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T cell differentiation and function Flashcards

1
Q

what are the different effector T cell populations?

A

cytotoxic CD8 cells

helper CD4 cells:
- Th1, Th2, Th17, Tfh, Treg

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2
Q

what controls T cell subset differentiation?

A

Signal 1, 2, 3
Signal 3 = cytokines from APCs
- epigenetic modifications can change CD4 cell fate
- hallmark TF control changes depending on subset

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3
Q

how is a T cell army generated?

A
  • Activated by APC presenting peptide-MHC
  • clonal expansion of pathogen-specific clones
  • differentiation of clones

contraction into either
- effector pool
- memory pool which are long-lived
- re-exposure results in faster response

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4
Q

why do T cells need to clonally expand?

A

TCRs are highly diverse – only small proportion will respond to specific pathogen
- once the TCR has bound its cognate antigen, it needs to expand to generate more T cells which recognise the same antigen

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5
Q

what is a naive T cell?

A

Naïve T cell has undergone thymic selection so have rearranged their alpha-beta TCRs
- received selection signals in thymus
– exist in periphery but haven’t been activated in secondary lymphoid tissues

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6
Q

what induces a naive T cell to expand?

A

Naïve T cells activated by antigen presentation in secondary lymphoid organs proliferate and differentiate before exiting
- secondary lymphoid organs are the major site of T cell activation

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7
Q

How do T cells home to secondary lymphoid tissues (SLTs)?

A

Naive T cells express chemokine receptors/lymph node homing receptors CCR7 and I-selectin – homing into lymph node

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8
Q

what happens to naive T cells when in SLT??

A

APCs (DCs) enter lymph node via lymphatics
– naive T cells screen their TCRs for peptide-MHC
- Some T cells will be activated by APC presented peptide = proliferation and differentiation of T cell
- Mature T cells leave lymph node and change their homing receptors to enter sites of inflammation

Different Th subsets have different homing receptors to enter different sites of inflammation

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9
Q

what are the important lymph node homing receptors? how are they expressed by different T cells?

A

CD62L/L-selectin
CCR7
- naive T cells have high levels of these
- mature effector memory T cells lose homing receptors to lymph nodes to enter other tissues
- central memory T cells retain homing receptors to circulate around lymph nodes

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10
Q

how do naive T cells enter the lymph node?

A

Naïve T cells can enter lymph node using CD62L/L-selectin to become activated and adhere before differentiating and exiting to the tissues

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11
Q

what signals are important for naive T cell activation?

A

Signal 1 – TCR binding MHC-peptide

Signal 2 – co-stimulation CD28 with CD80/86
- this is required for full activation
- delivers signals for metabolic reprogramming - changes metabolism from inactivate to active for proliferation and clonal expansion

Signal 3 – cytokines produced from APC – induces subset which naïve T cell differentiates to
- cytokine environment is crucial

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12
Q

what are CD8 T cells?

A

CD8 can take on hallmarks of CD4 subsets, but are mostly cytolytic killing cells which produce IFNy (similar to TH1)
- capacity to kill but can be similar to Th1 with regards to cytokines
- CD8 kill cells with peptide + MHCI

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13
Q

how do CD8 T cells kill cells?

A

Virally-infected cell contains viral peptides presented in MHCI
- CD8 specific for peptide forms synapse with infected cell and release perforin and granzymes into the cell – direct granules to immunosynapse and secrete - forms holes in membrane to directly lyse cell
- Or can express death receptors e.g. Fas ligand
- if target cell expresses Fas, can induce apoptosis of infected cell

Directly kills infected cell or induces apoptosis via death receptor signalling

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14
Q

what are the CD8 killing mechanisms?

A

Perforin and granzyme B = lytic mechanism

CCL5, IFNy, TNFa, IL-17 = non-lytic

Fas ligand = apoptotic/death mechanism

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15
Q

how do CD8 T cells become exhausted in cancer?

