Blockade of HVEM for Prostate Cancer Immunotherapy in Humanized Mice

Simple Summary Current immune checkpoint inhibitors have shown limitations for immunotherapy of prostate cancer. Thus, it is crucial to investigate other immune checkpoints to prevent disease progression in patients with prostate cancer. Here, we first show that the HVEM/BTLA immune checkpoint is associated with disease progression in patients. We then show that immunotherapy aimed at targeting HVEM reduced tumor growth twofold in vivo in a humanized mouse model of the pathology. The mode of action of the therapy was dependent on CD8+ T cells and is associated with improved T cell activation and reduced exhaustion. Finally, we demonstrated that HVEM expressed by the tumor negatively regulated the anti-tumor immune response. Our results indicate that targeting HVEM might be an attractive option for patients with prostate cancer. Abstract The herpes virus entry mediator (HVEM) delivers a negative signal to T cells mainly through the B and T lymphocyte attenuator (BTLA) molecule. Thus, HVEM/BTLA may represent a novel immune checkpoint during an anti-tumor immune response. However, a formal demonstration that HVEM can represent a target for cancer immunotherapy is still lacking. Here, we first showed that HVEM and BTLA mRNA expression levels were associated with a worse progression-free interval in patients with prostate adenocarcinomas, indicating a detrimental role for the HVEM/BTLA immune checkpoint during prostate cancer progression. We then showed that administration of a monoclonal antibody to human HVEM resulted in a twofold reduction in the growth of a prostate cancer cell line in NOD.SCID.gc-null mice reconstituted with human T cells. Using CRISPR/Cas9, we showed that the therapeutic effect of the mAb depended on HVEM expression by the tumor, with no effect on graft vs. host disease or activation of human T cells in the spleen. In contrast, the proliferation and number of tumor-infiltrating leukocytes increased following treatment, and depletion of CD8+ T cells partly alleviated treatment’s efficacy. The expression of genes belonging to various T cell activation pathways was enriched in tumor-infiltrating leukocytes, whereas genes associated with immuno-suppressive pathways were decreased, possibly resulting in modifications of leukocyte adhesion and motility. Finally, we developed a simple in vivo assay in humanized mice to directly demonstrate that HVEM expressed by the tumor is an immune checkpoint for T cell-mediated tumor control. Our results show that targeting HVEM is a promising strategy for prostate cancer immunotherapy.


Introduction
Immune escape by tumors is now considered a hallmark of cancer [1]. Many immune mechanisms may explain the loss of tumor control, including defective MHC function and expression, recruitment of suppressive immune cells, and expression of co-inhibitory the efficacy of various treatments for cancer immunotherapy [35]. Regardless of the mode of reconstitution of the human immune system in NSG mice (from progenitors or peripheral blood, as herein), all suffer from the same drawback that human T cells will be allogeneic to the tumor. This would prevent the tumor antigen-specific response to be accurately measured in humanized mice. Nevertheless, accumulating evidence indicates that, in addition to their different proportions in the naïve T cell repertoire, a high degree of similarity exists between antigen-specific and allogeneic-specific immune responses [36,37]. Furthermore, T cell response to the tumor is not restricted to antigenspecific T cells but can also imply bystander T cells, which are not specific but actively engaged in tumor control [38,39]. Thus, despite some limitations, humanized mice may still provide valuable clues on therapeutic strategies aimed at enhancing the allogeneic immune response mediated by human T cells to the tumor, with relevance to T cell-mediated tumor antigen-specific and bystander immune responses in cancer patients. In this study, we investigated the therapeutic potential of a monoclonal antibody targeting human HVEM in humanized mice and the underlying cellular and molecular mechanisms associated with therapeutic efficacy.

