Human Lymphatic Fibroblasts (HLF)

Expression of GARP and Integrins are Produced In Vitro by Human Endothelial Cells and LN Fibroblasts

GARP is expressed on regulatory T lymphocytes (Tregs) in human and murine tumors and activates TGF-β1, causing immunosuppression in tumor-bearing mice. However, its sources and role in lymph nodes (LNs) during metastasis are unclear. LNs, where immune responses occur, contain immune and non-immune cells like blood (BEC)/lymphatic (LEC) endothelial, fibroblastic, and perivascular cells (FRCs).

To find which non-immune cells express LRRC32 (the gene for GARP) in human LNs, Rouaud et al. analyzed single-cell RNA sequencing (scRNA-Seq) datasets from human LN samples. This identified LRRC32 expression in endothelial and perivascular cell subpopulations. They then checked GARP protein expression in primary cultures of human umbilical vein endothelial cells (HUVECs), human LECs, and human lymphatic fibroblasts (HLFs). Jurkat cells expressing human GARP served as a positive control. Western blotting showed similar GARP levels in all primary cells (Fig. 1a and Fig. 2). Flow cytometry confirmed GARP on the cell surface of HUVECs, LECs, and HLFs (Figu. 1b). Since integrins αVβ6 and αVβ8 can activate latent TGF-β1 presented by GARP, we checked their presence on these cells, along with the common integrin αVβ3. Flow cytometry detected significant levels of αV and β3 subunits in all three primary cell cultures (Fig. 2). β6 and β8 subunits were also present. Thus, in basal culture conditions, GARP, αVβ6, and αVβ8 are co-expressed on the surface of primary HUVEC, LEC, and HLF cultures.