A

Chronic antigen stimulation leads to exhausted fate of CD8
- limited killing and function so can’t kill tumours
- Immunotherapy can override exhaustion and induce effector function

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16
Q

what dictates what subset a CD4 cell differentiates to?

A

Signal 3 is crucial for differentiation
- Cytokines and CRs (cytokine receptors)
induce TFs to cause differentiation

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17
Q

how do dendritic cells control the environment for T cell differentiation?

A

APC: TCR-peptide-MHCII, costimulation (CD28 – CD80/86) induce activation and proliferation
- Cytokines from APC induce differentiation – innate immune system instructs T cell about nature of infection
- Can make Th1, Th2, Th17 polarising signals – instructions for response needed to be mount

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18
Q

what is the main cytokine produced by Th1 cells? What is its role?

A

Produce IFNy – activation of infected macrophages – important for intracellular pathogens which can persist in macrophages e.g. TB, Leishmania

IFNy signalling in macrophage induces ROS and respiratory burst – inflammatory reaction is tightly controlled – macrophage increases its killing capacity via Th1 release of IFNy

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19
Q

what induces differentiation of naive T cell to Th1?

A

IL-12 is the Th1 polarising cytokine:
- IL-12 signals via IL-12R to induce STAT4 signalling
– this upregulates TBET (master TF for TH1)
– TBET binds and promotes IFNy expression
- TBET also binds IL-12Rbeta2 gene - increases expression of IL-12R and IFNy
- IFNy can further upregulate IL-12Rbeta2 signalling – positive feedback
- TBET antagonises GATA3 TF

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20
Q

what induces differentiation of naive T cell to Th2?

A

IL-4 is the polarising cytokine for TH2
- IL-4 activates STAT6 to induce master GATA3 TF, - induces signature cytokine IL-4 to increase IL-4R expression

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21
Q

what is the main dogma for Th1/Th2 differentiation?

A

cytokine binds cytokine receptor
- this activates a transducing molecule, usually a STAT
- this induces a master TF
- the master TF induces signature cytokine production
- together, the TF and cytokine can control cytokine receptor expression in positive or negative feedback

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22
Q

what are the key functions of Th1 cells?

A

IFN-y stimulates infected macrophages to help control infection, by increasing:
- MHC expression
- Co-stimulatory molecule expression
- Nitric Oxide (NO) production
- Phagolysosome maturation
- TNF-a production

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23
Q

how do Th1 cells control macrophages?

A

Important for full activation of macrophage
- Th1 delivers IFNy and CD40L signals to bind to CD40 on macrophage
- causes engulfment and fusion of bacteria into phagolysosome
- induces upregulation of inducible nitric oxide synthase, mature phagolysosome, upregulate ROS to kill pathogen – augments intracellular pathogen killing

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24
Q

how are Th1 cells involved in a response to TB?

A

M. tuberculosis is “controlled” by Th1 cells activating infected macrophages:
- TB infects macrophage and resists ROS
- Th1 forms granulomas, where a collection of infected multi-nucleated giant cells (fused macrophages) are surrounded by Th1s
- Th1 can then provide IFNy for macrophage activation and prevent TB spread

