Induced TiRP melanomas resist immunotherapy

Immunotherapy based on checkpoint inhibitors anti-CTLA4 and anti-PD1 proved unable to alter the growth of tamoxifen-induced TiRP tumors, whether given alone or in combination (Fig. 1a). Because TiRP tumors express MAGE-type tumor antigen P1A, we also tested a preventive vaccination against P1A using an established prime/boost immunization scheme based on P1A-recombinant adenovirus and Semliki Forest virus (SFV), which induces high levels of P1A-specific CD8+ T cells and significant protection against a challenge injection of P1A-positive tumor cells6. However, immunization of TiRP mice before tumor induction with tamoxifen failed to prevent tumor onset, and this was not improved upon combination of vaccine with anti-CTLA4 and anti-PD1 (Fig. 1a). This was seemingly not due to loss of antigen expression, as the tumors that appeared in vaccinated mice still expressed high amounts of P1A transcripts (Supplementary Fig. 1). These results suggested a strong immunosuppressive mechanism preventing T-cell attack of TiRP tumors.

Fig. 1 Induced TiRP tumors resist immunotherapy. a Schedule for tumor induction and immunotherapy. TiRP mice (B10.D2;Ink4a/Arfflox/flox;TiRP+/+) injected twice with 4 mg 4OH-tamoxifen on day 0 and 15, were also injected as indicated with anti-CTLA4 (4 × 40 µg) and/or anti-PD1 (4 × 200 µg) and/or a prime/boost vaccine regimen of recombinant adenovirus (Adeno.Ii.P1A t ) and Semliki Forest virus (SFV.P1A) encoding the MAGE-type tumor antigen P1A. Tumor appearance and mice survival were monitored. The figure represents the cumulative data of three experiments. b Tumor-bearing TiRP mice and control mice (B10.D2;Ink4a/Arfflox/flox mice, which have the same genetic background as TiRP mice but lack the TiRP transgene) were immunized with the P1A vaccine as above, and the P1A-specific CD8+ T-cell response was monitored by FACS in spleen cells stimulated one week with P1A-peptide pulsed spleen cells, using antibodies to CD3ε and CD8α, and H-2Ld-P1A tetramers. Five mice were analyzed at each time point for each group. c TiRP mice were immunized simultaneously against P1A as above and against the irrelevant H-2Ld-restricted antigen P91A (mutated peptide) by intramuscular injections of P91A peptide in AS15 adjuvant52 one week apart during 1 month. Mice were then treated with 4OH-tamoxifen. At the time of tumor appearance, spleen cells were stimulated one week with P1A or P91A peptide-pulsed spleen cells, and P1A- or P91A-specific CD8+ T cells were quantified by FACS analysis using the relevant H-2Ld tetramers. Results are expressed as mean + s.e.m. Unpaired t-test, two-tailed c, *P < 0.05, **P < 0.01 Full size image

We then used H-2Ld-P1A tetramers to monitor P1A-specific CD8+ T cells in the spleen of vaccinated mice ﻿﻿stimulated one week in vitro with the P﻿1A peptide ﻿(LPYLGWLVF) (Fig. 1b)49﻿. All vaccinated mice developed an anti-P1A CD8+ response. In control mice without tumor, this response persisted until at least 70 days after vaccination. However, in mice developing a TiRP tumor, the P1A-specific CD8+ T-cell response plummeted to the levels of unimmunized mice at day 70. Hence, the presence of a TiRP tumor induced complete disappearance of the P1A-specific anti-tumor CD8+ response. We also observed that non-vaccinated mice developing a TiRP tumor spontaneously mounted a P1A-specific immune response, confirming that TiRP tumors express the P1A antigen and indicating that the tumor is immunogenic. However, as observed in vaccinated mice, this anti-tumor immune response did not persist. This lack of persistence was restricted to anti-tumor CD8+ T cells, because when we immunized mice against both P1A and an irrelevant antigen named P91A (an H-2Ld-restricted mutated peptide7), we observed that the CD8+ response against the latter antigen persisted in tumor-bearing mice (Fig. 1c). Moreover, tumor-bearing mice were able to mount a normal primary CTL response against minor histocompatibility antigens (Supplementary Fig. 2), indicating that tumor development did not induce a general CTL unresponsiveness, as reported previously in a spontaneous sporadic tumor mouse model8, 9. In the latter model, increased levels of TGFβ1 were measured in the serum of tumor-bearing mice, and correlated with the general T-cell unresponsiveness observed after a long latency period8, 9. Therefore, we also measured TGFβ1 levels in the serum of tumor-bearing TiRP mice (Supplementary Fig. 3). Total TGFβ1 levels were high in both groups and increased in tumor-bearing mice as compared to tumor-free mice. However, the levels of active TGFβ1 were low and similar in both groups, indicating that most of the seric TGFβ1 is in its latent, inactive form. Altogether, our data do not suggest a lack of general T-cell responsiveness, but rather indicate a selective suppression of tumor-specific CTL in tumor-bearing TiRP mice.

