Michaël Mingueneau - Academia.edu (original) (raw)

Papers by Michaël Mingueneau

Research paper thumbnail of Single-cell mass cytometry of TCR signaling: Amplification of small initial differences results in low ERK activation in NOD mice

Proceedings of the National Academy of Sciences, 2014

Signaling from the T-cell receptor (TCR) conditions T-cell differentiation and activation, requir... more Signaling from the T-cell receptor (TCR) conditions T-cell differentiation and activation, requiring exquisite sensitivity and discrimination. Using mass cytometry, a high-dimensional technique that can probe multiple signaling nodes at the single-cell level, we interrogate TCR signaling dynamics in control C57BL/6 and autoimmunity-prone nonobese diabetic (NOD) mice, which show ineffective ERK activation after TCR triggering. By quantitating signals at multiple steps along the signaling cascade and parsing the phosphorylation level of each node as a function of its predecessors, we show that a small impairment in initial pCD3ζ activation resonates farther down the signaling cascade and results in larger defects in activation of the ERK1/2-S6 and IκBα modules. This nonlinear property of TCR signaling networks, which magnifies small initial differences during signal propagation, also applies in cells from B6 mice activated at different levels of intensity. Impairment in pCD3ζ and pSLP76 is not a feedback consequence of a primary deficiency in ERK activation because no proximal signaling defect was observed in Erk2 KO T cells. These defects, which were manifest at all stages of T-cell differentiation from early thymic pre-T cells to memory T cells, may condition the imbalanced immunoregulation and tolerance in NOD T cells. More generally, this amplification of small initial differences in signal intensity may explain how T cells discriminate between closely related ligands and adopt strongly delineated cell fates.

Research paper thumbnail of Multiplicity and plasticity of Natural Killer cell signaling pathways

Natural killer (NK) cells express an array of activating receptors that associate with DAP12 (KAR... more Natural killer (NK) cells express an array of activating receptors that associate with DAP12 (KARAP), CD3zeta, and/or FcRgamma ITAM (immunoreceptor tyrosine-based activation motif)-bearing signaling subunits. In T and mast cells, ITAM-dependent signals are integrated by critical scaffolding elements such as LAT (linker for activation of T cells) and NTAL (non-T-cell activation linker). Using mice that are deficient for ITAM-bearing molecules, LAT or NTAL, we show that NK cell cytotoxicity and interferon-gamma secretion are initiated by ITAM-dependent and -independent as well as LAT/NTAL-dependent and -independent pathways. The role of these various signaling circuits depends on the target cell as well as on the activation status of the NK cell. The multiplicity and the plasticity of the pathways that initiate NK cell effector functions contrast with the situation in T cells and B cells and provide an explanation for the resiliency of NK cell effector functions to various pharmacologic inhibitors and genetic mutations in signaling molecules.

Research paper thumbnail of Multiplicity and plasticity of natural killer cell signaling pathways

Natural killer (NK) cells express an array of activating receptors that associate with DAP12 (KAR... more Natural killer (NK) cells express an array of activating receptors that associate with DAP12 (KARAP), CD3zeta, and/or FcRgamma ITAM (immunoreceptor tyrosine-based activation motif)-bearing signaling subunits. In T and mast cells, ITAM-dependent signals are integrated by critical scaffolding elements such as LAT (linker for activation of T cells) and NTAL (non-T-cell activation linker). Using mice that are deficient for ITAM-bearing molecules, LAT or NTAL, we show that NK cell cytotoxicity and interferon-gamma secretion are initiated by ITAM-dependent and -independent as well as LAT/NTAL-dependent and -independent pathways. The role of these various signaling circuits depends on the target cell as well as on the activation status of the NK cell. The multiplicity and the plasticity of the pathways that initiate NK cell effector functions contrast with the situation in T cells and B cells and provide an explanation for the resiliency of NK cell effector functions to various pharmacologic inhibitors and genetic mutations in signaling molecules.

