Alexandra Chloe-VillaniAlexandra-Chloe Villani, PhD, an investigator in the Krantz Family for Cancer Research and the Center for Immunology and Inflammatory Diseases at Massachusetts General Hospital and an assistant professor of Medicine at Harvard Medical School and Institute Member of the Broad Institute, is a corresponding author of a new study in Nature Medicine entitled Single-Cell Transcriptomic Analyses Reveal Distinct Immune Cell Contributions to Epithelial Barrier Dysfunction in Checkpoint Inhibitor Colitis.

Dr. Villani co-supervised together with Kerry Reynolds, MD, Clinical Director of the Inpatient Oncology Units and Director of the Severe Immunotherapy Complication Program. She is also an assistant professor of Medicine at Harvard Medical School.

This work was co-led by two postdoctoral fellows—Dr. Molly Fisher Thomas, a gastroenterologist physician scientist and immunologist who is now on faculty as an assistant professor at the Oregon Health & Science University, and Dr. Kamil Slowikowski who is a computational postdoctoral fellow in the Villani Lab.

What Question Were You Investigating with Your Study?

Dr. Villani explains the genesis of the study: “This study was designed to investigate which cellular, transcriptional and signaling programs are associated with the development of colitis in cancer patients being treated with immune checkpoint inhibitors (ICIs).”

ICIs targeting PD-1 and CTLA-4 have revolutionized the treatment of cancer and are approved for use in over half of all patients with cancer in the United States with over 80 FDA-approved indications.

ICIs regulate T cell activation and tolerance, allowing the immune system to overcome cancer’s evasion of immunogenicity and reestablish its ability to attack tumor tissues.

Beyond unleashing the immune system to attack tumor cells, ICIs can also cause inflammatory side effects the impact healthy organs. Nearly every organ system has been reported to be affected by these treatment side effects with a wide spectrum of severity, ranging from minor rashes and fevers to potentially deadly side effects such as bowel perforation or heart inflammation. We refer to these off-target complications as immune related adverse events (irAEs).

IrAEs can affect up to 90% of patients depending on the dose and combination therapy being used, with up to 55% of patients experiencing severe irAEs that require immunosuppressive therapy or cessation of ICI treatment.

The expanded use of ICIs across many cancer types has provided new hope for patients confronting what was once terminal cancer. However, as ICI use increases as they become the standard of care for many types of cancer, we also unfortunately anticipate an increasing incidence of irAEs unless we find solutions for how to recognize and treat these complications early and ideally prevent them for occurring. 

Little is known about the mechanistic underpinnings of irAEs, which has resulted in limited targeted treatment options that can mitigate irAEs while preserving the anti-tumor immune responses to ICI therapy. Unfortunately, patients with adverse events will have delayed or discontinued cancer treatments, and some will require high dose steroids that compromise anti-tumor immune responses and decrease patient survival.

Given the clinical implications of irAEs, a better understanding of the underlying mechanisms is crucial to preventing their occurrence and improving their treatment. This study published in Nature Medicine addresses these urgent and important clinical questions impacting patients on ICI treatments.

This study focused specifically on immune checkpoint related colitis – inflammation of the colon — which is the most common, severe irAE, though little is understood about its pathogenesis in part because mice given checkpoint inhibitors do not readily develop colitis. The authors referred to it as immune-related colitis (irColitis).

The authors sought to define the cell populations that could ultimately be tracked for diagnosis and to characterize associated signaling pathways and biological programs that could be manipulated therapeutically to resolve irAEs.

Dr. Thomas explains the important clinical implications of studying irAEs: “The study of irAEs will ultimately allow providers to risk-stratify patients who may develop organ specific toxicities, treat IRAEs with modalities that do not abrogate anti-tumor immune responses, and design less toxic, more effective immune-based therapies to treat cancer. As a gastroenterologist who clinically cares for patients with these toxicities, I know firsthand the complexities of choosing treatment modalities that mitigate patients’ gastrointestinal symptoms without interrupting their cancer care.

What Methods Did You Use?

The authors collected blood and tissue samples from patients with cancer who received ICI therapy and had new onset gastrointestinal symptoms such as diarrhea and/or abdominal pain. Their final colonoscopy-based patient cohort included 29 patients: 13 with checkpoint colitis, eight controls on checkpoint therapy without colitis, and eight healthy controls.

Dr. Slowikowski explained the overall strategy: “We leveraged single cell multi-omics experimental and computational strategies, paired T cell receptor (TCR) sequencing analysis, and multispectral microscopy to deeply analyze colon mucosal biopsies and matched blood specimens”.

The analysis sought to define cell populations that could ultimately be tracked for diagnosis and to nominate signaling pathways and biological programs that might eventually be targeted therapeutically to resolve irAEs or prevent their development.

What Were Your Findings?

Compared to healthy controls and controls on ICI therapy, those with irColitis had a significant expansion of CD8+ T cells that are resident to the intestinal tract (i.e. tissue-resident memory CD8+ T cells), CD8+ T cells that recirculate between blood and tissue, Th17 gene programs, CD4+ T regulatory cells, and inflammatory monocytes and macrophages.

The authors additionally observed that patients with irColitis had increased epithelial turnover and a strong epithelial, interferon-induced gene signature that was most pronounced at the top of epithelial crypts near the bowel lumen.

This signature was marked by the significant downregulation of aquaporin and solute carrier genes that promote water and solute absorption, respectively, by the colon epithelium.

These epithelial findings suggest increased barrier breakdown and decreased intestinal absorptive capacity, which likely contributes to disease morbidity.