Induced but not transplanted tumors resist adoptive cell therapy

To better track the fate of tumor-specific CD8+ T cells in this model, and to evaluate another clinically relevant immunotherapy approach, we resorted to adoptive transfer of P1A-specific CD8+ T cells isolated from mice transgenic for the anti-P1A T-cell receptor (TCR) (TCRP1A mice in the Rag1−/− B10.D2 background)10. Mice bearing induced Amela TiRP tumors received an intravenous injection of 10 million CD8+ T cells isolated from TCRP1A mice and stimulated 4 days in vitro (Fig. 2a). Strikingly, this adoptive cell therapy (ACT) was unable to alter the growth of induced TiRP tumors (Fig. 2b). In parallel, we used tumor cell line T429, which was previously established from an induced Amela TiRP tumor and grown in vitro4. We established isogenic transplanted tumors by subcutaneous injection of cells from a T429 clone, and we treated these mice with ACT as above. In contrast to induced tumors, those transplanted tumors were efficiently rejected by adoptively transferred TCRP1A CD8+ T cells (Fig. 2c). Similar results were obtained with distinct clones from line T429 and with tumors transplanted in the subcutaneous (Fig. 2d) or the intradermal space, which is the natural niche for melanoma development (Fig. 2e). Because of the identical genetic background of the tumor cells, this striking difference between induced and transplanted tumors likely resulted from differences in the tumor microenvironment, and underlines the relevance of autochthonous tumor models as opposed to transplanted tumor models for preclinical evaluation of cancer immunotherapy.

Fig. 2 Rejection of transplanted but not induced autochthonous tumors after adoptive transfer of tumor-specific CD8+ T cells. a Adoptive transfer protocol. Mice bearing induced autochthonous Amela TiRP tumors or transplanted isogenic tumors received intravenously 107 CD8+ T cells that were isolated from TCRP1A B10.D2 mice and activated 4 days in vitro by co-incubation with lethally irradiated cells expressing P1A and B7-1 (L1210.P1A.B7-1). b TiRP mice bearing induced Amela tumors 22–24 days after 4OH-tamoxifen injection received adoptive transfer (n = 32) of 107 TCRP1A CD8+ T cells activated in vitro for 4 days (red symbols). Black symbols show control mice (n = 30) receiving no T cells. Tumor volume is shown. (Data accumulated from two identical experiments). c Mice (B10.D2;Ink4a/Arfflox/flox) were injected subcutaneously with 2 × 106 cells from clone 11 of isogenic tumor line T429, which had previously been adapted to culture from an induced Amela TiRP tumor. After 36 days, mice received an intravenous injection of 107 activated TCRP1A CD8+ T cells (blue symbols). Control mice received no T cells (black symbols). Tumor volume was monitored. Results are shown from one representative experiment (n = 6/group) out of at least three performed. d Same as in panel c but using another clone (T429.6) from tumor line T429. Mice received 107 activated TCRP1A CD8+ T cells (green symbols) or not (black symbols) 18 days after tumor injection. Results are shown from one representative experiment (n = 6−7/group) out of at least three performed. e B10.D2;Ink4a/Arfflox/flox mice were injected intradermally (2 × 106 cells) with three distinct clones of tumor line T429. When tumors reached a size of about 400 mm3, mice received an intravenous injection of 107 activated TCRP1A CD8+ T cells (colored curves). Black symbols indicate control mice that received no T cells. Tumor growth was monitored. Individual growth curves are shown (8–10 mice/group). f Mice treated as in panels b–d were killed 22 days after T-cell transfer, and cells from the spleen and the draining lymph nodes were tested ex vivo (i.e. without in vitro﻿ stimulation) for the presence of P1A-specific T cells by FACS using H-2Ld-P1A tetramer. The number of mice analyzed is indicated for each group. Results are expressed as mean ± s.e.m Full size image

We then analyzed the persistence of TCRP1A CD8+ T cells in the spleen and lymph nodes of mice 22 days after ACT, using H-2Ld-P1A tetramers ex vivo. P1A-specific CTL were easily detected in tumor-free mice and in mice bearing transplanted tumors. However, they were undetectable in mice bearing an induced tumor (Fig. 2f). We then used TCR-specific clonotypic PCR as a more sensitive approach, but we also failed to detect TCRP1A T cells after 22 days in the spleen of mice bearing induced tumors, while we easily detected them in tumor-free and tumor-transplanted mice (Supplementary Fig. 4a). At this time point, TCRP1A T cells were also absent in most of the induced tumors themselves (Supplementary Fig. 4a). These results indicated that tumor-specific T cells were actively deleted in mice bearing induced Amela TiRP tumors, while they were not in mice bearing transplanted tumors.

