T-cell receptor signalling

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1 First Summer School: 31 Aug-4 Sep School of Mathematics, University of Leeds Theoretical and Experimental Immunology T-cell receptor signalling Klaus Okkenhaug, PhD The Babraham Institute Cambridge, UK.

2 The ultimate receptor tyrosine kinase

3 SH2 domain containing signalling proteins See also Schraven Curr Op Immunol 13:307

4 Crystal structures of the TcR-MHC General rule: CDR3 contacts peptide, CDR1, 2 contacts MHC

5 What the TcR sees MHC class I peptides MHC class II peptides

6 The TcR-CD3 complex is maintained by neutralising positive and negative charges

7 Does the TcR change shape upon antigen recognition? Evidence for conformational change: A Proline Rich motif in CD3ε becomes exposed and binds an Nck SH3 domain. However, neither the PR motif not Nck is essential for TcR signalling. Levin & Weiss, J Exp Med :

8 A model for how the TcR and CD8 interact with MHC-I The cytoplasmic domain of the coreceptor is associated with Lck CD3 TcRαβ Coreceptor: peptide CD8αβ (CD4 is the coreceptor for MHC II) MHCI

9 TcR can recognise a single pmhc complex APC MHC molecules loaded with peptide-biotin conjugates Only 1 peptide per MHC possible peptide-biotin binds Streptavidinphycoerythrin (SAv-PE) PE sufficiently bright to that a single molecule can be detected by microscopy The sensitivity of the TcR is equivalent to the photoreceptor of the eye (where one photon is sufficient to trigger a signal)! Huppa and Davis Nat Rev Immunol 2002

10 TcR: pseudodimer single pmhc sufficient CD4-MHCII interaction blocked >25 pmhc required Structural considerations argue against the TcR and CD4 binding same pmhc complex

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12 The Jurkat T cell leukaemia: an essential T cell signalling discovery tool Key genes discovered (or co-discovered) in this way: Lck ZAP-70 LAT CD45 Abraham and Weiss 2004

13 Mouse knockout mutants Gene targeting experiments allow investigators to ask what the consequences of removing a single element in signalling pathways on T cell signalling can be. Usually, the removal of key signalling molecules results in a developmental block shortly after TcRβ recombination. Knockout experiments have also revealed redundancies between related signalling proteins and have described proteins that are important later on in development.

14 T cells are educated in the thymus - but most of them fail 99% die at this stage 1% pass and become CD4 or CD8 T cells The 3 fates of double positive thymocytes Neglect: 98% die because they don t recognise MHC molecules. Negative selection: 1% die because they recognise MHC molecules too well. Pass: 1% recognise MHC molecules, but are able to restrain themselves.

15 Stepwise TcR signalling to the nucleus Recruitment of kinases (Lck ZAP70) Phosphorylation of substrates (LAT) Recruitment of additional enzymes to these substrates (PLC-γ) Generation of second-messenger signalling molecules (IP3, Ca2+). Triggering of signal transduction cascades (MAPK, NF-κB, NFAT). Gene transcription.

16 Non-receptor tyrosine kinases involved in TcR signalling Family PH TH *Rlk lacks PH domain Src Lck, Fyn Kinase Csk Csk SH2 Kinase Syk Syk, ZAP 70 SH2 Kinase Tec Itk, Tec, Rlk* SH3 SH2 Kinase SH3 SH2 SH2 SH3 Y Examples

17 CD28-PXXP Unc119-PXXP ZAP-70 (Tyr505 in Lck) (Tyr394 in Lck)

18 The CD45 tyrosine phosphatase CD45 Heavily glycosylated ectodomain DOMAIN 1 PTPase DOMAIN 2 Regulatory * Abundant transmembrane glycoprotein expressed on all nucleated haematopoietic cells * Ectodomain is alternatively spliced to generate up to 8 different isoforms. * No known physiologically relevant exogenous ligands * The tyrosine phosphatase (PTPase) activity is in Domain 1 of the cytoplasmic tail. * The homologous Domain 2 has a regulatory function. * Dephosphorylates Y394 and Y505 in Lck hence positive and negative regulator of T cell activation (Gatekeeper) CD45 / : Impaired DN DP and DP SP transitions.

