Intra-tumoral Treg Ablation Activates a CD4-NK Cell Axis to Control CD8 T cell Resistant Tumors

Tumors evade control by CD8+ T cells by losing MHC I expression, necessitating new approaches to improve immunotherapy. NK cells target MHC I-deficient cells but are insufficiently activated to control tumors without help. Here we uncovered a novel approach to control MHC I-deficient tumors by ablating Treg cells intratumorally, which incites potent antitumor NK cell responses without imparting lethal autoimmunity. IT-Treg ablation increased NK cell activation, maturation, expression of effector molecules, and anti-tumor cytotoxic activity. In this setting, conventional CD4+ T cells were required for NK cell activation, due to their production of IL-2 after interacting with conventional type 2 dendritic cells. These results demonstrate an opportunity to reinvigorate antitumor NK cell responses against tumors that evade CD8 T cell responses, by locally depleting Tregs. The findings also highlight the multicellular immunosuppression enforced by Tregs that extends beyond the control of CD8 T cell responses to tumors. Tumors from mice treated with PBS, IT-DT or ITDT + anti-CD4 were resected, and single cell suspensions were prepared. Tumor immune cells were enriched by layering on a lympholyte-M gradient then stained with DAPI, CD19-PECy5, F4/80-PECy5, Ter119-PECy5, CD8a-PECy5, CD3e-APC, CD45-FITC, CD4-BV605, and NK1.1-APC-Cy7 for 30 min on ice. NK cells and CD4 T cells were FACS sorted from tumors pooled from 2-4 mice per replicate, with a yield ranging from 20,000-200,000 cells, with three replicates total.