Induced tumors trigger apoptosis of tumor-specific T cells

To understand the fate of tumor-specific T cells, we then analyzed mice earlier, 4 days after ACT. At this time, both induced and transplanted tumors were infiltrated by CD8+ T cells (Fig. 3a), and transferred TCRP1A CD8+ T cells were easily detected in induced tumors by clonotypic PCR (Supplementary Fig. 4b). Ex vivo tetramer analysis further showed that transferred T cells were enriched in the tumors as compared to the spleen and draining lymph nodes, in both induced and transplanted tumors (Fig. 3b). These results indicated that transferred TCRP1A CD8+ T cells effectively migrated into the induced tumors in the first days after adoptive transfer. Furthermore, those tumor-infiltrating T cells expressed activation marker CD69, indicating that they recognized the P1A tumor antigen on tumor cells (Fig. 3c). To further evaluate the functional capacity of transferred T cells in the early days after ACT, we tested their ability to kill P1A-peptide pulsed target cells in an in vivo killing assay performed 3 days after transfer (Supplementary Fig. 5). We observed efficient killing of P1A-positive targets in mice bearing induced tumors, even though it was slightly reduced as compared with tumor-free mice. Thus, the results so far indicated that transferred TCRP1A CD8+ T cells retained their functional capacity, infiltrated the induced tumors, recognized their antigen and became activated. This raised the question: why did they fail to reject the induced tumors?

Fig. 3 In vivo apoptosis of tumor-infiltrating CD8+ T cells 4 days after transfer. a Mice bearing induced (n = 14) or transplanted (n = 13) tumors (500 mm3) received adoptive transfer of TCRP1A CD8+ T cells as in Fig. 2. Four days later, tumors were analyzed by FACS ex vivo for CD8+ T cells among living cells. b–d Draining lymph nodes (LN), spleens and tumors from tumor-bearing mice were analyzed 4 days after adoptive transfer of TCRP1A CD8+ T cells by ex vivo FACS staining for CD8 and H-2Ld-P1A tetramers b, CD69 c, and with Annexin V d (for b–d: n = 75 mice for induced tumors, n = 75 mice for T429.11, n = 32 mice for T429.6, n = 12 for tumor-free mice). e H-2Ld-P1A tetramer-negative CD8+ T cells infiltrating induced tumors or spleens from tumor-free mice were stained for Annexin V. Mice were identical to b–d (induced tumors: n = 75, spleens from tumor-free mice: n = 12). f Mice bearing induced (n = 4) or transplanted (n = 3) tumors (500 mm3) were transferred with activated TCRP1A CD8+ T cells. Four days after transfer, they received an i.v. injection of FLIVO (inhibitor-based pan-caspase probe) 4 h before killing. Apoptosis of TCRP1A CD8+ T cells was evaluated ex vivo by FACS staining for FLIVO. Mice receiving a non-targeting FLIVO control dye showed no staining of TCRP1A CD8+ T cells. g Slices (300 µm) of fresh tumor tissues were incubated with CMAC-stained TCRP1A CD8+ T cells for 24 h. Cryosections (7 μm) were stained for apoptosis using inhibitor-based active pan-caspase marker FLICA, and scanned with a MIRAX digital microscope. Data were quantified using Biopix software (n = 5 mice/group; three sections analyzed per mouse). h TiRP mice bearing either pigmented (Mela) or unpigmented (Amela) induced tumors were treated and analyzed as in b–d (n = 20 mice/group). Results are expressed as mean + s.e.m. Unpaired t-test, two-tailed a–h, *P < 0.05, **P < 0.01, ***P < 0.001. ****P < 0.0001 Full size image

We then observed that 4 days after adoptive transfer, a high proportion of P1A-specific CD8+ T cells infiltrating the induced tumors were apoptotic, as defined by staining with Annexin V (Fig. 3d). This was not the case in transplanted tumors (Fig. 3d). The non P1A-specific CD8+ T cells that infiltrated induced tumors were not stained with Annexin V (Fig. 3e). This suggested that induced tumors resisted immune rejection by triggering apoptosis of tumor-specific T cells. Because of the high background staining observed in spleen and lymph nodes with Annexin V, we used a series of other approaches to reveal apoptosis. 4 days after T-cell transfer, we injected mice with FLIVO, a fluorescent inhibitor-based pan-caspase marker that selectively binds active caspases and was developed for in vivo use11. After 4 h, we killed the mice and analyzed TIL by FACS after staining with H-2Ld-P1A tetramer. We observed that approximately 50% of P1A-specific TIL were stained with FLIVO in induced tumors, while only about 10% were in transplanted tumors (Fig. 3f). We also analyzed sections of those tumors by immunofluorescence, monitoring cells positive for FLIVO and for CellTracker Blue CMAC Dye, which was used to stain T cells before adoptive transfer. This confirmed the higher proportion of apoptotic TCRP1A CD8+ T cells in induced tumors (Supplementary Fig. 6a). Similar results were obtained with tumors from mice adoptively transferred but not treated with FLIVO, which were stained with antibodies to cleaved caspase-3 and to V-alpha8, the alpha chain of the TCR used in TCRP1A CD8+ T cells (Supplementary Fig. 6b, c). Lastly, we tried to recapitulate apoptosis induction by collecting tumors ex vivo and incubating freshly cut tumor slices with CMAC-labeled TCRP1A CD8+ T cells for 24 h. We then stained cryosections of these tumor slices with FLICA, a fluorescent inhibitor-based marker of activated pan-caspases, and again we observed more apoptotic TCRP1A CD8+ T cells in induced as compared with transplanted tumors (Fig. 3g).

