While cancer immunotherapy has transformed the treatment of many types of cancer, not all patients get the same benefit from these therapies. The results of a study led by scientists at Harvard Medical School and Dana-Farber Cancer Institute suggest that an individual’s gut microbiota may play a key role in immunotherapy treatment outcome.

The team’s study in mice identified how gut microbes enhance the body’s response to a common type of immunotherapy known as PD-1 checkpoint blockade, which is currently used for the treatment of 25 forms of cancer. The findings showed that specific gut bacteria can affect the activity of two immune molecules—PD-L2 and RGMb —as well as the interplay between them. The researchers also demonstrated that blocking the activity of either molecule, or this interplay between them, enhanced responses to cancer immunotherapy and optimized the body’s ability to detect and destroy cancer cells.

The study identifies the molecule RGMb as a previously unknown accomplice in sabotaging the body’s ability to spot and destroy tumors. RGMb, primarily known for its role in nervous system development, is also found on the surface of cancer-fighting T cells. Until now, however, no one knew it played a role in regulating T-cell responses to cancer immunotherapy.

If replicated in humans, the findings can inform the design of therapies that improve immunotherapy treatment outcomes, the researchers noted. “The engagement between PD-L2 and RGMb acts as a brake on cancer-fighting T cells, and our work shows that treatment with antibodies that block the interaction of PD-L2 with RGMb releases this brake and allows T cells to eradicate tumors,” said Arlene Sharpe, MD, PhD, the Kolokotrones University Professor at Harvard and chair of the Department of Immunology in the Blavatnik Institute at HMS.

“Our findings offer a critical clue into a complex puzzle and in doing so suggest concrete ways to enhance the potency of cancer immunotherapy and improve patient outcomes,” added co-first author Joon Seok Park, PhD, a postdoctoral research fellow in immunology in the Sharpe lab. “We propose a new approach to overcome the resistance to the current cancer immunotherapies by learning from gut bacteria that help our immune system to fight cancer.”

Sharpe co-led the research with Dennis Kasper, MD, the William Ellery Channing Professor of Medicine and professor of immunology at HMS, and Gordon Freeman, PhD, professor of medicine at HMS and Dana-Farber. Their results are reported in Nature, in a paper titled “Targeting PD-L2–RGMb overcomes microbiome-related immunotherapy resistance,” in which they concluded “These studies identify downregulation of the PD-L2–RGMb pathway as a specific mechanism by which the gut microbiota can promote responses to PD-1 checkpoint blockade. The results also define a potentially effective immunological strategy for treating patients who do not respond to PD-1 cancer immunotherapy.”

Critical to cancer’s survival and spread is its ability to evade the body’s immune defenses, and starting back in the 1990s, and Sharpe and Freeman performed some of the critical early work that elucidated how cancer manages to evade the immune system. Their work focused on PD-L1 and PD-L2, which reside on the surface of immune cells, with studies showing that when PD-L1 or PD-L2 interact with another molecule, PD-1, on the surface of T cells, the activity of T cells is kept in check. Under normal conditions, this interaction functions as a brake on T cells to ensure they do not mistakenly attack the body’s own cells and tissues.

Sharpe, Freeman, and others discovered that cancer exploits precisely this safety mechanism to evade detection and destruction by T cells. The cancer cells do so by expressing PD-L1 and PD-L2 on their surfaces, engaging with PD-1 and reining in the T cells. A form of immune checkpoint blockade (ICB) cancer immunotherapy is designed to block the interaction of PD-1 with PD-L1 or PD-L2,  and release the T cells’ attack against the cancer cells.

Such treatments, currently used for 25 forms of cancer, have revolutionized cancer care, but a subset of patients do not benefit from them. “The response rates for PD-1–PD-L1 blockade in approved indications range from 13% to 69% depending on the tumour types,” the team further explained. “There is substantial interest in understanding the factors that regulate the responsiveness to PD-1 inhibitors to develop strategies to benefit more patients.”