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25
what do defects in Th1 result in for TB?
dissemination of TB: - Deficiencies in Th1 cells results in reactivation of latent infections or inadequate control of de novo infection – TB can escape from granuloma
26
how are Th1 cells implicated in disease?
Th1 cells are also found at affected sites in autoimmune and inflammatory disease and can cause type 1 IFN response against self-antigens: - Multiple Sclerosis - Autoimmune thyroiditis - Rheumatoid Arthritis - Type I Diabetes - Psoriasis - Crohn’s Disease - Allograft rejection
27
what are Th2 cells important for?
Extracellular infections e.g. bacteria, helminths Produces IL-4: induces B cell isotype switch to IgE for mast cell activation against parasites
28
what is the signature differentiation of Th2 cells?
IL-4 is the main cytokine that induces TH2: - IL-4 upregulates GATA3 - GATA3 inhibits TBET due to IL-4 – forcing lineage choice via dose of TF to TH2 state - GATA3 shuts down IFNy expression and activates IL-4 expression (vice versa for TBET inhibiting IL-4 expression) – antagonises TH1 programme
29
how do TBET and GATA3 antagonise one another?
T-bet and Gata3 reciprocally antagonise Th1/Th2 differentiation T-bet will activate Ifng, repressing Il4 locus; - suppress Th2 programme Gata3 activates Il4, repressing Ifng - suppress Th1 programme
30
what are the synergistic/switch processes in Th1/Th2 differentiation?
TBET (gene is tbx21): - Synergistc effect: IL-12 signals via STAT4 to induce IFNy (early activation), - STAT4 induces TBET - TBET induces own expression and upregulates IFNy (later activation) – multiple pathways and TFs coverging on same fate Switch effect: TBET inhibits GATA3 expression and function - Once IFNy is in motion – TBET-IFNy reinforcement loop to stabilise TBET to commit to TH1 rather than TH2 (TH1 is a highly stable phenotype so will not revert to Th2)
31
what are the functions of Th2 cells?
secrete IL-4, IL-5, IL-9 and IL-13: - Target basophils, mast cells, eosinophils - IL-4 induce IgE class switch - Confers resistance to helminth – coats in IgE to crosslink mast cell - leads to degranulation and gut expulsion - Stimulate intestinal mucus secretion and peristalsis
32
how are Th2 cells implicated in disease?
- Implicated in hayfever – IgE response to grass pollen – mast cells crosslinked to allergen via IgE – histamine release - Asthma – release of I-L4,IL-13 - smooth muscle spasms, eosinophilic inflammation and hypersecretion of mucus Th2 drives allergic cell disease - unwanted effects of Th2-derived cytokines on innate immune effectors in airway
33
how does a Th17 fate differ from Th1/Th2?
Th1 and th2 can commit easily Th17 are highly plastic - Phenotype depends on environment, can become TH1 or TH2 if cytokine environment changes
34
what induces the differentiation of naive T cells into Th17?
polarising cytokines IL6 and TGFb, IL-1b and IL-21 induce TFs RORgt and RORa – these activate signature cytokines: - IL17A/F, IL-22, CCL20 Th17 is maintained by IL-23
35
what key cytokine does Th17 produce?
IL-17 protects against fungal and mucosal infection – increase neutrophil recruitment and production in BM via cytokine release, tighten epithelial barriers
36
what diseases have Th17 cells been implicated in?
Also implicated in autoimmunity, for example Multiple Sclerosis, Crohn’s Disease, Rheumatoid Arthritis
37
what are the key functions of Th17 cells?
- Produce IL17, IL22 – act on epithelial cells to produce antimicrobial peptides and prevent pathogen invasion – inhibit bacterial growth - IL-22 increase epithelial cell turnover to shed bacteria - produce G-CSF which acts on BM to increase neutrophil production - Recruit neutrophils via chemokines - Produce CCL20, which is recruitment chemokine for Th17 cells which express CCR6 – recruitment of more TH17 to amplify immunity
38
how are Th17 cells in an unstable lineage?
Unstable phenotype – highly plastic: - TH17 cells are between a naïve cell and TH1/TH2 - IL6 and TGFb induce TH17 - Cytokines instruct which CRs will be expressed by the T cell that makes them sensitive to certain fates
39
how many chains does the IL-12 receptor have?