We then performed ACT in pigmented (Mela) and unpigmented (Amela) induced TiRP tumors, and compared P1A-specific CD8+ TIL 4 days after transfer. We observed equal T-cell infiltration and activation, but T-cell apoptosis was restricted to Amela tumors, suggesting that the ability to induce T-cell apoptosis was linked to the inflammatory microenvironment that is typical of Amela tumors in this model (Fig. 3h)4.

TIL apoptosis is dependent on Fas-ligand

We then tried to understand what triggered apoptosis of anti-tumor T cells in induced tumors. Interferon-gamma (IFNγ) can play a dual role in the course of the immune response, contributing to an efficient response by promoting effector T-cell differentiation and MHC class I expression on the one hand, but also, on the other hand, inducing inhibitory molecules that contribute to the negative feedback of the immune response, including IDO, FasL and PDL112,13,14,15. Four days after T-cell transfer, induced tumors contained high levels of IFNγ mRNA and secreted protein, confirming the functionality of transferred T cells (Fig. 4a, b). To determine whether this IFNγ played a role in the induction of T-cell apoptosis, we injected an IFNγ-neutralizing antibody into mice bearing induced tumors 1 day before ACT. Four days after ACT, we observed a strong reduction in the number of apoptotic TILs, indicating the involvement of IFNγ in triggering T-cell apoptosis at the tumor site (Fig. 4c).

Fig. 4 Role of IFNγ in triggering apoptosis of tumor-specific CD8+ T cells. a Quantitative RT-PCR analysis of IFNγ mRNA expression in induced TiRP tumor tissues collected 4 days after ACT. Results normalized to β-actin are expressed relative to the level measured in control tumors that did not receive ACT (controls: n = 20; ACT: n = 24). b Fresh homogenates from induced tumors collected 4 days after ACT were cultured in vitro for 24 h and supernatants were tested by ELISA for the presence of IFNγ (controls: n = 22; ACT: n = 34). c Tumor-bearing mice received 0.5 mg neutralizing anti-IFNγ antibody i.p. 1 day before transfer of activated TCRP1A CD8+ T cells. Four days after transfer, dissociated tumor tissues were analyzed ex vivo by FACS for apoptosis of TCRP1A CD8+ T cells (controls: n = 23; anti-IFNγ: n = 9). d Quantitative RT-PCR analysis of FasL mRNA expression in induced TiRP tumor tissues collected 4 days after transfer of activated TCRP1A CD8+ T cells preceded or not by injection of neutralizing anti-IFNγ antibody as in a–c. Results normalized to β-actin mRNA level are expressed relative to the level measured in control tumors that did not receive TCRP1A CD8+ T-cell transfer (controls: n = 12; T-cell transfer: n = 11; T-cell transfer + anti-IFNγ: n = 8). e Quantitative RT-PCR analysis of FasL mRNA expression in induced TiRP tumor tissues (n = 19) as compared with T429.11 transplanted tumor tissues (n = 14). Results normalized to Gapdh are expressed relative to the level measured in transplanted tumors. Results are expressed as mean ± s.e.m. Unpaired t-test, two-tailed, *P < 0.05, **P < 0.01, ***P < 0.001. ****P < 0.0001 Full size image

