Checkpoint Blockade Rescues the Repressive Effect of Histone Deacetylases Inhibitors on γδ T Cell Function

Histone deacetylases (HDAC) are one of the key epigenetic modifiers that control chromatin accessibility and gene expression. Their role in tumorigenesis is well established and HDAC inhibitors have emerged as an effective treatment modality. HDAC inhibitors have been investigated for their specific antitumor activities and also clinically evaluated in treatment of various malignancies. In the present study, we have investigated the effect of HDAC inhibitors on the effector functions of human γδ T cells. HDAC inhibitors inhibit the antigen-specific proliferative response of γδ T cells and cell cycle progression. In antigen-activated γδ T cells, the expression of transcription factors (Eomes and Tbet) and effector molecules (perforin and granzyme B) were decreased upon treatment with HDAC inhibitors. Treatment with HDAC inhibitors attenuated the antitumor cytotoxic potential of γδ T cells, which correlated with the enhanced expression of immune checkpoints programmed death-1 (PD-1) and programmed death ligand-1 in γδ T cells. Interestingly, PD-1 blockade improves the antitumor effector functions of HDAC inhibitor-treated γδ T cells, which is reflected in the increased expression of Granzyme B and Lamp-1. This study provides a rationale for designing HDAC inhibitor and immune check point blockade as a combinatorial treatment modality for cancer.

Histone deacetylases (HDAC) are one of the key epigenetic modifiers that control chromatin accessibility and gene expression. Their role in tumorigenesis is well established and HDAC inhibitors have emerged as an effective treatment modality. HDAC inhibitors have been investigated for their specific antitumor activities and also clinically evaluated in treatment of various malignancies. In the present study, we have investigated the effect of HDAC inhibitors on the effector functions of human γδ T cells. HDAC inhibitors inhibit the antigen-specific proliferative response of γδ T cells and cell cycle progression. In antigen-activated γδ T cells, the expression of transcription factors (Eomes and Tbet) and effector molecules (perforin and granzyme B) were decreased upon treatment with HDAC inhibitors. Treatment with HDAC inhibitors attenuated the antitumor cytotoxic potential of γδ T cells, which correlated with the enhanced expression of immune checkpoints programmed death-1 (PD-1) and programmed death ligand-1 in γδ T cells. Interestingly, PD-1 blockade improves the antitumor effector functions of HDAC inhibitortreated γδ T cells, which is reflected in the increased expression of Granzyme B and Lamp-1. This study provides a rationale for designing HDAC inhibitor and immune check point blockade as a combinatorial treatment modality for cancer.
Keywords: gamma delta (γδ) T cells, phosphoantigen, histone deacetylases inhibitors, effector functions, programmed death-1, programmed death ligand-1 inTrODUcTiOn Gamma delta T cells, the enigmatic brethren of alpha beta (αβ) T cells were discovered coincidently during cloning the αβ T-cell receptor (TCR) locus (1). This small subset of T cells, γδ T cells constitute about 5-10% of the circulating T cell population, which express the variant form of TCR heterodimer (2). γδ T cells manifest the features of both innate and adaptive immunity (3). TheVγ9Vδ2 T cell subset of γδ T cells predominates in peripheral blood, and these cells play an important role in the defense against microbial pathogens, stressed cells, and tumor cells of various origin (4,5). γδ T cells differ from αβ T cells by their TCR gene usage, tissue tropism, and MHC-independent antigen recognition (6,7). γδ T-cells display broad functional plasticity, like regulatory potential, antigen-presenting capacity, B-cell helper activity, and have the potential for diverse cytokine production (8). γδ T cells recognize nonpeptide phosphoantigens such as isopentenyl pyrophosphate (IPP) or 4-hydroxy-3-methylbut-2-eneyl pyrophosphate (HMBPP), which are produced through the mevalonate pathway in mammalian cells or non-mevalonate/Rohmer pathway in non-mammalian cells, respectively (9). γδ T cells are also activated indirectly by aminobisphosphonates such as Zoledronate. Aminobisphosphonates inhibit the key enzyme of mevalonate pathway, farnesyl pyrophosphate synthase and lead to accumulation of IPP. Tumor cells treated with aminobisphophonates show increase in the intracellular level of IPP and, therefore, are easily targeted by γδ Tcells (10,11).
Activated γδ T cells are known to produce large amounts of the pro-inflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) as well as the chemokines MIP-1 (macrophage inflammatory protein) and RANTES (regulated on activation, normal T cell expressed, and secreted) (12). In addition, cytolytic mediators such as granzyme B and perforin are produced to induce specific lysis of cells with elevated phosphoantigen levels (13). Transcription factors like Eomes and Tbet are known to be expressed upon activation by γδ T cells and are essential for antitumor effector functions (14).