This study highlights critical features of epithelial-immune crosstalk and epithelial barrier dysfunction associated with the loss of PD-1 and CTLA-4-dependent tolerance. The authors also nominated potential therapeutic targets that could be leveraged to mitigate irColitis.

Their results also highlight that some of the T cell biology associated with irColitis was distinct from that observed in T cells that reside in tumors and are unleashed by ICI therapy to treat cancer. This is important as we seek to define biology that is distinct between anti-tumor immune responses and irAEs so that we can develop irAE mitigation strategies that will spare the anti-tumor immune response.

Dr. Thomas explains: “ Given the strong upregulation of CXCL13, IL17A and IL26 by CD8 T resident memory T cells in irColitis, we queried a published cancer meta-analysis dataset to determine the frequency of this signature in tumor-infiltrating CD8 T cells. We showed that IL17A and IL26 expression was limited and essentially absent from melanoma, which is a type of cancer frequently treated with immunotherapy.

Because IL-17 blockade may worsen IBD, and IL-26 can ameliorate murine colitis, the authors speculate that the IL-17/IL26 pathways may differentially impact tumor immune responses and colitis risk, so this should be an area of further investigation.

The authors also nominated therapeutic targets from FDA-approved medications and those in trial to treat IBD that may disrupt some of the targets we identified in irColitis. For example, they showed that TNF, ITGA4 and ITGB7 were particularly enriched in T cells associated with colitis, whereas ITGA4/ITGB7 ligands, the targets of vedolizumab were additionally expressed in fibroblasts and endothelial cells. S1P receptors, which are modified by ozanimod treatment for IBD, showed striking enrichment in ITGB2Hi CD8 T cells and endothelial cells associated with checkpoint colitis.

The authors also identified putative therapeutic targets of FDA-approved medications for non-colitis indications, including PCSK9 inhibitors, which decrease low density lipids and may suppress T cell activation. Compared to controls, patients with irColitis had higher PCSK9 in colon epithelial cells and a strong correlation between epithelial PCSK9 and CD8 T LDLR. Given differential effects of PCSK9 inhibition on attenuating colitis and promoting anti-tumor responses, future studies will have to determine if inhibiting versus promoting this ligand–receptor interaction could treat irColitis.

Importantly, the authors wanted to ensure that the scientific community could easily access the data and results. Dr. Slowikowski explains: “In this study, we adhered to the FAIR guiding principles for scientific data management and stewardship to improve the Findability, Accessibility, Interoperability and Reuse of our data by releasing a user-friendly interface for the scientific community to browser and query all our data – which is available at – and making all the data and source code readily available on this website. By doing so, we hope to empower the scientific community to design follow-up studies that will continue furthering our understanding of the biological determinants of irColitis”.

What are the Clinical Implications?

Dr. Kerry Reynolds explains the important clinical implications: “Immune checkpoint inhibitors have completely transformed oncology, instilling patients with hope and representing a monumental leap forward in our ability to treat cancer! These drugs are a good news story. However, their use can lead to off-target immune-related adverse events (IRAEs) like irColitis, which presents a significant challenge as we have witnessed for years. The insights from this groundbreaking paper are not just valuable but they are essential for cancer patients and the field of immuno-oncology. By meticulously examining the cellular, transcriptional, and signaling programs associated with IRAEs, this work has provided us with a comprehensive blueprint of the landscape, significantly advancing our understanding and paving the way for more effective treatment strategies. This deeper understanding is crucial for ensuring that our patients can continue to benefit from these life-saving immunotherapies!

Dr. Villani explains the importance of studying the biological drivers of immune-related adverse events: “The translational studies we are pursuing highlight improved therapeutic strategies treat irAEs so that we can keep patients with cancer on lifesaving immune treatments. Together with my colleague, Dr. Kerry Reynolds, we've created the Severe Immunotherapy Complications Service at Massachusetts General Hospital (MGH). Dr. Reynolds serves as Clinical Director of the inpatient cancer unit, while I oversee the translational research program.

This is a spectacular and committed multidisciplinary care unit consisting of teams of oncologists that work together with different medical specialists to help diagnose and tailor the care of cancer patients that present with suspected immune-related adverse events. As part of their care – and with patient consent – tissue and blood specimens are collected for researchers in my team to analyze and try to understand what cells are likely to drive the disease. This approach allows us to efficiently identify which of culprit, disease-causing cells could be targeted for future treatments. We are so grateful to the patients and their family for their willingness to partner with us in this research endeavor! None of this would be possible without them.”

Dr. Thomas explains: “In this study, we have discovered the mechanisms that drive toxicities in the colon. Some immune cell populations in the colon cause damage and other non-immune cells contribute to local inflammation. Excitingly, we've identified potential therapeutic targets that could help stop the migration of immune cells into the bowel to help treat not only irColitis but potentially other forms of gastrointestinal inflammation.”

What are the Next Steps?

Human research in patients with cancer is challenging given the heterogeneity of individuals’ genetic backgrounds, disease presentations, treatment history, and environmental exposures. Nonetheless, we have shown that it is possible to assemble and study specimens from patients who – despite their differences – present with similar symptoms and share tissue-specific disease features common to all individuals with checkpoint inhibitor colitis. Going forward, we are working towards developing human model systems that will enable us to mechanistically validate our findings and test the efficacy of putative therapeutic drug targets.

Dr. Villani explains: “Our research team is conducting parallel studies in other tissues – including the heart, liver, joints, lung, thyroid, neurologic system, endocrine system, and kidney – to understand the biology of irAEs across these diverse organ systems. By comparing irAEs across organs, we hope to define which cellular and molecular mechanisms are common versus unique to individual tissues. These studies will help improve the safety and efficacy of future ICI therapies.”