A number of genes induced by IFNγ encode proteins that can trigger lymphocyte apoptosis. These include IDO12, 15, PDL112, 16 and Fas-ligand17. IDO was only weakly expressed in this model, and only barely increased after T-cell transfer. In contrast, PDL1 was well expressed in this model4. However, it was not involved in TIL apoptosis because the injection of an anti-PD1 blocking antibody failed to prevent apoptosis of transferred T cells (Supplementary Fig. 7a). This was in line with the lack of therapeutic effect of PD1 blockade in this model (Fig. 1a). Expression of Fas-ligand (FasL) transcripts was observed in TiRP tumors, increased after T-cell transfer, and was dramatically reduced after IFNγ neutralization (Fig. 4d). Moreover, FasL expression was higher in induced TiRP tumors as compared to transplanted T429 tumors (Fig. 4e). We therefore investigated the Fas-FasL pathway as potentially involved in triggering apoptosis of tumor-infiltrating T cells. We first used siRNA to silence Fas in TCRP1A CD8+ T cells and make them insensitive to FasL-mediated apoptosis, and we optimized the conditions so that Fas silencing was effective for at least 7 days (Supplementary Fig. 8). We then transferred Fas-silenced TCRP1A CD8+ T cells into mice bearing induced TiRP tumors, and analyzed TIL apoptosis after 4 days, by FACS staining with Annexin V and for activated caspases (FLICA). We observed a strong reduction of apoptosis of Fas-silenced TIL (Fig. 5a). We obtained similar results when we incubated Fas-silenced TCRP1A CD8+ T cells 24 h in vitro with fresh slices of induced tumors (Fig. 5b). To confirm this result, we then used soluble Fas-Fc to neutralize FasL in vivo. We injected mice bearing induced tumors with 150 µg Fas-Fc every day starting 1 day before ACT. Again we observed a clear reduction in the number of apoptotic TILs after 4 days (Fig. 5c). Fas-Fc also reduced apoptosis of TCRP1A CD8+ T cells incubated in vitro with fresh slices of induced tumors (Fig. 5d). We conclude that the apoptosis of anti-tumor CD8+ T cells observed in induced tumors is mostly driven by FasL. Tumor necrosis factor alpha (TNFα) is another factor known to induce T-cell apoptosis18. Even though TNFα is not clearly induced by IFNγ, we tested its involvement in T-cell apoptosis in the TiRP model by injecting an anti-TNFα neutralizing antibody 1 day before and 2 days after ACT. We observed no reduction of T-cell apoptosis on day 4 after ACT (Supplementary Fig. 7b). These results supported a dominant role of FasL in triggering T-cell apoptosis in the TiRP model.

Fig. 5 TIL apoptosis is triggered by FasL. a Tumor-bearing TiRP mice were transferred with 107 activated TCRP1A CD8+ T cells treated with either control siRNA or Fas siRNA. Four days later, tumor tissues, draining lymph nodes and spleen were analyzed ex vivo by FACS for apoptosis of TCRP1A CD8+ T cells, using Annexin V (left) or pan-caspase marker FLICA (right) (n = 13 per group). b Fresh slices of induced TiRP tumors were incubated 24 h in vitro with TCRP1A CD8+ T cells treated with control or Fas siRNA. Apoptosis of TCRP1A CD8+ T cells was evaluated as in Fig. 3g (n = 5 mice/group; three sections analyzed per mouse). c Tumor-bearing TiRP mice transferred with 107 activated TCRP1A CD8+ T cells received daily injections of 150 µg soluble Fas-Fc starting 1 day before T-cell transfer. Four days later, tumor tissues, lymph nodes and spleen were analyzed ex vivo by FACS for apoptosis of P1A-specific CD8+ T cells, using Annexin V (left) or active pan-caspase marker FLICA (right). (n = 13 per group). d Fresh slices of induced TiRP tumors pre-incubated 4 h with soluble Fas-Fc (10 µg/ml) were incubated in vitro with TCRP1A CD8+ T cells and soluble Fas-Fc (10 µg/ml). Apoptosis was measured as in Fig. 3g (n = 5 mice/group; three sections analyzed per mouse). Unpaired t-test, two-tailed (a-d). Results are expressed as mean + s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001. ****P < 0.0001In figure 5 the panel (i) is explained in the legend but not mentioned in the figure. Please check.We changed (a-i) to (a-d) Full size image