Nucleosome is the basic structure of eukaryotic chromatin, composed of histones and DNA. Each nucleosome comprises 146 bp of DNA wrapped around an octamer of core histones (two H2A-H2B dimers and a H3-H4 tetramer) (15). Histone proteins are rich in basic amino acids lysine and arginine. It is through interaction with these histone proteins that massive DNA is packed inside the nucleus. The tails of histone proteins undergo different complex and coordinated posttranslational modifications like histone acetylation, methylation, phosphorylation, and ubiquitination. According to histone code hypothesis, these modifications are read by specific factors, which ultimately lead to downstream events (16). Histone modifications are reversible in nature and influence many fundamental biological processes. Histone acetylation are directed by histone modifying enzymes, histone acetyl transferases (HAT), and histone deacetylases (HDAC), which participate in potential cross-talk between different modifications (15). Normal physiological functions require a balance between HAT and HDAC. Abrupt alterations that skew this balance can give rise to different pathophysiological conditions like cancer (17,18).
Histone deacetylases inhibitors, including Trichostatin-A (TSA) and sodium valproate (VPA), can alter the acetylation of histones in chromatin and enhance gene transcription. In the recent decades HDAC inhibitors have received attention as antineoplastic treatment. Extensive evidence suggests that HDAC inhibitors play a role in antitumor immunity (19). HDAC inhibitors lead to growth arrest, induction of apoptosis, and differentiation in tumors. Pan HDAC inhibitors like VPA, TSA, and suberoylanilidehydroxamic acid (SAHA) target Class I (HDAC 1, 2, 3, and 8), Class II (HDAC 4, 5, 7, 9, 6, and 10) HDACs. Hence, their anticancer activities are pleotropic in nature, mediated by altering the expression of various genes that are regulated by class I and II HDACs. Additionally, they also target several non-histone proteins such as transcription factors (p53, E2f1) and cytoplasmic proteins (tubulin, hsp, β-catenin). Hyperacetylation of these histone and non-histone proteins brought about by HDAC inhibiton culminate in induction of cell-death pathways in cancer cells. Several studies have established effective tumor reduction in vitro as well as in vivo upon HDAC inhibitor treatment (20).
Moreover, HDAC inhibitors inhibit angiogenesis and increase the tumor cell antigenicity (21,22). HDAC inhibitors mediate elevated expression of antigens on tumor cells so that they can be easily targeted by immune cells (23,24). Due to these promising antitumor functions, HDAC inhibitors are now assessed in clinical trials and some of them have been approved for treatment (25,26). Recent reports have demonstrated that HDAC inhibitors enhance response to immune checkpoint blockade in triple negative breast cancer, lung adenocarcinoma, melanoma, and multiple myeloma (27)(28)(29)(30).
Although the impact of HDAC inhibitors on tumor cells is well studied, their effect on immune cells has recently surfaced. HDAC inhibitors have been shown to have a dual effect on immune cells, either enhancing their activation in cases of CD4 T cell and Tregs whereas dampening the effector functions of NK cells and CD8 T cells. HDAC inhibitors are also known to inhibit the cytotoxic potential of NK cells. HDAC inhibitors are also reported to downregulate the co-stimulatory molecules and cytokine signals in antigen-presenting cells (31). Previous studies have shown that HDAC inhibitor treated tumor cells are easily targeted by γδ T cells (32), but the impact of HDAC inhibitors on the functional responses of human γδ T cells are not well understood.
For successful immunotherapy, T cell responses are essential. Besides the TCR signal, co-stimulatory signal also determines the functional response of T cells. Co-stimulatory signal may be of positive or negative. Negative co stimulatory signals may be from different receptors like programmed death-1 (PD-1) and PD ligand-1 (PD-L1) interaction. PD-1 and PD-L1 are the members of immunoglobin family like that of CD28. Interaction of PD-1 and PD-L1 leads to functional impairment in T cells (33). It is well-known fact that tumors use this mechanism to escape the immune attack. Blocking antibodies for these immune check points can enhance antitumor responses, and these immune-modulating antibodies have achieved clinical success with FDA approved treatments for several malignancies (34). It has been shown that γδ T cells express PD-1 and PD-L1 and blocking of this signaling lead to increase in the antitumor potential of γδ T cells (35).
The present study focuses on investigating the direct impact of HDAC inhibitors on human γδ T cells. We have studied the effect of three different HDAC inhibitors, TSA, SAHA, and VPA on γδ T cells. We observed that HDAC inhibitors suppress the antigen-specific proliferative responses of γδ T cells and their antitumor effector functions by increasing the expression of immune checkpoints (PD-1 and PD-L1). The study further demonstrates that blocking of immune checkpoints on γδ T cells is capable of augmenting their antitumor cytotoxic potential. The present study will open new avenues in the field of cancer immunotherapy using HDAC inhibitors.

