Week 12 Flashcards
What is synthesized upon activation of a CTL?
Granzymes, perforin, TRAIL and Fas-L
also TNF, IFNg, some IL-2
cSMAC
Central supramolecular activation complex
Central portion of the immune synapse. Includes (in T cells) TCRs, CD2, CD4/8, CD28
pSMAC
peripheral supramolecular activation complex
Contains mostly LFA-1 and ICAMs
Three steps of CTL:target cell interactions
1) Adhesion - low affinity adhesion that shifts to high affinity upon TCR ligation. Synapse formation and reorientation of granules
2) Lethal hit - directional release of granule content
3) release (serial killing)
Cytolytic mechanisms of CTLs
- granule exocytosis - predominant pathway (FAST) perforin and granzymes
- expression of cell-surface TNF-family molecules (SLOW killing) Fas L, TRAIL, membrane TNF, lymphotoxin
- secretion of soluble toxic cytokines (SLOW) TNF, IFNg
heterogeneity of CD8 effector T cell population
At the peak of response, most effectors are KLRG1-hi and IL7Ra (CD127) low, and these are destined to die by apoptosis: Short-lived effectors (SLECs)
A fraction of effectors express KLRG1-lo and IL7Ra-hi, survive the contraction phase and go on to gnerate memory T cells: memory precursor effector cells (MPECs)
SLEC cell markers
KLRG1-hi, IL7Ra-low, CD122-hi, T-bet-hi
MPEC cell markers
KLRG1-lo, IL7Ra-hi, CD122-hi, T-bet-lo
What were the two possible mechanisms for CD8 T cell contraction?
- Competition for limited resources causes cell death after the peak of primary response (disproven through adoptive transfer)
- Contraction is ‘pre-programmed’ or cell-intrinsic and not regulated by competition
How was the resource competition theory for T cell contraction disproven?
Using OT-I bim-/- T cell adoptive transfer models. bim-/- do not undergo apoptosis after expansion. Co-transfering bim-/- and WT OT-I T cells into a mouse and immunizing with OVA showed the same kinetics of contraction for WT OT-I, suggesting that they were unaffected by the more limited resources
Four possible mechanisms for T effector cell diversification
- Separate precursor model
- Linear progression model (Differentiation states according to the cumulative history of signals encountered during infection)
- Signal-strength model
- Asymmetric cell fate model
What is the proposed asymmetric cell fate model for T effector cell diversification?
Through the interaction with the APC, the T cell is polarized. The daughter cell that arises from cellular content closer to the APC has a greater Teff potential, while the other daughter cell have greater Tmem potential
What are the four transcription factor ‘spectra’ that define less- and more-terminaly differentiated T cells?
less differentiated <> more differentiated
EOMES <> T-bet
Bcl-6 <> BLIMP1
ID3 <> ID2
STAT3 ‘activity’ <> STAT4 ‘activity’
Central memory T cell marker phenotype
CD62L+ CCR7+ high proliferative potential IL-2 non-cytolytic SLO-resident
Peripheral effector T cell memory phenotype
CD62L-CCR7- low proliferative potential inflammatory cytokines immediately cytolytic non-lymphoid tissue resident
Examples of persistent chronic infections
LCMV (clone 13), HCV, HBV, HIV, SIV
Inhibitory receptors/markers of exhausted T cells
PD1, Lag3, 2B4, Tim3, CD160
STAT signaling for various Th subsets
Th1 - STAT1
Th2 - STAT6
Th17 - STAT3
iTreg - STAT5
What are the steps for Th1 development?
1) intracellular microbes infect DCs and macrophages
2) infected APCs start producing IL-12, IFNg and more
3) IL-12 and IFNg induce Th1 differentiation
4) Th1 cells produce mostly IFNg, which inhibits the growth of intracellular bacteria
5) IFNg also inhibits the development of Th2 and Th17
What are the steps for Th2 development?
