Single cell sequencing reveals T follicular helper cells as the link between combination ICI therapy and the development of increased IRAEs.

person Rana Yakhshi Tafti Rosalind Franklin Medical School, Chicago, IL info_outline Rana Yakhshi Tafti, Willy Hugo, Alexandra Drakaki, Trevor E Angell, Melissa G Lechner

Authors person Rana Yakhshi Tafti Rosalind Franklin Medical School, Chicago, IL info_outline Rana Yakhshi Tafti, Willy Hugo, Alexandra Drakaki, Trevor E Angell, Melissa G Lechner Organizations Rosalind Franklin Medical School, Chicago, IL, UCLA Geffen School of Medicine, Los Angeles, CA, Division of Hematology/Oncology, UCLA David Geffen School of Medicine, Los Angeles, CA, USC Keck School of Medicine, Los Angeles, CA Abstract Disclosures Research Funding U.S. National Institutes of Health Aramont Charitable Foundation, Doris Duke Charitable Foundation Background: Targeting multiple checkpoints via the combination of individual immune checkpoint inhibitor (ICI) therapies has increased anti-tumor effects compared to single agent ICI regimens. Despite these benefits, Dual ICI therapies have a higher risk of immune toxicity and incidence of immune related adverse events (irAEs) compared to single agent immunotherapies. Of note, T follicular helper (TFH) cells have been shown to contribute to both spontaneous and, more recently, irAE autoimmunity. Given the association of TFH cells and Tertiary Lymphoid Structures (TLS) with ICI response, a potential reason for the increased effectiveness of Dual ICI in anti-cancer therapy, TFHs may also be involved in the pathogenesis and increased incidence of irAEs seen with Dual ICI therapy. Methods: To understand the underlying mechanism by which Dual ICI therapy may increase the incidence of irAEs, we obtained thyroid fine needle aspirates from 9 individuals with ICI-thyroiditis, one of the most common irAEs. We performed single-cell RNA sequencing (scRNA-Seq) and compared immune infiltrates of patients receiving anti-programmed cell death protein 1 (PD-1) or anti-programmed cell death ligand 1 (PD-L1) to Dual ICI patients who received PD-1 or PD-L1 in combination with anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4). Results: ScRNA-Seq analyses revealed a 3-fold expansion of TFH and T peripheral helper (TPH) cells in Dual ICI therapy compared to PD-1/L1 patients. CD4T cell trajectory analysis revealed a dominant transition among Dual ICI patients from a naïve SELL+ CCR7+ phenotype to the CXCR5+ TFH and later IL21+ IFNG+ TPH phenotypes (p < 0.0001). The differentiation involved an upregulation of IL21, CD40LG, BCL6, and CXCR5 genes within CD4 T cells. Differential gene analysis showed enrichment of Tertiary Lymphoid Structure (TLS) gene sets in B and TFH cell clusters indicative of TLS formation within which CD8 T, CD4 T TFH, and GC B cells interact. CD8 T cell analysis indicated a dominant expansion and differentiation towards a CXCL13+ IFNG+ FASLG+ phenotype in Dual ICI therapy compared to PD1/PDL1 monotherapy (p < 0.0001). Cell interaction analysis showed up-regulated TFH-mediated IL21 signaling and CD8 T cell-mediated CXCL13 signaling to BANK1 B cells in Dual ICI therapy. Conclusions: Dual ICI therapy led to up-regulation of activation markers and preferential differentiation of CD4 T cells towards TFH cells in addition to upregulation of TLS signature genes and signaling pathways in TFH, CD8 T, and B cells. Given the previously established association between TFHs and the pathogenesis of autoimmune diseases and irAEs, their increased numbers and activity in Dual ICI therapy may explain the greater toxicities seen.