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Invariant natural killer T cells regulate anti-tumor immunity by controlling the population of dendritic cells in tumor and draining lymph nodes

Invariant natural killer T cells regulate anti-tumor immunity by controlling the population of... Background: Invariant natural killer T (iNKT) cells are CD1d-restricted T cells, which respond rapidly to antigen recognition and promote development of anti-tumor immunity in many tumor models. Surprisingly, we previously found that mice deficient in iNKT cells developed spontaneous CD8 T cells responses partially effective at inhibiting metastases in mice bearing the 4T1 mammary carcinoma, and showed a markedly improved response to treatment with local radiotherapy and anti-CTLA-4 antibody compared to wild type (WT) mice. Methods: To understand the mechanisms of the immunosuppressive function of iNKT cells, dendritic cells (DCs) −/− were analyzed by immunohistochemistry and flow cytometry in WT and iNKT-deficient (iNKT ) mice. The effects of antibody-mediated blockade of CD1d on DC number and phenotype, priming of anti-tumor T cells, and tumor response to treatment with local radiotherapy and anti-CTLA-4 antibody were evaluated. To determine if the improved response to treatment in the absence of iNKT cells was independent from the immunotherapy employed, 4T1-tumor −/− bearing WT and iNKT mice were treated with local radiotherapy in combination with antibody-mediated CD137 co-stimulation. Results: DCs in 4T1 tumors and tumor-draining lymph nodes but not distant lymph nodes were significantly −/− reduced in WT mice compared to iNKT mice (p < 0.05), suggesting the selective elimination of DCs cross-presenting tumor-associated antigens by iNKT cells. Consistently, priming of T cells to a tumor-specific CD8 T cell epitope in mice −/− treated with radiotherapy and anti-CTLA-4 or anti-CD137 was markedly enhanced in iNKT compared to WT mice. CD1d blockade restored the number of DC in WT mice, improved T cell priming in draining lymph nodes and significantly enhanced response to treatment. Conclusions: Here we describe a novel mechanism of tumor immune escape mediated by iNKT cells that limit priming of anti-tumor T cells by controlling DC in tumors and draining lymph nodes. These results have important implications for the design of immunotherapies targeting iNKT cells. Keywords: Breast cancer, CD1d, CD137, CTLA-4, CD8 T-cells, Dendritic cells, Immunoregulation, Invariant NKT cells, Radiotherapy * Correspondence: Sandra.Demaria@nyumc.org Department of Pathology, New York University School of Medicine, New York, NY 10016, USA Department of Radiation Oncology, New York University School of Medicine, New York, NY 10016, USA Full list of author information is available at the end of the article © 2014 Pilones et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 2 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 Background Response to treatment with local radiotherapy and anti- Natural killer T (NKT) cells comprise a subset of lympho- CTLA-4 monoclonal antibody (mAb) was also markedly −/− cytes originating from a distinct developmental lineage [1] improved in iNKT compared to wild type (WT) mice which bridge innate and adaptive immunity and modulate with half of the mice rejecting completely the primary immune responses in autoimmunity, malignancies and in- irradiated tumor and lung metastases and showing long- fections [2]. Although initially identified by co-expression term survival compared to none of the WT mice [25]. of conventional αβ T-cell receptors (TCR) and markers These data implicated iNKT cells as major regulators of typically associated with natural killer (NK) cells [3], NKT the spontaneous as well as immunotherapy-elicited anti- are currently distinguished on the basis of CD1d restric- tumor immune response to 4T1 tumor. tion as well as specific usage of TCRα chains [4]. In both CD4 regulatory T cells (Treg) have been shown to limit mice and humans, most NKT cells express TCRs formed the development of anti-tumor T cells by killing DCs in by the rearrangement of a canonical α chain (Vα14 in tumor-draining lymph nodes (dLN) [26]. DCs are a major mice, Vα24 in humans) and a limited set of Vβ chains subset of CD1d antigen-presenting cells (APC) that inter- (Vβ.2, Vβ7, Vβ2in mice, Vβ11 in humans) and are com- act with iNKT cells. Therefore, we hypothesized that monly referred to as type I or invariant natural killer T iNKT cells may be limiting the anti-tumor immune re- (iNKT) cells [5,6]. A smaller NKT cell subset utilizes a sponse in 4T1 tumor-bearing mice by controlling the more diverse set of TCR αβ chains and is referred to as loco-regional population of DCs in the tumor and dLNs. type II or non-invariant NKT cells [7]. For the first time, we present evidence that WT mice have Identification of α-galactosylceramide (α-GalCer) as a reduced numbers of DCs in tumor and dLN compared to −/− strong agonist selective for iNKT cells [8] facilitated their iNKT mice, and reduced priming of tumor-specific characterization using α-GalCer-loaded CD1d tetramers CD8 T cells, as measured by gamma interferon (IFNγ) [9]. In several tumor models, iNKT cells were found to production in response to a H2-L -restricted peptide perform important immunosurveillance functions and derived from a tumor antigen. DC numbers and tumor- become key effectors of tumor rejection when activated specific CD8 T cell responses were at least partially re- by α-GalCer [10-13]. Expression of high levels of Fas stored by blocking CD1d, resulting in improved response Ligand, perforin, and granzyme B by iNKT cells underlies to immunotherapy. Overall, data demonstrate a novel their cytolytic activity against CD1d tumor cells [14] and mechanism of immune escape mediated by iNKT cells. myeloid cells with immunosuppressive function present in the tumor microenvironment [15]. In addition, iNKT cells Results exert anti-tumor functions by rapid and robust secretion Numbers of DCs infiltrating 4T1 tumors and dLN are −/− of cytokines that improve DC ability to cross-prime anti- lower in WT compared to iNKT mice tumor T cells [10,12,16,17] and enhance recruitment of We hypothesized that the interaction of iNKT cells with + + other effectors such as NK cells, CD4 T helper-1 (Th1) CD1d APC could modulate the ability of WT mice to and CD8 cytotoxic T (CTL) cells [13,18]. develop anti-tumor immune responses upon treatment. Experimental data in different systems indicate a func- To test this hypothesis we compared DCs in healthy and −/− −/− tional plasticity of iNKT cells. iNKT cells can promote tumor-bearing WT and iNKT mice. iNKT mice the polarization of adaptive immune responses towards were generated by targeted deletion of the Jα18 segment both Th1 and Th2 and can secrete immunosuppressive of the T-cell receptor (TCR) and show selective loss of cytokines [19]. The regulatory function of iNKT cells has the Vα14Jα18 TCR expressed by iNKT cells while other been demonstrated in multiple models of autoimmune lymphoid and myeloid populations are not affected [27]. diseases in which iNKT cells played essential roles in Consistently, there were no differences in the number of maintenance of tolerance [20-22]. The mechanisms that DCs present in LNs or their expression of maturation determine whether iNKT cells act to promote immune markers CD80 and CD86, including CD1d, between WT −/− activation or tolerance remain incompletely understood, and iNKT healthy mice (Figure 1A and B). In con- but the inflammatory context in which interactions of trast, after 4T1 tumor implantation there was a differ- iNKT cells with CD1d myeloid cells take place is thought ence in the numbers of DCs in the tumor-draining but to be a key factor [23,24]. not distant LNs (Figure 1C and D), with significantly The 4T1 mouse mammary carcinoma is a model for lower numbers of DCs present in WT compared to −/− triple negative breast cancer and shows an aggressive iNKT mice. Significantly lower numbers of DCs were behavior with rapid spread of metastatic cells to the lungs also found in the tumors growing in WT compared to −/− after subcutaneous injection. We previously found that iNKT mice (Figure 1E and F). Interestingly, while −/− −/− 4T1 tumor-bearing iNKT-deficient (iNKT ) mice devel- non-draining LNs of iNKT mice showed a DC density oped a spontaneous CD8 T cell response that was par- comparable to LNs of healthy mice, DC density was tially effective at controlling metastases in the lungs [25]. markedly increased in tumor-draining LNs, suggesting Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 3 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 AB C 30 100 300 20 200 10 100 0 0 -/- WT CD80 CD86 CD1d Day 13 Day 26 iNKT DE F Day 13 Day 26 40 50 0 0 Day 13 Day 26 Day 13 Day 26 Figure 1 iNKT cells regulate the number of DCs in 4T1 tumors and draining lymph nodes. (A, B) Lymph nodes collected from healthy WT −/− (black bars) and iNKT (white bars) mice (N = 3/strain). (A) Full-face lymph node sections were stained with anti-CD11c mAb and positive cells counted. (B) Dissociated lymph node cells were gated on CD11c + cells and expression of CD1d and co-stimulatory molecules CD80 and CD86 −/− determined by flow cytometry. (C-F) WT (black bars) and iNKT (white bars) mice were inoculated s.c. with 4T1 cells. On indicated days, tumor-draining lymph nodes (C), non-draining lymph nodes (D),and tumors (E, F) were excised, tissue sections stained with anti-CD11c mAb and positive cells counted. Numbers are the mean ± SD of 3 mice of each strain analyzed at each time point. (F) Representative fields (400× magnification). Results are representative of two experiments. p < 0.05. that even in the absence of treatment the presence of addition, we verified that administration of 20H2 mAb the growing tumor leads to some activation of the re- to mice did not cause the depletion of CD1d cells gional immune system. This finding is consistent with (Additional file 2: Figure S2) nor induce their stimula- the presence of a spontaneous anti-tumor CD8 T cell tion by reverse signaling (Additional file 3: Figure S3). −/− response in iNKT mice that was partially effective at Next, anti-CD1d mAb was given to 4T1 tumor-bearing controlling metastases in the lungs as we have previously WT mice starting on day 3 post-tumor inoculation and reported [25]. tumors and dLN analyzed at day 13, a time when tumors Overall, data suggest that in WT mice iNKT cells may are irradiated in treated mice and the availability of DCs control the population of DCs in the tumor and dLN that can cross-present the tumor antigens released by thus precluding the priming of anti-tumor T cells. radiation is critical [29,30]. CD1d blockade led to a sig- nificant increase in the total number of DCs present in tu- CD1d blockade restores DC numbers and improves mors and dLNs (Figure 2A). Both immunostimulatory tumor-specific T cell cross-priming in WT 4T1 CD8α + and CD8α- DC subsets were increased (Figure 2B), tumor-bearing mice whereas expression of activation markers CD40, CD70, Elimination of DCs in dLN has been shown to be one CD86, and of MHC Class I molecules in either subsets of mechanism whereby Treg suppress the anti-tumor im- tumor infiltrating DCs were not altered by CD1d blockade mune response [26]. To determine if iNKT cells could (Figure 2C-F). Therefore, data indicate that iNKT cells be responsible for the reduction in the number of DCs regulate DC numbers in 4T1 tumor-bearing WT mice via in tumors and dLN of WT mice we tested whether inhi- CD1d-mediated interactions. biting their interaction with DCs with a mAb that blocks To determine if blockade of CD1d resulted in im- CD1d [28] would lead to increased DC numbers. First, proved priming of anti-tumor CD8 T cells in dLN of we confirmed that the anti-CD1d mAb used (20H2) was WT mice treated with local radiotherapy and anti- able to inhibit the interaction of the DN32.D3 iNKT-like CTLA-4 mAb [25], we measured tumor antigen-specific hybridoma cells with α-GalCer-loaded DCs in vitro in a production of IFNγ by dLN cells stimulated with a CD8 dose-dependent manner (Additional file 1: Figure S1). In T cell epitope derived from the tumor antigen gp70 [31]. Number of CD11c+ / field Number of CD11c+ / field %ofCD11c+cells Number of CD11c+ / field Number of CD11c+/field -/- iNKT WT Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 4 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 