ScRNA-seq defines dynamic T-cell subsets in longitudinal colon and peripheral blood samples in immune checkpoint inhibitor-induced colitis

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 defined. To better understand the changing colon-specific 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 defined 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 inflammation and response to immune suppression. Our findings 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.

Keywords: CD8-Positive T-Lymphocytes; Immune Checkpoint Inhibitors; Immunotherapy; Programmed Cell Death 1 Receptor; T-Lymphocytes.

Figure 1

(A) Timeline of patient’s clinical signs and interventions (upper) and sample collections (lower). (B) Multiple random colon biopsies revealed diffuse involvement by chronic active colitis, with increased lamina propria plasma cells, and active crypt epithelial injury with apoptosis (C; arrows). (D) Subclustering of colon biopsy-derived CD3+ cells. (E) Proportion of total CD3+ cells in each subcluster for each biopsy sample and dot plot representing expression of select markers in each subcluster. (F) Subclustering of peripheral blood-derived CD3+ cells. (G) Proportion of cells in each subcluster for each blood sample. G. Proportion of total CD3+ cells in each subcluster for each blood sample and dot plot representing expression of select markers in each subcluster. ICI, immune checkpoint inhibitor; IFX, infliximab; IMDC, immune-mediated diarrhea and colitis; MAIT, mucosal-associated invariant T cell; Pred, prednisone; Tcm, central memory T cell; Teff, T effector cell; Teff-GZMK, GZMK-positive Teff; Tregs, regulatory T cells; Trm, resident memory T cell; tSNE, t-distributed stochastic neighbor embedding; VDZ, vedolizumab.

Figure 2

(A) Abundance of clonotypes by expansion status in blood. (B) The most prevalent clonotypes in the indicated clusters were visualized across colon samples via alluvial plot (compareClonotypes function in scRepertoire). (C) Select clonotypes shared between blood and colon at Tox-1 are represented for each of the indicated samples by alluvial plot. No Teff-GZMK or cycling cells were observed post-immune suppression (Cl. #, Clonotype number). (D) Shared clonotypes that were expanded in blood were visualized by alluvial plot across blood samples. (E) Clonotypes of interest were highlighted as above on the tSNE plot representing all four blood samples. (F) Differentially expressed genes for circulating-only clonotypes versus shared clonotypes. (G) Expression of genes involved in targetable T-cell trafficking and homing mechanisms in the indicated circulating clonotypes. MAIT, mucosal-associated invariant T cell; Teff, T effector cell; Teff-GZMK, GZMK-positive Teff; Trm, resident memory T cell; tSNE, t-distributed stochastic neighbor embedding.