γδ T cell separation
Heparinized peripheral blood was collected from healthy individuals. Peripheral blood mononuclear cells (PBMCs) were isolated by differential density gradient centrifugation using Ficoll Hypaque (Sigma-Aldrich, St. Louis, MO, USA). The study was approved by the Institutional Ethics Committee (TMC-IECIII Project no. 166) and written informed consent was obtained from the donors prior to collection of blood samples. The experimental conditions and procedures for handling blood samples were performed as per the biosafety guidelines of the Institute Biosafety Committee. In short, blood samples were handled in biosafety cabinets and personnel handling blood samples were vaccinated against Hepatitis B. γδ T cells were purified from PBMCs using immunomagnetic MicroBeads (Miltenyi Biotech, Bergish Gladbach, Germany) by positive selection, as per manufacturer's instructions. The purity of separated γδ T cells was >95% as confirmed by flow cytometry (FACS Aria, BD Biosciences, USA). Isolated γδ T cells were cultured in RPMI 1640 supplemented with 10% heat inactivated AB serum, 2 mM glutamine, and antibiotics.

cell Viability assay
The viability of γδ T cells upon treatment with HDAC inhibitors was evaluated with MTT assay and Annexin V and 7-AAD staining. Briefly, 0.1 × 10 6 γδ T cells, seeded in 96-well flat bottom plates (Nunc), were treated with the following HDAC inhibitors for the given concentration range: VPA (4-0.25 mM; Sigma-Aldrich), TSA (200-25 nM; Sigma-Aldrich), and SAHA (4-0.25 µM; Sigma-Aldrich) along with HDMAPP (1 nM; Echelon) and rIL-2 (50 IU/ml; Peprotech) for 72 h. γδ T cells treated only with HDMAPP (1 nM) and rIL-2 (50 IU/ml) were used as control. DMSO was used as vehicle control. Following 72 h of treatment, MTT (5 mg/ml) was added and incubated for 4 h at 37°C. After incubation, the spent medium was discarded, the formazan crystals were dissolved in DMSO, and the absorbance was measured at 570 nm by microplate reader (TECAN, Switzerland). Untreated γδ T cells were used as reference for calculating the viability. Concentrations of HDAC inhibitors, which had no impact on viability of γδ T cells were further validated by Annexin V and 7-AAD staining. The concentration of HDAC inhibitors showing viability more or equal to 90% in γδ T cells were used for all the further experimental procedures.
For cell cycle analysis, 1 × 10 6 γδ T cells treated with HDAC inhibitors VPA (0.5, 1, 2 mM), TSA (25, 50, 100 nM), and SAHA (0.25, 0.5, 1 µM) for 72 h or kept untreated, were harvested, and fixed by adding chilled 70% ethanol. Next day, cells were washed with PBS and stained with DNA intercalating dye propidium iodide (PI) along with RNAse A at a concentration of 40 and 10 µg/ml, respectively. Cells were incubated at room temperature for 30 min. The samples were acquired on FACS Calibur (BD Biosciences, USA) and analyzed using ModFit software.

Proliferation assay
Proliferation of γδ T cells was analyzed using 3 H-Thymidine (3HTdR) incorporation assay. A total of 5 × 10 4 γδ T cells were treated in the presence or absence of HDAC inhibitors VPA (0.5, 1, 2 mM), TSA (25, 50, 100 nM), and SAHA (0.25, 0.5, 1 μM) along with HDMAPP (1 nM) and rIL2 (50 IU/ml) for 72 h in 96-well tissue culture plates. The cultures were pulsed with 1 μCi [ 3 H] thymidine (Board of Radiation and Isotope Technology, Mumbai) 18 h prior to termination of the assay. Following the incubation, cells were transferred upon glass-wool filters using cell harvester (Perkin Elmer, UK). The radioactivity incorporated into the DNA was measured in a liquid beta scintillation counter (Packard, Meriden, CT, USA). Data were expressed as counts per minute (cpm).