Research paper thumbnail of Conditional density-based analysis of T cell signaling in single-cell data

Science, 2014

Quantitative characterization of T cell signaling. (A) The pCD3ζ-pSLP76 signaling interaction sho... more Quantitative characterization of T cell signaling. (A) The pCD3ζ-pSLP76 signaling interaction shown as (I) a scatterplot, (II) a kernel density estimate, and (III) by using a conditional DREVI method. (IV) Shape features are extracted and quantif ed. (B) DREVI plots of a signaling cascade downstream of TCR show the time-varying nature of edge shapes and strengths. (C) Edge strengths are quantif ed by using conditional DREMI.

Research paper thumbnail of Th2 Lymphoproliferative Disorders Resulting from Defective LAT Signalosomes

Novartis Foundation Symposia, 2006

LAT (linker for activation of T cells) is an integral membrane adaptor protein that constitutes i... more LAT (linker for activation of T cells) is an integral membrane adaptor protein that constitutes in T cells a major substrate of the ZAP-70 protein tyrosine kinase. LAT coordinates the assembly of a multiprotein signalling complex through phosphotyrosine-based motifs present within its intracytoplasmic segment. The resulting 'LAT signalosome' links the TCR to the main intracellular signalling pathways that regulate T cell development and T cell function. Early studies using transformed T cell lines suggested that LAT acts primarily as a positive regulator of T cell receptor (TCR) signalling. The partial or complete inhibition of T cell development observed in several mouse lines harbouring mutant forms of LAT was congruent with that view. More recently, LAT 'knock-ins' harbouring point mutations in the four COOH-terminal tyrosine residues, were found to develop lymphoproliferative disorders involving polyclonal T cells that produced high amounts of T helper-type 2 (Th2) cytokines. This unexpected finding revealed that LAT also constitutes a negative regulator of TCR signalling and T cell homeostasis. As discussed, the available data underscore that a novel immunopathology proper to defective LAT signalosome is likely taking shape.

Research paper thumbnail of LAT signaling pathology: an “autoimmune” condition without T cell self-reactivity

Trends in Immunology, 2010

Partial loss-of-function mutations in several molecules involved in T-cell receptor (TCR) signali... more 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.

Research paper thumbnail of Running head: Plasticity of NK signaling pathways

2 Summary Natural Killer (NK) cells express an array of activating receptors that associate with ... more 2 Summary Natural Killer (NK) cells express an array of activating receptors that associate with DAP12 (KARAP), CD3ζ and/or FcRγ ITAM (Immunoreceptor Tyrosine-based Activation Motif)bearing signaling subunits. In T and mast cells, ITAM-dependent signals are integrated by critical scaffolding elements such as LAT (linker for activation of T cells) and NTAL (non-T cell activation linker). Using mice that are deficient for ITAM-bearing molecules, LAT or NTAL, we show that NK cell cytotoxicity and interferon-γ secretion are initiated by ITAM-dependent and -independent as well as LAT/NTAL-dependent and -independent pathways. The role of these various signaling circuits depends on the target cell as well as on the activation status of the NK cell. The multiplicity and the plasticity of the pathways that initiate NK cell effector functions contrast with the situation in T cells and B cells and provide an explanation for the resiliency of NK cell effector functions to various pharmacological inhibitors and genetic mutations in signaling molecules.

Research paper thumbnail of Supplemental Data Loss of the LAT Adaptor Converts Antigen-Responsive T Cells into Pathogenic Effectors that Function Independently of the T Cell Receptor

Research paper thumbnail of LAT signaling pathology: an “autoimmune” condition without T cell self-reactivity

Trends in Immunology, 2010

Partial loss-of-function mutations in several molecules involved in T-cell receptor (TCR) signali... more 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.

