Ovarian granulosa cell tumor characterization identifies FOXL2 as an immunotherapeutic target

Granulosa cell tumors (GCT) are rare ovarian malignancies. Due to the lack of effective treatment in late relapse, there is a clear unmet need for novel therapies. Forkhead Box L2 (FOXL2) is a protein mainly expressed in granulosa cells (GC) and therefore is a rational therapeutic target. Since we identified tumor infiltrating lymphocytes (TILs) as the main immune population within GCT, TILs from 11 GCT patients were expanded, and their phenotypes were interrogated to determine that T cells acquired late antigen-experienced phenotypes and lower levels of PD1 expression. Importantly, TILs maintained their functionality after ex vivo expansion as they vigorously reacted against autologous tumors (100% of patients) and against FOXL2 peptides (57.1% of patients). To validate the relevance of FOXL2 as a target for immune therapy, we developed a plasmid DNA vaccine (FoxL2–tetanus toxin; FoxL2-TT) by fusing Foxl2 cDNA with the immune-enhancing domain of TT. Mice immunization with FoxL2-TT controlled growth of FOXL2-expressing ovarian (BR5) and breast (4T1) cancers in a T cell–mediated manner. Combination of anti–PD-L1 with FoxL2-TT vaccination further reduced tumor progression and improved mouse survival without affecting the female reproductive system and pregnancy. Together, our results suggest that FOXL2 immune targeting can produce substantial long-term clinical benefits. Our study can serve as a foundation for trials testing immunotherapeutic approaches in patients with ovarian GCT.


Introduction
Granulosa cell tumors (GCT) of the ovary are rare tumors accounting for less than 5% of all ovarian malignancies. Due to the relatively high recurrence rate, 30% of women diagnosed with GCT will ultimately die 10-30 years after their initial diagnosis (1,2). As GCT does not respond well to standard chemotherapy, novel therapeutic approaches are desperately needed.
Cancer immunotherapy aims to reprogram the patient's immune system to fight its own cancer cells via recognition of tumor antigens. Immunotherapeutic agents can be divided into active and passive categories. Active immunotherapies, such as cancer vaccines, aim to instruct the immune system to recognize and attack tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs) (3). Passive immunotherapies deal with adoptive transfer of ex vivo expanded cells (4) or exogenous administration of monoclonal antibodies. Adoptive T cell therapy (ACT) consists of isolating a cancer patient's T cells, followed by selection (in case of tumor-infiltrating lymphocytes [TILs]) (5) or engineering (in case of CAR-T cells) (6), ex vivo expansion, and infusion back into the patient. ACT of TILs was first tested in melanoma patients and demonstrated impressive objective response rates of over 40% and a complete remission rate of up to 24% (7). Although cancer vaccines targeting nonself antigens (e.g., Cervarix, Gardasil9) have produced exciting results in the clinic (8), cancer vaccines encoding self TAA have shown poor efficacy, with only a small fraction of patients experiencing an objective clinical response (9,10). Such limited efficacy is, in part, explained by the low immunogenicity of most TAAs, and accordingly, several strategies are being studied to boost immune responses. For example, in the case of plasmid-DNA cancer vaccines, in vivo electroporation increases DNA uptake, leading to enhanced antigen expression and concomitant increase in immune responses (11). Moreover, fusion of the antigen with the minimized domain of the C fragment of tetanus toxin (TT), has been used to elicit antigen-specific immune responses (12)(13)(14). Despite the limited efficacy Granulosa cell tumors (GCT) are rare ovarian malignancies. Due to the lack of effective treatment in late relapse, there is a clear unmet need for novel therapies. Forkhead Box L2 (FOXL2) is a protein mainly expressed in granulosa cells (GC) and therefore is a rational therapeutic target. Since we identified tumor infiltrating lymphocytes (TILs) as the main immune population within GCT, TILs from 11 GCT patients were expanded, and their phenotypes were interrogated to determine that T cells acquired late antigen-experienced phenotypes and lower levels of PD1 expression. Importantly, TILs maintained their functionality after ex vivo expansion as they vigorously reacted against autologous tumors (100% of patients) and against FOXL2 peptides (57.1% of patients). To validate the relevance of FOXL2 as a target for immune therapy, we developed a plasmid DNA vaccine (FoxL2-tetanus toxin; FoxL2-TT) by fusing Foxl2 cDNA with the immune-enhancing domain of TT. Mice immunization with FoxL2-TT controlled growth of FOXL2-expressing ovarian (BR5) and breast (4T1) cancers in a T cell-mediated manner. Combination of anti-PD-L1 with FoxL2-TT vaccination further reduced tumor progression and improved mouse survival without affecting the female reproductive system and pregnancy. Together, our results suggest that FOXL2 immune targeting can produce substantial long-term clinical benefits. Our study can serve as a foundation for trials testing immunotherapeutic approaches in patients with ovarian GCT. of many clinical trials targeting self TAA, vaccines remain an attractive anticancer modality. They generally represent a specific, off-the-shelf intervention that is well tolerated and could lead to durable responses because of immunological memory.
Immunotherapy efficacy depends on T cell availability and TAAs they target. An ideal antigen for immunotherapy should be uniquely expressed by neoplastic cells, display robust antigenicity, and participate in key cellular functions to prevent the selection of malignant clones losing expression. Thus, the identification of potentially novel TAAs is critically important in exploring the potential of vaccines and ACT in cancer. Forkhead box protein L2 (FOXL2), a member of the forkhead-winged helix family, is a highly conserved transcription factor involved in virtually all stages of ovarian development and function (15)(16)(17). According to the Human Protein Atlas, FOXL2 protein is exclusively found in the ovary and the endometrium, while its RNA is also observed in endocrine tissues at a significantly lower level than female tissues (18). Indeed, substantial FOXL2 expression has been reported in the pituitary glands (19). Misregulation of FOXL2 expression and the presence of a highly recurrent somatic mutation C402G (C134W), identified in 90%-97% of GCT (1, 2), contributes to the transformation of normal granulosa cells to a malignant state (20). Interestingly, FOXL2 expression levels increase in GCT (20), as well as in some breast (21) and cervical cancers (22). High expression of FOXL2 correlates with worse overall survival in GCT (23), possibly due to its antiapoptotic role (24).
