Kinetic proofreading through the multi-step activation of the ZAP70 kinase underlies early T cell ligand discrimination.

T cells recognize a few high-affinity antigens among a vast array of lower affinity antigens. According to the kinetic proofreading model, antigen discrimination properties could be explained by the gradual amplification of small differences in binding affinities as the signal is transduced downstream of the T cell receptor. Which early molecular events are affected by ligand affinity, and how, has not been fully resolved. Here, we used time-resolved high-throughput proteomic analyses to identify and quantify the phosphorylation events and protein-protein interactions encoding T cell ligand discrimination in antigen-experienced T cells. Although low-affinity ligands induced phosphorylation of the Cd3 chains of the T cell receptor and the interaction of Cd3 with the Zap70 kinase as strongly as high-affinity ligands, they failed to activate Zap70 to the same extent. As a result, formation of the signalosome of the Lat adaptor was severely impaired with low- compared with high-affinity ligands, whereas formation of the signalosome of the Cd6 receptor was affected only partially. Overall, this study provides a comprehensive map of molecular events associated with T cell ligand discrimination.

Quantitative interactomics in primary T cells unveils TCR signal diversification extent and dynamics.

The activation of T cells by the T cell antigen receptor (TCR) results in the formation of signaling protein complexes (signalosomes), the composition of which has not been analyzed at a systems level. Here, we isolated primary CD4+ T cells from 15 gene-targeted mice, each expressing one tagged form of a canonical protein of the TCR-signaling pathway. Using affinity purification coupled with mass spectrometry, we analyzed the composition and dynamics of the signalosomes assembling around each of the tagged proteins over 600 s of TCR engagement. We showed that the TCR signal-transduction network comprises at least 277 unique proteins involved in 366 high-confidence interactions, and that TCR signals diversify extensively at the level of the plasma membrane. Integrating the cellular abundance of the interacting proteins and their interaction stoichiometry provided a quantitative and contextual view of each documented interaction, permitting anticipation of whether ablation of a single interacting protein can impinge on the whole TCR signal-transduction network.

A hematopoietic cell-driven mechanism involving SLAMF6 receptor, SAP adaptors and SHP-1 phosphatase regulates NK cell education.

Activation of natural killer (NK) cells by hematopoietic target cells is controlled by the SLAM family of receptors and by the associated SAP family of adaptors. Here we found that SLAM receptors also enhanced NK cell activation by nonhematopoietic target cells, which lack ligands for SLAM receptors. This function was mediated by SLAMF6, a homotypic SLAM receptor found on NK cells and other hematopoietic cells, and was regulated by SAP adaptors, which uncoupled SLAM receptors from phosphatase SHP-1 and diminished the effect of SLAMF6 on NK cell responsiveness toward nonhematopoietic cells. Thus, in addition to their role in NK cell activation by hematopoietic cells, the SLAM-SAP pathways influence responsiveness toward nonhematopoietic targets by a process akin to NK cell 'education'.

Integrative biology of T cell activation.

The activation of T cells mediated by the T cell antigen receptor (TCR) requires the interaction of dozens of proteins, and its malfunction has pathological consequences. Our major focus is on new developments in the systems-level understanding of the TCR signal-transduction network. To make sense of the formidable complexity of this network, we argue that 'fine-grained' methods are needed to assess the relationships among a few components that interact on a nanometric scale, and those should be integrated with high-throughput '-omic' approaches that simultaneously capture large numbers of parameters. We illustrate the utility of this integrative approach with the transmembrane signaling protein Lat, which is a key signaling hub of the TCR signal-transduction network, as a connecting thread.

Quantitative proteomics analysis of signalosome dynamics in primary T cells identifies the surface receptor CD6 as a Lat adaptor-independent TCR signaling hub.

T cell antigen receptor (TCR)-mediated activation of T cells requires the interaction of dozens of proteins. Here we used quantitative mass spectrometry and activated primary CD4(+) T cells from mice in which a tag for affinity purification was knocked into several genes to determine the composition and dynamics of multiprotein complexes that formed around the kinase Zap70 and the adaptors Lat and SLP-76. Most of the 112 high-confidence time-resolved protein interactions we observed were previously unknown. The surface receptor CD6 was able to initiate its own signaling pathway by recruiting SLP-76 and the guanine nucleotide-exchange factor Vav1 regardless of the presence of Lat. Our findings provide a more complete model of TCR signaling in which CD6 constitutes a signaling hub that contributes to the diversification of TCR signaling.

