Ager, Casey R

ABSTRACT We report the optimization of a 38‐parameter spectral flow cytometry panel to identify, phenotype, and assess lineage plasticity of human regulatory T cells (Tregs). Tregs are an indispensable T cell lineage with pleiotropic tolerogenic functions whose activities contribute critically to numerous disease settings including cancer, autoimmunity, and infectious disease, among others. Phenotypic and functional heterogeneity within the Treg lineage has been appreciated, but the etiology and impact of Treg plasticity across disease states remain incompletely understood. Better tools are thus needed to deeply characterize human Treg phenotypic heterogeneity at the single cell level to advance discovery of Treg‐based biomarkers in disease and to assess the effects of Treg‐directed therapeutics. To this end, our 38‐parameter panel consists of a 13‐marker PBMC backbone module to broadly phenotype PBMCs while specifically discriminating Tregs, and a 25‐marker Treg phenotyping module to determine Treg differentiation state, activation profile, and lineage subtype. In contrast to many high‐parameter OMIPs that rely on surface staining only, we incorporated several intracellular targets in this panel. This afforded the opportunity to thoroughly evaluate binding characteristics of all antibodies in both pre‐fixation and post‐fixation and permeabilization settings; we identify several antibodies eligible for overnight post‐fixation staining that require substantially reduced titers as compared to traditional pre‐fixation staining, resulting in significant cost savings. Dimensionality reduction and semi‐supervised clustering on healthy donor PBMC‐derived Tregs profiled by our panel reveal up to 14 discrete Treg phenotypes. In sum, this panel enables deep phenotypic characterization of human Treg heterogeneity in peripheral blood specimens by spectral flow cytometry.

Abstract 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, IL1β 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-IL1β 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-IL1β reduces infiltration of polymorphonuclear MDSCs and TAMs. Combination treatment with anti-IL1β 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 IL1β blockade and ICI or TKI remodel the myeloid compartment through nonredundant, relatively T-cell–independent mechanisms. IL1β is an upstream mediator of adaptive myeloid resistance and represents a potential target for kidney cancer immunotherapy.