19 How can a phosphatase control positive and negative signals? T cell activation Lck Y505 dephosphorylation Lck Y394 dephosphorylation py394 Y505 py505 Y394 CD45 activity

20 Lck-Fyn redundancy? No signalling in Lck- Jurkats Lck-/- mice: impaired DN-DP transition. Fyn-/- mice: mild phenotype (partial Th2 defect see lecture 2). Lck-/-;Fyn-/-: complete DN-DP block (rescued by Fyn over-expression). Lck is primary SFK in T cells. Redundancy common in progenitors, reduced in mature cells

21 Fyn and Lck: structural differences Lck is constitutively associated with CD4 via Cys-Cys mediated interactions Lck is associated with the soluble plasma membrane, Fyn is raft associated Weak interaction between Fyn and CD3 (lipid mediated?). Different substrate specificities (consequence of catalytic differences or distinct subcellular localisation and hence substrate availability?) Palacios and Weiss, Oncogene 2004

22 CD3 chains have ITAM motifs bi-dentate YXXL/V X6-9 YXXL/V motifs present in CD3, ζ, Igα, Igβ ανδ Fcγ chains: Antigen recognition. tyrosine-phosphorylated ITAM provide docking sites for Syk or ZAP-70 Reth. Sem Immunol 1995

23 Cbl Vav py At this stage, the TcR can be considered a functional RTK, but is has still not recruited any effectors! py Lck py Hatada er al Nature 1995

24 ZAP-70 phosphorylates LAT -a transmembrane adapter protein 36 kda membrane bound phoshoprotein Lipid modified, found in rafts Absolutely essential for T cell activation; non redundant (DN->DP block). Binding sites for: 1X YLVV: PLCγ (Y6) 3X YXNX: Grb2/Gads (Y7-9) Knockin mutations of 4 C-terminal tyrosines (Y6-9) abrogates all signalling (=LATKO). Knockin mutation of Grb2/Gads binding Tyrosines (Y7-9)= similar to KO, except γ/δ phenotype Knockin mutation of PLCγ binding site (Y6) leads to impaired T cell development, also Th2 autoimmunity ZAP-70 is mainly responsible for LAT phosphorylation (though Lck and Itk can also phosphorylate LAT). LAT links TcR associated tyrosine kinase activity with cellular activation. Other ZAP-70 substrates include PLCγ and SLP-76 in the complex No function? 1

25 Grb2 Gads a conserved signalling pathway is diverted LAT Ras GDP SOS Ras GTP SH3 SH3 SH2 Grb 2 SH2 SH3 ADAP SH3 Gads SLP-76 Vav Raf Nck Itk Erk1/2 Transcription factors Ca2+, cytoskeletal changes

26 Two complementary pathways to Ras activation Ras is a small GTPase GTP-bound form is active, GDP-bound form is inactive RasGEFs catlyse the exchange of GDP to GTP Intrinsic GTPase activity catalyses return to GDP-bound state GAPs stimulate intrinsic GTPase activity Ras-GTP generated by RasGRP stimulates the GEF activity by Sos. LAT PIP2 Ras GDP SOS SH3 SH3 DAG + IP3 PLCγ PKC SH2 Grb 2 Ras GTP GDP Raf Ras GDP Ras-GAP Erk1/2 Ras GTP Transcription factors (Elk 1, SRF, AP 1, NFAT) Ras-GRP1 GDP

27 LAT and SLP-76 nucleate a large signalling complex NFκB SH 2 SH SH 2 3 SH 2 2 H S Jordan et al, Nat Immunol SKAP55

28 Vav: a signalling integrator Three isoforms: Vav1, 2, 3. T cells: Vav1>Vav3>>Vav2 The DH domain regulates Rac-GTP Rac-GTP activates the JNK and controls actin-cytoskeletal rearrangements Also important adapter functiondifficult to dissect! Turner- Bildau 2002

