signalling - cytokine, BCR, TCR

families of cytokine receptors

JAK-STAT - common g chain, IL6, IFN
NF-kB - IL1, TNFR, IL17
SMAD - TGFB

general JAK-STAT signalling

1. ligand binds receptor

2. JAKs bind cytosolic tail and phos

3. STATs bind via SH2 and JAKs phos

4. nuclear translocation of STAT dimer - binds GAS promoters

regulatory mechanisms of JAK-STAT signalling

PTPs
PIAS - nuclear STAT inhibitors
SOCS - cytosolic STAT inhibitors

IL2R family

low affinity - IL2Ra, doesn’t signal
intermediate affinity - IL2RB/g, in naive T, NK and Tmem
high affinity - IL2Ra/B/g, in Tregs and Teff
APC can provide IL2Ra/CD25

IL2R signalling in Th1

increases IL12R in naive CD4 cells - STAT5
IL12 from DCs binds and activates Tbet and IFNg transcription - STAT5/4/1

IL2R signalling Th2

increases STAT5 and IL4R expression in naive CD4 cells - STAT5
IL4 binds and activates GATA3 and further IL4 production - STAT5

IL2R signalling Th17

inhibits STAT3 and RORgT expression in naive CD4 cells
induces STAT3, RORgT and IL17 in mature Th17 cells - STAT3/5

IL2R signalling Treg

Foxp3 expression - STAT5

IL2R signalling Tfh cells

antagonises Tfh differentiation

mutation in common gamma chain

loss of chain leads to SCID
cured by BMT

JAK structure and function

SH2 domain - STAT binding
FERM domain - receptor binding
pseudokinase and kinase domains
receptor chain aggregation leads to STAT recruitment

STATs structure

coiled coil
DBD
SH2
transactivation
may exist in cytosol as antiparallel inactive dimers

IFNR family

T1IFN bind IFNAR1/2 complex
T2IFN bind IFNG1/2 complex
T3IFN bind IL10RB/IFNlR1 complex

all activate STAT1

SOCS

inhibit signalling
transcribed downstream of STATs
SH2 binds pY and KIR domain inhibits JAK activity
SOCS box recruits components of cullin5 E3 Ub ligase complex - degradation

filgotinib

JAK1 inhibitor used in RA

NF-kB family

p65 - C
RelB - NC
c-Rel - C
p100/p52 - NC
p105/p50 - C

speed of C vs NC

C = fast and transient

NC = slow and sustained

trigger of C vs NC

C = TNF, IL1, TLRs
NC = BAFF, CD40, LTBR

key kinase C vs NC

C = IKKB and NEMO
NC = IKKa

key regulator C vs NC

C = IkBa degradation
NC = NIK stabilisation

NF-kB dimer C vs NC

C = p65/p50
NC = RelB/p52

canonical NF-kB pathway

1. ligand binds receptor and recruits adaptors

2. activation of IKK complex - IKKa, b, NEMO

3. phosphorylation, Ub and degradation of IkBa

4. p65/p50 release to nucleus

5. binds kB enhancer elements and induces transcription

non-canonical NF-kB pathway

1. ligand binding and recruitment of TRAF proteins

2. TRAF3 degradation prevents NIK degradation

3. NIK phos and activates IKKa

4. IKKa phos p100 and degrades to p52

5. p52 binds RelB to form TF

IL17R family signalling

recruitment of act1 to receptor
TRAF6 - TAK1 - IKK - NF-kB

SMAD signalling

1. ligand binds TGFBR

2. TGFBRII phos TGFBRI which phos SMAD2/3

3. SMAD2/3 dissociate from SARA adn associate with SMAD4

4. complex moves to nucleus

regulation of SMAD signalling

SMAD7 and Smurf2 inhibit

TGFB and transcription

promotes RORgT and Foxp3
suppresses Th1 and Th2 differentiation
in presence of pro inflammatory signals - more Th17
IL6 activates STAT3 to drive Th17

mechanism of TGFB immunosuppression

CNS1 enhanced element binds SMADs at foxp3
downregulation of IL2 transcription
maintains T cell quiescence
blocks IL12 mediated activation of JAK2 and TYK2

TCR structure

complex is maintained via electrostatic charges
alpha/beta
CD3
zeta chains
CD4/CD8 co receptor

role of co receptor

not required for signalling but increases sensitivity
recruits Lck and increases mechanical stability

outcome of TCR signalling

activation, proliferation and differentiation of T cells

antigen dependent and independent TCR signalling

dependent - DP cells
independent - DN3-4 stage during pre TCR

overview of TCR signalling mechanism

1. ligand binding and complex clustering

2. Lck activation

3. Lck phos of zeta chains at ITAMs

4. ZAP70 recruitment

5. ZAP70 phos LAT and SLP76

6. recruitmentof PLCg and Grb2

TCR signalling - Lck activation

dephos of Y505 by CD45 - catalytic domain adopts active conf
autophos of Y394
CSK phos Y505 to inactivate
CD45 dephos Y394 to inactivate

TCR signalling - CD45

PTPase activity in domain 1 of cytosolic tail
domain 2 is regulatory
can also dephos zeta chain
KO = impaired DN-DP and DP-SP transition

TCR signalling - Lck mediated phosphorylation of zeta chains at ITAMs

mediates recruitment of Zap70
Lck can phosphorylate ZAP70 to further recruitment
Lck can bind pY319 on zeta for further positive feedback