In this preclinical study, we characterized the immune landscape of GCT and identified T lymphocytes as the main immune population in the tumor microenvironment (TME). We successfully expanded TILs from 11 GCT patients and demonstrated that lymphocytes acquired late antigen-experienced memory phenotypes that express low PD1 levels. For those patients whose viable tumor cells were available, we tested their TIL reactivity and concluded that 9 of 9 patients had at least 1 TIL culture robustly reacting against autologous tumors. Seven patients were also tested for reactivity against FOXL2 peptides, and we demonstrated that 4 of them (57.1%) possessed FOXL2-specific TILs, suggesting that FOXL2 is an ideal target in GCT. With the goal of validating the immunogenicity and effectiveness of FOXL2-targeted response, we developed a plasmid-DNA vaccine encoding murine Foxl2 that was able to reduce tumor progression in FOXL2-expressing ovarian and breast cancer models in a T cell-mediated manner. Combination of vaccination with anti-PD-L1 further suppressed tumor progression and improved mice survival without affecting female reproductive system and pregnancy.

Results
T lymphocytes is the main immune population within digested GCT. The composition of tumor immune cell infiltration impacts the outcome of several human malignancies, as well as the response to anticancer therapies (25). In this study, we used multiparametric flow cytometry ( Figure 1A) to quantify the number of helper (CD4 + ) and cytotoxic (CD8 + ) T cells as well as Tregs (CD4 + CD25 + FOXP3 + ) in GCT. We also develop a 9-color panel ( Figure 1, B-D) to carefully characterize myeloid cells, such as tumor-associated macrophages (TAMs), DC, and myeloid-derived suppressor cells (MDSC). Peripheral blood mononuclear cells (PBMCs) from healthy donors were also included. Analyses of 7 GCT specimens showed that 4.0% of total tumor single cells suspensions were CD8 + T cells, 3.3% were CD4 + T cells and 0.72% were CD4 + CD25 + FOXP3 + Tregs ( Figure 1E). Moreover, FACS staining indicated that both CD4 + and CD8 + T cells expressed increased levels of the activation marker PD1, which is suggestive of tumor-specific T cells (26,27), compared with circulating T cells (CD8 + PD1 + T cells; CD4 + PD1 + T cells, P < 0.05) ( Figure 1F). In ovarian cancer, it has been suggested that the effector/suppressor cell ratio may be a better indicator of outcome than individual T cell count (28). In ovarian GCT, we found a lower CD8 + T cells/Treg ratio than in healthy PBMCs (P = 0.067), likely contributing to an immunosuppressive tumor environment ( Figure 1G). Our results also showed that TAMs/monocytes (CD45 + CD14 + ) were the main myeloid population in GCT, accounting for 2.2% of total tumor single cell suspension ( Figure 1H). DCs were separated from the TAMs/monocytes based on CD14, HLA-DR, and CD11c markers (29) (CD45 + CD14 -HLA-DR + CD11c + ) and represented 0.27% of the total cell suspension. The MDSC populations (30) were marked as eMDSC (Lineage -CD11b + CD33 + ), amounting at 0.06%, and as PMN-MD-SC (CD45 + CD15 + CD14 -CD11b + ), amounting at 0.11% of the total tumor cell suspension in GCT (Figure 1H). Using comparative real-time PCR, we observed a 16-fold increase of PD-L1 in flash-frozen GCT compared with PBMCs or with a non-GCT malignancy (renal cell carcinoma; RCC) (Supplemental Figure 3A; supplemental material available online with this article; https://doi.org/10.1172/ jci.insight.136773DS1) (PBMCs vs. GCT, P = 0.05; non-GCT malignancy vs. GCT, not significant).
In conclusion, our results show that GCT is significantly infiltrated by helper and cytotoxic lymphocytes, which are possibly tumor specific. However, the relatively high proportion of PD1 + T cells, CD8 + T cells/ Treg ratio, and high TAMs/monocytes in the TME imply that GCT might establish immunosuppressive mechanisms to escape immune recognition.
Memory phenotype TILs expressing a low level of PD1 compose the major subset after REP. Most immunotherapies aim to boost the presence of tumor-reactive T cells within the solid tumor; therefore, we studied the feasibility of expanding T cells within GCT. Freshly resected tumors (n = 11) were minced into small fragments (~1-2 mm 3 ) and plated as 1 fragment per well in media containing IL-2 (31). After approximately 3 weeks, wells with about 1 × 10 6 cells were further expanded for 2 additional weeks using rapid expansion protocol (REP), allowing quick expansion of TILs to ~1 × 10 8 cells (32) (post-REP TILs). All 11 GCT samples were successfully expanded, analyzed, and cryopreserved. FACS phenotype demonstrated that virtually all pre-and post-REP TILs were CD3 + (data not shown). The proportion of CD4 + T cells was significantly higher than CD8 + T cells in both pre-REP (P < 0.0001) and post-REP TIL samples (P = 0.0007) (Figure 2A). Furthermore, both CD4 + and CD8 + T cells had significantly higher levels of PD1 before REP than those of PBMCs (CD4 + T cell, P < 0.0001; CD8 + T cell, P = 0.0023), though levels dropped significantly after REP to became statistically indistinguishable (CD4, P = 0.15; CD8, P = 0.72) from PBMCs ( Figure 2B) (PD1 + CD4 + T cell pre-REP vs. post-REP, P = 0.0002; PD1 + CD8 + T cell pre-REP vs. post-REP, P = 0.0007).