Research paper thumbnail of Lymphoproliferative disorders involving T helper effector cells with defective LAT signalosomes

Seminars in Immunopathology, 2010

coordinates the assembly of a multiprotein complex-the LAT signalosome-that links the T cell-spec... more coordinates the assembly of a multiprotein complex-the LAT signalosome-that links the T cell-specific and the ubiquitous components of the T cell antigen receptor (TCR) signaling pathway. The present review focuses on recent LAT knock-in mice that were found to develop lymphoproliferative disorders involving polyclonal CD4 + T cells that produced excessive amounts of T helper-type 2 cytokines. These mouse models revealed that LAT constitutes more than just a positive regulator of TCR signaling and plays a negative regulatory role that contributes to terminate antigen-driven T cell responses by exerting a repressive function on components of the TCR signaling cassette that lie upstream of LAT or function independently of LAT. In the absence of such a LAT-operated negative regulatory loop that is intrinsic to conventional CD4 + T cells and of no lesser importance than the extrinsic regulatory mechanisms mediated by regulatory T cells, physiologic, antigenspecific CD4 + T cell responses evolve into chronic proinflammatory responses that perpetuate themselves in a manner that does not depend on engagement of the TCR and that induce the production of massive amounts of antibodies and autoantibodies in a major histocompatibility complex-II-independent, "quasi-mitogenic" mode. As discussed, these data underscore that a novel immunopathology proper to defective LAT signalosomes is likely taking shape, and we propose to call it "LAT signaling pathology."

Research paper thumbnail of The proline-rich sequence of CD3ε controls T cell antigen receptor expression on and signaling potency in preselection CD4+CD8+ thymocytes

Nature Immunology, 2008

Antigen recognition by T cell antigen receptors (TCRs) is thought to &amp... more 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.

Research paper thumbnail of Transcriptional insights into the CD8+ T cell response to infection and memory T cell formation

Nature Immunology, 2013

After infection, many factors coordinate the population expansion and differentiation of CD8+ eff... more After infection, many factors coordinate the population expansion and differentiation of CD8+ effector and memory T cells. Using data of unparalleled breadth from the Immunological Genome Project, we analyzed the CD8+ T cell transcriptome throughout infection to establish gene-expression signatures and identify putative transcriptional regulators. Notably, we found that the expression of key gene signatures can be used to predict the memory-precursor potential of CD8+ effector cells. Long-lived memory CD8+ cells ultimately expressed a small subset of genes shared by natural killer T and γδ T cells. Although distinct inflammatory milieu and T cell precursor frequencies influenced the differentiation of CD8+ effector and memory populations, core transcriptional signatures were regulated similarly, whether polyclonal or transgenic, and whether responding to bacterial or viral model pathogens. Our results provide insights into the transcriptional regulation that influence memory formation and CD8+ T cell immunity.

Research paper thumbnail of Shared and distinct transcriptional programs underlie the hybrid nature of iNKT cells

Nature Immunology, 2012

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that act as critical regula... more Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that act as critical regulators of the immune response. To better characterize this population, we profiled iNKT cell gene expression during ontogeny and in peripheral subsets as part of the Immunological Genome Project (ImmGen). High-resolution comparative transcriptional analyses defined developmental and subset-specific iNKT cell gene expression programs. In addition, iNKT cells were found to share an extensive transcriptional program with natural killer (NK) cells, similar in magnitude to that shared with major histocompatibility complex (MHC)-restricted T cells. Strikingly, the NK-iNKT program also operated constitutively in γδT cells and in adaptive T cells following activation. Together, our findings highlight a core effector program regulated distinctly in innate and adaptive lymphocytes.

Research paper thumbnail of The transcriptional landscape of αβ T cell differentiation

Nature Immunology, 2013

nature immunology r e s o u r c e or selected checkpoints. Here we revisited the entire spectrum ... more nature immunology r e s o u r c e or selected checkpoints. Here we revisited the entire spectrum of differentiation states in the αβ T cell branch of the lymphoid tree from a transcriptional standpoint, using the breadth of ImmGen data sets. This analysis showed unexpected aspects of T cell differentiation and identified candidate genes whose coordinated regulation at certain transitions hinted at important functions during thymocyte selection and maturation.

