T-CELL MATURATION, ACTIVATION, AND

DIFFERENTIATION
T Lymphocytes
 all express CD3- antigen specific receptor (TCR)
  TCR, about 10% in blood
  TCR, about 90% in blood: ~2/3 express CD4, ~1/3 express CD8. All
mature T cells express one or the other
 CD4+ = T helper cells, regulatory T cells- Secrete cytokines
 CD8+ = cytotoxic T cells- Lyse infected cells, secrete cytokines
 Thymic output of naive T cells declines with age, and the thymus atrophies.
Therefore, older people have a reduced ability to respond to new infections.
However, the total number of T cells does not change, there are just more
memory cells.
 ANTIGEN RECOGNITION
 only recognize processed antigen presented at the surface of another cell
using T cell receptor
 • antigen is presented by an MHC molecule
 Where do T cells come from?
• Multipotent Lymphoid
Progenitors Migrate from the
Bone Marrow to the Thymus

• In the Thymus, the Lymphoid

Progenitors Differentiate to pre-T
Cells and are Educated to
Differentiate Self from Non-self

• Positively Selected T Cells

Emigrate from the Thymus to
Mediate and Effect the Cognate
Immune Response
The Thymus

• Full of lymphocytes, but no immune

response to infection
• T cell precursors; PROGENITOR
CELLS, develop in the bone marrow
and migrate towards the thymus in
the circulation
• Maturation of the Thymocytes occurs
from the CORTEX to the
MEDULLA
• Mature THYMOCYTES/T cells then
are transported out of the thymus and
around the body via the circulation
T cell development
 Progenitor T cells migrate to thymus
○ At about 8th or 9th week of gestation in
humans
 T cell maturation involves
rearrangements of the germ-line TCR
genes
 In thymus, thymocytes proliferate and
differentiate
Ikaros controls commitment to Lymphoid lineage

Ikaros - transcription factor that regulates

gene expression, particularly in the
commitment of precursor cells to the lymphoid
lineage.

Ikaros controls the expression of genes

involved in the development and function of T
cells, B cells, and natural killer cells, and its
expression is required for the proper
differentiation of lymphoid progenitor cells
into mature immune cells.

Notch 1 is required for T cell lineage

commitment
 Progenitor T cells
migrate from bone
marrow to thymus
 T cells can be grown
in vitro in absence of
thymic fragments
 Grown on bone
marrow stem cells
with Notch protein
 Notch protein is key
in determining T-
lineage specification
Development
1. T cells are CD4- and CD8- (they express neither; double negative)
2. In the cortex, the T cells express a TCR precursor (pre TCR; β + “surrogate” αTCR)
3. In the medulla, ~1010 different αβTCR’s created by gene rearrangements. The generated
TCRs will only express either CD4 or CD8
• Due to these random gene rearrangements, many of the generated T cells will be “SELF-
REACTIVE”, therefore these must be destroyed
T-cell Development
 Begins with arrival of small numbers of
lymphoid precursors migrating from blood to
thymus
○ When they do arrive in thymus, T-cell precursors don’t
express signature surface markers (CD3, CD4, and CD8)
○ Do not express RAG-1 or RAG-2 that are necessary for gene
rearrangement
○ During 3rd week development, differentiating T cells pass
through stages of development based on surface phenotypes
DN = Double negative
CD4- and CD8-

DP = Double positive
CD4+ and CD8+

C-kit – receptor for stem cell growth factor

CD44 – an adhesion molecule
CD25 - alpha chain of IL-2 receptor
DN1 Cells in the Thymus Are Not
Committed to become T Lymphocytes

 TCR loci are in

germline configuration
 Cell can differentiate to
become a B
lymphocyte, Natural
Killer cell, or Dendritic
cell (Michie et al, JI, 2000;
Ikawa et al, JEM,
1999;Sanchez et al, JEM,
1994)
The DN2 Subset of Thymocytes are More
Committed, but Not Quite Exclusive

 TCR loci are in

germline configuration
 DN2 thymocytes may
give rise to dendritic
cell(Moore et al, 1995; Wu et al
1996; Ardavin et al, Nature 1993;
Shortman et al, Imm Rev. 1998)

 DN2 thymocytes no
longer differentiate to
the NK cell or B
lymphocyte lineages
DN3 Cells are Committed to the T Lineage

 Downregulation of
CD44 expression
 Upregulation of
RAG genes
 V-D-J
recombination of
TCR chain locus
 Expression of pre-
TCR chain
After Assembly of a Functional pre-TCR

 Shut down of TCR rearrangement; TCR

allelic exclusion
 Onset of proliferation/expansion
 Differentiation to DN4, CD8ISP, and then
DP
 Targeted Gene Mutants Unable to
Mature to the DP Stage