The lymphoid lineage-specific actin-uncapping protein Rltpr is essential for costimulation via CD28 and the development of regulatory T cells.

Although T cell activation can result from signaling via T cell antigen receptor (TCR) alone, physiological T cell responses require costimulation via the coreceptor CD28. Through the use of an N-ethyl-N-nitrosourea-mutagenesis screen, we identified a mutation in Rltpr. We found that Rltpr was a lymphoid cell-specific, actin-uncapping protein essential for costimulation via CD28 and the development of regulatory T cells. Engagement of TCR-CD28 at the immunological synapse resulted in the colocalization of CD28 with both wild-type and mutant Rltpr proteins. However, the connection between CD28 and protein kinase C-θ and Carma1, two key effectors of CD28 costimulation, was abrogated in T cells expressing mutant Rltpr, and CD28 costimulation did not occur in those cells. Our findings provide a more complete model of CD28 costimulation in which Rltpr has a key role.

ARHGAP45 controls naïve T- and B-cell entry into lymph nodes and T-cell progenitor thymus seeding.

T and B cells continually recirculate between blood and secondary lymphoid organs. To promote their trans-endothelial migration (TEM), chemokine receptors control the activity of RHO family small GTPases in part via GTPase-activating proteins (GAPs). T and B cells express several RHO-GAPs, the function of most of which remains unknown. The ARHGAP45 GAP is predominantly expressed in hematopoietic cells. To define its in vivo function, we describe two mouse models where ARHGAP45 is ablated systemically or selectively in T cells. We combine their analysis with affinity purification coupled to mass spectrometry to determine the ARHGAP45 interactome in T cells and with time-lapse and reflection interference contrast microscopy to assess the role of ARGHAP45 in T-cell polarization and motility. We demonstrate that ARHGAP45 regulates naïve T-cell deformability and motility. Under physiological conditions, ARHGAP45 controls the entry of naïve T and B cells into lymph nodes whereas under competitive repopulation it further regulates hematopoietic progenitor cell engraftment in the bone marrow, and T-cell progenitor thymus seeding. Therefore, the ARGHAP45 GAP controls multiple key steps in the life of T and B cells.

LymphoAtlas: a dynamic and integrated phosphoproteomic resource of TCR signaling in primary T cells reveals ITSN2 as a regulator of effector functions.

T-cell receptor (TCR) ligation-mediated protein phosphorylation regulates the activation, cellular responses, and fates of T cells. Here, we used time-resolved high-resolution phosphoproteomics to identify, quantify, and characterize the phosphorylation dynamics of thousands of phosphorylation sites in primary T cells during the first 10 min after TCR stimulation. Bioinformatic analysis of the data revealed a coherent orchestration of biological processes underlying T-cell activation. In particular, functional modules associated with cytoskeletal remodeling, transcription, translation, and metabolic processes were mobilized within seconds after TCR engagement. Among proteins whose phosphorylation was regulated by TCR stimulation, we demonstrated, using a fast-track gene inactivation approach in primary lymphocytes, that the ITSN2 adaptor protein regulated T-cell effector functions. This resource, called LymphoAtlas, represents an integrated pipeline to further decipher the organization of the signaling network encoding T-cell activation. LymphoAtlas is accessible to the community at: https://bmm-lab.github.io/LymphoAtlas.

The proline-rich sequence of CD3epsilon controls T cell antigen receptor expression on and signaling potency in preselection CD4+CD8+ thymocytes.

Antigen recognition by T cell antigen receptors (TCRs) is thought to 'unmask' a proline-rich sequence (PRS) present in the CD3epsilon cytosolic segment, which allows it to trigger T cell activation. Using 'knock-in' mice with deletion of the PRS, we demonstrate here that elimination of the CD3epsilon PRS had no effect on mature T cell responsiveness. In contrast, in preselection CD4+CD8+ thymocytes, the CD3epsilon PRS acted together with the adaptor protein SLAP to promote CD3zeta degradation, thereby contributing to downregulation of TCR expression on the cell surface. In addition, analysis of CD4+CD8+ thymocytes of TCR-transgenic mice showed that the CD3epsilon PRS enhanced TCR sensitivity to weak ligands. Our results identify previously unknown functions for the evolutionarily conserved CD3epsilon PRS at the CD4+CD8+ developmental stage and suggest a rather limited function in mature T cells.

A THEMIS:SHP1 complex promotes T-cell survival.

