RECEPTORS OF T AND B

LYMPHOCYTES-GENETIC
REARRANGEMENT, CLONAL
SELECTION THEORIES
MANISHA SARKAR
M.SC MEDICAL BIOCHEMISTRY
3RD YEAR

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T CELL DEVELOPMENT

3. Expansion of cells that

have successfully
1. Commitment of 2. Initiation of antigen
rearranged one of their T-
hematopoietic precursors receptor gene
cell receptor genes (a
to the T-cell lineage, rearrangements, and
process called β-
selection).

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DEVELOPMENT
OF T CELLS
EXPRESSING
NOTCH LIGANDS

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SCHEMATIC
PRESENTATION
OF T CELL
DEVELOPMENT

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 STAGES OF FORMATION
The most immature, CD4-, CD8 -(double negative, DN)
thymocytes pass through several stages

(DN1-DN4), during which they commit to the T-cell lineage and

begin to rearrange their T-cell receptor (TCR) gene loci. Those

that successfully rearrange their TCR β chain proliferate, initiate

rearrangement of their TCR α chains, and become CD4 +CD8+
(double positive, DP) thymocytes, which dominate the thymus.

DP thymocytes undergo negative and positive selection in the

thymic cortex. 5
STAGES OF FORMATION
Positively selected thymocytes continue to mature and migrate to
the medulla, where they are subject to another round of negative
selection to self antigens that include tissue-specific proteins.

Mature T cells express either CD4 or CD8 (single positive, SP) and
leave the thymus with the potential to initiate an immune
response.

Although most thymocytes develop into conventional αβ TCR CD4

or CD8 T cells, some thymocytes develop into other cell lineages,
including lymphoid dendritic cells, γδ TCR T cells, natural killer T
(NKT) cells, regulatory T (T ) cells, 6
SECOND PHASE OF T CELL DEVELOPMENT
• The second phase of T-cell development is largely dependent on T-cell
receptor interactions and brings cells to maturity from the CD4+ CD8+ stage
to the CD4 or CD8 (single-positive, SP) stage.
• The events include the following:
1. Positive selection: Selection for those cells whose T-cell receptors engage
self-MHC,
2. Negative selection: Selection against those cells whose T-cell receptors bind
too strongly to self-peptide/MHC combinations, and
3. Lineage commitment: Commitment of thymocytes to effector cell lineages,
including CD4 helper or CD8 cytotoxic populations, as well as CD4 regulatory
T cells.
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 DOUBLE NEGATIVE THYMOCYTE
DEVELOPMENT

After arriving in the thymus from the bone marrow

where they proliferate and begin to express their T-
via blood vessels at the corticomedullary boundary,
cell receptors. T cells that survive the stringent
T-cell precursors encounter Notch ligands, which are
selection processes mature and migrate into the
abundantly expressed by the thymic epithelium. T-
thymic medulla before exiting where they arrived, at
cell precursors then travel to the outer thymic
the corticomedullary junction.
cortex,

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WHAT IS THIS DOUBLE NEGATIVE
THYMOCYTE
• During the time it takes cells to develop to maturity in the thymus (1
to 3 weeks), thymocytes pass through a series of stages defined by
changes in their surface phenotype .
• The earliest T cells lack detectable CD4 and CD8 and are therefore
referred to as doublenegative (DN) cells.
• DN T cells can be further divided into four subsets, DN1-DN4, based
on the presence or absence of other cell surface molecules, including
c-Kit (CD117), the receptor for stem cell growth factor; CD44, an
adhesion molecule; and CD25, the a chain of the interleukin (IL)-
2receptor

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SCHEMATIC PRESENTATION OF T CELL
DEVELOPMENT

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CONTD..

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Thymocytes Express Either αβ or γδ T Cell
Receptors

Vertebrates generate two broad categories of T cells: those that

express TCR α and β chains and those that express TCR γ and δ chains.

TCR-αβ cells are the dominant participants in the adaptive immune

response in secondary lymphoid organs.

However, TCR-γδ cells also play an important role, particularly in

protecting our barrier tissues from outside infection.

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CONTD..

• Rearrangement of the β, γ, and δ loci begins during the DN2 stage. To become an
αβ T cell, a cell must generate a TCR β protein chain—an event that depends on

a single in-frame V(D)J rearrangement.

• To become a γδ cell, however, a thymocyte must generate two functional

proteins that depend on two separate in-frame rearrangement events.

