Lactobacillus johnsonii N6.2 Phospholipids Induce T Cell Anergy upon Cognate Dendritic Cell Interactions

<b>Background/Objectives</b>: <i>Lactobacillus johnsonii</i> N6.2 is a gut symbiont with probiotic properties. <i>L. johnsonii</i> N6.2 delayed the progression of type 1 diabetes (T1D) in diabetic-prone rats. The probiotic intake demonstrated immune cell modulation in healthy volunteers, leading to improved wellness and fewer reported symptoms like headaches and abdominal pain. These systemic immune-modulating benefits are attributed to <i>L. johnsonii</i> N6.2's bioactive fractions, including extracellular vesicles (EVs) and purified phospholipids (PLs). We have previously shown that <i>L. johnsonii</i> N6.2 PLs modulate dendritic cell (DC) function towards a regulatory-like phenotype. Here, we further characterize the immune regulatory effects of <i>L. johnsonii</i> N6.2 PLs on adaptive immunity, specifically upon DC and T cell interactions. We hypothesized that PL-stimulated DCs suppress T cell-mediated responses to maintain tolerance in intra- and extra-intestinal sites. <b>Methods</b>: Bone marrow-derived dendritic cells (BMDCs) were generated from Sprague-Dawley rats and stimulated with <i>L. johnsonii</i> N6.2 PLs. Isogenic T cells were isolated from PBMCs obtained via terminal exsanguination. In vitro cellular assays, co-culture experiments, gene expression analysis by qRT-PCR, and flow cytometry assays were conducted to assess the immune regulatory effects of <i>L. johnsonii</i> N6.2 PLs. <b>Results</b>: The PL-stimulated BMDCs upregulated DC regulatory markers and exhibited an immature-like phenotype with reduced surface expression of maturation markers but increased surface migratory molecules (ICAM-1). These BMDCs presented immunosuppressive functions upon cognate T cell interactions and in the presence of TCR stimulation. Specifically, PL-stimulated BMCDs suppressed Th1 effector function and induced the expression of T cell anergy-related genes after co-culturing for 72 h. <b>Conclusions</b>: This study highlights the immune regulatory capacity of <i>L. johnsonii</i> N6.2's bioactive components on adaptive immunity, specifically that of purified PLs on DC:T cell-mediated responses leading to immunosuppression. Our findings suggest that <i>L. johnsonii</i> N6.2-purified PLs play a role in regulating adaptive immunity, offering potential benefits for managing immune-related diseases like T1D.