AB C 60 10 Isotype ** 100 PBS α-CD1d α-CD1d 40 * ** 1.0 0.5 0.0 0 0 dLN Tumor CD11c+ CD11c+ CD11c+ CD11c+ CD8α+ CD8α- CD8α+ CD8α- DE F 60 60 40 40 20 20 0 0 CD11c+ CD11c+ CD11c+ CD11c+ CD11c+ CD11c+ CD8α+ CD8α- CD8α+ CD8α- CD8α+ CD8α- Figure 2 Blocking CD1d enhances DC numbers in 4T1 tumor-bearing WT mice. WT mice were inoculated with 4T1 cells on day 0. On days 3, 7 and 11 mice were given either PBS (A) or Isotype mAb (B-F) (black bars) or anti-CD1d mAb (A-F) (white bars) i.p. (A) Tumors and dLN were excised on day 13, and tissue sections stained with anti-CD11c mAb. Numbers are the mean ± SD of 3 mice/group. Results are representative of two experiments. (B-F) In a separate experiment using the same blocking regimen, tumors were digested for flow cytometry analysis of dissociated cells (N = 9/group). To obtain sufficient material, each sample was prepared by pooling tumors from 3 mice, and 3 independent samples/group analyzed. (B) Samples were gated on CD45 + CD11c + cells, and analyzed for expression of (B) CD8α,and (C) CD40, (D) CD86, * ** (E) CD70 and (F) MHC Class I molecules. p < 0.05, p < 0.005. No IFNγ production was detected in dLNs from WT Overall, data strongly support the conclusion that mice that were untreated or treated with RT or anti- iNKT cells perform a regulatory function and hinder the CTLA-4 alone (Figure 3). However, the combination of development of anti-tumor T cells in 4T1 tumor-bearing RT and anti-CTLA-4 induced significant tumor-specific mice by controlling DC population that cross-present IFNγ production (p < 0.005 versus all other groups), as tumor-derived antigens in dLNs. expected given the therapeutic synergy we have previ- ously shown with this combination [25,29,32]. Import- CD1d blockade improves response to treatment with antly, the response was significantly enhanced by radiotherapy and anti-CTLA-4 in WT mice blockade of CD1d (mean IFN-γ 248.50 ± 67.41 pg/mL in Next, we investigated whether the improved anti-tumor RT + anti-CTLA-4 + anti-CD1d versus 88.62 ± 19.80 pg/ T cell response seen in the presence of CD1d blocking mL in RT + anti-CTLA-4, p < 0.005). Interestingly, tumor mAb results in improved tumor response to treatment. antigen-specific IFN-γ production was detected in dLNs To this end, WT 4T1 tumor-bearing mice were treated −/− from untreated iNKT mice (33.08 ± 10.04 pg/mL for with RT + anti-CTLA-4 in the presence or absence of AH1-A5 versus 9.65 ± 10.86 pg/mL for pMCMV, p = 0.05) CD1d blockade (Figure 4A). Treatment with RT and anti- (Figure 3), a finding consistent with the ability of these CTLA-4 was effective at controlling the primary irradiated mice to inhibit lung metastases in a CD8-dependent man- tumor and extending significantly mice survival compared ner [25]. This response was not affected by radiotherapy to control (median survival 41 versus 32.5 days, p < 0.05) or anti-CTLA-4 as single treatment, but was dramatically (Figure 4B and C), as previously shown [29]. However, no increased by their combination (696.12 ± 109.22 pg/mL, complete tumor regression was achieved and all mice p < 0.001 versus control). Remarkably, IFNγ response eventually succumbed to metastases. CD1d blockade did of dLN cells from mice treated with radiotherapy + not have any effect by itself on tumor growth or survival, −/− anti-CTLA-4 was seven fold higher in iNKT com- but it improved control of the irradiated tumor (p < 0.05) pared to WT mice. and survival (p < 0.05) of mice treated with RT and anti- %CD86 Number of CD11c+ / field %CD70 %CD45+ cells %MHC I %CD40 Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 5 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 −/− WT and iNKT mice and were not enhanced by CD1d *** blockade in WT mice. Taken together, these results indicate that iNKT cells ** can actively suppress the development and/or function of 500 *** anti-tumor T cells and impair response to anti-CTLA-4 immunotherapy in 4T1 tumor-bearing mice. AH-1-A5 ** iNKT cells regulate the response of 4T1 tumor-bearing pMCMV mice to the combination of local radiotherapy and CD137 costimulation To determine whether negative regulation by iNKT cells could influence the response to a different immunother- apy, we tested treatment with local radiotherapy in com- -/- bination with an agonistic anti-CD137 (4-1BB) mAb BALB/c WT iNKT Strain (Figure 7A). CD137 is a member of the tumor necrosis α-CTLA4 - + + + - + + - receptor superfamily that is upregulated shortly after RT - - +- + + - + + T-cell activation [33]. CD137 ligation delivers a strong α-CD1d -- - - + - -- - survival signal to T-cells, stimulates their effector func- Figure 3 CD1d blockade enhances IFN-γ response by tion and promotes their differentiation into memory tumor-specific cells generated by RT + anti-CTLA-4 blockade. −/− cells [34-37]. Importantly, we previously demonstrated WT or iNKT mice were treated with local tumor radiotherapy in two fractions of 12 Gy given on days 13 and 14 post-tumor inoculation. that the combination of radiotherapy and CD137 costi- Mice received anti-CTLA-4 mAb on days 15, 18 and 21. Some mice mulation was significantly more effective than each treat- additionally received anti-CD1d mAb on days 3, 7 and 11 post tumor ment alone in improving survival of mice with the inoculation. Cells from tumor-draining lymph nodes were collected on intracranial GL261 glioma achieving tumor eradication in day 23 and stimulated with feeder cells pre-loaded with irrelevant the majority of mice [38]. peptide pMCMV (open circles) or AH-1-A5 (filled circles). Secreted IFN-γ −/− in the supernatant was measured 48 hours later. Each symbol In both WT and iNKT mice anti-CD137 mAb given represents an individual mouse. Bars indicate the mean ± SD of as a single agent did not enhance survival compared to * ** *** 5mice/group. p < 0.05, p < 0.005, p < 0.0005. control untreated mice (median survival in WT mice: 39 days in control vs 40 days in anti-CD137 group, p = −/− CTLA-4 (Figure 4B and C). Complete tumor regression 0.36; iNKT mice: 42 days in control vs 46 days in was seen in 25% of the mice and these mice survived anti-CD137 group, p = 0.33, Additional file 4: Figure S4). long-term and rejected a challenge of 4T1 cells 150 days Consistent with previous observations that radiotherapy after the initial inoculum (data not shown). Interestingly, alone does not inhibit 4T1 lung metastases [29], median −/− CD1d blockade did not change significantly intra-tumoral survival in WT or iNKT mice given radiotherapy was levels of TNF-α, IL-10, and IL-4 in mice treated with radi- not enhanced significantly (WT mice: 42 days, p = 0.75 −/− ation and anti-CTLA-4 (Figure 5B-D). IL-17 was un- compared to control; iNKT mice: 40 days, p = 0.98 detectable in all conditions (data not shown). However, compared to control). In contrast, radiotherapy and anti- CD1d blockade enhanced significantly the levels of IFNγ CD137 in combination significantly prolonged survival in induced in tumors by treatment with radiation and anti- WT (median survival 56 days, p < 0.05 compared to con- −/− CTLA-4 (Figure 5A), suggesting that iNKT cells hinder trol) and iNKT (median survival 60 days, p < 0.005 therapeutically effective Th1 responses in WT mice. Con- compared to control) mice (Figure 7C). Importantly, no −/− sistently, intra-tumoral levels of IFNγ were high in iNKT complete tumor regression was achieved in WT mice mice treated with radiation and anti-CTLA-4 and were treated with this combination and all mice eventually suc- not further increased by CD1d blockade. Similarly, IFNγ cumbed to metastases. In contrast, complete tumor re- was produced at significantly higher levels by spleen cells gression and long-term survival was achieved in 50% of −/− from mice treated with radiation and anti-CTLA-4 upon iNKT mice treated with radiotherapy + anti-CD137 −/− ex vivo activation in both WT and iNKT mice, and it (Figure 7C). Furthermore, all of the long-term regressors was further increased by CD1d blockade in WT but not were able to reject a tumorigenic challenge of viable 4T1 −/− iNKT mice (Figure 6A). Interestingly, while IL-10 levels cells 120 days after the initial tumor implantation, indicat- were low and were not affected by any of the treatments, ing that they had developed a long-lasting protective im- IL-4 was produced at significantly higher levels by spleen munological memory to the tumor (Figure 7D). To cells of mice treated with RT + anti-CTLA-4 compared to determine if blockade of CD1d in the context of radio- control (Figure 6B and C). However, this effect was inde- therapy and CD137 costimulation resulted in improved pendent from iNKT cells since IL-4 levels were similar in priming of anti-tumor CD8 T cells in dLN of WT mice IFNg (pg/mL) Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 6 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 A Treatment Schedule: Day 15 Day 18 Day 21 Day 0 Day 11 Day 13 Day 14 Day 3 Day 7 α-CD1d 4T1 tumor 12 Gy RT α-CTLA4 inoculation B C 900 100 PBS 800 α-CD1d ** RT+ α-CTLA4 ** PBS RT+ α-CTLA4 α-CD1d ** + α-CD1d RT+α-CTLA4 ** RT+α-CTLA4 + α-CD1d 0/8 2/8 0 0 10 15 20 25 30 35 10 20 30 40 50 60 70 80 100 160 Days Post Tumor Inoculation Days Post Tumor Inoculation Figure 4 CD1d blockade improves response of WT mice to treatment with radiotherapy and anti-CTLA-4. (A) WT 4T1 tumor-bearing mice (N = 8 mice/group) were treated with local tumor radiotherapy in two fractions of 12 Gy given on days 13 and 14 post-tumor inoculation. Mice received anti-CTLA-4 mAb on days 15, 18 and 21. Some mice additionally received anti-CD1d mAb on days 3, 7 and 11 post tumor inoculation. (B) Tumor growth is shown for each group until the last day when all mice were alive, day 28 for control and anti-CD1d treated groups, and day 35 for RT + anti-CTLA-4-treated groups. Fractions indicate the number of mice showing complete tumor regression over the total in each group. (C) Surviving tumor-free mice were followed until day 150. Data shown are from one of 2 independent experiments performed with similar * ** results. p < 0.05, p < 0.005. similar to that observed during anti-CTLA-4 blockade have previously shown that the combination of local (Figure 3), we measured tumor antigen-specific produc- radiotherapy with anti-CTLA-4 induces a CD8 T cell- tion of IFNγ by dLN cells. While mice treated with anti- mediated anti-tumor response effective at controlling CD137 monotherapy did not show any peptide-specific the irradiated tumor as well as systemic metastases lead- IFNγ production, treatment with the combination of RT ing to increased survival of 4T1 tumor-bearing mice and anti-CD137 induced significant tumor-specific IFNγ [29,32,40]. However, cures were rarely seen and most −/− production in both WT and iNKT mice (Figure 8). Im- mice eventually succumbed to the tumor. Intriguingly, −/− portantly, the response was markedly higher in iNKT response to radiotherapy and anti-CTLA-4 was mark- mice and was significantly enhanced by in vivo blockade edly improved in mice lacking iNKT cells, achieving of CD1d in WT (mean IFN-γ 109.03 ± 13.85 pg/mL in cures in 50% of the mice [25]. The improved response RT + anti-CD137 + anti-CD1d versus 71.58 ± 17.06 pg/mL could not be attributed to altered immunogenicity of −/− in RT + anti-CD137, p < 0.05) but not iNKT−/− mice. 4T1 cells in iNKT mice and could not be recapitu- Overall, these results indicate that iNKT cells hinder lated in WT mice by administration of α-GalCer, a po- priming of tumor-specific CD8 T cells independent of tent activator of iNKT cells anti-tumor activity in many the immune response modifier used in combination with tumor models [12,13,41-44]. Here we show that the im- radiotherapy to treat 4T1 tumor-bearing mice. proved response of 4T1 tumor-bearing mice seen in the absence of iNKT cells is not unique to anti-CTLA-4- Discussion based immunotherapy. Co-stimulation of T cells by anti- The 4T1 mouse mammary carcinoma is considered a CD137 in combination with radiotherapy was similarly −/− model for the aggressive triple negative subtype of hu- more effective in iNKT than WT mice and resulted in man breast cancer [39]. 