1 Mann JE, et al. J Immunother Cancer 2023;11:e007358. doi:10.1136/jitc-2023-007358 Open access ScRNA-seq defnes dynamic T-cell subsets in longitudinal colon and peripheral blood samples in immune checkpoint inhibitor-induced colitis Jacqueline E Mann, 1 Liliana Lucca, 2 Matthew R Austin, 1 Ross D Merkin, 1 Marie E Robert, 3 Badr Al Bawardy, 4 Khadir Raddassi, 2 Lilach Aizenbud, 1 Nikhil S Joshi, 5 David A Hafer, 2,5 Clara Abraham, 4 Kevan C Herold, 5,6 Harriet M Kluger 1 To cite: Mann JE, Lucca L, Austin MR, et al. ScRNA-seq defnes dynamic T-cell subsets in longitudinal colon and peripheral blood samples in immune checkpoint inhibitor- induced colitis. Journal for ImmunoTherapy of Cancer 2023;11:e007358. doi:10.1136/ jitc-2023-007358 ► Additional supplemental material is published online only. To view, please visit the journal online (http://dx.doi.org/10. 1136/jitc-2023-007358). Accepted 12 July 2023 For numbered affliations see end of article. Correspondence to Dr Harriet M Kluger; Harriet.Kluger@Yale.edu Case report © Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. ABSTRACT Immune checkpoint inhibitors (ICIs) are increasingly being used to manage multiple tumor types. Unfortunately, immune-related adverse events affect up to 60% of recipients, often leading to treatment discontinuation in settings where few alternative cancer therapies may be available. Checkpoint inhibitor induced colitis (ICI-colitis) is a common toxicity for which the underlying mechanisms are poorly defned. To better understand the changing colon-specifc and peripheral immune environments over the course of progression and treatment of colitis, we collected blood and colon tissue from a patient with Merkel cell carcinoma who developed colitis on treatment with pembrolizumab. We performed single-cell RNA sequencing and T-cell receptor sequencing on samples collected before, during and after pembrolizumab and after various interventions to mitigate toxicity. We report T-cells populations defned by cytotoxicity, memory, and proliferation markers at various stages of colitis. We show preferential depletion of CD8+ T cells with biologic therapy and nominate both circulating and colon-resident T-cell subsets as potential drivers of infammation and response to immune suppression. Our fndings highlight the need for further exploration of the colon immune environment and rationalize future studies evaluating biologics for ICI-colitis, including in the context of ICI re-challenge. INSIGHTS This study suggests that distinct colon-resident and circulating T-cell populations, defined by cytotoxicity markers and granzyme K expres- sion, respectively, may contribute to ICI-colitis pathogenesis and treatment response. BACKGROUND Immune-related adverse events (irAEs) impede long-term disease control in many immune checkpoint inhibitor (ICI)-treated patients. ICI-colitis, a common irAE, often necessitates ICI discontinuation and use of immune suppressants which can diminish tumor rejection. 1 While mechanisms under- lying ICI-induced colitis (ICI-colitis) are incompletely defined, immune cell subsets enriched in ICI-colitis colon tissue have been identified, including expanded CD8+ effector and memory T-cells. 2 Understanding the dynamics of these populations in ICI- colitis is crucial for clinical interventions, and circulating markers could enable non-invasive irAE monitoring. We, therefore, present longitudinal single-cell RNA sequencing (scRNA-seq) analyses of peripheral blood and colon samples from an ICI-colitis patient treated with corticosteroids and antibodies against tumor necrosis factor (TNF)-α and integrin-α 4 β 7 . RESULTS A man in his 70s presented with a growing lesion on his left buttock. Biopsy revealed Merkel cell carcinoma (MCC). Wide local excision revealed a 6 cm wide (pT3) tumor with clear margins. Sentinel nodes were uninvolved. After 6 months he developed in-transit and lymph node metastases. He received three cycles of ICI (pembrolizumab) and developed ICI-colitis (figure 1A). Oral budesonide was started. Five weeks later, sigmoidoscopy revealed severe, diffuse inflammation (figure 1B,C). He received high-dose corticosteroids without improve- ment, and after 3 days received anti-TNF-α (infliximab) with clinical improvement. He completed a 34-day corticosteroid taper and repeat sigmoidoscopy revealed moderately persistent colitis. After another infliximab dose and three doses of anti-integrin-α 4 β 7 (vedolizumab) his symptoms resolved. A third sigmoidoscopy revealed mildly persistent on November 27, 2023 by guest. Protected by copyright. http://jitc.bmj.com/ J Immunother Cancer: first published as 10.1136/jitc-2023-007358 on 16 August 2023. Downloaded from