The interplay between the immune system and the gut microbiota has been the focus of Kasper’s work for many years. His lab has identified not only mechanisms of regulation but also specific microbial molecules and microbial enzymes responsible for modulating the immune system.

The notion that gut microbes could affect cancer immunotherapy is not entirely new. Recent studies have found tantalizing clues about the role that gut microbes play in immunotherapy treatment outcomes, and indicated that the gut microbiota is a crucial regulator of anti-tumour immunity during immune checkpoint inhibitor therapy. “Studies have shown that the gut microbiota can modulate the efficacy of PD-1 pathway inhibitors in cancer,” the team noted. “These findings have stimulated the investigation of probiotic bacteria and faecal transplants to promote anti-tumour responses to PD-1 immunotherapy.” However, to date, it hasn’t been understood exactly how the microbiome impacts on immunotherapy success. “Current understanding of how the gut microbiota can enhance cancer immunotherapy is still at an early stage,” the authors stated. “Our goal was to identify targetable immunological mechanisms by which the gut microbiome regulates anti-tumour effects of cancer immunotherapy.”

For their newly reported study the team used mice whose colons were seeded with gut microbiota from patients with cancer. Some of those patients had responded well to immunotherapy, while others had not experienced much benefit. The responses of the mice to immunotherapy mimicked the treatment response in the humans whose gut microbes now lived in the animals’ intestines.

Comparing the immune system profiles of the two groups of mice, the researchers identified tell-tale differences in various immune cells involved in cancer detection and destruction. The findings suggested that gut microbiota altered the immune cells’ behavior and, therefore, response to immunotherapy.

Mice seeded with gut microbes from patients that had themselves responded well to cancer immunotherapy had lower levels of PD-L2 on a antigen-presenting cells (APCs). These immune cells play a critical role in rallying the body’s immune defenses by patrolling the body for pathogens or tumors and presenting these foreign or abnormal proteins to T cells for destruction. Conversely, mice seeded with gut microbes from patients who had demonstrated a poor response to immunotherapy showed increased levels of the PD-L2 molecule.

To tease out the effect of specific gut microbes, the researchers treated groups of mice with broad-spectrum antibiotics, which kill gut bacteria. Antibiotic-treated mice did not respond to immunotherapy that blocked the PD-1 molecule. These mice, however, had high levels of PD-L2, the other molecular brake that typically acts through PD-1. Animals that had a robust response to the same treatment had lower levels of PD-L2.

Intrigued that PD-1 blockade did not work, the researchers hypothesized that PD-L2 acts as a brake on T cells, not through PD-1 alone but through another molecular accomplice. The researchers turned their attention to RGMb, which was of interest because the Freeman lab had previously shown that RGMb and PD-L2 regulated immune tolerance in lungs.

When the scientists treated the mice that had not responded to anti-PD-1 therapy alone using antibodies that blocked RGMb, these animals experienced both an increase in cancer-fighting T cells and rapid overall improvement. “The interplay between the microbiota and immune cells in the anticancer response just got clearer, and with the identification of RGMb as PD-L2’s molecular accomplice, we have another target for cancer immunotherapy,” Freeman said. The authors stated, “The requirement for RGMb or PD-L2 blockade to potentiate the effect of PD-1 blockade in GF mice suggests that the PD-L2 inhibitory effect may be largely through RGMb–PD-L2 interactions and not PD-1–PD-L2 interactions.”

Further analyses showed that the interaction between RGMb and PD-L2 depended on the composition of gut microbes. The researchers found that certain gut microbes could affect the levels of both molecules.

Mice with cancer whose intestines had been seeded with certain gut microbes had levels of RGMb on their T cells six times lower than animals with microbe-free guts and responded to anti-PD-L1 or anti-PD-1 therapy. In comparison, mice with depleted gut microbiota did not respond to these treatments and had higher levels of RGMb on their T cells, especially on T cells that had infiltrated their tumors.