4T1 cells are highly metastatic protective anti-tumor recall responses. These data fur- and relatively resistant to many immunotherapies once ther establish a negative regulatory role of iNKT cells in established, including anti-CTLA-4 treatment [29]. We the 4T1 tumor model. Tumor volume (mm ) Percent survival Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 7 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 AB 400 2000 IFNγ TNF-α 300 1500 200 1000 100 500 0 0 CD IL-10 IL-4 -/- -/- Strain BALB/c WT iNKT Strain BALB/c WT iNKT α-CTLA4 - + - + + + + + α-CTLA4 - + - + + + + + - - RT - + + - + + RT - - - + + - + + -- - + - - - α-CD1d -- - + - - - + α-CD1d + −/− Figure 5 CD1d blockade enhances intratumoral IFN-γ response induced by RT + anti-CTLA-4 blockade. WT and iNKT mice were treated with local tumor radiotherapy in two fractions of 12 Gy given on days 13 and 14 post-tumor inoculation. Mice received anti-CTLA-4 mAb on days 15, 18 and 21. Some mice additionally received anti-CD1d mAb or isotype mAb on days 3, 7 and 11 post tumor inoculation. Tumors were harvested at day 22 and concentration of (A) IFN-γ, (B) TNF-α, (C) IL-10 and (D) IL-4 were determined. Bars indicate the mean ± SD of 4 mice/group. p < 0.05. To understand the mechanisms of immune regulation their ability to kill DCs ex vivo, but human iNKT cells by iNKT cells we examined DC, which are largely CD1d , have been shown to kill DCs in vitro [52], and it is pos- and are the APCs required for cross-presentation of sible that a similar mechanism is responsible for DC re- tumor antigens to T cells [45,46]. Surprisingly, there was a duction in 4T1 tumor-bearing mice. reduction in the number of DCs present in the tumor and Blockade of CD1d in WT mice increased not only locor- −/− dLN of WT mice compared to iNKT mice (Figure 1). egional DC numbers, but also the priming of tumor- Downregulation of surface CD11c molecules has been re- specific CD8 T cells in dLN and the overall tumor ported in TLR-activated mouse DCs [47]. We did not see response to treatment with radiotherapy and anti- any difference by flow cytometry in the levels of CD11c in CTLA-4 mAb, suggesting that temporarily “disabling” DCs isolated from non-draining LN, dLN and tumors iNKT cells was sufficient to enhance Th1 type anti- (not shown), suggesting that CD11c downregulation in- tumor immunity. The increased production of IFNγ duced by activation of DCs in the tumor of WT mice is in the tumor and spleen of WT mice treated with not likely to account for locoregional reduction of DCs. RT + anti-CTLA-4 in the presence of CD1d blockade Moreover, blocking the interaction of iNKT cells with DCs supports this interpretation. with anti-CD1d mAb led to an increase in both CD8α + The overall better anti-tumor immunity and the high −/− (immunostimulatory) and CD8α- DCs in WT mice with- levels of IFNγ seen in treated iNKT mice could be out affecting their activation state as measured by expres- due to a larger population of immunostimulatory DC sion of costimulatory molecules (Figure 2). Overall, data presenting tumor-derived antigens, but may also reflect indicate that iNKT cells regulate DCs numbers in the skewing of the immune system that develops in BALB/c tumor and dLN. mice in the absence of iNKT cells towards Th1 rather Clearance of APC has been proposed as a mechanism than Th2 immunity [53]. IL-4 producing iNKT cells to prevent unrestrained T-cell activation and prolifera- (NKT2) were recently described and shown to be the tion, which is beneficial in preventing systemic auto- dominant iNKT subset in the thymus of BALB/c mice, immunity [48] but detrimental for anti-tumor responses and were implicated in determining the Th2 dominance of [49]. Several immune cells, including Tregs [26], NK this mouse strain [53]. Among tumor-infiltrating iNKT cells [50] and CD8 CTLs [49,51], have been implicated cells, we found a larger percentage (6%) that expressed in direct elimination of DCs. We were unable to isolate markers (CD27 + CD122-) shown to define iNKT2 subset, tumor-infiltrating iNKT in sufficient quantities to test while only 0.1% had markers of iNKT1 (CD27 + CD122+) pg/mL pg/mL pg/mL pg/mL Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 8 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 In the spleen IL-4 was increased similarly in treated WT ** −/− and iNKT mice (Figure 6C). IFN-γ iNKT cell role in promoting immune tolerance has been ** established in several experimental conditions, including ** autoimmune disease models, organ transplant and toler- ance to oral antigens [23]. While multiple mechanisms have been suggested to play a role, to our knowledge con- trol of DCs numbers by iNKT cells has not been previ- ously documented in immune tolerance models or in the setting of cancer [23,24,54,55]. However, evidence that hu- IL-10 man iNKT cells can kill DCs in vitro [52], suggests that such mechanism could contribute to immunosuppression in some tumors and be responsible, at least in part, for the lack of clinical activity of α-GalCer in cancer patients 10 [56,57]. Consistent with this hypothesis, administration of α-GalCer to 4T1 tumor-bearing mice at day 15 post- tumor inoculation failed to show any effect by itself or in combination with immunotherapy [25]. By this time, DCs C are low in tumors and dLN (Figure 1), and the remaining IL-4 DCs may be insufficient to present α-GalCer and/or may have acquired a tolerogenic phenotype. ** ** The identity of the CD1d ligand(s) presented by DCs to iNKT cells in 4T1 tumors and dLN is unknown. The endogenous lipids presented by CD1d in steady state conditions are likely to be non-antigenic or elicit toler- ance. However, metabolic changes in the tumor may lead -/- Strain BALB/c WT iNKT to altered lipid biosynthesis and generation of qualitatively or quantitatively modified CD1d ligands capable of trig- α-CTLA4 - + - + + + + + gering a different functional response of iNKT cells, fur- - - RT - + + - + + ther modulated by the inflammatory environment [23]. -- + - - - α-CD1d Improved understanding of the relationship between lipid Figure 6 CD1d blockade enhances systemic IFN-γ response metabolism in cancer and immune regulation will pave −/− induced by RT + anti-CTLA-4 blockade. WT or iNKT mice were the way to the design of more effective immunotherapies treated with local tumor radiotherapy in two fractions of 12 Gy that can enlist the power of iNKT cells to elicit strong given on days 13 and 14 post-tumor inoculation. Mice received anti-CTLA-4 mAb on days 15, 18 and 21. Some mice additionally anti-tumor immunity [58]. received anti-CD1d mAb or isotype mAb i.p. on days 3, 7 and 11 post tumor inoculation. Spleen were harvested at day 22 and Conclusions concentration of (A) IFN-γ, (B) IL-10 and (C) IL-4 were measured in In this study we show that local reduction of DCs medi- supernatants of PMA + ionomycin stimulated cells. Bars indicate the ** ated by iNKT cells regulates the response to immunother- mean ± SD of 4 mice/group. p < 0.005. apy in a mouse model of aggressive breast cancer. The elimination of APC that cross-present tumor-derived anti- gens in dLNs was shown previously to be one mechanism cells (Additional file 5: Figure S5). However, the majority of immunosuppression mediated by Tregs [26]. However, was CD27-CD122-, a phenotype associated with IL-17 iNKT cells were believed to consistently perform anti- production in the thymus [53]. Since IL-17 was undetect- tumor activities, while immunosuppressive functions were able in 4T1 tumors, defining the functional differentiation ascribed to the non-invariant subset of NKT cells [59]. of iNKT cells in the tumor microenvironment may require Here we demonstrate for the first time a novel mechanism different markers than in the thymus. Importantly, com- of immune escape mediated by iNKT cells. parison between IL-4 levels produced in the tumor and These results have important implications for the de- −/− spleen of tumor-bearing WT and iNKT mice did not sign of immunotherapies targeting iNKT cells (reviewed reveal significant differences consistent with differential in [58]). CD1d blockade, or the iNKT-depleting antibody Th2 polarization. Intratumoral IL-4 levels were higher in NKTT120 recently developed for inflammatory diseases −/− treated WT than iNKT mice, but unlike IFNγ,IL-4 [60], could have a role in treatment of cancers in which levels were not modulated by CD1d blockade (Figure 5D). iNKT acquire immunosuppressive functions. pg/mL pg/mL pg/mL Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 9 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 A Treatment Schedule: Day 0 Day 13 Day 14 Day 15 Day 18 Day 21 4T1 tumor 12 Gy RT α-CD137 inoculation B C D 800 100 WT: Control Naive WT (n=5) WT: RT+α-CD137 -/- -/- * Surviving iNKT (n=3) 80 iNKT :Control 600 -/- ** iNKT :RT+ α-CD137 WT: Control 60 WT: RT+α-CD137 -/- iNKT :Control * -/- iNKT :RT+ * α-CD137 0/5 3/6 0 20 40 60 80 100 10 15 20 25 30 35 10 15 20 25 30 35 40 Days Post Tumor Inoculation Days Post Tumor Inoculation Days Post Tumor Inoculation Figure 7 Response of 4T1 tumor-bearing mice to treatment with local radiotherapy and anti-CD137 mAb is improved in the absence −/− of iNKT cells. WT or iNKT mice were injected s.c. with 4T1 cells and randomly assigned to treatment groups (N = 5-6 mice/group) on day 13 when tumors became palpable. Local tumor radiotherapy (RT) in two fractions of 12 Gy was given on days 13 and 14 post-tumor inoculation and anti-CD137 mAb on days 15, 18 and 21. (A) Treatment schema. (B) Tumor growth over time. Fractions indicate the number of mice showing −/− complete tumor regression. (C) Kaplan-Meier survival curves. (D) Tumor growth in naïve WT mice and iNKT mice that rejected tumors and were challenged on day 120 with 4T1 cells. Data is representative of 2 independent experiments. p < 0.05. Methods Animals Cells, antibodies and reagents Six to eight-week old WT BALB/c mice were purchased −/− 4T1 is a BALB/c-derived mammary carcinoma that is from Taconic. iNKT (Vα14Jα18-deficient) mice [27] in highly metastatic and mimics the behavior of triple the BALB/c background obtained from M. Taniguchi were negative human breast cancer [39,61]. 4T1 cells were bred at Berg Animal Facility at NYU School of Medicine grown in complete medium consisting of DMEM and used between 6 to 8 weeks of age. All experiments (Invitrogen) supplemented with 2 mol/L L-glutamine, were approved by the Institutional Animal Care and Use 100 U/mL penicillin, 100 μg/mL streptomycin, 2.5 × Committee of NYU Langone Medical Center. −5 10 mol/L 2-mercaptoethanol and 10% fetal bovine serum (BioWest). Cells were confirmed to be free of In vivo treatment mycoplasma contamination using a mycoplasma de- Mice were inoculated subcutaneously (s.c.) with 5 × 10 tection kit (Sigma). DN32.D3 hybridoma cells, kindly 4T1 cells and randomly assigned to treatment groups provided by Masaki Terabe from the NCI Branch of 13 days later when tumors reached an average diameter the National Institutes of Health, were grown in supple- of 5 mm. Radiotherapy was given as previously described mented RPMI (Invitrogen) [62]. Anti-CTLA-4 hamster [25] with some modifications. Lightly anesthetized mice monoclonal antibody (Clone 9H10) and anti-CD1d rat were positioned on a dedicated plexiglass tray with only monoclonal antibody (Clone 20H2) were purchased the tumor area exposed to radiation while the rest of the from BioXCell (West Lebanon, NH). Control hamster body was protected by lead shielding. Radiotherapy was IgG (isotype control for anti-CTLA-4) was purchased given using the Small Animal Radiation Research Plat- from Jackson Immunoresearch Laboratories. Control rat form (SARRP) (Gulmay Medical, Suwanee, GA) [63] in IgG1 (isotype control for anti-CD1d) was purchased from two doses of 12 Gy each on days 13 and 14 post tumor BioXCell. A rat IgG2a mAb against mouse CD137 (BMS- inoculation. Control hamster IgG and anti-CTLA-4 or 469492, clone 1D8) was provided for these studies by anti-CD137/4-1BB mAbs were given at 200 μg i.p. at 1, Bristol-Myers Squibb (Princeton, NJ). 