Understanding the complexity of γδ T-cell subsets in mouse and human

γδ T cells are increasingly recognized as having important functional roles in a range of disease scenarios such as infection, allergy, autoimmunity and cancer. With this has come realization that γδ cells are not a homogeneous population of cells with a single physiological role. Instead, ever increasing complexity in both phenotype and function is being ascribed to γδ cell subsets from various tissues and locations, and in both mouse and human. Here, we review this complexity by describing how diverse γδ cell subsets are generated in the murine thymus, and how these events relate to subsequent γδ subset function in the periphery. We then review the two major γδ cell populations in human, highlighting the several similarities of Vδ1(+) cells to certain murine γδ subsets, and describing the remarkable functional plasticity of human Vδ2(+) cells. A better understanding of this spectrum of γδ cell phenotypes should facilitate more targeted approaches to utilise their tremendous functional potential in the clinic.     

Functional and metabolic dichotomy of murine γδ T cell subsets in cancer immunity

γδ T cells can display a plethora of immune functions, but recent studies have highlighted their importance, in multiple disease models, as sources of the pro-inflammatory cytokines, IL-17A (IL-17), and IFN-γ. These are produced by distinct murine effector γδ T cell subsets that diverge during thymic γδ T cell development. Among the multiple roles these subsets play in peripheral tissues, a striking dichotomy has emerged at tumor sites: whereas IFN-γ⁺ γδ T cells inhibit tumor cell growth, IL-17⁺ γδ T cells promote tumor progression and metastasis formation. In this review, we discuss the main lines of evidence, mostly from preclinical studies in mouse models, for this functional dichotomy in cancer immunity. We further highlight very recent advances in our understanding how metabolic sources and pathways can impact on the balance between IFN-γ⁺ and IL-17⁺ γδ T cells in the tumor microenvironment, which opens a new exciting avenue to explore toward the application of γδ T cells in cancer immunotherapy.     

Diversity of γδ T-cell antigens

In the last two decades, it has become clear that γδ T cells recognize a diverse array of antigens including self and foreign, large and small, and peptidic and non-peptidic molecules. In this respect, γδ antigens as a whole resemble more the antigens recognized by antibodies than those recognized by αβ T cells. Because of this antigenic diversity, no single mechanism—such as the major histocompatibility complex (MHC) restriction of αβ T cells—is likely to provide a basis for all observed T-cell antigen receptor (TCR)-dependent γδ T-cell responses. Furthermore, available evidence suggests that many individual γδ T cells are poly-specific, probably using different modes of ligand recognition in their responses to unrelated antigens. While posing a unique challenge in the maintenance of self-tolerance, this broad reactivity pattern might enable multiple overlapping uses of γδ T-cell populations, and thus generate a more efficient immune response.     

Antigen-restricted γδ T-cell receptors?

After more than two decades of investigation, the biological role of the γδ T-cell receptors (TCRs) remains elusive. In fact, a theory of ligand recognition is still lacking that accounts for their adaptable structure, their peripheral selection, and the observed responses of γδ T cells, which do not require immunization but only include cells sharing germline-encoded components of the TCR. Assuming that all γδ T cells recognize ligands by a common mechanism, we now propose that germline-encoded components of the γδ TCRs provide for the specific recognition of a select set of antigenic determinants (Ags) which appear on the cell surface in various molecular associations. Furthermore, we hypothesize that the adaptivity of the γδ TCRs serves to increase affinity for the molecules with which these Ags associate rather than for the Ags themselves. Here we outline this hypothetical mechanism and discuss its possible implications for thymic selection and potential for complementing known innate and adaptive mechanisms of immune defense.     

