Effector Cytokines in Mucosally-Primed Lymphocytes Specific for Commensals and Pathogens

Protection against infection requires memory T helper cells supporting either cell mediated immunity or immunoglobulin production. We hypothesized that naive CD4+ T cells, primed transmucosally by either commensal or invasive microorganisms, proliferate and differentiate into memory T cells with distinct patterns of cytokine expression. Objectives: Determine whether intranasal inoculation of Lactobacillus murinus or Streptococcus pyogenes, both expressing the T cell epitope ovalbumin323-339, induce clonal expansion and differential cytokine expression in adoptively transferred ovalbumin-specific TCR-transgenic T cells. Methods: Peripheral lymph node and spleen lymphocytes (10^6) from TCR-transgenic DO11.10 donor mice stained with the vital dye CFSE were transferred intravenously into recipient BALB/c mice. OVA+streptococci (1.5X10^8) were inoculated intranasally once, whereas 4x10^9 OVA+lactobacilli were applied daily for 14 days. Spleen mononuclear cells (10^7/mL/well) were stimulated with either PBS, 10µg/mL ConcanavalinA or 20µg/mL ovalbumin323-339 for 3 hours at 37C. Washed cells were incubated with a bi-specific mAb recognizing anti-CD45 on leukocytes and the other specificity recognizing either IL-2, IFNγ, IL-10 or IL-4. These cytokines, released by ovalbumin–specific T cells, were then detected by a second PE-labeled anti-cytokine mAb in sandwich. Ovalbumin-specific T cells were analyzed by FACS after enrichment by positive selection with streptavidin-magnetic beads and a biotinylated anti-clonotypic TCR mAb recognizing the ovalbumin-specific TCR. Results: Compared to L. murinus, S. pyogenes priming induced higher proliferation in ovalbumin-specific T cell (P=0.03), similar number of IL-2-releasing (P=0.32) and higher number of IFNg-releasing (P=0.02) memory T cells when rechallenged in vitro with ovalbumin323-339. Additionally, L. murinus priming reduced the number of IFNγ-releasing ovalbumin-specific and endogenous T cells (P<0.05) after activation with ConcanavalinA. Conclusion: Unlike pathogens, commensals appear to preferentially induce regulatory cells rather than stimulating mucosal T cells to generate IFNγ-producing memory cells. Without mature mucosal anti-commensal memory T cells commensal microorganisms may be enabled to persist in their ecological niche. NIH/NIDCR supported DE014371.