Research paper thumbnail of STAT6 Deletion Converts the Th2 Inflammatory Pathology Afflicting LatY136F Mice into a Lymphoproliferative Disorder Involving Th1 and CD8 Effector T Cells

The Journal of Immunology, 2009

Research paper thumbnail of Th2 Lymphoproliferative Disorder of LatY136F Mutant Mice Unfolds Independently of TCR-MHC Engagement and Is Insensitive to the Action of Foxp3+ Regulatory T Cells

The Journal of Immunology, 2008

mice) develop a fast-onset lymphoproliferative disorder involving polyclonal CD4 T cells that pro... more mice) develop a fast-onset lymphoproliferative disorder involving polyclonal CD4 T cells that produce massive amounts of Th2 cytokines and trigger severe inflammation and autoantibodies. We analyzed whether the Lat Y136F pathology constitutes a bona fide autoimmune disorder dependent on TCR specificity. Using adoptive transfer experiments, we demonstrated that the expansion and uncontrolled Th2-effector function of Lat Y136F CD4 cells are not triggered by an MHC class II-driven, autoreactive process. Using Foxp3EGFP reporter mice, we further showed that nonfunctional Foxp3 ؉ regulatory T cells are present in Lat Y136F mice and that pathogenic Lat Y136F CD4 T cells were capable of escaping the control of infused wild-type Foxp3 ؉ regulatory T cells. These results argue against a scenario where the Lat Y136F pathology is primarily due to a lack of functional Foxp3 ؉ regulatory T cells and suggest that a defect intrinsic to Lat Y136F CD4 T cells leads to a state of TCR-independent hyperactivity. This abnormal status confers Lat Y136F CD4 T cells with the ability to trigger the production of Abs and of autoantibodies in a TCR-independent, quasi-mitogenic fashion. Therefore, despite the presence of autoantibodies causative of severe systemic disease, the pathological conditions observed in Lat Y136F mice unfold in an Ag-independent manner and thus do not qualify as a genuine autoimmune disorder.

Research paper thumbnail of Thymic negative selection is functional in NOD mice

Journal of Experimental Medicine, 2012

Based on analyses of multiple TCR transgenic (tg) models, the emergence of pathogenic T cells in ... more Based on analyses of multiple TCR transgenic (tg) models, the emergence of pathogenic T cells in diabetes-prone NOD mice has been ascribed to a failure to censure autoreactive clones in the thymus. In contrast, using isolated and preselected thymocytes, we show that nonobese diabetic (NOD) genetic variation impairs neither clonal deletion nor downstream transcriptional programs. However, we find that NOD genetic variation influences /-lineage decisions promoted by early expression of tg -TCRs at the double-negative (DN) stage. In B6 and other genetic backgrounds, tg -TCRs behave like -TCRs and commit a large fraction of DNs toward the -lineage, thereby decreasing the size of the double-positive (DP) pool, which is efficiently positively and negatively selected. In NOD DNs, -TCR signalosomes instead behave like pre-TCRs, resulting in high numbers of DPs competing for limited selection niches, and poor positive and negative selection. Once niche effects are neutralized in mixed bone marrow chimeras, positive and negative selection are equally efficient on B6 and NOD backgrounds. Biochemical analysis revealed a selective defect in the activation of Erk1/2 downstream of NOD -TCR signalosomes. Therefore, NOD genetic variation influences /-lineage decisions when the -TCR heterodimer is prematurely expressed, but not the process of negative selection.

Research paper thumbnail of Loss of the LAT Adaptor Converts Antigen-Responsive T Cells into Pathogenic Effectors that Function Independently of the T Cell Receptor

Immunity, 2009

Despite compromised T cell antigen receptor (TCR) signaling, mice in which tyrosine 136 of the ad... more 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 (LatY136F 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.

Research paper thumbnail of Loss of the LAT Adaptor Converts Antigen-Responsive T Cells into Pathogenic Effectors that Function Independently of the T Cell Receptor

Research paper thumbnail of Tonic ubiquitylation controls T-cell receptor: CD3 complex expression during T-cell development

The EMBO Journal, Feb 11, 2010

Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell developmen... more Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3ɛ proline-rich sequence, Lck, c-Cbl, and SLAP, which collectively trigger the dynamin-dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3ζ-monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T-cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T-cell development.