cytotoxicity assay
Cytotoxic potential of γδ T cells against panel of tumor cell lines, oral tumor cell line (AW13516), colon tumor cell line (COLO-205), and B lymphoblastic cell line (Raji) was performed using lactate dehydrogenase (LDH) release assay as described previously (37). Tumor cell lines were treated for 18 h with zoledronate (100 µM; Sigma-Aldrich). γδ T cells were treated with HDMAPP (1 nM) and rIL-2 (50 IU/ml) in presence and absence of HDAC inhibitors, VPA (2 mM), TSA (100 nM), and SAHA (1 µM) for 72 h at 37°C were used as effectors. Additionally, for PD-1 blockade, anti-PD1 antibody (3 µg/ml) was added to HDAC inhibitor treated γδ T cells for 72 h at 37°C and were also used as effectors. Briefly, tumor cell lines were cocultured with effectors at 40:1 effector target (E/T) ratio for 4 h at 37°C in 96-well plates (Nunc, Denmark). After 4 h of coculture, an aliquot of 50 µl of media was used in LDH cytotoxic assay using the LDH cytotoxic assay kit (Thermo Fisher Scientific, USA) according to manufactures protocol. γδ T cell cytotoxicity was defined as % specific lysis = Experimental value -Effector cells spontaneous control − Target cells spontaneous control/Target Cell Maximum Control − Target cells spontaneous control.

statistical analysis
Data analysis was done by Student's t-test using GraphPad Prism software (GraphPad Software Inc., CA, USA). The comparative CT method was applied in the quantitative real time RT-PCR according to 2 −(ΔΔCt) method. Results were indicated as means ± SE and considered significant at p < 0.05. hDac inhibitors inhibit the antigen-Driven Proliferation and cell cycle Progression of γδ T cells γδ T cell show robust proliferation when stimulated with phosphoantigen (HDMAPP) in presence of rIL2. In order to investigate the effect of HDAC inhibitors on proliferation of γδ T cells, γδ T cells were stimulated with phosphoantigen HDMAPP and rIL2 in the presence or absence of different concentration of HDAC inhibitors (VPA; 0.5-2 mM, TSA; 25-100 nM, and SAHA; 0.25-1 μM) and proliferation was monitored using 3 H thymidine incorporation assay. γδ T cells showed robust proliferative responses to phosphoantigen HDMAPP in presence of rIL-2, compared to unstimulated γδ T cells. However, in the presence of various concentrations of VPA, TSA, and SAHA, the proliferative responses of γδ T cells were significantly reduced in a concentration-dependent manner ( Figure  S3A in Supplementary Material), with maximum decrease in proliferation of γδ T cells observed at higher concentration of HDAC inhibitors, VPA 2 mM, TSA 100 nM, and SAHA 1 µM, respectively. Further, we also evaluated the role of HDAC inhibitors on cell cycle progression of γδ T cells. Freshly isolated γδ T cells were stimulated with HDMAPP and rIL2 in presence or absence of different concentrations of HDAC inhibitors. Upon stimulation with HDMAPP and rIL2, significant number of γδ T cells were in S phase and G2/M phase. However, upon treatment of HDAC inhibitors, γδ T cells were arrested in G0/ G1 phase (Figures S3B,C in Supplementary Material). This inhibition of cell cycle progression in γδ T cells upon HDAC inhibitor treatment was reflected in the increased expression of p53 and its downstream target p21, suggesting that HDAC inhibitors impede the G0/G1-S phase transition in γδ T cells in p53-dependent manner (Figures 1A-C) and (Figures 1D-I).
hDac inhibitors regulate cytokine Production and activation in γδ T cells γδ T cells upon activation secrete copious amount of cytokines such as IFN-γ and TNF-α (36). We examined the effect of HDAC inhibitors on expression of these cytokines in γδ T cells. Marked increase in the expression of cytokines IFN-γ and TNF-α was observed upon stimulation of γδ T cells with HDMAPP and rIL2 compared to unstimulated γδ T cells. Expression of IFN-γ (Figures 2A,B) and TNF-α (Figures 2C,D) was decreased significantly when treated with HDAC inhibitors TSA, SAHA, and VPA. This inhibition was observed both at mRNA and protein levels. It was observed that inhibition of cytokine expression was concentration dependent for HDAC inhibitors. We also evaluated the effect of HDAC inhibitors on the expression of early activation marker CD69 and late activation marker CD25 on γδ T cells. Treatment of γδ T cells with HDAC inhibitors led to decrease in the expression of early activation (Figures 3A,B) and late activation marker on γδ T cells (Figures 3C,D). The expression of these activation markers on γδ T cells were significantly reduced in a concentration-dependent manner, with maximum decrease at VPA 2 mM, TSA 100 nM, and SAHA 1 µM, respectively. Percentage of γδ T cells positive for these markers was also less in HDAC inhibitor treated γδ T cells as compared to untreated γδ T cells. To investigate the role of HDAC inhibitors on the expression of other activating receptors like NKG2D, CD16, and inhibitory receptors like KIR2DL2/3 (CD158b), γδ T cells were treated with HDAC inhibitors VPA 2 mM, TSA 100 nM, and SAHA 1 µM for 72 h. We found that HDAC inhibitor-treated γδ T cells show decreased expression of NKG2D ( Figure S4A in Supplementary Material) as compared to untreated γδ T cells. On the contrary, γδ T cells treated with HDAC inhibitors VPA 2 mM, TSA 100 nM, and SAHA 1 µM show increase in the expression of inhibitory receptor KIR2DL2/3 (CD158b) ( Figure S4B in Supplementary Material). However, we did not observe any change in CD16 expression ( Figure S4C in Supplementary Material). Collectively, the data advocate the role of HDAC inhibitors in abating the expression of activation markers (CD69, CD25, NKG2D) and cytokine (IFN-γ, TNF-α) production in γδ T cells.

