Suppressive activity of Vδ2⁺ γδ T cells on αβ T cells is licensed by TCR signaling and correlates with signal strength

T lymphocytes are divided into two subsets by their expression of T cell antigen receptors (TCRs): αβ T cells (combination of an α chain and a β chain) and γδ T cells (combination of a γ chain and a δ chain). γδ T cells constitute a major T cell population in the epithelial tissues but represent a rare (typically 1–15%) T cell population in the peripheral circulation with the majority (50–95%) of γδ T cells carrying a Vγ9Vδ2 TCR [1]. A unique and specific feature of human Vγ9Vδ2 T cells (Vδ2⁺ T cells) is their TCR-dependent recognition of phosphoantigens (PAgs), metabolites of the phosphorylated isoprenoid pathway. Because of γδ T cells’ distinctive features—potent antitumor effect and independence from MHC restriction—a special interest has been taken in the application of Vδ2⁺ T cells in cancer immunotherapy [2, 3]. However, phase II trials for evaluating the efficacy of adoptive transfer and in vivo expansions of Vδ2⁺ T cells have resulted in limited clinical response in solid tumors thus far, even though many clinically unresponsive patients exhibited sustained Vδ2⁺ T-cell activation and proliferation [4‐13]. Therefore, the mechanism which prevents Vδ2⁺ T cells from eliciting long-lasting antitumor effects in vivo needs to be elucidated. γδ T cells are known to inhibit or suppress the maturation and/or activation of other immune cells under certain conditions [14, 15]. In particular, their interaction with effector αβ T cells is of great interest with respect to a potential regulatory function of γδ T cells in cancer [16‐18].

A tumor-infiltrating immunosuppressive Vδ2⁺ T cell population has been identified in multiple types of solid cancers [19, 20]; an in vitro study suggested that tumor-infiltrating γδ T cells inhibit αβ T cell activation via PD-1/PD-L1 ligation [19]. Yet, consensus has not been reached on whether or under which condition(s) Vδ2⁺ T cells exert suppressive function on αβ T cells. Casetti et al. showed that up to 30% of Vδ2⁺ T cells express the Foxp3 transcription factor when they are activated by isopentyl pyrophosphate (IPP) in the presence of IL-15 and TGF-β. Foxp3⁺-enriched Vδ2⁺ T cells, but not positively freshly isolated Vδ2⁺ T cells, displayed regulatory/immunosuppressive activity on αβ T cells when co-cultured with autologous PBMCs in the presence of anti-CD3/anti-CD28 mAb [21]. In the study of Peters et al. [22] neither IL-2 nor the combination of TGF-β1 and IL-15 induced regulatory functions in PAg-expanded γδ T cells on Staphylococcus aureus enterotoxin-stimulated CD4⁺CD25^- T cells. On the other hand, γδ T cells initially activated by anti-CD3/anti-CD28 in the presence of TGF-β and IL-15 suppressed CD4⁺CD25^- T cells although Foxp3 in γδ T cells was downregulated after transient expression. In contrast to Casetti’s paper, it was also reported that positively freshly isolated γδ T cells, which are Foxp3-negative, can potently suppress the in vitro proliferation of CD4⁺ T cells in the presence of anti-CD3/anti-CD28 mAb stimulation in the co-culture [22, 23]. In addition, Traxlmayr et al. [24] demonstrated that in the presence of antigen presenting cells, Vδ2⁺ T cells stimulated with IPP, but not negatively freshly isolated Vδ2⁺ T cells, can inhibit the proliferation of CD4⁺ and CD8⁺ αβ T cells reacting to strong recall antigens or allo-antigens. Combining these findings, Peters et al. [18, 22] suggested that γδ T cells exert their suppressive function only in the presence of anti-CD28 stimulation or antigen-presenting cells and that anti-CD28 stimulation rather than Foxp3 expression correlates closely with the suppressive capacity of γδ T cells. Moreover, as discussed by Wesch’s group, Foxp3 expression in suppressive human peripheral blood-derived Vδ2⁺ T cells cannot be detected with the Treg-specific 259D mAb [22] but can be identified with the PCH101 mAb that does not correlate with suppressive function [25, 26]. Clarity on this issue could be derived from methylation studies of the gene [27]. Taken together, it is still controversial as to whether Foxp3 expression is critical, or whether PAg stimulation is sufficient or additional cytokines are necessary for Vδ2⁺ T cells to exhibit cell-contact dependent suppression.

