Purpose: Laminin a5 (Lama5) is associated with immunity and regulates T cell entry and responses within the lymph node (LN). We hypothesized that stromal Lama5 depletion would impair T cell activation in the LN, thereby suppressing allograft rejection.
Methods: Lama5 conditional KO (KO) mice with depletion of Lama5 in LN stromal cells and wild type littermate control (WT) received BALB/c donor-specific splenocytes (DST), or DST plus anti-CD40L mAb for immunity and tolerance induction, respectively. T cell receptor transgenic CD4+ TEa cells recognizing donor I-Ed presented by recipient I-Ab (Ea antigen) were transferred into recipients; and TEa cell migration and differentiation were measured. BALB/c donor hearts were transplanted to WT and KO mice, and graft survival was measured.
Results: Lama5 KO LN had greater numbers of Treg in the T cell zones compared to WT. More dendritic cells were also detected in the KO cortical ridge (CR) and around high endothelial venules (HEV). CCL19, CCL21 and CXCL12 transcripts were increased in KO fibroblastic reticular cells (FRC); and CXCL12 and CCL21 protein expression were increased in the CR and around HEV, respectively. ICAM-1 and VCAM-1 transcripts but not MAdCAM-1 were increased in KO FRC; and VCAM-1 protein was increased in CR and HEV. More antigen specific TEa cells migrated into KO LN under both immune and tolerant conditions, and these cells localized to the CR and around the HEV. TEa cell activation to CD44hiCD69+ cells was lower in KO LN, indicating Lama5 depletion prevented T cell activation. Under both tolerance and immune conditions, TEa cell differentiation toward Foxp3+ Treg and IL-17+ Th17 was altered, leading to a higher Foxp3:Th17 ratio in KO than WT. Low dose tacrolimus (2 mg/kg/d) treated KO cardiac allograft recipients had significantly prolonged graft survival (mean survival time (MST) 89 days vs 27.5 days in WT, p<.002). Similarly, low dose anti-CD40L mAb treated KO recipients also had prolonged allograft survival (MST 155 vs 91 days, p=0.07). Blocking Lama5 receptor anti-a6 integrin or anti-aDG with mAbs had additive effects to genetic depletion of Lama5. With low dose tacrolimus, graft survival was prolonged with anti-a6 integrin (MST from 28.5 to 67.5 days, P<0.005) and anti-aDG (MST from 19 to 63.5 days, p<0.001).
Conclusions: Depleting stromal Lama5 promoted antigen-specific CD4 T cell migration to the LN cortical ridge, suppressed T cell activation, and channeled T cell differentiation from inflammatory to suppressive regulatory phenotypes. Depleting stromal Lama5 also promoted Treg induction and inhibited Th17 differentiation, creating a tolerogenic niche to enhance cardiac allograft survival. Targeting Lama5 on stromal cells of LN could lay the groundwork for establishing innovative tolerogenic approaches to improve cardiac graft survival.