• Probability therefore favors the single event and, in fact, T cells are at least three
times more likely to become TCR-αβ cells than TCR-γδ cells.
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CONTD..

• TCR-γδ T-cell generation is also regulated developmentally. TCR-γδ cells

are the first T cells that arise during fetal development, in a wave, and
provide a very important protective function perhaps even prior to birth.

• However, production of γδ T cells declines after birth, and the TCR-γδ T-

cell population represents only 0.5% of all mature T cells in the periphery
of an adult animal.

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COURSE OF APPEARANCE

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GENES ENCODING αβ or γδ T CELL

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VDJ
REARANGEME
NT IN TCR

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V(D) J REARRANGEMENT FOLLOWS 23/12
RULE

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TCR- DIVERSITY

TCRs are diversified via V(D)J recombination

TCR alpha chains contain V and J segments.

TCR beta chains contain v, D, J segments.

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 DN Thymocytes Undergo β-Selection, Which
Results in Proliferation and Differentiation
• Double-negative (DN) thymocytes that have successfully rearranged their TCR β
chains are valuable, and are identified and expanded via a process known as β-
selection .
• This process involves a protein that is uniquely expressed at this stage of
development: a 33-kDa invariant glycoprotein known as the pre-Tα chain.
• Pre-T acts as a surrogate for the real TCR α chain, which has yet to rearrange,
and assembles with a successfully rearranged and translated β chain, as well as
CD3 complex proteins.
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CONTD..
• This immature TCR/CD3 complex is known as the pre-T-cell receptor
(pre-TCR) and acts as a sensor by initiating a signal transduction
pathway.
• The signaling that the pre-TCR complex initiates is dependent on
many of the same T cell–specific kinases used by a mature TCR.

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Pre-TCR signaling at the DN3 stage triggers the
following sequence of events:

• 1. Maturation to the DN4 stage (c-Kit CD44 CD25 )

• 2. Rapid proliferation in the subcapsular cortex of the thymus

• 3. Suppression of further rearrangement of TCR β-chain genes, resulting in allelic

exclusion of the β-chain locus

• 4. Development to the CD4 CD8 double-positive (DP) stage

• 5. Cessation of proliferation

• 6. Initiation of TCR α-chain rearrangement

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Activation of naive and effector T cells by antigen.

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Phases of T cell responses.

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Role of
costimulation and
helper T cells in
the differentiation
of CD8+ T
lymphocytes.

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Functions of
costimulators in T
cell activation.

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 Role of CD40in T cell activation.
• Naive T cells are activated by peptide-MHC
complexes on antigen-presenting cells (APCs)
previously activated by the binding of pathogen-
associated molecular patterns (PAMP) to Toll-like
receptors (TLRs).
• CD40L engages CD40 on the APC and may
stimulate the expression of B7 molecules and the
secretion of cytokines that activate T cells.
• Thus, CD40L on the T cells makes the APCs
"better" APCs, and the B7 and CD40 pathways
stimulate each other.

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Intracellular
signaling
events during
T cell
activation.

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 The Ras-MAP kinase pathway in T cell
activation

• ZAP-70 that is activated by antigen

recognition phosphorylates
membrane-associated adapter proteins
(such as LAT).
• which then bind another adapter, Grb-
2, which provides a docking site for
the GTP/GDP exchange factor Sos.
• Sos converts Ras.GDP to Ras.GTP.

• Ras.GTP activates a cascade of

enzymes, which culminates in the
activation of the MAP kinase ERK
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Mechanisms of action of the
inhibitory receptor
CTLA-4.

CTLA-4 may inhibit T cell

responses by competitively
preventing
CD28 binding to B7
costimulators (A).

or by generating
inhibitory signals (e.g. by
associated phosphatases)
that attenuate
activation via the TCR and
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CD28 (B).
Positive and Negative Selection

• Positive selection, which selects for those thymocytes bearing

receptors capable of binding self-MHC molecules with low affinity,
resulting in MHC restriction.

• Negative selection, which selects against thymocytes bearing

receptors with high affinity for self-MHC/self-peptide complexes,
resulting in self-tolerance.

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POSITIVE AND
NEGATIVE SELECTION

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Positive Selection Ensures MHC Restriction

• In the thymic cortex, newly generated CD4 CD8 thymocytes first

browse the surfaces of cortical thymic epithelial cells (cTECs), which
are unique in their ability to mediate positive selection.
• If a DP thymocyte expresses a TCR that recognizes a self-MHC/self-
peptide complex on the surface, it will undergo positive selection, a
process that induces both survival and differentiation of DP
thymocytes.