In the thymus, differences in signal strength dictate αβ versus γδ lineage choice through modulation of lineage specific transcription factors, while other signaling pathways that integrate with TCR signaling impact the resulting lineage outcome through altering activity of key proteins [28]. In light of this, it seemed likely that in the periphery, graded signals downstream of the TCR may result in differential functional maturation of T cell effector subpopulations while, at the same time, environmental cues such as cytokines might further modulate TCR signaling strength and effector function. The purpose of the present study therefore was to elucidate the role of the TCR in the acquisition of suppressive properties of peripheral human Vδ2⁺ T cells on autologous αβ T cells, specifically to address whether and how graded TCR stimulation and or cytokines control regulatory activities of Vδ2⁺ T cells. We examined the effect of proliferation inhibition and apoptosis induction mediated by negatively or positively freshly isolated Vδ2⁺ T cells obtained from healthy donors in comparison with those stimulated with IL-12/IL-18 (TCR bypass) + IL-15 and those after prolonged exposure to IPP with or without Th1 or Th2 cytokines. In addition, we tested the suppressive activity of Vδ2⁺ T cells in the presence or absence of a PD-1 blocking antibody. Next, to determine whether physiologic stimuli, such as the direct contact with tumor cells, affect the suppressive activity of Vδ2⁺ T cells, we exposed Vδ2⁺ T cells to a glioblastoma cell line (U251) or a melanoma cell line (SK-Mel-28) and subsequently examined these Vδ2⁺ T cells in mixed lymphocyte cultures (MLC) with anti-CD3/anti-CD28 stimulated autologous αβ T cells. Finally, we investigated the suppressive activity of Vδ2⁺ T cells in the presence or absence of CD28 stimulation. By employing Vδ2⁺ T cells that had experienced a range of stimuli, from none at all to supraphysiological levels, we identified conditions under which Vδ2⁺ T cells exerted suppressive effects on autologous αβ T cells.

Tumor-infiltrating γδ T cells have been considered the most favorable prognostic marker in many types of cancers [55]. While the adoptive transfer of ex vivo-expanded Vδ2⁺ T cells and even the adoptive transfer of allogeneic γδ T cells shows objective effects, complete remissions still remain anecdotical and the necessary repetitive in vivo activation shows limited responses [56]. The present study, therefore, examined γδ T cells’ suppressive behavior as one potential mechanism preventing Vδ2⁺ T cells from exerting long-lasting antitumor effects in vivo. Current in vitro and in vivo strategies employ pharmacological drugs targeting the Vδ2⁺ TCR for the induction of activation, proliferation, and tumor-lytic function of γδ T cells. By taking a closer look at the role of the TCR in this process, we demonstrate that the suppressive activity of Vδ2⁺ T cells on autologous αβ T cells correlates with the strength of γδ TCR signal and that the functional differentiation of γδ T cells into a suppressive phenotype is fine-tuned by the integration of signals arising from other environmental cues, such as cytokines. More specifically, negatively freshly isolated Vδ2⁺ T cells, TCR-bypass cytokine-stimulated negatively isolated Vδ2⁺ T cells, TCR-crosslinked freshly isolated Vδ2⁺ T cells, and IPP-stimulated TCR-crosslinked Vδ2⁺ T cells exhibit increasing inhibitory activities on αβ T cell proliferation, from the weakest to the strongest stimulation setting in this order. In contrast, direct contact with BTN3A1-expressing cells in vitro alone is not a sufficiently strong γδ TCR-stimulation to bestow suppressive activity on Vδ2⁺ T cells.

