Altered interactions between circulating and tissue-resident CD8 T cells with the colonic mucosa define colitis associated with immune checkpoint inhibitors

Abstract

Therapeutic blockade of co-inhibitory immune receptors PD-1 and CTLA-4 has revolutionized oncology, but treatments are limited by immune-related adverse events (IRAEs). IRAE Colitis (irColitis) is the most common, severe IRAE affecting up to 25% of patients on dual PD-1 and CTLA-4 inhibition. Here, we present a systems biology approach to define the cell populations and transcriptional programs driving irColitis. We collected paired colon mucosal biopsy and blood specimens from 13 patients with irColitis, 8 healthy individuals, and 8 controls on immune checkpoint inhibitors (ICIs), and analyzed them with single-cell/nuclei RNA sequencing with paired TCR and BCR sequencing, multispectral fluorescence microscopy, and secreted factor analysis (Luminex). We profiled 299,407 cells from tissue and blood and identified 105 cell subsets that revealed significant tissue remodeling in active disease. Colon mucosal immune populations were dominated by tissue-resident memory (Trm) ITGAE-expressing CD8 T cells representing a phenotypic spectrum defined by gene programs associated with T cell activation, cytotoxicity, cycling, and exhaustion. CD8 Trm and effector CD4 T cells upregulated type 17 immune programs (IL17A, IL26) and Tfh-like programs (CXCL13, PDCD1). We also identified for the first time an increased abundance of two KLRG1 and ITGB2-expressing CD8 T cell populations with circulatory cell markers, including a GZMK Trm-like population and a CX3CR1 population that is predicted to be intravascular. These two populations were more abundant in irColitis patients treated with dual PD-1/CTLA-4 inhibition than those receiving anti-PD-1 monotherapy. They also had significant TCR sharing with PBMCs, suggesting a circulatory origin. In irColitis we observed significant epithelial turnover marked by fewer LGR5-expressing stem cells, more transit amplifying cells, and upregulation of apoptotic and DNA-sensing programs such as the cGAS-STING pathway. Mature epithelial cells with top crypt genes upregulated interferon-stimulated pathways, CD274 (PD-L1), anti-microbial genes, and MHC-class II genes, and downregulated aquaporin and solute-carrier gene families, likely contributing to epithelial cell damage and absorptive dysfunction. Mesenchymal remodeling was defined by increased endothelial cells, both in irColitis patients and specifically in patients on dual PD-1/CTLA-4 blockade. Cell-cell communication analysis identified putative receptor-ligand pairs that recruit CD8 T cells from blood to inflamed endothelium and positive feedback loops such as the CXCR3 chemokine system that retain cells in tissue. This study highlights the cellular and molecular drivers underlying irColitis and provides new insights into the role of CTLA-4 and PD-1 signaling in maintaining CD8 Trm homeostasis, regulating CD8 T recruitment from blood, and promoting epithelial-immune crosstalk critical to gastrointestinal immune tolerance and intestinal barrier function.

A Novel Potential Role for Monocytes Revealed by Single Cell Analysis of Immunotherapy Induced Immune Related Adverse Events

“…In melanoma patients, it has been shown that untreated patients with untreated metastatic disease have decreased CD14 + monocytes in the peripheral blood compared to healthy controls, a finding recapitulated in our analysis, and that the phenotypes of these monocytes may be a predictor of disease response to CBI [ 22 ]. Although limited data exists thus far on monocyte dynamics in tissue during irAEs, early analyses of colitis patients point to an influx of monocytes with a phenotype resembling that of the CD14 + population reported here [ 23 ]. While additional studies are needed, the existing data clearly suggests an important role for these monocyte populations in irAE development.…”

Effects of immune‐related adverse events (irAEs) and their treatment on antitumor immune responses

“…As our understanding of irAE mechanisms in patients evolve, it will help to identify which, if any, animal models most accurately recapitulate organ-specific irAEs in patients and inform the next generation of irAE models that can be used to test the relationship between irAE therapeutics and antitumor efficacy. 53 As T cells are thought to be central to the antitumor responses driven by ICIs 4,9 and are found to predominate the immune cell infiltrate in irAE tissue biopsies across organ systems, 8,10,11,13,16,23,38,[54][55][56][57] many studies have looked at the relationship of T cells in irAEs and paired tumors. While central and peripheral tolerance mechanisms cause many autoreactive T cells to become anergic or die during Tcell development, autoreactive T cells are still commonly found in humans 14,44 and are anticipated to play a role in the development of irAEs.…”

“…Depending on the drugs and doses used, 66%–96% of patients will have side effects from ICIs, and 7%–59% of patients will experience severe irAEs, which are also commonly referred to as “high‐grade” irAEs 6,7 . While the prevailing hypothesis in the field suggests that irAEs are due to a loss of self‐tolerance, 8,9 the mechanistic drivers of irAEs are only beginning to emerge 10–16 . As murine tumor models typically utilized to study ICI efficacy do not develop severe irAEs, 17 insights into the relationship between irAEs and antitumor immunity have been guided by clinical observations and translational research.…”

“…6,7 While the prevailing hypothesis in the field suggests that irAEs are due to a loss of self-tolerance, 8,9 the mechanistic drivers of irAEs are only beginning to emerge. [10][11][12][13][14][15][16] As murine tumor models typically utilized to study ICI efficacy do not develop severe irAEs, 17 insights into the relationship between irAEs and antitumor immunity have been guided by clinical observations and translational research. Currently, the management of severe irAEs involves discontinuing ICI therapy and administering glucocorticoids and other immunosuppressive medications, [18][19][20][21] which may adversely impact antitumor immunity.…”

Rheumatic complications of checkpoint inhibitors: Lessons from autoimmunity

“…These T cells are activated (expressing IFNG and HAVCR2 ), proliferative, and are more clonal 38,39 . Phenotypically similar activated PDCD1 hi CXCL13 + CD8A + T cells have been found to be enriched in irAE‐colitis, 40 again expressing high IFNG and HAVCR2 but, in contrast to those in irArthritis, also coexpressing Th17 cytokines IL17A and IL26 . The same population is enriched in checkpoint inhibitor pneumonitis, again overexpressing IL17A and IFNG but also here expressing CSF2 41 .…”