THEMIS is critical for conventional T-cell development, but its precise molecular function remains elusive. Here, we show that THEMIS constitutively associates with the phosphatases SHP1 and SHP2. This complex requires the adapter GRB2, which bridges SHP to THEMIS in a Tyr-phosphorylation-independent fashion. Rather, SHP1 and THEMIS engage with the N-SH3 and C-SH3 domains of GRB2, respectively, a configuration that allows GRB2-SH2 to recruit the complex onto LAT. Consistent with THEMIS-mediated recruitment of SHP to the TCR signalosome, THEMIS knock-down increased TCR-induced CD3-ζ phosphorylation, Erk activation and CD69 expression, but not LCK phosphorylation. This generalized TCR signalling increase led to augmented apoptosis, a phenotype mirrored by SHP1 knock-down. Remarkably, a KI mutation of LCK Ser59, previously suggested to be key in ERK-mediated resistance towards SHP1 negative feedback, did not affect TCR signalling nor ligand discrimination in vivo. Thus, the THEMIS:SHP complex dampens early TCR signalling by a previously unknown molecular mechanism that favours T-cell survival. We discuss possible implications of this mechanism in modulating TCR output signals towards conventional T-cell development and differentiation.

Fit αß T-cell receptor suppresses leukemogenesis of Pten-deficient thymocytes.

Signaling through the αßT cell receptor (TCR) is a crucial determinant of T-cell fate and can induce two opposite outcomes during thymocyte development: cell death or survival and differentiation. To date, the role played by T-cell receptor in the oncogenic transformation of developing T cells remains unclear. Here we show that human primary T-cell acute lymphoblastic leukemias expressing an αßT cell receptor are frequently deficient for phosphatase and tensin homolog protein (PTEN), and fail to respond strongly to T-cell receptor activation. Using Pten-deficient T-cell acute lymphoblastic leukemia mouse models, we confirm that T-cell receptor signaling is involved in leukemogenesis. We show that abrogation of T-cell receptor expression accelerated tumor onset, while enforced expression of a fit transgenic T-cell receptor led to the development of T-cell receptor-negative lymphoma and delayed tumorigenesis. We further demonstrate that pre-tumoral Pten-deficient thymocytes harboring fit T-cell receptors undergo early clonal deletion, thus preventing their malignant transformation, while cells with unfit T-cell receptors that should normally be deleted during positive selection, pass selection and develop T-cell acute lymphoblastic leukemias. Altogether, our data show that fit T-cell receptor signaling suppresses tumor development mediated by Pten loss-of-function and point towards a role of Pten in positive selection.

Loss of the LAT adaptor converts antigen-responsive T cells into pathogenic effectors that function independently of the T cell receptor.

Despite compromised T cell antigen receptor (TCR) signaling, mice in which tyrosine 136 of the adaptor linker for activation of T cells (LAT) was constitutively mutated (Lat(Y136F) mice) accumulate CD4(+) T cells that trigger autoimmunity and inflammation. Here we show that equipping postthymic CD4(+) T cells with LATY136F molecules or rendering them deficient in LAT molecules triggers a lymphoproliferative disorder dependent on prior TCR engagement. Therefore, such disorders required neither faulty thymic T cell maturation nor LATY136F molecules. Unexpectedly, in CD4(+) T cells recently deprived of LAT, the proximal triggering module of the TCR induced a spectrum of protein tyrosine phosphorylation that largely overlapped the one observed in the presence of LAT. The fact that such LAT-independent signals result in lymphoproliferative disorders with excessive cytokine production demonstrates that LAT constitutes a key negative regulator of the triggering module and of the LAT-independent branches of the TCR signaling cassette.

Co-recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR-induced ubiquitylation.

T-cell receptor (TCR) signaling is essential for the function of T cells and negatively regulated by the E3 ubiquitin-protein ligases CBL and CBLB Here, we combined mouse genetics and affinity purification coupled to quantitative mass spectrometry to monitor the dynamics of the CBL and CBLB signaling complexes that assemble in normal T cells over 600 seconds of TCR stimulation. We identify most previously known CBL and CBLB interacting partners, as well as a majority of proteins that have not yet been implicated in those signaling complexes. We exploit correlations in protein association with CBL and CBLB as a function of time of TCR stimulation for predicting the occurrence of direct physical association between them. By combining co-recruitment analysis with biochemical analysis, we demonstrated that the CD5 transmembrane receptor constitutes a key scaffold for CBL- and CBLB-mediated ubiquitylation following TCR engagement. Our results offer an integrated view of the CBL and CBLB signaling complexes induced by TCR stimulation and provide a molecular basis for their negative regulatory function in normal T cells.

