Meningeal macrophages protect against viral neuroinfection.

The surface of the central nervous system (CNS) is protected by the meninges, which contain a dense network of meningeal macrophages (MMs). Here, we examined the role of tissue-resident MM in viral infection. MHC-II- MM were abundant neonatally, whereas MHC-II+ MM appeared over time. These barrier macrophages differentially responded to in vivo peripheral challenges such as LPS, SARS-CoV-2, and lymphocytic choriomeningitis virus (LCMV). Peripheral LCMV infection, which was asymptomatic, led to a transient infection and activation of the meninges. Mice lacking macrophages but conserving brain microglia, or mice bearing macrophage-specific deletion of Stat1 or Ifnar, exhibited extensive viral spread into the CNS. Transcranial pharmacological depletion strategies targeting MM locally resulted in several areas of the meninges becoming infected and fatal meningitis. Low numbers of MHC-II+ MM, which is seen upon LPS challenge or in neonates, corelated with higher viral load upon infection. Thus, MMs protect against viral infection and may present targets for therapeutic manipulation.

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 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.

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.

Design, immunogenicity, and efficacy of a pan-sarbecovirus dendritic-cell targeting vaccine.

BACKGROUND: There is an urgent need of a new generation of vaccine that are able to enhance protection against SARS-CoV-2 and related variants of concern (VOC) and emerging coronaviruses. METHODS: We identified conserved T- and B-cell epitopes from Spike (S) and Nucleocapsid (N) highly homologous to 38 sarbecoviruses, including SARS-CoV-2 VOCs, to design a protein subunit vaccine targeting antigens to Dendritic Cells (DC) via CD40 surface receptor (CD40.CoV2). FINDINGS: CD40.CoV2 immunization elicited high levels of cross-neutralizing antibodies against SARS-CoV-2, VOCs, and SARS-CoV-1 in K18-hACE2 transgenic mice, associated with viral control and survival after SARS-CoV-2 challenge. A direct comparison of CD40.CoV2 with the mRNA BNT162b2 vaccine showed that the two vaccines were equally immunogenic in mice. We demonstrated the potency of CD40.CoV2 to recall in vitro human multi-epitope, functional, and cytotoxic SARS-CoV-2 S- and N-specific T-cell responses that are unaffected by VOC mutations and cross-reactive with SARS-CoV-1 and, to a lesser extent, MERS epitopes. INTERPRETATION: We report the immunogenicity and antiviral efficacy of the CD40.CoV2 vaccine in a preclinical model providing a framework for a pan-sarbecovirus vaccine. FUNDINGS: This work was supported by INSERM and the Investissements d'Avenir program, Vaccine Research Institute (VRI), managed by the ANR and the CARE project funded from the Innovative Medicines Initiative 2 Joint Undertaking (JU).

STAT6 deletion converts the Th2 inflammatory pathology afflicting Lat(Y136F) mice into a lymphoproliferative disorder involving Th1 and CD8 effector T cells.

Mutant mice in which tyrosine 136 of linker for activation of T cells (LAT) was replaced with a phenylalanine (Lat(Y136F) mice) develop a lymphoproliferative disorder involving polyclonal CD4 effector T cells that produce massive amounts of IL-4 and trigger severe Th2 inflammation. Naive CD4 T cells can themselves produce IL-4 and thereby initiate a self-reinforcing positive regulatory loop that involves the STAT6 transcription factor and leads to Th2 polarization. We determined the functional outcome that results when Lat(Y136F) T cells differentiate in the absence of such STAT6-dependent regulatory loop. The lack of STAT6 had no effect on the timing and magnitude of the lymphoproliferative disorder. However, in Lat(Y136F) mice deprived of STAT6, the expanding CD4 T cell population was dominated by Th1 effector cells that triggered B cell proliferation, elevated IgG2a and IgG2b levels as well as the production of autoantibodies. In contrast to Lat(Y136F) mice that showed no CD8 T cell expansion, the CD8 T cells present in Lat(Y136F) mice deprived of STAT6 massively expanded and acquired effector potential. Therefore, the lack of STAT6 is sufficient to convert the Th2 lymphoproliferative disorder that characterizes Lat(Y136F) mice into a lymphoproliferative disorder that is dominated by Th1 and CD8 effector T cells. The possibility to dispose of a pair of mice that differs by a single gene and develops in the absence of deliberate immunization large numbers of Th cells with almost reciprocal polarization should facilitate the identification of genes involved in the control of normal and pathological Th cell differentiation.

Deletion of the LIME adaptor protein minimally affects T and B cell development and function.

LIME (Lck-interacting membrane protein) is a transmembrane adaptor that associates with the Lck and Fyn protein tyrosine kinases and with the C-terminal Src kinase (Csk). To delineate the role of LIME in vivo, LIME-deficient mice were generated. Although Lime transcripts were expressed in immature and mature B and T cells, the absence of LIME impeded neither the development nor the function of B and T cells. TCR transgenic mice deprived of LIME showed, however, a 1.8-fold enhancement in positive selection. Since B cells and activated T cells express LIME and the related adaptor NTAL, mice lacking both adaptors were generated. Double-deficient mice showed no defect in the development and function of B and T cells, and the lack of LIME had no effect on the autoimmune syndrome that develops in aged NTAL-deficient mice. In contrast to a previous report, we further showed that this autoimmune syndrome develops in the absence of T cells. Therefore, our in vivo results refute all the previous roles postulated for LIME on the basis of studies of transformed B and T cells and demonstrate that LIME has no seminal role in the signaling cassette operated by antigen receptors and coreceptors.

Visualization of the earliest steps of gammadelta T cell development in the adult thymus.

The checkpoint in gammadelta cell development that controls successful T cell receptor (TCR) gene rearrangements remains poorly characterized. Using mice expressing a reporter gene 'knocked into' the Tcrd constant region gene, we have characterized many of the events that mark the life of early gammadelta cells in the adult thymus. We identify the developmental stage during which the Tcrd locus 'opens' in early T cell progenitors and show that a single checkpoint controls gammadelta cell development during the penultimate CD4- CD8- stage. Passage through this checkpoint required the assembly of gammadelta TCR heterodimers on the cell surface and signaling via the Lat adaptor protein. In addition, we show that gammadelta selection triggered a phase of sustained proliferation similar to that induced by the pre-TCR.