CD69 Role on T Cell Priming Figure 5. CD69 deficiency on T cells does not affect Ag-specific T cell proliferation in Ag-draining LN. A. Purified CD69+/+ or CD692/2 OT-I CD8+ T cells were CFSE stained and transferred into recipients receiving the indicated doses of OVA and 5 mg of LPS subcutaneously in a posterior footpad. The percentage of proliferated OT-I CD8+ T cells was analyzed 42h later in the popliteal LN. Pool of two experiments, with 1 (dose 0) to 4 (doses 0.1–10 mg) mice per point. Bars represent SD. B. As in A, but mice received 10 mg of OVA with or without 5 mg of LPS in the footpad. C. Purified CD69+/+ or CD692/2 RAG22/2 DO10.11 CD4+ T cells were transferred into Balb/c mice receiving 1 mg of OVA subcutaneously in the footpad. 3 days later the popliteal LN were analyzed for the percentage of proliferated cells within DO10.11 CD4+ T cells. doi:10.1371/journal.pone.0048593.g005 min). The light-density fraction was treated with anti-CD3 (KT3- 1.1), anti-Thy1-1 (T24/31.7), anti-Ly6G (IA8), anti-CD19 (ID3) and anti-erythrocyte (TER119), all home-produced, followed by immunomagnetic depletion with sheep anti-ratIgG coated Dyna- beads (Dynal Biotech, Oslo, Norway). Where indicated, DC were stained with home produced anti-CD11c, -CD45RA and -Bst2 and further purified into conventional DC (cDC, CD11chi CD45RA2 Bst22) or plasmacytoid DC (pDC, CD11cint CD45RA+ Bst2+) with a FACS Aria sorter (Becton Dickinson). In vitro cultures For T-DC co-cultures, OT-I and OT-II LN T cells were negatively selected using MAbs against CD11b (M1/70), F4/80, Ter-119, Gr-1 (RB6), MHC class II (M5/114), and CD4 (GK 1.5) or CD8 (YTS 169.4), respectively, followed by incubation with anti-rat IgG-coupled magnetic beads (Dynal Biotech). T cell preparations were 90–95% pure. T cells were stained with 5 mM carboxyfluorescein succinimidyl ester (CFSE). DC were plated at 56103 per well in U-bottom 96 well plates and cultured in complete media with the indicated conditions, in duplicate. Where indicated anti-CD69 2.2 or IgG1 control were added at 10 mg/ml. DC were co-cultured with 56104 OT-I CD8+ or OT-II CD4+ per well for 2 or 3 days respectively. After that, cells were stained with anti-Va2, PI and anti-CD8 or anti-CD4 MAbs. For cultures of OT-I RAG12/2 cells with peptide doses, whole LN and spleen cells were labeled with 2 mM CFSE and plated at 106 cells per well in 96 well flat-bottom plates and were cultured with the indicated peptide doses, 10 mg/ml of anti-CD69 2.2 or IgG1 and with or without 1 mg/ml LPS for 3 days. For cultures of CD69+/+ or CD692/2 OT-I cells with peptide doses, CD8+ T cells were negatively purified using CD8+ T cell isolation Kit and MACs columns (Miltenyi Biotec, Auburn, CA, USA), stained with 2 mM CFSE and cultured at 0.56106 cells per well together with 0.56105 CD69+/+ antigen presenting cells (the positive fraction) in 96 well flat-bottom plates with the indicated peptide doses for 3 days. Cells were then stained for CD8 and CD25 and analyzed by flow cytometry. 2.56104 Callibrite beads (Becton Dickinson) were added to each sample, and samples were acquired until 5x103 beads had been collected. In vivo cell transfers OT-I CD8+ T cells or DO10.11 CD4+ T cells were purified from CD69+/+ or CD692/2 OT-I or DO10.11 mice, respectively, with CD8+ or CD4+ T cell isolation Kit and MACs columns (Miltenyi Biotec). After that, they were stained with 5 mM CFSE and injected intravenously into C57BL/6 or Balb/c recipients, PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e48593