idly and vacantly, utterly
indifferent to the prostrate and broken-hearted family before them!
Not a look of sympathy or pity, not even a glance of recognition cast
in the direction of the prostrate forms! This is the very real illustration
in the living priest, of the pictorial representation of the priest of the
Buddhist religion given above! The Buddhist system is devoid of
hope, pity, consolation, or even ordinary human sympathy. It is as
heartless as its own stony or brazen images of Gautama so
universally worshiped. Men become like their gods.
Festivities at a Poungyi’s Cremation

CHAPTER XII
Outline of Christian Missions in Burma
C HRISTIANITY entered Burma with the incoming of traders and

Governments from the West, as it did in the other portions of
Southern Asia. Some of these traders and some of these
representatives of nominally Christian Governments were
unscrupulous adventurers. They were seldom men of strict moral
integrity, much less spiritual Christians. They would not measure
very high by present-day social standards, much less by the lofty
standards of the gospel. They were Christians in name only. The
Portuguese gained some footing in Burma, as in all this tropical
world, in the sixteenth century, and continued with varying influence
for nearly two hundred years. They gained a foothold in Burma, and
built a city called Syrian, and still bearing that name, just across the
Pegu River from the present site of Rangoon. Here, however, they
met utter defeat and destruction at the hands of the great Alombra,
the founder of the Burmese Dynasty, of which King Thebaw,
deposed at Mandalay in 1886, was the last sovereign. This great
king completely overthrew the Portuguese Government, and with it
the only form of Christianity then known among them, that
represented by the Roman Catholic Church of that day.
These venturesome Portuguese carried with them their religious
observances, and their priests always were active missionary
agents. They built a church at Syrian, the well-defined ruins of which
still remain. They made converts among the Burmans. Whether their
methods in converting the Burmese were as unscrupulous as the
Portuguese traders’ methods were, we have no detailed account.
But in this matter we are not left in much doubt, as their attempts to
convert the Burmese could not have been much different from that
employed in India at the same period, which unhappily we know only
too well were in utter disregard of the true Christian spirit. But these
missionaries of the Catholic Church counted their converts by
possibly the hundred thousand.
These foreigners on their shores were then, as all are now,
regarded as representatives of the Christian religion. In Asia every
man is regarded as an adherent of some religion. Little account is
taken of whether he represents his faith or misrepresents it. His
Government and his personal and social life are supposed to flow
from his religion; so that the Christian religion, as represented by the
Portuguese, was probably despised as cordially by the Buddhists as
their commercial prestige and Government authority were hated by
the Burmese people. And when they made war to the extermination
of the Portuguese settlements and their fortified city, and sunk their
ships in the river, they also dealt a fatal blow to Catholic missions in
Burma. There remains little trace of their old-time teaching. This is
very unfortunate; for it is only just to say that even the Catholicism of
two centuries ago taught more truth concerning God and God’s
dealings with men than Buddhism ever did in its best state. Nominal
Christianity, even when half idolatrous, kept the name of God alive in
the minds of men, while they waited for a better day in which the
gospel would be preached in its purity.
With the beginning of the nineteenth century the British began to
get control of Burma, and this advantage has been followed by
further acquisitions of territory until in 1886 Upper Burma was
annexed, and so the whole land has come under the English flag.
During this century, with its better Governmental conditions, the
Catholic Church has re-established its institutions in all parts of the
province. For many years its growth was slow, and even now, in
numbers, it is far exceeded by the Baptist mission. But in the last ten
years there has been a marked growth in the Catholic community.
This has been to some extent by immigrants from India, who are
Catholics. But the Church has been aggressive in every respect. Its
priests are multiplying in the cities, and they also have occupied
strategic points in the interiors. They are especially far-seeing in the
acquisition of great properties in cities like Rangoon and in building

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Mathematical Computational and

Experimental T Cell Immunology

Current Problems in Experimental and Computational

Theoretical and Computational Physics of Gas Discharge

Differential Equations Mathematical Modeling and

Experimental and Computational Investigations in

Computational and Experimental Simulations in

A Mathematical Modeling Framework to Simulate and

Analysis of Legal Argumentation Documents A

Mathematical, Computational Intelligence and

ISBN 978-3-030-57203-7 ISBN 978-3-030-57204-4 (eBook)

© Springer Nature Switzerland AG 2021

1 Cytokine Receptor Signaling and CD4/CD8 Lineage Choice

10 Experimental and Mathematical Approaches to Quantify

Megan A. Luckey and Jung Hyun Park

M. A. Luckey · J. H. Park ()

© Springer Nature Switzerland AG 2021 1

1.2 T Cell Development and CD4/CD8 Lineage Choice

It was thus surprising when it was discovered that the downregulation of

1.3 Cytokine Requirement in T Cell Development

restore proper thymocyte differentiation in the absence of γc signaling [60, 81].

1.4 Cytokine Signaling in the CD4/CD8 Lineage Choice

Pre-selection DP thymocytes are precluded from intra-thymic cytokine signaling

1.5 Cytokine-Driven Molecular Circuitry of CD4/CD8

lineage-specifying cytokine would contribute to CD8 cell differentiation. However,

1.6 IL-7 Receptor Expression during T Cell Development

1.7 Cytokine Signaling in the Post-Selection Maturation

1.8 Conclusion and Perspectives

The generation of effector T cells in peripheral tissues is an actively investigated

1. Abe A, Tani-ichi S, Shitara S, Cui G, Yamada H, Miyachi H, Kitano S, Hara T, Abe R,

3. Akashi K, Kondo M, von Freeden-Jeffry U, Murray R, Weissman IL (1997) Bcl-2 rescues T

Sahamoddin Khailaie, Philippe A. Robert, and Michael Meyer-Hermann

The recognition of conserved features among pathogens is not sufficient to face

© Springer Nature Switzerland AG 2021 21

2.1.1 Affinity Model of Thymic Selection

The strength of binding between two proteins (affinity) is commonly represented

2.1.2 Selection of Regulatory T Cells

2.2.1 Representation of Proteins and Their Binding Affinity

M(C)={mj } with mj =pj −qj +1 s.t.

2.2.2 TCR-pMHC Interaction Surface

ApMHC = A(TCR, pMHC). (2.5)

AMHC = A(TCR, MHC). (2.6)

In a similar fashion, the affinity of a TCR to a single self-peptide (sp) is computed

Asp = A(TCR, sp). (2.7)

The separation of TCR-pMHC interaction affinity to MHC and peptide affinities

2.2.3 Signal Integration

2.2.3.1 Fate Decision Requires Multiple Interactions

Each T cell carries a unique TCR protein sequence, expressed as thousands of

2.2.3.2 Integrated TCR Signal

Here, a randomly generated TCR is assigned to each agent (a thymocyte) at birth

where Ai is the TCR-pMHCi interaction affinity computed by Eq. (2.4).

2.2.4 Simulation Settings: Thymocyte-APC Interactions

We consider a set of Nsp random non-identical sequences as the self-peptide pool,

2.2.4.1 T Cells and In Silico Scanning of APCs

At any given time-point, we assign a random interaction event between thymocytes

Table 2.1 Thymic selection model: parameter descriptions and values

2.2.5 Dynamics of Integrated TCR Signal and Fate

creates a high avidity thymocyte-APC interaction; or, consecutive moderate affinity