Julie C. Ribot

CD27 is a thymic determinant of the balance between interferon-gamma- and interleukin 17-producing gammadelta T cell subsets.

The production of cytokines such as interferon-γ and interleukin 17 by αβ and γδ T cells influences the outcome of immune responses. Here we show that most γδ T lymphocytes expressed the tumor necrosis factor receptor family member CD27 and secreted interferon-γ, whereas interleukin 17 production was restricted to CD27− γδ T cells. In contrast to the apparent plasticity of αβ T cells, the cytokine profiles of these distinct γδ T cell subsets were essentially stable, even during infection. These phenotypes were established during thymic development, when CD27 functions as a regulator of the differentiation of γδ T cells at least in part by inducing expression of the lymphotoxin-β receptor and genes associated with trans-conditioning and interferon-γ production. Thus, the cytokine profiles of peripheral γδ T cells are predetermined mainly by a mechanism involving CD27.

Identification of Regulatory Foxp3+ Invariant NKT Cells Induced by TGF-β

Invariant NKT (iNKT) cells were shown to prevent the onset of experimental autoimmune encephalomyelitis in mice following administration of their specific TCR agonist α-galactosylceramide. We found that this protection was associated with the emergence of a Foxp3+ iNKT cell population in cervical lymph nodes. We demonstrate that the differentiation of these cells is critically dependent on TGF-β in both mice and humans. Moreover, in vivo generation of Foxp3+ iNKT cells was observed in the TGF-β–rich environment of the murine gut. Foxp3+ iNKT cells displayed a phenotype similar to that of Foxp3+ regulatory T cells, and they suppress through a contact-dependent, glucocorticoid-induced TNFR-mediated mechanism. Nevertheless, Foxp3+ iNKT cells retain distinctive NKT cell characteristics, such as promyelocytic leukemia zinc finger protein expression and preferential homing to the liver following adoptive transfer, where they stably maintained Foxp3 expression. Our data thus unveil an unexpected capacity of iNKT cells to acquire regulatory functions that may contribute to the establishment of immunological tolerance.

Epithelial and dendritic cells in the thymic medulla promote CD4+Foxp3+ regulatory T cell development via the CD27-CD70 pathway

CD27–CD70 signals are required for optimal development of natural regulatory T cells from the thymus.

CD70–CD27 interactions provide survival and proliferative signals that regulate T cell receptor-driven activation of human γδ peripheral blood lymphocytes

Human Vγ9Vδ2 T cells are potent anti‐tumor lymphocytes that specifically respond to pyrophosphate (phospho‐) antigens, which constitute the basis of current γδ T‐cell‐based immunotherapy strategies. Despite a clear involvement of the TCR, the costimulation requirements of Vγ9Vδ2 T cells remain ill‐defined. Here, we show that the expression of the CD27 receptor by the vast majority of Vγ9Vδ2 peripheral blood lymphocytes endows them with enhanced proliferative capacity upon ligation by its unique ligand CD70, a tumor necrosis factor superfamily member expressed on lymphoma B‐cells but also on TCR‐activated γδ T cells. Moreover, Vγ9Vδ2 T‐cell treatment with soluble recombinant CD70 induced calcium signals and increased transcription of anti‐apoptotic Bcl2a1 and cell‐cycle‐promoting Cyclin D2 genes. We further demonstrate that the manipulation of CD70–CD27 interactions significantly impacted on Vγ9Vδ2 T‐cell survival, proliferation and cytokine secretion, in both loss‐of‐function and gain‐of‐function experiments. Thus, CD27 coreceptor signals strongly promoted the expansion of Th1‐biased, CD27+ Vγ9Vδ2 peripheral blood lymphocytes in the context of TCR‐mediated stimulation with phosphoantigens. These data collectively establish a novel role for the CD70–CD27 axis in human γδ T‐cell activation and hence open new perspectives for its modulation in clinical settings.

B7–CD28 Costimulatory Signals Control the Survival and Proliferation of Murine and Human γδ T Cells via IL-2 Production

γδ T cells play key nonredundant roles in immunity to infections and tumors. Thus, it is critical to understand the molecular mechanisms responsible for γδ T cell activation and expansion in vivo. In striking contrast to their αβ counterparts, the costimulation requirements of γδ T cells remain poorly understood. Having previously described a role for the TNFR superfamily member CD27, we since screened for other nonredundant costimulatory receptors in γδ T cell activation. We report in this article that the Ig superfamily receptor CD28 (but not its related protein ICOS) is expressed on freshly isolated lymphoid γδ T cells and synergizes with the TCR to induce autocrine IL-2 production that promotes γδ cell survival and proliferation in both mice and humans. Specific gain-of-function and loss-of-function experiments demonstrated a nonredundant function for CD28 interactions with its B7 ligands, B7.1 (CD80) and B7.2 (CD86), both in vitro and in vivo. Thus, γδ cell proliferation was significantly enhanced by CD28 receptor agonists but abrogated by B7 Ab-mediated blockade. Furthermore, γδ cell expansion following Plasmodium infection was severely impaired in mice genetically deficient for CD28. This resulted in the failure to mount both IFN-γ–mediated and IL-17–mediated γδ cell responses, which contrasted with the selective effect of CD27 on IFN-γ–producing γδ cells. Our data collectively show that CD28 signals are required for IL-2–mediated survival and proliferation of both CD27+ and CD27− γδ T cell subsets, thus providing new mechanistic insight for their modulation in disease models.