hDac inhibitors suppress the expression of Transcription Factors eomes and Tbet in γδ T cells
Eomes and Tbet are two main T-box transcription factors expressed in T cells. They are the main transcription factors, which regulate the effector functions of CD8 T cells through the expression of effector genes perforin and granzyme B (39,40). Besides CD8 T cells, γδ T cells also express Eomes and Tbet (41). Upon activation with phosphoantigen (HDMAPP) and rIL2, γδ T cells show increased expression of these two transcription factors. We hypothesized that HDAC inhibitors may have an impact on the expression of these two transcription factors in γδ T cells. Therefore, the role of HDAC inhibitors was analyzed in regulating expression of Eomes and Tbet in γδ T cells activated with phosphoantigen (HDMAPP) and rIL2. γδ T cells treated with HDAC inhibitors showed decrease in the expression of Eomes and Tbet at both mRNA (Figures 4A,B) and protein level (Figure 4C). Cell lysates of γδ T cells, stimulated with HDMAPP after treatment with HDAC inhibitors at different concentrations for 72 h were prepared and p53, p21 proteins were detected. β-actin was used as loading control. Densitometry quantification of p53 (D-F) and p21 (g-i) expression in γδ T cells upon treatment with VPA, TSA, and SAHA, relative to β-actin. hDac inhibitors inhibit the antitumor cytotoxic Potential of γδ T cells To evaluate the role of HDAC inhibitors in modulation of antitumor potential of γδ T cells, we analyzed the expression of effector molecules Perforin and Granzyme B in γδ T cells at mRNA and protein level. Perforin and Granzyme B are the effector molecules, which are responsible for the antitumor functions of CD8 and γδ T cells (42,43). Freshly isolated γδ T cells activated with phosphoantigen HDMAPP and rIL2 show increased expression of these two effector genes; however, γδ T cells activated in presence of HDAC inhibitors showed decrease in the expression of perforin (Figures 5A-C) and granzyme B. (Figures 5D-F).
Maximum effect on the expression of perforin and granzyme B was observed with VPA 2 mM, TSA 100 nM, and SAHA 1 µM. These concentrations of HDAC inhibitors were used in further cytotoxicity experiments. We next evaluated whether decrease in expression of effector molecules perforin and granzyme B are regulated by histone modifications in γδ T cells. To investigate this, we checked the total histone H3 and H4 acetylation in γδ T cells treated with HDAC inhibitors VPA 2 mM, TSA 100 nM, and SAHA 1 µM. We observed that the total level of H3 acetylation and H4 acetylation increases in γδ T cells after treatment of Zoledronate, an aminobisphonate drug, inhibits the enzyme farnesyl pyrophosphate synthase in the mevalonate pathway leading to accumulation of IPP, which stimulates γδ T cell activation via TCR signaling. γδ T cells treated with different HDAC inhibitors showed significant decrease in their cytotoxic potential against zoledronate treated tumor targets (AW13516, COLO 205, and Raji) (Figures 6D-F). It was observed that all the three HDAC inhibitors (VPA, TSA, and SAHA) significantly inhibited the ability of γδ T cells to kill zoledronate treated tumor cell lines.  Figures 7A,B) and PD-L1 (Figures 7C,D) on HDMAPP and rIL-2 activated γδ T cells was observed after treatment with VPA (2 mM), TSA (100 nm), and SAHA (1 µM).

hDac inhibitors abrogate the effector Functions of γδ T cells by Upregulating the immune checkpoint Proteins PD-1 and PD-l1
To assess the role of PD1/PD-L1 signaling in HDAC inhibitor treated γδ T cells, γδ T cells were activated with HDMAPP and rIL2, treated or untreated with HDAC inhibitors for 72 h. PD-1 blocking antibody was added at the start of culture. After 72 h, these γδ T cells were cultured with zoledronate-treated tumor cell lines AW13516, COLO-205, and Raji for 4 h at E/T ratio of 4:1.
