Intratumoral Lactobacillus johnsonii Enhances Sensitivity to PD-1 Blockade by Inducing CD8+ T-cell Expansion in Hepatocellular Carcinoma

Although surgical resection is an effective intervention for early-stage hepatocellular carcinoma (HCC), postoperative recurrence remains a major clinical hurdle. Delving into the mechanisms underlying relapse and pinpointing potential therapeutic targets are imperative for improving outcomes for patients with HCC. By comparing the microbiota composition in patients with early- and non-relapsing HCC, we identified that Lactobacillus was enriched in patients with relapse-free HCC, serving as an independent prognostic predictor of disease-free survival. Higher levels of intratumoral Lactobacillus johnsonii correlated with an increased abundance of IFNγ+PD-1+CD8+ T cells. Single-cell RNA sequencing, transcriptomic profiling of intratumoral CD45+ immune cells, and in vitro functional assays demonstrated that L. johnsonii preferentially enhanced this cytotoxic-exhausted T-cell population. Nicotinic acid (NA) served as a key metabolite derived from L. johnsonii that expanded IFNγ+PD-1+CD8+ T cells and upregulated effector (granzyme B) and exhaustion (CTLA4) markers. Mechanistically, both L. johnsonii and NA activated the NF-κB pathway, leading to increased IFNγ production and upregulation of the transcription factor NR4A2, which in turn sustained PD-1 expression on CD8+ T cells. Combining L. johnsonii or NA with anti-PD-1 therapy synergistically inhibited tumor relapse and tumor growth in immunocompetent or humanized mice. Crucially, the antitumor efficacy of L. johnsonii was CD8+ T cell-dependent, as depletion abolished its activity. This work unveils a mechanism by which L. johnsonii and its metabolite NA enrich intratumoral IFNγ+PD-1+CD8+ T cells, thereby reshaping the immune microenvironment to potentiate immunotherapy efficacy and suppress HCC recurrence. SIGNIFICANCE: Lactobacillus johnsonii produces nicotinic acid that expands intratumoral IFNγ+PD-1+CD8+ T cells, which prevents hepatocellular carcinoma relapse and enhances the efficacy of anti-PD-1 therapy.