*Purpose: Fibroblastic reticular cells (FRCs) support lymph node (LN) structure by producing various functional extracellular matrix (ECM) components. We hypothesized that FRCs and their ECM laminin α4 (Lama4) contributes to the LN vasculature and conduit development and function.

*Methods: CCL19/iDTR mice with diphtheria toxin (DT)-inducible FRC depletion were used for investigating the contribution of FRCs to LN structure and cellularity. FRC derived Lama4 conditional KO (Lama4 KO) mice were used for studying the specific role of FRC-Lama4. Dextran-FITC (40 kD) was injected s.c. to visualize the conduit system in draining LNs (dLNs).

*Results: Depleting FRCs resulted in fewer and smaller high endothelial venules (HEVs) with decreased expression of peripheral node addressin (PNAd). Treg, CD3⁺, CD4⁺, and CD8⁺ T cell abundance all decreased in the LN cortical ridge (CR) after depleting FRCs. FRC depletion decreased CXCL12, CCL21, and IL-33 in the CR and around HEVs. Depleting FRC-Lama4 also decreased HEVs in size and numbers. Lama4 KO LNs had lower density of the conduits, which were also smaller and sparser than WT controls. Moreover, podoplanin (Pdpn) expression in Lama4 KO LN regions, including CR, HEV, T-zone, and medullary regions, was lower than WT, indicating a reduced reticular network. Lama4 KO LNs expressed lower levels of the mitotic marker Ki67 in the FRC-supported regions, suggesting decreased proliferation of FRCs. Lama4 KO FRCs expressed more p16 and β-galactosidase compared to WT, indicating that FRCs lacking Lama4 were more prone to senescence. The p16 signal was highly colocalized with the FRC fiber ER-TR7, suggesting the p16 signal is attributed to FRCs but not other cell types. Compared to WT, the Lama4 KO FRCs had fewer viable annexin V^–7-AAD^– (live) cells, but more annexin V⁺7-AAD^– (early apoptotic) and annexin V⁺7-AAD⁺ (late apoptotic) populations, indicating that FRCs lacking Lama4 were prone to apoptosis.

*Conclusions: FRC derived Lama4 is required for maintenance of LN structure, including HEVs, conduits, and T cell survival factors. FRCs and the Lama4 they produce play important roles in maintaining LN conduit numbers, density, size, and structure. FRC-Lama4 serves these functions through supporting FRC proliferation and inhibiting their senescence and apoptosis. Manipulation of FRC or their stromal fibers identifies novel ways to regulate immunity and tolerance.

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