Arnaud Delpoux

La plupart des lymphocytes T régulateurs CD4+ Foxp3+ (Tregs) sont générés dans le thymus. Plusieurs études suggèrent fortement que le développement des Tregs est dû à la forte affinité de leur TCR pour le soi (complexes « peptide du soi-CMH II ») présenté dans le thymus. Après avoir migré à la périphérie, les Tregs continuent d’interagir avec le soi et l’expression de Foxp3 est considérée comme nécessaire et suffisante pour maintenir le programme de transcription nécessaire aux fonctions suppressives de ces cellules en périphérie. En utilisant deux models expérimentaux chez la souris, notre étude démontre l’importance des interactions continues avec le soi pour maintenir les capacités suppressives des Tregs à la périphérie. L’absence d’interactions avec le soi aboutit rapidement à une altération du phénotype des Tregs, de leur capacité à produire certaines cytokines et modifie également leur signature transcriptionnelle. De façon intéressante, nous avons observé que l’absence d’interaction avec le soi n’affecte pas le niveau d’expression de Foxp3 mais que la reconnaissance du soi induit une signature transcriptionnelle unique et des caractéristiques fonctionnelles qui ne sont pas liées à Foxp3. Dans une seconde étude, nous avons mis en évidence que, chez la souris jeune adulte, l’expression de Ly-6C permet d’identifier deux sous-populations de Tregs distinctes présentant des différences phénotypiques et fonctionnelles. En particulier, nous avons observé que les Tregs Ly-6C- présentent un phénotype plus activé et régulateur que leurs homologues Ly-6C+ et que seules les premières sont fonctionnelles in vitro et in vivo. Nous avons également montré un lien étroit entre expression de Ly-6C et autoréactivité, les Tregs Ly-6C- recevant plus de signaux du TCR que les Tregs Ly-6C+. Finalement, nous avons observé que seuls les Tregs Ly-6C- se maintiennent à la périphérie avec le temps, suggérant l’existence d’une sélection périphérique permettant la survie préférentielle des Tregs les plus fonctionnels. Au cours de ma thèse, nous avons ainsi pu démontrer que les interactions avec le soi étaient indispensables et nécessaires pour la fonctionnalité, le phénotype et l’homéostasie des Tregs.

In response to an intracellular infectious agent, the immune system produces a specific cellular response as well as a T cell–dependent Ab response. Precursor T cells differentiate into effector T cells, including Th1 cells, and T follicular helper (TFH) cells. The latter cooperate with B cells to form germinal centers and induce the formation of Ab-forming plasmacytes. One major focal point for control of T cell differentiation is the transcription factor BCL6. In this study, we demonstrated that the Bcl6 gene is regulated by FOXO1-binding, cis-acting sequences located in a highly conserved region of the first Bcl6 intron. In both mouse and human T cells, deletion of the tandem FOXO1 binding sites increased the expression of BCL6 and enhanced the proportion of TFH cells. These results reveal a fundamental control point for cellular versus humoral immunity.

In response to infection, pathogen-specific CD8 T cells differentiate into functionally diverse effector and memory T cell populations critical for resolving disease and providing durable immunity. Through small-molecule inhibition, RNAi studies, and induced genetic deletion, we reveal an essential role for the chromatin modifier and BET family member BRD4 in supporting the differentiation and maintenance of terminally fated effector CD8 T cells during infection. BRD4 bound diverse regulatory regions critical to effector T cell differentiation and controlled transcriptional activity of terminal effector–specific super-enhancers in vivo. Consequentially, induced deletion of Brd4 or small molecule–mediated BET inhibition impaired maintenance of a terminal effector T cell phenotype. BRD4 was also required for terminal differentiation of CD8 T cells in the tumor microenvironment in murine models, which we show has implications for immunotherapies. Taken together, these data reveal an unappreciated requirement for BRD4 in coordinating activity of cis regulatory elements to control CD8 T cell fate and lineage stability.

Significance: Memory CD8 T lymphocytes are a long-lived population of immune cells that provide formidable protection against infections and malignancy and are thus often central to vaccine and cancer immunotherapy efficacy. Here we refine the identity, developmental relationships, and functional roles of memory CD8 T cells in mice through delineation of a discrete population that confers robust protection against reinfection and exhibits an unexpected molecular program bearing typically divergent characteristics of short-lived effector cells and long-lived memory T cells. Mass cytometry and single-cell RNA-sequencing analyses revealed an analogous population of cells in humans. These findings hold broad implications for both informing immunotherapy treatments and predicting their efficacy.

