IL-17+ γδ T cells as kick-starters of inflammation.

Shortly after the discovery of interleukin 17 (IL-17)-producing CD4+ helper T cells (TH17 cells), it was found that γδ T cells can also secrete large amounts of this pro-inflammatory cytokine. A decade later, it is now known that IL-17+ γδ T cells (γδ17 T cells) are often the main providers of IL-17A in various models of inflammatory diseases, while they also contribute to protective immune responses to infectious organisms. Due to an intricate thymic program of differentiation, γδ17 T cells are able to respond faster than TH17 cells do and thus predominate in the early stages of inflammatory responses. Here we review the current knowledge of the development, activation and pathophysiological functions of γδ17 T cells, aiming to increase the awareness in the community of the therapeutic potential of this 'other side' of IL-17-mediated immune responses.

TCR signal strength controls thymic differentiation of discrete proinflammatory γδ 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 αß 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.

The multisensory regulation of unconventional T cell homeostasis.

Unconventional T cells typically group γδ T cells, invariant Natural Killer T cells (NKT) and Mucosal Associated Invariant T (MAIT) cells. With their pre-activated status and biased tropism for non-lymphoid organs, they provide a rapid (innate-like) and efficient first line of defense against pathogens at strategical barrier sites, while they can also trigger chronic inflammation, and unexpectedly contribute to steady state physiology. Thus, a tight control of their homeostasis is critical to maintain tissue integrity. In this review, we discuss the recent advances of our understanding of the factors, from neuroimmune to inflammatory regulators, shaping the size and functional properties of unconventional T cell subsets in non-lymphoid organs. We present a general overview of the mechanisms common to these populations, while also acknowledging specific aspects of their diversity. We mainly focus on their maintenance at steady state and upon inflammation, highlighting some key unresolved issues and raising upcoming technical, fundamental and translational challenges.

MicroRNA-181a restricts human γδ T cell differentiation by targeting Map3k2 and Notch2.

γδ T cells are a conserved population of lymphocytes that contributes to anti-tumor responses through its overt type 1 inflammatory and cytotoxic properties. We have previously shown that human γδ T cells acquire this profile upon stimulation with IL-2 or IL-15, in a differentiation process dependent on MAPK/ERK signaling. Here, we identify microRNA-181a as a key modulator of human γδ T cell differentiation. We observe that miR-181a is highly expressed in patients with prostate cancer and that this pattern associates with lower expression of NKG2D, a critical mediator of cancer surveillance. Interestingly, miR-181a expression negatively correlates with an activated type 1 effector profile obtained from in vitro differentiated γδ T cells and miR-181a overexpression restricts their levels of NKG2D and TNF-α. Upon in silico analysis, we identify two miR-181a candidate targets, Map3k2 and Notch2, which we validate via overexpression coupled with luciferase assays. These results reveal a novel role for miR-181a as critical regulator of human γδ T cell differentiation and highlight its potential for manipulation of γδ T cells in next-generation immunotherapies.

γδ-T cells promote IFN-γ-dependent Plasmodium pathogenesis upon liver-stage infection.

Cerebral malaria (CM) is a major cause of death due to Plasmodium infection. Both parasite and host factors contribute to the onset of CM, but the precise cellular and molecular mechanisms that contribute to its pathogenesis remain poorly characterized. Unlike conventional αß-T cells, previous studies on murine γδ-T cells failed to identify a nonredundant role for this T cell subset in experimental cerebral malaria (ECM). Here we show that mice lacking γδ-T cells are resistant to ECM when infected with Plasmodium berghei ANKA sporozoites, the liver-infective form of the parasite and the natural route of infection, in contrast with their susceptible phenotype if challenged with P. berghei ANKA-infected red blood cells that bypass the liver stage of infection. Strikingly, the presence of γδ-T cells enhanced the expression of Plasmodium immunogenic factors and exacerbated subsequent systemic and brain-infiltrating inflammatory αß-T cell responses. These phenomena were dependent on the proinflammatory cytokine IFN-γ, which was required during liver stage for modulation of the parasite transcriptome, as well as for downstream immune-mediated pathology. Our work reveals an unanticipated critical role of γδ-T cells in the development of ECM upon Plasmodium liver-stage infection.

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-gamma and interleukin 17 by alphabeta and gammadelta T cells influences the outcome of immune responses. Here we show that most gammadelta T lymphocytes expressed the tumor necrosis factor receptor family member CD27 and secreted interferon-gamma, whereas interleukin 17 production was restricted to CD27(-) gammadelta T cells. In contrast to the apparent plasticity of alphabeta T cells, the cytokine profiles of these distinct gammadelta 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 gammadelta T cells at least in part by inducing expression of the lymphotoxin-beta receptor and genes associated with trans-conditioning and interferon-gamma production. Thus, the cytokine profiles of peripheral gammadelta T cells are predetermined mainly by a mechanism involving CD27.