HVEM and BTLA Were Associated with Lower Progression-Free Intervals in Prostate Cancer Patients
Analysis of TCGA (The Cancer Genome Atlas) revealed that HVEM mRNA was expressed at high levels in the PRAD (PRostate ADenocarcinomas) dataset (normalized log 2 counts > 10) and higher levels in primary tumors than in patient-matched normal tissues ( Figure 1A). In contrast, BTLA was expressed at low (normalized log 2 counts < 3) and similar levels in the tumor and patient-matched normal tissues ( Figure 1B). For patients with Gleason scores of 6 or 7 (low-grade adenocarcinomas), above-median expression of HVEM was associated with a lower progression-free interval (PFI) over 5 years, whereas this was not observed for BTLA ( Figure 1C). In contrast, HVEM mRNA expression levels were not correlated with good or poor PFI in patients with more advanced disease (Gleason scores of 8 or 9), but above-median expression of BTLA was associated with a lower PFI ( Figure 1D). Overall, the above-median expression of HVEM and BTLA together was a poor prognostic factor for PFI irrespective of the clinical score ( Figure 1E). Interestingly, this was not observed for the PD1/PD-L1 co-inhibitory signaling pathway ( Figure 1F). These results indicate a detrimental role of HVEM/BTLA in PCa at all stages of the disease and suggest that blocking the HVEM/BTLA pathway might be an attractive option for PCa patients. Accordingly, we focused the rest of the study on PCa cell lines in vivo.

Targeting HVEM with a mAb Improved Tumor Control in Humanized Mice
We first determined whether targeting HVEM with a mAb could impact tumor growth in vivo. For this, we implanted prostate cancer cell lines in NSG mice and grafted human PBMCs a few days later. No differences in tumor growth were observed in mice grafted with the prostate cancer cell line DU145, which did not express HVEM (Figure 2A). In contrast, a twofold reduction in tumor growth was observed in mice grafted with the HVEM-positive PC3 prostate cancer cell line ( Figure 2B). The presence of human cells was mandatory for the efficacy of the mAb since tumor growth was unaffected by the treatment in non-humanized NSG mice ( Figure S1A). The lack of an effect in non-humanized mice also indicates that the mAb did not directly kill the PC3 cell line in vivo. To formally demonstrate that HVEM on the tumor was required for therapeutic efficacy of the mAb, we generated an HVEM-deficient PC3 cell line (clone 1B11) using CRISPR-Cas9 ribonucleoprotein (RNP) transfection. Treatment with the mAb was completely inefficient on the 1B11 clone in humanized mice ( Figure 2C), showing that HVEM expression by the tumor was mandatory for the therapeutic efficacy of the mAb. Notably, the mAb was also effective at controlling tumor growth in humanized mice grafted with the HVEM-positive melanoma cell line Gerlach but not with the HVEM-negative triple-negative breast cancer (TNBC) cell  Figure S1B,C). Altogether, these results indicate that the therapeutic efficacy of targeting HVEM was independent of the tumor tissue origin but rather strictly dependent on HVEM expression by the tumor.

Targeting HVEM with a mAb Improved Tumor Control in Humanized Mice
We first determined whether targeting HVEM with a mAb could impact tum growth in vivo. For this, we implanted prostate cancer cell lines in NSG mice and grafte human PBMCs a few days later. No differences in tumor growth were observed in mi grafted with the prostate cancer cell line DU145, which did not express HVEM ( Figu  2A). In contrast, a twofold reduction in tumor growth was observed in mice grafted wi the HVEM-positive PC3 prostate cancer cell line ( Figure 2B). The presence of human ce

