Sphingosine 1-phosphate engages multiple T cell and lymphatic endothelial cell receptors to regulate lymphocyte migration.

Programmed death-1 (PD-1) and its ligand PD-L1 are checkpoint molecules which regulate immune responses. Little is known about their functions in T cell migration and there are contradictory data about their roles in regulatory T cell (Treg) function. Here we show activated Tregs and CD4 effector T cells (Teffs) use PD-1/PD-L1 and CD80/PD-L1, respectively, to regulate transendothelial migration across lymphatic endothelial cells (LECs). Antibody blockade of Treg PD-1, Teff CD80 (the alternative ligand for PD-L1), or LEC PD-L1 impairs Treg or Teff migration in vitro and in vivo. PD-1/PD-L1 signals through PI3K/Akt and ERK to regulate zipper junctional VE-cadherin, and through NFκB-p65 to up-regulate VCAM-1 expression on LECs. CD80/PD-L1 signaling up-regulates VCAM-1 through ERK and NFκB-p65. PD-1 and CD80 blockade reduces tumor egress of PD-1^high fragile Tregs and Teffs into draining lymph nodes, respectively, and promotes tumor regression. These data provide roles for PD-L1 in cell migration and immune regulation.

Lymph node (LN) fibroblastic reticular cells (FRCs) define LN niches and regulate lymphocyte homeostasis through producing diverse extracellular matrix (ECM) components. We examined the role of ECM laminin α4 (Lama4) using FRC-Lama4 conditional KO Pdgfrb-Cre^–/– × Lama4^fl/fl mice. Single-cell RNA-sequencing (scRNA-Seq) data showed the promoter gene Pdgfrb was exclusively expressed in FRCs. Depleting FRC-Lama4 reduced Tregs and dendritic cells, decreased high endothelial venules, impaired the conduit system, and downregulated T cell survival factors in LNs. FRC-Lama4 depletion impaired the homing of lymphocytes to LNs in homeostasis and after allografting. Alloantigen-specific T cells proliferated, were activated to greater degrees in LNs lacking FRC-Lama4, and were more prone to differentiate into effector phenotypes relative to the Treg phenotype. In murine cardiac transplantation, tolerogenic immunosuppression was not effective in FRC-Lama4 recipients, which produced more alloantibodies than WT. After lung transplantation, FRC-Lama4–KO mice had more severe graft rejection with fewer Tregs in their LNs. Overall, FRC-Lama4 critically contributes to a tolerogenic LN niche by supporting T cell migration, constraining T cell activation and proliferation, and promoting Treg differentiation. Hence, it serves as a therapeutic target for immunoengineering.

Lymph node stromal cells (LNSCs) regulate immunity through constructing lymphocyte niches. LNSC-produced laminin α5 (Lama5) regulates CD4⁺ T cells but the underlying mechanisms of its functions are poorly understood. Here we show that depleting Lama5 in LNSCs resulted in decreased Lama5 protein in the LN cortical ridge (CR) and around high endothelial venules (HEVs). Lama5 depletion affected LN structure with increased HEVs, upregulated chemokines, and cell adhesion molecules, and led to greater numbers of Tregs in the T cell zone. Mouse and human T cell transendothelial migration and T cell entry into LNs were suppressed by Lama5 through the receptors α6 integrin and α-dystroglycan. During immune responses and allograft transplantation, depleting Lama5 promoted antigen-specific CD4⁺ T cell entry into the CR through HEVs, suppressed T cell activation, and altered T cell differentiation to suppressive regulatory phenotypes. Enhanced allograft acceptance resulted from depleting Lama5 or blockade of T cell Lama5 receptors. Lama5 and Lama4/Lama5 ratios in allografts were associated with the rejection severity. Overall, our results demonstrated that stromal Lama5 regulated immune responses through altering LN structures and T cell behaviors. This study delineated a stromal Lama5–T cell receptor axis that can be targeted for immune tolerance modulation.

The pleiotropic functions of lymphotoxin (LT)β receptor (LTβR) signaling are linked to the control of secondary lymphoid organ development and structural maintenance, inflammatory or autoimmune disorders, and carcinogenesis. Recently, LTβR signaling in endothelial cells has been revealed to regulate immune cell migration. Signaling through LTβR is comprised of both the canonical and non-canonical-nuclear factor κB (NF-κB) pathways, which induce chemokines, cytokines, and cell adhesion molecules. Here, we focus on the novel functions of LTβR signaling in lymphatic endothelial cells for migration of regulatory T cells (Tregs), and specific targeting of LTβR signaling for potential therapeutics in transplantation and cancer patient survival.