IL-23 drives differentiation of peripheral γδ17 T cells from adult bone marrow-derived precursors.

Pro-inflammatory interleukin (IL)-17-producing γδ (γδ17) T cells are thought to develop exclusively in the thymus during fetal/perinatal life, as adult bone marrow precursors fail to generate γδ17 T cells under homeostatic conditions. Here, we employ a model of experimental autoimmune encephalomyelitis (EAE) in which hematopoiesis is reset by bone marrow transplantation and demonstrate unequivocally that Vγ4+ γδ17 T cells can develop de novo in draining lymph nodes in response to innate stimuli. In vitro, γδ T cells from IL-17 fate-mapping reporter mice that had never activated the Il17 locus acquire IL-17 expression upon stimulation with IL-1ß and IL-23. Furthermore, IL-23R (but not IL-1R1) deficiency severely compromises the induction of γδ17 T cells in EAE, demonstrating the key role of IL-23 in the process. Finally, we show, in a composite model involving transfers of both adult bone marrow and neonatal thymocytes, that induced γδ17 T cells make up a substantial fraction of the total IL-17-producing Vγ4+ T-cell pool upon inflammation, which attests the relevance of this novel pathway of peripheral γδ17 T-cell differentiation.

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.

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 αß 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.

Tumor-Infiltrating T Cells in Skin Basal Cell Carcinomas and Squamous Cell Carcinomas: Global Th1 Preponderance with Th17 Enrichment-A Cross-Sectional Study.

Basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) are high-incidence, non-melanoma skin cancers (NMSCs). The success of immune-targeted therapies in advanced NMSCs led us to anticipate that NMSCs harbored significant populations of tumor-infiltrating lymphocytes with potential anti-tumor activity. The main aim of this study was to characterize T cells infiltrating NMSCs. Flow cytometry and immunohistochemistry were used to assess, respectively, the proportions and densities of T cell subpopulations in BCCs (n = 118), SCCs (n = 33), and normal skin (NS, n = 30). CD8+ T cells, CD4+ T cell subsets, namely, Th1, Th2, Th17, Th9, and regulatory T cells (Tregs), CD8+ and CD4+ memory T cells, and γδ T cells were compared between NMSCs and NS samples. Remarkably, both BCCs and SCCs featured a significantly higher Th1/Th2 ratio (~four-fold) and an enrichment for Th17 cells. NMSCs also showed a significant enrichment for IFN-γ-producing CD8+T cells, and a depletion of γδ T cells. Using immunohistochemistry, NMSCs featured denser T cell infiltrates (CD4+, CD8+, and Tregs) than NS. Overall, these data favor a Th1-predominant response in BCCs and SCCs, providing support for immune-based treatments in NMSCs. Th17-mediated inflammation may play a role in the progression of NMSCs and thus become a potential therapeutic target in NMSCs.

Differentiation and activation of γδ T Lymphocytes: Focus on CD27 and CD28 costimulatory receptors.

γδ T lymphocytes are major providers of the pro-inflammatory cytokines interferon-γ (IFN-γ) and interleukin-17 (IL-17) at early stages of (auto)immune responses. We and others have recently described the phenotype and differentiation requirements of two distinct murine γδ T cell subsets producing either IFN-γ or IL-17. Here we summarize our current understanding of the molecular mechanisms that control γδ T cell differentiation, which is programmed in the thymus, and peripheral activation upon infection. We focus on the costimulatory receptors CD27 and CD28, which play independent and non-redundant roles in the physiology of γδ T cells in mice and in humans.

The IL-17A-neutrophil axis promotes epithelial cell IL-33 production during nematode lung migration.

The early migratory phase of pulmonary helminth infections is characterized by tissue injury leading to the release of the alarmin interleukin (IL)-33 and subsequent induction of type 2 immune responses. We recently described a role for IL-17A, through suppression of interferon (IFN)-γ, as an important inducer of type 2 responses during infection with the lung-migrating rodent nematode Nippostrongylus brasiliensis. Here, we aimed to investigate the interaction between IL-17A and IL-33 during the early lung migratory stages of N. brasiliensis infection. In this brief report, we demonstrate that deficiency of IL-17A leads to impaired IL-33 expression and secretion early in infection, independent of IL-17A suppression of IFN-γ. Neutrophil-depletion experiments, which dramatically reduce lung injury, revealed that neutrophils are primarily responsible for the IL-17A-dependent release of IL-33 into the airways. Taken together, our results reveal an IL-17A-neutrophil-axis that can drive IL-33 during helminth infection, highlighting an additional pathway by which IL-17A regulates pulmonary type 2 immunity.

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.

Identification of regulatory Foxp3+ invariant NKT cells induced by TGF-beta.

