Identification and regulation of circulating tumor TCR-matched cytotoxic CD4+ lymphocytes by KLRG1 in bladder cancer

While cytotoxic CD4+ tumor-infiltrating lymphocytes have anti-cancer activity in patients, whether these can be non-invasively monitored and how these are regulated remains obscure. By matching single cells with T cell receptors (TCR) in tumor and blood of bladder cancer patients, we identified distinct pools of tumor-matching cytotoxic CD4+ T cells in the periphery directly reflecting the predominant antigenic specificities of intratumoral CD4+ TILs. On one hand, the granzyme B (GZMB)-expressing cytotoxic CD4+ subset proliferated in blood in response to PD-1 blockade, but was separately regulated by the killer cell lectin-like receptor G1 (KLRG1) which inhibited their killing by interacting with E-cadherin. Conversely, a clonally related, granzyme K (GZMK)-expressing circulating CD4+ population demonstrated basal proliferation and a memory phenotype that may result from activation of GZMB+ cells, but was not directly mobilized by PD-1 blockade. As KLRG1 marked the majority of circulating tumor TCR-matched cytotoxic CD4+ T cells, this work nominates KLRG1 as a means to isolate them from blood and provide a window into intratumoral CD4+ recognition, as well as a putative regulatory receptor to mobilize the cytolytic GZMB+ subset for therapeutic benefit. Our findings also underscore the ontogenic relationship of GZMB- and GZMK-expressing populations and the distinct cues that regulate their activity. For single-cell analysis of circulating immune responses, the sample set was available matched blood samples from 6 of 7 patients with localized, muscle-invasive bladder cancer whose tumor and normal adjacent tissue (NAT) scRNAseq was previously published (Oh DY, Kwek SK et al. Cell 2020). These patients included 3 patients ("Anti-PD-L1 B", "Anti-PD-L1 C", "Anti-PD-L1 D") who were treated with neoadjuvant atezolizumab prior to cystectomy on a clinical trial (NCT02451423, see "Clinical trial design"); 1 patient treated with neoadjuvant chemotherapy before cystectomy as standard of care; and 2 patients untreated with any systemic therapy before cystectomy as standard of care. Blood samples from an additional patient from the neoadjuvant trial ("Anti-PD-L1 E"), as well as three separately sequenced technical replicates from a single healthy donor, were also analyzed but without matching tumor data. For standard of care, there was a single blood draw the same day as surgery. For trial patients, a baseline blood draw was taken prior to starting (pre-treatment) and after neoadjuvant atezolizumab (post-treatment) for the trial. All post-treatment blood draws except 1 were prior to surgery, and most were within 1-2 weeks of the surgery date. scRNAseq/TCRseq were conducted as described below and as previously published, and jointly analyzed with the previously published tumor scRNAseq/TCRseq data. All available blood timepoints, and a single tumor timepoint (surgery), were combined for clustering and initial analysis. Connected CD4+ between the blood and TILs were identified in the blood based on matching, paired TCR sequences in single cells, and downstream analyses characterized these circulating, tumor-matching cytotoxic CD4+ T cells. *************************************************************** Raw files for human/patient samples were not submitted to GEO due to concerns about submitting personally identifiable sequence data for open access. ***************************************************************