1) Granulocytes and mast cells recognize helminths
2) Activated mast cells and granulocytes produce IL-4
3) IL-4 induces Th2 differentiation
4) Th2 cells produce mostly IL-4, IL-5, and IL-13, which coordinate mucosal defenses against worms
5) IL-4 inhibits Th1 and Th17 development
What are the steps for Th17 development?
1) APCs recognize bacteria and fungi in the extracellular space
2) Activated APC produce IL-1, IL-6, IL-23, TGFb and others
3) All of these cytokines together induce Th17 differentiation
4) Th17 cells produce mainly IL-17 and IL-22, which strongly stimulate inflammation in the mucosa
5) IL-23 and TGFB (mainly) suppress Th1 and Th2 differentiation
Three subsets of CD8 Tmems
Tcm
Tem
Trm
Trm markers that can distinguish them from infiltrating Tem cells in an infection
CD103 and CD69.
What are limitations of measuring cytokines in the blood?
Since the majority of cytokines act in a autocrine or paracrine manner, circulating cytokines rarely reflect the cytokines in the tissues.
An exception is IL-6, which can act as an endocrine
Homeostatic/anti-inflammatory cytokines
IL-10, TGFB, IL-1RA
inflammatory cytokines
TNF, IL-1, IL-6, (IL-8 (chemokines)), Type I interferons (IFNa/B)
cytokines involved in the transition to acquired immunity
IL-12 (Th1)
IL-1, IL-6, IL-23 (Th17)
IL-6, TGFB (Tregs)
cytokines involved in growth and differentiation factors
IL-2, IL-4, IL-7, IL-15, IL-21
T cell effector cytokines
IFNg (Th1)
IL-4 (Th2)
IL-17 (Th17)
Macrophage activating cytokine
IFNg
B cell-activating cytokines
IL-4, IL-5, IL-6, IL-21
Eosinophil and/or mast cell activating cytokines
IL-3, IL-4, IL-13, IL-5
Signaling module for IL-1 and IL-18 family cytokines
TIR domains and MyD88 signaling, leading to AP-1 and NFkB.
Canonical pro-inflammatory transcription factor
NFkB, activates expression of IL-1, TNF etc.
Signaling module for type I and type II cytokines
JAK/STAT pathway
What are the two main modes of receptor activation upon ligand binding?
Conformational change of receptor, or induced proximity.
Both lead to an induction of enzymatic activity intracellularly, which propagates the signal
Basic model for JAK/STAT signaling pathway
Before receptor ligation, receptors are freely floating throughout the cell membrane. Ligation will bring together two subunits of the receptor, and the associated JAK kinases will phosphorylate tyrosines on one another. This phosphorylation event increases its catalytic activity, bringing it above the background dephosphorylation activity, and the JAK kinases will then phosphorylate the receptor itself. This becomes a docking site for transcription factor STATs. Upon binding to phosphorylated receptors, the STATs are then phosphorylated by the JAKs, activating them and allowing for translocation to the nucleus.
What does STAT stand for?
signal transducer and activation of transcriptions
4 JAK kinases
Jak1, Jak2, Jak3, and Tyk2
Where are Jaks located before signaling is activated?
Jaks are already associated with their receptors
7 different STATs
STAT1-6 and STAT5A/5B
Which STAT is the most immune-specific and inducible?
STAT4
Downstream signaling of IFNg receptor
Heterodimer of IFNGR1 and IFNGR2 is brought into proximity with another heterodimer by IFNg binding. The associated Jak1s then activate one another and phosphorylate STAT1, which then dimerizes with another pSTAT1 to enter into the nucleus
What do STATs bind to?
GAS elements in the DNA (but it is complicated)
Through what domains do STATs dimerize?
SH2-pY reciprocal interactions
How is specificity for different STATs conferred?
All have an SH2 domain that recognizes phosphorylated tyrosine, but the amino acids that surround that tyrosine (three positions away) confer specificity to specific SH2 domains and thus specific STATs
How do activated STATs access DNA?
Unactivated STATs are monomers that are located in the cytoplasm. Activation (phosphorylation) and dimerization of STATs lead to conformational change that reveals a cryptic nuclear localization signal, which allows for its translocation to the nucleus.