Invariant NKT (iNKT) cells were shown to prevent the onset of experimental autoimmune encephalomyelitis in mice following administration of their specific TCR agonist alpha-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-beta in both mice and humans. Moreover, in vivo generation of Foxp3(+) iNKT cells was observed in the TGF-beta-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.

Cutting edge: adaptive versus innate receptor signals selectively control the pool sizes of murine IFN-γ- or IL-17-producing γδ T cells upon infection.

γδ T lymphocytes are commonly viewed as embracing properties of both adaptive and innate immunity. Contributing to this is their responsiveness to pathogen products, either with or without the involvement of the TCR and its coreceptors. This study clarifies this paradoxical behavior by showing that these two modes of responsiveness are the properties of two discrete sets of murine lymphoid γδ T cells. Thus, MyD88 deficiency severely impaired the response to malaria infection of CD27((-)), IL-17A-producing γδ T cells, but not of IFN-γ-producing γδ cells. Instead, the latter compartment was severely contracted by ablating CD27, which synergizes with TCRγδ in the induction of antiapoptotic mediators and cell cycle-promoting genes in CD27((+)), IFN-γ-secreting γδ T cells. Hence, innate versus adaptive receptors differentially control the peripheral pool sizes of discrete proinflammatory γδ T cell subsets during immune responses to infection.

Searching for "signal 2": costimulation requirements of γδ T cells.

T cell activation requires the integration of signals that arise from various types of receptors. Although TCR triggering is a necessary condition, it is often not sufficient to induce full T-cell activation, as reflected in cell proliferation and cytokine secretion. This has been firmly demonstrated for conventional αß T cells, for which a large panel of costimulatory receptors has been identified. By contrast, the area remains more obscure for unconventional, innate-like γδ T cells, as the literature has been scarce and at times contradictory. Here we review the current state of the art on the costimulatory requirements of γδ T cell activation. We highlight the roles of members of the immunoglobulin (like CD28 or JAML) or tumour necrosis factor receptor (like CD27) superfamilies of coreceptors, but also of more atypical costimulatory molecules, such as NKG2D or CD46. Finally, we identify various areas where our knowledge is still markedly insufficient, hoping to provoke future research on γδ T cell costimulation.

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

Gammadelta 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 gammadelta-T-cell-based cancer therapies. Thus, the regulation of gammadelta-T-cell activity is of great biological and clinical relevance. Here, we show that murine CD4+CD25+ alphabeta T cells, the vast majority of which express the Treg marker, Foxp3, abolish key effector functions of gammadelta T cells, namely the production of the pro-inflammatory cytokines, IFN-gamma 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 gammadelta T cells, results in the induction of an anergic state in gammadelta 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 gammadelta T cells are regulated.

γδ T cells in tissue physiology and surveillance.

γδ T cells are a unique T cell subpopulation that are rare in secondary lymphoid organs but enriched in many peripheral tissues, such as the skin, intestines and lungs. By rapidly producing large amounts of cytokines, γδ T cells make key contributions to immune responses in these tissues. In addition to their immune surveillance activities, recent reports have unravelled exciting new roles for γδ T cells in steady-state tissue physiology, with functions ranging from the regulation of thermogenesis in adipose tissue to the control of neuronal synaptic plasticity in the central nervous system. Here, we review the roles of γδ T cells in tissue homeostasis and in surveillance of infection, aiming to illustrate their major impact on tissue integrity, tissue repair and immune protection.

Microbiota-dependent expansion of testicular IL-17-producing Vγ6+ γδ T cells upon puberty promotes local tissue immune surveillance.

γδT cells represent the majority of lymphocytes in several mucosal tissues where they contribute to tissue homoeostasis, microbial defence and wound repair. Here we characterise a population of interleukin (IL) 17-producing γδ (γδ17) T cells that seed the testis of naive C57BL/6 mice, expand at puberty and persist throughout adulthood. We show that this population is foetal-derived and displays a T-cell receptor (TCR) repertoire highly biased towards Vγ6-containing rearrangements. These γδ17 cells were the major source of IL-17 in the testis, whereas αß T cells mostly provided interferon (IFN)-γ in situ. Importantly, testicular γδ17 cell homoeostasis was strongly dependent on the microbiota and Toll-like receptor (TLR4)/IL-1α/IL-23 signalling. We further found that γδ17 cells contributed to tissue surveillance in a model of experimental orchitis induced by intra-testicular inoculation of Listeria monocytogenes, as Tcrδ-/- and Il17-/- infected mice displayed higher bacterial loads than wild-type (WT) controls and died 3 days after infection. Altogether, this study identified a previously unappreciated foetal-derived γδ17 cell subset that infiltrates the testis at steady state, expands upon puberty and plays a crucial role in local tissue immune surveillance.