Potent anti-tumor immunity and reversal of CD8 T cell exhaustion by spatially and functionally targeting Treg cells in the tumor microenvironment

Regulatory T cells (Tregs) are a key mediator of resistance to cancer immunotherapy, including anti-PD-(L)1 immune checkpoint blockade (ICB). Tregs perpetually infiltrate tumors and hamper effective anti-tumor immunity. The mechanisms driving Treg infiltration into the tumor microenvironment (TME) and the consequence on CD8+ T cell exhaustion remains elusive. Herein, we report that heat shock protein gp96 (GRP94) is indispensable for Treg infiltration into the tumor, primarily through gp96’s roles in chaperoning integrins. Among various gp96-dependent integrins, we found that only LFA-1 (αL integrin) but not integrin αV, CD103 (αE) or β7 was required for Treg homing into the tumors. Loss of Treg infiltration into the TME by genetically deleting gp96/LFA-1 potently induces rejection of multiple ICB-resistant murine cancer models in a CD8+ T cell dependent manner without loss of self-tolerance. Moreover, gp96 deletion impeded Treg activation primarily by suppressing IL-2/phosphorylated STAT5 (pSTAT5) signaling pathway, which also contributes to tumor regression. Notably, by a mechanism in part through competing for IL-2 in the TME, intra-tumoral Tregs prevent activation of CD8+ tumor-infiltrating lymphocytes (TILs), drive TOX induction and induce bona fide CD8+ T cell exhaustion. By contrast, Treg ablation leads to a striking CD8+ T cell activation without TOX induction, demonstrating clear uncoupling of the two processes. Our study reveals that the gp96/LFA-1 axis plays a fundamental role in Treg biology and intratumoral CD8+ T cell exhaustion. We suggest that, Treg-specific targeting of gp96/LFA-1 represents a novel strategy for cancer immunotherapy without inflicting autoinflammatory conditions. 1×106 of splenic GFP+ Tregs were purified from WT (n=4) and KO mice (n=4) via FACS isolation. RNA was extracted using RNeasy Micro Kit (Qiagen) following its standard protocol and RNA degradation and contamination was monitored on 1% agarose gels. For RNA sequencing, libraries were prepared using NEBNext® Ultra TM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and paired-end reads were generated. All samples were prepared at the same time and sequenced on the same lane. Original image data file from high-throughput sequencing platforms (like Illumina) is transformed to sequenced reads (called Raw Data or Raw Reads) by CASAVA base recognition (Base Calling). Raw data in FASTQ format were processed through fastp. In this step, clean data (clean reads) were obtained by removing reads containing adapter and poly-N sequences and reads with low quality from raw data.