Targeting NKT cells and PD-L1 pathway results in augmented anti-tumor responses in a melanoma model
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- Durgan, K., Ali, M., Warner, P. et al. Cancer Immunol Immunother (2011) 60: 547. doi:10.1007/s00262-010-0963-5
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Invariant or Type 1 NKT cells (iNKT cells) are a unique population of lymphocytes that share characteristics of T cells and natural killer (NK) cells. Various studies have shown that positive costimulatory pathways such as the CD28 and CD40 pathways can influence the expansion and cytokine production by iNKT cells. However, little is understood about the regulation of iNKT cells by negative costimulatory pathways. Here, we show that in vivo activation with α-GalCer results in increased cytokine production and expansion of iNKT cells in the absence of programmed cell death ligand-1 (PD-L1, B7-H1, and CD274). To study whether PD-L1 deficiency on NKT cells would enhance antigen-specific T-cell responses, we utilized CD8+ OT-1 OVA transgenic T cells. α-GalCer enhanced the expansion and cytokine production of OT-1 CD8+ cells after adoptive transfer into wild-type recipients. However, this expansion was significantly enhanced when OT-1 CD8+ T cells were adoptively transferred into PD-L1−/− recipients. To extend these results to a tumor model, we used the B16 melanoma system. PD-L1−/− mice given dendritic cells loaded with antigen and α-GalCer had a significant reduction in tumor growth and this was associated with increased trafficking of antigen-presenting cells and CD8+ T cells to the tumors. These data demonstrate that abrogating PDL1:PD-1 interactions during the activation of iNKT cells amplifies an anti-tumor response when coupled with DC vaccination.
KeywordsCostimulationProgrammed cell death-1Programmed cell death ligand-1TumorInvariant NKT cellsT cells
Natural Killer T cells (NKT cells) are a unique population of lymphocytes that share characteristics of T cells and natural killer (NK) cells. The key features of NKT cells are a biased T-cell receptor (TCR) gene expression, restriction by CD1d molecules on antigen-presenting cells, and rapid production of a spectrum of cytokines following activation. In numerous murine models, NKT cells have been demonstrated to be important in: (1) protection against infectious pathogens and diseases, (2) immune surveillance of tumors, (3) induction and control of autoimmunity, and (4) asthma (reviewed in ). It has become apparent that both human and murine NKT cells represent a heterogeneous population of cells, with functional differences ascribed to differential expression of cell-surface markers such as CD4, CD8, and chemokine receptors . It has also been shown that CD1d-restricted NKT cells can be subdivided into Vα14-Jα18+ α-GalCer-reactive Type I NKT cells, termed invariant NKT cells (iNKT), and α-GalCer non-reactive Type II NKT cells (reviewed in ). Type II NKT cells produce IL-13 and may downregulate immune responses by inducing myeloid cells to produce TGF-β. Several factors may influence the activation and function of NKT cells: (1) cytokine environment, (2) interacting antigen-presenting cell, (3) subset of NKT cell or anatomical location, or (4) costimulatory interactions . Thus, while all subsets of NKT cells are not well defined, the ability of these cells to influence the adaptive immune response is well accepted .
The rapid response to antigen by iNKT cells has led to the concept that these cells, unlike conventional T cells, may not require secondary signals to become activated. However, there is growing evidence that costimulation may play a role in the activation of iNKT cells. Murine and human iNKT cells constitutively express CD28 and rapidly upregulate CD40L(CD154) on activation [4, 5]. It also has been shown that blocking antibodies to B7, CD28, CD40, and CD154 inhibits the α-GalCer-induced proliferation of murine iNKT cells . In addition, several studies have shown a role for CD40:CD40L and B7:CD28 pathways in enhancing cytokine production by iNKT cells in vitro and in vivo [4, 7, 8]. In contrast, other studies have shown no role for these pathways in the initial cytokine production by iNKT cells but costimulation was important for the expansion and subsequent systemic cytokine production by NKT cells [9, 10]. Recent studies indicate that the B7:CD28 pathway might be important in the development of iNKT cells [11, 12]. Newly discovered costimulatory molecules also have been implicated in the function of iNKT cells. Interestingly, blockade of the inducible costimulatory molecule (ICOS) has been shown to inhibit α-GalCer-induced cytokine production and anti-tumor effects of NKT cells . Furthermore, iNKT cells from ICOS−/− mice produced less IL-4 and IL-13 and failed to induce airway hyper-reactivity in a murine model of asthma . The PD-1 pathway has been shown to be involved partially in anergy induced by α-GalCer, and the CD28 pathway is important in overcoming the PD-1 inhibition on iNKT cells [11, 15–17]. Therefore, similar to the activation of conventional T cells, it appears that iNKT cells have the same constraints for costimulation and negative regulatory signals.