Tumor Control Was Dependent on CD8 + T Cells in Anti-HVEM-Treated Mice
To investigate the mode of action of the mAb, we determined the composition of human CD45 + cells in the PC3 tumor and the spleens of treated and untreated animals by flow cytometry. To determine the efficiency of human cell reconstitution independent of technical variability linked to sample preparation, we first established the frequencies of human CD45 + cells relative to the added frequencies of murine and human CD45 + cells ( Figure S2A). Human CD45 + cells represented close to 100% of all CD45 + cells in the spleen ( Figure S2B). Among hCD45 + cells, CD3 + T cells represented 90% ( Figure S2C), in which CD4 + and CD8 + cells represented 60% and 30%, respectively ( Figure S2D). Likewise, human CD45 + cells represented 90% of all CD45 + cells in the tumor ( Figure S2E). Among hCD45 + cells, CD3 + T cells represented more than 98% ( Figure S2F), in which CD4 + and CD8 + cells represented 40% and 60%, respectively ( Figure S2G). We noticed that the CD4 to CD8 ratio was inverted in the tumor relative to the spleen ( Figure S2D-G), indicating that CD8 + T cells might have a preferential tropism to the PC3 tumor in humanized mice, as previously described [40]. Importantly, treatment did not affect any of these proportions. In contrast, we observed an increase in the frequencies of both CD4 + and CD8 + T cells expressing the proliferation marker Ki67 ( Figure 3A,B). Additionally, CD8 but not CD4 T cell numbers (represented as z-scores to normalize the numbers across experiments) were significantly increased in the anti-HVEM-treated group relative to the control group ( Figure 3C), suggesting a role for CD8 + T cells in tumor control. However, the frequencies of CD8 + and CD4 + T cells expressing the cytotoxic molecules granzyme B or perforin were not elevated in the tumor or spleen of the anti-HVEM group ( Figure S3). To directly determine the contribution of CD8 + T cells to tumor control in anti-HVEM-treated mice, we investigated tumor growth in mice depleted of human CD8 + T cells. Depletion of CD8 + T cells was efficient in most of the mice analyzed, as assessed by the frequencies of CD4 − CD3 + cells remaining after treatment ( Figure S4). Depletion of CD8 + T cells reversed the effect of the anti-HVEM mAb ( Figure 3D), showing that tumor control was dependent on CD8 + T cells.

Treatment with the Anti-HVEM mAb Did Not Increase Graft vs. Host Disease Nor the Number or Proliferation of Human T Cells
Our observations thus far are consistent with the hypothesis that targeting HVEM on the tumor releases an inhibitory signal on CD8 + T cells, allowing their proliferation in situ. We wanted to rule out the possibility that the mAb behaves as an agonist, directly activating HVEM + CD8 + T cells in vivo, leading to better tumor control. One prediction of this hypothesis would be that GVHD occurring in NSG mice grafted with the tumor and reconstituted with human T cells would be exacerbated following anti-HVEM administration. However, despite the impact of the mAb on tumor growth (Figure 2), we observed similar weight loss and mortality in anti-HVEM or isotype control-treated mice ( Figure 4A,B). Furthermore, in contrast to TILs, the activation of human T cells in the spleens was the same in both groups, as judged by similar numbers of CD4 + and CD8 + T cells and similar frequencies of Ki67 + cells ( Figure 4C,D). Thus, anti-HVEM therapy in humanized mice reduced the growth of HVEM + tumors by a mechanism that was independent of a systemic agonist effect of the mAb on human T cells.

mRNA Enrichment Analysis Showed Increased Activation and Decreased Immuno-Suppression in TILs of Anti-HVEM-Treated Mice
To better characterize the anti-tumor immune response following mAb treatment, we established a list of differentially expressed genes (DEG) in sorted hCD45 + TILs from mice treated with the anti-HVEM mAb or its isotype control. For the analysis, 287 genes with a raw count higher than 55 and an absolute fold-change of at least 20% were set to be differentially expressed. Among the 287 genes, 145 were upregulated with a log 2 FC > 0.26, and 142 were downregulated (log 2 FC < −0.3) in HVEM-treated mice relative to isotype-treated controls ( Figure 5A). Several interleukins and chemokines genes signing T cell activation were enriched in the treated group, such as LTA, IL22, IL32, CCL5, and CCL4. Of note, GZMB and PRF1 were among the genes with the highest levels of expression, but the difference between the groups was weak, confirming our observation by flow cytometry ( Figure S3). Gene set enrichment analysis (GSEA) identified the "JAK-STAT signaling pathway" signature as significantly and positively enriched in TILs of HVEMtreated mice ( Figure 5B). In addition, upregulated genes in the anti-HVEM group were enriched in members of several ontologies related to lymphocyte activation, including the tumor necrosis factor-mediated signaling pathway, cytokine and chemokine binding and activity, and T cell receptor complex ( Figure S5). On the other hand, some genes belonging to immunosuppressive pathways were downregulated in HVEM-treated TILs such as ENTPD1 (CD39); IL10; and the co-inhibitory receptors BTLA, TIGIT, LAG3, and HAVCR2 (TIM3), as well as the "don't eat me" receptor CD47 ( Figure 5A). We verified by flow cytometry that BTLA was indeed down modulated on human T cells following anti-HVEM treatment ( Figure S6). In addition, GSEA showed that the "immunoregulatory interactions between a lymphoid and a non-lymphoid cell" signature was significantly repressed in the DEG signature ( Figure 5C), signing altered adhesion and motility of TILs. Overall, anti-HVEM treatment was associated with profound modifications of TILs, with increased expression of genes belonging to activation and proliferation signaling pathways and decreased expression of genes signing an exhausted phenotype.

