The human gut symbiont Lacticaseibacillus (L.) casei (previously Lactobacillus casei) is under intense research due to its wide range of immunomodulatory effects on the human host. Dendritic cells (DCs) are crucial players in the direct and indirect communication with lactobacilli in the gastrointestinal tract. Here, we demonstrate that human monocyte-derived DCs (moDCs) are able to engulf L. casei BL23, in which the intact bacterial cell wall and morphology have a key role. The absence of the bacterial cell-wall-degrading enzyme, Lc-p75, in L. casei cells causes remarkable morphological changes, which have important consequences in the phagocytosis of L. casei by moDCs. Our results showed that the Lc-p75 mutation induced defective internalization and impaired proinflammatory and T-cell-polarizing cytokine secretion by bacteria-exposed moDCs. The T helper (Th) 1 and Th17 cell activating capacity of moDCs induced by the mutant L. casei was consequently reduced. Moreover, inhibition of the phagocytosis of wild-type bacteria showed similar results. Taken together, these data suggested that formation of short bacterial chains helps to exert the potent immunomodulatory properties of L. casei BL23.

Immune responses are highly complex and intricately regulated processes involving immune and non-immune cells in close direct and indirect contact with each other. These cells are highly sensitive to environmental signals, including factors derived from microbiota. Here, we demonstrate that the human microbiota member Lactobacillus casei (L. casei)-derived cell-free supernatant (CFS) enhances the sensitivity of mesenchymal-stromal-cell-like (MSCI) cells to viral stimuli and induces the development of dendritic cells (DCs) with anti-inflammatory and antiviral properties via pretreated MSCl cells. Our results showed that the production of INFβ and CXCL10 by MSCl cells upon viral stimulation was dependent on the presence of L. casei-derived extracellular vesicles in CFS during pretreatment. Moreover, L. casei CFS and/or poly (I:C)-conditioned MSCI cells altered the differentiation process of freshly isolated monocytes, as well as the developing DCs’ phenotype and functional activities, such as cytokine and chemokine secretion. Taken together, L. casei CFS contains factors which contribute to the pronounced antiviral response of MSCI cells, avoiding the development of inflammation via the induction of differentiation of anti-inflammatory DCs that retain their antiviral properties.

Partially preserved function of recombination-activating genes (RAG) 1 and 2 can be linked to divergent and hard-to-predict clinical and immunological phenotypes, even among close relatives. It is unclear how intrinsic RAG hypomorphism interacts with extrinsic environmental pressures to shape clinical trajectory. We dissect this phenomenon in a unique cohort of 18 Old Order Mennonite individuals with a novel missense founder RAG1 variant (p.C176F) with 25.8% recombinase activity of wild-type protein. After studying a myriad of markers, we demonstrate varying severity and progression of clinical disease (predominantly in females) that paralleled decline in CMV-specific immune competence, break in tolerance with hallmark presence of antibodies to IFNα, a TH1/IFNγ-driven inflammatory milieu with elevated serum markers (CXCL9/10), expansion of follicular helper T cells and clonal B cells enriched for autoreactive-prone VH4.34 B cell receptor. BCR and TCR repertoires carry the fingerprints of both extrinsic and RAG-driven intrinsic changes. Through this ‘experiment of nature’, our cohort demonstrated that partial RAG deficiency (pRD) is not a monolithic disease but the result of compounded RAG intrinsic and extrinsic events. Overall, this study uses multi-omics approaches to dissect the contribution of a hypomorphic RAG variant to variable severity of clinical disease and immunological dysregulation at cellular and molecular level. Emerging biomarkers of a multi-omic roadmap should allow monitoring of progression of clinical disease and guide who and when to treat with hematopoietic stem cell transplant in pRD. One sentence summary: A unique cohort of pediatric and adult US Mennonite patients with shared novel partial RAG defect and variable penetrance of disease allow correlation of clinical severity with RAG-intrinsic and extrinsic factors driving plasticity in T and B cell competence to eliminate infections and tolerance to self.