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

While 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 HCC patient outcomes. By comparing the microbiota composition in patients with early-relapsing and non-relapsing HCC, we identified that Lactobacillus was enriched in relapse-free HCC patients, serving as an independent prognostic predictor of disease-free survival. Higher levels of intratumoral Lactobacillus johnsonii (L. johnsonii) correlated with an increased abundance of IFN-γ+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 IFN-γ+PD-1+CD8+ T cells and upregulated effector (GZMB) and exhaustion (CTLA-4) markers. Mechanistically, both L. johnsonii and NA activated the NF-κB pathway, leading to increased IFN-γ 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 anti-tumor 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 IFN-γ+PD-1+CD8+ T cells, thereby reshaping the immune microenvironment to potentiate immunotherapy efficacy and suppress HCC recurrence.