Dendritic cells (DCs) are central regulators of innate and adaptive immunity and are classified into conventional DCs (cDCs) and plasmacytoid DCs (pDCs). The study tested whether two lactic acid bacterial strains—Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (OLL1073R-1) and Streptococcus thermophilus OLS3059 (OLS3059)—activate human DC subsets when added to peripheral blood mononuclear cells (PBMCs).
PBMCs were incubated with heat-treated bacteria (1 × 10^6 cells/mL) for 24 hours. Flow cytometry measured expression of activation markers CD86, CD40 and HLA-DR on gated cDC and pDC fractions. Both OLL1073R-1 and OLS3059 significantly increased CD86, CD40 and HLA-DR expression in pDCs, while antigen-presentation markers on cDCs did not show the same increase. Given prior reports that regulatory T cells can suppress cDC surface marker expression in mixed cultures, intracellular IL-12 staining was used as an alternative cDC activation readout; both strains significantly increased the proportion of IL-12–expressing cDCs. These results indicate that OLL1073R-1 and OLS3059 activate both DC subsets in PBMC cultures.
To determine whether activation is direct or mediated by other PBMC components, cDCs and pDCs were differentiated from human cord blood CD34(+) hematopoietic progenitor cells, sorted, and stimulated with OLL1073R-1. After 24 hours, both isolated cDCs and pDCs showed significant upregulation of CD86 and CD40; HLA-DR increased significantly in pDCs and trended upward in cDCs. The fraction of cells expressing these markers was elevated. These data demonstrate that OLL1073R-1 can directly activate human cDCs and pDCs independent of other immune cell types.
To capture early gene expression changes, mRNA-seq was performed on isolated cDCs and pDCs after 6 hours of OLL1073R-1 stimulation. Differential expression analysis identified 411 upregulated and 261 downregulated genes in cDCs, and 387 upregulated and 192 downregulated genes in pDCs (criteria: |log2 fold change| > 0.5 and p < 0.05). Enrichment analyses of upregulated genes in each subset highlighted immune-related pathways and regulation of cellular processes, indicating that OLL1073R-1 triggers transcriptional programs consistent with activation and immune signaling in both DC subsets.
The investigators examined the contribution of each DC subset to cytokine release and T cell activation by depleting cDCs or pDCs from PBMCs prior to stimulation. PBMCs, cDC-depleted PBMCs (CD11c–), and pDC-depleted PBMCs (CD304–) were stimulated with OLL1073R-1 with or without influenza A (H1N1) antigen. Key observations:
Type I interferons (IFN-α2a and IFN-β) induced by OLL1073R-1 required both cDCs and pDCs; depletion of either subset reduced production. This indicates cooperative roles of cDCs and pDCs in type I IFN responses to the bacterial stimulus.
Enhancement of effector-memory CD4+ T cell (CD4+ TEM) IFN-γ responses to influenza antigen was dependent on cDCs but not pDCs when cultures were stimulated with OLL1073R-1, implying a dominant role for cDCs in promoting antigen-specific T cell effector function.
IL-12 measured in culture supernatants and intracellular IL-12 staining in cDCs supported the importance of cDC-derived IL-12 in shaping T helper responses.
These functional assays link DC activation by OLL1073R-1 to biologically relevant antiviral immune outputs, including type I interferons and enhanced CD4+ T cell responses to a viral antigen.
Phagocytic uptake of OLL1073R-1 by DCs in mixed PBMC cultures was assessed using pH-sensitive labeling of bacteria and flow cytometry. Labeled OLL1073R-1 was detected within DC fractions after 24 hours, consistent with bacterial uptake by both cDCs and pDCs. Cytokine quantification in supernatants used ELISA for IL-12 and S-PLEX assays for IFN-α2a and IFN-β; assay minimum detection limits were reported. Experimental results showed measurable increases in these cytokines following bacterial stimulation, supporting the flow cytometry and transcriptomic findings.
OLL1073R-1 and OLS3059 were cultured under strain-appropriate conditions, washed, heat-treated at 75°C for 1 hour, and stored at −80°C prior to use. Bacteria counts were determined from suspensions prior to heat treatment. PBMC stimulations used RPMI 1640 with 10% fetal bovine serum at 37°C and 5% CO2. cDCs and pDCs were differentiated from human cord blood CD34(+) progenitors over three weeks with cytokines including IL-3, thrombopoietin and Flt3-L, then sorted for isolation. Flow cytometry panels included lineage markers, HLA-DR, CD11c, CD123, CD304, CD86, CD40 and intracellular cytokine stains. mRNA-seq libraries were prepared and sequenced on Illumina platforms and analyzed using standard pipelines; differentially expressed genes were defined by log2 fold change and p-value thresholds.
Normality was tested by Shapiro-Wilk. Comparisons used Student’s t-test or Dunnett’s test for normally distributed data and Mann-Whitney U test for non-normal data. All tests were two-sided with p < 0.05 considered significant. Individual plots represent cells derived from single donors. The authors interpreted concordant phenotypic, transcriptional and functional data to support that both OLL1073R-1 and OLS3059 activate human DC subsets and thereby enhance antiviral immune responses.
The study was funded by Meiji Holdings Co., Ltd., Japan, and the authors are employees of Meiji Holdings Co., Ltd. The authors declared no other competing interests. All relevant data are reported within the manuscript and its Supporting Information files.
Heat-treated strains OLL1073R-1 and OLS3059 directly activate human cDCs and pDCs, trigger transcriptional programs related to immune regulation, and promote production of type I interferons and IL-12. cDCs are essential for enhanced effector-memory CD4+ T cell responses to viral antigen, while both DC subsets contribute to type I IFN production. These mechanisms provide a cellular basis for previously observed antiviral effects associated with consumption of yogurt fermented with these strains.