Abstract 5630: Intratumoral regulatory T cell clones originate from the draining lymph nodes regulatory T cells
Authors: Aoki Hiroyasu , Shimizu Haruka , Shichino Shigeyuki , Tsunoda Mikiya , Ogiwara Haru , Hori Shohei , Matsushima Kouji , Ueha Satoshi 
Published: June 2022

Forkhead box P3 (Foxp3)-positive intratumoral regulatory T (Treg) cells play critical roles in suppressing anti-tumor immune responses. It is reported that human tumors accumulate more Tregs than peripheral blood. However, it remains unclear whether intratumoral Tregs are primarily derived from Tregs in tumor-draining lymph nodes (dLNs) or are converted from conventional CD4+ T cells (Tconvs) in the transforming growth factor-beta -rich tumor microenvironment.Because each T-cell clone has its unique T-cell receptor (TCR), tracking the TCRs of the tumor Treg repertoire in the dLN Treg, dLN Tconv, and tumor Tconv enables us to identify the primary source of intratumoral Tregs. GFP+ hCD2+ CD25high and GFP- hCD2+ CD25int Tregs, which we defined as mature and immature Tregs, respectively, are distinguished from Tconvs in Foxp3-GDL Tg × Foxp3hCD2 Treg reporter mice having the Foxp3-GFP-diphtheria toxin receptor transgene and Foxp3-human CD2/CD52 knocked-in locus. On day 14 after subcutaneous injection of the Lewis lung carcinoma cell line, CD4+ Treg subsets and Tconvs in the tumor and dLN were collected for TCR sequencing, and the TCR repertoire overlap between these populations was quantified.We found that approximately 70%-80% of the Treg repertoire in the tumor overlapped with that in the dLN. Overlap between tumor Treg and tumor Tconv or dLN Tconv was at most 30% in tumor Tregs, significantly smaller than the tumor Treg-dLN Treg overlap. Moreover, single-cell TCR/RNA sequencing analysis exhibited that the tumor and dLN Treg clusters shared a significant number of T-cell clones, with few clones shared with tumor or dLN Tconv clusters. To examine whether dLN Tregs were the source of tumor Tregs even when they were actively repopulating, GFP+ mature Tregs were transiently depleted by administrating diphtheria toxin to tumor bearing mice. Mature tumor Tregs were completely depleted by day 2 and recovered rapidly through day 5 after DT administration. Analysis of the TCR repertoire of repopulated Treg and Tconv subsets revealed that repertoire overlap between tumor Treg and dLN or tumor Tconv was small and that tumor Treg-dLN Tregs overlap remained dominant, proposing that the repopulated intratumoral Tregs were also mainly derived from dLN Tregs. These results demonstrated that dLN-tumor mobilization was the primary source of intratumoral Tregs and that Tregs converted from Tconvs comprised only a small fraction of the intratumoral Tregs in our model. This finding suggests that Treg clones that suppress anti-tumor responses in the tumor also inhibit the priming of tumor-reactive T cells in the dLN, which can limit the efficacy of tumor site-specific Treg depletion therapy.

Citation Format: Hiroyasu Aoki, Haruka Shimizu, Shigeyuki Shichino, Mikiya Tsunoda, Haru Ogiwara, Shohei Hori, Kouji Matsushima, Satoshi Ueha. Intratumoral regulatory T cell clones originate from the draining lymph nodes regulatory T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5630.