Cd3 percp sp 34 2

PBMCs, peripheral lymph nodes, spleen and bone marrow cells were labeled with a combination of the following mAbs: CD3 PerCP (SP 34–2), CD4 PerCP (L-200), CD8 PerCP (SK1), CD8 APC (SK1), CD95 FITC (DX2), CD95 APC (DX2), and CD28PE (CD28.2) (BD Pharmingen, San Jose, CA). For chimerism analyses, we used an anti-MHC class I HLA mAb (H38, One Lambda, Inc, CA) reacting specifically with certain MHC class I antigens. The recipient and donor pairs were chosen based on their differential reactivity to this mAb. The fluorescence of the stained samples was analyzed using FACS Calibur and FACS Scan flow cytometers and Cell Quest Software (BD), or FlowJo software. For assessing the effect of CTLA4Ig on memory T cell functions, the monoclonal antibodies anti-CD16, anti-CD95, anti-CD4 and anti-CD8 were used to purify CD4 Tmems (CD16⁻CD4⁺CD95⁺) and CD8 Tmems(CD16⁻CD8⁺CD95⁺) using a FACS Vantage cell sorter (BD Immunocytometry System), then those purified populations were tested for their IFNγ production in ELISPOT assay as previously described (11 (link), 12 (link)). The purity of sorted cells was consistently > 95% as previously described.

Peripheral blood mononuclear cells (PBMCs), peripheral lymph nodes, spleen, and bone marrow cells were labeled with a combination of the following mAbs: CD3 PerCP (SP 34-2), CD4 PerCP (L-200), CD8 PerCP (SK1), CD8 APC (SK1), CD95 FITC (DX2), CD95 APC (DX2), and CD28PE (CD28.2) (BD Pharmingen, San Jose, CA). Lymphocytes from the animals treated with anti-CD8 mAbs were stained with anti-CD8-PE (DK25, Dako, Inc., Carpenteria, CA). For chimerism analyses, we used an anti-MHC class I HLA mAb (H38, One Lambda, Inc, CA) reacting specifically with donor MHC class I antigens. To assess intracellular protein expression of FOXP3 (PCH101), Ki67 (B56) and CD152 (BNI3), cells were permeabilized using fixation/permeabilization solution (eBioscience). The fluorescence of the stained samples was analyzed using a FACS Calibur flow cytometer and FlowJo software. For assessing memory cell function, fresh PBMCs were gated on lymphocytes and sorted into CD16⁻CD95⁻CD28⁺ naïve and CD16⁻CD95⁺CD28^low/high memory T cell populations using a FACS Vantage cell sorter (BD Immunocytometry System). The purity of sorted cells was consistently > 95%, as previously described (32 (link)).

Peripheral blood cells were stained with the following mAbs two times a week after transplantation: CD56 PE (B159), CD95 PE (DX2), HLA-I PE (DX17), CD3 PerCP (SP34–2), CD4 BV510 (L200), CD4 APC (L200) (BD Biosciences, San Jose, CA); CD8 APC (BW135.80), CD20 APC-Vio770 (LT20), CD25 Pacific Blue (BC96) (BioLegend), CD45RA APC-Vio770 (T6D11), CD11b Viogreen (M1/70.15.11.5), FOXP3 (236A/E7) (Invitrogen), HLA-I PE-Vio770 (REA230) (Miltenyi Biotec Inc. San Diego, CA); CD28 BV421 (CD28.2) and CD8 BV421 (RPA-T8), (BioLegend Inc. San Diego, CA). Anti-Bw6 FITC (H38, One Lambda, Canoga Park, CA) was used to assess donor chimerism. Tregs were defined as CD3+/CD4+/CD25^high/FoxP3+. The same analysis was performed on liver tissue and lymph nodes on sacrifice. Data was acquired on a FACS Canto II Flow Cytometer (BD Bioscience) and analyzed using FloJo Software (TreeStar Inc. Ashland, OR).