2: IL-12beta2 - provides sensing of IL-12 IL-12beta1
40
what is the function of IL-12R?
it phosphorylates STAT4 to induce TBET and IFNy - IFNy signals back via IFNyR to increase expression of IL-12Rbeta2 positive feedback - Th1 increases expression of IL-12R to increase sensitivity to IL-12
41
what IL-12 receptor subunit do Th17 cells use? how does this affect differentiation?
TH17 use common chain IL-12RB1 paired with IL-23R: - When IFNy is low – IL-23 signals by STAT3 to reinforce TH17 fate - When IFNy is high – IFNy upregulates IL-12RB2 so more susceptible to IL-12 – TH17 becomes TH1
42
what regulates Th17 to Th1 lineage plasticity?
Chromatin structures and modifications: Epigenetics – modifications that can be heritable but don’t directly change DNA sequence
43
what are the two key chromatin modifications in Th17 plasticity?
H3K27me3 modification: compaction of chromatin - DNA is tightly wound up, so not accessible for TFs – turns off gene expression H3K4me3: relaxation of chromatin - unwinding of DNA – TFs can access – active gene transcription – gene turned on
44
how is TBET modified to enable Th17 plasticity?
Naïve T cell has both H3K27me3 and H3K4me3 modifications at TBET – bivalent chromatin - gene is poised to choose different differentiation fate (same in stem cells) - Gene could be expressed or turned off depending on signal
45
what modification occurs in TBET during Th1 differentiation?
- For Th1: TBET locus loses K27 modification – DNA unwinds and opens IFNy locus whilst shutting of RORc (RORyt gene) and IL-17 - reinforces TH1 fate
46
what modification occurs in TBET for Th17 differentiation?
TH17 cell opens RORc (gene encoding RORyt) and expresses IL-17, but doesn’t change TBET modification – plastic - retains the TBET bivalency – Th17 could open the TBET programme in the future to become TH1 in the right cytokine environment
47
how can cytokines induce Th17 conversion?
Th17 mainitained with IL-23 cytokine (IL-12RB1 paired with IL-23R) If IL-12 and IFNy are added, IFNy production in cell increases, showing TH1 development from TH17 – IFNy upregulates IL-12RB2 chain to pair with beta1 chain - cell is now sensitive to IL-12 - IL-12 induces STAT4, which means TBET locus can be opened from poised state, becomes TH1 over time
48
what cytokines and TFs activate Th17?
TGFB, IL-23, IL-21 – STAT3, RORgt, RORa – promotes TH17
49
what cytokines and TFs inhibit Th17?
IFNYR or IL-12R induce STAT4 for TBET and IFNy – induced from TH17 to TH1 – inhibits TH17
50
is the plasticity of Th17 favourable?
TH17 useful for initial dealing with EC pathogen, when pathogen is phagocytosed, switch to TH1 to deal with it intracellularly and increase macrophage killing – gives flexibility to immune response
51
can Th17 switch to Th2 phenotype?
yes - if exposed to IL-4 - activates GATA3 - this is less common than Th1
52
what are T follicular helper cells?
Important for GC response – somatic hypermutation and isotype switching to improve BCR affinity and antibody production - generated in secondary lymphoid tissues - deliver help and instruction to B cells - high affinity antibody response
53
what induces the differentiation of naive T cell into TFH?
Naïve T cell primed by APC: - Induced by high signal 1 persistance and IL-21 - IL-21 binds to IL-21R to induce STAT3, which activates BCL-6 - BCL-6 causes upregulation of CXCR5 to induce the T cell to migrate to B cell follicle – Tfh exerts function here to interact with B cells
54
what is the function of TFH cells?
Provide helper signals in GC: - T cell sees peptide-MHCII - B cells uptake native antigen and present on MHCII – B cells compete for T cell help for high affinity and survival - cognate interaction - B cell with high affinity BCR obtain more T cell help and can survive to expand - form memory B cells or high-affinity plasma cells
55
how do TFH cells induce B cell activation and expansion?
TFH provides IL-21 and CD40L co-stim molecule: - signal 1 via TCR and MHCII-peptide - signal 2 via CD40-CD40L - AP1/NFAT drive B cell activation - NF-KB and STAT3 drives B cell clonal expansion Deletion of these leads to poor GC reaction – TFH help is crucial to provide IL-21 which induces STAT3 in B cell
