The document outlines the processes of T-cell maturation, activation, and differentiation, highlighting key stages such as early development, selection processes in the thymus, and the mechanisms of positive and negative selection. It explains the activation of T cells mediated by the TCR-CD3 complex and discusses the importance of additional signaling for full activation. The role of apoptosis in eliminating autoreactive thymocytes and maintaining T-cell populations is also emphasized.

1. T-cell Maturation, Activation, &
Differentiation
BT6602
Unit 2
Dr K.Geetha
Associate Professor
Department of Biotechnology
Kamaraj College of Engg & Tech
Madurai

2. Overview
• Early Development
• T-Cell Repertoire Selection Processes
• Late Development
• Activation
• Differentiation
• Cell Death

4. • 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

5. The development of T cells

6. Figure 7-8 part 1 of 2
The cellular organization of the human Thymus

7. Figure 7-9
The cellular organization of the human Thymus

8. • 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

9. 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) – Double Negatives (DN)
• Do not express RAG-1 or RAG-2 that are
necessary for gene rearrangement

10. Figure 7-12
Changes in cell surface molecules throughout T-cell maturation in the Thymus

11. T-cell Development
• During 3 week development, differentiating
T cells pass through stages of
development based on surface
phenotypes

12. DN= double
negative
Cell-Surface
Molecules
•c-Kit:
Receptor for
stem cell
growth
factor
•CD44:
Adhesion
Molecule
•CD25:
Alpha-Chain
of the IL-2
receptor

13. TCR is now in the
Double Positive
(DP) State

15. Positive Selection
• Results in MHC restriction
• Mechanism:
– Immature thymocytes cluster with MHC
molecules on the cortical cells of the thymus
• If TCR interacts with MHC  protective signal
results that prevents apoptosis.
• If TCR does not interact with MHC  no protective
signal and apoptosis occurs.
• Result? Only reactive thymocytes survive.

16. Negative Selection
• Ensures self-tolerance
• Weeds out High affinity thymocytes
• Mechanism:
– APC’s bearing MHC’s interact with thymocytes
• If avidity is too strong  thymocyte undergoes
apoptosis.
• Details unknown…
• Result? Only self-tolerant thymocytes
survive.
Avidity (functional affinity) is the accumulated strength of multiple affinities.

17. • T cell development is expensive for host
• 98% of all thymocytes do not mature, die by
apoptosis within thymus

18. 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

19. T cell Activation
• 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

20. T cell Activation
• 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

23. Signal-transduction pathways
associated with T-cell activation.
(a) Phospholipase C (PLC) is activated
by phosphorylation. Active PLC
hydrolyzes a phospholipid component of
the plasma membrane to generate the
second messengers, DAG and IP3.
(b) Protein kinase C (PKC) is activated
by DAG and Ca2. Among the numerous
effects of PKC is phosphorylation of
IkB, a cytoplasmic protein that binds the
transcription factor NF-kB and prevents
it from entering the nucleus.
Phosphorylation of IkB releases NF-kB,
which then translocates into the nucleus.
(c) Ca2-dependent activation of
calcineurin. Calcineurin is a
Ca2/calmodulin dependent phosphatase.
IP3 mediates the release of Ca2 from the
endoplasmic reticulum. Ca2 binds the
protein calmodulin, which then
associates with and activates the
Ca2/calmodulin-dependent phosphatase
calcineurin. Active calcineurin removes
a phosphate group from NFAT, which
allows this transcription factor to
translocate into the nucleus.

24. Activation of the small G protein, Ras.
Signals from the T-cell receptor result in
activation of Ras via the action of specific
guanine nucleotide exchange factors
(GEFs) that catalyze the exchange
of GDP for GTP. Active Ras causes a
cascade of reactions that result in the
increased production of the transcription
factor Fos.
Following their phosphorylation, Fos and
Jun dimerize to yield the transcription
factor AP-1. Note that all these pathways
have important effects other than the
specific examples shown in the figure.

25. Chokingly complex- what to
remember
• Importance of CD3
• Phosphorylation activates proteins
• Cascade
• G proteins,
• 2nd messengers
• Gene activation
• Ongoing proliferation- IL2 and its receptor.

26. “Signal 1 and 2”- TCR activation
isn’t the whole story
• TCR activation is necessary, but not
sufficient, to produce activation. It is called
“signal 1”.
• The T cell also needs “signal 2”- CD28-B7
interaction.
• Its absence produces clonal anergy
TH-cell recognition of an antigenic peptide–MHC complex
sometimes results in a state of nonresponsiveness called
clonal anergy, marked by the inability of cells to proliferate
in response to a peptide-MHC complex

27. Second Signal

28. •Superantigens (SAgs) are a class of antigens that cause non-specific activation of T-
cells resulting in polyclonal T cell activation and massive cytokine release.
•SAgs are produced by some pathogenic viruses and bacteria most likely as a defense
mechanism against the immune system.

29. 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)

31. Treg Cells
• Shown to inhibit proliferation of other T
cells in vitro
• CD4+CD25+
• Shown to inhibit development of
autoimmune diseases.
• maintain tolerance to self-antigens.

33. 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

34. Cell Death!
• How does apoptosis occur during
thymocyte selection??
• Specialized Protease called “Caspases”
• Every cell produces these proteases which
are maintained in an inactive form
• Must get activated to undergo apoptosis

35. 2 Pathways
Death
Signal!!!
(Apoptosis Inducing Factor)
BID: BH3 Interacting Domain
Apaf-1: Apoptotic Protease Activating Factor 1

36. THANK YOU