HVEM Was an Immune Checkpoint during Anti-Tumor T Cell Immune Response in Humanized Mice
A reduction in tumor growth in the absence of HVEM was already apparent for the isotype-treated group, as shown in Figure 2B,C. Thus, we directly compared the growth of HVEM-positive or HVEM-negative PC3 cells in NSG mice with or without the addition of human PBMCs (Figure 6). Both cell lines grew equally well in non-humanized NSG mice ( Figure 6A), showing that HVEM deficiency did not affect in vivo tumor development per se. In contrast, tumor growth of the 1B11 clone was reduced twofold compared to that of the parental PC3 cell line in humanized mice ( Figure 6B). Furthermore, T cell proliferation was significantly increased in the absence of HVEM from the tumor ( Figure 6C). Thus, removing HVEM from the tumor released a brake on the allogeneic T cell response to the tumor, demonstrating that HVEM was an immune checkpoint under these experimental conditions.

HVEM Was an Immune Checkpoint during Anti-Tumor T Cell Immune Response in Humanized Mice
A reduction in tumor growth in the absence of HVEM was already apparent for the isotype-treated group, as shown in Figure 2B,C. Thus, we directly compared the growth of HVEM-positive or HVEM-negative PC3 cells in NSG mice with or without the addition of human PBMCs ( Figure 6). Both cell lines grew equally well in non-humanized NSG mice ( Figure 6A), showing that HVEM deficiency did not affect in vivo tumor development per se. In contrast, tumor growth of the 1B11 clone was reduced twofold compared to that of the parental PC3 cell line in humanized mice ( Figure 6B). Furthermore, T cell proliferation was significantly increased in the absence of HVEM from the tumor ( Figure 6C). Thus, removing HVEM from the tumor released a brake on the allogeneic T cell response to the tumor, demonstrating that HVEM was an immune checkpoint under these experimental conditions.

Discussion
Here, we report improved tumor control by human T cells in vivo following administration of a mAb to HVEM. Moreover, we deciphered the mode of action of the mAb in vivo using complementary technologies. Furthermore, we propose a simple in vivo assay for immune checkpoint discovery and validation. To our knowledge, the present report is the first to combine CRISPR/Cas9-mediated deletion of putative checkpoints with the assessment of tumor growth in humanized mice. One limitation of the assay is that PBMC-humanized mice are mostly reconstituted with T cells, as shown herein, limiting the usefulness of the assay to T cell-specific immune checkpoints. Another limitation is the allogeneic nature of the immune response to the tumor in humanized mice, which would only be circumvented in PDX models with autologous human immune cells, an ongoing effort but still challenging to set up [41]. Despite these limitations, we believe that our in vivo assay will be of great help in investigating other candidates in more advanced models of humanized mice, that is, mice reconstituted with human hematopoietic progenitors.