We examined peripheral Vδ2⁺ T cells with four different magnitudes of TCR stimulation. First, we showed that negatively freshly isolated Vδ2⁺ T cells (no stimulation) do not possess suppressive activity in accordance with Traxlmayr and colleagues’ findings [24]. While these authors used anti-CD4, CD16, TCRαβ cocktail antibodies for depleting unwanted cells, a custom-made kit for the isolation of untouched γδ T cells (omitting anti-CD16 antibody from the commercially available cocktail of antibodies) was used in the present study to avoid losing CD16⁺ Vδ2⁺ T cells because this fraction consists of 10–40% of peripheral Vδ2⁺ T cells in healthy donors [57]. In addition, we depleted Vδ1⁺ T cells to specifically analyze Vδ2⁺ T cells. Therefore, we believe that we studied the most purified (> 99%) untouched Vδ2⁺ T cells. Next, we found that TCR-bypass stimulation (TCR-independent cytokine-mediated stimulation) does not confer significant immunosuppressive function to negatively freshly isolated Vδ2⁺ T cells. To our knowledge, this is the first study to test the impact of TCR-bypass stimulation on the suppressive effect of truly untouched Vδ2⁺ T cells. Third, our study demonstrates that positively freshly isolated Vδ2⁺ T cells (a single γδ TCR stimulation) potently suppress the proliferation of co-cultured autologous αβ T cells that have been stimulated with anti-CD3/anti-CD28 mAb in whole PBMCs. Casetti et al. [21] concluded that positively isolated Vδ2⁺ T cells do not suppress the proliferation of anti-CD3/anti-CD28 mAb-stimulated PBMCs, while Peters et al. and Kuhl et al. showed that positively freshly isolated pan γδ T cells suppress the proliferation of CD4⁺ T cells in response to anti-CD3/anti-CD28 mAb stimulation [22, 23]. These conflicting results might be explained by the fact that Casetti et al. used not fresh PBMCs, but buffy coats and bead-sorted γδ T cells that had been frozen for use at later time points. Of utmost importance, however, is our new finding that crosslinking the γδ TCR merely once leads to a remarkable suppressive function of Vδ2⁺ T cells. Finally, we reveal that IPP-stimulated Vδ2⁺ T cells (strongest and continuous γδ TCR stimulation) exhibit the most robust suppressive activity. Intriguingly, untouched Vδ2⁺ T cells do not suppress autologous αβ T cells even after exposure to BTN3A1⁺ tumor cells. As tumor-infiltrating γδ T cells are thought to be the most favorable prognostic marker in many types of cancers [55], this finding suggests that tumor cells do not license suppressive functions of γδ T cells. On the other hand, in vivo or ex vivo stimulation of Vδ2⁺ T cells via pharmacologic drugs for activation and expansion may dampen the anti-tumor reactivity of αβ T cells indirectly by reducing their number, which points to a thus far unappreciated limitation to adoptive Vδ2⁺ T cell therapy for cancer patients. However, since T cells with high capacity of proliferation and those with high potential of cytokine-production or anti-tumor cytolytic function are different populations, we cannot conclude whether γδ T cells dampen αβ T cell responses per se. They may actually enhance the cytolytic potential of suppressed αβ T cells, which we did not examine in this study.