Despite being implicated in non-lymphoid tissues, non-recirculating T cells may also exist in secondary lymphoid organs (SLO). However, a detailed characterization of this lymphoid-resident T cell pool has not yet been done. Here we show that a substantial proportion of CD4 regulatory (Treg) and memory (Tmem) cells establish long-term residence in the SLOs of specific pathogen-free mice. Of these SLOs, only T cell residence within Peyer’s patches is affected by microbiota. Resident CD4 Treg and CD4 Tmem cells from lymph nodes and non-lymphoid tissues share many phenotypic and functional characteristics. The percentage of resident T cells in SLOs increases considerably with age, with S1PR1 downregulation possibly contributing to this altered homeostasis. Our results thus show that T cell residence is not only a hallmark of non-lymphoid tissues, but can be extended to secondary lymphoid organs.

Upon infection with an intracellular pathogen, cytotoxic CD8⁺ T cells develop diverse differentiation states characterized by function, localization, longevity, and the capacity for self-renewal. The program of differentiation is determined, in part, by FOXO1, a transcription factor known to integrate extrinsic input in order to specify survival, DNA repair, self-renewal, and proliferation. At issue is whether the state of T cell differentiation is specified by initial conditions of activation or is actively maintained. To study the spectrum of T cell differentiation, we have analyzed an infection with mouse cytomegalovirus, a persistent-latent virus that elicits different cytotoxic T cell responses characterized as acute resolving or inflationary. Our results show that FOXO1 is continuously required for all the phenotypic characteristics of memory-effector T cells such that with acute inactivation of the gene encoding FOXO1, T cells revert to a short-lived effector phenotype, exhibit reduced viability, and manifest characteristics of anergy.

Significance: Acute infection with intracellular pathogen results in the expansion and effector differentiation of pathogen-specific CD8⁺T cells, most of which die after pathogen clearance. The factors and steps controlling CD8⁺T cell differentiation in response to acute infection are not fully understood. We find FOXO1 and TCF7 drive postinfection immune memory characteristics in cells expressing low TIM3, where TCF7 actively down-regulates the cytotoxic molecule GZMB and reduces terminally differentiated KLRG1^higheffector abundance. We further report the creation of a continuum of CD8⁺T cell phenotypes, ranging from terminal effector cells to long-lived immune-memory cells, dependent on FOXO1 and TCF7. We conclude FOXO1 and TCF7 are essential to oppose cytotoxic/terminal differentiation programs and enable full phenotypic diversity of postinfection CD8⁺T cells.

CD4 regulatory T cells (Tregs) can be subdivided into two subsets according to Ly-6C expression in the periphery. Phenotypic analysis, imaging, and adoptive-transfer experiments of peripheral Ly-6C⁻ and Ly-6C⁺ Tregs reveal that the nonexpression of Ly-6C by ∼70% of peripheral Tregs depends on TCR signaling events. Interestingly, Ly-6C⁻ Tregs express higher surface amounts of key immunosuppressive molecules than do Ly-6C⁺ Tregs and produce constitutively anti-inflammatory cytokines. In line with their phenotype, Ly-6C⁺ Tregs exhibit poor suppressive capacities in vitro and in vivo. Finally, although Ly-6C⁻ Tregs maintain their numbers with age, Ly-6C⁺ Tregs gradually disappear. Altogether, our data strongly suggest that both the survival and suppressive functions of peripheral CD4 Tregs rely on their ability to receive strong TCR signals.

Upon activation, naive CD4 T cells differentiate into a variety of T-helper-cell subsets characterized by different cytokine production and functions. Currently, lineage commitment is considered to depend mostly on the environmental context to which naive CD4 T cells are exposed. Here we challenge this model based on the supposed homogeneity of the naive CD4 T-cell compartment. We show that peripheral naive CD4 T cells can be subdivided into two subsets according to Ly-6C expression. Furthermore, the two newly defined subsets (Ly-6C⁻ and Ly-6C⁺ naive CD4 T cells) are not equal in their intrinsic ability to commit into the induced regulatory T-cell lineage. Finally, phenotypic analysis, imaging and adoptive transfer experiments reveal that Ly-6C expression is modulated by self-recognition, allowing the dichotomization of the naive CD4 T-cell compartment into two cell subsets with distinct self-reactivity. Altogether, our results show that naive CD4 T cells with the highest avidity for self are prone to differentiate into regulatory T cells.