56
what TF do TFH cells express?
BCL-6 hallmark TF for TFH – BCL-6 induces SAP protein which enables B and TFH to stabilise physical interaction
57
why is linked recognition important for TFH and B cells?
- TFH recognises processed antigen - B cell recognises native antigen and can internalise whole pathogen and process peptides to load onto MHC - TFH and B cell look at the same thing in different ways - TFH can then provide help - B cells with better BCR are more likely to uptake antigen and present to TFH – competition drives GC
58
what cytokines to TFH cells produce?
different cytokines induce B cell to switch to different antibody classes - IL-21 important for IgA, IgG isotype switch - IL-4 important for IgE - IFNy can induce IgG2A (neutralising antibody)
59
where are Tregs derived from?
Derived from two major sources: - Some thymic-derived Treg selected against self-antigen - Peripheral-derived Treg (pTreg/iTreg) generated as part of any peripheral immune response - can be generated from naive T cells
60
how are pTregs induced?
they can arise in gut, particularly in response to TGFb and retinoic acid in response to commensal bacteria or to harmless food antigens
61
what TF do most Tregs express?
FOXP3 - promotes Treg phenotype
62
what is the function of Tregs?
- control autoimmune responses that have been generated from lack of central tolerance - control immune responses against commensal bacteria or innocuous, abundant antigens - also help to resolve inflammation after infection
63
how do Tregs perform their function?
Treg suppress immune responses through: - Cytokine consumption e.g. sequester IL-2 with CD25 and prevent effector cells proliferating - Suppressive cytokine release (e.g. TGF-beta, IL-10, IL-35) - anti-inflammatory cytokines Suppression of antigen presenting cell function – inhibitory signalling, removal of co-stimulatory molecules, via CTLA-4, LAG3 etc - cytotoxicity - can kill dendritic cells
64
what is the function of AIRE?
Autoimmune regulator: - Aire regulates ectopic gene expression in the thymus - mTECs express AIRE – promotes expression of self-antigens from peripheral body tissues - Negative selection = deletion of autoreactive T cells which bind self-antigens strongly - AIRE promotes Treg development to suppress responses against self-antigens
65
what happens if AIRE is absent?
In absence of AIRE, T cells reactive to tissue-specific antigens mature and leave the thymus
66
does every T cell have the capacity to be autoreactive?
yes, every T cell has reacted against MHC - some potential of self recognition - but these T cells bind weakly to MHC - there is a sweetspot for MHC binding of high affinity that induces Treg differentiation rather than deletion
67
how is AIRE important for Tregs?
AIRE can promote Treg development against self-antigens - fail-safe mechanism - repertoire of cells adopt regulatory lineage to suppress responses against self-antigen
68
what happens if Foxp3 is deleted?
autoimmune disease
69
what is FoxP3?
Both thymic-derived Treg and inducible Treg express FoxP3 - although activated effector T cells transiently up-regulate expression - FoxP3 is a “master regulator” transcription factor that controls the expression of a group of genes involved in Treg function and phenotype
70
what genetic evidence is there for Foxp3 as a critical immunoregulatory TF?
Scurfy mice have a spontaneous loss-of-function mutation that affects FoxP3 function – they develop a fatal autoimmune like disease with hyperproliferative T cells - scurfy males die within 3 weeks - gene mutation on X chromosome - double A insertion in Fxp3, causing LOF mutation and truncation of Foxp3 - enlarged lymph node - loss of control
71
what is IPEX?
In humans with Immunodysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome, a rare disorder caused by mutations in the FOXP3 gene - there are defects in Treg function - IPEX is analogous to scurfy - Severity depends on where mutation is in foxp3 – can affect DNA binding, truncation, spectrum of phenotypes but usually autoimmunity
72
how does foxp3 function as a TF? how can this be seen experimentally?
