Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification of a new subset of lymph node stromal cells involved in regulating plasma cell homeostasis

doi: 10.1073/pnas.1712628115

Figure Lengend Snippet: Medullary FRCs have a unique phenotype and cytokine profile and expand in number during lymph node swelling. (A–D) FACS analysis of CD45−CD35− cells from pLNs of naive or OVA/Mont-immunized mice at the indicated time points. (A) Dot plots in Upper row show the gating of FRCs (pdpn+CD31−) versus BECs (pdpn−CD31+) and LECs (pdpn+CD31+) from naive pLNs. The expression of MAdCAM and BP3 in FRCs distinguishes three FRC subpopulations: MRCs, TRCs, and MedRCs. Shown at Right is the control staining when anti-MAdCAM antibody was not added. Dot plots in Lower row show a representative staining of the three FRC subsets in naive versus activated LNs, with the percentages indicated for each population (n ≥ 3). (B) Number of total FRCs and of each of the three FRC subsets per pLN upon immunization. (C) Representative histograms showing the level of BrdU incorporation in the three FRC subsets (OVA/Mont d5.5). Numbers indicate the percentage of BrdU+ cells. Black line, anti-BrdU antibody; dashed line, isotype control antibody on total FRCs. (D) Representative histograms displaying the frequency of CCL21- and CXCL13-expressing cells among the three FRC subsets found in activated pLNs. Black line, specific antibody; dashed line, no primary antibody on total FRCs. (E) The three FRC subsets and CD31+ cells (LECs/BECs) were sorted from activated pLNs and normalized transcript levels of indicated cytokine transcripts measured by qRT-PCR (means ± SD, n = 4). (F) ISH analysis for indicated cytokine transcripts (green) or the sense control (for CXCL12). (G) pLN sections stained with the indicated antibodies. The boxed areas are represented Below at higher magnification. (H) Multiparameter flow cytometry-based clustering of FRC (CD45−31−pdpn+) subsets from naive pLNs using the TSNe algorithms (FlowJo) to display seven parameters (FCS, SSC, Pdpn, BP3, MAdCAM-1, CXCL13, and CCL21) in a 2D representation. Colored areas were added manually. (Scale bar, 100 µm.) Results in A–D, F, and G are representative of at least three independent mice. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The boxed areas are represented Below at higher magnification. ( H ) Multiparameter flow cytometry-based clustering of FRC (CD45 − 31 − pdpn + ) subsets from naive pLNs using the TSNe algorithms (FlowJo) to display seven parameters (FCS, SSC, Pdpn, BP3, MAdCAM-1, CXCL13, and CCL21) in a 2D representation.

Techniques: Expressing, Staining, BrdU Incorporation Assay, Quantitative RT-PCR, Flow Cytometry