In the proper context, radiotherapy can promote antitumor immunity. It is unknown if elective nodal irradiation (ENI), a strategy that irradiates tumor-associated draining lymph nodes (DLN), affects adaptive immune responses and combinatorial efficacy of radiotherapy with immune checkpoint blockade (ICB). We developed a preclinical model to compare stereotactic radiotherapy (Tumor RT) with or without ENI to examine immunologic differences between radiotherapy techniques that spare or irradiate the DLN. Tumor RT was associated with upregulation of an intratumoral T-cell chemoattractant chemokine signature (CXCR3, CCR5-related) that resulted in robust infiltration of antigen-specific CD8 effector T cells as well as FoxP3 regulatory T cells (Tregs). The addition of ENI attenuated chemokine expression, restrained immune infiltration, and adversely affected survival when combined with ICB, especially with anti-CLTA4 therapy. The combination of stereotactic radiotherapy and ICB led to long-term survival in a subset of mice and was associated with favorable CD8 effector-to-Treg ratios and increased intratumoral density of antigen-specific CD8 T cells. Although radiotherapy technique (Tumor RT vs. ENI) affected initial tumor control and survival, the ability to reject tumor upon rechallenge was partially dependent upon the mechanism of action of ICB; as radiotherapy/anti-CTLA4 was superior to radiotherapy/anti-PD-1. Our results highlight that irradiation of the DLN restrains adaptive immune responses through altered chemokine expression and CD8 T-cell trafficking. These data have implications for combining radiotherapy and ICB, long-term survival, and induction of immunologic memory. Clinically, the immunomodulatory effect of the radiotherapy strategy should be considered when combining stereotactic radiotherapy with immunotherapy. .

◥Purpose: Intratumoral immunosuppression mediated by myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) represents a potential mechanism of immune checkpoint inhibitor (ICI) resistance in solid tumors. By promoting TAM and MDSC infiltration, IL1b may drive adaptive and innate immune resistance in renal cell carcinoma (RCC) and in other tumor types.Experimental Design: Using the RENCA model of RCC, we evaluated clinically relevant combinations of anti-IL1b plus either anti-PD-1 or the multitargeted tyrosine kinase inhibitor (TKI), cabozantinib. We performed comprehensive immune profiling of established RENCA tumors via multiparameter flow cytometry, tumor cytokine profiling, and single-cell RNA sequencing (RNA-seq). Similar analyses were extended to the MC38 tumor model.Results: Analyses via multiparameter flow cytometry, tumor cytokine profiling, and single-cell RNA-seq showed that anti-IL1b reduces infiltration of polymorphonuclear MDSCs and TAMs. Combination treatment with anti-IL1b plus anti-PD-1 or cabozantinib showed increased antitumor activity that was associated with decreases in immunosuppressive MDSCs and increases in M1-like TAMs.Conclusions: Single-cell RNA-seq analyses show that IL1b blockade and ICI or TKI remodel the myeloid compartment through nonredundant, relatively T-cell-independent mechanisms. IL1b is an upstream mediator of adaptive myeloid resistance and represents a potential target for kidney cancer immunotherapy.

BackgroundProstate cancer responds poorly to current immunotherapies. Epigenetic therapies such as BET Bromodomain inhibition can change the transcriptome of tumor cells, possibly making them more immunogenic and thus susceptible to immune targeting.MethodsWe characterized the effects of BET bromodomain inhibition using JQ1 on PD-L1 and HLA-ABC expression in two human prostate cell lines, DU145 and PC3. RNA-Seq was performed to assess changes on a genome-wide level. A cytotoxic T cell killing assay was performed in MC38-OVA cells treated with JQ1 to demonstrate increased immunogenicity. In vivo experiments in the Myc-Cap model were conducted to show the effects of JQ1 administration in concert with anti-CTLA-4 checkpoint blockade.ResultsHere, we show that targeting BET bromodomains using the small molecule inhibitor JQ1 decreased PD-L1 expression and mitigated tumor progression in prostate cancer models. Mechanistically, BET bromodomain inhibition increased MHC I expression and increased the immunogenicity of tumor cells. Transcriptional profiling showed that BET bromodomain inhibition regulates distinct networks of antigen processing and immune checkpoint molecules. In murine models, treatment with JQ1 was additive with anti-CTLA-4 immunotherapy, resulting in an increased CD8/Treg ratio.ConclusionsBET Bromodomain inhibition can mediate changes in expression at a genome wide level in prostate cancer cells, resulting in an increased susceptibility to CD8 T cell targeting. These data suggest that combining BET bromodomain inhibition with immune checkpoint blockade may have clinical activity in prostate cancer patients.