Blockade of PD-1 in HDAC inhibitor treated HDMAPP activated γδ T cells rescued the expression of effector molecules Lamp-1 (CD107a) ( Figure 8A) and granzyme B ( Figure 8B) as compared to only HDAC inhibitor treated γδ T cells. To further evaluate the role of HDAC inhibitors on the PD1/PD-L1 signaling axis in γδ T cells, we did the similar experiment by coculturing the effectors and above mentioned tumor targets to analyze the cytotoxic potential by LDH release assay at a ratio of 40:1 for 4 h. Blocking of PD-1 in HDMAPP-activated γδ T cells treated with HDAC inhibitorsVPA (2 mM), TSA (100 nM), and SAHA (1 µM) improves the cytolytic potential of γδ T cells as compared to γδ T cells treated with HDAC inhibitor only ( Figure 8C). Thus, the results shows that blockade of PD-1 and PD-L1 signaling in HDAC inhibitor treated γδ T cells rescue their effector functions.

DiscUssiOn
γδ T cell immunotherapy has become the emerging lead in the landscape of cancer immunotherapies due to their distinctive immune features and potent antitumor effector functions. They have been extensively targeted against diverse tumors such as melanoma, renal cell carcinoma, as well as B cell malignancies and have shown promising results in clinical settings (44). While these therapies have encountered modest clinical success, they have to overcome certain challenges such as limited availability of γδ T cells and rapid exhaustion upon repeated in vitro activation. Hence, combinational approaches have been envisaged with chemotherapeutics, monoclonal antibodies, small molecule inhibitors, etc. Newer treatment modality may include combining γδ T cell immunotherapy with antitumor drugs and other immune-modulating antibodies. Epigenetic dysregulation is one of the hallmarks of cancer. Hence, epigenetic modifiers such as HDAC inhibitors are being comprehensively explored for their anticancer potential. Besides anticancer properties, HDAC inhibitors have also shown promising results in controlling the other pathological conditions such as neurological disorders and viral infections and are well tolerated  (45,46). Currently, VPA along with other short-chain fatty acids HDAC inhibitors are being clinically evaluated as anticancer drugs (47). HDAC inhibitors employ wide range of antitumor mechanisms such as induction of apoptosis, senescence, differentiation, or inhibition of cell cycle (48,49). Vorinostat (SAHA), is among the first HDAC inhibitor to be approved by United   States Food and Drug Administration (FDA) for the treatment of relapsed and refractory cutaneous T-cell lymphoma (50). Although HDAC inhibitors are approved for hematological malignancies, but clear proof-of-concept data for the clinical efficacy of HDAC inhibitors in solid tumors remains to be established (51). Recent studies have demonstrated that HDAC inhibitors exhibit higher therapeutic efficiency when combined with other antineoplastic agents (52). Hence, there is growing interest in exploring other combined therapeutic strategies with HDAC inhibitors. Emerging evidence suggest that HDAC play a crucial role in T cell differentiation and effector functions. A number of studies have demonstrated that HDAC inhibitors suppress the immune response of T cells in severe inflammatory conditions and induce tolerance in organ transplantation (53). Specifically, HDAC inhibitors have shown to induce the regulatory T cell (Tregs) generation or stabilization of Tregs in inflammatory microenvironment due to which they have shown promising responses in experimental colitis (54). HDAC inhibitors increase the immunogenicity of tumors by increasing the expression of tumor antigens recognized by the immune cells. The antitumor responses of cytotoxic T lymphocytes like γδ T cells are mediated through recognition of stress molecules (ULBP, HSPs) or danger signals like MICA/B expressed on tumor cells by class of activating receptors known as NKG2D (55)(56)(57). Studies have demonstrated that HDAC inhibitors upregulate the NKG2D ligands on tumor cells, thereby sensitizing tumor cells to cytotoxicity mediated by γδ T cells in bladder cancer as well as NK cells in other malignancies such as osteosarcoma, pancreatic cancer, and multiple myeloma (32,(58)(59)(60). However, the causal effect of HDAC inhibitors on immune scenario is not well investigated and is contradictory. Several studies have shown that HDAC inhibitors affect each immune subset distinctly either leading to activation as in the case of CD4 T cells and CD8 T cells or by abrogating the effector functions of cells such as NK cell (61)(62)(63). Furthermore, for a particular immune cell type, the nature of immune regulation differs based on the type of HDAC inhibitor (64,65). A recent study demonstrated that NKG2D expression in NK cells is inhibited by VPA (66).