4 and 7 days after completion of radiotherapy. To block Tumorvolume (mm ) ± SD Percent survival Tumor volume(mm ) ± SD Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 10 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA) at a 1/2,000 dilution in 1% BSA for 30 minutes. AH-1-A5 To amplify the signal, slides were subject to two rounds of pMCMV biotin-tyramide amplification (PerkinElmer, Melville, NY) using manufacturer’s suggested protocol. The slides were counterstained with DAPI, mounted with Vectashield and images obtained using a Nikon Eclipse 800 deconvolution *** microscope. Positive cells were counted from at least 3 randomly selected fields (20× magnification). Flow cytometry Lymph nodes and minced tumors were digested using a -/- Strain BALB/c WT iNKT cocktail of 1.67 Wünsch U/mL Liberase TL (Roche) and 0.2 mg/mL DNAse (Roche) as previously described α-CD137 - - + + + + + [40,64]. After lysis of red blood cells single cell suspen- RT - - + + + + + sions were filtered through a 40-μm nylon cell strainer. -- - α-CD1d - - + + Live cells were distinguished using Fixable Viability Dye Figure 8 CD1d blockade enhances production of IFN-γ eFluor 660 (eBioscience) prior to flow staining. All sam- by tumor-specific cells generated by RT + anti-CD137 ples were incubated with anti-mouse CD16/32 (Fc block) −/− co-stimulation. WT or iNKT mice were treated with local for 10 minutes followed by staining for various surface tumor radiotherapy in two fractions of 12 Gy given on days 13 markers for 30 minutes at 4°C. The following antibodies and 14 post-tumor inoculation. Mice received anti-CD137 mAb on days were used (all purchased from eBiosciences): PE-Cy7- 15, 18 and 21. Some mice additionally received anti-CD1d mAb or isotype control on days 3, 7 and 11 post tumor inoculation. Cells from conjugated anti-mouse CD11c, Alexa Fluor 700-conjugated tumor-draining lymph nodes were collected on day 23 and stimulated anti-mouse CD86, PE-conjugated anti-mouse CD80, PE- with feeder cells pre-loaded with irrelevant peptide pMCMV (open conjugated anti-mouse CD1d, PE-conjugated anti-mouse circles) or AH-1-A5 (filled circles). Secreted IFN-γ in the supernatant was CD70, FITC-conjugated anti-mouse CD40 and Alexa measured 48 hours later. Each symbol represents an individual mouse. * *** Fluor 700-conjugated anti-mouse MHC Class I (H-2Kd/ Bars indicate the mean ± SD of 3–5 mice/group. p <0.05, p <0.0005. Dd), FITC-conjugated anti-mouse CD27, PECy7-conjugated anti-mouse CD3ε, APC-conjugated anti-mouse CD122, CD1d in vivo mice were given three doses of anti-CD1d Alexa Fluor 700-conjugated anti-mouse CD4. iNKT cells mAb (Clone 20H2) at 100 μg i.p. on days 3, 7 and 11 were identified by staining with mouse CD1d/PBS-57 or post tumor inoculation. Tumors were measured every CD1d/unloaded tetramers (NIH Tetramer Core Facility) 2–3 days until death or sacrifice when tumor dimensions for 30 minutes at room temperature [65]. All samples exceeded 5% of body weight or if animals showed signs were analyzed using a BD LSR II flow cytometer and of significant pain or distress due to metastatic disease. FlowJo version 8.7.5 (Tree Star). In some experiments, mice that eradicated the tumor after treatment and remained tumor-free for at least 120 days were inoculated in the contralateral flank with Ex vivo cytokine production 4T1 cells and tumor growth monitored. A group of Single cell suspensions (3 × 10 per well) from draining −/− naïve mice was similarly challenged with 4T1 cells as lymph nodes of 4T1 tumor-bearing WT or iNKT mice control. were cultured in complete T-cell medium in 48-well tissue culture plates with irradiated (12 Gy) feeder cells (3 × 10 ) Immunohistochemistry prepared from the spleen of healthy BALB/c mice 4T1 tumors and dLNs were harvested on indicated days, pre-loaded with tumor-derived peptides (AH-1-A5) or fixed in freshly prepared 4% paraformaldehyde for 1 hr at an H-2L -binding irrelevant peptide (pMCMV) used at a 4°C and incubated overnight in 30% sucrose. Tumors were final concentration of 1 μg/mL. Peptide sequences are then frozen in optimum cutting temperature (OCT) SPSYAYHQF (AH-1-A5) [66], and YPHFMPTNL (pMCMV) medium and stored at −80°C. Sections (5 μm) were treated [66]. After 48 hours, supernatants were collected and with 3% H O in PBS for 30 minutes in order to eliminate stored at −80°C. Secreted IFN-γ was measured from 2 2 endogenous peroxide activity. After blocking in 4% ham- duplicate wells using Flowcytomix kit (eBioscience, ster serum for an hour and three washes with PBS, anti- SanDiego,CA).Background IFN-γ production from mouse CD11c eFluor 615 (eBioscience, San Diego, CA) supernatants of cells cultured in complete T-cell media was applied at a 1/100 dilution in 1% BSA for 1 hour, alone was subtracted. Spleen cell suspensions (10 ) from followed by three washes and incubation in HRP- 4T1 tumor-bearing mice were cultured in a 24-well tissue IFNg(pg/mL) Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 11 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 culture plate and stimulated with PMA + ionomycin Cell Additional file 4: Figure S4 Local tumor radiotherapy and CD137 Stimulation Cocktail Mix (eBioscience) for 48 hours. co-stimulation used alone are ineffective in prolonging survival of −/− 4T1 tumor-bearing mice. WT and iNKT mice were injected s.c. with 4T1 cells and randomly assigned to treatment groups (n = 5-6/group) on day 13 when tumors became palpable. Local tumor radiotherapy (RT) Intratumoral cytokines was given in two fractions of 12 Gy on days 13 and 14 post-tumor Freshly harvested tumors were immediately frozen in li- inoculation. Mice received anti-CD137 mAb on days 15, 18 and 21. quid nitrogen and stored at −80°C. To isolate total protein, Kaplan-Meier curves were generated from survival data of each mouse calculated as time (days) from date of inoculation until death or euthanasia. tissues were thawed on ice, minced and homogenized in Median survival (days) in each treatment group is indicated in parenthesis. lysis buffer (10 mM Tris–HCl pH8.0, 150 mM NaCl, 1% Data is representative of 2 independent experiments. NP-40, 10% glycerol, 5 mM EDTA) and protease inhibitor Additional file 5: Figure S5 iNKT subset compartments in 4T1 cocktail (Roche). Total volume (μL) of lysis buffer used tumors and tumor-draining lymph nodes. WT mice were injected s.c. with 4T1 cells. On day 15, tumor samples from three mice were pooled was equal to 5× weight of tumor tissue (mg). Lysates were and digested to obtain single cell suspensions. (A) Intratumoral iNKT cells sonicated in 4°C using a 7-minute regimen of 30s intermit- were identified using mouse CD1d/PBS-57 tetramers. Expression of surface tent on and off. Samples were spun at 14,000 rpm for CD122 and CD27 antigens were assessed on CD3+ CD1d/PBS-57 tetramer + populations to identify iNKT1 (CD27 + CD122+) and iNKT2 (CD27-CD122+) 15 minutes at 4°C, supernatants collected and protein con- subsets. (B) The relative abundance of iNKT cells in the tumor and draining centrations determined using Bradford assay. Samples lymph nodes is shown as a percentage of total CD3 cells. (C) The relative were stored at −80°C. Cytokines were measured from du- abundance of iNKT1 (white bars) and iNKT2 (black bars) subsets is shown as a percentage of total CD3+ CD1d/PBS-57 tetramer + cells. Bars indicate the plicates wells using multi-analyte Flowcytomix kit. ** mean ± SD of three samples. p < 0.005. Statistical analysis Abbreviations Random coefficients regression (RCR) was used to assess α-GalCer: Alpha galactosylceramide; APC: Antigen presenting cells; BMDC: Bone the effect of treatment on tumor growth. The dependent marrow-derived dendritic cells; CD: Cluster of differentiation; CTL: Cytotoxic T lymphocytes; DCs: Dendritic cells; dLN: Draining lymph node; Gy: Gray; variable was the natural log of tumor volume at all avail- −/− IFN-γ: Interferon gamma; IHC: Immunohistochemistry; iNKT : Invariant natural able times; log volumes were used since the change in killer T cell-deficient; i.p.: Intraperitoneal; mAb: Monoclonal antibody; log volume over the course of follow-up was well ap- MHC: Major histocompatibility complex; ndLN: Non-draining lymph node; NK: Natural killer; PBS: Phosphate buffered saline; PMA: Phorbol 12-myristate proximated as linear within each treatment arm. Log rank 13-acetate; RT: Radiotherapy; s.c.: Subcutaneous; TAA: Tumor-associated tests were used to compare treatment arms in terms of sur- antigens; Th1: T-helper type 1; Th2: T-helper type 2; Tregs: Regulatory T cells; vival. The Kaplan-Meier method was used to estimate me- WT: Wild type. dian survival times and the log cumulative hazard transformation was used to derive 95% confidence limits for Competing interests The authors declare that they have no competing interests. median survival in each arm. All reported p values are two- sided and statistical significance defined as p < 0.05. SAS 9.3 Authors’ contributions (SAS Institute, Cary, NC) was used for all computations. KAP designed and performed all experiments and wrote the manuscript. JA carried out in vitro studies and immunostaining and contributed to writing the manuscript. SD conceived and designed the study with KAP and wrote Additional files the manuscript. JSB performed statistical analysis. All authors read and approved the final manuscript. Additional file 1: Figure S1 Anti-CD1d mAb blocks cytokine production by DN32.D3 NKT-like hybridoma cells stimulated with Authors’ information α-GalCer-loaded DCs. CD11c bone-marrow derived dendritic cells KAP started his postdoctoral fellowship at NYU School of Medicine and is (BMDC) from healthy BALB/c mice were plated in 24-well tissue culture 4 currently an associate research scientist in the department of Pathology. JA is plate (2×10 /well) and incubated with 100 ng/mL α-GalCer overnight, an undergraduate student at NYU College of Arts and Sciences. JSB has over washed extensively and incubated overnight with fresh media containing 20 years experience as research biostatistician and is currently associate anti-CD1d at indicated final concentrations. Cells were washed again 5 professor of Radiology at NYU School of Medicine. SD holds dual before the addition of DN32.D3 hybridoma cells (10 /well). Supernatants appointments as professor of Pathology and Radiation Oncology at NYU were collected three days later and cytokines measured. School of Medicine and is Co-leader of the Cancer Immunology program of Additional file 2: Figure S2 Anti-CD1d mAb does not deplete the NYU Cancer Institute. CD1d-expressing cells in vivo. Healthy WT mice (N = 4/group) were given isotype or anti-CD1d mAb i.p. (200 μg/mouse) every 4 days for a total of 3 doses. Two days after the last dose, mice were euthanized and Acknowledgments single cell suspensions from digested spleens collected for flow staining KAP was supported by a Postdoctoral Research Fellow Award (W81XWH-10- of (A) DC, (B) myeloid cells, (C) B-cells and (D) macrophage populations. 1-0007) from the US Department of Defense Breast Cancer Research Program. S. Demaria is supported by grants from the Chemotherapy Additional file 3: Figure S3 Anti-CD1d mAb does not induce Foundation, the US Department of Defense Breast Cancer Research Program stimulation of CD1d-expressing APCs. Splenocytes from naïve WT −/− (W81XWH-11-1-0532), and the Breast Cancer Alliance. The Histopathology or iNKT mice (n = 4/group) were stimulated in vitro with 10 μg/mL and Flow Cytometry Core Facilities of the Laura and Isaac Perlmutter α-CD1d or isotype control mAbs for 48 hours. As a positive control, Cancer Center at the NYU Langone Medical Center are partially supported splenocytes were stimulated with LPS (1 μg/mL). Supernatants were by NIH/NCI P30CA016087. The authors wish to thank Maria Jure-Kunkel collected and measured for secreted (A) IL-10, (B) IFN-γ, (C) IL-12p70 (Bristol-Myers) for anti-CD137 mAb. We acknowledge the NIH Tetramer Core and (D) IL-4. Facility (contract HHSN272201300006C) for provision of CD1d tetramers. Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 12 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 Author details 17. 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Hegde S, Jankowska-Gan E, Roenneburg DA, Torrealba J, Burlingham WJ, • Inclusion in PubMed, CAS, Scopus and Google Scholar Gumperz JE: Human NKT cells promote monocyte differentiation into • Research which is freely available for redistribution suppressive myeloid antigen-presenting cells. J Leukoc Biol 2009, 86:757–768. 56. Giaccone G, Punt CJ, Ando Y, Ruijter R, Nishi N, Peters M, von Blomberg BM, Submit your manuscript at Scheper RJ, van der Vliet HJ, van den Eertwegh AJ, Roelvink M, Beijnen J, www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal for ImmunoTherapy of Cancer Springer Journals

Invariant natural killer T cells regulate anti-tumor immunity by controlling the population of dendritic cells in tumor and draining lymph nodes

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Springer Journals
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Copyright © 2014 by Pilones et al.; licensee BioMed Central Ltd.
