Gut microbiota-mediated tryptophan metabolism plays dual-effects in diffuse large B-cell lymphoma via regulating 3-IAA/5-HTP balance and antitumor immunity Free

Diffuse large B-cell lymphoma (DLBCL), the most common aggressive lymphoma, exhibits limited immunotherapeutic response due to its immunosuppressive microenvironment. Dysbiotic gut microbiota and dysregulated tryptophan metabolism contribute to DLBCL pathogenesis, yet their combined role in driving immunosuppression remains uncharacterized.

Syngeneic A20 lymphoma models were used to evaluate tryptophan-enriched diets administered 7 days pre- or post-tumor inoculation. Clinical validation integrated metabolomic and metagenomic profiling of DLBCL/healthy cohorts. In vitro cell cocultures and multi-omics (16S rRNA-seq, serum metabolomics, tumor RNA-seq, immune function assays) elucidated mechanisms.

We first performed diet-context experiments by feeding mice with tryptophan before tumor inoculation (Pre-Trp) or after tumor inoculation (Post-Trp). Interestingly, we found that Pre-Trp intervention exerted significant tumor-suppressive effects whereas Post-Trp exposure of tryptophan potentiated obvious tumor progression. Next, we performed targeted tryptophan metabolomics analysis on peripheral blood from Pre-Trp mice, Post-Trp mice and control mice. Among these differential metabolites, indole-3-acetic acid (3-IAA) and indole-3-carbaldehyde (I3C) increased in the Pre-Trp group, indicating enhanced microbiota-driven indole metabolism. Conversely, 5-hydroxytryptophan (5-HTP) and kynurenine (Kyn) decreased in the Post-Trp group, suggesting suppressed metabolism and a shift of host tryptophan metabolism toward the 5-HTP/Kyn pathway.

To further validate the clinical significance of tryptophan metabolism in DLBCL patients, we conducted metabolomics analysis on DLBCL patients. The results revealed significantly downregulated microbiota-dependent metabolites (3-IAA and I3C) and obviously elevated levels of 5-HTP in DLBCL patients. Subsequently, when we administered the four metabolites by gavage to the mice, we found that 3-IAA or I3C significantly suppressed tumor progression. Conversely, 5-HTP or KYN promoted tumor progression. Flow cytometry analysis of tumor tissue showed significantly increased proportions of IFN-γ⁺CD8⁺ T cells and GZMB⁺CD8⁺ T cells in the 3-IAA group compared with the control group, while no significant change occurred in the I3C group. Meanwhile, significantly decreased proportions of IFN-γ⁺CD8⁺ T cells and GZMB⁺CD8⁺ T cells were observed in the 5-HTP group, with no change in the KYN group.

RNA-seq and functional validation revealed that 3-IAA enhanced CD8⁺ T cell function by alleviating NET-mediated immunosuppression without direct T cell activation. Conversely, 5-HTP directly impaired CD8⁺ T cells through PI3K-AKT-NF-κB suppression, exhaustion marker upregulation, and effector molecule downregulation.

We further investigated microbial determinants of metabolic dysregulation. DLBCL patients exhibited significant depletion of tryptophan catabolism commensals (Ruminococcus, Lachnospiraceae, Blautia), which strongly correlated with 3-IAA deficiency and 5-HTP accumulation. Functional validation via fecal microbiota transplantation (FMT) recapitulated dysbiosis and abolished protection. Gut microbiota integrity was essential for the bidirectional effects of tryptophan dietary intervention, while the loss of tryptophan-metabolizing bacteria in the clinical cohort was directly associated with DLBCL progression.

Given the central role of microbiota in driving tryptophan metabolic dysregulation, we systematically explored pectin-mediated reprogramming strategies. Pectin supplementation significantly restored gut microbial α-diversity. This microbial remodeling normalized the 3-IAA/5-HTP metabolic ratio, effectively reversing immunosuppressive metabolite imbalances. Notably, pectin reactivated CD8⁺ T cell functionality, as evidenced by increased cytotoxic effectors (GZMB⁺CD8⁺ T cells and IFNγ⁺CD8⁺ T cells) and reduced exhaustion markers (PD-1⁺CD8⁺ T cells).

Our study establishes a “Time-Microbiota-Immune” paradigm in DLBCL, demonstrating that identical dietary inputs exert opposing effects based on intervention timing. The microbiota acts as a central processor converting tryptophan into 3-IAA (protective) or 5-HTP (pro-tumorigenic), with dysbiosis skewing this equilibrium toward immunosuppression. The actionable “pectin + tryptophan” strategy reprograms metabolic-immune crosstalk, offering a clinically translatable approach to potentiate lymphoma therapy.