Foxp3 suppresses effector cell cytokines: - Autoreactive T cell can be switched off via foxp3 – prevents release of cytokines such as IL-2, IFNy, IL-4 Transduce T cells with control GFP vector or FOXP3-GFP vector - Measure cytokine release - Presence of foxp3 suppresses the cytokines
73
how does the strength of the TCR signal determine differentiation or apoptosis in thymic development?
low affinity: TCR cannot bind anything, including MHC – no survival signal so die by neglect in thymus due to non-functional ab-TCR moderate: TCR can interact with MHC-peptide so can survive Shoulder: some cells become Treg – goldilocks binding, strong but not too strong High affinity = too strong binding to MHC = apoptosis due to self-reactivity
74
how does TCR signal strength govern Foxp3+ Treg differentiation?
As TCR signal strength increases, cells can avoid negative selection by upregulation of FOXP3 or CD25 (IL-2 receptor): - IL-2 can signal via CD25 to upregulate FOXP3 and differentiate to Treg to avoid negative selection Cell with too strong TCR binding will die, or die due to lack of IL-2 cytokine stimulation
75
what genes in T cells are sensitive to TCR signal strength?
NR4A family of receptors which are rapidly upregulated upon TCR signalling – correlates with strength and duration of TCR signal - can be used as a surrogate marker as a proxy for TCR signal
76
how can NR4A be used to measure TCR signal strength?
- NR4A1 gene with GFP construct - TCR signal induces GFP signal via NR4A1 OT-1 TCR transgenic cell specific for OVA albumin: - Can change OVA antigen peptide sequence to weaken affinity to TCR - can use self-peptide and negative control which shouldn't bind - As signal strength lowers due to changing the OVA sequence, GFP expression falls – indicates lower TCR signalling
77
what signal strength is induced by Tregs as shown by NR4A1-GFP?
All T cells have TCR signal within thymus Treg progenitor (CD25 high) has highest TCR signal Tregs have high TCR signal in thymus and in periphery – self-antigen specific and stop immune response to APC presentation of self-antigen
78
how do Treg TCR signals compare to T conventional cells?
Tregs receive stronger and/or longer TCR signals compared to conventional T cells: - Double positive TCRb-low have low NR4A1, low NR4A3 – weak signal - DP TCRb-positive have some NR4A1 and not much NR4A3 - Single positive CD8 and CD4 increase in NR4A1/3 - CD25 high precursors have high NR4A1/3 – high TCR signal – NR4A3 requires stronger TCR signal
79
what is the two-stage model for thymic Treg development?
Naïve T cell with strong TCR signal can directly induce FOXP3 to become Treg Or Strong TCR signal can induce CD25 expression, which leads to IL-2 being produced from the pre-Treg and binding to CD25 - this induces FOXP3 expression via autocrine signalling to become Treg
80
why are NR4A TFs important?
NR4A TFs have important function, as well as for studying signal strength: - they control how Tregs translate signal strength and accumulate at promoter to induce FOXP3 - Also involved in negative selection – too much accumulation induces apoptosis
81
what happens when NR4A family is deleted?
NR4A family 1-3 – functional redundancy - foxp3 double positive in thymus - Deletion of NR4A leads to lack of foxp3 development in thymus – no Treg development and can induce Scufy phenotype in mice - clearly important for TCR signal strength translation into Foxp3 expression
82
summarise thymic Treg development:
mTEC presents self antigen to a CD4 SP thymocyte - too high binding = clonal deletion - moderate binding = survival signals and enter periphery - goldilocks binding with IL-2 induces Treg development and Foxp3 expression
83
how are peripheral Tregs induced?
pTregs generated against low affinity self-antigen e.g. commensal antigen, food antigen in the periphery - in the gut, there is a lot of TGFb and retinoic acid - in the absence of danger signals or inflammation, these cytokines can induce CD4 development to pTregs down the Foxp3 pathway
84
how can pTreg development occur in immune privileged sites? how can this be studied?