The impact of cytokines on γδ T cells, especially on the suppressive function of γδ T cells, is largely unknown. In this study, we show that IL-12 in the presence of a strong TCR signal decreases the inhibitory effect on αβ T cell proliferation by Vδ2⁺ T cells yet very slightly increases apoptosis induction by Vδ2⁺ T cells (Fig. 1a and Supplementary Figure S1). An interesting study by Traxlmayr et al. [24] showed that IL-12 enhanced the suppressive activity of γδ T cells against memory T cells, stimulated by recall antigens, but not against naïve T cells, responding to neoantigens. Combined together, the modulation of the features of Vδ2⁺ T cells by IL-12 is still controversial and warrants further investigation. In addition, as shown in Fig. 1a and Supplementary Figure S1, MLC data suggest that TGF-β alleviates the suppressive activity of IPP-stimulated Vδ2⁺ T cells both in proliferation inhibition and apoptosis induction. From the literature we know that TGF-β suppresses the effector function of γδ T cells [58‐60] and inhibits IL-12-induced proliferation and activation of T cells [61]; therefore, it is likely that IL-12 and TGF-β also counter-regulate and cancel out the TCR-mediated activation status of Vδ2⁺ T cells in the current study. Moreover, we show that IL-15 enhances the suppressive activity of IPP-stimulated Vδ2⁺ T cells both via inhibiting proliferation and inducing apoptosis. This finding is in accordance with a previous report demonstrating that the addition of IL-15 to γδ T cell cultures results in a more activated phenotype, a higher proliferative capacity, and increased cytotoxicity of γδ T cells [38]. Most noteworthy, however, is that IL-15 signaling involves Lck and MAPK in its downstream cascade [40], mimics T cell receptor crosslinking in the induction of gene expression, and shares overall downstream events with anti-CD3 stimulation in memory T cells, thus is fortifying TCR signaling [62].

Immunotherapeutic strategies that aim to activate and expand Vδ2⁺ T cells in vitro or in vivo stimulate Vδ2⁺ γδ T cells via their TCR to maximize their cytotoxic properties, dependent on cytotoxic mediators. Due to the observed dichotomy in Vδ2⁺ T cells, i.e., that a TCR signal induces both anti-tumoral as well as a suppressive phenotype, we investigated the role of apoptosis induction in the regulatory function of Vδ2⁺ T cells. Consistent with results shown by Peters et al., we found the contribution of apoptosis marginal compared to that of proliferation inhibition (Fig. 1a and Supplementary Figure S1). Peters et al. examined the relevance of Fas/FasL and TRAIL/TRAILR in suppressive assays simultaneously by using blocking antibodies for these apoptosis cascades and did not detect any effect on overall suppressive activity [22].

Thus, although we and others have identified an array of conditions that create a suppressive phenotype in Vδ2⁺ γδ T cells, the molecules involved still remain largely unknown. While extensive research has convincingly established the key contribution of costimulation on αβ T cell functions, the functional relevance of co-stimulatory/co-inhibitory molecules on γδ T cells in the context of suppressive activity as well as a role for CD28 in the acquisition of a suppressive phenotype of Vδ2⁺ γδ T cells remains to be defined. CD28 is expressed by 40–60% of freshly isolated human peripheral γδ T cells, but downregulated after TCR signaling so that very few (< 10%) activated γδ T cells express CD28 [63‐65]. CTLA-4 is expressed only marginally before and after PAg-stimulation on the cell surface [63]. Our data confirm the downregulation of CD28 in Vδ2⁺ T cells after TCR signaling, i.e., in this study after IPP/IL-15-stimulation, and also the only marginal expression of CTLA-4 before and after IPP/IL-15-stimulation (Supplementary Figure S9). In contrast, PD-1 and PD-L1 expression were significantly upregulated by the IPP-containing regimen. By using blocking antibodies, Peters et al. showed that CD28-CTLA-4/CD80-CD86 and PD-1-PD-L1 interactions are involved in the suppressive activity of Vδ2⁺ γδ T cells. Our experiments clearly confirm a role for the PD-1/PD-L1 axis. This finding also supports the notion that the TCR is decisive in inducing regulatory function in γδ T cells, as PD-L1 is positively regulated by TCR stimulation. We also demonstrate that CD28 stimulation on Vδ2⁺ T cells is not essential for Vδ2⁺ T cells to acquire suppressive function on autologous αβ T cells. However, since PD-1/PD-L1 interaction explains only about 50% of suppressive function of Vδ2⁺ T cells, CD28 stimulation on αβ T cells by suppressive Vδ2⁺ T cells is of relevance and needs to be elucidated as well as the role of other inhibitory receptors such as Tim-3, Lag-3 or TIGIT.

