Expression and regulation. Thymocytes, T cells and B-1a cells Developmentally regulated. Activated T cells Intracellular pool reservoir

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1 Supplementary information Table 1 Additional negative regulators of T-cell-receptor-induced signalling Negative regulator Membrane receptors CD5 1-4 Localization and key structural features Three scavenger receptor cysteine-rich domains ITAM-like, ITIM-like Expression and regulation Thymocytes, T and B-1a Developmentally regulated Proposed interactors and mechanism Intracellular ligands: LCK, SHP1, CKII, CAMKII, CAMKIV Negative regulation/tuning of TCR signalling Phenotype of gene-knockout mice Slightly altered positive and negative selection of thymocytes Increased thymocyte proliferation Increased TCR-induced Ca 2+ flux CTLA4 5-7 PD1 5, 8 9, 10 BTLA Plasma Enzymes 11, 12 CD45 Plasma 13, 14 CD148 Plasma Mainly 15, 16 SHP2 (PTPN11) PTPN22 (PEP, LYP) 15, Ig-domain homodimeric Critical YVKM motif in the intracellular region CD28/CTLA4 family member One ITIM, one ITSM CD28/CTLA4 family member Two ITIMs and one GRB2 motif Three fibronectin type III domains One PTP domain and one PTP- like domain in the intracellular region Nine fibronectin type III domains in the extracellular region One PTP domain in the intracellular region cytoplasmic Tyrosine phosphatase related to SHP1 Cytoplasmic PTP-domain containing a C-terminal PEST motif Activated T Intracellular pool reservoir Activated T, B and macrophages Activated T and T H 1 High levels in haematopoietic Alternative spliced isoforms Activated T Widely distributed, also in non-haematopoietic Widely distributed Interacts with inhibitory receptors and adaptors Haematopoietic Upregulated after TCR stimulation Extracellular ligands : CD80, CD86 Intracellular ligands: SHP1, SHP2, PP2A Antagonizes stop signal and CD28 signalling Extracellular ligands: PDL1, PDL2 Intracellular ligands: SHP1, SHP2 Broad negative regulator of the immune response Extracellular ligands: Herpesvirus cell entry mediator Intracellular ligands: SHP1, SHP2 A positive regulator of LCK High levels negatively regulate LCK by dephosphorylating ptyr394lck Dephosphorylates LAT and PLCγ1 when overexpressed Positively regulates SRF in B and myeloid GAB2 Dephosphorylates some TCR signalosome components (?) CSK, c-cbl, GRB2 Dephosphorylates FYN and LCK on activation loop Dephosphorylates ZAP70 Spontaneous T-cell activation and lymphoproliferative disease CTLA4 abnormalities are associated with autoimmune disease susceptibility in humans Lupus-like glomerulonepritis in C57BL/6 mice Dilated cardiomyopathy in BALB/c mice Increased numbers of CD8 + memory T Increased TCR-induced proliferation Increased susceptibility to autoimmunity Severe block of thymocyte differentiation Development of a few mature T that do not respond to TCR stimulation Humans lacking CD45 expression develop severe combined immunodeficiency Altered B-cell and myeloid-cell, but not T-cell, development Decreased LYN activity in CD45 -/- CD148 -/- B and myeloid Decreased thymic cellularity and TCR-induced ERK activation Decreased numbers of effector and memory T in aged mice Spontaneous germinal-centre development Increased TCR-induced LCK activation in memory T SNPs associated with type 1 diabetes and rheumatoid arthritis susceptibility

2 DGKα 20 DGKζ 21 DRAK , 24 ERK1/ERK2 Cytoplasmic Cytoplasm, DGKα: EF-hand, C1 DGKζ: C1, ankyrin repeats Cytoplasmic and/or nuclear Serine/threonine kinase C-terminal regulatory domain and/or nuclear Serine/threonine kinase, Member of the MAPK family DGKα regulated by Ca 2+, lipids and Tyr DGKζ regulated by lipids and Ser Ubiquitous and upregulated in thymic maturation TCR-induced Ca2 + - dependent auto Ubiquitous -Involved in the activation of transcription factors Converts diacylglycerol to phosphatidic acid Decreases recruitment of RASGRP1 and RAS activation No substrate or interacting protein identified to date Phosphorylates Thr155 of LAT, impairing recruitment of PLCγ1 Increased proliferation and cytokine production T Increased RAS and ERK activation Resistance to anergy induction Increased positive selection of CD4 + thymocytes Increased numbers of memory-like T Decreased susceptibility to autoimmunity Increased Ca 2+ flux and ERK activation Decreased T-cell requirement for co-stimulation Widespread effects Defective thymocyte maturation SLAT 25 Scaffolds/adaptors PAG (CBP) , 29 LIME Trans 29, 30 NTAL (LAT2, LAB) Trans LAX31 26, 32 SIT Trans 31, 33 GAB2 Cytoplasmic Guanine nucleotide exchange factor for RAC/CDC42 Trans, related to LAT Trans Not present in GEMs Related to LAT Not present in GEMs N-glycosylated disulphide-bound homodimer PH domain, c-met binding domain, Proand Thymocytes and T Increased in T H 2 Decreased in T H 1 TCR-induced Co-localizes with TCR at the immunological synapse Ubiquitous Dephosphorylated on Tyr after TCR stimulation Associated with FYNT Predominantly T, B and macrophages CD4-induced Tyr Activated T, B, NK and mast Activated T and B Lymphocyte-specific Tyr phosphorylated after TCR stimulation Widely expressed Upregulated in activated T Decreases ZAP70 TCRζ association Inhibits SFK activation through CSK recruitment CSK, LCK, FYN Antagonizes LAT function Mechanism unknown Sequesters GRB2, GADS and PI3K Mechanism unknown SHP2, CSK, GRB2 SHP2, PI3K, LAT Constitutive association with GADS and GRB2 Decreased Ca 2+ release from ER stores Decreased DN1 thymocyte expansion No alteration of T-cell development and activation Increased positive selection No effector B-cell development Small increase in positive selection Hyperactivated T and autoimmune syndrome in aged mice* Increased Ca 2+ flux, MAPK and AKT activation Increased T-cell and B-cell survival Spontaneous germinal-centre formation Increased homeostatic proliferation of CD8 + T Decreased TCR-mediated ZAP70 activation Increased TCR-induced IL-2 production and proliferation