It has been demonstrated that memory phenotype TILs, specifically the central memory (T CM ) subset, is required for effective ACT therapy (33). Surface expression of CD27 and CD45RA markers can be used to differentiate T cells into 4 subtypes: effector (T E , CD27 − CD45RA + ), naive (T N , CD27 + CD45RA + ), T CM (CD27 + CD45RA − ), and effector memory (T EM , CD27 − CD45RA − ) (34). Similar to what has been reported (34), healthy PBMCs -used as control population -showed a typical distribution composed largely of T N , followed by T CM , T E , and hardly any T EM (Figure 2, C and D). In GCTs, the CD4 + T cells in pre-REP TILs were predominantly memory T cells (32.8% T CM and 60.6% T EM ), which transitioned after-REP to become almost entirely T EM (91.7%) (P < 0.0001) ( Figure 2C). The CD8 + T cells in pre-REP TILs, however, showed a more heterogeneous phenotype (18.9% T E , 41.5% T N , 10.4% T CM , and 25.5% T EM ) ( Figure 2D). Similar to the CD4 + subset, the CD8 + T cells transitioned to a memory phenotype (25.7% T CM and 64.1% T EM ) during REP, but interestingly, both the T CM and T EM significantly increased after REP compared with pre-REP (CD8 + T CM , P < 0.0001; CD8 + T EM , P < 0.0001) ( Figure 2D). Percentages of PD1 + CD8 + T cells and PD1 + CD4 + T cells in pre-and post-REP (n = 10 patients) cultures, as well as in healthy PBMCs (n = 6 patients). (C) and (D) Percentage of T cells subtypes in pre-and post-REP (n = 10 patients) cultures and in healthy PBMCs (n = 6). Each point represents an independent TIL fragment (or culture). Mean ± SEM is shown. Tukey's multiple comparison tests were performed. *P < 0.05, **P < 0.01, ***P < 0.001.
Selection of TILs' cultures for ACT requires screening of tumor-reactive T cells. To assess TIL reactivity, IL-2-rested TILs (post-REP) were cocultured overnight with autologous tumor cells, and ELISA was used to determine IFN-γ secretion, a marker of lymphocyte activation. Out of the 11 patients, only 9 had viable tumor cells to test T cell reactivity. Remarkably, all 9 patients tested had at least 1 TIL culture showing increased IFN-γ production after exposure to autologous tumor cells ( Figure 3 and Supplemental Figure  1A). IFN-γ production was usually decreased with the addition of anti-MHC class I (anti-MHCI) blocking antibody, indicating that tumor recognition by TILs was MHCI mediated. For some samples, pre-REP TILs were also screened for tumor reactivity. Data from patient 2493 show that, after REP, IFN-γ production significantly increased compared with the pre-REP TILs, possibly due to a higher CD8 + /CD4 + T cell ratio observed after REP in this sample (Supplemental Figure 1B). Ability to kill target cells was also tested using TILs and primary GCT cells both derived from patient 2522, the only patient whose tumor cells stably grew in vitro (Supplemental Figure 2).
Altogether, these results indicate that TILs from GCT can easily be expanded to large numbers, making them suitable for ACT approaches. After REP, the CD4 + TILs become entirely T EM , while the CD8 + counterpart maintain a pronounced T CM phenotype, which is important for an effective ACT. Moreover, TILs decrease PD1 expression during REP and vigorously respond to autologous tumors, possibly demonstrating reversion from immune dysfunction to a more effective status.
Expanded TILs recognize epitopes of FOXL2 protein. Since endogenous FOXL2 is upregulated in GCTs (Supplemental Figure 3B) and 90%-97% of GCTs contain a somatic mutation (FOXL2 C134W ) that can lead to the generation of a neoantigen, we investigated whether patients spontaneously possess reactive TILs against FOXL2. To test this hypothesis, we developed a FOXL2 peptide library encompassing the entire human FOXL2 C134W protein, including the mutation C134W, divided in 4 pools: pool A (sequences spanning aa 1-103), pool B (aa 93-195), pool C (aa 185-287), and pool D (aa 277-376). To this end, we measured IFN-γ production by ELISA and intracellular staining (ICS) after overnight coculture of IL-2-rested TILs with autologous PBMCs pulsed with FOXL2 peptides pools. As revealed in Figure 4, A and B, 7 patients were tested -those with available PBMCs -and 4 of them (57.1%) possessed at least 1 TIL fragment specific to FOXL2 pools. Moreover, IFN-γ ICS performed on sample 2406 #1 showed CD8 + T cells reacting against pool C and pool D ( Figure 4C), validating the ELISA. Figure 4B summarizes the normalized values of TIL reactivity sorted by peptide pools. Because most patients recognized pool D, pool D was deemed a hot-spot region likely to contain immunodominant epitopes. Interestingly, limited reactivity was observed against pool B, which contained peptides covering the C134W mutation. Stimulation of PBMCs from healthy donors with the FOXL2 peptide library revealed a lack of T cell activation (data not shown), suggesting that, in physiological conditions, FOXL2 protein does not induce spontaneous adaptive immune response. Altogether, the results suggest that T cell-infiltrating GCTs are reactive against autologous tumors and that part of such reactivity is targeted toward FOXL2 epitopes.