The greatest challenge to an effective immune response against tumors is finding ways to overcome the immunosuppressive effect of the tumor itself. To address this factor, combination strategies to target tumors through the immune system have become increasingly prevalent. The knowledge that iNKT function can be modulated by secondary signals and their ability to influence adaptive immunity through cognate interactions with antigen-presenting cells has led us to examine whether the blockade of PD-L1:PD-1 pathways together with NKT cell activation will lead to an effective anti-tumor CD8+ T-cell response in a solid tumor model. In this study, we show that the PD-1 pathway negatively regulates iNKT expansion, and abrogation of the PD-1 pathway results in a further expansion of antigen-specific responses. Our results are the first to show that combining NKT cell activation with inhibition of negative costimulation can affect the growth of B16 cells in a solid tumor model.
Materials and methods
C57BL6 and OT-II mice were purchased from Jackson Laboratory (Bar Harbor, ME). PD-L1-deficient (−/−) mice were previously described . OT-I CD45.1/2 heterozygous mice and Vα14/Jα18-deficient mice were kindly provided by Dr. Keith Elkon (University of Washington) and Dr. M Taniguchi (RIKEN Research Center for Allergy and Immunology), respectively. All animals were maintained under SPF conditions at the University of Washington in accordance with approved protocols and IACUC standards.
Antibodies and reagents
mCD1d/PBS57 and unloaded mCD1d tetramers were obtained from NIAID MHC tetramer core facility. α-GalCer (KRN7000) and vehicle were initially provided by Kirin Pharma Company and then obtained from Avanti Polar Lipids Inc (Alabaster, AL). Anti-PD-L1 (M1H5), anti-PD-1 (RMPI-30), anti-PD-L2 (TY25), anti-CD4 (L3T3), anti-NK1.1 (PK136), anti-CD19 (eBio1D3), anti-CD8 (53-6.7), anti-CD45.1 (A20), anti-CD28 (37.51), anti-CD3 (17A2), and anti-V alpha 2 TCR (B20.1) were obtained from eBiosciences (San Diego, CA). Anti-V beta 5.1,5.2 TCR was obtained from BD Biosciences (San Diego, CA). All cytokine primary and detection antibodies for ELISA, ELISPOT, and intracellular staining were obtained from eBiosciences. OVA323–339 and OVA257–264 peptides were obtained from AnaSpec (San Jose, CA).
iNKT cell activation in vivo and in vitro
Mice were given 2 μg α-GalCer or vehicle i.p. After 2 h, splenocytes were isolated and treated with 1× RBC lysis buffer (eBiosciences). A total of 2 × 105 cells were resuspended in RPMI 1640 (Mediatech Herndon,VA) supplemented with 10% fetal calf serum, l-glutamine (2 mM), penicillin (100 U/ml), streptomycin 100 mg/ml, HEPES (10 mM), and 2-mercaptoethanol (50 mM) (all from Mediatech) and cultured in cytokine-coated ELISPOT plates for 18 h. For intracellular staining, splenocytes were isolated and cultured with monesin (eBiosciences) for 2 h. Cytokine analysis was preformed using Perm and Fix solutions from eBiosciences.
To study expansion of iNKT cells, mice were given 2 μg α-GalCer or vehicle i.p. After 3 or 7 days, splenocytes were isolated and NKT cell expansion analyzed by flow cytometry using mCD1d/PBS57 tetramer, anti-CD4, and anti-CD19. Unloaded mCD1d tetramer was used as a control.