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Medicine & Public Health; Oncology
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2051-1426
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10.1186/s40425-014-0037-x
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25349699
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Abstract

Background: Invariant natural killer T (iNKT) cells are CD1d-restricted T cells, which respond rapidly to antigen recognition and promote development of anti-tumor immunity in many tumor models. Surprisingly, we previously found that mice deficient in iNKT cells developed spontaneous CD8 T cells responses partially effective at inhibiting metastases in mice bearing the 4T1 mammary carcinoma, and showed a markedly improved response to treatment with local radiotherapy and anti-CTLA-4 antibody compared to wild type (WT) mice. Methods: To understand the mechanisms of the immunosuppressive function of iNKT cells, dendritic cells (DCs) −/− were analyzed by immunohistochemistry and flow cytometry in WT and iNKT-deficient (iNKT ) mice. The effects of antibody-mediated blockade of CD1d on DC number and phenotype, priming of anti-tumor T cells, and tumor response to treatment with local radiotherapy and anti-CTLA-4 antibody were evaluated. To determine if the improved response to treatment in the absence of iNKT cells was independent from the immunotherapy employed, 4T1-tumor −/− bearing WT and iNKT mice were treated with local radiotherapy in combination with antibody-mediated CD137 co-stimulation. Results: DCs in 4T1 tumors and tumor-draining lymph nodes but not distant lymph nodes were significantly −/− reduced in WT mice compared to iNKT mice (p < 0.05), suggesting the selective elimination of DCs cross-presenting tumor-associated antigens by iNKT cells. Consistently, priming of T cells to a tumor-specific CD8 T cell epitope in mice −/− treated with radiotherapy and anti-CTLA-4 or anti-CD137 was markedly enhanced in iNKT compared to WT mice. CD1d blockade restored the number of DC in WT mice, improved T cell priming in draining lymph nodes and significantly enhanced response to treatment. Conclusions: Here we describe a novel mechanism of tumor immune escape mediated by iNKT cells that limit priming of anti-tumor T cells by controlling DC in tumors and draining lymph nodes. These results have important implications for the design of immunotherapies targeting iNKT cells. Keywords: Breast cancer, CD1d, CD137, CTLA-4, CD8 T-cells, Dendritic cells, Immunoregulation, Invariant NKT cells, Radiotherapy * Correspondence: Sandra.Demaria@nyumc.org Department of Pathology, New York University School of Medicine, New York, NY 10016, USA Department of Radiation Oncology, New York University School of Medicine, New York, NY 10016, USA Full list of author information is available at the end of the article © 2014 Pilones et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 2 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 Background Response to treatment with local radiotherapy and anti- Natural killer T (NKT) cells comprise a subset of lympho- CTLA-4 monoclonal antibody (mAb) was also markedly −/− cytes originating from a distinct developmental lineage [1] improved in iNKT compared to wild type (WT) mice which bridge innate and adaptive immunity and modulate with half of the mice rejecting completely the primary immune responses in autoimmunity, malignancies and in- irradiated tumor and lung metastases and showing long- fections [2]. Although initially identified by co-expression term survival compared to none of the WT mice [25]. of conventional αβ T-cell receptors (TCR) and markers These data implicated iNKT cells as major regulators of typically associated with natural killer (NK) cells [3], NKT the spontaneous as well as immunotherapy-elicited anti- are currently distinguished on the basis of CD1d restric- tumor immune response to 4T1 tumor. tion as well as specific usage of TCRα chains [4]. In both CD4 regulatory T cells (Treg) have been shown to limit mice and humans, most NKT cells express TCRs formed the development of anti-tumor T cells by killing DCs in by the rearrangement of a canonical α chain (Vα14 in tumor-draining lymph nodes (dLN) [26]. DCs are a major mice, Vα24 in humans) and a limited set of Vβ chains subset of CD1d antigen-presenting cells (APC) that inter- (Vβ.2, Vβ7, Vβ2in mice, Vβ11 in humans) and are com- act with iNKT cells. Therefore, we hypothesized that monly referred to as type I or invariant natural killer T iNKT cells may be limiting the anti-tumor immune re- (iNKT) cells [5,6]. A smaller NKT cell subset utilizes a sponse in 4T1 tumor-bearing mice by controlling the more diverse set of TCR αβ chains and is referred to as loco-regional population of DCs in the tumor and dLNs. type II or non-invariant NKT cells [7]. For the first time, we present evidence that WT mice have Identification of α-galactosylceramide (α-GalCer) as a reduced numbers of DCs in tumor and dLN compared to −/− strong agonist selective for iNKT cells [8] facilitated their iNKT mice, and reduced priming of tumor-specific characterization using α-GalCer-loaded CD1d tetramers CD8 T cells, as measured by gamma interferon (IFNγ) [9]. In several tumor models, iNKT cells were found to production in response to a H2-L -restricted peptide perform important immunosurveillance functions and derived from a tumor antigen. DC numbers and tumor- become key effectors of tumor rejection when activated specific CD8 T cell responses were at least partially re- by α-GalCer [10-13]. Expression of high levels of Fas stored by blocking CD1d, resulting in improved response Ligand, perforin, and granzyme B by iNKT cells underlies to immunotherapy. Overall, data demonstrate a novel their cytolytic activity against CD1d tumor cells [14] and mechanism of immune escape mediated by iNKT cells. myeloid cells with immunosuppressive function present in the tumor microenvironment [15]. In addition, iNKT cells Results exert anti-tumor functions by rapid and robust secretion Numbers of DCs infiltrating 4T1 tumors and dLN are −/− of cytokines that improve DC ability to cross-prime anti- lower in WT compared to iNKT mice tumor T cells [10,12,16,17] and enhance recruitment of We hypothesized that the interaction of iNKT cells with + + other effectors such as NK cells, CD4 T helper-1 (Th1) CD1d APC could modulate the ability of WT mice to and CD8 cytotoxic T (CTL) cells [13,18]. develop anti-tumor immune responses upon treatment. Experimental data in different systems indicate a func- To test this hypothesis we compared DCs in healthy and −/− −/− tional plasticity of iNKT cells. iNKT cells can promote tumor-bearing WT and iNKT mice. iNKT mice the polarization of adaptive immune responses towards were generated by targeted deletion of the Jα18 segment both Th1 and Th2 and can secrete immunosuppressive of the T-cell receptor (TCR) and show selective loss of cytokines [19]. The regulatory function of iNKT cells has the Vα14Jα18 TCR expressed by iNKT cells while other been demonstrated in multiple models of autoimmune lymphoid and myeloid populations are not affected [27]. diseases in which iNKT cells played essential roles in Consistently, there were no differences in the number of maintenance of tolerance [20-22]. The mechanisms that DCs present in LNs or their expression of maturation determine whether iNKT cells act to promote immune markers CD80 and CD86, including CD1d, between WT −/− activation or tolerance remain incompletely understood, and iNKT healthy mice (Figure 1A and B). In con- but the inflammatory context in which interactions of trast, after 4T1 tumor implantation there was a differ- iNKT cells with CD1d myeloid cells take place is thought ence in the numbers of DCs in the tumor-draining but to be a key factor [23,24]. not distant LNs (Figure 1C and D), with significantly The 4T1 mouse mammary carcinoma is a model for lower numbers of DCs present in WT compared to −/− triple negative breast cancer and shows an aggressive iNKT mice. Significantly lower numbers of DCs were behavior with rapid spread of metastatic cells to the lungs also found in the tumors growing in WT compared to −/− after subcutaneous injection. We previously found that iNKT mice (Figure 1E and F). Interestingly, while −/− −/− 4T1 tumor-bearing iNKT-deficient (iNKT ) mice devel- non-draining LNs of iNKT mice showed a DC density oped a spontaneous CD8 T cell response that was par- comparable to LNs of healthy mice, DC density was tially effective at controlling metastases in the lungs [25]. markedly increased in tumor-draining LNs, suggesting Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 3 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 AB C 30 100 300 20 200 10 100 0 0 -/- WT CD80 CD86 CD1d Day 13 Day 26 iNKT DE F Day 13 Day 26 40 50 0 0 Day 13 Day 26 Day 13 Day 26 Figure 1 iNKT cells regulate the number of DCs in 4T1 tumors and draining lymph nodes. (A, B) Lymph nodes collected from healthy WT −/− (black bars) and iNKT (white bars) mice (N = 3/strain). (A) Full-face lymph node sections were stained with anti-CD11c mAb and positive cells counted. (B) Dissociated lymph node cells were gated on CD11c + cells and expression of CD1d and co-stimulatory molecules CD80 and CD86 −/− determined by flow cytometry. (C-F) WT (black bars) and iNKT (white bars) mice were inoculated s.c. with 4T1 cells. On indicated days, tumor-draining lymph nodes (C), non-draining lymph nodes (D),and tumors (E, F) were excised, tissue sections stained with anti-CD11c mAb and positive cells counted. Numbers are the mean ± SD of 3 mice of each strain analyzed at each time point. (F) Representative fields (400× magnification). Results are representative of two experiments. p < 0.05. that even in the absence of treatment the presence of addition, we verified that administration of 20H2 mAb the growing tumor leads to some activation of the re- to mice did not cause the depletion of CD1d cells gional immune system. This finding is consistent with (Additional file 2: Figure S2) nor induce their stimula- the presence of a spontaneous anti-tumor CD8 T cell tion by reverse signaling (Additional file 3: Figure S3). −/− response in iNKT mice that was partially effective at Next, anti-CD1d mAb was given to 4T1 tumor-bearing controlling metastases in the lungs as we have previously WT mice starting on day 3 post-tumor inoculation and reported [25]. tumors and dLN analyzed at day 13, a time when tumors Overall, data suggest that in WT mice iNKT cells may are irradiated in treated mice and the availability of DCs control the population of DCs in the tumor and dLN that can cross-present the tumor antigens released by thus precluding the priming of anti-tumor T cells. radiation is critical [29,30]. CD1d blockade led to a sig- nificant increase in the total number of DCs present in tu- CD1d blockade restores DC numbers and improves mors and dLNs (Figure 2A). Both immunostimulatory tumor-specific T cell cross-priming in WT 4T1 CD8α + and CD8α- DC subsets were increased (Figure 2B), tumor-bearing mice whereas expression of activation markers CD40, CD70, Elimination of DCs in dLN has been shown to be one CD86, and of MHC Class I molecules in either subsets of mechanism whereby Treg suppress the anti-tumor im- tumor infiltrating DCs were not altered by CD1d blockade mune response [26]. To determine if iNKT cells could (Figure 2C-F). Therefore, data indicate that iNKT cells be responsible for the reduction in the number of DCs regulate DC numbers in 4T1 tumor-bearing WT mice via in tumors and dLN of WT mice we tested whether inhi- CD1d-mediated interactions. biting their interaction with DCs with a mAb that blocks To determine if blockade of CD1d resulted in im- CD1d [28] would lead to increased DC numbers. First, proved priming of anti-tumor CD8 T cells in dLN of we confirmed that the anti-CD1d mAb used (20H2) was WT mice treated with local radiotherapy and anti- able to inhibit the interaction of the DN32.D3 iNKT-like CTLA-4 mAb [25], we measured tumor antigen-specific hybridoma cells with α-GalCer-loaded DCs in vitro in a production of IFNγ by dLN cells stimulated with a CD8 dose-dependent manner (Additional file 1: Figure S1). In T cell epitope derived from the tumor antigen gp70 [31]. Number of CD11c+ / field Number of CD11c+ / field %ofCD11c+cells Number of CD11c+ / field Number of CD11c+/field -/- iNKT WT Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 4 