DNA immunization with FoxL2-TT breaks immune tolerance to FOXL2 in 3 different strains of mice. To study the in vivo effectiveness of FOXL2 targeting, we developed a plasmid-DNA vaccine encoding murine FOXL2. Plasmid vaccines are closed circular DNA expression vectors designed to deliver antigens encoded under a strong promoter. Our DNA vaccine was generated using a pVAX plasmid vector that encoded the codon-optimized mouse Foxl2 C389G cDNA, which harbored the C130W mutation (corresponding to C134W in human) and was fused in frame with the first domain of the C fragment of the TT sequence (TT 865-1120), used as an immune enhancer (11, 14, 35) (Supplemental Figure 4A, left panel). Fusion of FOXL2 and TT was confirmed by Western blot (WB) (Supplemental Figure 4B). The resulting construct (FoxL2-TT) was injected 3 times at weekly intervals in healthy C57BL/6, BAL-B/c, and Tg (HLA-A2.1) mice. Priming/boost injections were followed by in vivo electroporation. One week after the last immunization, splenocytes harvested from vaccinated mice were stimulated with the mouse FOXL2 peptide library, consisting of pools A (aa 1-103), B (aa 93-195), C (aa 185-286), and D (aa 276-375). IFN-γ ELISpot indicated that the majority of T cell epitopes in BALB/c were contained within pool D ( Figure 5A), whereas spleen-derived T cells from vaccinated C57BL/6 and Tg (HLA-A2.1) mice exhibited reactivity against both pools C and D ( Figure 5C and Supplemental Figure  5A). IFN-γ ICS confirmed the ELISpot results and further showed that, in BALB/c, both CD4 + and CD8 + T cells reacted against pool D ( Figure 5B), whereas CD8 + but not CD4 + T cells reacted against pools C and D in C57BL/6 ( Figure 5D). FoxL2-TT immunization did not induce any FOXL2 C130W -specific T cell response, as highlighted by the lack of reactivity against pool B, which contained the peptides harboring the C130W mutation. By individually using each single peptide from pools C and D, we found multiple immunodominant reactive peptides including #54 (FOXL2 213-227 ); #61, #62, and  Table 1) indicating ASYGPYSRV (FOXL2 249-257 ), contained within peptides #62 and #63, as the best H-2Kb-restricted binder in C57BL/6. Finally, IFN-γ ICS showed that both CD4 + T cells and CD8 + T cells were activated by peptide #72 and #73, suggesting their binding to MHCI and MHCII in BALB/c (Supplemental Figure 5B). Together, these data confirm the immunogenicity of FOXL2 and indicate that it is possible to break the immune tolerance to FOXL2 in different animal strains and elicit T cells response.
FoxL2-TT immunization suppresses tumor growth in FOXL2-expressing ovarian and breast cancer models. Using Realtime PCR, we first measured the endogenous Foxl2 expression in different mouse organs and found Foxl2 mostly expressed in the ovary ( Figure 6A), paralleling human data (Human Protein Atlas; ref. 18). Due to the lack of any suitable GCT model (36) to test in vivo vaccine's efficacy, we attempted to identify FOXL2 expression in several tumor cell lines. In line with a FOXL2 expression pattern in human female tissues (18, 21), we found a very limited level of Foxl2 cDNA in 2 ovarian cancer cell lines (BR5 and ID8) and 1 breast cancer cell line (4T1) (Supplemental Figure 6). The lack of Foxl2 expression prompted us to overexpress mutated Foxl2 C389G in BR5 and 4T1 cancer cell lines using a pCMV6-A-PURO vector (Supplemental Figure 4A, right panel). Significant expression of FOXL2 was confirmed by WB and FACS (Supplemental Figure 4B and Supplemental Figure 4, C and D, left panels), and no difference in both in vitro (not shown) and in vivo tumor progression between the WT and the FOXL2-expressing cell lines was observed (Supplemental Figure 4, C and D, right panels). We then tested the specificity of vaccine-primed T cells to recognize FOXL2-expressing cells. To this end, we vaccinated FVB mice with FoxL2-TT, magnetically isolated T cells from splenocytes and exposed BR5-FOXL2 and BR5 WT to the T cells. IFN-γ ELISpot assay revealed that overexpression of FOXL2 in BR5 significantly increased T cell activation (P = 0.002) compared with BR5 WT ( Figure 6B). Similarly, BALB/c mice were vaccinated with FoxL2-TT and with a control vector encoding an irrelevant antigen fused with TT (TEM1-TT plasmid-DNA vaccine; ref. 11). 4T1-FOXL2 and 4T1 WT cells were then exposed to T cells isolated from vaccinated mice. Results from Figure 6C show that T cells from FoxL2-TT-vaccinated mice, but not from TEM1-TT-vaccinated mice, recognized the 4T1-FOXL2 cells (P = 0.047) but not 4T1 WT parental cells.
To assess the in vivo antitumor effects of FOXL2 immunization, tumor cells were inoculated s.c., and 3-5 days later, mice were given 3 weekly vaccine injections followed by in vivo electroporation. Therapeutic FoxL2-TT vaccination suppressed tumor progression compared with control vector (4T1, P = 0.0062; BR5, P = 0.0028) in BR5-FOXL2 and 4T1-FOXL2 tumor-bearing mice (Figure 6, D and F) and improved mouse survival (BR5, P < 0.02; 4T1, P < 0.04) (Supplemental Figure 7). In line with in vitro results, vaccination of mice bearing BR5 WT failed to significantly slow down tumor progression (Supplemental Figure 8, A  and B), indicating an antigen-specific effect of the vaccine. Analysis of the TME by flow cytometry revealed heavy infiltration of CD8 + and CD4 + T cells after vaccination in BR5-FOXL2 (CD8 + , P < 0.02; CD4 + , P < 0.003) ( Figure 6E) and 4T1-FOXL2 (CD8 + , P < 0.008; CD4 + , P < 0.002) tumors ( Figure 6G) versus control constructs. In the 4T1-FOXL2 model, we also studied the phenotypes (Supplemental Figure 9, A and B) of both bulk and FOXL2-reactive T cells, and we found that T N concentrate in the periphery (spleen and lymph node [LN]) whereas T EM accumulate within the tumor, mirroring our human T cell data. In contrast, FOXL2-reactive T cells were typically effector memory.
