AML: Acute Myeloid Leukemia; CNS: Central Nervous System; DST: Donor-Specific Transfusion; EAE: Experimental Autoimmune Encephalomyelitis; GVHD: Graft-Versus-Host Disease; GVT: Graft-Versus-Tumor; HSCT: Hematopoietic Allogeneic Stem Cell Transplantation; LN: Lymph Node; Th1: T helper type 1; Treg: Regulatory CD4 T cell

Gamma-delta (γδ) T cells belong to a rare population of T cells in mice lymphoid tissue and human peripheral blood. On the contrary, the epithelial and mucosal barriers are enriched with γδ T cell population, where they perform unique immune functions, help in tissue repair, and maintain homeostasis. These tissue-resident γδ T cells possess the characteristics of innate and adaptive immune cells, enabling them to act as a bridge between the two. γδ T cells exist as various independent subsets which are shown to perform distinct functions during disease conditions. The molecular intricacies involved in the development, differentiation, and distribution of γδ T cell subsets are covered by us and others [1]. γδ T cells constitute about 1-10% of T cells in circulation and are highly enriched on epithelial and mucosal sites [2,3]. γδ T cells have also been shown to perform effector functions during infection, cancer, and autoimmunity, which have been thoroughly reviewed in recent publications [4,5]. The role of γδ T cells is very well explored in infection, cancer, and autoimmunity but well explored in transplantation. It has been perceived that γδ T cells may have a possible function in graft-versus-tumor (GVT) without driving the graft-versus-host disease (GVHD) in hematopoietic allogeneic stem cell transplantation (HSCT) [6]. Minculescu et al. showed a protective role of γδ T cells in relapse-free survival and GVHD in patients with a high frequency of γδ T cells in circulation [7]. In newly diagnosed acute myeloid leukemia (AML) patients, an increased frequency of CD25⁺CD127^loVδ2⁺ γδ T cells was reported in the bone marrow of patients and show an immunosuppressive function [8]. Proteasome inhibitor Bortezomib is shown to enhance the cytotoxic function of ex-vivo expanded γδ against AML and T-cell acute lymphoblastic leukemia (T-ALL) [9]. γδ T cells perform their functions by direct cytotoxic effect through death receptor (FAS-FASL, TRAIL-TRAIL receptor or through perforin/granzyme) or by secreting the soluble molecules such as IFN-γ, TNF-α, IL-4, IL-13, IL-17, and IL-22 [2,5]. γδ T cell subsets are also proposed to be used as adaptive cellular therapy in some cancers [10-12]. The role of some of the γδ T cell subsets is listed in Table 1. The function of various subsets of γδ T cells and the effect of various tolerogenic regimens on the γδ T cells in the transplantation still need to be explored.

