The pericyte and stromal cell marker CD248 (endosialin) is required for efficient lymph node expansion

Abstract

CD248 is a cell surface receptor that specifically identifies fibroblasts and pericytes during development and in association with cancer and inflammation. However, its function is poorly defined and its role in lymphoid organs not studied. Here, we used (4-hydroxy-3-nitrophenyl)acetyl chicken gamma-globulin immunisation and mice lacking CD248 to study whether CD248 modulates popliteal LN (pLN) expansion and subsequent immune responses. We have found that CD248 is required for complete pLN expansion but not for co-ordination of B and T cell compartmentalisation or antibody production following (4-hydroxy-3-nitrophenyl)acetyl chicken gamma-globulin immunisation. In vitro, we show that CD248 expression in human MG63 stromal cells and mouse embryonic fibroblasts leads to a pro-proliferative and pro-migratory phenotype. This correlates with a proliferating CD248(+) population observed in vivo during pLN expansion. Taken together, these data highlight a role for CD248 in secondary lymphoid organ remodelling during adaptive immune responses.

Figure 1

CD248 expression is associated with initial pLN expansion. PCR for WT and KO CD248 alleles (left) and RT-PCR for CD248 (right) confirmed genotype of WT and CD248 KO-derived MEF (A). WT and CD248 KO MEF stained with P13 (green), vimentin (red) and Hoechst (blue). Bar=50 μm (B). Expression of CD248 (green) post-NP-CGG immunisation was tracked in WT pLN by immunofluorescence. Bars=200 μm (C). At 7 days post-NP-CGG immunisation, WT pLN were stained with CD248 (green), CD11b (red) and gp38 (blue) to analyse stromal subsets. Bars=200 μm in main images and 50 μm in inserts (D).

Figure 2

CD248 is required for pLN expansion but not antibody production following NP-CGG immunisation. WT and CD248 KO pLN were weighed post NP-CGG immunisation. Data are mean (+SEM) from three experiments, each with at least three replicates. p-Values calculated by Student’s non-paired t-test (A). Organisation of B-cell follicles (B) and T-cell zones (C) in pLN from unimmunised and day 21 post-NP-CGG immunisation WT and CD248 KO mice were analysed by immunofluorescence. Bars=200 μm. Sera antibody titres of innate (IgM) and adaptive (IgG1) antibody specific for NP and CGG were analysed by ELISA. Data shown as a ratio of mean antibody titre:mean pLN weight (n=4) (D).

Figure 3

CD248 regulates proliferation and migration, but not differentiation. Transfection of MG63 cells with CD248 was confirmed by flow cytometry (A). Migration of MG63 cells in the absence or presence of PDGF (left, n=4) and WT and CD248 KO MEF (right, n=2) were assayed using a Dunn chamber (B). Proliferation of MG63 (left, n=3) and MEF (right, n=6) was analysed by ³H uptake (C). Stromal proliferation within pLN was analysed by immunohistochemical staining of CD248 (blue) and BrdU (red) at day 0 (top) and day 7 (bottom) post-NP-CGG immunisation, bars=200 μm. Black box indicates area of interest with magnified image right, bars=50 μm (D). WT (top) and CD248 KO (bottom) MEF were driven to differentiate into adipocytes (left, green), chondrocytes (middle, blue) and osteocytes (right, red). Bars=100 μm (E). Bars in (B) and (C) show mean (±SEM), with p-values calculated by Student’s non-paired t-test.