Food-Derived Limosilactobacillus fermentum GR-3 Enhances Anti–PD-1 Immunotherapy Efficacy

Immune checkpoint blockade (ICB) has improved the cancer treatment, its application remains constrained by heterogeneous response rates and immune-related adverse events. While gut microbiome modulation represents a promising avenue to optimize ICB, rationally selected probiotics with defined mechanistic actions are critically lacking. Here, we selected Lactobacillus fermentum GR-3 (L. fermentum GR-3), a food-derived strain pre-screened for exceptional gastrointestinal resilience (maintaining > 107 CFU/mL), antioxidant activity (87.4% DPPH-Scavenging efficiency), and immunomodulatory potential, as an oral combination strategy to enhance anti-PD-1 therapy. In the MC38 murine colorectal cancer model, oral L. fermentum GR-3 synergistically enhanced ICB efficacy, yielding significantly greater tumor suppression than anti-PD-1 monotherapy (51.7% volume reduction vs. untreated controls), while attenuating therapy-associated weight loss. Locally within the tumor microenvironment (TME), the combination therapy promoted dendritic cell maturation (MHC-II⁺ DCs reaching 56.0%), favored pro-inflammatory macrophage polarization (M1 increased to 20.8%, while M2 reduced to 2.57%), and suppressed regulatory T cell infiltration (Tregs decreased to 2.32%). These shifts strongly correlated with enhanced cytotoxic responses, with Granzyme B⁺ CD8⁺ T cells reaching 40.4% (doubling the effect of PD-1 monotherapy). Mechanistically, L. fermentum GR-3 colonization partially reversed tumor-associated gut dysbiosis, enriching short-chain fatty acid (SCFA)-producing consortia (e.g., Lachnospiraceae and Bacteroides) and increasing intestinal SCFA concentrations (e.g., propionic and butyric acids recovering to 70.13 and 26.07 μM/g, respectively)). This microbial-metabolic shift was accompanied by upregulated expression of mucosal tight junction genes (ZO-1, Occludin). Furthermore, L. fermentum GR-3 facilitated a balanced immune response. Systemically, it alleviated oxidative stress and buffered peripheral inflammatory signals, contributing to immune homeostasis in non-tumor tissues (spleen and colon). Collectively, these findings position L. fermentum GR-3 as a grounded, translatable strategy to simultaneously enhance ICB efficacy and improve therapeutic tolerability.