Romain Roncagalli

The proline-rich sequence of CD3ε 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 CD3ε 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 CD3ε PRS had no effect on mature T cell responsiveness. In contrast, in preselection CD4+CD8+ thymocytes, the CD3ε PRS acted together with the adaptor protein SLAP to promote CD3ζ 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 CD3ε PRS enhanced TCR sensitivity to weak ligands. Our results identify previously unknown functions for the evolutionarily conserved CD3ε PRS at the CD4+CD8+ developmental stage and suggest a rather limited function in mature T cells.

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.

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

Significance Mechanisms controlling immune reactivity prevent excessive inflammation and autoimmunity, but generally dampen antitumor activity. The tumor necrosis factor alpha-induced protein 3 gene encoding the A20 protein, a key molecule controlling NF-κB activation, has been linked to the development of multiple inflammatory pathologies in humans, some of which are recapitulated in mice with selective deletion of A20 in myeloid, dendritic, or B cells. Here, mice with selective deletion of A20 in mature conventional T cells presented no detectable pathology. CD8 T cells from these mice showed increased antigen sensitivity with enhanced production of IL-2 and IFNγ. Importantly, A20-deleted CD8 T cells possessed heightened antitumor activity in vivo. Targeting this gene in adoptively transferred CD8 T cells could represent a promising mechanism to achieve tumor rejection. 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.

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.

CD6 modulates thymocyte selection and peripheral T cell homeostasis

Orta-Mascaró, Lozano, and collaborators provide the first analysis of CD6-deficient mice, showing that this molecule modulates T cell receptor signaling and the threshold for thymocyte and peripheral T cell subset selection.

The scaffolding function of the RLTPR protein explains its essential role for CD28 co-stimulation in mouse and human T cells

In two complementary papers, Casanova, Malissen, and collaborators report the discovery of human RLTPR deficiency, the first primary immunodeficiency of the human CD28 pathway in T cells. Together, the two studies elucidate the largely (but not completely) overlapping roles of RLTPR in CD28 signaling in T and B cells of humans and mice.

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.

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.

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.