Fibroblastic FLT3L supports lymph node dendritic cells in the interfollicular niche

Dendritic cell (DC) homeostasis is maintained in secondary lymphoid organs (SLOs) by Fms-like tyrosine kinase 3 ligand (FLT3L). The specific niche providing this DC growth factor within human and mouse SLOs is unclear. Here, we show that Gremlin1 (GREM1)-expressing lymph node fibroblastic reticular cells (FRCs) support DC homeostasis via provision of FLT3L. Grem1-expressing FRCs co-localize with DCs and express FLT3L/Flt3l in human and mouse lymph nodes. Using a new genetic model, we provide evidence that FLT3L produced by GREM1+ FRCs maintains lymph node DC precursors (preDCs), and both conventional (cDCs) and plasmacytoid DCs (pDCs). Spatial transcriptomics and cytofluorometry reveal that GREM1+ FRC-derived FLT3L supports not only proliferation, but also survival of lymph node preDCs and cDCs within the interfollicular zone (IFZ). Functionally, loss of GREM1+ FRC-derived FLT3L impairs cDC activation of antigen-specific T cell responses to both immunization and infection. These findings provide key mechanistic insights underlying stromal cell support of DC homeostasis and function.Dendritic cells were isolated by FACS from lymph nodes of 4x wild-type (Grem1 wt/ki Flt3l wt/wt) and 4x FLT3L floxed (Grem1 wt/ki Flt3l fl/fl) mice for single-cell profiling (total 8). Samples were indexed using TotalSeq-A hashtag antibodies (B0101-B0108). Stromal, myeloid and lymphoid cell compartments were isolated by FACS from lymph nodes of 4x wild-type (Grem1 wt/ki Flt3l wt/wt) and 4x FLT3L floxed (Grem1 wt/ki Flt3l fl/fl) for single-cell profiling (total 8). For spatial profiling, lymph nodes were isolated from 4x wild-type (Grem1 wt/ki Flt3l wt/wt) and 4x FLT3L floxed (Grem1 wt/ki Flt3l fl/fl) mice for spatial transcriptomics profiling using the Visium assay (10X Genomics).

树突状细胞（Dendritic cell, DC）稳态在次级淋巴器官（secondary lymphoid organs, SLOs）中由Fms样酪氨酸激酶3配体（Fms-like tyrosine kinase 3 ligand, FLT3L）维持。目前尚不明确人类与小鼠次级淋巴器官中提供该DC生长因子的特异性生态位。本研究证实，表达Gremlin1（GREM1）的淋巴结成纤维网状细胞（fibroblastic reticular cells, FRCs）可通过分泌FLT3L维持DC稳态。表达Grem1的FRCs与DC共定位，并在人类与小鼠淋巴结中表达FLT3L/Flt3l。本研究利用新型遗传模型，证实GREM1阳性FRC分泌的FLT3L可维持淋巴结DC前体细胞（preDCs）、经典DC（conventional cDCs）及浆细胞样DC（plasmacytoid DCs, pDCs）的稳态。空间转录组学与细胞荧光计数分析显示，GREM1阳性FRC来源的FLT3L不仅可促进滤泡间区（interfollicular zone, IFZ）内淋巴结DC前体细胞与经典DC的增殖，还能维持其存活。功能实验表明，缺失GREM1阳性FRC来源的FLT3L会削弱DC对免疫接种与感染引发的抗原特异性T细胞应答的激活能力。本研究为基质细胞支持DC稳态与功能的核心机制提供了关键见解。
本研究通过荧光激活细胞分选术（Fluorescence-Activated Cell Sorting, FACS）从4只野生型（Grem1 wt/ki Flt3l wt/wt）与4只FLT3L条件性敲除（Grem1 wt/ki Flt3l fl/fl）小鼠的淋巴结中分离树突状细胞，用于单细胞测序分析（总计8例样本）。样本采用TotalSeq-A标签抗体（B0101-B0108）进行索引标记。本研究同时通过FACS从上述基因型小鼠的淋巴结中分离基质细胞、髓系细胞与淋巴系细胞群体，用于单细胞测序分析（总计8例样本）。空间转录组分析部分：研究采用Visium检测技术（10X Genomics）对上述基因型小鼠的淋巴结开展空间转录组学分析。