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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.

Despite ongoing improvements in regimens to prevent allograft rejection, most cardiac and other types of allografts eventually succumb to chronic vasculopathy, interstitial fibrosis, or endothelial changes, and eventually graft failure. Adaptive immune-mediated damage is considered the primary cause of long-term graft failure, though the events leading to chronic rejection are still poorly understood. Gut microbiota is a known driving force in immune dysfunction. We previously showed that gut microbiota dysbiosis profoundly influences the outcome of vascularized cardiac allografts and subsequently identified biomarker species or ″bacterial determinants″ associated with these differential graft outcomes (anti-inflammatoryBifidobacterium pseudolongum(Bifido) and pro-inflammatoryDesulfovibrio desulfuricans(Desulfo)). In this study, we further detailed the multifaceted immunomodulatory properties of the pro-tolerogenic bacterial species over time, using our clinically relevant model of allogenic heart transplantation. This model simultaneously accounts for the immunomodulatory properties of antibiotics and tacrolimus immunosuppression, in addition to bacterial determinants. We observed thatBifidoinduced an early anti-inflammatory phenotype within 7 days, whileDesulforesulted in a pro-inflammatory phenotype, defined by alterations in leukocyte distribution and lymph node (LN) structure. These effects were further modulated by inhibition of myeloid cell migration by CCR2 inhibition, supporting the notion that bacterial determinants regulate innate immunity which subsequently influences adaptive immunity. Indeed,in vitroresults showed thatBifidoandDesulfoacted directly on primary innate immune cells. However, by 40 days after treatment, these two bacterial determinants were associated with mixed effects in their impact on LN architecture and immune cell composition. These dynamic effects suggest a critical role for early bacterial determinant-triggered immunological events such as innate immune cell engagement and LN architectural changes in the subsequent modulation of pro-tolerant versus pro-inflammatory immune responses in organ transplant recipients.

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