PMN-MDSC trigger TIL apoptosis

We then compared the cellular composition of the tumor tissue of induced (Amela) and transplanted tumors. Both tumor types contained CD45+ cells (hematopoietic origin), which were more abundant in induced tumors. This likely resulted from a significant enrichment of induced tumors in polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSC) (P < 0.0001, t-test), while monocytic myeloid-derived suppressor cells (M-MDSC) were equally represented in both tumor types (Fig. 6a). MDSC are a subset of immature myeloid cells that can be recruited at tumor sites and display immunosuppressive activity19. This PMN-MDSC enrichment was observed in Amela but not in Mela tumors (Fig. 6b). As compared with M-MDSC, PMN-MDSC produced more ROS, expressed less iNOS and produced less NO, in line with previous reports20, 21 (Supplementary Fig. 9). They also expressed higher levels of arginase, another immunosuppressive factor that is often expressed in M-MDSC but also in PMN-MDSC20,21,22,23. We confirmed the immunosuppressive function of these PMN-MDSC by showing their ability to suppress in vitro proliferation and cytolytic activity of anti-tumor CD8+ T cells at a 1/1 ratio (Fig. 6c, b). Moreover, when we established transplanted tumors by co-injecting T429.11 tumor cells with PMN-MDSC (isolated from induced tumors) at a 4 to 1 ratio and then treated mice with ACT, the co-injected tumors resisted immune rejection, while control tumors were rejected (Fig. 6e). These results confirmed the key role of PMN-MDSC in resistance of tumors to immunotherapy in this model. To explain the higher recruitment of PMN-MDSC in induced as compared with transplanted tumors, we measured the expression of a series of cytokines and chemokines known to play a role in the recruitment or differentiation of MDSC24, 25. We found that most of them were expressed at a higher level in induced tumors as compared with transplanted tumors (Fig. 6f, g). Among these factors, Csf3, Cxcl1 and Cxcl5 are known to specifically recruit PMN-MDSC24,25,26. Although Ccl2, Cxcl12 and TGFβ are mostly known for their ability to recruit M-MDSC, they can also promote accumulation of PMN-MDSC in some tumor settings21, 24,25,26. In addition, it has been suggested that M-MDSC recruited at the tumor site can be subsequently converted into PMN-MDSC in the tumor microenvironment27. These results could therefore explain the accumulation of PMN-MDSC in induced Amela TiRP tumors, and corroborate our previous observations of increased expression of such factors, including Ccl2, Cxcl5 and Ccl7, in Amela TiRP tumors as compared to Mela TiRP tumors, which recruit much less MDSC3, 4. Interestingly, overexpression of CCL2 and CCL7 was also reported in human metastatic melanomas that resist anti-PD1 therapy, and is a cardinal feature of the IPRES signature5.

Fig. 6 PMN-MDSC are enriched in induced Amela TiRP tumors. a Cellular analysis of the tumor tissue. Induced Amela tumors (n = 27) and transplanted T429.11 (n = 25) or T429.6 (n = 18) tumors (500 mm3) were homogenized and analyzed by FACS for the proportion of CD45+ cells (left panel) and MDSC of the polymorphonuclear type (PMN-MDSC: Gr-1h, CD11b+, Ly6C−/lo Ly6G+) or the monocytic type (M-MDSC: Gr-1lo/int, CD11b+, Ly6Ch and Ly6G-). b Same analysis as in Fig. 5a comparing pigmented (Mela, n = 16) and unpigmented (Amela, n = 16) induced TiRP tumors. c PMN-MDSC isolated from induced Amela TiRP tumors were co-cultured with activated TCRP1A CD8+ T cells for 3 days. CD8+ T-cell proliferation was evaluated by measuring 3H-thymidine incorporation. Two independent experiments, in triplicates. d PMN-MDSCs isolated from induced Amela TiRP tumors were co-cultured for 3 days with activated TCRP1A CD8+ T cells. CD8+ T cells were then isolated and their ability to kill P1A-positive P815 cells (clone P511) was evaluated in a standard chromium release assay. Two independent experiments, in triplicates. e Melanoma cells T429.11 were mixed with PMN-MDSC isolated from induced Amela TiRP tumors at a 4/1 ratio and injected into the left flank of B10.D2;Ink4a/Arfflox/flox mice. The right flank of the mice received the tumor cells without MDSC. ACT was performed when the left tumor size reached around 500 mm3 (n = 8). f and g Expression of cytokines and chemokines potentially involved in MDSC recruitment was analyzed by quantitative RT-PCR analysis in tumor tissues from induced Amela TiRP tumors (n = 19) and from transplanted T429.11 tumors (n = 14). Gapdh was used as an endogenous control to normalize each sample. Results are expressed as mean ± s.e.m. Unpaired t-test, two-tailed a, b, f, g. Two-way ANOVA e. *P < 0.05, **P < 0.01, ***P < 0.001. ****P < 0.0001 Full size image

Having shown that TIL apoptosis in the TiRP model was mediated by FasL, we then analyzed FasL expression on cells from the tumor tissue to determine which cell type was responsible for inducing TIL apoptosis. We consistently observed high levels of FasL on MDSC (Fig. 7a, b). Lower levels of FasL were also observed on endothelial cells and, although inconsistently, on tumor cells. This expression profile was identical whether we analyzed tumors collected before or after ACT (Fig. 7a, b). FasL expression was equally high on PMN-MDSC and M-MDSC (Fig. 7c), but only the former were enriched in induced as compared with transplanted tumors (Fig. 6a). This result suggested that PMN-MDSC were responsible for TIL apoptosis. This conclusion was supported by in vitro experiments showing that PMN-MDSC induced apoptosis of co-cultured TCRP1A CD8+ T cells, the extent of which was reduced by FasL neutralization with soluble Fas-Fc (Fig. 7d, e). Moreover, TIL apoptosis was reduced in vivo when we depleted PMN-MDSC by intra-tumoral injection of anti-Ly6G antibody (Fig. 7f).