To determine if FoxL2-TT immune response was T cell mediated and to assess whether adoptive transfer of FOXL2-specific T cells would be efficacious in controlling growth of establish tumors, we performed ACT of T cells from immunized mice to recipient tumor-bearing mice. Transfer of CD3 + and CD8 + T cells was able to significantly suppress the progression of tumors expressing FOXL2, compared with control CD3 + T cells (4T1, CD3 + P < 0.001, CD8 + P = 0.012; BR5, CD3 + P < 0.02, CD8 + P = 0.05). Moreover, transfer of CD4 + T cells inhibited tumor progression in BR5-FOXL2, but not in 4T1-FOXL2, compared with control CD3 + T cells (4T1, nonsignificant; BR5 CD4 + , P < 0.03) (Supplemental Figure 10, A and B).
Cumulatively, these results demonstrate the therapeutic impact of FOXL2-specific T cells in 2 tumor models expressing FOXL2. Moreover, ACT experiments also demonstrate that transfer of FOXL2-restricted T cells controls tumor progression, indicating a T cell-mediated effect of the vaccine.
Combination of FoxL2-TT DNA immunization and anti-PD-L1 further suppresses tumor progression. Although immunization with Foxl2-TT plasmid-DNA significantly reduced tumor progression, we hypothesized that the vaccine's efficacy could be further improved via combination therapy. Since increased PD1 expression was observed both in TIL of human GCT ( Figure 2B) and on mouse intratumor CD4 + and CD8 + T cells (Figure 7, A and B), we combined PD1/PD-L1 inhibition with FoxL2-TT vaccination in the 4T1-FOXL2 model. To this end, we followed the same immunization protocol described above, but this time, an anti-PD-L1 was administered for 4 times, every 3 days, starting at day 12. Mice receiving the combination of vaccine and anti-PD-L1 suppressed tumor progression ( Figure 7C) (FoxL2-TT plus anti-PD-L1 vs. FoxL2-TT, P = 0.0042) and improved mice survival compared with vaccination (P < 0.007) or anti-PD-L1 (P < 0.001) monotherapies ( Figure 7D). Combination therapy also increased FOXL2-restricted immune response in the spleen and LN ( Figure 7E) and improved anti-FOXL2 T cell infiltration in the tumor (Figure 7F). In conclusion, adding anti-PD-L1 to FoxL2-TT vaccination significantly improved the antitumor effect and mice survival compared with monotherapy.
FoxL2-TT DNA vaccination does not affect mouse reproductive system and pregnancy. As FOXL2 expression is confined in the reproductive organs and inflammation influences pregnancy (37), we investigate whether FoxL2-TT immunization would affect female gestation. Female mice were immunized with FoxL2-TT or TT vaccine with or without anti-PD-L1. To assess potential inflammation and toxicity due to T cell activation, we performed H&E staining in the ovary, fallopian tubes, and uterus and found that vaccination does not induce inflammatory infiltrates, indicating that FOXL2-specific immune response is not directed to normal tissues ( Figure 8A).
Embryo implantation and placentation require a careful immunological balance. Intrauterine inflammation (IUI) is strongly associated with preterm birth, with clinical evidence of inflammation in up to 40% of preterm births (37). Furthermore, exposure to IUI during prenatal development is a known risk factor for adverse neurodevelopmental outcomes in offspring (38). Therefore, to test the possible impact of FoxL2-TT immunization on pregnancy, female mice were immunized with FoxL2-TT or TT vaccine, allowed to bred with healthy males, and monitored during 4 cycles of pregnancy.
No effects on time to gestation , total litter size, and pup weight at birth compared with control immunization were observed ( Figure 8B) (11). Collectively, FoxL2-TT vaccination appears to have no untoward effects on the mouse reproductive system.

Discussion
Because the immune system plays a critical role in tumor growth in virtually all solid tumors, exploring the immune landscape of GCT can guide development of novel immunotherapeutic approaches where effective treatment is lacking. The TME is composed of multiple immune cell types known to hamper lymphocyte-mediated attack, including Tregs, TAMs, and MDSCs. Several studies demonstrated that accumulation of Tregs and, in particular, a lower CD8 + T cell/Treg ratio within the tumor, correlates with poor clinical outcome in many cancers (39). In our GCT cohort, TILs were the most abundant immune cell type in the TME. In line with other malignancies (26), PD1 expression was consistently increased on TILs compared with T cells within PBMCs. The CD8 + T cell/Treg ratio in the tumor decreased compared with healthy PBMCs, suggesting that Tregs participate to the immunosuppressive environment of GCT. MDSCs are a heterogeneous cell population characterized by the ability to suppress T cells and NK cell function (40), as well as the ability to limit DC cross-presentation (41). Two studies in RCC (42) and colorectal cancer (43) found intratumor MDSCs to amount at about 5% and 2.99% of total tumor cell suspension, respectively. In our cohort of patients, the percentage of MDSCs  A and B) T cells from spleen and tumor suspension were stained to assess PD1 expression level. Each data point represents 1 mouse. Mean ± SEM is shown. (C and D) BALB/c were injected s.c. with 2.5 × 10 5 4T1-FOXL2 and, 5 days later, injected 3 times with FoxL2-TT or TT vaccines followed by electroporation. Data are shown as mean ± SEM (n = 6-8 mice per group). Two-way ANOVA analyses were performed for tumor growth experiments (C). For the Kaplan-Meier analysis (D), cutoff values for tumor volume of 1000 mm 3 for 4T1 were set to assess mice expiration. Long-rank test was performed to estimate statistical significance. (E and F) A total of 1 × 10 6 cells (spleen, LN, and tumor suspension) from vaccinated BALB/c mice were stimulated overnight with BALB/c-reactive peptide #73 and tested by ELISpot (E) and ICS (F). Bar graphs illustrating number of IFN-γ spots (E) and percentage of IFN-γ-secreting cells (F). Each data point represents 1 mouse. Mean ± SEM is shown.