29 PLCγ activate three parallel signalling pathways PLCγ DAG + IP3 PIP2 RasGRP PKC Ca2+ Ras Erk NF-κB NFAT Gene transcription (Cell cycle, cytokines, etc)

30 NFκB Thome Nat Rev Immunol, 2004

31 Calcium signalling in T cells

32 Calcium signalling in T cells Ca2+ Orai (also known as CRACM1) time 2nd phase (long) STIM1 Ca2+ IP3 STIM1 ER hand detects low Ca2+ Ca2+ 1 phase (transient) st Ca2+ IP3R Ca2+ SERCA ER thapsigargin

33 Calcium $ignalling Cyclosporine and FK506 are pro-drugs that inhibit calcineurin FK506/ (Tacrolimus) FKBP Ca2+ Cyclosporine A Cyclophilin Calcineurin (a phosphatase) Pi NFAT-P (retained in cytoplasm) PPi GSK3 Cyclosporine annual sales: > $1 billion NFAT NFAT ATP Target genes Nucleus

34 Signalling to the nucleus TcR PTK LAT Gads Grb2 SLP76 Calcineurin PKC Vav GEF Ca2+ PLCγ Sos Ras-GRP Carma Rac (GADD45γ) Ras Bcl10 MAPKKK MEKK MEKK4 Raf Malt MAPKK MKK4/7 MKK3/6 MEK1/2 IKK JNK p38 GTPase MAPK ERK1/2 IkBα Elk cjun Key transcription factors: cfos AP1 NFkB Cytokine transcription NFAT *p38 pathway may be induced principally by cytokines (IL12/18) (Yang et al, Nat Immunol 2001)

35 Central tolerance Signal strength CD8 CD4 Death by neglect Positive selection Treg development Negative selection TcR affinity/avidity AIRE ensures that the entire genome is expressed at low levels in the thymus. Strong Lck signal favours CD4 development Medium Lck signal favours CD8 development

36 Peripheral activation Signal strength CD8 CD4 T cell activation T cell survival/ priming Death by neglect? TcR affinity/avidity

37 The steep threshold problem In the thymus, the decision to positively select or negatively select is of fundamental importance. Failure to negatively select cells can lead to autoimmunity, excessive negative selection could deplete the available repertoire of useful T cells. In the periphery (lymph nodes, spleen), the decision of a T cells to be activated or not has profound consequences for health. T cells need to recognise peptides from pathogens with extremely high sensitivity, yet

38 Analogue to digital conversion of TcR signals Unstable transition state 0 Affinity of pmhc for TcR threshold I # of T cells that respond threshold # of T ces that respond The abundance and affinity of peptide-mhc complexes (pmhc) exist in a continuum. The existence of a threshold of response and a steep ensuing curve (rather than a linear relationship between ligand density/affinity) may be explained by an analogue to digital conversion (other explanations are also possible). Problem: the difference between a low and high affinity peptide is relatively small. Therefore, a small change in affinity may need to results in a substantial increase in the probability that a cell responds. Number of pmhc per APC (assuming affinity exceeds a threshold)

39 The OT1 TcR recognises a peptide from chicken ovalbumin presented by MHC1 molecules Optimal sequence: SIINFEKL (by definition a foreign antigen) SIINFEKL stimulates deletion of thymocytes or activation of peripheral T cells. Mutants of SIINFEKL can be agonist, partial agonist or antagonist. The character of the peptide depends on the half life (t½) life of the interaction between the OT1 TcR and the pmhc complex.

40 Sharp boundary effect for negative selection SIINFEKL SIIQFEKL SIIQFERL SIITFEKL SIIQFEHL SIINFEQL SIIGFEKL EIINFEKL

41 Negative and positive selecting peptides differentially localise key signalling proteins Neg Pos Neg Pos

42 0 Affinity of pmhc for TcR threshold I # of T ces that respond threshold # of T ces that respond How can you achieve the sharp threshold effect that regulates signal protein activity and/or localisation? I 0 Affinity of pmhc for TcR

43 Positive feedback loop: X+Y=Z Rapid accumulation of Z (a self reinforcing circuit) Negative feedback loop: X+Y=Z Rapid depletion of X, and hence Z (a self limiting circuit) Positive and negative feedback loops are non-trivial to characterise using genetic approaches. Eg, if X were knocked out, there would be no Z produced and hence not feedback observed. If Z were knocked out, X+Y might not have any effect and feedback still not observed.

