Microbial short-chain fatty acids modulate CD8+ T cell responses and improve adoptive immunotherapy for cancer

The gut microbiota significantly impacts the efficacy of cancer immunotherapies, particularly immune checkpoint inhibitors (ICIs) and adoptive cell therapies using tumor-specific CD8+ CTLs. While certain bacteria like *Akkermansia muciniphila* and some *Bifidobacterium* strains have shown promise in enhancing anti-tumor responses, the role of soluble microbial molecules and metabolites remains less understood. Previous research has identified butyrate, propionate, and acetate as major microbial metabolites (SCFAs) that can modulate T cell function. Butyrate, in particular, is linked to protection against autoimmune processes. However, the therapeutic potential of ex vivo treatment of CTLs – endogenous or genetically engineered – with bacterial metabolites to augment their anti-tumor reactivity is largely unexplored. Pentanoate, produced by low-abundant commensals such as *Megasphaera massiliensis*, is a SCFA of particular interest due to its previously demonstrated impact on CD4+ T lymphocyte function. This study investigates the influence of bacterial SCFAs, focusing on pentanoate and butyrate, on the efficacy of adoptive T cell therapy using antigen-specific CD8+ CTLs and CAR T cells.

Existing literature highlights the significant influence of the gut microbiota on cancer immunotherapy. Studies have shown that specific bacterial species and their metabolites can modulate immune responses. For example, *Akkermansia muciniphila* and certain *Bifidobacterium* strains have demonstrated the ability to improve the efficacy of immune checkpoint inhibitors. The metabolite inosinic acid, produced by specific commensal bacteria, has also been shown to enhance ICI therapy. SCFAs, including butyrate, propionate, and acetate, have been implicated in the regulation of T cell responses, with butyrate specifically linked to the expansion of Tregs and the improvement of effector CD4+ T cell function. Butyrate's role in protecting against autoimmune processes and graft-versus-host disease has also been documented. While the microbiota's impact on CD8+ T cell generation is recognized, less is known about the direct effects of soluble microbial metabolites on adoptive T cell therapy.

The study employed both in vitro and in vivo experiments using murine models. In vitro, CTLs and CAR T cells were treated with various SCFAs (pentanoate, butyrate, propionate, acetate) and the effects on cytokine production (IFN-γ, TNF-α), HDAC activity, and mTOR activity were assessed using flow cytometry, ELISA, and western blotting. Optimal SCFA concentrations were determined to maximize effects while minimizing toxicity. In vivo, the anti-tumor activity of SCFA-treated antigen-specific CD8+ CTLs and ROR1-targeting CAR T cells was evaluated in syngeneic murine melanoma and pancreatic cancer models. Tumor growth, T cell infiltration, and cytokine expression were measured. The study also investigated the role of HDAC class I and II enzymes and the mTOR pathway using specific inhibitors. Similar experiments were conducted using human CD8+ T cells and CAR T cells to explore the translational potential of the findings. The study carefully controlled for potential confounding factors, such as the use of supernatants from various commensal bacteria to isolate the effects of the SCFAs.

The study's key findings demonstrate that pentanoate and butyrate significantly enhance the anti-tumor activity of CTLs and CAR T cells. Specifically: * Treatment with pentanoate and butyrate increased the production of effector cytokines (IFN-γ and TNF-α) in both CTLs and CAR T cells. * These SCFAs inhibited class I HDAC activity, leading to epigenetic changes that contribute to enhanced CTL function. * Pentanoate stimulated mTOR activity, which plays a crucial role in T cell metabolism and effector function. This effect was shown to be independent of GPR41/GPR43 SCFA receptors. * In vivo studies using murine melanoma and pancreatic cancer models confirmed that pentanoate and butyrate-treated CTLs and CAR T cells exhibited superior anti-tumor activity compared to untreated cells, resulting in reduced tumor volume and increased numbers of effector T cells in tumor-draining lymph nodes. * The positive effects were also observed in experiments using human CD8+ T cells and CAR T cells, suggesting a translational potential of these findings for human cancer immunotherapy. * The study also showed that the positive effects of pentanoate on CAR T cells includes enhanced proliferation and increased CD25 expression. Inhibition of glycolysis abolished pentanoate-induced CD25 upregulation, highlighting the importance of metabolic reprogramming.

This research provides strong evidence that the microbial metabolites pentanoate and butyrate can significantly improve the efficacy of adoptive cell therapy for cancer. The mechanisms underlying these effects involve both epigenetic modulation through HDAC inhibition and metabolic reprogramming through mTOR activation. The findings highlight a previously underappreciated aspect of the microbiota-immune system interaction and suggest new avenues for improving cancer immunotherapy. The observation that these effects translate to human T cells suggests potential clinical applications. The study's results support the further investigation of SCFA-based therapies to enhance the efficacy of cellular immunotherapies, potentially combining SCFA administration with adoptive cell transfer strategies.

This study demonstrates that the SCFAs pentanoate and butyrate significantly enhance the anti-tumor activity of CTLs and CAR T cells through metabolic and epigenetic reprogramming. These findings suggest that SCFAs, particularly pentanoate, have considerable therapeutic potential for improving the efficacy of adoptive cell therapy for cancer. Future research should focus on optimizing the administration of these SCFAs in clinical settings and exploring their potential use in combination with other immunotherapeutic strategies.

The study primarily used murine models, and while human T cell studies were included, further validation in human clinical trials is necessary to confirm these findings. The specific mechanisms by which pentanoate and butyrate modulate mTOR activity require additional investigation. The study focused on specific SCFAs; investigating other SCFAs and their combinations could reveal additional therapeutic opportunities.