Iris of eye = immune privileged site - TCR-transgenic mouse specific for iris proteins - CD90.2 congenic marker to stain with antibody and see these cells when transferred into host - Foxp3 GFP reporter – identify Tregs as green when they express Foxp3 - Sort GFP-negative cells (no Tregs) which are specific for iris protein - inject GFP-negative cells into eyes of recipient mice - Collect eyes Donor GFP-negative cells convert to foxp3-GFP within the eye – peripheral treg generation
85
what are other Treg subsets?
Tr1 Tfr
86
what are Tr1 cells?
TR1 cells: produced by repetitive stimulation of TH1 cells in the presence of IL-10/IL-27 - Tr1 cells are FoxP3-negative and require Eomes for their differentiation - signature cytokine is IL-10
87
how can Tr1 differentiation be studied?
Stimulate Th1 with antigen in vivo: - As dose increases, IL-10 is switched on more
88
what cytokine and TFs are important for inducing Tr1 cells? how do Tr1 cells arise?
IL-27 is important: - TFs cMAF and BLIMP are important for inducing IL-10 promoter - Arise due to chronic, repeated self-antigen or microbial antigen in absence of danger signal and costimulation – avoid immune response to this and cause tolerance
89
what are T follicular regulatory cells (TFR)?
Share features of both Treg and Tfh - CXCR5, PD-1hi, FoxP3 - Identified in both mice and humans - enter and regulate germinal centre responses
90
how does Foxp3 expression differ in naturally occurring vs inducible Tregs?
Thymic derived nTreg constitutively express FoxP3, whereas those generated in the periphery are induced to express FoxP3
91
how is Foxp3 expression controlled?
Specific non-coding DNA elements in the FoxP3 gene are required for either the nTreg or inducible Treg cells - Controlling when and maintaining FOXP3 expression is crucial - dictate when Foxp3 is turned on or off
92
what are the conserved non-coding DNA elements of Foxp3? how can they be studied?
Sequence homology across species: - Non coding and coding exons - Promoter is conserved - Conserved 3 non-coding DNA sequences (CNS1-3) - Can delete each one and see how each affects foxp3
93
what happens when CNS3 is deleted from FOXP3?
Mouse K/O of intronic CNS3 and compared to intact FOX3: - CNS3 K/O induced a large drop in Treg foxp3 expression in the thymus - Loss of CNS3 did not lead to a big drop in Tregs in the spleen - CNS3 must control how thymic Tregs express foxp3 in thymus – enhance Treg differentiation if CNS3 is expressed
94
what is the role of CNS3 in foxp3 expression?
CNS3 controls de novo thymic foxp3 - CNS3 must control how thymic Tregs begin to express foxp3 in thymus – enhance Treg differentiation if CNS3 is expressed - CNS3 binds Rel (NF-KB family TF) upon strong TCR signal in the thymus - pTregs can still be generated without CNS3
95
what happens when CNS1 is deleted from Foxp3?
Deletion of CNS1: - Thymic Tregs are unchanged - Transfer experiment in another mouse – deletion of CNS1 means that Tregs in periphery are lost - CNS1 important for peripheral Treg development
96
what is the role of CNS1 in Treg development?
CNS1 important for peripheral Treg development - not important for thymic population
97
what happens when CNS2 is deleted from Foxp3?
- loss of CNS2 caused no difference in thymic selection of Tregs - not affecting thymic Treg selection - In spleen or lymph node, CNS2 K/O caused loss of foxp3 expression and loss of Tregs
98
what is the role of CNS2 in Treg maintenance?
CNS2 depends on methylation status: - Forms memory module - CNS2 has lots of CpG methylated motifs - Methylation inhibits expression – winds up chromatin - In precursor Tregs, DNA is tightly wound, but as foxp3 is expressed, there is demethylation, so DNA unwinds and allows TFs to bind - Methylation prevents function = K/O of FOXP3 - When Tregs proliferate, they upregulate foxp3 – remembering foxp3 - CNS2-deficient Tregs proliferate, but they lose FOXP3 – forget to maintain foxp3 when CNS2 is lost
99
what are the key sites on the foxp3 gene?
promoter: binds Foxo and NR4A (corresponds to TCR signalling) CNS1: pTreg generation - AP-1, NFAT, Smad3 CNS2: Treg stability - Runx ,Foxp3, c-rel - Methylation/loss of CNS2 means Treg loses ability to keep foxp3 expressed when dividing CNS3: thymic Treg generation - Foxo and C-rel
100
why do Tregs express Foxp3?
