*Purpose: Fibroblastic reticular cells (FRC) support lymph node (LN) structure and can regulate T cell function through the CD40-CD40L pathway. Our previous work showed that disrupting the interaction between CD40 on FRCs and CD40L on T cells alters immunity by preventing T cell communication with FRCs. We hypothesized that depleting FRC CD40 will affect the FRC regulatory effects on leukocytes in LNs.

*Methods: We created an FRC-CD40 knockout (KO) by crossing CD40^flox/flox with Pdgfrb-Cre mice to generate CD40 KO in LN FRCs. Immunohistochemistry and flow cytometry were used to analyze stromal cells and immune cells.

*Results: Compared to wildtype littermate controls (WT), CD40KO mice had larger LNs with a trend for greater numbers of CD4 T cells, CD8 T cells, B cells, dendritic (DCs), and macrophages and a statistically significant increase in the percentage of CD4 T cells in the LN. CD40 KO mice had increased CD40L expression on LN CD4 T cells, CD8 T cells, and B cells, suggesting that these subsets normally interact with FRC CD40. Thymic CD4 T cells also had increased CD40L expression after depleting FRC-CD40. CD40 KO mice had fewer total Tregs in the LNs, but greater total Tregs in the thymus, suggesting changes in maturation and migration. There were also fewer thymic derived Tregs (Foxp3+ Helios+) and more peripherally induced Tregs (Foxp3+ Helios-) in LNs and more exTregs (Foxp3+ CD25- ) in LNs in CD40 KO compared to WT. Immunohistochemistry showed that Foxp3 Tregs were decreased in all regions of the LN in CD40 KO compared to WT. By flow cytometry, CD40 KO mice had increased CD80 expression in LN conventional cDCs and increased MHC II in thymic cDCs, suggesting DC activation. Immunohistochemistry showed an overall decrease in the number of cDCs around the high endothelial venules (HEVs) and cortical ridge of CD40 KO mice compared to WT, although plasmacytoid DCs (pDCs) were unchanged. CD40 KO mice had less podoplanin and CD31 around the high endothelial venules (HEV) and the cortical ridge (CR) of the LN, suggesting altered maturation and function of the HEV.

*Conclusions: These results showed that depleting FRC-CD40 affects homeostasis in the LN niche. FRC-derived CD40 signaling maintains LN homeostatic structure in HEVs and contributes to the LN cellularity and positioning, including CD4 T cells and Tregs. Moreover, homeostatic activation of T and B lymphocytes and DCs are also affected by FRC-CD40, suggesting CD40 in FRCs is a potential target for modulating T and B cell responses.

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