Regulatory T (Treg) cells expressing the transcription factor FOXP3 are essential for the maintenance of immunologic self-tolerance but play a detrimental role in most cancers due to their ability to suppress antitumor immunity. The phenotype of human circulating Treg cells has been extensively studied, but less is known about tumor-infiltrating Treg cells. We studied the phenotype and function of tumor-infiltrating Treg cells in ovarian cancer and melanoma to identify potential Treg cell-associated molecules that can be targeted by tumor immunotherapies. The phenotype of intratumoral and circulating Treg cells was analyzed by multicolor flow cytometry, mass cytometry, RNA-seq, and functional assays. Treg cells isolated from ovarian tumors displayed a distinct cell surface phenotype with increased expression of a number of receptors associated with TCR engagement, including PD-1, 4-1BB, and ICOS. Higher PD-1 and 4-1BB expression was associated with increased responsiveness to further TCR stimulation and increased suppressive capacity, respectively. Transcriptomic and mass cytometry analyses revealed the presence of Treg cell subpopulations and further supported a highly activated state specifically in ovarian tumors. In comparison, Treg cells infiltrating melanomas displayed lower FOXP3, PD-1, 4-1BB, and ICOS expression and were less potent suppressors of CD8 T-cell proliferation. The highly activated phenotype of ovarian tumor-infiltrating Treg cells may be a key component of an immunosuppressive tumor microenvironment. Receptors that are expressed by tumor-infiltrating Treg cells could be exploited for the design of novel combination tumor immunotherapies. .

Cancers arising from germline DNA mismatch repair deficiency or polymerase proofreading deficiency (MMRD and PPD) in children harbour the highest mutational and microsatellite insertion–deletion (MS-indel) burden in humans. MMRD and PPD cancers are commonly lethal due to the inherent resistance to chemo-irradiation. Although immune checkpoint inhibitors (ICIs) have failed to benefit children in previous studies, we hypothesized that hypermutation caused by MMRD and PPD will improve outcomes following ICI treatment in these patients. Using an international consortium registry study, we report on the ICI treatment of 45 progressive or recurrent tumors from 38 patients. Durable objective responses were observed in most patients, culminating in a 3 year survival of 41.4%. High mutation burden predicted response for ultra-hypermutant cancers (>100 mutations per Mb) enriched for combined MMRD + PPD, while MS-indels predicted response in MMRD tumors with lower mutation burden (10–100 mutations per Mb). Furthermore, both mechanisms were associated with increased immune infiltration even in ‘immunologically cold’ tumors such as gliomas, contributing to the favorable response. Pseudo-progression (flare) was common and was associated with immune activation in the tumor microenvironment and systemically. Furthermore, patients with flare who continued ICI treatment achieved durable responses. This study demonstrates improved survival for patients with tumors not previously known to respond to ICI treatment, including central nervous system and synchronous cancers, and identifies the dual roles of mutation burden and MS-indels in predicting sustained response to immunotherapy.

Human papillomavirus (HPV) is the main etiological factor for cervical cancer development. HPV is also associated with other anogenital and oropharyngeal tumors. HPV associated tumors are frequent and constitute a public health problem, mainly in developing countries. Therapy against such tumors is usually excisional, causing iatrogenic morbidity. Therefore, development of strategies for new therapies is desirable. The tumor microenvironment is essential for tumor growth, where inflammation is an important component, displaying a central role in tumor progression. Inflammation may be a causal agent, suppressor of anti-tumor T cell responses, or may have a role in angiogenesis, drug resistance, and metastasis. The aim of this work was to investigate the role of HPV transformed cells in the tumor microenvironment and tumor effects on myeloid populations in lymphoid organs in the host. We used experimental models, where we injected cervical cancer derived cell lines in immunodeficient mice, comparing HPV positive, SiHa, and HeLa cells (HPV 16 and HPV18, respectively), with HPV negative cell line, C33A. Our data shows that HPV positive cell lines were more efficient than the HPV negative cell line in leukocyte recruitment to the tumor microenvironment and increase in myeloid cell proliferation in the bone marrow and spleen. We also observed that HPV positive cells lines expressed significantly higher levels of IL-6 and IL-8, while C33A expressed significantly higher levels of IL-16 and IL-17. Finally, in spite of cytokine secretion by tumor cells, leukocytes infiltrating SiHa and HeLa tumors displayed almost negligible STAT3 and no NFκB phosphorylation. Only the inflammatory infiltrate of C33A tumors had NFκB and STAT3 activated isoforms. Our results indicate that, although from the same anatomical site, the uterine cervix, these cell lines display important differences regarding inflammation. These results are important for the design of immunotherapies against cervical cancer, and possibly against HPV associated tumors in other anatomical sites.