Similarly, mice whose guts were seeded with microbiota from patients who had demonstrated poor treatment responses also had higher levels of RGMb, a finding suggesting that patients who do not mount a good response to cancer immunotherapy harbor higher levels of RGMb on their T cells, which in turn interferes with their immune cells’ antitumor response.

Disabling the activity of either PD-L2 or RGMb was sufficient to preserve T cells’ antitumor activity and ensured a robust response to PD-L1 and PD-1 therapy. Remarkably, blocking the activity of PD-L2 led to a potent antitumor response in animals treated with another form of cancer immunotherapy known as dendritic cell therapy. This observation suggests that modulating PD-L2 activity holds promise for boosting the body’s response to multiple types of cancer immunotherapy. “ … by investigating how the gut microbiome modulates anti-tumour immunity, we identified attenuation of PD-L2 and RGMb expression as an immunoregulatory mechanism that informs a possible therapeutic strategy to overcome resistance to PD-1 blockade,” the investigators noted. “Antibody-mediated blockade of the PD-L2–RGMb pathway or conditional deletion of RGMb in T cells combined with an anti-PD-1 or anti-PD-L1 antibody promotes anti-tumour responses in multiple mouse tumour models that do not respond to anti-PD-1 or anti-PD-L1 alone (germ-free mice, antibiotic-treated mice and even mice colonized with stool samples from a patient who did not respond to treatment).”

Altering the composition of the gut microbiota in different groups of mice revealed that one organism, C. cateniformis, suppressed PD-L2 levels and rendered immunotherapy more effective in mice with cancer. “Monocolonization with C. cateniformis was sufficient to promote a response to anti-PD-L1 therapy and suppress PD-L2 expression …”. The researchers acknowledged that, given that the human gut is home to thousands of bacterial species, this single microbe is probably not the only organism capable of regulating antitumor immunity. “Previous studies have shown that bacteria from different phyla can have overlapping immunoregulatory consequences, and strains of the same species can produce different immunoregulatory Outcomes,” they wrote. “Consequently, C. cateniformis is probably not the only strain in the human microbiota that can mediate this effect.”

Nevertheless, the finding suggests that specific microbial molecules might be harnessed in the form of small-molecule drugs to augment the immune system’s ability to control cancer. Such treatments could supplement or be an alternative to traditional antibody-based cancer immunotherapy.

A small-molecule approach would have the added appeal of being cheaper to develop and store and easier to deliver into the body, Sharpe noted. Small-molecule medicines are generally given as pills, while cancer immunotherapy is administered in the form of intravenously infused antibodies.

The researchers caution that while their work reveals a critical piece of the puzzle, it is likely only one of several ways in which the immune system and the microbiome interact in cancer. “This is likely only the beginning of the story,” said Francesca Gazzaniga, PhD, co-first author on the study and a former postdoctoral researcher in the Kasper lab, now assistant professor of pathology at HMS and principal investigator at Massachusetts General Hospital. “Cancer, the immune system, and the microbiome are astoundingly complex individually, but when you put these systems together, the resulting interplay is exponentially more intricate.”

“There are likely many other ways in which the microbiome can affect cancer immunity in general and cancer immunotherapy in particular,” Kasper said. “With this work, we’ve found a whole new way of looking at how the gut microbiota affects not only the efficacy of cancer treatments but cancer immunity in general.”

In conclusion, the team noted, “Blockade of PD-L2–RGMb interactions combined with anti-PD-1 or anti-PD-L1 therapy can overcome microbiome-dependent resistance to monotherapies with PD-1 pathway inhibitors and provides a new strategy for treating patients who do not respond to PD-1 cancer immunotherapy. Our study provides an innovative approach to identify new cancer immunotherapy targets using the gut microbiome as a discovery platform.”

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