Lymph node fibroblastic reticular cells preserve a tolerogenic niche in allograft transplantation through laminin α4
Lushen Li, Marina W. Shirkey, Tianshu Zhang, Wenji Piao, Xiaofei Li, Jing Zhao, Zhongcheng Mei, Yizhan Guo, Vikas Saxena, Allison Kensiski, Samuel J. Gavzy, Yang Song, Bing Ma, Jing Wu, Yanbao Xiong, Long Wu, Xiaoxuan Fan, Holly Roussey, Meng Li, Alexæander S. Krupnick, Reza Abdi, Jonathan S. Bromberg
Lushen Li, Marina W. Shirkey, Tianshu Zhang, Wenji Piao, Xiaofei Li, Jing Zhao, Zhongcheng Mei, Yizhan Guo, Vikas Saxena, Allison Kensiski, Samuel J. Gavzy, Yang Song, Bing Ma, Jing Wu, Yanbao Xiong, Long Wu, Xiaoxuan Fan, Holly Roussey, Meng Li, Alexæander S. Krupnick, Reza Abdi, Jonathan S. Bromberg
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Research Article Cell biology

Lymph node fibroblastic reticular cells preserve a tolerogenic niche in allograft transplantation through laminin α4

Abstract

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–/– × Lama4fl/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.

Authors

Lushen Li, Marina W. Shirkey, Tianshu Zhang, Wenji Piao, Xiaofei Li, Jing Zhao, Zhongcheng Mei, Yizhan Guo, Vikas Saxena, Allison Kensiski, Samuel J. Gavzy, Yang Song, Bing Ma, Jing Wu, Yanbao Xiong, Long Wu, Xiaoxuan Fan, Holly Roussey, Meng Li, Alexæander S. Krupnick, Reza Abdi, Jonathan S. Bromberg

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Figure 6

FRC-Lama4 depletion affects lymphocyte entry into LNs.

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FRC-Lama4 depletion affects lymphocyte entry into LNs. (A) 107 CD45.1+ s...
(A) 107 CD45.1+ splenocytes transferred i.v. into CD45.2+ WT and FRC-Lama4–KO recipients. After 1 hour, LNs were harvested and total migrated CD45.1+ cells, and CD45.1+ CD4+ T, CD8+ T cells, B cells, cDCs, pDCs, and Foxp3-GFP+ tTregs were counted in each LN. Gating strategy (upper) and data summary (lower) of migrated cells. (B and C) 2 × 106 CFSE+ iTregs and 2 × 106 eFlour 670+ CD4+ T cells transferred i.v. to FRC-Lama4–KO and WT mice. After 16 hours, LNs were harvested and transferred cells measured. (B) Flow cytometry gating strategy (left, values show percentage); number of transferred naive CD4+ T cells and iTregs relative to 106 total CD4+ T cells in LNs. (C) LN cryosections for CD4+ and iTregs and ER-TR7. Original magnification, ×20. Scale bar: 100 μm. Quantification of naive CD4+ T cells and iTregs in CR and HEV. (D) 107 CD45.1+ splenocytes transferred i.v. into CD45.2+ WT and FRC-Lama4–KO recipients. Eighteen hours later, recipients received 100 μg anti-CD62L mAb or isotype i.v. After an additional 18 hours, transferred cells in LNs were analyzed. Gating strategy (upper) and data summary (lower) of migrated cell frequency in recipient LNs. (A–D) Values in gating strategy show percentages. Representative of 3 independent experiments with 3 mice/group, 5 LNs/mouse, 3 sections/LN, and 3 to 5 fields/section. Student’s unpaired 2-tailed t test for 2-group comparisons. Two-way ANOVA with multiple comparisons test. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. P < 0.05 was considered significant.