Revisiting the Timing of Action of the PAG Adaptor Using Quantitative Proteomics Analysis of Primary T Cells.

The protein tyrosine kinase LCK plays a key role in TCR signaling, and its activity is dynamically controlled by the tyrosine kinase C-terminal Src kinase (CSK) and the tyrosine phosphatase CD45. CSK is brought in contiguity to LCK via binding to a transmembrane adaptor known as phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG). The lack of a blatant phenotype in PAG-deficient mice has impeded our understanding of the mechanisms through which PAG exerts its negative-regulatory role in TCR signaling. We used quantitative mass spectrometry and both thymocytes and CD4(+) T cells from mice in which a tag for affinity purification was knocked in the gene coding for PAG to determine the composition and dynamics of the multiprotein complexes that are found around PAG over 5 min of activation. Most of the high-confidence interactions that we observed were previously unknown. Using phosphoproteomic analysis, PAG showed low levels of tyrosine phosphorylation in resting primary mouse CD4(+) T cells; the levels of tyrosine phosphorylation increased and reached a maximum 2 min after stimulation. Analysis of the dynamics of association of the protein tyrosine phosphatase PTPN22 and lipid phosphatase SHIP-1 with PAG following T cell activation suggests that both cooperate with CSK to terminate T cell activation. Our findings provide a model of the role for PAG in mouse primary CD4(+) T cells that is consistent with recent phosphoproteomic studies of the Jurkat T cell line but difficult to reconcile with former biochemical studies indicating that PAG is constitutively phosphorylated in resting T cells and rapidly dephosphorylated once the TCR is engaged.

The tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20) imposes a brake on antitumor activity of CD8 T cells.

The transcription factor NF-κB is central to inflammatory signaling and activation of innate and adaptive immune responses. Activation of the NF-κB pathway is tightly controlled by several negative feedback mechanisms, including A20, an ubiquitin-modifying enzyme encoded by the tnfaip3 gene. Mice with selective deletion of A20 in myeloid, dendritic, or B cells recapitulate some human inflammatory pathology. As we observed high expression of A20 transcripts in dysfunctional CD8 T cells in an autochthonous melanoma, we analyzed the role of A20 in regulation of CD8 T-cell functions, using mice in which A20 was selectively deleted in mature conventional T cells. These mice developed lymphadenopathy and some organ infiltration by T cells but no splenomegaly and no detectable pathology. A20-deleted CD8 T cells had increased sensitivity to antigen stimulation with production of large amounts of IL-2 and IFNγ, correlated with sustained nuclear expression of NF-κB components reticuloendotheliosis oncogene c-Rel and p65. Overexpression of A20 by retroviral transduction of CD8 T cells dampened their intratumor accumulation and antitumor activity. In contrast, relief from the A20 brake in NF-κB activation in adoptively transferred antitumor CD8 T cells led to improved control of melanoma growth. Tumor-infiltrating A20-deleted CD8 T cells had enhanced production of IFNγ and TNFα and reduced expression of the inhibitory receptor programmed cell death 1. As manipulation of A20 expression in CD8 T cells did not result in pathologic manifestations in the mice, we propose it as a candidate to be targeted to increase antitumor efficiency of adoptive T-cell immunotherapy.

In the absence of its cytosolic domain, the CD28 molecule still contributes to T cell activation.

The CD28 costimulatory receptor has a pivotal role in T cell biology as this molecule amplifies T cell receptor (TCR) signals to provide an efficient immune T cell response. There is a large debate about how CD28 mediates these signals. Here, we designed a CD28 gene-targeted knock-in mouse strain lacking the cytoplasmic tail of CD28. As is the case in CD28-deficient (CD28 knock-out) mice, regulatory T cell homeostasis and T cell activation are altered in these CD28 knock-in mice. Unexpectedly, the presence of a CD28 molecule deprived of its cytoplasmic tail could partially induce some early activation events in T cells such as signaling events or expression of early activation markers. These results unravel a new mechanism of T cell costimulation by CD28, independent of its cytoplasmic tail.

Control of CD8 T cell proliferation and terminal differentiation by active STAT5 and CDKN2A/CDKN2B.