Fig. 7 PMN-MDSC induce CD8+ T-cell apoptosis through Fas-ligand. a FasL expression was analyzed by FACS on cell homogenates from two representative induced Amela TiRP tumors, having received ACT or not 4 days earlier. Top panel: whole cell population. Second panel: MDSC (Gr1+ CD11b+). Third panel: Endothelial cells (CD31+). Fourth panel: Tumor cells (P1A+, CD45-). Fifth panel: Other cells (Gr1−, CD11b−, CD31−, P1A−). Sixth panel: isotype control. b Mean fluorescence intensity (MFI) of FasL expression of indicated cells obtained as in Fig. 6a (induced tumor with ACT: n = 12; induced tumor without ACT: n = 12). c Comparison of mean fluorescence intensity (MFI) of FasL expression by M-MDSC and PMN-MDSC (induced tumors without ACT: n = 8). d Apoptosis of activated TCRP1A CD8+ T cells upon co-incubation for 24 h at the indicated ratio with PMN-MDSC isolated from induced Amela TiRP tumors. Purity of PMN-MDSC cells: 80–90%. Five independent experiments, each in duplicate. e Apoptosis of activated TCRP1A CD8+ T cells was prevented by adding soluble Fas-Fc (10 µg/ml) to PMN-MDSC 1 h before and during the 24 h co-incubation with activated TCRP1A CD8+ T cells. Three independent experiments, each in duplicate. f Ex vivo analysis of apoptosis of TCRP1A CD8+ T cells in induced Amela TiRP tumors 4 days after adoptive transfer, in mice that were depleted of Ly6Gh cells by intra-tumoral injection of anti-Ly6G antibody (n = 42) or isotype control (n = 33) (3 injections of 200 µg every 3 days, starting 4 days before adoptive transfer). The right panel shows the efficiency of depletion in the same mice. Results are expressed as mean + s.e.m. Unpaired t-test, two-tailed b–e. *P < 0.05, **P < 0.01, ***P < 0.001. ****P < 0.0001 Full size image

To determine whether in vivo FasL neutralization could increase the efficacy of ACT, we treated mice bearing induced tumors with soluble Fas-Fc starting one week before ACT, and followed tumor growth. We observed better tumor control in mice receiving ACT combined with Fas-Fc, as compared with mice receiving ACT alone (Fig. 8a, b). Even though Ly6G-antibody mediated depletion of PMN-MDSC combined with ACT showed some effect (Fig. 8b), when combined with Fas-Fc and ACT, PMN-MDSC depletion did not further improve tumor control (Fig. 8a, b), indicating that FasL-mediated apoptosis was the dominant suppressive mechanism of PMN-MDSC in this model system. Those results indicate that FasL neutralization can improve the efficacy of immunotherapy based on ACT.

Fig. 8 FasL neutralization increases the efficacy of immunotherapy. a, b Mice bearing Amela TiRP tumors received i.p. injections of soluble Fas-Fc and/or anti-Ly6G antibody, starting when tumor size reached 500 mm3, and repeated twice a week. ACT was applied 3 days after the first injection, and tumor volume was monitored (Mean ± s.e.m, n = 10 mice/group). c Mice treated as in (a, b) received anti-CTLA4 and anti-PD1 antibodies i.p. 1 day after the first injection of Fas-Fc/anti-Ly6G, and then twice a week for a total of four injections (Mean ± s.e.m; isotype: n = 10; CTLA4/PD1: n = 9; CTLA4/PD1/Ly6G/Fas-Fc: n = 10; this experiment was run together with the one described in (a, b) and the isotype control group is identical). d Mice bearing transplanted melanomas T429.11 received i.p. injections of anti-Ly6G and/or Fas-Fc starting when tumors became palpable, and repeated twice a week. 1 day later they received i.p. injections of anti-CTLA4 and anti-PD1, repeated twice a week for a total of four injections (Mean ± s.e.m; isotype: n = 7; CTLA4/PD1: n = 7; CTLA4/PD1/Fas-Fc: n = 10, CTLA4/PD1/Ly6G/Fas-Fc: n = 10; data pooled from two independent experiments). Depletion of Ly6G+ cells in tumors was checked after killing a–d. Two-way ANOVA a–d, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 Full size image

