Purpose: To characterize the role of B cells on human papilloma virus (HPV)-associated cancer patient outcomes and determine the effects of radiation and PD-1 blockade on Bcell populations.Experimental Design: Tumor RNA-sequencing data from over 800 patients with head and neck squamous cell carcinoma (HNSCC) and cervical cancer, including a prospective validation cohort, was analyzed to study the impact of B-cell gene expression on overall survival (OS). A novel murine model of HPV þ HNSCC was used to study the effects of PD-1 blockade and radiotherapy on B-cell activation, differentiation, and clonality including analysis by single-cell RNA-sequencing and B-cell receptor (BCR)sequencing. Human protein microarray was then used to quantify B-cell-mediated IgG and IgM antibodies to over 16,000 proteins in the serum of patients treated on a clinical trial with PD-1 blockade.Results: RNA-sequencing identified CD19 and IGJ as novel Bcell prognostic biomarkers for 3-year OS (HR, 0.545; P < 0.001). PD-1 blockade and radiotherapy enhance development of memory B cells, plasma cells, and antigen-specific B cells. BCRsequencing found that radiotherapy enhances B-cell clonality, decreases CDR3 length, and induces B-cell somatic hypermutation. Single-cell RNA-sequencing identified dramatic increases in B-cell germinal center formation after PD-1 blockade and radiotherapy. Human proteome array revealed enhanced IgG and IgM antibody responses in patients who derived clinical benefit but not those with progressive disease after treatment with PD-1 blockade.Conclusions: These findings establish a key role for B cells in patient outcomes and responses to PD-1 blockade in HPVassociated squamous cell carcinomas and demonstrate the need for additional diagnostics and therapeutics targeting B cells.

This article reports a patient with a rare metastatic, chemotherapy‐refractory neuroendocrine carcinoma who was treated with stereotactic body radiation therapy (SBRT) combined with anti‐programmed cell death protein 1 antibody. The novel treatment modality of SBRT combined with a checkpoint inhibitor is discussed, as well as the implications of molecular profiling and tumor mutational burden as potential predictors of response.

◥ Macrophages (MÈ) play a critical role in tumor growth, immunosuppression, and inhibition of adaptive immune responses in cancer. Hence, targeting signaling pathways in MÈs that promote tumor immunosuppression will provide therapeutic benefit. PI3Kg has been recently established by our group and others as a novel immuno-oncology target. Herein, we report that an MÈ Syk-PI3K axis drives polarization of immunosuppressive MÈs that establish an immunosuppressive tumor microenvironment in in vivo syngeneic tumor models. Genetic or pharmacologic inhibition of Syk and/or PI3Kg in MÈs promotes a proinflammatory MÈ phenotype, restores CD8 þ T-cell activity, destabilizes HIF under hypoxia, and stimulates an antitumor immune response. Assay for transposaseaccessible Chromatin using Sequencing (ATAC-seq) analyses on the bone marrow-derived macrophages (BMDM) show that inhibition of Syk kinase promotes activation and binding of NF-kB motif in Syk MC-KO BMDMs, thus stimulating immunostimulatory transcriptional programming in MÈs to suppress tumor growth. Finally, we have developed in silico the "first-in-class" dual Syk/ PI3K inhibitor, SRX3207, for the combinatorial inhibition of Syk and PI3K in one small molecule. This chemotype demonstrates efficacy in multiple tumor models and represents a novel combinatorial approach to activate antitumor immunity.

There is a critical need to understand mechanisms of resistance and to develop combinatorial strategies to improve responses to checkpoint blockade immunotherapy (CBI). Here, we uncover a novel mechanism by which the human papillomavirus (HPV) inhibits the activity of CBI in head and neck squamous cell carcinoma (HNSCC). Using orthotopic HNSCC models, we show that radiation combined with anti-PD-L1 immunotherapy significantly enhanced local control, CD8 þ memory T cells, and induced preferential T-cell homing via modulation of vascular endothelial cells. However, the HPV E5 oncoprotein suppressed immune responses by downregulating expression of major histocompatibility complex and interfering with antigen presentation in murine models and patient tumors. Furthermore, tumors expressing HPV E5 were rendered entirely resistant to anti-PD-L1 immunotherapy, and patients with high expression of HPV16 E5 had worse survival. The antiviral E5 inhibitor rimantadine demonstrated remarkable single-agent antitumor activity. This is the first report that describes HPV E5 as a mediator of resistance to anti-PD-1/PD-L1 immunotherapy and demonstrates the antitumor activity of rimantadine. These results have broad clinical relevance beyond HNSCC to other HPV-associated malignancies and reveal a powerful mechanism of HPV-mediated immunosuppression, which can be exploited to improve response rates to checkpoint blockade.Significance: This study identifies a novel mechanism of resistance to anti-PD-1/PD-L1 immunotherapy mediated by HPV E5, which can be exploited using the HPV E5 inhibitor rimantadine to improve outcomes for head and neck cancer patients.

Bone marrow (BM) failure syndrome encompasses a group of disorders characterized by BM stem cell dysfunction, resulting in varying degrees of hypoplasia and blood pancytopenia, and in many patients is autoimmune and inflammatory in nature. The important role of T helper 1 (Th1) polarized CD4+ T cells in driving BM failure has been clearly established in several models. However, animal model data demonstrating a functional role for CD8+ T cells in BM dysfunction is largely lacking and our objective was to test the hypothesis that CD8+ T cells play a non-redundant role in driving BM failure. Clinical evidence implicates a detrimental role for CD8+ T cells in BM failure and a beneficial role for Foxp3+ regulatory T cells (Tregs) in maintaining immune tolerance in the BM. We demonstrate that IL-2-deficient mice, which have a deficit in functional Tregs, develop spontaneous BM failure. Furthermore, we demonstrate a critical role for CD8+ T cells in the development of BM failure, which is dependent on the cytokine, IFNγ. CD8+ T cells promote hematopoietic stem cell dysfunction and depletion of myeloid lineage progenitor cells, resulting in anemia. Adoptive transfer experiments demonstrate that CD8+ T cells dramatically expedite disease progression and promote CD4+ T cell accumulation in the BM. Thus, BM dysregulation in IL-2-deficient mice is mediated by a Th1 and IFNγ-producing CD8+ T cell (Tc1) response.