Human γδ Thymocytes Are Functionally Immature and Differentiate into Cytotoxic Type 1 Effector T Cells upon IL-2/IL-15 Signaling

Cytotoxicity and IFN-γ production by human γδ T cells underlie their potent antitumor functions. However, it remains unclear where and how human γδ T cells acquire these key effector properties. Given the recent disclosure of a major contribution of the thymus to murine γδ T cell functional differentiation, in this study we have analyzed a series of human pediatric thymuses. We found that ex vivo–isolated γδ thymocytes produced negligible IFN-γ and lacked cytolytic activity against leukemia cells. However, these properties were selectively acquired upon stimulation with IL-2 or IL-15, but not IL-4 or IL-7. Unexpectedly, TCR activation was dispensable for these stages of functional differentiation. The effects of IL-2/IL-15 depended on MAPK/ERK signaling and induced de novo expression of the transcription factors T-bet and eomesodermin, as well as the cytolytic enzyme perforin, required for the cytotoxic type 1 program. These findings have implications for the manipulation of γδ T cells in cancer immunotherapy.

Inhibition of murine γδ lymphocyte expansion and effector function by regulatory αβ T cells is cell-contact-dependent and sensitive to GITR modulation

γδ T cells are highly cytolytic lymphocytes that produce large amounts of pro‐inflammatory cytokines during immune responses to multiple pathogens. Furthermore, their ability to kill tumor cells has fueled the development of γδ‐T‐cell‐based cancer therapies. Thus, the regulation of γδ‐T‐cell activity is of great biological and clinical relevance. Here, we show that murine CD4+CD25+ αβ T cells, the vast majority of which express the Treg marker, Foxp3, abolish key effector functions of γδ T cells, namely the production of the pro‐inflammatory cytokines, IFN‐γ and IL‐17, cytotoxicity, and lymphocyte proliferation in vitro and in vivo. We further show that suppression is dependent on cellular contact between Treg and γδ T cells, results in the induction of an anergic state in γδ lymphocytes, and can be partially reversed by manipulating glucocorticoid‐induced TNF receptor‐related protein (GITR) signals. Our data collectively dissect a novel mechanism by which the expansion and pro‐inflammatory functions of γδ T cells are regulated.

TCR signal strength controls thymic differentiation of discrete proinflammatory [gamma][delta] T cell subsets

The mouse thymus produces discrete γδ T cell subsets that make either interferon-γ (IFN-γ) or interleukin 17 (IL-17), but the role of the T cell antigen receptor (TCR) in this developmental process remains controversial. Here we show that Cd3g+/− Cd3d+/− (CD3 double-haploinsufficient (CD3DH)) mice have reduced TCR expression and signaling strength on γδ T cells. CD3DH mice had normal numbers and phenotypes of αβ thymocyte subsets, but impaired differentiation of fetal Vγ6+ (but not Vγ4+) IL-17-producing γδ T cells and a marked depletion of IFN-γ-producing CD122+ NK1.1+ γδ T cells throughout ontogeny. Adult CD3DH mice showed reduced peripheral IFN-γ+ γδ T cells and were resistant to experimental cerebral malaria. Thus, TCR signal strength within specific thymic developmental windows is a major determinant of the generation of proinflammatory γδ T cell subsets and their impact on pathophysiology.

Five Layers of Receptor Signaling in γδ T-Cell Differentiation and Activation.

The contributions of γδ T-cells to immunity to infection or tumors critically depend on their activation and differentiation into effectors capable of secreting cytokines and killing infected or transformed cells. These processes are molecularly controlled by surface receptors that capture key extracellular cues and convey downstream intracellular signals that regulate γδ T-cell physiology. The understanding of how environmental signals are integrated by γδ T-cells is critical for their manipulation in clinical settings. Here, we discuss how different classes of surface receptors impact on human and murine γδ T-cell differentiation, activation, and expansion. In particular, we review the role of five receptor types: the T-cell receptor (TCR), costimulatory receptors, cytokine receptors, NK receptors, and inhibitory receptors. Some of the key players are the costimulatory receptors CD27 and CD28, which differentially impact on pro-inflammatory subsets of γδ T-cells; the cytokine receptors IL-2R, IL-7R, and IL-15R, which drive functional differentiation and expansion of γδ T-cells; the NK receptor NKG2D and its contribution to γδ T-cell cytotoxicity; and the inhibitory receptors PD-1 and BTLA that control γδ T-cell homeostasis. We discuss these and other receptors in the context of a five-step model of receptor signaling in γδ T-cell differentiation and activation, and discuss its implications for the manipulation of γδ T-cells in immunotherapy.

Foxp3 induction in human and murine thymus precedes the CD4+ CD8+ stage but requires early T-cell receptor expression

The thymus generates a T‐cell lineage dedicated to immune regulation, ‘naturally occurring’ regulatory T cells, best specified by the forkhead family transcription factor Foxp3. Here, we have conducted a parallel study in humans and mice where we have dissected the earliest stages of Foxp3 induction during thymocyte development. By analyzing a large collection of 21 human thymuses we show that Foxp3 can be consistently detected in CD4 immature single positive thymocytes that precede the CD4(+)CD8(+) (double positive, DP) stage. The reduced levels of CD3 expression found at this stage of human thymocyte development raise the question of TCR (T‐cell receptor) requirement for Foxp3 induction. We further show that, in mice, Foxp3 expression was also detected in pre‐DP thymocytes of TCRα‐sufficient but not in TCRα‐deficient animals, genetically showing the TCR dependence of Foxp3 expression at pre‐DP stages of T‐cell development.