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 AB C 60 10 Isotype ** 100 PBS α-CD1d α-CD1d 40 * ** 1.0 0.5 0.0 0 0 dLN Tumor CD11c+ CD11c+ CD11c+ CD11c+ CD8α+ CD8α- CD8α+ CD8α- DE F 60 60 40 40 20 20 0 0 CD11c+ CD11c+ CD11c+ CD11c+ CD11c+ CD11c+ CD8α+ CD8α- CD8α+ CD8α- CD8α+ CD8α- Figure 2 Blocking CD1d enhances DC numbers in 4T1 tumor-bearing WT mice. WT mice were inoculated with 4T1 cells on day 0. On days 3, 7 and 11 mice were given either PBS (A) or Isotype mAb (B-F) (black bars) or anti-CD1d mAb (A-F) (white bars) i.p. (A) Tumors and dLN were excised on day 13, and tissue sections stained with anti-CD11c mAb. Numbers are the mean ± SD of 3 mice/group. Results are representative of two experiments. (B-F) In a separate experiment using the same blocking regimen, tumors were digested for flow cytometry analysis of dissociated cells (N = 9/group). To obtain sufficient material, each sample was prepared by pooling tumors from 3 mice, and 3 independent samples/group analyzed. (B) Samples were gated on CD45 + CD11c + cells, and analyzed for expression of (B) CD8α,and (C) CD40, (D) CD86, * ** (E) CD70 and (F) MHC Class I molecules. p < 0.05, p < 0.005. No IFNγ production was detected in dLNs from WT Overall, data strongly support the conclusion that mice that were untreated or treated with RT or anti- iNKT cells perform a regulatory function and hinder the CTLA-4 alone (Figure 3). However, the combination of development of anti-tumor T cells in 4T1 tumor-bearing RT and anti-CTLA-4 induced significant tumor-specific mice by controlling DC population that cross-present IFNγ production (p < 0.005 versus all other groups), as tumor-derived antigens in dLNs. expected given the therapeutic synergy we have previ- ously shown with this combination [25,29,32]. Import- CD1d blockade improves response to treatment with antly, the response was significantly enhanced by radiotherapy and anti-CTLA-4 in WT mice blockade of CD1d (mean IFN-γ 248.50 ± 67.41 pg/mL in Next, we investigated whether the improved anti-tumor RT + anti-CTLA-4 + anti-CD1d versus 88.62 ± 19.80 pg/ T cell response seen in the presence of CD1d blocking mL in RT + anti-CTLA-4, p < 0.005). Interestingly, tumor mAb results in improved tumor response to treatment. antigen-specific IFN-γ production was detected in dLNs To this end, WT 4T1 tumor-bearing mice were treated −/− from untreated iNKT mice (33.08 ± 10.04 pg/mL for with RT + anti-CTLA-4 in the presence or absence of AH1-A5 versus 9.65 ± 10.86 pg/mL for pMCMV, p = 0.05) CD1d blockade (Figure 4A). Treatment with RT and anti- (Figure 3), a finding consistent with the ability of these CTLA-4 was effective at controlling the primary irradiated mice to inhibit lung metastases in a CD8-dependent man- tumor and extending significantly mice survival compared ner [25]. This response was not affected by radiotherapy to control (median survival 41 versus 32.5 days, p < 0.05) or anti-CTLA-4 as single treatment, but was dramatically (Figure 4B and C), as previously shown [29]. However, no increased by their combination (696.12 ± 109.22 pg/mL, complete tumor regression was achieved and all mice p < 0.001 versus control). Remarkably, IFNγ response eventually succumbed to metastases. CD1d blockade did of dLN cells from mice treated with radiotherapy + not have any effect by itself on tumor growth or survival, −/− anti-CTLA-4 was seven fold higher in iNKT com- but it improved control of the irradiated tumor (p < 0.05) pared to WT mice. and survival (p < 0.05) of mice treated with RT and anti- %CD86 Number of CD11c+ / field %CD70 %CD45+ cells %MHC I %CD40 Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 5 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 −/− WT and iNKT mice and were not enhanced by CD1d *** blockade in WT mice. Taken together, these results indicate that iNKT cells ** can actively suppress the development and/or function of 500 *** anti-tumor T cells and impair response to anti-CTLA-4 immunotherapy in 4T1 tumor-bearing mice. AH-1-A5 ** iNKT cells regulate the response of 4T1 tumor-bearing pMCMV mice to the combination of local radiotherapy and CD137 costimulation To determine whether negative regulation by iNKT cells could influence the response to a different immunother- apy, we tested treatment with local radiotherapy in com- -/- bination with an agonistic anti-CD137 (4-1BB) mAb BALB/c WT iNKT Strain (Figure 7A). CD137 is a member of the tumor necrosis α-CTLA4 - + + + - + + - receptor superfamily that is upregulated shortly after RT - - +- + + - + + T-cell activation [33]. CD137 ligation delivers a strong α-CD1d -- - - + - -- - survival signal to T-cells, stimulates their effector func- Figure 3 CD1d blockade enhances IFN-γ response by tion and promotes their differentiation into memory tumor-specific cells generated by RT + anti-CTLA-4 blockade. −/− cells [34-37]. Importantly, we previously demonstrated WT or iNKT mice were treated with local tumor radiotherapy in two fractions of 12 Gy given on days 13 and 14 post-tumor inoculation. that the combination of radiotherapy and CD137 costi- Mice received anti-CTLA-4 mAb on days 15, 18 and 21. Some mice mulation was significantly more effective than each treat- additionally received anti-CD1d mAb on days 3, 7 and 11 post tumor ment alone in improving survival of mice with the inoculation. Cells from tumor-draining lymph nodes were collected on intracranial GL261 glioma achieving tumor eradication in day 23 and stimulated with feeder cells pre-loaded with irrelevant the majority of mice [38]. peptide pMCMV (open circles) or AH-1-A5 (filled circles). Secreted IFN-γ −/− in the supernatant was measured 48 hours later. Each symbol In both WT and iNKT mice anti-CD137 mAb given represents an individual mouse. Bars indicate the mean ± SD of as a single agent did not enhance survival compared to * ** *** 5mice/group. p < 0.05, p < 0.005, p < 0.0005. control untreated mice (median survival in WT mice: 39 days in control vs 40 days in anti-CD137 group, p = −/− CTLA-4 (Figure 4B and C). Complete tumor regression 0.36; iNKT mice: 42 days in control vs 46 days in was seen in 25% of the mice and these mice survived anti-CD137 group, p = 0.33, Additional file 4: Figure S4). long-term and rejected a challenge of 4T1 cells 150 days Consistent with previous observations that radiotherapy after the initial inoculum (data not shown). Interestingly, alone does not inhibit 4T1 lung metastases [29], median −/− CD1d blockade did not change significantly intra-tumoral survival in WT or iNKT mice given radiotherapy was levels of TNF-α, IL-10, and IL-4 in mice treated with radi- not enhanced significantly (WT mice: 42 days, p = 0.75 −/− ation and anti-CTLA-4 (Figure 5B-D). IL-17 was un- compared to control; iNKT mice: 40 days, p = 0.98 detectable in all conditions (data not shown). However, compared to control). In contrast, radiotherapy and anti- CD1d blockade enhanced significantly the levels of IFNγ CD137 in combination significantly prolonged survival in induced in tumors by treatment with radiation and anti- WT (median survival 56 days, p < 0.05 compared to con- −/− CTLA-4 (Figure 5A), suggesting that iNKT cells hinder trol) and iNKT (median survival 60 days, p < 0.005 therapeutically effective Th1 responses in WT mice. Con- compared to control) mice (Figure 7C). Importantly, no −/− sistently, intra-tumoral levels of IFNγ were high in iNKT complete tumor regression was achieved in WT mice mice treated with radiation and anti-CTLA-4 and were treated with this combination and all mice eventually suc- not further increased by CD1d blockade. Similarly, IFNγ cumbed to metastases. In contrast, complete tumor re- was produced at significantly higher levels by spleen cells gression and long-term survival was achieved in 50% of −/− from mice treated with radiation and anti-CTLA-4 upon iNKT mice treated with radiotherapy + anti-CD137 −/− ex vivo activation in both WT and iNKT mice, and it (Figure 7C). Furthermore, all of the long-term regressors was further increased by CD1d blockade in WT but not were able to reject a tumorigenic challenge of viable 4T1 −/− iNKT mice (Figure 6A). Interestingly, while IL-10 levels cells 120 days after the initial tumor implantation, indicat- were low and were not affected by any of the treatments, ing that they had developed a long-lasting protective im- IL-4 was produced at significantly higher levels by spleen munological memory to the tumor (Figure 7D). To cells of mice treated with RT + anti-CTLA-4 compared to determine if blockade of CD1d in the context of radio- control (Figure 6B and C). However, this effect was inde- therapy and CD137 costimulation resulted in improved pendent from iNKT cells since IL-4 levels were similar in priming of anti-tumor CD8 T cells in dLN of WT mice IFNg (pg/mL) Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 6 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 A Treatment Schedule: Day 15 Day 18 Day 21 Day 0 Day 11 Day 13 Day 14 Day 3 Day 7 α-CD1d 4T1 tumor 12 Gy RT α-CTLA4 inoculation B C 900 100 PBS 800 α-CD1d ** RT+ α-CTLA4 ** PBS RT+ α-CTLA4 α-CD1d ** + α-CD1d RT+α-CTLA4 ** RT+α-CTLA4 + α-CD1d 0/8 2/8 0 0 10 15 20 25 30 35 10 20 30 40 50 60 70 80 100 160 Days Post Tumor Inoculation Days Post Tumor Inoculation Figure 4 CD1d blockade improves response of WT mice to treatment with radiotherapy and anti-CTLA-4. (A) WT 4T1 tumor-bearing mice (N = 8 mice/group) were treated with local tumor radiotherapy in two fractions of 12 Gy given on days 13 and 14 post-tumor inoculation. Mice received anti-CTLA-4 mAb on days 15, 18 and 21. Some mice additionally received anti-CD1d mAb on days 3, 7 and 11 post tumor inoculation. (B) Tumor growth is shown for each group until the last day when all mice were alive, day 28 for control and anti-CD1d treated groups, and day 35 for RT + anti-CTLA-4-treated groups. Fractions indicate the number of mice showing complete tumor regression over the total in each group. (C) Surviving tumor-free mice were followed until day 150. Data shown are from one of 2 independent experiments performed with similar * ** results. p < 0.05, p < 0.005. similar to that observed during anti-CTLA-4 blockade have previously shown that the combination of local (Figure 3), we measured tumor antigen-specific produc- radiotherapy with anti-CTLA-4 induces a CD8 T cell- tion of IFNγ by dLN cells. While mice treated with anti- mediated anti-tumor response effective at controlling CD137 monotherapy did not show any peptide-specific the irradiated tumor as well as systemic metastases lead- IFNγ production, treatment with the combination of RT ing to increased survival of 4T1 tumor-bearing mice and anti-CD137 induced significant tumor-specific IFNγ [29,32,40]. However, cures were rarely seen and most −/− production in both WT and iNKT mice (Figure 8). Im- mice eventually succumbed to the tumor. Intriguingly, −/− portantly, the response was markedly higher in iNKT response to radiotherapy and anti-CTLA-4 was mark- mice and was significantly enhanced by in vivo blockade edly improved in mice lacking iNKT cells, achieving of CD1d in WT (mean IFN-γ 109.03 ± 13.85 pg/mL in cures in 50% of the mice [25]. The improved response RT + anti-CD137 + anti-CD1d versus 71.58 ± 17.06 pg/mL could not be attributed to altered immunogenicity of −/− in RT + anti-CD137, p < 0.05) but not iNKT−/− mice. 4T1 cells in iNKT mice and could not be recapitu- Overall, these results indicate that iNKT cells hinder lated in WT mice by administration of α-GalCer, a po- priming of tumor-specific CD8 T cells independent of tent activator of iNKT cells anti-tumor activity in many the immune response modifier used in combination with tumor models [12,13,41-44]. Here we show that the im- radiotherapy to treat 4T1 tumor-bearing mice. proved response of 4T1 tumor-bearing mice seen in the absence of iNKT cells is not unique to anti-CTLA-4- Discussion based immunotherapy. Co-stimulation of T cells by anti- The 4T1 mouse mammary carcinoma is considered a CD137 in combination with radiotherapy was similarly −/− model for the aggressive triple negative subtype of hu- more effective in iNKT than WT mice and resulted in man breast cancer [39]. 