Similar to what is shown by Peters et al. [22], no correlation between Foxp3 expression and suppressive activity of Vδ2⁺ T cells was observed in the present study. On the contrary, the population with the least suppressive activity among PAg-stimulated Vδ2⁺ T cells (IPP/IL-15/IL-12 stimulation) had the highest Foxp3 expression and vice versa (IPP/IL-15 stimulation). This suggests that Foxp3 is a transient activation marker rather than a regulatory marker analogous to Foxp3 expression in activated human CD4 αβ T helper cells [66, 67]. Instead, as mentioned above we detected a significant correlation between PD-L1 protein expression and the suppressive activity of IPP-stimulated Vδ2⁺ T cells. This supports the finding that the interaction between PD-1 on αβ T cells and its ligand PD-L1 on γδ T cells restrains αβ T cell activation [19]. Interestingly, we demonstrate that PD-L1 expression is induced on γδ T cells by TCR-bypass stimulation as well as by PAgs [68] or by co-culture with αβ T cells as reported [22], but it is not significantly induced by a single strong TCR signal via TCR crosslinking (Fig. 2a bottom). Consequently, although our finding suggests that PD-L1 expression on Vδ2⁺ T cells can be a marker to predict their suppressive activity and respective blocking antibodies massively reduced inhibitory function, this prediction is not applicable to TCR-bypass-stimulated Vδ2⁺ T cells, which express high PD-L1 but exhibit no suppression, or positively freshly isolated Vδ2⁺ T cells, which have only marginal expression of PD-L1 but possess high suppressive activity. On the other hand, it is already known that activated γδ T cells express CD80/86 [49]. Although IPP-stimulated γδ T cells express CD80/86, which supports the priming and proliferation of αβ T cells, they also express PD-L1, the marker that can suppress αβ T cell proliferation. This, as well as inconsistency between PD-L1 expression and suppressive function, points to a potential regulatory mechanism hierarchically upstream of these molecules. Whether these regulatory mechanism(s) are on the effector cell or the target cell remains to be shown.

The finding that BTN3A1-exposed Vδ2⁺ T cells, like untouched and TCR-bypass-stimulated Vδ2⁺ T cells, did not exhibit any suppressive behavior, although BTN3A1 is suggested to be the natural Vδ2 TCR ligand, illuminates our incomplete understanding of what truly activates γδ T cells and those factors that contribute to Vδ2⁺ TCR signaling and thus (suppressive) effector differentiation in vivo. If assuming suppression resulting from crosslinking the TCR is 100%, IL-15 was able to significantly increase suppressive behavior further by 35% while other cytokines, such as TGF-β and IL-12, down-modulated inhibitory activity distinctively. This denotes a decisive role of environmental cues in (suppressive) effector differentiation of γδ T cells. The excellent performance of Vδ2⁺ T cells in terms of viability throughout the experimental procedures and the consistently distinct modulation of the TCR signal by cytokine stimulation strongly suggest that these TCR signals represent a physiological range of activation and function. Until we have identified the physiological (co-)ligand(s) and factors that contribute to Vδ2⁺ T-cell activation, we are unable to delineate what specifically promotes suppressive γδ T cells in vivo.

In summary, here we show for the first time that the suppressive activity of Vδ2⁺ T cells on αβ T cells is dependent on a TCR signal and correlates with its strength (Fig. 6). Accordingly, suppression correlates with and is dependent on a molecule that is also regulated via TCR stimulation: PD-L1. In the presence of a strong Vδ2⁺ TCR signal, functional maturation into a suppressive phenotype can be positively or negatively modulated by microenvironmental cues such as cytokines. Since direct contact with BTN3A1-expressing tumor cells alone does not bestow suppressive activity onto Vδ2⁺ T cells, further studies are needed to comprehensively identify what exactly activates Vδ2⁺ T cells and how activation translates into various specific effector functions. Thus, we first need to identify and understand its complete functional spectrum before we can utilize the full potential of the Vδ2⁺ effector T cell population in clinical settings.