Research paper thumbnail of Single-cell mass cytometry of TCR signaling: Amplification of small initial differences results in low ERK activation in NOD mice

Proceedings of the National Academy of Sciences, 2014

Signaling from the T-cell receptor (TCR) conditions T-cell differentiation and activation, requir... more Signaling from the T-cell receptor (TCR) conditions T-cell differentiation and activation, requiring exquisite sensitivity and discrimination. Using mass cytometry, a high-dimensional technique that can probe multiple signaling nodes at the single-cell level, we interrogate TCR signaling dynamics in control C57BL/6 and autoimmunity-prone nonobese diabetic (NOD) mice, which show ineffective ERK activation after TCR triggering. By quantitating signals at multiple steps along the signaling cascade and parsing the phosphorylation level of each node as a function of its predecessors, we show that a small impairment in initial pCD3ζ activation resonates farther down the signaling cascade and results in larger defects in activation of the ERK1/2-S6 and IκBα modules. This nonlinear property of TCR signaling networks, which magnifies small initial differences during signal propagation, also applies in cells from B6 mice activated at different levels of intensity. Impairment in pCD3ζ and pSLP76 is not a feedback consequence of a primary deficiency in ERK activation because no proximal signaling defect was observed in Erk2 KO T cells. These defects, which were manifest at all stages of T-cell differentiation from early thymic pre-T cells to memory T cells, may condition the imbalanced immunoregulation and tolerance in NOD T cells. More generally, this amplification of small initial differences in signal intensity may explain how T cells discriminate between closely related ligands and adopt strongly delineated cell fates.

Research paper thumbnail of Multiplicity and plasticity of Natural Killer cell signaling pathways

Natural killer (NK) cells express an array of activating receptors that associate with DAP12 (KAR... more Natural killer (NK) cells express an array of activating receptors that associate with DAP12 (KARAP), CD3zeta, and/or FcRgamma ITAM (immunoreceptor tyrosine-based activation motif)-bearing signaling subunits. In T and mast cells, ITAM-dependent signals are integrated by critical scaffolding elements such as LAT (linker for activation of T cells) and NTAL (non-T-cell activation linker). Using mice that are deficient for ITAM-bearing molecules, LAT or NTAL, we show that NK cell cytotoxicity and interferon-gamma secretion are initiated by ITAM-dependent and -independent as well as LAT/NTAL-dependent and -independent pathways. The role of these various signaling circuits depends on the target cell as well as on the activation status of the NK cell. The multiplicity and the plasticity of the pathways that initiate NK cell effector functions contrast with the situation in T cells and B cells and provide an explanation for the resiliency of NK cell effector functions to various pharmacologic inhibitors and genetic mutations in signaling molecules.

Research paper thumbnail of Multiplicity and plasticity of natural killer cell signaling pathways

Natural killer (NK) cells express an array of activating receptors that associate with DAP12 (KAR... more Natural killer (NK) cells express an array of activating receptors that associate with DAP12 (KARAP), CD3zeta, and/or FcRgamma ITAM (immunoreceptor tyrosine-based activation motif)-bearing signaling subunits. In T and mast cells, ITAM-dependent signals are integrated by critical scaffolding elements such as LAT (linker for activation of T cells) and NTAL (non-T-cell activation linker). Using mice that are deficient for ITAM-bearing molecules, LAT or NTAL, we show that NK cell cytotoxicity and interferon-gamma secretion are initiated by ITAM-dependent and -independent as well as LAT/NTAL-dependent and -independent pathways. The role of these various signaling circuits depends on the target cell as well as on the activation status of the NK cell. The multiplicity and the plasticity of the pathways that initiate NK cell effector functions contrast with the situation in T cells and B cells and provide an explanation for the resiliency of NK cell effector functions to various pharmacologic inhibitors and genetic mutations in signaling molecules.