Dimerization-induced conformational changes also allow the STATs to recognize and bind to DNA.
Do STATs form homo- or heterodimers upon activation?
It depends on the specificity conferred by the receptor. If only STAT1 is recruited to the activated receptor (such as in IFNgR signaling), then only STAT1 homodimers are formed. Other times (such as IL6R signaling), both STAT1 and STAT3 are recruited and activated, and so you will get a mixture of STAT1/3 homodimers and STAT1:STAT3 heterodimers.
Do Jaks confer specificity of STATs?
No. The Jaks are mostly there just to phosphorylate things. The specificity of STATs is conferred by the amino acid three positions away from the pY on the receptor through specific SH2 domain interactions on the STAT.
IFNa/BR STATs
STAT1-4
IFNgR STAT
STAT1
IL-2R STAT
STAT5
IL-4R STAT
STAT6
IL-6 STATs
STAT1/3
IL-10 STAT
STAT3
IL-12 STAT
STAT4
Which two STATs seem to have opposite activites?
STAT1 and STAT3 (IFNg and IL-10, respectively)
Why do IFNa/B receptors activate so many STATs?
It could be that the pYXXZ amino acid does not confer very good specificity and is thus promiscuously activating multiple STATs
But the better explanation is that the IFNAR has at least six pY motifs that are phosphorylated.
What is the general model for cytokine receptor heterodimers?
One chain will often confer the specificity for the specific ligand and is considered to be the binding chain (alpha chain), while the other chain confers the signaling machinery and is considered to be the signaling chain (Beta chain).
Signaling chains are often shared among different cytokine receptors.
What is one way that cytokine signaling is regulated by individual cells?
Proteases on the cell surface can cleave cytokine receptors to make soluble cytokine receptors, which can circulate and serve as a sink for available cytokines
soluble IL6R is one exception, as it will still activate the signaling.
What is the key event in STAT activation?
C-terminal tyrosine phosphorylation, which leads to dimerization, nuclear translocation and DNA binding.
Domains of STAT proteins
N-terminal domain, DNA-binding domain, linker domain, SH2 domain, pY domain, ACT (transcription activation domain), pS domain
What are the two phosphorylation domains of STATs and how do they differ functionally?
phosphorylation of tyrosine (pY) determines whether or not the STAT is activated. phosphorylation of the serine (by MAPK) can fine-tune transcriptional activity
Function of STAT1/2:IRF9
anti-viral, downstream from IFNa/B
Function of STAT1
activation: chemokines, Ag presentation, synergy with TLRs, TNF, M1 mqs. Downstream of IFNg.
Function of STAT5
minimal function. Downstream of IL-2R
STAT6 function
homeostasis, tissue remodeling, angiogenesis, “M2”, anti-helminth. Downstream of IL-4R.
STAT3 function
suppression (downregulates TNF, IL-6). Opposes STAT1 signaling. Downstream of IL-10
STAT4 function
unknown, downstream of IL-12.
What pathogen is associated with STAT1 -/- mice or patients?
Tuberculosis
Which STATs are involved in Th1 differentiation?
STAT1, STAT4
What STAT is involved in Treg differentiation?
STAT5
What STAT is involved in Th2 differentiation?
STAT6
Which STATs are involved in Th17 differentiation?
STAT1, STAT3
Which genes are targeted by STAT1?
many IFNg-inducible genes (IRF-1, MHC, C3, iNOS, FcgRI, B7-2)
Which genes are targeted by Stat3?
suppresses cytokine production, ‘stem-ness’
Which gene is targeted by Stat4?
IFNg
Which gene is targeted by Stat5?
IL-2Ra (CD25)
Which genes are targted by Stat6?
Igh epsilon, CD23, MHC II,
What are the main differences in Stat signaling between IFNg and IFNa/B receptor ligation?
IFNgR ligation leads to STAT1 dimerization and nuclear localization, and transcription of inflammatory genes.
IFNa/B ligation leads to Stat1:Stat2 heterodimerization, which cannot bind to DNA well without IRF9, binding to a very different site than STAT1 dimers.