Immunotherapy is a treatment for many types of cancer, primarily due to deep and durable clinical responses mediated by immune checkpoint blockade (ICB) 1,2 . Prostate cancer is a notable exception in that it is generally unresponsive to ICB. The standard treatment for advanced prostate cancer is androgen-deprivation therapy (ADT), a form of castration (CTX). ADT is initially effective, but over time patients eventually develop castrationresistant prostate cancer (CRPC). Here, we focused on defining tumor-cell intrinsic factors that contribute to prostate cancer progression and resistance to immunotherapy.We analyzed cancer cells isolated from castration-sensitive and castration-resistant prostate tumors, and discovered that castration resulted in significant secretion of Interleukin-8 (IL-8) and it's likely murine homolog Cxcl15. These chemokines drove subsequent intra-tumoral infiltration with polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), promoting tumor progression.PMN-MDSC infiltration was abrogated when IL-8 was deleted from prostate cancer epithelial cells using CRISPR/Cas9, or when PMN-MDSC migration was blocked with antibodies against the IL-8 receptor CXCR2. Blocking PMN-MDSC infiltration in combination with anti-CTLA-4 delayed the onset of castration resistance and increased the density of polyfunctional CD8 T cells in tumors. Taken together, our findings establish castration-mediated IL-8 secretion and subsequent PMN-MDSC infiltration as a key suppressive mechanism in the

23 24 post-castration. We used the MCRedAL prostate cancer cell line; an RFP expressing 66 version of the Myc-Cap cell line characterized by MYC overexpression 15 . Like human 67 prostate cancer, MCRedAL tumors are initially castration-sensitive (CS), but castration-68 resistance (CR) develops approximately 30 days after castration (Extended Data Fig. 1a). 69 Pre-and post-ADT tumor cells were sorted to > 96% purity (Extended Data Fig. 1b) and 70 analyzed ( Fig. 1a-b and Extended Data Fig. 1c). A number of cytokine and chemokine 71 transcripts were significantly up-regulated post-ADT ( Fig. 1b right), including Cxcl15, a 72 CXC chemokine with a conserved ELR motif (Extended Data Table 1), which is the likely 73 murine homolog of human IL-8 (CXCL8) [16][17][18][19] . qRT-PCR and ELISA assays confirmed the 74 upregulation of Cxcl15 post-ADT at the protein level (Extended Data Fig. 1d). In addition 75 to the chemokines above, GSEA revealed the upregulation of several pro-inflammatory 76 pathways post-ADT ( Fig. 1c). In vitro experiments using the human androgen-responsive 77 LNCaP cell line corroborated a role for these pro-inflammatory signals, showing that in 78 the absence of androgen, TNFα upregulated IL-8 expression in a dose-dependent 79 manner ( Fig. 1d left); while AR signaling in the absence of inflammation did not affect IL-80 8 expression ( Fig. 1d right). These data led to the hypothesis that AR signaling directly 81 suppresses IL-8 expression in prostate cancer cells. We performed in silico ChIP-Seq 82 analyses using human LNCaP cells (GSE83860) and found AR binding at the IL-8 83 promoter in the presence of the potent androgen dihydrotestosterone (DHT; Fig. 1e top). 84This androgen dependent binding was verified by ChIP-qRT-PCR ( Fig. 1f). 85To further explore the role of AR in IL-8 regulation, we interrogated RNA polymerase 86 binding and transcription marks found at sites of active promoters 20 . In the presence of 87 DHT, binding of RNA polymerase II (pol II), phosphorylated serine 2 RNA polymerase II 88 5 (pSer2 pol II) and histone H3 tri-methyl Lys4 (H3K4me3) to the IL-8 locus were 89 substantially reduced, consistent with reduced transcriptional activity ( Fig. 1f). 90Conversely, pSer2 pol II binding to the promoter of the well-established AR-regulated 91 gene PSA (KLK3), was significantly increased in the presence of DHT as expected 92 (Extended Data Fig. 1e). Consistent with a role for inflammation, TNFα significantly 93 increased p65 binding at the IL-8 (CXCL8) promoter in LNCaP cells (Fig. 1e bottom). No 94 significant binding of AR was detected at the promoters of the chemokines CXCL1, 95 CXCL2, CXCL5 or CXCL12 (Extended Data Fig. 1f). These data suggest that AR directly 96 suppresses IL-8 expression through repressive AR binding to the IL-8 promoter. Taken 97 together, we found that IL-8 transcription is up-regulated by pro-inflammatory signaling, 98 and down-regulated by AR signaling (Fig. 1g). 99 We next investigated the effects of ADT on the expression of Cxcl15 in vivo, using RNA 1...