CD8 T cells used in adoptive immunotherapy may be manipulated to optimize their effector functions, tissue-migratory properties and long-term replicative potential. We reported that antigen-stimulated CD8 T cells transduced to express an active form of the transcription factor signal transducer and activator of transcription 5 (STAT5CA) maintained these properties upon adoptive transfer. We now report on the requirements of STAT5CA-expressing CD8 T cells for cell survival and proliferation in vivo. We show that STAT5CA expression allows for greater expansion of T cells in vivo, while preserving dependency on T-cell-receptor-mediated tonic stimulation for their in vivo maintenance and return to a quiescent stage. STAT5CA expression promotes the formation of a large pool of effector memory T cells that respond upon re-exposure to antigen and present an increased sensitivity to γc receptor cytokine engagement for STAT5 phosphorylation. In addition, STAT5CA expression prolongs the survival of what would otherwise be short-lived terminally differentiated KLRG1-positive effector cells with up-regulated expression of the senescence-associated p16(INK) (4A) transcripts. However, development of a KLRG1-positive CD8 T cell population was independent of either p16(INK) (4A) or p19(ARF) expression (as shown using T cells from CDKN2A(-/-) mice) but was associated with expression of transcripts encoding p15(INK) (4B) , another protein involved in senescence induction. We conclude that T-cell-receptor- and cytokine-dependent regulation of effector T cell homeostasis, as well as mechanisms leading to senescent features of a population of CD8 T cells are maintained in STAT5CA-expressing CD8 T cells, even for cells that are genetically deficient in expression of the tumour suppressors p16(INK) (4A) and p19(ARF) .

Computational modeling of the main signaling pathways involved in mast cell activation.

A global and rigorous understanding of the signaling pathways and cross-regulatory processes involved in mast cell activation requires the integration of published information with novel functional datasets into a comprehensive computational model. Based on an exhaustive curation of the existing literature and using the software CellDesigner, we have built and annotated a comprehensive molecular map for the FcεRI signaling network. This map can be used to visualize and interpret high-throughput expression data. Furthermore, leaning on this map and using the logical modeling software GINsim, we have derived a qualitative dynamical model, which recapitulates the most salient features of mast cell activation. The resulting logical model can be used to explore the dynamical properties of the system and its responses to different stimuli, in normal or mutant conditions.

Proteomic analysis of the SH2 domain-containing leukocyte protein of 76 kDa (SLP76) interactome in resting and activated primary mast cells [corrected].

We report the first proteomic analysis of the SLP76 interactome in resting and activated primary mouse mast cells. This was made possible by a novel genetic approach used for the first time here. It consists in generating knock-in mice that express signaling molecules bearing a C-terminal tag that has a high affinity for a streptavidin analog. Tagged molecules can be used as molecular baits to affinity-purify the molecular complex in which they are engaged, which can then be studied by mass spectrometry. We examined first SLP76 because, although this cytosolic adapter is critical for both T cell and mast cell activation, its role is well known in T cells but not in mast cells. Tagged SLP76 was expressed in physiological amounts and fully functional in mast cells. We unexpectedly found that SLP76 is exquisitely sensitive to mast cell granular proteases, that Zn(2+)-dependent metalloproteases are especially abundant in mast cells and that they were responsible for SLP76 degradation. Adding a Zn(2+) chelator fully protected SLP76 in mast cell lysates, thereby enabling an efficient affinity-purification of this adapter with its partners. Label-free quantitative mass spectrometry analysis of affinity-purified SLP76 interactomes uncovered both partners already described in T cells and novel partners seen in mast cells only. Noticeably, molecules inducibly recruited in both cell types primarily concur to activation signals, whereas molecules recruited in activated mast cells only are mostly associated with inhibition signals. The transmembrane adapter LAT2, and the serine/threonine kinase with an exchange factor activity Bcr were the most recruited molecules. Biochemical and functional validations established the unexpected finding that Bcr is recruited by SLP76 and positively regulates antigen-induced mast cell activation. Knock-in mice expressing tagged molecules with a normal tissue distribution and expression therefore provide potent novel tools to investigate signalosomes and to uncover novel signaling molecules in mast cells.

LAT signaling pathology: an "autoimmune" condition without T cell self-reactivity.

Partial loss-of-function mutations in several molecules involved in T-cell receptor (TCR) signaling result in inflammation and autoimmunity. How can mutations that reduce TCR signaling output, paradoxically lead to immune pathology? This review summarizes experiments demonstrating that mutations in the linker for activation of T cells (LAT) predispose toward aberrant T cell responses to antigen in the presence of normal thymic selection. In the absence of LAT, antigen-specific T cells give rise to self-perpetuating pro-inflammatory responses and induce the production of autoantibodies independently of TCR engagement. Therefore, some pathological conditions called "autoimmune" might not result from the presence of self-reactive T cells, but from defective mechanisms that normally keep T cell activation in check.