Discussion
Here, we report improved tumor control by human T cells in vivo following administration of a mAb to HVEM. Moreover, we deciphered the mode of action of the mAb in vivo using complementary technologies. Furthermore, we propose a simple in vivo assay for immune checkpoint discovery and validation. To our knowledge, the present report is the first to combine CRISPR/Cas9-mediated deletion of putative checkpoints with the assessment of tumor growth in humanized mice. One limitation of the assay is that PBMC-humanized mice are mostly reconstituted with T cells, as shown herein, limiting the usefulness of the assay to T cell-specific immune checkpoints. Another limitation is the allogeneic nature of the immune response to the tumor in humanized mice, which would only be circumvented in PDX models with autologous human immune cells, an ongoing effort but still challenging to set up [41]. Despite these limitations, we believe that our in vivo assay will be of great help in investigating other candidates in more advanced models of humanized mice, that is, mice reconstituted with human hematopoietic progenitors.
We show that the HVEM/BTLA checkpoint could be exploited for therapy in humanized mice using a mAb to human HVEM. We found that HVEM expression by the tumor was necessary and sufficient to elicit tumor control by the mAb since it had no effect on HVEM-negative cell lines and no agonist activity on human T cells. Park et al. showed in a syngeneic mouse model that transfecting an agonist scFv anti-HVEM in tumor cells resulted in increased T cell proliferation, improved IFN-γ and IL-2 production and improved tumor control [24]. In addition to the species differences, the discrepancy with our results could be explained by the fact that T cells are strongly activated in huPBMC mice [42]. The downregulation of HVEM expression upon activation [43] may have limited the binding of the anti-HVEM antibody to T cells in our model. Thus, it remains possible that the mAb would behave differently in humans. In contrast, BTLA is upregulated upon T cell activation [44], increasing the susceptibility of T cells to inhibition by HVEM + tumor cells [16,18,20,45]. The opposite was observed in the tumor microenvironment following treatment, with an increase in HVEM and a reduction of BTLA gene and protein expression, with a concomitant increase in LTA and LIGHT, two other ligands for HVEM. It is important to note that the binding sites of LIGHT and BTLA differ on HVEM [46]. Thus, the anti-HVEM mAb might have limited inhibition of activated T cells through blockade of HVEM binding with BTLA but not with the other ligands that are T cell activators. An alternative possibility would be that LIGHT and LTα in their soluble forms inhibit the interaction of HVEM with BTLA [47]. As of today, reciprocal regulation of HVEM and BTLA has not been reported but our observation is reminiscent of earlier findings showing reciprocal regulation of HVEM by LIGHT [43].
Previous studies in mice have also shown that inhibiting HVEM expression on the tumor or its interaction with its ligands has a positive effect on T cells. Injection of a plasmid encoding a soluble form of BTLA (to compete with endogenous BTLA for HVEM) was associated with an increase in inflammatory cytokine production by TILs and a decrease in anti-inflammatory cytokines at the RNA level [25]. Similarly, vaccination with a tumorassociated antigen was more efficient if HVEM interactions with its ligands were blocked by HSV-1 gD, allowing regression of a large tumor mass [48]. Moreover, silencing HVEM expression in the tumors with siRNA was also associated with an increase in CD8 T cells and inflammatory cytokine production in a murine colon carcinoma model [18]. In addition, the use of siRNA to HVEM on ovarian cancer in vitro promoted T cell proliferation and TNF-α and IFN-γ production [49]. Numerous results from our study also support increased T cell activation in the absence of HVEM/BTLA signaling: (i) TILs from mice treated with anti-HVEM were enriched in ontologies signing activation by cytokines, chemokines, and signaling pathways that are well-known inducers of proliferation, differentiation, migration, and apoptosis; (ii) comparison between TILs from mice treated with the anti-HVEM or isotype control mAb also highlighted decreased expression of many co-inhibitory receptor genes (BTLA, TIGIT, LAG3, and HAVCR2 [50,51]) or with immunosuppressive functions (ENTPD1 and IL10), suggesting a lower exhaustion status; and (iii) TILs from anti-HVEM-treated mice or the HVEM-null PC3 cell line had increased frequencies of Ki67 + cells relative to controls, signing improved proliferation. Altogether, these results strongly suggest that HVEM expressed by the tumor negatively regulated T cell-mediated control of tumor growth.
Interestingly, we observed that CD8 + T cell depletion in vivo favored the growth of PC3 tumors in anti-HVEM-treated mice. It might be inferred from this result that CD8 + T cells would also play a role in the absence of the treatment. However, a major role for human CD4 + T cells in PC3 tumor control in NSG mice has been reported [40]. In this study, CD4 + or CD8 + T cells were depleted before injection into mice. Despite depletion, a significant proportion of CD8 T cells were still present in the tumor although they were absent from the spleen, casting doubt on the interpretation of these results. It would be interesting to evaluate the role of CD4 + and CD8 + T cells in the absence of treatment in our model. Notably, a similar implication of CD8 + T cells on tumor control in humanized mice was reported for the TNBC MDA cell line treated with the anti-PD1 pembrolizumab [52]. Overall, we propose a model in which treatment with the anti-HVEM mAb would block the HVEM/BTLA inhibitory signaling on CD8 + TILs that would increase their proliferation and numbers, associated with a reduction of their exhausted phenotype, ultimately leading to better tumor control.