was inferior to what was reported in RCC and colorectal cancer, underling the great disparity in the distribution and phenotypes of MDSCs in human cancers (29). TAMs represent diverse and heterogeneous populations of cells characterized by considerable plasticity. According to FACS analysis, they were the most prominent myeloid population in GCT. Finally, DC are the most powerful antigen-presenting cells and are critical in immune response initiation and development (44). It has been demonstrated that, although antigen presentation by DCs in the TME can be profoundly impaired (45), infiltration of tumors by DCs has often been linked to favorable prognosis in ovarian cancer (46) and other malignancies (47). DCs can be distinguished from TAMs based on low CD14 expression levels (29). In conclusion, our study shows that GCT is significantly infiltrated with multiple immune cell populations, likely affecting GCT responsiveness to therapies and patient outcomes.
ACT has shown remarkable efficacy, including overall response rates of 40% in metastatic melanoma, 90% in acute lymphoblastic leukemia, and 40% in chronic lymphoblastic leukemia (48). Because GCTs are heavily infiltrated by T cells, we investigated the feasibility of expanding TILs from freshly resected ovarian GCT. ACT efficacy depends upon both quantity and quality of the expanded lymphocytes. For instance, Radvanyi et al. demonstrated that the number of CD8 + T cells within the infused TILs are of critical importance in mediating tumor regression and increasing patient survival (49). However, the ratio between CD4: and CD8 in expanded TILs depends on the type of tumor as well as the culturing methods and can result in a heterogeneous ratio within the same study. For instance, while Dudley et al. (50) reported that most melanoma TILs cultures were predominantly CD8 + , others showed no consistent frequency of CD4 and CD8 among the expanded cultures (51). In our study, expanded TILs were mostly CD4 + T cells, averaging to 58%, which is similar to pancreatic cancer according to Hall et al. (52). In line with other studies reporting a decreased PD1 expression after long-term TILs expansion (53,54), we observed decreased PD1 expression on TILs after REP in both CD4 + and CD8 + T cells, probably due to the reduced proliferative potential of exhausted (PD1 + ) T cells (55). On the contrary, Li et al. did not observe any difference in PD1 expression during REP in melanoma (56).
Preclinical and clinical studies (33,57) have established that the adoptive transfer of early effector T CM CD8 + T cells, which possess higher levels of CD27, provides superior immunity compared with the transfer of late antigen-experienced (CD62L -CCR7 -CD27 -) T EM (33,58). In our study, we used CD45RA and CD27 markers to determine the subtype of expanded TILs (59,60). We observed that the CD4 + T cells became predominantly T EM after REP, as characterized by downregulation of the CD27 costimulatory marker. On the other hand, the CD8 + T cell population acquired expression of CD27 after REP, resulting in a small but significant increase in the central memory phenotype. The latter observation might emerge as contradictory to what has been observed in other TIL expansion studies, as these mostly found losses of CD28 and CD27 after extensive culturing of T cells during REP (57,61). However, expansion of TILs from different solid tumors might follow different dynamics; hence, reported observations in other studies might not be relevant in GCT.
When multiple independent TIL cultures are generated from a single tumor and screened for tumor recognition, they often exhibit multiple patterns of reactivity (61). In GTC, not all the TIL culture derived from an individual tumor showed the same level of reactivity, but importantly, all the patients demonstrated at least 1 tumor-reactive culture. The reactivity indicates that T cells are functionally active after REP and recognize 1 or more tumor antigen. In some samples, REP increased TILs' ability to react to the autologous tumor (i.e., patient 2493), a feature likely caused by increased CD8 + T cell numbers during REP. By using anti-MHCI blocking antibody, we were able to abrogate T cell activation in some TIL cultures, which indicated that tumor recognition was MHCI mediated. Importantly, tumor cells from patient 2522 successfully grew in vitro and allowed us to assess cytotoxicity of autologous expanded TILs, further validating their functionality. As previously achieved in other malignancies (56), we aimed to define the antigen recognized by T cells. FOXL2 is a marker for granulosa cells (17) and consistently contains a somatic nonsynonymous mutation (1,2), and its overexpression correlates with worse survival in GCT (23). Hence, we tested the reactivity of TILs against FOXL2-derived peptides, and we observed that 4 of 7 GCT patients showed spontaneous adaptive response against FOXL2, indicating that FOXL2 is a target of GCT. Given that FOXL2 expression has been reported in some breast (21) and cervical (22) cancers, we don't exclude that, in addition to being a marker and target in GCT, FOXL2 could be a potential target in those malignancies where its expression is elevated. The peptide-based approach facilitates detection of CD8 + and CD4 + T cells, and it has been suggested to provide higher sensitivity in mapping immune dominant epitopes than current prediction software (62). Our data suggest that GCT is an immunogenic tumor that often encloses functional tumor-reactive FOXL2-specific T cells within the repertoire of expanded TILs.
