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T-cell Maturation
What allows T cell maturation?
- Direct contact with thymic epithelial cells
- Influence of thymic hormones
- Growth factors (cytokines, CSF)
T cell maturation
T cell progenitor DN DP SP 2ry (Subcapsular (Cortex) (Medulla) lymphoid zone) organs
THYMUS
The earliest T cell precursors in the thymus:
- Express Thy-1 (mice)
- Have not yet rearranged TCR loci
- Do NOT express CD4 or CD
- Are called “double negatives”
MARKERS:
- C-KIT- Receptor for Stem Cell Growth Factor
- CD44 - Adh. Molecule. Homing to thymus
- CD25 - Alpha chain of IL-2 receptor
Double Negative
Time Course of Appearance in Thymus
Humans = 8-9 weeks
- Most double negative thymocytes will give rise to αβαβαβαβ T cells (in mice and humans).
- Some will differentiate into γδγδγδγδ T cells.
- The developmental pathway of γδγδγδγδ T cells is not well defined.
-Double negative thymocytes first express Pre- Tα (surrogate chain)
-They then undergo β chain locus re-arrangement.
- Once a β chain is produced, a pre-TCR is expressed
- Early markers are NOT expressed at this stage
1) Made productive TCR β chain re-arrangement
2) Signals for proliferation (similar β chain) and maturation
3) Suppresses further β chain re-arrangement (allelic
exclusion)
4) Signals for TCR αααα chain to undergo re-arrangement
4) Induces development to CD4+8+ (double positive) stage
Why is Pre-TCR important?
- Following proliferation, α- chain locus rearrangement occurs.
If a productive rearrangement is made, an α/β TCR is expressed on the cell surface.
DOUBLE POSITIVE STAGE
Kuby Figure 10-
Cells undergo positive and negative selection.
Those that fail either selection undergo apoptosis.
Those that pass the selection step lose EITHER CD4 or CD becoming “single positives”
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These mature single positive cells leave the thymus.
Positive and Negative Selection of T cells:
- Positive selection: occurs in the cortex and allows only those T cells that are able to bind to self-MHC molecules in the thymus to mature
Positive selection results in self MHC restriction.
- Negative selection: occurs in the medulla and removes T cells whose TCR strongly recognize (high affinity) self-MHC (or self self-peptides plus self MHC). Die of apoptosis within the thymus.
Negative selection results in self-tolerance (to some extent).
Summary:
MHC Restriction Self Tolerance POSITIVE NEGATIVE
Figure 9.
Signals through the TCR, CD3 and CD4/8 activate a protein tyrosine kinase (PTK) cascade
- PTK = enzyme that phosphorylates the amino acid tyrosine (Y) in ITAMs
- PHOSPHATASES = enzymes that remove inhibitory phosphates
Signals through the TCR, CD3 and CD4/ activate a PTK cascade
- CD4/8 are associated with a cytoplasmic tyrosine kinase enzyme – Lck (lymphocyte kinase)
1.- TCR-MHC-Peptide activates the phosphathase CD 2.- CD45 activates Fyn and Lck
3.- Activated Fyn and Lck phosphorylate ITAMs in CD3 chains
4.- Phosphorylated ITAM motifs on the CD3zz chains become a docking site for the PTK ZAP- 70.
5.- Binding of CD4 to MHC allows Lck to phosphorylate and activate ZAP-70 to become an active PTK.
- Activation of ZAP-70 initiates a cascade of events that
results in LAT activation and phosphorylation (activation)
of phospholypase C (PLCγγγγ1).
- PLCγγγγ1 converts phosphatidyl inositol 4,5-biphosphate (PIP 2 )
to diacylglycerol (DAG) and inositol 1,4,5-triphosphate
(IP 3 ):
PIP 2 DAG and IP 3
- IP 3 triggers the release of calcium from intracellular storage
vesicles into the cytosol, thus raising cytoplasmic calcium
levels.
The calcium:
1) synergizes with DAG to activate protein kinase C
(PKC)
2) PKC activates the transcription factor NF-κB
PLCγγγγ 1
The calcium:
- and PKC activate IkB kinase (IKK), which phosphorylates IkB, releasing the transcription factor NF-kB – which translocates to the nucleus.
- acts together with calmodulin to activate calcineurin (phosphatase)
- Calcineurin activates the cytoplasmic component of the transcription factor NFAT (NFATc), causing it to translocate to the nucleus, where it combines with NFATn
TRANSCRIPTION FACTORS- lead to gene transcription, cell proliferation and differentiation.