Research paper thumbnail of Conditional density-based analysis of T cell signaling in single-cell data

Science, 2014

Quantitative characterization of T cell signaling. (A) The pCD3ζ-pSLP76 signaling interaction sho... more Quantitative characterization of T cell signaling. (A) The pCD3ζ-pSLP76 signaling interaction shown as (I) a scatterplot, (II) a kernel density estimate, and (III) by using a conditional DREVI method. (IV) Shape features are extracted and quantif ed. (B) DREVI plots of a signaling cascade downstream of TCR show the time-varying nature of edge shapes and strengths. (C) Edge strengths are quantif ed by using conditional DREMI.

Research paper thumbnail of Th2 Lymphoproliferative Disorders Resulting from Defective LAT Signalosomes

Novartis Foundation Symposia, 2006

LAT (linker for activation of T cells) is an integral membrane adaptor protein that constitutes i... more LAT (linker for activation of T cells) is an integral membrane adaptor protein that constitutes in T cells a major substrate of the ZAP-70 protein tyrosine kinase. LAT coordinates the assembly of a multiprotein signalling complex through phosphotyrosine-based motifs present within its intracytoplasmic segment. The resulting 'LAT signalosome' links the TCR to the main intracellular signalling pathways that regulate T cell development and T cell function. Early studies using transformed T cell lines suggested that LAT acts primarily as a positive regulator of T cell receptor (TCR) signalling. The partial or complete inhibition of T cell development observed in several mouse lines harbouring mutant forms of LAT was congruent with that view. More recently, LAT 'knock-ins' harbouring point mutations in the four COOH-terminal tyrosine residues, were found to develop lymphoproliferative disorders involving polyclonal T cells that produced high amounts of T helper-type 2 (Th2) cytokines. This unexpected finding revealed that LAT also constitutes a negative regulator of TCR signalling and T cell homeostasis. As discussed, the available data underscore that a novel immunopathology proper to defective LAT signalosome is likely taking shape.

Research paper thumbnail of LAT signaling pathology: an “autoimmune” condition without T cell self-reactivity

Trends in Immunology, 2010

Partial loss-of-function mutations in several molecules involved in T-cell receptor (TCR) signali... more 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.

Research paper thumbnail of Running head: Plasticity of NK signaling pathways

2 Summary Natural Killer (NK) cells express an array of activating receptors that associate with ... more 2 Summary Natural Killer (NK) cells express an array of activating receptors that associate with DAP12 (KARAP), CD3ζ and/or FcRγ ITAM (Immunoreceptor Tyrosine-based Activation Motif)bearing signaling subunits. In T and mast cells, ITAM-dependent signals are integrated by critical scaffolding elements such as LAT (linker for activation of T cells) and NTAL (non-T cell activation linker). Using mice that are deficient for ITAM-bearing molecules, LAT or NTAL, we show that NK cell cytotoxicity and interferon-γ secretion are initiated by ITAM-dependent and -independent as well as LAT/NTAL-dependent and -independent pathways. The role of these various signaling circuits depends on the target cell as well as on the activation status of the NK cell. The multiplicity and the plasticity of the pathways that initiate NK cell effector functions contrast with the situation in T cells and B cells and provide an explanation for the resiliency of NK cell effector functions to various pharmacological inhibitors and genetic mutations in signaling molecules.

Research paper thumbnail of Supplemental Data Loss of the LAT Adaptor Converts Antigen-Responsive T Cells into Pathogenic Effectors that Function Independently of the T Cell Receptor

Research paper thumbnail of LAT signaling pathology: an “autoimmune” condition without T cell self-reactivity

Trends in Immunology, 2010

Partial loss-of-function mutations in several molecules involved in T-cell receptor (TCR) signali... more 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.