To explore whether FasL neutralization can also increase the efficacy of immunotherapy based on immune checkpoint inhibitors, we first set up experiments in which we treated mice bearing induced TiRP tumors with ACT combined with anti-CTLA4 and anti-PD1 antibodies. The addition of immune checkpoint inhibitors failed to improve tumor rejection, unless it was combined with FasL neutralization and Ly6G-depletion (Fig. 8c). This improved tumor control was, however, similar to the one observed with ACT combined with Fas-Fc ± anti-Ly6G (Fig. 8a, b). These results indicate that TiRP tumors display additional immunosuppressive mechanisms that make them insensitive to immune checkpoint inhibitors. To further explore potential synergy between immune checkpoint inhibitors and FasL neutralization, we treated mice bearing transplanted T429.11 tumors with anti-CTLA4 and anti-PD1. We observed no tumor growth inhibition (Fig. 8d). However, when we combined anti-CTLA4 and anti-PD1 with Fas-Fc we observed a significant tumor growth inhibition (P = 0.0004, two-way ANOVA). When we also depleted PMN-MDSC in those mice we observed no further increased tumor growth inhibition, indicating that MDSC do not contribute other major immunosuppressive mechanisms in this setting. Although their recruitment is lower than in induced TiRP tumors, MDSC are also present in transplanted T429.11 tumors (Fig. 6a) and they express high levels of FasL (FasL MFI was 2879 and 2758 for transplanted and induced tumors, respectively, mean of 10 mice each). Therefore, they seem to contribute to resistance to immune checkpoint therapy, although it is possible that other FasL-expressing cells also contribute. Altogether, these results indicate that FasL neutralization has the potential to improve the efficacy of immunotherapy based not only on adoptive cell therapy but also on immune checkpoint inhibitors.

A striking feature of this immunosuppressive mechanism is its antigen-specific nature: only anti-tumor CD8+ T cells are affected by apoptosis and disappear. This specificity likely results from the fact that antigenic activation strongly increases Fas expression at the surface of CD8+ T cells. This was confirmed when we isolated spleen cells from TiRP-tumor-bearing mice and incubated them with L1210.P1A.B7-1 cells to activate P1A-specific T cells: after 48 h, we compared Fas expression on CD8+ T cells that were P1A-specific or not (Supplementary Fig. 10a). Fas expression was much higher on P1A-specific CD8+ T cells, which were the only CD8+ T cells that were activated in these experimental conditions, as indicated by CD69 expression. Moreover, when we incubated these T cells with FasL, only P1A-specific CD8+ T cells underwent apoptosis (Supplementary Fig. 10b). We also observed that IFNγ further increased Fas expression on activated CD8+ T cells (Supplementary Fig. 4c). The latter finding likely contributes to explain the reduced TIL apoptosis we observed in mice treated with IFNγ-neutralizing antibodies (Fig. 4c). As FasL is already expressed by MDSC before ACT, and therefore probably does not depend on IFNγ produced by TIL, the pro-apoptotic effect of IFNγ in this setting likely results from increased expression, on activated T cells themselves, of both Fas and FasL, according to the well-described phenomenon of activation-induced cell death (AICD), an immune checkpoint process that prevents excessive T-cell activity by inducing Fas/FasL-mediated suicide/fratricide killing of activated T cells28, 29. AICD, however, is expected to occur in transplanted tumors, as well as in induced tumors, and therefore cannot account for the increased TIL apoptosis observed in induced tumors, which rather appears to be triggered by FasL-expressing MDSC that are enriched in these tumors.

Relevance to human tumors

To determine whether FASLG expression was associated with disease progression in human tumors, we used The Cancer Genome Atlas (TCGA) database to compare the survival of patients bearing tumors expressing different levels of FasL. In most tumor types, high FASLG tran﻿script levels were associated with a relatively better survival than low levels. This difference was statistically significant in cutaneous melanoma (P = 0.000014), head-and-neck squamous cell carcinoma (P = 0.011) and breast carcinoma (P = 0.0078) (Fig. 9a–c). In sharp contrast, high FASLG expression in renal cell carcinoma (P = 0.04) and uveal melanoma (P = 0.000059) was associated with significantly worse prognosis (Fig. 9d, e). It is noteworthy that these two tumor types are also those that diverge from most other malignancies by their shorter survival associated with higher TIL infiltration30,31,32. We therefore considered the possibility that FASLG expression in human tumors was in fact associated with T-cell infiltration. Consistently, expression of FASLG in the main TCGA tumor types was strongly correlated with the levels of T-cell-specific transcripts such as IFNG (shown for melanoma in Fig. 9f), CD3E and CD8B (not shown). FASLG transcript levels in tumors thus reflect TIL abundance and activity, in line with the selective expression of FasL in activated T cells, and cannot be used as an independent prognostic factor. Interestingly, a similar correlation with TIL infiltration was observed for the transcript levels of IDO1 and PD-L1, two well-known immune checkpoints that are induced by T-cell activation and involved in adaptive tumoral resistance, as ascertained by the clinical benefit obtained with specific inhibitors12.