Most of the studies have focused on investigating the impact of HDAC inhibitors on tumor cell lines and immune cells other than γδ T cells. Report by Suzuki et al. demonstrated that the antitumor effect of γδ T cells on bladder cancer was enhanced by treatment with VPA (32). The study focuses only on the impact of HDAC inhibitor, VPA on bladder cancer cell line. VPA leads to increase in the expression of MICA and MICB, which are recognized by NKG2D receptor on γδ T cells. The study does not explain the direct effect of HDAC inhibitors on γδ T cells. Earlier study by Kabelitz et al. reported that HDAC inhibitor VPA induces differential modulation of cell surface markers on γδ T cells compared to αβ T cells (67). Although the study shows the direct effect of VPA on γδ T cells, the functional responses of γδ T cells were not investigated in detail. In the present study, we have used three different HDAC inhibitors to delineate their effect on the functional responses of pure and sorted population of γδ T cells. We used clinically relevant concentrations of VPA, TSA, and SAHA in our study, which have been used in in vitro studies by other investigators (68,69). We showed that three different HDAC inhibitors used suppressed the antitumor effector functions of γδ T cells.
We observed that γδ T cells activated with the phosphoantigen, HDMAPP in the presence of HDAC inhibitors showed decreased proliferative potential. One of the mechanism by which HDAC inhibitors exhibit their anticancer properties is through induction of cell differentiation and cell cycle arrest at G1 phase (48,49). Besides affecting histone proteins, these inhibitors also have several non-histone protein substrates like p53, p21, Rb, and E2F1 in tumors (70,71). On the other hand, it was demonstrated that downmodulation of p53 in T cells enhances their antigenspecific proliferative response and also augments antitumor cytotoxic functions (72,73). Studies from our lab have shown that CD3-activated T cells upon activation show robust proliferative capacity and decreased expression of p53 and its downstream target p21 (74). Thus, the decrease in the antigen-specific proliferative response of γδ T cells in presence of HDAC inhibitors incited us to look for effect of HDAC inhibitors on cell cycle  progression and expression of cell cycle regulators p53 and its downstream target p21. Decrease in the proliferation of γδ T cells in presence of HDAC inhibitors was associated with the increase in the expression of p53 and its downstream target p21. γδ T cells show increased expression of activation markers CD69 and CD25 when activated with phosphoantigens (36,75). We observed that HDAC inhibitors inhibit the expression of CD69 and CD25 activation markers. CD25 is the high-affinity IL-2 receptor subunit and IL-2 signaling is necessary for the proliferation of T cells. It would be logical to conclude that HDAC inhibitors abrogate the IL-2 signaling and thus inhibit the proliferation of γδ T cells.
We have used three different HDAC inhibitors VPA, TSA, and SAHA at different concentrations and they showed varied effects on expression of all the γδ T cell markers we studied. The likely explanation for the differences observed in their effects could be their structural diversity and also the biological activities they exert may be cell-type dependent. Activated γδ T cells express Tbet and eomesodermin (Eomes) transcription factors. The T-box transcription factors T-bet and Eomes are important for acquisition of effector functions in cytotoxic T cells including γδ T cells (41,76). Eomes and T-bet are highly homologous transcription factors and have cooperative and redundant functions in regulating the expression of different genes involved in the effector functions of CD8 T cells and activated natural killer cells. T-bet and Eomes regulate the expression of perforin, Granzyme-B, and IFN-γ by binding to promoter regions of these effector genes (14,39). Knowing that HDAC inhibitors decrease the activation and proliferation of γδ T cells, we further hypothesized that HDAC inhibitors may modulate the effector functions of γδ T cells by affecting the expression of transcription factors Eomes and T-bet. We observed that treatment of γδ T cells with HDAC inhibitors lead to decrease in the expression of Eomes and T-bet. To further establish impact of HDAC inhibitors on the antitumor cytotoxic function of γδ T cell, we used panel of tumor cell lines (AW13516, COLO-205, and Raji) treated with zoledronate as target cell line in cytotoxicity assay. Previous work from our laboratory and others has demonstrated that tumor cells treated with zoledronate are aggressively killed by γδ T cells (10,77). Our data demonstrate that treatment of HDAC inhibitors retard the ability of γδ T cells to kill zoledronate-treated tumor targets. Further, we proved that this inhibition of cytotoxic potential of γδ T cells was due to decrease in the expression of perforin and granzyme-B in these cells.
The activation of T cells initiated through T cell receptor is regulated by balance between co-stimulatory and inhibitory signals (immune checkpoints). Imbalance between these signals lead to different pathological conditions like tumor. Majority of the tumors use these immune checkpoints such as PD-1 or its ligand PD-L1 to escape from the immune surveillance. Immune check point inhibitors have revolutionized the field of tumor immunotherapy (34). Besides surgery, radiation, and chemotherapy, immune check point inhibitors have surfaced as an important immunotherapeutic approach for cancer treatment. Due to their promising antitumor effects in experimental animal models, preclinical studies and successful clinical trials, immune check point inhibitors have been now approved by the U.S Food and Drug Administration (FDA) for treatment of different malignancies. PD-1/PD-L1 blocking strategy has led to tumor regression in patients with melanoma, renal cell carcinoma, nonsmall cell lung cancer, and bladder cancer (78)(79)(80)(81)(82).
