*Purpose: Lymph node (LN) stromal laminin α4 (Lama4) and α5 (Lama5) are associated with tolerance and immunity, respectively. We created a Lama4 conditional knockout (KO) mouse to investigate the role of Lama4 in LN stromal cells, hypothesizing that this model would be pro-inflammatory and complement our pro-tolerogenic Lama5 KO mouse.

*Methods: Loxp sites were inserted into the Lama4 gene using CRISPR/Cas9 and confirmed by genotyping and sequencing. Lama4^flox/flox mice were crossed with Pdgfrb-cre mice to create a conditional KO in LN stromal cells. Stromal cells, lymphocytes, and chemokines were analyzed by immunohistochemistry and flow cytometry.

*Results: Lama4 mRNA transcripts were significantly depleted in KO fibroblastic reticular cells (FRC), while blood endothelial cells (BEC) and lymphatic endothelial cells (LEC) were unaffected. Immunohistochemistry showed a marked decrease in the expression Lama4 protein around the high endothelial venules (HEV) and the cortical ridge (CR) of the LN, associated with a decreased Lama4:Lama5 protein ratio compared to wild-type (WT) controls. CCL21, CCL19, and CXCL12 mRNA transcripts were downregulated in KO FRC, and immunohistochemistry confirmed decreased CXCL12 and CCL21 around the HEV and CR. There was also decreased expression of VCAM-1 and ICAM-1 in the CR. Lama4 depletion did not affect the percentage of CD4, CD8, B cells, or dendritic cells in the LN and spleen. Flow cytometry also did not indicate a change in the total percentage of regulatory T cells (Treg) in LNs, but immunohistochemistry showed microanatomic domain differences in the distribution of Treg between WT and KO. In particular, Foxp3+ Treg were decreased in the T cell zone, CR, and HEV of KO compared to WT controls.

*Conclusions: The Lama4 conditional KO model effectively depleted Lama4 in FRC without affecting other stromal cell subsets, organogenesis, and overall health. Lama4 depletion made remarkable alterations to chemokines and cell adhesion molecules, which may impact immune cell trafficking into the LNs. The current study confirmed that the Lama4 KO mice are inverse to and consistent with our Lama5 KO mice, making this an ideal model to precisely delineate the roles of laminins in the alloimmune response.

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