Abstract
Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs) between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.
Highlights
While increased enzyme concentration was required to digest the lymph nodes, once digested, their stromal composition was markedly similar to mice. These results suggest that murine lymph node stromal cell techniques may be applicable to human studies
Histology and wholeorgan PCR, or in vivo systems involving lymphocytic readouts have been the field’s standard. These have lead to several important advances regarding the role of lymph node stromal cell subsets in steady-state tolerance and immunity; there are many unexplored questions of direct biological and medical relevance
In testing whether cross-talk between lymphocytes and stroma was required for the expansion of particular stromal subsets, we found that fibroblastic reticular cells (FRCs), lymphatic endothelial cells (LECs), Blood endothelial cells (BECs), and double negative” (DN) stroma were present at normal proportions in Rag−/− mice, showing no requirement for T or B cells in their development or expansion
Summary
Lymph nodes are important secondary lymphoid organs located at lymphatic intersections in the body, allowing efficient interaction between antigen-presenting cells and naïve T and B cells and promoting effective initiation of immune responses (Warnock et al, 1998; Mebius, 2003; Katakai et al, 2004a,b; Sixt et al, 2005; Bajenoff et al, 2006; Junt et al, 2008). The lymph node capsule is essentially an enlarged lymphatic vessel constructed from lymphatic endothelial cells (LECs), which carry interstitial fluid to the node and empty it into the parenchyma. It filters through the paracortex and cortex, eventually reaching efferent lymphatics in the medulla, which convey lymph further upstream (Mebius, 2003; Willard-Mack, 2006). Blood endothelial cells (BECs) construct cortical blood vessels and capillaries, including high endothelial venules specialized to attract naïve T cells from the bloodstream (Mebius, 2003; Willard-Mack, 2006)
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