Conclusions
The recent success of ICI for cancer immunotherapy (anti-CTLA-4, anti-PD-1/PD-L1) has confirmed the hypothesis that the immune system can control many cancers, but disappointing results were obtained for PCa [4] which is in line with our observation that PD1/PD-L1 is not associated with lower PFI in PCa patients. In light of the promising results reported herein, anti-HVEM therapy might be combined with ICI and/or chemotherapy to further enhance anti-tumor immunity in PCa.

Preparation of Human Peripheral Mononuclear Cells
Human peripheral blood was obtained from Etablissement Francais du Sang (EFS) after obtaining informed consent from the donor. Human peripheral blood mononuclear cells (PBMC) were isolated using a Biocoll density gradient (Biochrom GmbH, Berlin, Germany). Cells were washed in PBS 3% FCS and diluted to the appropriate concentration in 1× PBS before injection into mice.

Humanized Mice Tumor Model
All animals used were NSG mice (stock = 005557) purchased from the Jackson Laboratory (Bar Harbor, ME, USA). To assess therapeutic activity, 8-20-week-old males and females NSG mice were injected subcutaneously with 2 × 10 6 tumor cells. One week later, mice were irradiated (2 Gy) and grafted the same day with 2 × 10 6 huPBMC by retro-orbital injection. This experimental protocol was inspired by our previous work on setting up the lower limit of grafted PBMCs to limit GVHD in NSG mice [53]. Four to five days after transplantation, the anti-huHVEM antibody or isotype control was injected intraperitoneally at 2 mg/kg. General health status, body weight, and survival of mice were monitored every 3-4 days to evaluate GVHD progression. Mice with overt signs of GVHD, such as weight loss greater than 20% of their initial weight, hunched back, ruffled fur, and reduced mobility, were immediately sacrificed. For CD8 depletion, the optimal conditions and efficacy assessments have been previously described [54]. Briefly, mice were injected intraperitoneally with 10 mg/kg of the anti-CD8 MT807R1 (rhesus recombinant IgG1 provided by the Nonhuman Primate Reagent Resource [55]) or the isotype control (clone DSPR1) the day following humanization.

Antibodies
The clone 18.10 has been described previously [56]. Briefly, 18.10, a murine IgG1 anti-human HVEM mAb, was produced as ascites and purified by protein A binding and elution with the Affi-gel Protein A MAPS II Kit (Bio-Rad, Marnes-La-Coquette, France). A Mouse IgG1 isotype control (clone MOPC-21 clone) was purchased from BioXCell (West Lebanon, NH, USA).

Cell Lines
PC3 (non-hormonal-dependent human prostate cancer cells) and DU145 (prostate cancer cells) were grown in high-glucose DMEM supplemented with 10% FCS, L-glutamine, and antibiotics (penicillin/streptomycin, ThermoFisher, Les Ulis, France). The PC3 cell line was genetically authenticated before the initiation of the experiments (Eurofins). All cells were confirmed to be free of mycoplasmas before injection into mice using the MycoAlert detection kit (Lonza, Basel, Switzerland). Tumor growth was monitored using an electronic caliper, and volumes were determined using the following formula: [(length × width 2 )/2]. The animals were sacrificed and analyzed 21 days after the humanization.