Express FOXP3 to suppress their own potential effector programme – prevent cytokine production by Treg against self-antigen - stop expression of IL-2 and IFNy
101
what are the different functions of Tregs?
1. release inhibitory cytokines 2. Cytolysis of APCs 3. Metabolic disruption of effectors 4. disruption of APC presentation
102
what inhibitory cytokines to Tregs release?
TGFb at membrane – cell-cell inhibition OR secretion of TGFb IL-10 – anti-inflammatory
103
how do Tregs induce cytolysis of APCs?
Cytolysis via granzymes and perforin into APC – prevent self-antigen presentation
104
how do Tregs disrupt the metabolism of effector T cells?
IL-2 sink: – Tregs express CD25, a high affinity IL-2R - IL-2 is important early in T cell activation before differentiation - NFAT/AP1 heterodimer is induced following TCR and co-stimulation - these can induce IL-2 expression, leading to autocrine proliferation of T cells - CD25 can mop up IL-2 to prevent effector T cell activation by NFAT and AP1 Target adenosine metabolism: - Tregs express CD39 and CD73 which breakdown ATP to anti-inflammatory adenosine which can inhibit T cells
105
how do Tregs disrupt APC antigen presentation?
CTLA-4 on Treg binds with higher affinity CD80/86 compared to CD28 – Treg internalise co-stim molecules from APC and degrades to prevent co-stimulation LAG3 binds MHCII and inhibits it Instruct DC to produce IDO to inhibit T cell – indirect inhibition of T cell via APC
106
how can Treg function suppression of effectors be studied?
See Treg activity via inhibition of T cell proliferation: label cells with CSFE dye that binds cell contents - as cell divides, fluorescence is halved - dilutes the signal Stimulate effector T cells and see clear division peaks via CSFE: - Titrating T effector with Tregs - all effector T cells divide when Tregs are absent - As more Tregs are added, majority of effector cells do not divide
107
how are Tregs important in cancer?
Tumours have high Treg to CD8 ratio – poor prognosis
108
how can the crucial role of CTLA-4 be studied?
CTLA4 has higher affinity for CD80/86 on APC compared to CD28: - If CTLA4 is K/O in general, mice die in 3 weeks – important to control T cell activation - if CTLA4 deleted in only FOXP3 cells via Foxp3-Cre system – all mice die, but slower – critical for negative regulation of T cells
109
how does CTLA-4 function?
CTLA-4 is expressed constitutively in Treg When activated, conventional T cells also express CTLA-4, where it is thought to negatively regulate their activation or CTLA-4 can block and rip CD80/86 from APCs
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how can CTLA-4 function against APCs be studied?
Red = cells with CTLA4 Green = cells with CD80/86 - Mix cells – yellow spots form with CTLA-4 cell – molecules have bound - Yellow complexes have been internalised – - CTLA4 leads to CD80/86 removal from APC to be internalised and degraded in Treg – removal of costimulatory molecules – prevents T cell proliferation
111
what happens if CTLA-4 is mutated? how is this studied?
Heterozygous mutation in CTLA4 is enough to give phenotype: - Patients with CTLA-4 mutation have less foxp3, less CD25, less functional Tregs - Treg suppression assay: higher the value, the more suppressive the Tregs are - took Tregs from healthy donor and from patient with mutation in one CTLA4 - Healthy Treg = 80-90% inhibition of effectors - Patient Treg = inhibition of effectors is reduced – CTLA-4 is crucial for function
112
what is the function of TGFb on Tregs?
TGFb can be held at Treg membrane in inactive form: - Tregs can release enzymes that make TGFb become active and anti-inflammatory - Suppress inflammation
113
how can Tregs be tissue-specific?
depending on local environments, Tregs can adapt to the tissue they are in e.g. gut, lung - will retain core signature of suppression - Can upregulate other TFs associated with TH1 e.g. TBET upregulation to express CXCR3 to home to regulate TH1 responses - This enables Tregs to express TFs of helper cells, enabling them to home to sites where effector cells function to regulate them

Molecular Mechanisms (9 decks)

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