Regulation of T Cell Priming by Lymphoid Stroma Figure 1. Inhibition of Initial T cell priming by Lymph node Stromal Populations. (a) Three cell lines were isolated representing FRC (FRC.5: CD312/gp38+)), LEC (LEC.6: CD31+gp38+), and BEC (BEC.7: CD31+gp382). (b) Contrast images of each line showing differences in morphology consistent with their in vivo function. (c) 105 CFSE-labeled OTI T cells were incubated in 96-well round-bottom wells in the presence of 104 BMDCs either unpulsed (NS) or pulsed (+SL8) with the indicated concentrations of SL8 peptides. 56103 stromal cells were added where indicated. Cells were analyzed by FACS after 48 hours. (d) CFSE proliferation assays were performed identically to (a) but included freshly purified gp38+CD312 FRC or an equal number of unfractionated total (including T cells, B cells, etc.) from collagenase digestion of lymph nodes. (e) Proliferation assays were performed identically to those in (c,d) but included mouse embryonic fibroblasts (MEF) or NIH-3T3 cells as 3rd party cell types. All experiments are representative of at least 3 similar trials. doi:10.1371/journal.pone.0026138.g001 passaged up to 10 times from each freeze and re-sorted between major freezes. These lines displayed distinct morphological features, consistent with their functions in situ; FRCs featuring very extended lamellopodia on plastic or glass surfaces and both LECs and BECs having elongated phenotypes with long extension (Fig 1b) and a high propensity to form side-by-side ‘bundles’ of cells in 2D cultures (not shown). We subsequently tested the effects of each line, as a 5% cellular proportion, when co-cultured with a standard stimulation culture of na¨ıve CD8 T cells, using monoclonal T cells derived from lymph nodes of OTI TCR transgenic mice mixed with in-vitro generated bone-marrow derived dendritic cells (BMDCs). T cells were labeled with CFSE to track cell division and, as shown in Fig 1c, in the absence of their cognate antigen, SIINFEKL (SL8), they did not divide, regardless of the presence of lymph node stromal lines. When BMDCs were pulsed with SIINFEKL peptide, T cells typically underwent multiple rounds of cell division within 48 hours. However, addition of FRC, LEC or BEC PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e26138