To analyze purified iNKT cells, splenocytes were depleted of B cells (anti-CD19) and CD8+ T cells (anti-CD8) using Biomag beads (Qiagen, Valencia, CA) and mCD1d/PBS57 tetramer+ cells were sorted. Purified iNKT cells (5 × 104) were cultured with 5 × 104 dendritic cells and 100 ng/ml α-GalCer. Supernatants were harvested after 5 days and analyzed for cytokines.
Dendritic cell isolation
Bone marrow-derived dendritic cells were prepared by depletion of bone marrow cells with anti-CD3, anti-CD19, and Gr-1. Cells were grown in GM-CSF and IL-4 for 8 days, and CD11c+ cells were isolated by anti-CD11c beads and magnetic-activated cell sorting separation (MACS) columns (Miltenyi Biotec, Auburn, CA) or cell sorting. For cytokine production, mice were given 2 μg α-GalCer i.p. After 8 h, spleens were isolated and treated with collagenase. CD11c+ cells were isolated from spleens using anti-CD11c beads as above CD11c+ cells were cultured for 48 h and IL-12 production analyzed by ELISA.
Adoptive transfer studies
Purified iNKT cells were isolated as above. A total of 2 x105 NKT cells were adoptively transferred into Vα14-/Jα18-deficient mice (NKT type I-deficient mice). Two days later, mice were given α-GalCer or vehicle i.p and cytokine production analyzed by ELISPOT.
For antigen-specific studies, OT-I or OT-II cells were isolated by positive selection using anti-CD8 or anti-CD4 beads, respectively; 1 × 106 OT-I or OT-II cells were adoptively transferred into mice. The following day, mice were given OVA peptide (50 μg/ml) together with 2 μg α-GalCer or vehicle i.v. Four days later, splenocytes were isolated and stained with anti-CD45.1, anti-Vα2, and anti-Vβ5.1 antibodies. Splenocytes were restimulated with 1 μg/ml of OVA peptide and supernatants analyzed by ELISA.
CD11c+ cells were loaded with 200 ng/ml α-GalCer or vehicle overnight and further cultured for 2 h with TRP-2181–188 peptide. A total of 5 × 105 dendritic cells were injected into their respective host, i.e., C57BL6 dendritic cells into C57BL6 mice or PD-L1−/− dendritic cells into PD-L1−/− mice. For the metastasis model, mice were challenged with 5 × 105 B16-F1 cells i.v (ATCC) 2 days later. After 14 days, mice were killed and lungs removed and placed in formalin. Tumor nodules were counted by light microscopy. For effector function, splenocytes were expanded for 7 days with TRP-2181–188 peptide and IL-2 and then cultured with B16 cells for a further 24 h. IFN-γ production was measured by ELISA. In the subcutaneous model, 2 × 105 B16-F1 melanoma cells were injected subcutaneously (s.c) in the right flank of mice and tumor growth was measured by calipers and analyzed by diameter (length × width) every 7 days. Mice were killed after 3 weeks or before the tumor on an individual mouse reached a diameter of 20 mm2, and results were expressed as mean ± SD.
