In contrast, thymocyte profiles of either B7-2 Collection 7 or CD28.FL single transgenics were much like those of wildtype mice, consistent with the need to introduce altered expression of both CD28 and its ligand in order to alter thymocyte development. Open in a separate window Figure 5 Altered thymocyte profile in CD28.FL/B7-2 double transgenic mice. B7-2 Collection 7, CD28.FL, CD28.TM and CD28. Y189F transgenic mice and TCR, CD3? and Rag-2 knockout mice were generated as previously explained (5C8) and managed in our breeding facility at Bioqual Inc. (Rockville, MD). All mice were used at between 8 and 12 weeks of age. Antibodies Anti-CD4, CD8, TCR (H57-597), CD45.1 (Ly5.2), CD45.2 (Ly5.1), CD90.1 (Thy1.1), B220, TCR(GL3), Mac-1, NK1.1, GR-1, CD11c, TER-119, and CD28 Abs were purchased from BD PharMingen (San Diego, CA). Anti-B7-2 (GL1) Ab was generated as explained previously (9). Circulation cytometric analysis Single cell suspensions were prepared from thymus and resuspended in FACS buffer (0.2% BSA, 0.01% sodium azide in HBSS without Rabbit polyclonal to CXCL10 phenol red). Anti-FcR mAb 24G2 (blocks FcII and FcIII) was added to prevent FcR-mediated binding of mAb, and cells were then incubated with directly conjugated FITC- and PE-labeled mAbs for 30 min. Following considerable washing, cells were incubated with biotin-labeled mAb and strepavidin-Cy-Chrome conjugate sequentially. Viable cells were analyzed by FACScan (Becton Dickinson, San Jose, CA) using CellQuest software. Four color staining was performed using FITC-labeled mAb, PE-labeled mAb, Cy-5-labeled mAb, biotinylated-mAb, and strepavidin-Alexa 594 conjugate (Molecular Probes, Eugene, OR). Viable cells, as determined by PI exclusion, were then analyzed on a dual-laser FACStar Plus (Becton Dickinson). Detection of intracellular TCR was performed as explained by manufacturers instructions (BD Biosciences). Briefly, cells were fixed in Cytofix/Cytoperm answer for 20 min at room temperature, washed in PermWash buffer, then incubated with FITC-labeled anti-TCR (H57-597) mAb or an isotype control for 30 min, followed by considerable washes in PermWash buffer before analysis on a FACScan. Bone marrow chimeras Radiation bone marrow chimeras were prepared as explained previously (10). CD28.FL/CD3? KO B7-2 Collection 7/CD3? KO chimeras were generated by reconstituting lethally irradiated (950 rad) B7-2 Collection 7 transgenic mice on a CD3? KO background with 107 T-depleted bone marrow cells from Cav 2.2 blocker 1 CD28 FL transgenics on a CD3? KO background. Immunohistology Sections (6 m) of OCT-embedded frozen tissue were air flow dried for 30 min and then incubated at least 2 hr with optimal dilutions of the primary antibodies rat anti-B7-2 (clone GL1, BD Biosciences PharMingen, San Diego, CA) and rabbit polyclonal anti-keratin 14 (Covance Research Products, Berkeley, CA). Tissues were washed and, after an amplification step for B7-2 with goat anti-rat IgG, immunoreactivity was detected with fluorochrome-conjugated donkey anti-rabbit IgG FITC and donkey anti-goat IgG Texas Red (Jackson ImmunoResearch Laboratories, West Grove, PA). Analysis was performed with an Cav 2.2 blocker 1 Olympus Provis AX70 microscope (Olympus, Melville, NY) and Cav 2.2 blocker 1 images were taken with a SPOT RT Color Video camera and SPOT Imaging Software (Diagnostic Devices, Sterling Heights, MI). Results Thymic expression patterns of CD28 and B7-2 in wildtype mice In order to understand the role that regulated expression of costimulatory molecules may play during thymic development, we first examined CD28 levels on thymic subpopulations from wild type B6 mice. As has previously been explained (1), we find that CD28 is expressed at the highest levels on DP cells and lower on CD4+ and CD8+ SP cells (Fig 1A). Analysis of CD28 expression on DN thymocytes revealed that CD28 levels are low on DN1, DN2 and the majority of DN3 cells as compared to an isotype-matched control antibody but are elevated on a subpopulation of DN3 cells and are uniformly high on DN4 thymocytes (Fig. 1B). Interestingly, when the CD25+ DN thymocytes (DN2 and DN3) were examined for expression of CD28 and TCR, we find that those cells expressing high levels of CD28 also express TCR (Fig. 2A) suggesting that there may be an important developmental link between expression of the pre-TCR and CD28. To determine whether the increase in CD28 expression levels observed in DN thymocytes which Cav 2.2 blocker 1 express TCR could result from signaling through the nascent pre-TCR, we tested the ability of anti-CD3 treatment to induce CD28 up-regulation in Rag KO Cav 2.2 blocker 1 mice. Treatment of Rag.