Research paper thumbnail of Lymphoproliferative disorders involving T helper effector cells with defective LAT signalosomes

Seminars in Immunopathology, 2010

coordinates the assembly of a multiprotein complex-the LAT signalosome-that links the T cell-spec... more coordinates the assembly of a multiprotein complex-the LAT signalosome-that links the T cell-specific and the ubiquitous components of the T cell antigen receptor (TCR) signaling pathway. The present review focuses on recent LAT knock-in mice that were found to develop lymphoproliferative disorders involving polyclonal CD4 + T cells that produced excessive amounts of T helper-type 2 cytokines. These mouse models revealed that LAT constitutes more than just a positive regulator of TCR signaling and plays a negative regulatory role that contributes to terminate antigen-driven T cell responses by exerting a repressive function on components of the TCR signaling cassette that lie upstream of LAT or function independently of LAT. In the absence of such a LAT-operated negative regulatory loop that is intrinsic to conventional CD4 + T cells and of no lesser importance than the extrinsic regulatory mechanisms mediated by regulatory T cells, physiologic, antigenspecific CD4 + T cell responses evolve into chronic proinflammatory responses that perpetuate themselves in a manner that does not depend on engagement of the TCR and that induce the production of massive amounts of antibodies and autoantibodies in a major histocompatibility complex-II-independent, "quasi-mitogenic" mode. As discussed, these data underscore that a novel immunopathology proper to defective LAT signalosomes is likely taking shape, and we propose to call it "LAT signaling pathology."

Research paper thumbnail of The proline-rich sequence of CD3ε controls T cell antigen receptor expression on and signaling potency in preselection CD4+CD8+ thymocytes

Nature Immunology, 2008

Antigen recognition by T cell antigen receptors (TCRs) is thought to &amp... more 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.

Research paper thumbnail of Transcriptional insights into the CD8+ T cell response to infection and memory T cell formation

Nature Immunology, 2013

After infection, many factors coordinate the population expansion and differentiation of CD8+ eff... more After infection, many factors coordinate the population expansion and differentiation of CD8+ effector and memory T cells. Using data of unparalleled breadth from the Immunological Genome Project, we analyzed the CD8+ T cell transcriptome throughout infection to establish gene-expression signatures and identify putative transcriptional regulators. Notably, we found that the expression of key gene signatures can be used to predict the memory-precursor potential of CD8+ effector cells. Long-lived memory CD8+ cells ultimately expressed a small subset of genes shared by natural killer T and γδ T cells. Although distinct inflammatory milieu and T cell precursor frequencies influenced the differentiation of CD8+ effector and memory populations, core transcriptional signatures were regulated similarly, whether polyclonal or transgenic, and whether responding to bacterial or viral model pathogens. Our results provide insights into the transcriptional regulation that influence memory formation and CD8+ T cell immunity.

Research paper thumbnail of Shared and distinct transcriptional programs underlie the hybrid nature of iNKT cells

Nature Immunology, 2012

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that act as critical regula... more Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that act as critical regulators of the immune response. To better characterize this population, we profiled iNKT cell gene expression during ontogeny and in peripheral subsets as part of the Immunological Genome Project (ImmGen). High-resolution comparative transcriptional analyses defined developmental and subset-specific iNKT cell gene expression programs. In addition, iNKT cells were found to share an extensive transcriptional program with natural killer (NK) cells, similar in magnitude to that shared with major histocompatibility complex (MHC)-restricted T cells. Strikingly, the NK-iNKT program also operated constitutively in γδT cells and in adaptive T cells following activation. Together, our findings highlight a core effector program regulated distinctly in innate and adaptive lymphocytes.

Research paper thumbnail of The transcriptional landscape of αβ T cell differentiation

Nature Immunology, 2013

nature immunology r e s o u r c e or selected checkpoints. Here we revisited the entire spectrum ... more nature immunology r e s o u r c e or selected checkpoints. Here we revisited the entire spectrum of differentiation states in the αβ T cell branch of the lymphoid tree from a transcriptional standpoint, using the breadth of ImmGen data sets. This analysis showed unexpected aspects of T cell differentiation and identified candidate genes whose coordinated regulation at certain transitions hinted at important functions during thymocyte selection and maturation.