Having demonstrated that FOXL2 is a shared TAA in GCT and that patients often possess FOXL2-specific T cells, we developed the FoxL2-TT plasmid-DNA vaccine (14) and tested feasibility and efficacy of immunotherapy targeting FOXL2. We fused the mouse Foxl2 sequence with TT to enhance immunogenicity through several possible mechanisms. The whole C fragment of TT activates DCs to secrete cytokines involved in CD4 + T cell activation (12,35). In addition, fragment C contains a universal T helper epitope (p30) effective across different MHCII haplotypes in mice and humans and elicits strong CD4 + responses (63). To further enhance the vaccine's efficacy, injections of the plasmid-DNA were followed by in vivo electroporation, which increases transfection and generates localized inflammation (11), and it has facilitated translation of DNA vaccines in clinical trials (9). Our vaccine also used full-length antigen rather than short peptides, thus including all the possible epitopes present within FOXL2 and bypassing MHC restriction. All these attributes likely contributed to the generation of potent anti-FOXL2 immune responses that we observed targeted against FOXL2 peptide's pools C and D in C57BL/6, BALB/c, and Tg (HLA-A2.1) mice. Lack of reactivity to FOXL2 C130W indicate that the epitopes containing the mutation are not immunogenic in mice, as they are not in human, probably due to the inability of the peptide to strongly bind MHC molecules.
A handful of mouse models of ovarian GCT have been developed and are summarized by Kim et al. (36). As reported, there is not any GCT cell line syngeneic for BALB/c, C57BL/6, or FVB mice that also expresses FOXL2. In order to find suitable models to test our vaccine, we screened several tumor cell lines, and in line with FOXL2 expression restriction in female organs, we found only modest levels of Foxl2 cDNA in 2 ovarian cancer cell lines (BR5 and ID8) and 1 breast cancer cell line (4T1). The latter observation is in agreement with a study by Wegman et al., who demonstrated that FOXL2 is expressed in some breast cancer patients (21). Because of the very low endogenous level of Foxl2, we decided to overexpress the mutated form of Foxl2 to generate BR5-FOXL2 and 4T1-FOXL2 cell lines. Using a plasmid-DNA vaccine encoding an irrelevant antigen (TEM1) not expressed by 4T1 cells (11), we proved that solely T cells primed by the FoxL2-TT vaccination, and not by irrelevant antigenic portions of the vector, actively recognize the FOXL2-expressing tumor. In the BR5 model, a basal response by FOXL2-restricted T cells against WT cells was observed, likely due to the moderate Foxl2 cDNA expression in this model. We tested the therapeutic potential of FoxL2-TT vaccination and showed significant tumor control in both tumor models. Moreover, FoxL2-TT vaccination was ineffective against the BR5 WT cell line, proving the specificity of our approach. Immunostaining of FOXL2 + tumors in vaccinated mice revealed heavy infiltration of CD8 + and CD4 + T cells. Given the increased T cell infiltration within the tumor after vaccination, we hypothesized that the vaccine-induced antitumor effect was T cell mediated. To this end, we used adoptive transfer of T cells from vaccinated mice into tumor-bearing mice and showed that a subset of T cells affected tumor progression in BALB/c and C57BL/6 mice. Moreover, we demonstrated that, in preclinical models, ACT was efficacious in controlling tumor progression of established FOXL2-expressing tumors. These findings together with our human TIL results are encouraging and suggest a possible clinical impact of T cell-based therapy in GCT patients. Like human TILs, mouse T cells within the 4T1-FOXL2 tumor express high levels of PD1. Since the PD1/PD-L1 is one of the major axes used by tumors to escape cancer immune attack, we hypothesized that the vaccine's efficacy could be further improved via combination therapy. Addition of anti-PD-L1 to FoxL2-TT vaccination further suppressed tumor growth and improved mice survival compared with monotherapies. Because vaccination induces reactivity against self-FoxL2 epitopes potentially raising concern about toxicity, we investigate whether FoxL2-TT immunization would alter healthy ovarian follicles or damage reproductive female organs. Notably, FoxL2-TT vaccination does not seem to affect female reproductive system and pregnancy.
Cumulatively, our data underline GCT as immunologically "hot" tumor, heavily infiltrated by T cells, which can be expanded and reinvigorate (64) in vitro, preserving their antitumor activity and their ability to target FOXL2 antigen. Immunization with a plasmid-DNA vaccine encoding murine Foxl2-tt confirms immunogenicity of FOXL2 and controls tumor growth of FoxL2-expressing tumors without affecting female reproductive organs. Our preclinical data set in GCT can serve as foundation for clinical development of immunotherapeutic approaches for patient with ovarian GCT

Methods
Patients and sample preparation. Tissues from 11 ovarian GCT patients (Table 1) were collected with informed patient consent at the Penn Medicine Hospital of the University of Pennsylvania. Blood was collected directly into polypropylene tube containing heparin, and PBMCs were separated using Ficol (MilliporeSigma) density gradient centrifugation and cryopreserved in freezing media containing 10% DMSO (Thermo Fisher Scientific) and 90% FBS (Invitrogen). Tumor samples were collected and transported directly to the laboratory for processing. Single tumor cell suspensions from GCT patients were . LIVE/DEAD cell stain kit (Invitrogen, L34966) was also used. For staining of FOXL2 overexpression, anti-FOXL2 antibody (Novus, NBP2-22473) was used together with secondary Alexa Fluor 594 antibody anti-rabbit IgG (Invitrogen, A11012). Transcription factor staining buffer set (eBioscience, 00-5523-00) was used to permeabilize cells and stain for all the intracellular markers.
