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Programmed death-1 (PD-1) and its ligand PD-L1 are checkpoint molecules which regulate immune responses. Little is known about their functions in T cell migration and there are contradictory data about their roles in regulatory T cell (Treg) function. Here we show activated Tregs and CD4 effector T cells (Teffs) use PD-1/PD-L1 and CD80/PD-L1, respectively, to regulate transendothelial migration across lymphatic endothelial cells (LECs). Antibody blockade of Treg PD-1, Teff CD80 (the alternative ligand for PD-L1), or LEC PD-L1 impairs Treg or Teff migration in vitro and in vivo. PD-1/PD-L1 signals through PI3K/Akt and ERK to regulate zipper junctional VE-cadherin, and through NFκB-p65 to up-regulate VCAM-1 expression on LECs. CD80/PD-L1 signaling up-regulates VCAM-1 through ERK and NFκB-p65. PD-1 and CD80 blockade reduces tumor egress of PD-1high fragile Tregs and Teffs into draining lymph nodes, respectively, and promotes tumor regression. These data provide roles for PD-L1 in cell migration and immune regulation.

The beneficial effects attributed to Bifidobacterium are largely attributed to their immunomodulatory capabilities, which are likely to be species- and even strain-specific. However, their strain-specificity in direct and indirect immune modulation remain largely uncharacterized. We have shown that B. pseudolongum UMB-MBP-01, a murine isolate strain, is capable of suppressing inflammation and reducing fibrosis in vivo. To ascertain the mechanism driving this activity and to determine if it is specific to UMB-MBP-01, we compared it to a porcine tropic strain B. pseudolongum ATCC25526 using a combination of cell culture and in vivo experimentation and comparative genomics approaches. Despite many shared features, we demonstrate that these two strains possess distinct genetic repertoires in carbohydrate assimilation, differential activation signatures and cytokine responses signatures in innate immune cells, and differential effects on lymph node morphology with unique local and systemic leukocyte distribution. Importantly, the administration of each B. pseudolongum strain resulted in major divergence in the structure, composition, and function of gut microbiota. This was accompanied by markedly different changes in intestinal transcriptional activities, suggesting strain-specific modulation of the endogenous gut microbiota as a key to immune modulatory host responses. Our study demonstrated a single probiotic strain can influence local, regional, and systemic immunity through both innate and adaptive pathways in a strain-specific manner. It highlights the importance to investigate both the endogenous gut microbiome and the intestinal responses in response to probiotic supplementation, which underpins the mechanisms through which the probiotic strains drive the strain-specific effect to impact health outcomes.

Fibroblastic reticular cells (FRCs) play important roles in tolerance by producing laminin α4 (Lama4) and altering lymph node (LN) structure and function. The present study revealed the specific roles of extracellular matrix Lama4 in regulating LN conduits using FRC-specific KO mouse strains. FRC-derived Lama4 maintained conduit fiber integrity, as its depletion altered conduit morphology and structure and reduced homeostatic conduit flow. Lama4 regulated the lymphotoxin β receptor (LTβR) pathway, which is critical for conduit and LN integrity. Depleting LTβR in FRCs further reduced conduits and impaired reticular fibers. Lama4 was indispensable for FRC generation and survival, as FRCs lacking Lama4 displayed reduced proliferation but upregulated senescence and apoptosis. During acute immunization, FRC Lama4 deficiency increased antigen flow through conduits. Importantly, adoptive transfer of WT FRCs to FRC Lama4–deficient mice rescued conduit structure, ameliorated Treg and chemokine distribution, and restored transplant allograft acceptance, which were all impaired by FRC Lama4 depletion. Single-cell RNA sequencing analysis of LN stromal cells indicated that the laminin and collagen signaling pathways linked crosstalk among FRC subsets and endothelial cells. This study demonstrated that FRC Lama4 is responsible for maintaining conduits by FRCs and can be harnessed to potentiate FRC-based immunomodulation.