Anti ctla 4 antibody clone uc10 4f10 11

Human CD86 C-terminally tagged with enhanced GFP (pCD86-EGFP, Addgene) was sub-cloned into a modified version of the MSCV2.2 retroviral plasmid in which the IRES-GFP cassette was removed. This plasmid was transfected into Plat-E packaging cells to produce retrovirus that was used to transduce NIH/3T3 fibroblasts. The resultant NIH/3T3 transfectants were sorted (FACS Aria Fusion, Becton Dickinson) for uniform GFP expression. CD4⁺ T cells from the spleen of B6 mice were enriched using CD4 MicroBeads (Miltenyi Biotec) according to the manufacturer's protocol. Enriched CD4⁺ T cells were co-cultured with CD86-GFP expressing NIH/3T3 cells at a 1:1 ratio for 2 h at 37°C. Where indicated, 100 µg/ml of anti-CTLA-4 antibody (clone UC10-4F10-11, Bio X Cell) or 25 nM Bafilomycin A1 (Sigma-Aldrich) was added to the culture. After incubation, NIH/3T3 cells remained adherent while CD4⁺ T cells were removed, stained, and analyzed by flow cytometry.

Mice were treated intraperitoneally (i.p.) with anti–CTLA-4 antibody (clone UC10-4F10-11, BioXCell) or Armenian hamster IgG isotype control (BioXcell). Treatment schedule was as follows: 300 μg of either antibody was injected i.p. in each mouse on days 0 and 2 after the first topical application of S. epidermidis. For CD4⁺ and CD8⁺ depletion, mice were treated i.p. with 200 μg of anti-CD4 antibody (GK1.5, BioXCell), 200 μg of anti-CD8 antibody (clone 2.43, BioXCell) or both antibodies on days −2, −1, 0, and 3 after first topical application of S. epidermidis.

CD80-mCherry.Rag2^−/− mice (12–14 weeks old, male and female) were injected intravenously on Day 0 with 8x10⁶ CD4⁺ T cells purified from wild-type BALB/c lymph nodes. 500 µg of anti-CTLA-4 antibody (clone UC10-4F10-11, BioXCell) or hamster IgG isotype control antibody (clone PIP, Bio X Cell) was administered via intraperitoneal injection on Day 0, Day 3, and Day 6. PBS or Fc.Mut24 was also administered via intraperitoneal injection on Day 3. Spleen cells were analyzed by flow cytometry on Day 7.