Initial TIL culture. TIL expansion was performed as described (50). Briefly, freshly resected GCT were minced into ~1-2 mm 3 fragments and placed in 24-well plates with 2 mL of TIL complete medium (CM) containing 3000 IU/mL of recombinant human IL-2 (rhIL-2; PEPROTECH), and GCT TILs were allowed to extravasate from the tissue. TIL were expanded in vitro for 3-5 weeks in CM consisting of RPMI 1640, 25 mmol/L HEPES, pH 7.2, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mmol/l-glutamine, and 5.5 × 10 −5 mol/L β-mercaptoethanol, supplemented with 10% human serum (Sigma-Aldrich, H4522). Half of the medium was replaced in all wells no later than 1 week after culture initiation and then twice weekly. Cell density was maintained at about 1 × 10 6 cells/mL, and cells were split into 2 daughter wells when needed. Each initial well was considered an independent TIL culture (fragment) and maintained separately from the others. When individual fragments reached about 2 × 10 6 , FACS analysis was performed and the REP was implemented. REP. REP expansion was performed as previously described (51). The REP used anti-CD3 antibody (Miltenyi Biotec, clone OKT3, 130-093-387) and rhIL-2 in the presence of irradiated, allogeneic feeder cells at a 200:1 ratio of feeder cells to TIL cells. Frozen PBMC feeder cells were obtained each time from 3 different healthy donors, thawed, washed, resuspended in CM, and irradiated (50 Gy). A total of 2 × 10 8 PBMC, OKT3 antibody (30 ng/mL), CM (75 mL), AIM V media (Thermo Fisher Scientific, 75 mL), and 1 × 10 6 TIL cells were combined into a 175 cm 2 tissue culture flask. Flasks were incubated upright at 37°C in 5% CO 2 , and rhIL-2 was added at 3000 IU/mL on day 2. On day 5 and day 10, 120 mL of culture supernatant was removed by aspiration, and media was replaced with a 1:1 mixture of CM/AIM V containing 3000 IU/mL IL-2. REP typically was allowed to proceed for 2 weeks, and cell expansion was monitored throughout. At the end of REP, cells were analyzed phenotypically by FACS, tested functionally by ELISA or ICS, and finally cryopreserved.
IFN-γ ELISA. To test TIL reactivity against autologous tumors, cocultures were established at a 1:1 ratio using 3 × 10 5 to 5 × 10 5 expanded TILs (fresh, never frozen) and autologous primary tumor cell suspensions that had been cryopreserved in 10% DMSO (Corning) and 90% FBS (Invitrogen, 16000044). To test TIL reactivity against FOXL2, cocultures of rhIL-2-rested TILs (1 × 10 5 ) and autologous PBMC (1 × 10 5 ) were incubated overnight with 1 μg/mL of individual peptide or peptides pools. For all the assays, TILs (after REP) were allowed to rest from rhIL2 and anti-CD3 stimulation for 7 days before coculture. Single tumor cell suspensions were CD45 depleted using EasySep depletion kit (Stemcell Technologies, 18259) before coculture. Where class I blocking experiments were performed, anti-HLA-ABC (BioLegend, clone W6/W32, 311402) was added to the tumor cells at 10 μg/mL and incubated for 30 minutes at room temperature before setting up the coculture. The incubation was carried out at 37°C overnight before the supernatant was harvested and analyzed for IFN-γ production using ELISA (ELISA MAX, BioLegend, 430104). To improve data presentation, IFN-γ values were normalized by subtracting the negative control (i.e., T cells alone) from the "Tumor + T cells" values or "T cells + PBMCs" values.
Cytotoxic assay. On day 1, target cells (GCT cell line and OVCAR-5) were stained with CellTrace CFSE (Invitrogen) following manufacturer's recommendation and plated in a 24-well plate at 1 × 10 5 /well. The day afterward, increasing ratios of TILs were added to the wells, and the plate was incubated overnight. On day 3, cells were collected and stained with LIVE/DEAD fixable violet dead cells stain kit (Invitrogen, L34964) and with anti-CD3 (eBioscience; clone 17A2, 46-0032-82). Cells were analyzed on a FACSCanto flow cytometer using FlowJo software.
WB. WB was performed with total cell lysate using antibody specific for FOXL2 (Abcam, ab5096), β-actin rabbit antibody (Cell Signaling Technology, 064967S). Membranes were incubated with a 1:2000 dilution of HRP-conjugated antibody rabbit to goat IgG (Abcam; ab97100) and anti-rabbit IgG (Cell Signaling Technology; 7074P2) and developed with the ECL system.
Statistics. For comparisons of more than 2 groups, we used Tukey's multiple comparison tests. For all other comparisons, 2-tailed Student's t tests using a pooled estimate of the variance were used. For all the mice experiments, sample sizes were chosen based on pilot experiments and our experience with similar experiments. For tumor progression experiments, we used the 2-way ANOVA (or mixed model) and the 2-tailed Student's t tests. *P < 0.05, **P < 0.01, ***P < 0.001. Study approval. All animal studies were approved by the IACUC and University Laboratory Animal Resources at the University of Pennsylvania. Mice were treated in accordance with University of Pennsylvania guidelines. Patients were collected under a research protocol that was approved by the University of Pennsylvania IRB.

Author contributions
SP helped design the studies, performed the experiments, analyzed the data, and drafted the manuscript. JLT provided human GCT samples and edited the manuscript. FS provided GCT samples and edited the manuscript. EG helped with patients and edited the manuscript. MUH performed experiments. RD assisted in study design and edited the manuscript. RB assisted patients and edited the manuscript. MAM helped in conceiving the project and writing the manuscript. AF conceived the experiments, supervised the project, and wrote the manuscript.