To study iNKT function in vivo, α-GalCer (2 μg) or vehicle was administrated i.p to PD-L1−/− or wild-type mice. After 2 or 4 h, splenocytes were isolated and cytokine production was analyzed by ELISPOT. After α-GalCer activation in vivo, there was an increase in the number of cells producing IFN-γ, IL-4, and IL-17 in PD-L1−/− mice when compared to wild-type mice (Fig. 2b). These results suggest that blocking the PD-L1 pathway influences Th1-, Th2-, and Th17-producing subsets of NKT cells. In addition, the ability of PD-L1−/−NKT cells to activate dendritic cells in vivo was investigated. PD-L1−/− and wild-type mice were treated with α-GalCer for 8 h and CD11c+ cells were isolated and IL-12 production analyzed by ELISA 48 h later. As shown in Fig. 2c, PD-L1−/− DC produced more IL-12 compared to wild-type DC after α-GalCer treatment of iNKT cells. To study the intrinsic role of PD-L1 deficiency on NKT cells in vivo, PD-L1−/− or wild-type NKT cells were sorted and adoptively transferred to iNKT-deficient (Jα18 deficient) mice. After 2 days, recipient mice were given α-GalCer i.p, and 2 h later, splenocytes were isolated and analyzed. As shown in Fig. 2d, adoptively transferred PD-L1−/− iNKT cells produced more cytokines than wild-type iNKT cells. iNKT cells are unique in their ability to rapidly produce cytokines after TCR engagement. This is due in part to the fact that NKT cells store preformed mRNA for various cytokines (reviewed in [19, 20]). To investigate whether PD-L1 deficiency affects the initial cytokine burst by iNKT cells, mice were given α-GalCer for 1 h and splenocytes were isolated and intracellular staining was preformed. There was no difference in cytokine production between PD-L1 and wild-type iNKT cells at this early time point indicating that the PD-L1 pathway had no role in the initial cytokine production by iNKT cells (Fig. 2e).
NKT activation can cause bystander activation of other cell types, assessed by an increase in CD69 expression . Wild-type and PD-L1−/− B cells, CD8+ T cells, and NK cells, but not CD4+ T cells upregulated CD69 expression after α-GalCer treatment (Supplementary Fig. 2B). PD-L1−/− B cells expressed higher levels of CD69 after α-GalCer treatment compared to wild-type B cells (Supplementary Fig. 2B). It has been shown that NK cells are rapidly activated after α-Galcer treatment of iNKT cells . A hallmark of this activation is IFN-γ production by NK cells. As PD-L1 and PD-1 are expressed on NK cells after activation (Supplementary data Fig. 2C), we investigated the trans-activation of NK cells in PD-L1−/− and wild-type mice after in vivo administration of α-GalCer. NK cells were intracellularly stained for IFN-γ production directly ex vivo. As previously shown, wild-type NK cells produced IFN-γ after α-GalCer treatment. Surprisingly, there was a slight but significant decrease in IFN-γ production by PD-L1−/− NK cells (Supplementary Fig. 2D).
To study the effect of iNKT activation on CD4+ T-cells responses, we adoptively transferred OT-II CD4+ T cells into wild-type and PD-L1−/− mice, and injected the mice with OVA peptide and α-GalCer or control vehicle. There was a significant expansion when α-GalCer was used with OVA peptide in wild-type and PD-L1−/− mice, compared to mice injected with vehicle plus OVA. While wild-type recipients given OVA peptide and α-GalCer had a higher average expansion of OT-II CD4+ T cells, compared to PD-L1−/− mice treated in the same manner, it was not statistically significant (Fig. 4c). However, OT-II CD4 T cells activated with α-GalCer and OVA peptide from PD-L1−/− recipients produced more IFN-γ than when activated in their wild-type counterparts (Fig. 4d). This indicates that PD-L1 deficiency on iNKT cells can result in an enhanced antigen-specific response, although there appears to be a more profound effect on CD8+ T cells.