44 Transient or low affinity interactions is abortive because Shp1 is activated and it inhibits Lck. Lck 394 CD Lck Shp1 phosphorylation of Shp1 by Lck results in Shp1 binding to the Lck SH2 domain and dephosphorylating Lck Ras Erk Sustained signalling leads to a positive feed forward loop as the interaction between Shp1 and Lck is inhibited.

45 The response to increased ligand is switch like The Erk response acts like a high gin digital amplifier.

46 Transient or low affinity interactions is abortive because Shp1 is activated and it inhibits Lck. Lck 394 CD45 Lck 505 Shp1 phosphorylation of Shp1 by Lck results in Shp1 binding to the Lck SH2 domain and dephosphorylating Lck Ras Sustained signalling leads to a positive feed forward loop as the interaction between Shp1 and Lck is inhibited. Erk Agonists induce Ras-Erk signalling and hence eliminate neg feedback. Antagonists are peptides with shorter half lives. These fail to engage Erk and instead activate Shp1. The Shp1-signal spreads hence blocking activation by subsequent agonist ligands.

47 Two complementary pathways to Ras activation Ras is a small GTPase GTP-bound form is active, GDP-bound form is inactive RasGEFs catlyse the exchange of GDP to GTP Intrinsic GTPase activity catalyses return to GDP-bound state GAPs stimulate intrinsic GTPase activity Ras-GTP generated by RasGRP stimulates the GEF activity by Sos. LAT PIP2 Ras GDP SOS SH3 SH3 DAG + IP3 PLCγ PKC SH2 Grb 2 Ras GTP GDP Raf Ras GDP Ras-GAP Erk1/2 Ras GTP Transcription factors (Elk 1, SRF, AP 1, NFAT) Ras-GRP1 GDP

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50 Two complementary pathways to Ras activation Ras is a small GTPase GTP-bound form is active, GDP-bound form is inactive RasGEFs catlyse the exchange of GDP to GTP Intrinsic GTPase activity catalyses return to GDP-bound state GAPs stimulate intrinsic GTPase activity Ras-GTP generated by RasGRP stimulates the GEF activity by Sos. LAT PIP2 Ras GDP SOS SH3 SH3 DAG + IP3 PLCγ PKC SH2 Grb 2 Ras GTP GDP Raf Ras GDP Ras-GAP Erk1/2 Ras GTP Transcription factors (Elk 1, SRF, AP 1, NFAT) Ras-GRP1 GDP

51 T cell activation occurs through sequential stages over a h period Given that T cells make multiple, often sustained contacts with DCs, there are several opportunities to make meaningful immune synapses which may affect differentiation.

52 Can the hysteresis model for Ras activation contribute to signalling memory?

53 Concluding remarks Due to the nature of the ligands and biology of the response, an analogue to digital conversion process appears to be essential. Several independent studies have identified the Ras-Erk pathway as a node that can mediate this conversion. Positive and negative feedback loops help create stable on or off states. Biochemical techniques and genetics have been used to identify the components of the signalling networks. Mathematical modelling is a fundamental tool used to predict how feedback loops respond to different agonist affinities and/or avidities. Mathematical modelling also makes predictions that can be tested experimentally.

54 Reading list Stefanova Germain: TCR ligand discrimination is enforced by competing ERK positive and SHP-1 negative feedback pathways. Nat Immunol : Altan-Bonnet Germain: Modelling T cell antigen discrimination based on feedback control of digital Erk responses. PLOS biology : Daniels Palmer: Thymic selection threshold defined by compartmentalisation of Ras/MAPK signalling. Nature : Das Weiss, Chakrobarty and Roose: Digital signalling and hysteresis characterises ras activation in lymphocytes. Cell : Prasad Chakrobarty. Origin of the sharp boundary that discriminates positive and negative selection of thymocytes. PNAS :