Knowledge of immune cell phenotypes in the tumor microenvironment is essential for understanding mechanisms of cancer progression and immunotherapy response. We profiled 45,000 immune cells from eight breast carcinomas, as well as matched normal breast tissue, blood, and lymph nodes, using single-cell RNA-seq. We developed a preprocessing pipeline, SEQC, and a Bayesian clustering and normalization method, Biscuit, to address computational challenges inherent to single-cell data. Despite significant similarity between normal and tumor tissue-resident immune cells, we observed continuous phenotypic expansions specific to the tumor microenvironment. Analysis of paired single-cell RNA and T cell receptor (TCR) sequencing data from 27,000 additional T cells revealed the combinatorial impact of TCR utilization on phenotypic diversity. Our results support a model of continuous activation in T cells and do not comport with the macrophage polarization model in cancer. Our results have important implications for characterizing tumor-infiltrating immune cells.

TPS3098 Background: Regulatory T cells (Treg) are a subset of CD4 T cells that preserve immune homeostasis by the establishment and maintenance of peripheral tolerance. This suppressive function however, limits anti-tumor immune responses and represents a critical obstacle to immunotherapy. Multiple preclinical models have demonstrated effective anti-tumor immunity by targeting Treg cells. Human tumor-infiltrating Treg cells express high levels of the chemokine receptor CCR4. Based on these findings we developed a Phase I/II clinical trial investigating the activity of Mogamulizumab, a fully humanized, IgG1, monoclonal, depleting, anti-CCR4 antibody in patients with advanced solid organ malignancies. Methods: Eligible patients include those with histologically confirmed solid organ malignancies, adequate performance status, and organ function who are not eligible for, have declined, or have failed standard treatment. Up to 24 patients will be enrolled in the phase I study and the dose of Mogamulizumab will be escalated according to a standard 3+3 design. Mogamulizumab will be administered weekly for the first month and then every two weeks for up to a year or until progressive disease or unacceptable toxicity. The primary study end points are safety/tolerability and identification of the maximum tolerated dose (MTD), which will be taken forward in a phase II study. Secondary objectives include evaluating clinical activity as assessed by tumor response (RECIST v1.1 and Immune-related Response Criteria), progression-free survival, and overall survival. Exploratory objectives include determining the extent of tumor and peripheral blood Treg cell depletion as well as the correlative immunologic changes. Tumor biopsies will be available both pre-treatment and during treatment. The first two dose escalation cohorts have been accrued. The phase II study will continue to evaluate the safety and preliminary efficacy of mogamulizumab at the MTD in 48 patients with triple negative breast cancer, gastric adenocarcinoma, and non-small cell lung cancer. Clinical trial information: NCT02281409. Clinical trial information: NCT02281409.

Regulatory T (Treg) cells reside in lymphoid organs and barrier tissues where they control different types of inflammatory responses. Treg cells are also found in human cancers, and studies in animal models suggest that they contribute to cancer progression. However, properties of human intratumoral Treg cells and those present in corresponding normal tissue remain largely unknown. Here, we analyzed features of Treg cells in untreated human breast carcinomas, normal mammary gland, and peripheral blood. Tumor-resident Treg cells were potently suppressive and their gene-expression pattern resembled that of normal breast tissue, but not of activated peripheral blood Treg cells. Nevertheless, a number of cytokine and chemokine receptor genes, most notably CCR8, were upregulated in tumor-resident Treg cells in comparison to normal tissue-resident ones. Our studies suggest that targeting CCR8 for the depletion of tumor-resident Treg cells might represent a promising immunotherapeutic approach for the treatment of breast cancer.