ZAP-70 also phosphorylates and activates LAT
and SLP-
- SLP-76 binds and activates PLC-γ , GEF and Tec Kinases
- GEF activates the Ras pathway resulting in formation of the
Jun/Fos, a component of the AP-1 transcription factor.
- NFATn and Jun/Fos bind to sites in the regulatory region of
the IL-2 gene and increase transcription of IL-2.
- The expression of >70 genes is increased within 4 hr of T cell
activation.
- The potent immunosuppressive drugs cyclosporin and
FK506 act by inhibiting the activation of calcineurin X
NF-AT!
ADAPTOR PROTEINS: 1) Serve as links for proteins, 2) Promote assembly of membrane proteins
ADAPTOR PROTEINS - SLP76 (SH 2 -domain containing leukocyte protein of 76 KDa and LAT (Linker of activated T cells).
The interaction of CD28 with B7 sends
additional activation signals.
• CTLA-4 is not initially expressed, but is expressed
after T cell activation.
• CTLA-4 has a higher affinity for B7 than CD28.
• Interaction of CTLA-4 with B7 is thought to down-
regulate T cell activation.
• There are actually two related molecules: B7.1 and
B7.
Expressed after T cell activation
Expressed in resting and activated T cells
Signal 2
Superantigens
• Proteins produced by pathogens
• Not processed by antigen presenting cells
• Intact protein binds to the β variable region
on TCR of T cells and to MHC class II on
antigen presenting cells (APC)
• Large numbers of activated T cells and APC
release cytokines having pathological
effects
Super Antigens
Consequences:
• Because they cross-link the Vβ domain of the
TCR with the Vα domain of the MHC-II, this
results in non-specific proliferation and
activation.
• Over production of Th cytokine leading to
systemic toxicity (IFN-γ, TNF-α) and
inflammatory mediators.
• Deletion (negative selection) of thymocytes
bearing Vβ domains recognized by the super
antigen---- beneficial?
Signal 1
Signal 2
↑IL-
↑IL-2R
Clonal expansion
- Outcome of CD4 T cells
- Th 0 will give rise to at least 3 Th populations
- Th populations differentiated based on cytokine production
Th
Suppressor T cells
- The old questions revisited!
- First described in the 70’s made CD
the
candidate for Ts cells
- Now it is believed that Ts cells are CD4+
cells with the phenotype CD4+CD25+.
- Several potential applications: 1)
suppression tissue rejection; 2) treatment
of allergies or autoimmune diseases; 3)
enhance response to vaccines; etc
Why are DC better APCs?
Cell death (apoptosis)
- Several apoptotic pathways
- Fas-FasL pathways
- Fas and its ligand FasL are
induced upon T cell activation
Pro-Caspase 3
Fas
FasL
FADD
Pro-Caspase 8
Caspase 8
Caspase 3
CAD
I-CAD
I-CAD
DNA Damage
GOAL:
- Regulation of T cell numbers,
- Removal of “turned off” T cells
γδγδγδγδ T cells
- Perhaps somewhat analogous to the B-1 B cell…evolved to respond to specific microbial antigens?
- B-1 B cells also known as CD5 B cells. Make IgM antibodies to polysaccharide antigens without T cell help
- During development in mice, temporally ordered waves of γδγδγδγδ T cells using specific V gene segments are produced. This may represent a “predefined” set of Ag specificities designed to deal with common environmental pathogens
- May be able to recognize “native” antigen without needing presentation of Ag on MHC or MHC-like molecules.
- They lack CD4 or CD8. Able to develop in the absence of Thymus
Gamma/delta (γδγδγδγδ) T cells
- make up a variable proportion of total T cells in different specie
- relatively low in humans and mice (~5%) ; high in ruminants (up to 75%)
- predominantly found in epithelial sites (lung, intestine etc) in mice and humans (rather than in secondary lymphoid tissues - where αβαβαβαβ T cells predominate)
- may undergo maturation outside the thymus
- do NOT express CD4 or CD8 (though some occasionally express CD8 alpha/alpha homodimers).
The End
Figure 9.
The calcium:
- Synergizes with DAG to activate protein kinase C (PKC)
- and Ca2+^ and calmodulin act together to increase the activity of calcineurin
Calcineurin activates the cytoplasmic component of the transcription factor NFAT (NFATc), causing it to translocate to the nucleus, where it combines with NFATn Calcineurin and PKC synergistically activate IκB kinase (IKK), which phosphorylated IκB, releasing the transcription factor NFκB – which translocates to the nucleus. Meanwhile, activation of the Ras pathway results in formation of the Jun/Fos (AP-1) transcription factor.