Two functionally distinct subsets of IL-17 producing γδ T cells

The γδ T cells play an important role in both mice and humans as a source of the cytokine IL-17, which is key for immune resistance to certain pathogens. In mice, most of these IL-17 producers, termed γδT-17 cells, actually comprise two distinct types: those expressing an invariant Vγ6Vδ1⁺ TCR and those expressing a Vγ4⁺ TCR. Murine γδT-17 cells acquire an inherent bias to produce IL-17 and other “type 17” cytokines during thymic development. The similarities and differences between the two mouse γδT-17 types are reviewed here, and the potential implications of their differences are discussed. There is some evidence that two distinct TCR-defined IL-17-producing γδ T cell subsets also exist in humans, but unlike the mouse γδT-17 cells, these cells are probably not imprinted with an IL-17 bias during thymic development, but rather acquire an IL-17 bias in the periphery.     

Fast-acting γδ T-cell subpopulation and protective immunity against infections

Unique Vγ2Vδ2 (Vγ9Vδ2) T cells existing only in human and non-human primates, account for the majority of circulating γδ T cells in human adults. Vγ2Vδ2 T cells are the sole γδ T-cell subpopulation capable of recognizing the microbial (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) produced by selected pathogens during infections. Recent seminal studies in non-human primate models have demonstrated that the unique HMBPP-specific Vγ2Vδ2 T cells are fast-acting, multi-functional, and protective during infections. This article reviews the recent seminal observations of Vγ2Vδ2 T cells in protective mechanisms against tuberculosis and other infections.     

Artesunate enhances γδ T-cell-mediated antitumor activity through augmenting γδ T-cell function and reversing immune escape of HepG2 cells

Objective: To explore the effect and mechanism of artesunate on γδ T cell-mediated antitumor immune responses against hepatoma carcinoma cells (HepG2) in vitro.

Methods: Human γδ T cells or HepG2 were respectively treated with artesunate, subjected to co-culture as appropriate, and the following assays were subsequently conducted: CCK8 to examine cell viability; LDH release assay to detect the killing effect of γδ T cells on HepG2 cells; flow cytometry to examine the expression of perforin (PFP) and granzyme B (GraB) of γδ T cells; ELISA to evaluate the levels of TGF-β1 and IL-10 in the collected supernatant of HepG2 cells pretreated with artesunate; and Western blot analysis to examine Fas, FasL, STAT3, p-STAT3 expression of HepG2 cells induced by artesunate.?

Results: The results showed that the cytotoxicity effect of γδ T cells pretreated with artesunate on HepG2 cells was augmented via elevating the expression of GraB in γδ T cells. Furthermore, treatment with artesunate reversed the inhibition of HepG2 cells on γδ T cells by reducing the secretion of TGF-β1 in HepG2 cells supernatant and enhanced the antitumor effect of γδ T cells against HepG2 cells through increasing the expression of Fas on HepG2 cells, which may be attributed to the inhibition of STAT3 signaling protein.

Conclusion: Artesunate has several mechanisms for augmenting the antitumor immune responses mediated by γδ T cells. These results suggested artesunate may be an efficacious agent in the treatment of hepatocellular carcinoma.     

Relative expression of γδ T-cell receptor gene families detected by RT-PCR and capillary electrophoresis

γδ T cells are intensively studied because their function in infection, allergy, autoimmune disease, cancer and post-transplant period is not yet fully understood. PCR-based techniques were established to study the γ variable (Vγ) and δ variable (Vδ) gene families. PCR product evaluation is routinely carried out by Southern blot analysis or the third complementarity-determining region spectratyping, but a fast and simple assessment of Vγ and Vδ gene family expression is missing. The aim of our study was to test capillary electrophoresis as a potential method for evaluating the composition of the γδ T-cell population. This report provides optimized PCR conditions for γδ T-cell receptor amplification. Further, it describes the utilization of capillary electrophoresis in the Agilent 2100 Bioanalyzer to evaluate the relative expression of Vγ and Vδ gene families after their amplification. An application of the methodology to peripheral blood mononuclear cell samples from patients during haemato-oncological treatment is shown. The described methodology is fast and simple to operate and is therefore suitable as a first screening of the γδ T-cell population composition in tissues of interest.