To investigate the mechanism by which NKT activation and lack of PD-1 interactions results in decreased tumor growth, we analyzed the tumor-infiltrating lymphocytes (TIL) by flow cytometry. Tumors were excised and weighed, and there was a signification reduction in the weight of tumors from PD-L1−/− mice given dendritic cells loaded with TRP-2 compared to wild-type mice given the same treatment (Fig. 6b). There was significant increase in IFN-γ-producing cells in the tumors of PD-L1−/− mice given TRP-2 and α-GalCer-pulsed dendritic cells compared to wild-type mice (Fig. 6c). However, there was no difference between the number of IFN-γ-producing cells in the tumors of PD-L1−/− mice given dendritic cells loaded with TRP-2 and vehicle or TRP-2 and α-GalCer, suggesting that IFN-γ production is not the mechanism behind decreased tumor load in PD-L1−/− mice treated with α-GalCer (Fig. 6c). On investigation of the cell types within tumors, there was a significant increase in dendritic cells (CD11chigh+ cells), macrophages (CD11b+), NK cells (NK1.1+), and CD8+ T cells in PD-L1−/− mice, in contrast to wild-type mice (Fig. 6d). Data are presented as the number of cells per gram of tumor tissue . Surprisingly, there also was a significant increase in CD4+ Foxp3+ T-regulatory cells (Tregs) and this may account for the incomplete clearance of tumors in PD-L1−/− mice. To test the efficacy of NKT cell activation and PDL1 abrogation in a therapeutically relevant setting, mice were given dendritic cells loaded with TRP-2 and α-GalCer 2 or 4 days after the B16 inoculation. PD-L1−/− mice given dendritic cells 4 days after B16 inoculation had a small but significant decrease in tumor growth (Fig. 6e). Although there was an increase in antigen-presenting cells, there was not an increase in CD8+ T cells necessary for the clearance of the tumor (Fig. 6f). This data imply that in a therapeutic setting, as with other models of combination therapy, Treg depletion may be required .
Several studies have established that the PD-L1:PD-1 pathway is involved in negatively regulating conventional T cells. However, PD-L1 is expressed on a variety of innate lymphocytes and the examination of the function of this pathway on innate cells has not been well characterized. In this study, we clearly demonstrate that the PD-L1 pathway inhibits the expansion and the cytokine production of iNKT cells. In addition, deficiency of PD-L1 on iNKT cells influenced an antigen-specific response even with intact PD-1 molecules on CD8+ T cells (Fig. 4). Furthermore, tumor-specific T cells were enhanced in the presence of NKT cell activation and antigen in PD-L1-deficient mice. These results indicate that negative pathways on innate cells play an important role in controlling a subsequent adaptive immune response and therefore warrant further investigation.
It has been shown previously that PD-1 pathway is involved in anergy induction of iNKT cells after repeated α-GalCer stimulation [16, 17]. These studies did not specifically address the expansion of iNKT after initial α-GalCer treatment; however, data presented by Parekh et al. showed a marginal increase in iNKT numbers by α-GalCer treatment in PD-1-deficient mice. This discrepancy might be due to the fact that PD-L1 deficiency removes two interactions, between PD-L1 and PD-1 as well as CD80, therefore eliminating the negative PD-1 pathway and allowing the engagement of CD80 and CD28. As the balance of signals through PD-L1/PD-1 and CD28 pathways are counterbalanced in the expansion of iNKT cells, blocking of PD-L1 rather than PD-1 would be more effective . The study of iNKT cells in PD-1/B7 double-deficient mice, PD-L1-, and PD-1-deficient mice will give a clearer understanding of the contribution of the PD-1 pathway in regulating the expansion of iNKT cells . Previous studies have revealed that PD-L1−/−mice have enhanced T-cell responses in the presence of the second ligand for PD-1, PD-L2 (B7-DC, CD273) [18, 37]. Both PD-L1 and PD-L2 (B7-DC, CD273) are upregulated by α-GalCer on dendritic cells (Supplementary Fig. 3A and data not shown). Therefore, these results would indicate that the lack of PD-L1 on iNKT cells is important for heterotypic interactions between PD-L1 and PD-1 on iNKT cells as seen in conventional T cells. This is supported by the fact that adoptive transfer of PD-L1−/− iNKT cells into iNKT-cell-deficient recipients, where PD-L1 is only deficient on the iNKT cells, leads to increased cytokine production compared to the adoptively transferred wild-type NKT cells. We are the first to show that the PD-L1 pathway negatively regulates IL-17 production by iNKT cells and therefore blocking the PD-L1 pathway not only has the potential to manipulate Th1 and Th2 subsets but also Th17-producing cells.
