Positive regulation of Vav1 by Themis controls CD4 T cell pathogenicity in a mouse model of central nervous system inflammation.

The susceptibility to autoimmune diseases is conditioned by the association of modest genetic alterations which altogether weaken self-tolerance. The mechanism whereby these genetic interactions modulate T-cell pathogenicity remains largely uncovered. Here, we investigated the epistatic interaction of two interacting proteins involved in T Cell Receptor signaling and which were previously associated with the development of Multiple Sclerosis. To this aim, we used mice expressing an hypomorphic variant of Vav1 (Vav1R63W), combined with a T cell-conditional deletion of Themis. We show that the combined mutations in Vav1 and Themis induce a strong attenuation of the severity of Experimental Autoimmune Encephalomyelitis (EAE), contrasting with the moderate effect of the single mutation in each of those two proteins. This genotype-dependent gradual decrease of EAE severity correlates with decreased quantity of phosphorylated Vav1 in CD4 T cells, establishing that Themis promotes the development of encephalitogenic Tconv response by enhancing Vav1 activity. We also show that the cooperative effect of Themis and Vav1 on EAE severity is independent of regulatory T cells and unrelated to the impact of Themis on thymic selection. Rather, it results from decreased production of pro-inflammatory cytokines (IFN-γ, IL-17, TNF and GM-CSF) and reduced T cell infiltration in the CNS. Together, our results provide a rationale to study combination of related genes, in addition to single gene association, to better understand the genetic bases of human diseases.

Eomes-Dependent Loss of the Co-activating Receptor CD226 Restrains CD8+ T Cell Anti-tumor Functions and Limits the Efficacy of Cancer Immunotherapy.

CD8+ T cells within the tumor microenvironment (TME) are exposed to various signals that ultimately determine functional outcomes. Here, we examined the role of the co-activating receptor CD226 (DNAM-1) in CD8+ T cell function. The absence of CD226 expression identified a subset of dysfunctional CD8+ T cells present in peripheral blood of healthy individuals. These cells exhibited reduced LFA-1 activation, altered TCR signaling, and a distinct transcriptomic program upon stimulation. CD226neg CD8+ T cells accumulated in human and mouse tumors of diverse origin through an antigen-specific mechanism involving the transcriptional regulator Eomesodermin (Eomes). Despite similar expression of co-inhibitory receptors, CD8+ tumor-infiltrating lymphocyte failed to respond to anti-PD-1 in the absence of CD226. Immune checkpoint blockade efficacy was hampered in Cd226-/- mice. Anti-CD137 (4-1BB) agonists also stimulated Eomes-dependent CD226 loss that limited the anti-tumor efficacy of this treatment. Thus, CD226 loss restrains CD8+ T cell function and limits the efficacy of cancer immunotherapy.

Foxo3 Transcription Factor Drives Pathogenic T Helper 1 Differentiation by Inducing the Expression of Eomes.

The transcription factor Foxo3 plays a crucial role in myeloid cell function but its role in lymphoid cells remains poorly defined. Here, we have shown that Foxo3 expression was increased after T cell receptor engagement and played a specific role in the polarization of CD4+ T cells toward pathogenic T helper 1 (Th1) cells producing interferon-γ (IFN-γ) and granulocyte monocyte colony stimulating factor (GM-CSF). Consequently, Foxo3-deficient mice exhibited reduced susceptibility to experimental autoimmune encephalomyelitis. At the molecular level, we identified Eomes as a direct target gene for Foxo3 in CD4+ T cells and we have shown that lentiviral-based overexpression of Eomes in Foxo3-deficient CD4+ T cells restored both IFN-γ and GM-CSF production. Thus, the Foxo3-Eomes pathway is central to achieve the complete specialized gene program required for pathogenic Th1 cell differentiation and development of neuroinflammation.

A Natural Variant of the T Cell Receptor-Signaling Molecule Vav1 Reduces Both Effector T Cell Functions and Susceptibility to Neuroinflammation.

The guanine nucleotide exchange factor Vav1 is essential for transducing T cell antigen receptor signals and therefore plays an important role in T cell development and activation. Our previous genetic studies identified a locus on rat chromosome 9 that controls the susceptibility to neuroinflammation and contains a non-synonymous polymorphism in the major candidate gene Vav1. To formally demonstrate the causal implication of this polymorphism, we generated a knock-in mouse bearing this polymorphism (Vav1R63W). Using this model, we show that Vav1R63W mice display reduced susceptibility to experimental autoimmune encephalomyelitis (EAE) induced by MOG35-55 peptide immunization. This is associated with a lower production of effector cytokines (IFN-γ, IL-17 and GM-CSF) by autoreactive CD4 T cells. Despite increased proportion of Foxp3+ regulatory T cells in Vav1R63W mice, we show that this lowered cytokine production is intrinsic to effector CD4 T cells and that Treg depletion has no impact on EAE development. Finally, we provide a mechanism for the above phenotype by showing that the Vav1R63W variant has normal enzymatic activity but reduced adaptor functions. Together, these data highlight the importance of Vav1 adaptor functions in the production of inflammatory cytokines by effector T cells and in the susceptibility to neuroinflammation.

