A post-transcriptional regulatory checkpoint restrains acquisition of cytotoxic activity in tumor-infiltrating CD4+ T cells
Tumor-infiltrating cytotoxic CD4+ T cells (TCTX) are a promising therapeutic target, but how their activity is regulated is poorly understood. Here, we show that a post-transcriptional checkpoint restrains TCTX activity in suppressive tumor microenvironments. In untreated tumors, poised TCTX express high levels of Gzmb mRNA but produce little Granzyme B (GzmB) protein. Differentiation into poised TCTX is regulated by the Blimp-1-Bcl6 axis and requires type I interferon signaling. GzmB protein is produced by tumor-infiltrating TCTX upon treatment with TREG-depleting aCTLA-4 antibodies and following blockade of LAG-3 and PD-1. These treatments downregulate Zfp36l1, encoding an RNA binding protein. Deletion of Zfp36l1 and its paralog Zfp36 increases GzmB protein in CD4+ T cells in untreated tumors and reduces tumor mass. These results demonstrate that TCTX activity is restrained in tumors through LAG-3 and PD-1 and the ZFP36 family of RNA binding proteins. We propose that these pathways represent targets for therapeutic intervention. Overall design: We wished to analyse the transcriptome of tumor infiltraing CD4+ T cells in the murine tumor model MCA205, in both untreated and anti-CTLA-4 treated conditions. To do so, we carried out single cell RNA sequencing of FACS sorted CD3+ T cells taken from MCA205 tumors treated with  anti-CTLA-4 on days 6, 9 and 11 post tumor inoculation or left untreated.