3 24, 31 GRAP Cytoplasmic 31, 34 ALX Cytoplasmic Related to GRB2 One SH2 domain Pro-rich motifs TCR-induced Tyr Lymphoid LAT, HPK1, SAM68 Increased TCR-induced IL-2, ERK activation, FOS expression Predominantly T and LAX Constitutive p38 kinase activation B Increased TCR-induced IL-2 production and TCR-induced Tyr proliferation c-cbl, CBL-B , 41 SLAP, SLAP2 Plasma--bound Cytoplasmic Tyr-kinase-binding domain, RING finger domain Pro-rich motifs, Leu-zipper/UBA domain Myristoylation motif, SH3 and SH2 domains: homology to LCK, C-terminal unique domain Ubiquitous; c-cbl preferentially expressed by thymocytes, CBL-B more abundant in mature T E3 ubiquitin ligases, RINGtype SLAP expressed by lymphoid tissues, DP thymocytes SLAP2 expression is haematopoietic-cell specific Recruited to TCR signalosome by binding to ZAP70, SLAP or GRB2/LAT Promotes ubiquitylation and degradation of TCRζ, LCK, PI3K (p85), VAV1 c-cbl, ZAP70 Regulates TCR/CD3 and ZAP70 expression c-cbl -/- : lymphoid hyperplasia, increased positive selection of CD4 + T ; decreased TCR/CD3 downmodulation; increased TCR-induced Tyr Cbl-b -/- : increased T-cell proliferation and IL-2 production; increased CD28-independent activation; susceptibility to autoimmunity c-cbl -/- Cbl-b -/- : severe autoimmune syndrome; decreased viability Slap -/- : increased positive selection; increased TCR and CD5 expression by DP thymocytes *Observed only in one knockout line. CAMK, calmodulin-dependent kinase; CBL, casitas B-lineage lymphoma; CDC42, cell-division cycle 42; CKII, casein kinase II; CTLA4, cytotoxic T- lymphocyte antigen 4; DKG, diacylglycerol kinase; DN, double negative; DP, double positive; ERK, extracellular-signal-regulated kinase; GAB2, growth-factor-receptor-binding protein 2 (GRB2)-associated binding protein 2; GEMs, glycosphingolipids-enriched s; GRAP, GRB2-related adaptor protein; HPK1, haematopoietic progenitor kinase 1; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibition motif; ITSM, immunoreceptor tyrosine-based switch motif; LAT, linker for activation of T ; LAX, linker for activation of X, where X denotes an as yet unidentified cell; LIME, LCK-interacting protein; MAPK, mitogen-activated protein kinase; NK, natural killer; NTAL, non-t-cell activation linker; PAG, phosphoprotein associated with GEMs; PI3K, phosphoinositide 3-kinase; PD1, programmed cell death 1; PDL1, PD ligand 1; PH, pleckstrin homology; PLCγ, phospholipase Cγ; PP2A, protein phosphatase 2A; PTP, protein tyrosine phosphatase; RING, really interesting new gene; SHP1, SRC homology 2 (SH2)-domain-containing protein tyrosine phosphatase 1; SIT, SHP2-interacting trans adaptor protein; SFK; SRC family kinases; SLAP, SRC-like-adaptor protein; SNPs; single nucleotide polymorphisms; SRF, serum response factor; TCR, T-cell receptor; T H, T helper cell; UBA, ubiquitin-associated; ZAP70, ζ-chain-associated protein kinase of 70 kda. Bibliography 1. Lozano, F. et al. CD5 signal transduction: positive or negative modulation of antigen receptor signaling. Crit Rev Immunol 20, (2000). 2. Tarakhovsky, A. et al. A role for CD5 in TCR-mediated signal transduction and thymocyte selection. Science 269, (1995). 3. Smith, K. et al. Sensory adaptation in naive peripheral CD4 T. J Exp Med 194, (2001).

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