The idea that iNKT cells can bridge the gap between the innate and adaptive immune response is well accepted although the exact mechanism by which this may occur is not clear. α-GalCer treatment results in the maturation of dendritic cells as well as the release of various cytokines from iNKT cells. In our study, we do not show any difference in the maturation of dendritic cells as defined by the expression of CD80, CD86, and CD40, between wild-type and PD-L1−/− mice. However, PD-L1−/− dendritic cells and NKT cells produce more cytokines than wild-type dendritic cells and iNKT cells and this factor might account for the increased expansion of OVA-specific CD8+ T cells. Interestingly, there was an increase in the bystander activation of B cells but not NK cells when PD-L1−/− mice were given α-GalCer in the context of antigen presentation. The ability of iNKT cell activation to target B cells as well as dendritic cells might be an important factor in the adjuvant effect of α-GalCer.
Blockade of negative regulators of the CD28 superfamily is a potent strategy for the design of therapeutic cancer vaccines due to powerful inhibitory role they play on the activation of naïve and effector T cells. It was originally shown in mice that some tumors expressing B7 were rejected with anti-CTLA-4 blockade [38, 39]. Since regression of tumors with anti-CTLA-4 was not as successful with poorly immunogenic tumors such as B16, combination therapy was employed to facilitate a robust immune response. Clinical trials for melanoma and prostate cancer are ongoing but autoimmune adverse reactions have been associated with clinical response with anti-CTLA-4 treatment. In our studies, PD-L1−/− mice had reduced tumor burden when dendritic cells were activated with α-GalCer and preloaded with antigen. It has been reported that dendritic cells loaded with α-GalCer inhibited tumor growth in a metastasis but not a subcutaneous B16 model . Our data shows that combining abrogation of the PD-L1 pathway together with iNKT activation can overcome the immunosuppressive effects of B16-F1 cells. B16-F1 cells express PD-L1 (Supplementary data Fig. 4) and therefore our combination therapy reduced tumor growth even in the presence of PD-L1 on the tumor. In addition, PD-L1-deficient mice did not show signs of autoimmune depigmentation suggesting that PD-L1 blockade did not lead to an autoimmune response. The lack of autoimmune disease also has been observed when PD-L1 blockade with combined with Gvax or Fvax [40, 41]. This would imply that blocking the PD-L1 pathway in therapeutic setting would result in fewer autoimmune side effects. This is further evidence for independent and non-overlapping roles that the CTLΑ-4 and PD-L1 pathways may play regulating the cells within the immune system. It is still unclear whether these pathways may target T cells at various effector phases.
We observed increased Tregs within the TIL of PD-L1−/− mice that have reduced tumor growth and there was no difference in the CD8+T cell:Treg ratio of wild-type and PD-L1-deficient mice. This result is somewhat surprising and counterintuitive, as two groups have shown that the PD-L1 pathway induces Tregs in vitro [42, 43]. However, a recent study showed that PD-L1 blockade increased Tregs in a mesothelioma tumor model and required CD4+ T-cell removal plus anti-PD-L1 treatment to reduce tumor growth . In the tumor setting with high TGF-β concentrations within the tumors, Tregs may be induced more readily by PD-L1 blockade. Notably, Fvax in combination with anti-PDL1 blockade increased Tregs in TIL whereas Fvax plus anti-PD-L1 and anti-CTLA-4 decreases Tregs in TIL , showing that the PD-1 and CTLA pathways target Tregs in different ways. Inclusion of an anti-Treg regime with NKT cell activation may result in further clearance of tumors in PD-L1-deficient mice. We detected increased antigen-presenting cells in the TIL of PD-L1 mice which may be due to α-GalCer treatment, as there was an non-significant increased number of antigen-presenting cells in the TIL of PD-L1−/− mice given dendritic cells activated with α-GalCer compared to wild-type mice treated the same (data not shown). Collectively, these results gives supporting evidence that the adjuvant effects of α-GalCer together with antigen and PD-L1 blockade can augment tumor responses. Furthermore, in a clinical setting, the outcome of PD-L1 blockade might result in fewer side effects, compared to the anti-CTLA-4 blockade.
We would like to thank Pei Fan for her excellent technical support. This study was funded by National Institute of Health 5R21A1066135 (YEL and PW) and Leukemia and Lymphoma society 3321-03 (YEL).