Enhancing CAR-T Cell Therapy for Non-Hodgkin Lymphoma through Gut Microbiota and Butyrate

Introduction. The gut microbiota is crucial for the development and function of the immune system and is considered a significant factor in the efficacy of immunotherapies, including CAR-T cells. However, the mechanisms of this influence remain unclear. Short Chain Fatty Acids (SCFAs), metabolites produced through microbial fermentation, play a role in regulating metabolic, endocrine, and immune functions.

Methods. Stool, serum samples, and clinical data were collected from 83 NHL patients (65 DLBCL, 16 FL and 3 others) before CD19 CAR-T treatment. Patients were classified as responders (achieving complete remission or partial response) or non-responders (exhibiting refractory or stable disease). The gut microbiota was characterized through 16S rRNA gene sequencing using the Ion 16S Metagenomics Kit and analyzed with QIIME2 and the SILVA138 database. Serum SCFA concentrations were measured using single quadrupole GC-MS. Variables were categorized based on the maximally selected rank statistic. T cells from healthy donors were isolated and activated before being transduced with a CD19-41BB-CD3z CAR lentiviral vector. CAR-T cells were expanded and exposed to the SCFA butyrate for 12 days. Their cytotoxic potential was determined through coculture with the Raji cell line and functionally characterized using flow cytometry and RNAseq.

Results. First, a multivariate analysis (MVA) was conducted to identify factors affecting patient progression-free survival (PFS). We found that a higher number of non-prophylactic antibiotic lines administered in the 8 weeks prior to infusion was associated with worse survival outcomes (HR = 1.61; 95% CI, 1.12-2.30; P = 0.013). Interestingly we observed that patients with higher antibiotic exposure had a higher proportion of CD4⁺ T cells (P = 0.039), increased IL-10 levels in serum (P = 0.004), and a trend towards lower IL-2 and TNFα levels. Similarly, the gut microbiota of these patients showed reduced richness (P = 0.010) and α-diversity (P = 0.017). Interestingly, we observed that patients with lower microbiota indices of richness and α-diversity exhibited worse PFS (P = 0.003 and P < 0.001, respectively).

To assess the role of the gut microbiota in response, we analyzed and compared their taxonomic composition, finding that responder patients had a significantly higher abundance of SCFA-producing bacterial groups including Prevotella, Ruminococcus, Terrisporobacter and Butyricicoccus. Next, we measured the circulating concentration of SCFAs in the patients' serum. MVA revealed that lower butyrate concentrations were associated with poorer survival (HR = 6.08; 95% CI, 1.22-30.4; P = 0.028).

We then exposed CAR-T cells to 0.5 mM butyrate and found that they exhibited greater specific lysis compared to their respective controls without stimulation (P = 0.009). These butyrate-stimulated CAR-T cells showed higher expression of activation markers (P = 0.008) and co-stimulatory molecules (P = 0.022), an increased percentage of transduction (P = 0.018), a lower percentage of senescent cells (P = 0.028) and a more undifferentiated phenotype (P = 0.008). Through whole transcriptome sequencing we identified 155 differentially expressed genes in the butyrate-stimulated CAR-T cells, with 145 upregulated and 10 downregulated genes. Gene set enrichment analysis revealed that butyrate-exposed CAR-T cells showed significant enrichment in pathways related to chemokine response, cytokine production or T cell mediated immunity.

Conclusions. These results shows that multiple lines of non-prophylactic antibiotics before CD19 CAR-T therapy negatively impact outcomes by causing dysbiosis. The analysis of the gut microbiota identified butyrate as a key prognostic factor, which enhances CAR-T cell efficacy through phenotypic and transcriptomic changes, and reinforce the role of the microbiota in CAR-T cell therapy outcomes.

Acknowledgment. This work was supported by a grant PID2021-123056OA-I00 funded by MCIN/AEI/10.13039/501100011033 and ERDF A way of making Europe, Universidad Complutense de Madrid (PR27/21-031), AECC (Accelerator Award and Ideas Semilla, IDEAS20014LINA) and CRIS Contra el Cancer Foundation (2021/0096 and 2024/0031). R.G.V holds a Formación de Profesorado Universitario (FPU19/04933) grant from the Ministry of Science, Innovation and Universities of Spain Government.