4T1 cells are highly metastatic protective anti-tumor recall responses. These data fur- and relatively resistant to many immunotherapies once ther establish a negative regulatory role of iNKT cells in established, including anti-CTLA-4 treatment [29]. We the 4T1 tumor model. Tumor volume (mm ) Percent survival Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 7 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 AB 400 2000 IFNγ TNF-α 300 1500 200 1000 100 500 0 0 CD IL-10 IL-4 -/- -/- Strain BALB/c WT iNKT Strain BALB/c WT iNKT α-CTLA4 - + - + + + + + α-CTLA4 - + - + + + + + - - RT - + + - + + RT - - - + + - + + -- - + - - - α-CD1d -- - + - - - + α-CD1d + −/− Figure 5 CD1d blockade enhances intratumoral IFN-γ response induced by RT + anti-CTLA-4 blockade. WT and iNKT mice were treated with local tumor radiotherapy in two fractions of 12 Gy given on days 13 and 14 post-tumor inoculation. Mice received anti-CTLA-4 mAb on days 15, 18 and 21. Some mice additionally received anti-CD1d mAb or isotype mAb on days 3, 7 and 11 post tumor inoculation. Tumors were harvested at day 22 and concentration of (A) IFN-γ, (B) TNF-α, (C) IL-10 and (D) IL-4 were determined. Bars indicate the mean ± SD of 4 mice/group. p < 0.05. To understand the mechanisms of immune regulation their ability to kill DCs ex vivo, but human iNKT cells by iNKT cells we examined DC, which are largely CD1d , have been shown to kill DCs in vitro [52], and it is pos- and are the APCs required for cross-presentation of sible that a similar mechanism is responsible for DC re- tumor antigens to T cells [45,46]. Surprisingly, there was a duction in 4T1 tumor-bearing mice. reduction in the number of DCs present in the tumor and Blockade of CD1d in WT mice increased not only locor- −/− dLN of WT mice compared to iNKT mice (Figure 1). egional DC numbers, but also the priming of tumor- Downregulation of surface CD11c molecules has been re- specific CD8 T cells in dLN and the overall tumor ported in TLR-activated mouse DCs [47]. We did not see response to treatment with radiotherapy and anti- any difference by flow cytometry in the levels of CD11c in CTLA-4 mAb, suggesting that temporarily “disabling” DCs isolated from non-draining LN, dLN and tumors iNKT cells was sufficient to enhance Th1 type anti- (not shown), suggesting that CD11c downregulation in- tumor immunity. The increased production of IFNγ duced by activation of DCs in the tumor of WT mice is in the tumor and spleen of WT mice treated with not likely to account for locoregional reduction of DCs. RT + anti-CTLA-4 in the presence of CD1d blockade Moreover, blocking the interaction of iNKT cells with DCs supports this interpretation. with anti-CD1d mAb led to an increase in both CD8α + The overall better anti-tumor immunity and the high −/− (immunostimulatory) and CD8α- DCs in WT mice with- levels of IFNγ seen in treated iNKT mice could be out affecting their activation state as measured by expres- due to a larger population of immunostimulatory DC sion of costimulatory molecules (Figure 2). Overall, data presenting tumor-derived antigens, but may also reflect indicate that iNKT cells regulate DCs numbers in the skewing of the immune system that develops in BALB/c tumor and dLN. mice in the absence of iNKT cells towards Th1 rather Clearance of APC has been proposed as a mechanism than Th2 immunity [53]. IL-4 producing iNKT cells to prevent unrestrained T-cell activation and prolifera- (NKT2) were recently described and shown to be the tion, which is beneficial in preventing systemic auto- dominant iNKT subset in the thymus of BALB/c mice, immunity [48] but detrimental for anti-tumor responses and were implicated in determining the Th2 dominance of [49]. Several immune cells, including Tregs [26], NK this mouse strain [53]. Among tumor-infiltrating iNKT cells [50] and CD8 CTLs [49,51], have been implicated cells, we found a larger percentage (6%) that expressed in direct elimination of DCs. We were unable to isolate markers (CD27 + CD122-) shown to define iNKT2 subset, tumor-infiltrating iNKT in sufficient quantities to test while only 0.1% had markers of iNKT1 (CD27 + CD122+) pg/mL pg/mL pg/mL pg/mL Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 8 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 In the spleen IL-4 was increased similarly in treated WT ** −/− and iNKT mice (Figure 6C). IFN-γ iNKT cell role in promoting immune tolerance has been ** established in several experimental conditions, including ** autoimmune disease models, organ transplant and toler- ance to oral antigens [23]. While multiple mechanisms have been suggested to play a role, to our knowledge con- trol of DCs numbers by iNKT cells has not been previ- ously documented in immune tolerance models or in the setting of cancer [23,24,54,55]. However, evidence that hu- IL-10 man iNKT cells can kill DCs in vitro [52], suggests that such mechanism could contribute to immunosuppression in some tumors and be responsible, at least in part, for the lack of clinical activity of α-GalCer in cancer patients 10 [56,57]. Consistent with this hypothesis, administration of α-GalCer to 4T1 tumor-bearing mice at day 15 post- tumor inoculation failed to show any effect by itself or in combination with immunotherapy [25]. By this time, DCs C are low in tumors and dLN (Figure 1), and the remaining IL-4 DCs may be insufficient to present α-GalCer and/or may have acquired a tolerogenic phenotype. ** ** The identity of the CD1d ligand(s) presented by DCs to iNKT cells in 4T1 tumors and dLN is unknown. The endogenous lipids presented by CD1d in steady state conditions are likely to be non-antigenic or elicit toler- ance. However, metabolic changes in the tumor may lead -/- Strain BALB/c WT iNKT to altered lipid biosynthesis and generation of qualitatively or quantitatively modified CD1d ligands capable of trig- α-CTLA4 - + - + + + + + gering a different functional response of iNKT cells, fur- - - RT - + + - + + ther modulated by the inflammatory environment [23]. -- + - - - α-CD1d Improved understanding of the relationship between lipid Figure 6 CD1d blockade enhances systemic IFN-γ response metabolism in cancer and immune regulation will pave −/− induced by RT + anti-CTLA-4 blockade. WT or iNKT mice were the way to the design of more effective immunotherapies treated with local tumor radiotherapy in two fractions of 12 Gy that can enlist the power of iNKT cells to elicit strong given on days 13 and 14 post-tumor inoculation. Mice received anti-CTLA-4 mAb on days 15, 18 and 21. Some mice additionally anti-tumor immunity [58]. received anti-CD1d mAb or isotype mAb i.p. on days 3, 7 and 11 post tumor inoculation. Spleen were harvested at day 22 and Conclusions concentration of (A) IFN-γ, (B) IL-10 and (C) IL-4 were measured in In this study we show that local reduction of DCs medi- supernatants of PMA + ionomycin stimulated cells. Bars indicate the ** ated by iNKT cells regulates the response to immunother- mean ± SD of 4 mice/group. p < 0.005. apy in a mouse model of aggressive breast cancer. The elimination of APC that cross-present tumor-derived anti- gens in dLNs was shown previously to be one mechanism cells (Additional file 5: Figure S5). However, the majority of immunosuppression mediated by Tregs [26]. However, was CD27-CD122-, a phenotype associated with IL-17 iNKT cells were believed to consistently perform anti- production in the thymus [53]. Since IL-17 was undetect- tumor activities, while immunosuppressive functions were able in 4T1 tumors, defining the functional differentiation ascribed to the non-invariant subset of NKT cells [59]. of iNKT cells in the tumor microenvironment may require Here we demonstrate for the first time a novel mechanism different markers than in the thymus. Importantly, com- of immune escape mediated by iNKT cells. parison between IL-4 levels produced in the tumor and These results have important implications for the de- −/− spleen of tumor-bearing WT and iNKT mice did not sign of immunotherapies targeting iNKT cells (reviewed reveal significant differences consistent with differential in [58]). CD1d blockade, or the iNKT-depleting antibody Th2 polarization. Intratumoral IL-4 levels were higher in NKTT120 recently developed for inflammatory diseases −/− treated WT than iNKT mice, but unlike IFNγ,IL-4 [60], could have a role in treatment of cancers in which levels were not modulated by CD1d blockade (Figure 5D). iNKT acquire immunosuppressive functions. pg/mL pg/mL pg/mL Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 9 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 A Treatment Schedule: Day 0 Day 13 Day 14 Day 15 Day 18 Day 21 4T1 tumor 12 Gy RT α-CD137 inoculation B C D 800 100 WT: Control Naive WT (n=5) WT: RT+α-CD137 -/- -/- * Surviving iNKT (n=3) 80 iNKT :Control 600 -/- ** iNKT :RT+ α-CD137 WT: Control 60 WT: RT+α-CD137 -/- iNKT :Control * -/- iNKT :RT+ * α-CD137 0/5 3/6 0 20 40 60 80 100 10 15 20 25 30 35 10 15 20 25 30 35 40 Days Post Tumor Inoculation Days Post Tumor Inoculation Days Post Tumor Inoculation Figure 7 Response of 4T1 tumor-bearing mice to treatment with local radiotherapy and anti-CD137 mAb is improved in the absence −/− of iNKT cells. WT or iNKT mice were injected s.c. with 4T1 cells and randomly assigned to treatment groups (N = 5-6 mice/group) on day 13 when tumors became palpable. Local tumor radiotherapy (RT) in two fractions of 12 Gy was given on days 13 and 14 post-tumor inoculation and anti-CD137 mAb on days 15, 18 and 21. (A) Treatment schema. (B) Tumor growth over time. Fractions indicate the number of mice showing −/− complete tumor regression. (C) Kaplan-Meier survival curves. (D) Tumor growth in naïve WT mice and iNKT mice that rejected tumors and were challenged on day 120 with 4T1 cells. Data is representative of 2 independent experiments. p < 0.05. Methods Animals Cells, antibodies and reagents Six to eight-week old WT BALB/c mice were purchased −/− 4T1 is a BALB/c-derived mammary carcinoma that is from Taconic. iNKT (Vα14Jα18-deficient) mice [27] in highly metastatic and mimics the behavior of triple the BALB/c background obtained from M. Taniguchi were negative human breast cancer [39,61]. 4T1 cells were bred at Berg Animal Facility at NYU School of Medicine grown in complete medium consisting of DMEM and used between 6 to 8 weeks of age. All experiments (Invitrogen) supplemented with 2 mol/L L-glutamine, were approved by the Institutional Animal Care and Use 100 U/mL penicillin, 100 μg/mL streptomycin, 2.5 × Committee of NYU Langone Medical Center. −5 10 mol/L 2-mercaptoethanol and 10% fetal bovine serum (BioWest). Cells were confirmed to be free of In vivo treatment mycoplasma contamination using a mycoplasma de- Mice were inoculated subcutaneously (s.c.) with 5 × 10 tection kit (Sigma). DN32.D3 hybridoma cells, kindly 4T1 cells and randomly assigned to treatment groups provided by Masaki Terabe from the NCI Branch of 13 days later when tumors reached an average diameter the National Institutes of Health, were grown in supple- of 5 mm. Radiotherapy was given as previously described mented RPMI (Invitrogen) [62]. Anti-CTLA-4 hamster [25] with some modifications. Lightly anesthetized mice monoclonal antibody (Clone 9H10) and anti-CD1d rat were positioned on a dedicated plexiglass tray with only monoclonal antibody (Clone 20H2) were purchased the tumor area exposed to radiation while the rest of the from BioXCell (West Lebanon, NH). Control hamster body was protected by lead shielding. Radiotherapy was IgG (isotype control for anti-CTLA-4) was purchased given using the Small Animal Radiation Research Plat- from Jackson Immunoresearch Laboratories. Control rat form (SARRP) (Gulmay Medical, Suwanee, GA) [63] in IgG1 (isotype control for anti-CD1d) was purchased from two doses of 12 Gy each on days 13 and 14 post tumor BioXCell. A rat IgG2a mAb against mouse CD137 (BMS- inoculation. Control hamster IgG and anti-CTLA-4 or 469492, clone 1D8) was provided for these studies by anti-CD137/4-1BB mAbs were given at 200 μg i.p. at 1, Bristol-Myers Squibb (Princeton, NJ). 