TCR signalling - ZAP70 phos LAT and SLP76

LAT is lipid modified and found in rafts
tyrosines 6-9 in LAT tail bind PLCg and Grb2

transcriptional outcomes

PLCg - PIP2 - IP3/DAG - calcium signalling triggers NFAT - IL2 production
Grb2/DAG - ras - AP-1 - clonal expansion
DAG - PKC - CBM - NF-kB - survival
CD28/ICOS - PI3K - mTOR - differentiation

models for TCR triggering

dimerisation and aggregation
conformational changes of chains
kinetic segregation

model 1 - dimerisation and aggregation

similar to RTKs
bivalent Abs are known to activate TCRs
insufficient pMHC complexes for this to be true

model 2 - conformational changes

FRET studies confirm
inhibition of CSK is enough to stimulate some signal events - activation of Lck without ligand involvement

model 3 - kinetic segregation

imaging and mutation studies support
rapid signalling may not support

TCR signalling - PLCg pathway

1. PLCg → IP3 + PIP2

2. IP3 binds IP3R in ER membrane = rapid calcium release

3. when calcium runs out, STIM binds ORAI and lets calcium in from exc space

4. IP3R can couple to mitochondria to deliver calcium to increase ATP production

5. ATP can stimulate further calcium signalling extrinsically

cyclosporine

interferes with calcium signalling
binds cyclophilin, inhibits calcineurin ppase
blocks NFAT activation as calcineurin can’t dephos
reduced cytokine gene transcription

used in transplants

NF-kB activation in TCR signalling

1. DAG activates PKCtheta

2. BCL10 filaments bind CARMA through CARD domains, MALT acts as scaffold

3. complex Ub by LUBAC

4. signals TRAF6 and IKK complex

importance of NF-kB pathway in T cell development

naive T cells - canonical NF-kB active for clonal expansion and TNFR induction
effector and memory T cells - non canonical NF-kB maintains pools

CD28

binds SH2 and SH3 domains
PXXP is essential
RLTPR links to CARMA - NF-kB
phosphorylation links to PI3K - mTOR pathway

CD28 deficiency

susceptibility to skin HPV but no others
may not be essential

may be compensated by CD2

ICOS

binds PI3K only but at higher affinity than CD28
required for Tfh generation and B cell CSR

anergy

no stimulation TCR triggering
NFAT pathway over AP-1/MAPK = negative regulatory program with E3 ligases

BCR structure

associates with IgB and a with ITAMs - responsible for signalling

roles of BCR

developmental selection of pre B cells
B cell survival
Ag presentation
selection of mature B cells

tonic signalling

pre BCR drives non specific signalling
Iga and B bind IgH, suppress rag and VDJh, proliferation and differentiation, increase VDJl

agonist dependent signalling

antigen presentation
antibody production

components of BCR signalling

adaptors - BLNK(SLP65)
lipid metabolising enzymes - activate PI3K and PLCg
negative regulators - SHP1, SHIP

BCR antigen dependent signalling overview

1. antigen induced BCR cross linking

2. Lyn mediated ITAM phos

3. Lyn phos activates CD19

4. Syk docking and activation of signalosome

5. transcriptional activation

BCR signalling - lyn mediated ITAM phosphorylation

allows Syk docking

BCR signalling - lyn phos activates CD19

amplification step
lyn positively regulates - ITAMs and CD19
and negatively regulates - CD22, FcRIIB
CD19 enhances activation through complement - C3d binds CD21 which activates CD19 on mature B cells
PI3K docks on phos CD19

Syk docking

phosphorylates SLP65 which acts as signalosome scaffold
BTK recruited by PIP3 where it phosphorylates and activates PLCg
PLCg activates IP3 and DAG pathways
Grb2 docks

BCR signalling - transcriptional programs

BTK - PLCg - IP3/DAG - NFAT and CMB NF-kB
Grb2 - MAPK - AP-1
CD19 - PI3K - PIP3 - mTOR

negative regulation of BCR signalling

FcRIIB binds Fcs and switches off antibodies
ITIM motifs recruit SHIP1 and SHP1 to reverse phosphorylation and attenuate signalling

lipid second messengers in BCR signalling

PI3K = PIP2→PIP3
PIP3 = Akt/mTORC1 activation and differentiation, foxo inhibition inhibits RAG and AID
PIP2 = gene transcription through PLCg/DAG/IP3 axis

role of PI3K signalling in B cells

expression of Pax5 - foxo1 and IRF4 inhibited so Pax5 free to be expressed
move past pre BCR - PI3K initiates BLNK activation and downregulation of pre BCR
antagonises apoptosis - decreases BIM levels
mature B cell survival - tonic signalling

PI3K signalling changes with antigen affinity

medium affinity antigen = high PI3K activity = high PIP3 = plasma cell differentiation
low affinity antigen = low PI3K activity = low PIP3 = GC SHM = high affinity plasma and memory cells

levels of PI3K activity across B cell development

low during VDJ recombination
medium in pre BCR and follicular B cell
high in BCR signalling due to antigen
low in CSR

CD40

B cell co stimulatory receptor required for CSR
relies on canonical and non canonical NF-kB signalling
no CD40 = hyper IgM syndrome

XLA

BTK mutation - R82C disrupts PH domain and interaction with PIP3
X linked
failed pre BCR signalling = no calcium or transcriptional activation
developmental arrest at pre B cell stage
absence of peripheral B cells and antibody = susceptibility to encapsulated bacteria

APDS

overactive GOF mutation in PI3Kd in catalytic or regulatory subunit
chronic Akt activation
combination of T cell, B cell and neutrophil defects

druggable with lenosilib

B cell lymphomas - B-ALL

Bcr-Abl fusion leads to overactive TK
PI3K pathway stimulation - survival and proliferation