Recent reports have shown that tumors associated with PD-1 expressing NK cells show poor survival (83). PD-1/PD-L1 signaling axis along with NKG2D signaling axis determine effector response of NK cells. Blockade of PD1/PD-L1 signaling cascade in NK cells along with other antitumor drugs have shown promising responses in cancer patients (84). This study supports our observation that HDAC inhibitors modulate the effector functions of human γδ T cells against tumors via PD1/ PD-L1 signaling axis. We observed that γδ T cells show increased expression of immune check points PD1 and PD-L1 upon HDAC inhibitor treatment.
A report by Garcia-Diaz et al. have shown that induction of PD-L1 and PD-L2 on tumor cells is regulated via IFN-γ (85). In the present study, we have demonstrated that HDAC inhibitors decrease the expression of IFN-γ and TNF-α in antigen-activated γδ T cells. It has been demonstrated that Tbet transcription factor binds to PD-1 promoter and mediates the suppression of PD-1 expression (86). In the present study, we have shown that upon HDAC inhibitor treatment of γδ T cells, Tbet protein and mRNA is decreased significantly indicating that less Tbet may be available to bind PD-1 promoter to suppress PD-1 expression. This mechanism may explain the IFN-γ independent mechanism of PD-1 expression on γδ T cells.
Activated γδ T cells are known to express PD-1, which was investigated by Iwaski et al., on expanded γδ T cells population. They found that γδ T cells express PD-1 rapidly from day 3 of induction and PD-1 + γδ T cells exhibit attenuated effector functions and decreased cytotoxicity against PD-L1 expressing tumors. However, they observed that zoledronate treatment to tumor cells, which induces IPP release along with PD-L1 blockade, rescued the γδ T cell cytotoxicity (35). While our study also confirms that blocking of PD-1 in γδ T cells increases the antitumor cytotoxic potential, our study reports on the effect of HDAC inhibitors on the freshly isolated γδ T cells activated with antigen for 72 h, whereas Iwaski group used γδ T cells already in activation state for their experimental purposes. Another interesting study by Castella et al. explores the multifunctional role of zoledronate in augmenting γδ T cells responses against multiple myeloma. In this study, zoledronate-treated autologous DCs were found to efficiently activate γδ T cells and enhance their cytotoxic functions against myeloma cells. Additionally, zoledronate was also shown to promote antitumor immunity via suppression of regulatory T cell function, downregulation of PD-L1 expression on DCs, and increased proliferation of tumor antigen-specific CD8 T cells. Although, their study has effectively demonstrated role of zoledronate in enhancing antitumor responses γδ T cells, it is specific only to multiple myeloma and uses zoledronate expanded γδ T cells from patient PBMNCs (87). Converse to our observation, they found that DC-activated γδ T cells did not express PD-1, this might be due to the immune modulation by zoledronate, which needs further exploration.
We observed that blockade of PD1/PD-L1 signaling partially restores the antitumor cytotoxic function of γδ T cells in the presence of HDAC inhibitors, which reflected in increased expression of effector molecules granzyme B and Lamp-1. Wei et al. have demonstrated that PD-1 ligation dramatically shifts the doseresponse curve, making CD8+ Tcells much less sensitive to TCR generated signals (88). Although, this was shown in CD8+ αβ T cells, it may also apply to γδ T cells. Thus, PD-1 ligation affects TCR signaling and thereby reduces the cytotoxic function of γδ T cells. The role of other activating receptors such as NKG2D interacting with MICA/B and inhibitory receptors KIR2DL2/3 (CD158b) cannot be ignored and it explains the incomplete restoration of cytotoxic effector function γδ T cells upon PD-1 blocking.
Our results implicate that HDAC inhibitors along with the immune checkpoint modulating antibodies could be developed as combination immunotherapy to treat different malignancies. Thus, in future, this strategy may be applied for overcoming the limitations of HDAC inhibitor-based cancer therapies. The underlying mechanistic link of PD-1/PD-L1 may be targeted in developing more efficacious combination γδ T cell-based therapies in the future.

eThics sTaTeMenT
The study was approved by the Institutional Ethics Committee of ACTREC-TMC. All subjects gave written informed consent in accordance with the Declaration of Institutional Ethics Committee, ACTREC-TMC. aUThOr cOnTriBUTiOns SC supervised, contributed conceptionally, and helped to write the manuscript. SB conducted experiments and wrote the manu script. DV helped in conducting experiments. All authors contributed in final approval of manuscript.