Cd28pe cd28.2

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)).

PBMCs, peripheral lymph nodes, spleen and bone marrow cells were labeled with a combination of the following mAbs: CD3 PerCP-Cy5.5 (SP 34-2), CD4 PerCP-Cy5.5 (L200), CD8 PerCP (SK1), CD8 APC (SK1), CD95 FITC (DX2), CD95 APC (DX2), and CD28 PE (CD28.2) (BD Pharmingen, San Jose, CA), CD20 PerCP (2H7) (Biolegend, Inc., San Diego, CA) and Foxp3 APC (236A/E7) (eBioscience, Inc., San Diego, CA). Lymphocytes from the animals treated with anti-CD8 mAb were stained with anti-CD8 PE mAb (DK25, Dako, Inc., Carpinteria, CA). For chimerism analyses, we used an antibody that reacts specifically with certain MHC class I antigens (H38, One Lambda, Inc., Canoga Park, CA). The transplant pairs were chosen based on their differential reactivity to this mAb. The presence of CD31 and CD45RA on T cells was detected using clones WM59 and 5H9, respectively (BD Pharmingen). The fluorescence of the stained samples was analyzed using BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA) and FlowJo (TreeStar, Inc., Ashland, OR) 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 populations using a FACSVantage cell sorter (BD Biosciences). The purity of sorted cells was consistently >95%, as previously described (19 (link)).