NanoString nCounter Expression Assay
For the NanoString ® experiment, 14-to 15-week-old NSG mice were humanized and treated with anti-HVEM or isotype. On day 28 post-humanization, tumors were harvested and TILs were isolated as described above. To maximize mRNA recovery, we pooled TILs by treatment groups (four mice in the anti-HVEM group and five in the isotype control group). Cells were then stained with a viability dye (eF506) and anti-hCD45-APC (HI30, BioLegend). Live hCD45 + cells were sorted using an Aria II cell sorter. After centrifugation, the cells were suspended in RLT buffer (Qiagen, Les Ulis, France) before freezing at −80 • C until analysis. Data were normalized using NanoString's intrinsic negative and positive controls according to the normalization approach of the nSolver analysis software (Nanostring, Seattle, WA, USA).

Bioinformatics Analysis
For ontology enrichment analysis, only genes upregulated by the treatment were analyzed using the enrichment analysis visualization Appyter to visualize Enrichr results [57]. The list of DEG (up-and downregulated) was ranked by fold-change for pre-ranked GSEA. Enrichment was performed with the C2 Canonical Pathways v.7.4 gene set using the GSEA 4.1.0 Linux desktop application [58] from the Broad Institute. With that workflow, a false discovery rate (FDR) or a family-wise error rate (FWER) less than 0.25 is deemed "significant". The Cancer Genome Atlas (TCGA) database was analyzed using the Xena browser (http://xena.ucsc.edu, accessed on 9 June 2021) provided by the University of California (Santa Cruz, CA, USA) [59]. The Prostate Adenocarcinoma (PRAD) dataset was used with subsequent filterings on TNFRSF14, BTLA, PDCD1, and CD274 mRNA expression levels and Gleason clinical scores.

Statistical Analysis
All statistical tests were performed using Prism v.8 (GraphPad Inc., La Jolla, CA, USA) or JASP v.0.14.3 (available at https://jasp-stats.org, accessed on 9 June 2021). The nature of the statistical test used to compare results is indicated in each figure legend. When necessary, the p-values of these tests are indicated in the figure panels. The statistical power of the analyses (alpha) was set arbitrarily at 0.05.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/cancers13123009/s1, Figure S1: Anti-HVEM therapy in humanized mice, Figure S2: Human cell reconstitution in spleen and PC3 tumor of humanized mice, Figure S3: Granzyme B and perforin expression and frequencies in T cells in humanized mice, Figure S4: CD8 depletion efficiency in humanized mice, Figure S5: Enrichment analysis in TILs of anti-HVEM-treated mice. Figure   Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of Sorbonne University (protocol code 18/EFS/033). Mice were bred in our animal facility under specific pathogen-free conditions following current European legislation. All protocols were approved by the Ethics Committee for Animal Experimentation Charles Darwin (Ce5/2012/025).

Informed Consent Statement:
Human peripheral blood mononuclear cells were collected from healthy adult volunteers by Etablissement Français du Sang. Informed consent was obtained from all the subjects involved in the study.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author.

Acknowledgments:
The authors would like to thank Olivier Bregerie, Flora Issert, and Doriane Foret for taking care of our mice; Pukar KC for technical help; Sylvaine Just-Landi for preparing the 18.10 mAb; Armanda Casrouge and Claude Baillou for cell sorting; and Benoit Salomon for critical reading of the manuscript. The results shown here are partly based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga.

Conflicts of Interest: D.
O. declares competing interests as being the co-founder and shareholder of Imcheck Therapeutics, Alderaan Biotechnology, and Emergence Therapeutics, and has research funds from Imcheck Therapeutics, Alderaan Biotechnology, Cellectis, and Emergence Therapeutics.