An Epistatic Interaction between Themis1 and Vav1 Modulates Regulatory T Cell Function and Inflammatory Bowel Disease Development.

The development of inflammatory diseases depends on complex interactions between several genes and various environmental factors. Discovering new genetic risk factors and understanding the mechanisms whereby they influence disease development is of paramount importance. We previously reported that deficiency in Themis1, a new actor of TCR signaling, impairs regulatory T cell (Treg) function and predisposes Brown-Norway (BN) rats to spontaneous inflammatory bowel disease (IBD). In this study, we reveal that the epistasis between Themis1 and Vav1 controls the occurrence of these phenotypes. Indeed, by contrast with BN rats, Themis1 deficiency in Lewis rats neither impairs Treg suppressive functions nor induces pathological manifestations. By using congenic lines on the BN genomic background, we show that the impact of Themis1 deficiency on Treg suppressive functions depends on a 117-kb interval coding for a R63W polymorphism that impacts Vav1 expression and functions. Indeed, the introduction of a 117-kb interval containing the Lewis Vav1-R63 variant restores Treg function and protects Themis1-deficient BN rats from spontaneous IBD development. We further show that Themis1 binds more efficiently to the BN Vav1-W63 variant and is required to stabilize its recruitment to the transmembrane adaptor LAT and to fully promote the activation of Erk kinases. Together, these results highlight the importance of the signaling pathway involving epistasis between Themis1 and Vav1 in the control of Treg suppressive function and susceptibility to IBD development.

The p.Arg63Trp polymorphism controls Vav1 functions and Foxp3 regulatory T cell development.

CD4(+) regulatory T cells (T(reg) cells) expressing the transcription factor Foxp3 play a pivotal role in maintaining peripheral tolerance by inhibiting the expansion and function of pathogenic conventional T cells (T(conv) cells). In this study, we show that a locus on rat chromosome 9 controls the size of the natural T(reg) cell compartment. Fine mapping of this locus with interval-specific congenic lines and association experiments using single nucleotide polymorphisms (SNPs) identified a nonsynonymous SNP in the Vav1 gene that leads to the substitution of an arginine by a tryptophan (p.Arg63Trp). This p.Arg63Trp polymorphism is associated with increased proportion and absolute numbers of T(reg) cells in the thymus and peripheral lymphoid organs, without impacting the size of the T(conv) cell compartment. This polymorphism is also responsible for Vav1 constitutive activation, revealed by its tyrosine 174 hyperphosphorylation and increased guanine nucleotide exchange factor activity. Moreover, it induces a marked reduction in Vav1 cellular contents and a reduction of Ca(2+) flux after TCR engagement. Together, our data reveal a key role for Vav1-dependent T cell antigen receptor signaling in natural T(reg) cell development.

Transcription factor Foxo3 controls the magnitude of T cell immune responses by modulating the function of dendritic cells.

Foxo transcription factors regulate cell cycle progression, cell survival and DNA-repair pathways. Here we demonstrate that deficiency in Foxo3 resulted in greater expansion of T cell populations after viral infection. This exaggerated expansion was not T cell intrinsic. Instead, it was caused by the enhanced capacity of Foxo3-deficient dendritic cells to sustain T cell viability by producing more interleukin 6. Stimulation of dendritic cells mediated by the coinhibitory molecule CTLA-4 induced nuclear localization of Foxo3, which in turn inhibited the production of interleukin 6 and tumor necrosis factor. Thus, Foxo3 acts to constrain the production of key inflammatory cytokines by dendritic cells and to control T cell survival.

Foxo1 links homing and survival of naive T cells by regulating L-selectin, CCR7 and interleukin 7 receptor.