4 and 7 days after completion of radiotherapy. To block Tumorvolume (mm ) ± SD Percent survival Tumor volume(mm ) ± SD Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 10 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA) at a 1/2,000 dilution in 1% BSA for 30 minutes. AH-1-A5 To amplify the signal, slides were subject to two rounds of pMCMV biotin-tyramide amplification (PerkinElmer, Melville, NY) using manufacturer’s suggested protocol. The slides were counterstained with DAPI, mounted with Vectashield and images obtained using a Nikon Eclipse 800 deconvolution *** microscope. Positive cells were counted from at least 3 randomly selected fields (20× magnification). Flow cytometry Lymph nodes and minced tumors were digested using a -/- Strain BALB/c WT iNKT cocktail of 1.67 Wünsch U/mL Liberase TL (Roche) and 0.2 mg/mL DNAse (Roche) as previously described α-CD137 - - + + + + + [40,64]. After lysis of red blood cells single cell suspen- RT - - + + + + + sions were filtered through a 40-μm nylon cell strainer. -- - α-CD1d - - + + Live cells were distinguished using Fixable Viability Dye Figure 8 CD1d blockade enhances production of IFN-γ eFluor 660 (eBioscience) prior to flow staining. All sam- by tumor-specific cells generated by RT + anti-CD137 ples were incubated with anti-mouse CD16/32 (Fc block) −/− co-stimulation. WT or iNKT mice were treated with local for 10 minutes followed by staining for various surface tumor radiotherapy in two fractions of 12 Gy given on days 13 markers for 30 minutes at 4°C. The following antibodies and 14 post-tumor inoculation. Mice received anti-CD137 mAb on days were used (all purchased from eBiosciences): PE-Cy7- 15, 18 and 21. Some mice additionally received anti-CD1d mAb or isotype control on days 3, 7 and 11 post tumor inoculation. Cells from conjugated anti-mouse CD11c, Alexa Fluor 700-conjugated tumor-draining lymph nodes were collected on day 23 and stimulated anti-mouse CD86, PE-conjugated anti-mouse CD80, PE- with feeder cells pre-loaded with irrelevant peptide pMCMV (open conjugated anti-mouse CD1d, PE-conjugated anti-mouse circles) or AH-1-A5 (filled circles). Secreted IFN-γ in the supernatant was CD70, FITC-conjugated anti-mouse CD40 and Alexa measured 48 hours later. Each symbol represents an individual mouse. * *** Fluor 700-conjugated anti-mouse MHC Class I (H-2Kd/ Bars indicate the mean ± SD of 3–5 mice/group. p <0.05, p <0.0005. Dd), FITC-conjugated anti-mouse CD27, PECy7-conjugated anti-mouse CD3ε, APC-conjugated anti-mouse CD122, CD1d in vivo mice were given three doses of anti-CD1d Alexa Fluor 700-conjugated anti-mouse CD4. iNKT cells mAb (Clone 20H2) at 100 μg i.p. on days 3, 7 and 11 were identified by staining with mouse CD1d/PBS-57 or post tumor inoculation. Tumors were measured every CD1d/unloaded tetramers (NIH Tetramer Core Facility) 2–3 days until death or sacrifice when tumor dimensions for 30 minutes at room temperature [65]. All samples exceeded 5% of body weight or if animals showed signs were analyzed using a BD LSR II flow cytometer and of significant pain or distress due to metastatic disease. FlowJo version 8.7.5 (Tree Star). In some experiments, mice that eradicated the tumor after treatment and remained tumor-free for at least 120 days were inoculated in the contralateral flank with Ex vivo cytokine production 4T1 cells and tumor growth monitored. A group of Single cell suspensions (3 × 10 per well) from draining −/− naïve mice was similarly challenged with 4T1 cells as lymph nodes of 4T1 tumor-bearing WT or iNKT mice control. were cultured in complete T-cell medium in 48-well tissue culture plates with irradiated (12 Gy) feeder cells (3 × 10 ) Immunohistochemistry prepared from the spleen of healthy BALB/c mice 4T1 tumors and dLNs were harvested on indicated days, pre-loaded with tumor-derived peptides (AH-1-A5) or fixed in freshly prepared 4% paraformaldehyde for 1 hr at an H-2L -binding irrelevant peptide (pMCMV) used at a 4°C and incubated overnight in 30% sucrose. Tumors were final concentration of 1 μg/mL. Peptide sequences are then frozen in optimum cutting temperature (OCT) SPSYAYHQF (AH-1-A5) [66], and YPHFMPTNL (pMCMV) medium and stored at −80°C. Sections (5 μm) were treated [66]. After 48 hours, supernatants were collected and with 3% H O in PBS for 30 minutes in order to eliminate stored at −80°C. Secreted IFN-γ was measured from 2 2 endogenous peroxide activity. After blocking in 4% ham- duplicate wells using Flowcytomix kit (eBioscience, ster serum for an hour and three washes with PBS, anti- SanDiego,CA).Background IFN-γ production from mouse CD11c eFluor 615 (eBioscience, San Diego, CA) supernatants of cells cultured in complete T-cell media was applied at a 1/100 dilution in 1% BSA for 1 hour, alone was subtracted. Spleen cell suspensions (10 ) from followed by three washes and incubation in HRP- 4T1 tumor-bearing mice were cultured in a 24-well tissue IFNg(pg/mL) Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 11 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 culture plate and stimulated with PMA + ionomycin Cell Additional file 4: Figure S4 Local tumor radiotherapy and CD137 Stimulation Cocktail Mix (eBioscience) for 48 hours. co-stimulation used alone are ineffective in prolonging survival of −/− 4T1 tumor-bearing mice. WT and iNKT mice were injected s.c. with 4T1 cells and randomly assigned to treatment groups (n = 5-6/group) on day 13 when tumors became palpable. Local tumor radiotherapy (RT) Intratumoral cytokines was given in two fractions of 12 Gy on days 13 and 14 post-tumor Freshly harvested tumors were immediately frozen in li- inoculation. Mice received anti-CD137 mAb on days 15, 18 and 21. quid nitrogen and stored at −80°C. To isolate total protein, Kaplan-Meier curves were generated from survival data of each mouse calculated as time (days) from date of inoculation until death or euthanasia. tissues were thawed on ice, minced and homogenized in Median survival (days) in each treatment group is indicated in parenthesis. lysis buffer (10 mM Tris–HCl pH8.0, 150 mM NaCl, 1% Data is representative of 2 independent experiments. NP-40, 10% glycerol, 5 mM EDTA) and protease inhibitor Additional file 5: Figure S5 iNKT subset compartments in 4T1 cocktail (Roche). Total volume (μL) of lysis buffer used tumors and tumor-draining lymph nodes. WT mice were injected s.c. with 4T1 cells. On day 15, tumor samples from three mice were pooled was equal to 5× weight of tumor tissue (mg). Lysates were and digested to obtain single cell suspensions. (A) Intratumoral iNKT cells sonicated in 4°C using a 7-minute regimen of 30s intermit- were identified using mouse CD1d/PBS-57 tetramers. Expression of surface tent on and off. Samples were spun at 14,000 rpm for CD122 and CD27 antigens were assessed on CD3+ CD1d/PBS-57 tetramer + populations to identify iNKT1 (CD27 + CD122+) and iNKT2 (CD27-CD122+) 15 minutes at 4°C, supernatants collected and protein con- subsets. (B) The relative abundance of iNKT cells in the tumor and draining centrations determined using Bradford assay. Samples lymph nodes is shown as a percentage of total CD3 cells. (C) The relative were stored at −80°C. Cytokines were measured from du- abundance of iNKT1 (white bars) and iNKT2 (black bars) subsets is shown as a percentage of total CD3+ CD1d/PBS-57 tetramer + cells. Bars indicate the plicates wells using multi-analyte Flowcytomix kit. ** mean ± SD of three samples. p < 0.005. Statistical analysis Abbreviations Random coefficients regression (RCR) was used to assess α-GalCer: Alpha galactosylceramide; APC: Antigen presenting cells; BMDC: Bone the effect of treatment on tumor growth. The dependent marrow-derived dendritic cells; CD: Cluster of differentiation; CTL: Cytotoxic T lymphocytes; DCs: Dendritic cells; dLN: Draining lymph node; Gy: Gray; variable was the natural log of tumor volume at all avail- −/− IFN-γ: Interferon gamma; IHC: Immunohistochemistry; iNKT : Invariant natural able times; log volumes were used since the change in killer T cell-deficient; i.p.: Intraperitoneal; mAb: Monoclonal antibody; log volume over the course of follow-up was well ap- MHC: Major histocompatibility complex; ndLN: Non-draining lymph node; NK: Natural killer; PBS: Phosphate buffered saline; PMA: Phorbol 12-myristate proximated as linear within each treatment arm. Log rank 13-acetate; RT: Radiotherapy; s.c.: Subcutaneous; TAA: Tumor-associated tests were used to compare treatment arms in terms of sur- antigens; Th1: T-helper type 1; Th2: T-helper type 2; Tregs: Regulatory T cells; vival. The Kaplan-Meier method was used to estimate me- WT: Wild type. dian survival times and the log cumulative hazard transformation was used to derive 95% confidence limits for Competing interests The authors declare that they have no competing interests. median survival in each arm. All reported p values are two- sided and statistical significance defined as p < 0.05. SAS 9.3 Authors’ contributions (SAS Institute, Cary, NC) was used for all computations. KAP designed and performed all experiments and wrote the manuscript. JA carried out in vitro studies and immunostaining and contributed to writing the manuscript. SD conceived and designed the study with KAP and wrote Additional files the manuscript. JSB performed statistical analysis. All authors read and approved the final manuscript. Additional file 1: Figure S1 Anti-CD1d mAb blocks cytokine production by DN32.D3 NKT-like hybridoma cells stimulated with Authors’ information α-GalCer-loaded DCs. CD11c bone-marrow derived dendritic cells KAP started his postdoctoral fellowship at NYU School of Medicine and is (BMDC) from healthy BALB/c mice were plated in 24-well tissue culture 4 currently an associate research scientist in the department of Pathology. JA is plate (2×10 /well) and incubated with 100 ng/mL α-GalCer overnight, an undergraduate student at NYU College of Arts and Sciences. JSB has over washed extensively and incubated overnight with fresh media containing 20 years experience as research biostatistician and is currently associate anti-CD1d at indicated final concentrations. Cells were washed again 5 professor of Radiology at NYU School of Medicine. SD holds dual before the addition of DN32.D3 hybridoma cells (10 /well). Supernatants appointments as professor of Pathology and Radiation Oncology at NYU were collected three days later and cytokines measured. School of Medicine and is Co-leader of the Cancer Immunology program of Additional file 2: Figure S2 Anti-CD1d mAb does not deplete the NYU Cancer Institute. CD1d-expressing cells in vivo. Healthy WT mice (N = 4/group) were given isotype or anti-CD1d mAb i.p. (200 μg/mouse) every 4 days for a total of 3 doses. Two days after the last dose, mice were euthanized and Acknowledgments single cell suspensions from digested spleens collected for flow staining KAP was supported by a Postdoctoral Research Fellow Award (W81XWH-10- of (A) DC, (B) myeloid cells, (C) B-cells and (D) macrophage populations. 1-0007) from the US Department of Defense Breast Cancer Research Program. S. Demaria is supported by grants from the Chemotherapy Additional file 3: Figure S3 Anti-CD1d mAb does not induce Foundation, the US Department of Defense Breast Cancer Research Program stimulation of CD1d-expressing APCs. Splenocytes from naïve WT −/− (W81XWH-11-1-0532), and the Breast Cancer Alliance. The Histopathology or iNKT mice (n = 4/group) were stimulated in vitro with 10 μg/mL and Flow Cytometry Core Facilities of the Laura and Isaac Perlmutter α-CD1d or isotype control mAbs for 48 hours. As a positive control, Cancer Center at the NYU Langone Medical Center are partially supported splenocytes were stimulated with LPS (1 μg/mL). Supernatants were by NIH/NCI P30CA016087. The authors wish to thank Maria Jure-Kunkel collected and measured for secreted (A) IL-10, (B) IFN-γ, (C) IL-12p70 (Bristol-Myers) for anti-CD137 mAb. We acknowledge the NIH Tetramer Core and (D) IL-4. Facility (contract HHSN272201300006C) for provision of CD1d tetramers. Pilones et al. Journal for ImmunoTherapy of Cancer 2014, 2:37 Page 12 of 13 http://www.immunotherapyofcancer.org/content/2/1/37 Author details 17. Yang YF, Tomura M, Ono S, Hamaoka T, Fujiwara H: Requirement for Department of Pathology, New York University School of Medicine, New IFN-gamma in IL-12 production induced by collaboration between v York, NY 10016, USA. Department of Radiology, New York University School (alpha)14(+) NKT cells and antigen-presenting cells. Int Immunol 2000, of Medicine, New York, NY 10016, USA. Department of Radiation Oncology, 12:1669–1675. New York University School of Medicine, New York, NY 10016, USA. New 18. Crowe NY, Smyth MJ, Godfrey DI: A critical role for natural killer T cells in York University School of Medicine, Alexandria Center for Life Sciences, 450 immunosurveillance of methylcholanthrene-induced sarcomas. J Exp Med East 29th St, Room 324B, New York, NY 10016, USA. 2002, 196:119–127. 19. Godfrey DI, Kronenberg M: Going both ways: immune regulation via Received: 3 March 2014 Accepted: 11 September 2014 CD1d-dependent NKT cells. J Clin Invest 2004, 114:1379–1388. 20. 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