Discussion:
The pathogeneses of ICI toxicities are poorly understood, with few known risk factors, biomarkers or tailored therapies. As a result, clinicians cannot identify those at greatest risk or intervene to alter therapy before morbidity occurs. For ICI-related colitis diagnostic and treatment algorithms are being derived from classical forms of IBD, and have not been completely validated in this new clinical setting.
Here we applied multiparameter flow cytometry to the study of gut-infiltrating and circulating lymphocytes from melanoma patients treated with combination ipilimumab and nivolumab therapy in the hope of finding novel insights into disease pathogenesis.
We began by directly investigating the profile of gut-infiltrating lymphocytes in affected patients (Cohort 1). Here we found IN-COL was characterised by a predominantly CD8+T cell lymphocytosis, with the proportion of activated memory CD8+ T cells greater than observed in active UC, and supports earlier reports suggesting T cell checkpoint-inhibitor-related colitis is dominated by CD8+ T cells . The high proportion of Treg evident in the gastrointestinal tissue of patients with UC was not evident in IN-COL and we propose this is due to Treg regulatory mechanisms involving high expression of CTLA-4 . IN-COL was associated with a higher proportion of activated and granzyme B+ MAIT cells compared with IN-NAE, changes that were not found in active UC, again highlighting the distinct pathology of IN-COL. We confirmed higher rates of MAIT cell activation in IN-COL using MAIT TCR-specific tetramer staining and paired peripheral blood and gut-isolated mononuclear cell samples from the same patients.
The comparison of IN-COL and UC patients in Cohort 1 (Table Ic) is important. The IN-COL patients were older than UC patients and the time since first diagnosis of colitis was much shorter (1 versus 48 months). Despite this, the IN-C and UC cohorts scored comparably on both the UCEIS endoscopic and Nancy histopathological grading system, two scales commonly used to score severity of UC. The higher levels of CD4+and CD8+ T cell and MAIT cell activation measurable by flow cytometry in IN-COL did not correspond with higher UCEIS or Nancy scores. Therefore, the significant cellular changes found in IN-COL cannot be simply seen as a function of colitis severity.
We sought to understand if changes concordant with those seen in local tissue could be detected in the peripheral blood. We found therapy results in a widespread “on-treatment” activation of memory T cells, however these were not predictive of colitis. We additionally determined that patients with IN-COL could also have pre-existing lower circulating MAIT cells than those in the IN-NAE group, raising the possibility that a pre-existing reduction of peripheral MAIT cells might be a risk factor for IN-COL, as similarly described in other autoimmune diseases of the gastrointestinal tract . However, this proved an unreliable predictor in Cohort 3 where the IN-NAE group also had low levels of circulating MAIT cells. The differences between the IN-NAE groups in Cohort 2 and Cohort 3 could not be explained by significant differences in age, sex or serum LDH. There is a potential confounder in that the IN-NAE patients in Cohort 2 were of higher melanoma stage compared with Cohort 3.
IN-COL was not associated with high levels of circulating plasmablasts, a point of distinction from UC, possibly suggesting there is less of a role of pathogenic B-cells in IN-COL. Analysis of gut-derived mononuclear cells demonstrated that IN-COL was associated with high rates of predominantly activated memory CD8+ T-cells and granzyme B+ MAIT cells, with a low proportion of Treg in a cellular pathology that was distinct from both IN-NAE and UC groups.
Our work provides further evidence that CD8+ T cells are the predominant cell type in IN-COL. Given that cytotoxic CD8+ T cells are thought to be the major target of ICI, it appears that their activation in IN-COL leads to off-target collateral damage. Important questions remain, including why IN-COL occurs in some patients and not others, if there is any mechanism to predict this toxicity prior to treatment, and why steroid and infliximab-refractory cases occur. Our finding that both CD8+ T cells and MAIT cells are activated in IN-COL appears to provide important information as to the initiating antigen and suggests an interaction with the intestinal microbiome.
MAIT cells provide an important link between microbes and cellular immunity, being directly activated by vitamin B2 metabolites generated from bacteria, or indirectly from cytokines (e.g. IFN-α and TNF- α). Whilst we are unable to confirm that low circulating MAIT cell counts definitively correlate with subsequent risk of IN-COL, activated MAIT cells were only seen in the gut-isolated mononuclear cells of IN-COL and not IN-NAE patients. This raises important questions that centre around if specific microbes are involved in MAIT cell activation in the setting of IN-COL, and how MAIT cell trafficking is regulated, particularly between the blood and gut. There is literature suggesting a host’s microbiome can impact on the efficacy of ICI therapyand the development of associated colitis that is relevant to this field and that we are exploring in a prospective trial. Additionally, there is data supporting the efficacy of faecal microbiota transplant for ICI-colitis, suggesting that replacement of microbiome species can ameliorate disease, and one mechanism of this might be through reducing the provision of species-specific bacterial-derived MAIT cell ligands.
The presence of the riboflavin operon in bacteria identifies species that can activate MAIT cells through their TCR. The activation of MAIT cells in this setting raises a compelling link between host microbiome, MAIT cells and IN-COL that warrants further investigation, especially in regard to whether these cells contribute to tissue damage. A report investigating colitis in the setting of ipilimumab monotherapy found higher levels of riboflavin (vitamin B2) in the microbiome was associated with protection from colitis , providing a possible link to MAIT cells (that are activated by riboflavin metabolites) as protective in ICI-associated colitis.
MAIT cells can be activated by their TCR or cytokines (IL-12, IL-15, IL-18, IFNα/β, TNFα) or synergistically by both. We require further work to determine which pathways are leading to the activation of MAIT cells in IN-COL and whether this activity contributes to tissue damage .
This work provides an important first report on changes in gut-derived and peripheral-blood mononuclear cells that occur in ICI-associated colitis, but our approach has several limitations. This work constitutes retrospective and cross-sectional analysis conducted in relatively small numbers of patients that requires confirmation in larger studies. We also cannot comment at this stage on whether these changes occur in anti-PD-1 monotherapy-associated colitis.
We are currently recruiting to a prospective single centre study of oncology patients who have PBMC and GMNC collection, flexible sigmoidoscopy with gastrointestinal biopsy, and microbiome analysis prior to commencement of ICI therapy, and at the time of subsequent development of related colitis. We aim to determine the mechanisms by which MAIT cells become activated in the gastrointestinal tract. Here we demonstrate that the levels of activated MAIT cells in the circulation are not a good indicator of the levels in the colon, and further investigation into how circulating MAIT cells relate to tissue-based populations would be of value. It is also unclear at this stage whether MAIT cell activation contributes to tissue damage or is part of a homeostatic repair mechanism. It is hoped that further understanding the mechanisms that activate memory CD8+ T cells and MAIT cells in the gastrointestinal tract of patients with ICI-related colitis will lead to the development of improved and novel targeted therapies.