Research paper thumbnail of STAT6 Deletion Converts the Th2 Inflammatory Pathology Afflicting LatY136F Mice into a Lymphoproliferative Disorder Involving Th1 and CD8 Effector T Cells

The Journal of Immunology, 2009

Research paper thumbnail of Th2 Lymphoproliferative Disorder of LatY136F Mutant Mice Unfolds Independently of TCR-MHC Engagement and Is Insensitive to the Action of Foxp3+ Regulatory T Cells

The Journal of Immunology, 2008

mice) develop a fast-onset lymphoproliferative disorder involving polyclonal CD4 T cells that pro... more mice) develop a fast-onset lymphoproliferative disorder involving polyclonal CD4 T cells that produce massive amounts of Th2 cytokines and trigger severe inflammation and autoantibodies. We analyzed whether the Lat Y136F pathology constitutes a bona fide autoimmune disorder dependent on TCR specificity. Using adoptive transfer experiments, we demonstrated that the expansion and uncontrolled Th2-effector function of Lat Y136F CD4 cells are not triggered by an MHC class II-driven, autoreactive process. Using Foxp3EGFP reporter mice, we further showed that nonfunctional Foxp3 ؉ regulatory T cells are present in Lat Y136F mice and that pathogenic Lat Y136F CD4 T cells were capable of escaping the control of infused wild-type Foxp3 ؉ regulatory T cells. These results argue against a scenario where the Lat Y136F pathology is primarily due to a lack of functional Foxp3 ؉ regulatory T cells and suggest that a defect intrinsic to Lat Y136F CD4 T cells leads to a state of TCR-independent hyperactivity. This abnormal status confers Lat Y136F CD4 T cells with the ability to trigger the production of Abs and of autoantibodies in a TCR-independent, quasi-mitogenic fashion. Therefore, despite the presence of autoantibodies causative of severe systemic disease, the pathological conditions observed in Lat Y136F mice unfold in an Ag-independent manner and thus do not qualify as a genuine autoimmune disorder.

Research paper thumbnail of Thymic negative selection is functional in NOD mice

Journal of Experimental Medicine, 2012

Based on analyses of multiple TCR transgenic (tg) models, the emergence of pathogenic T cells in ... more Based on analyses of multiple TCR transgenic (tg) models, the emergence of pathogenic T cells in diabetes-prone NOD mice has been ascribed to a failure to censure autoreactive clones in the thymus. In contrast, using isolated and preselected thymocytes, we show that nonobese diabetic (NOD) genetic variation impairs neither clonal deletion nor downstream transcriptional programs. However, we find that NOD genetic variation influences /-lineage decisions promoted by early expression of tg -TCRs at the double-negative (DN) stage. In B6 and other genetic backgrounds, tg -TCRs behave like -TCRs and commit a large fraction of DNs toward the -lineage, thereby decreasing the size of the double-positive (DP) pool, which is efficiently positively and negatively selected. In NOD DNs, -TCR signalosomes instead behave like pre-TCRs, resulting in high numbers of DPs competing for limited selection niches, and poor positive and negative selection. Once niche effects are neutralized in mixed bone marrow chimeras, positive and negative selection are equally efficient on B6 and NOD backgrounds. Biochemical analysis revealed a selective defect in the activation of Erk1/2 downstream of NOD -TCR signalosomes. Therefore, NOD genetic variation influences /-lineage decisions when the -TCR heterodimer is prematurely expressed, but not the process of negative selection.

Research paper thumbnail of Loss of the LAT Adaptor Converts Antigen-Responsive T Cells into Pathogenic Effectors that Function Independently of the T Cell Receptor

Immunity, 2009

Despite compromised T cell antigen receptor (TCR) signaling, mice in which tyrosine 136 of the ad... more 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 (LatY136F 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.

Research paper thumbnail of Loss of the LAT Adaptor Converts Antigen-Responsive T Cells into Pathogenic Effectors that Function Independently of the T Cell Receptor

Research paper thumbnail of Tonic ubiquitylation controls T-cell receptor: CD3 complex expression during T-cell development

The EMBO Journal, Feb 11, 2010

Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell developmen... more Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3ɛ proline-rich sequence, Lck, c-Cbl, and SLAP, which collectively trigger the dynamin-dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3ζ-monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T-cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T-cell development.