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CONTD..
• Remarkably, over 90% of CD4 CD8 thymocytes never engage the
MHC/peptides they encounter with their TCRs.
• Either they have not generated a functional TCR-αβ combination, or
the combination does not bind MHC/peptide complexes with
sufficient affinity.
• These cells have “failed” the positive selection test, do not receive
survival signals through their TCRs and, within 3 to 4 days, undergo a
form of apoptosis referred to as death by neglect.

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Relationship of tcr
affinity and selection

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Negative Selection (Central Tolerance) Ensures
Self-Tolerance
• Autoreactive CD4 CD8 thymocytes with high-affinity receptors for self-
MHC/self-peptide combinations are potentially dangerous to an
organism, and many are killed by negative selection in the thymus.

• Negative selection is defined broadly as any process that rids OF

autoreactive clones and is responsible for central tolerance.

• Errors in the negative selection process are responsible for a host of

autoimmune disorders, including type 1 diabetes 36
THEORIES RELATED TO
T CELL

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Proposed model of lineage committment

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• According to the kinetic signaling model, the decision to commit to the CD4 or CD8 lineage is
based on the continuity of the TCR signal that a thymocyte receives.
• Positive selection results in down-regulation of CD8 on all thymocytes.
• This will not alter the intensity of a TCR/CD4/MHC class II signal, and cells receiving this signal
will continue development to the CD4 SP lineage. However, down-regulation of CD8
diminishes (interrupts) a TCR/CD8/MHC class I signal, an experience that sends a cell toward
the CD8 lineage. IL-7 signals are required to “seal” CD8 lineage commitment.
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 Transcription Factors Th-POK and Runx3 Regulate
Lineage Commitment
• Th-POK and Runx3 have taken center stage as transcription factors required for CD4
and CD8 commitment, respectively.
• Both Th-POK and Runx3 act, at least in part, by suppressing genes involved in
differentiation to the other lineage.
• Th-POK activates expression of CD4 and inhibits expression of genes that regulate CD8
differentiation, including genes encoding CD8 itself and Runx3.
• Reciprocally, Runx3 activates expression of CD8 and inhibits expression of genes
encoding CD4 and Th-POK.
• The sequence of events and full cast of players that regulate lineage commitment at
the genomic level are still under investigation; there is clearly more to come.

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HOW REGULATORY T CELLS INACTIVE
TRADITIONAL T CELLS

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TERMINATION OF T CELL RESPONSE

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Humoral immune response

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Phases of humoral immune response

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Primary and secondary immune response

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SIGNAL
TRANSDUCTION
BY BCR COMPLEX

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ROLE OF COMPLEMENT
IN B CELL ACTIVATION

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EARLY AND LATE EVENTS IN HUMORAL
RESPONSE

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INTERACTION OF HELPER TCELL AND
B CELL

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MECHANISM OF T CELL MEDIATED B
CELL ACTIVATION

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B CELL DEVELOPMENT

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HSC s AND B
CELL
PROGENITOR

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FACTORS REGULATING B CELL
DEVELOPMENT

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transitional B cells can enter
splenic B-cell follicles and
recirculate

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Transitional B cells undergo
positive and negative
selection in the spleen.

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EVENTS OCCURING
IN LYMPHOID
GERMINAL CENTRES

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IG HEAVY CHAIN ISOTYPE SWITCHING

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IMMUNOGLOBULIN WITH VDJ REGION

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 ENZYMES INVOLVED IN RECOMBINATION

Joined by enzymes –
Rag- 1, 2 ( recombinase Artimis , Ku70/80 , dNA
Causes double strand
associated gene) binds Forms hair pin loop in dependent protein
break between
to RSS( recombination segment end. kinase , ligase ,
segment and RSS.
signal sequence) terminal
deoxynuleotidase.

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SCHEMATIC
PRESENTATION OF V(D)J
REARRANGEMENT

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MOLECULAR
MECHANISM OF
HEAVY CHAIN
SWITCHING

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Based on cytokine , class switching happens

1. IL4- Ig G1 , Ig E
2. TGF beta- IgG2b , IgA
3. IL5 – Ig A
4. IFN gamma – IgG2a , IgG 3

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THANK YOU

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REFERENCES
• - Kuby Immunology-W. H. Freeman 8th edition(2018)
• Abul K. Abbas - Cellular and Molecular Immunology-W.B. Saunders
Company (2007)

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