Pre-TCR is just there to indicate that the cell has rearranged TCRb
in frame so that these differentiate into DP
T Lymphocyte Maturation in the Thymus
T cell Activation
 Initiated by TCR-CD3 complex with processed antigen on MHC molecule
○ CD8+ cells with Class I
○ CD4+ cells with Class II
 Initiates cascade of biochemical events
○ Inducing resting T cell to enter cell cycle, proliferate, differentiate into
memory and effector T cells
 Cascade of biochemical events leading to gene expression:
 Interaction of signal and molecule (example: TCR + MHC and antigen)
 Generation of “second messenger” that diffuses to other areas of cell
 Protein kinases and protein phosphatases are activated or inhibitied
 Signals are amplified by enzyme cascades
 Gene products after activation
○ Immediate genes (1/2 hour of recognition)
 Transcription factors (c-Myc, NFAT, NF-κB)
○ Early genes (1-2 hours from recognition): IL-2, IL2R, IL-6, IFN-γ
○ Late genes (more than 2 days later): Encode adhesion molecules
T-cell differentiation
 T-cell differentiation is the process by which immature T cells, called
thymocytes, mature into functional T cells with specific immune
functions.
 This process occurs in the thymus and is characterized by several
stages of development, including positive and negative selection, as
well as the acquisition of T cell receptor (TCR) specificity and effector
functions.
 Positive selection occurs when thymocytes interact with self-MHC
molecules presented by thymic stromal cells and receive signals to
survive and mature.
 Negative selection involves the deletion of T cells that have high affinity
for self-antigens to avoid autoimmunity.
 T-cell differentiation results in the formation of CD4+ T cells and CD8+
T cells, which have distinct functions in immune regulation and defense
against pathogens.
Selection process in thymus

 Positive selection
○ Survival of only T cells whose TCRs recognize
self-MHC molecules
 Negative selection
○ Eliminates T cells that react too strongly with
self MHC or MHC with self-peptides
Positive Selection
Negative Selection
 Positive selection is the process of
 The cells that survive the positive
movement of double-positive T cells
selection move into the medulla
(CD4+ and CD8+) to the cortex, where
and undergo negative selection,
they encounter self-antigens.
which eliminates thymocytes with
 The thymic cortal epithelial cells a high affinity for self-antigens.
express self-antigens on MHC
 The cells that interact too strongly
molecules where the T cells interact
with the self-antigens receive an
with the molecules. The cells that do
apoptotic signal resulting in cell
not interact with the molecules strongly
death.
enough die whereas others with high
affinity to MHC cells survive.  During the same process,
however, some cells are selected
 A large portion of the developing
to form Treg cells.
thymocyte die during the process,
which lasts for number of days.  The cells that successfully
complete the selection process
 In the positive selection, CD4+ T cells
exit the thymus as mature naïve
interact with class II MHC molecules
T cells.
and CD8+ T cells interact with class I
MHC molecules.
Selection
Occurs during interaction with macrophages and dendritic cells within
the thymus. Only useful cells leave the thymus.
Pre TCR checkpoint
- Is the new β chain functional?
- No: Death by APOPTOSIS
- Yes: Survival and development to CD4+ CD8+ αβ TCR+
Post TCR checkpoint
- Is the αβ TCR functional?
- Is the αβ TCR dangerous/autoreactive?
- Useless: cannot see MHC – die by apoptosis
- Dangerous: see “self”, i.e. host molecules – receive signal to die by apoptosis, i.e.
NEGATIVE SELECTION
- Useful: binds weakly to MHC molecule – receive signal to survive, i.e.
POSITIVE SELECTION
- Note: only 5% of thymocytes survive selection
Fates Awaiting a DP Thymocyte

Default
T cell development is expensive for host
(95%) 98% of all thymocytes do not mature, die by
apoptosis within thymus
T-Cell Differentiation
 CD4+ and CD8+ cells leave thymus and enter circulation in
G0 phase
○ Naïve cells (condensed chromatin, little cytoplasm)
○ About twice as many CD4+
 Naïve cell recognized MHC-antigen complex
○ Initiated primary response
○ After 48 hours, enlarges into blast cell and undergoes
repeated rounds of cell division
○ Differentiate into:
- Effector cells – cytokine secretion, B-cell help
- Memory cells – long lived, respond with heightened
activity (secondary response)
Treg Cells
 Shown to inhibit proliferation of other T
cells in vitro
 CD4+CD25+
 Shown to inhibit development of
autoimmune diseases
Cell Death and T Cell Populations

 Apoptosis plays critical role

 Deletion of potentially autoreactive
thymocytes
 Deletion of T cell populations after activation
○ Fas and FasL pathway to induce self death