Foxo transcription factors have a conserved role in the adaptation of cells and organisms to nutrient and growth factor availability. Here we show that Foxo1 has a crucial, nonredundant role in T cells. In naive T cells, Foxo1 controlled the expression of the adhesion molecule L-selectin, the chemokine receptor CCR7 and the transcription factor Klf2, and its deletion was sufficient to alter lymphocyte trafficking. Furthermore, Foxo1 deficiency resulted in a severe defect in interleukin 7 receptor alpha-chain (IL-7Ralpha) expression associated with its ability to bind an Il7r enhancer. Finally, growth factor withdrawal induced a Foxo1-dependent increase in Sell, Klf2 and Il7r expression. These data suggest that Foxo1 regulates the homeostasis and life span of naive T cells by sensing growth factor availability and regulating homing and survival signals.

A role for VAV1 in experimental autoimmune encephalomyelitis and multiple sclerosis.

Multiple sclerosis, the most common cause of progressive neurological disability in young adults, is a chronic inflammatory disease. There is solid evidence for a genetic influence in multiple sclerosis, and deciphering the causative genes could reveal key pathways influencing the disease. A genome region on rat chromosome 9 regulates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. Using interval-specific congenic rat lines and association of single-nucleotide polymorphisms with inflammatory phenotypes, we localized the gene of influence to Vav1, which codes for a signal-transducing protein in leukocytes. Analysis of seven human cohorts (12,735 individuals) demonstrated an association of rs2546133-rs2617822 haplotypes in the first VAV1 intron with multiple sclerosis (CA: odds ratio, 1.18; CG: odds ratio, 0.86; TG: odds ratio, 0.90). The risk CA haplotype also predisposed for higher VAV1 messenger RNA expression. VAV1 expression was increased in individuals with multiple sclerosis and correlated with tumor necrosis factor and interferon-gamma expression in peripheral blood and cerebrospinal fluid cells. We conclude that VAV1 plays a central role in controlling central nervous system immune-mediated disease and proinflammatory cytokine production critical for disease pathogenesis.

Functional and genetic analysis of two CD8 T cell subsets defined by the level of CD45RC expression in the rat.

Differential cytokine production by T cells plays an important role in the outcome of the immune response. We show that the level of CD45RC expression differentiates rat CD8 T cells in two subpopulations, CD45RC(high) and CD45RC(low), that have different cytokine profiles and functions. Upon in vitro stimulation, in an Ag-presenting cell-independent system, CD45RC(high) CD8 T cells produce IL-2 and IFN-gamma while CD45RC(low) CD8 T cells produce IL-4, IL-10, and IL-13. In vitro, these subsets also exhibit different cytotoxic and suppressive functions. The CD45RC(high)/CD45RC(low) CD8 T cell ratio was determined in Lewis (LEW) and Brown-Norway (BN) rats. These two rat strains differ with respect to the Th1/Th2 polarization of their immune responses and to their susceptibility to develop distinct immune diseases. The CD45RC(high)/CD45RC(low) CD8 T cell ratio is higher in LEW than in BN rats, and this difference is dependent on hemopoietic cells. Linkage analysis in a F(2)(LEW x BN) intercross identified two quantitative trait loci on chromosomes 9 and 20 controlling the CD45RC(high)/CD45RC(low) CD8 T cell ratio. This genetic control was confirmed in congenic rats. The region on chromosome 9 was narrowed down to a 1.2-cM interval that was found to also control the IgE response in a model of Th2-mediated disorder. Identification of genes that control the CD45RC(high)/CD45RC(low) CD8 T cell subsets in these regions could be of great interest for the understanding of the pathophysiology of immune-mediated diseases.

Identification of a novel natural regulatory CD8 T-cell subset and analysis of its mechanism of regulation.

The immune system contains natural regulatory T cells that control the magnitude of the immune response during physiologic and pathologic conditions. Although this suppressive function was historically attributed to CD8 T cells, most recent reports have focused on natural regulatory CD4 T cells. In the present study, we describe a new subset of natural CD8 regulatory T cells in normal healthy animals. This subset expresses low levels of CD45RC at its surface (CD45RC(low)); produces mainly interleukin-4 (IL-4), IL-10, and IL-13 cytokines upon in vitro stimulation; expresses Foxp3 and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4); and is not cytotoxic against allogeneic targets. This subset suppresses the proliferation and differentiation of autologous CD4 T cells into type-1 cytokines producing T cells after stimulation with allogeneic accessory cells. We also provide evidence that this regulatory subset mediates its suppression by cell-to-cell contact and not through secretion of suppressive cytokines. Finally, the regulatory activity of CD8 CD45RC(low) cells is also demonstrated in vivo in a rat model of CD4-dependent graft-versus-host disease. Collectively, these data demonstrate for the first time that freshly isolated rat CD8 CD45RC(low) T cells contain T cells with regulatory properties, a result that enlarges the general picture of T-cell-mediated regulation.