Recently, Giri et al., showed that treatment of DST and anti-CD40L promotes regulatory Vγ2⁺ γδ T cells and prolongs skin allograft survival [13]. Murine γδ T cells can recognize surface-expressed proteins directly without the major histocompatibility complex molecules. Because our results showed that the depletion of Vγ2⁺ T cells under tolerogenic conditions affects allograft survival, but not the syngeneic grafts. This suggest that Vγ2⁺ cells’ actions are mediated toward alloantigen and not towards the nonspecific injury associated with transplantation protocols. Further investigation is still required to establish the molecular nature of γδ T cells specificity towards alloantigen. Depleting Vγ2⁺ γδ T cells led to increased MHC-II^highF4/80⁺ macrophage migration, which induces efficient alloantigen presentation and T cell priming. TCR and CD40L signaling are reported to promote the proliferation of γδ T cells and the expression of IFN-γ in an IL-2–independent manner [14]. Hence, the blockade of CD40L signaling impacted IFN-γ producing ability of γδ T cells and supported the proliferation of a specific subset that showed enhanced regulatory functions. During bacterial and virus infection, IFN-γ produced by γδ T cells is reported to promote CD4⁺ T cells’ differentiation toward the Th1 lineage [15,16]. We also observed that naive Vγ2⁺ γδ T cells could independently induce the differentiation of Th1 cells, which was suppressed in the presence of tolerized Vγ2⁺ γδ T cells (Figure 1). In support, in-vivo depletion of Vγ2⁺ γδ T cells in tolerogen-treated mice significantly increased the expression of IFN-γ in CD4⁺ T cells, CD8⁺ T cells, and Vγ1.1⁺ γδ T cells. Extracellular adenosine triphosphate produced during tissue damage or surgery during transplantation is pro-inflammatory. The membrane-associated enzymes CD39 and CD73 convert inflammatory adenosine triphosphate to noninflammatory adenosine. CD39⁺/CD73⁺ γδ T cells function as regulatory cells [17]. In our study, we observed increased CD39⁺ γδ T cells in the LN and the skin allografts of tolerized recipients (Figure 1). CD4⁺Foxp3⁺ Tregs are critical for the induction of tolerance, and their increased frequency correlates to improved allograft survival [18]. We observed that depletion of Vγ2⁺ γδ T cells in tolerogen-treated mice reduced the percentage of CD4⁺Foxp3⁺ Tregs in the spleen. A study using the autoimmune keratitis model also reported that the differentiation of Foxp3⁺Tregs in the animal is dependent on γδ T cells, and TCRδ^–/–mice have a reduced number of peripheral CD4⁺CD25⁺Foxp3⁺ Tregs [19]. However, the direct interaction between γδ T cells and Tregs and the molecular mechanism promoting the tolerance need detailed investigation. Emerging reports also suggest the involvement of γδ T cells during transplant rejection and spontaneous tolerance. IL-17 produced by γδ T cells is shown to mediate allograft rejection by inducing neutrophil recruitment and CD4⁺Th17 differentiation [20]. IL-17 produced by Vγ4⁺ γδ T cells facilitated the accumulation of mature DCs and priming of effector T cells, causing the rejection of H-Y minor antigen-mismatched skin allograft [21]. Consequently, the depletion of γδ T cells prolonged the allograft survival by reducing IL-17-mediated pathology and enhancing the accumulation of CD4⁺Foxp3⁺ Tregs into the allograft [18,22]. Activated V γ2⁺ γδ T cells have been shown to produce high levels of IFN-γ and perforin, thereby preventing the development of B16 melanoma in mice. γδ Tregs were also reported to suppress acute Graft-versus-host disease (GVHD) during allogeneic peripheral blood stem cell transplantation [23]. Regulatory γδ T cells secrete IL-4 and IL-10, which suppress Th1 differentiation of CD4 T cells, prolonging the survival of kidney and skin allografts [24,25].

Figure 1. DST plus anti-CD40L (tolerogen) promotes the generation of tolerogenic γδ T cells which in turn inhibits the generation of T-bet⁺ Th1 CD4 T cells and enhances the differentiation of Foxp3⁺ Treg CD4 T cells. Regulatory CD4 T cells and γδ T cells also migrate to the allograft and promote allograft survival.

γδ T cells are relatively new to be recognized for their non-redundant functions across many disease conditions. But their unique potential for performing effector and regulatory functions has made them a potent target for clinical research. However, detailed studies on crosstalk with other immune cells at the site of inflammation and in secondary lymphoid tissues need to be investigated. Although the regulatory function of γδ T cells is reported in several publications, detailed signals and molecular interaction that brings the regulatory plasticity are unknown and need future investigation. Our study strengthens the role of regulatory γδ T cells, which might prove helpful in designing better strategies for tolerance and immunosuppression. Clinical trials with γδ T cell therapy against cancer are ongoing and establishing their important contributions to human health.

SG received Senior Research Fellowship from the Indian Council of Medical Research, Government of India. GL received grants from the Department of Biotechnology (grant numbers BT/PR15533/MED/30/1616/2015 and BT/PR14156/BRB/10/1515/2016) and the Department of Science and Technology (DST/SJF/LSA-01/2017-18), Government of India.