Clone rm4 5

Splenocytes were prepared as indicated above. Stimulation assays were performed using 1 × 10⁶ live splenocytes per well in 96-well U-bottom plates. Pools of overlapping peptides spanning the entire coding sequences of brachyury, CEA and MUC1 were synthesized as 15-mers with 11-amino acid overlaps (JPT GmbH) and lyophilized peptide pools were dissolved in Dimethyl sulfoxide (DMSO). Similarly constructed peptide pools corresponding to SIV-Vif and SIV-Nef served as off-target controls. Splenocytes in R10 media (RPMI 1640, 10% fetal bovine serum, and antibiotics) were stimulated by the addition of peptide pools at 2 μg/mL/peptide for 6 h at 37°C and 5% CO₂, with protein transport inhibitor (GolgiStop, BD) added 2 hours into the incubation. Stimulated splenocytes were then stained for lymphocyte surface markers CD8α and CD4, fixed, permeabilized, and then stained for the intracellular accumulation of IFN-γ and TNF-α. Antibodies against mouse CD8α (clone 53–6.7), CD4 (clone RM4–5), IFN-γ (clone XMG1.2), and TNF-α (clone MP6-XT22) were purchased from BD and staining was performed in the presence of anti-CD16/CD32 (clone 2.4G2). Flow cytometry was performed using an Accuri C6 Flow Cytometer (BD) and analyzed in BD Accuri C6 Software.

Lungs were excised, minced, and enzymatically digested. Lymphocytes were enriched using a histopaque gradient, diluted to 2 × 10⁶/100 μL, and incubated with a nonspecific binding blocking reagent cocktail of anti-mouse CD16/CD32 (2.4G2), normal mouse, and normal rat serum (Jackson ImmunoResearch) for 10 minutes. Cells were then stained with fluorochrome-labeled antibodies against mouse CD4 (BD Biosciences catalog 740208; clone RM4-5) and CD25 (BD Biosciences catalog 551071; clone PC61), then fixed prior to intracellular staining for the mouse transcription factors Gata3 (BioLegend catalog 653810; clone 16E10A23), T-bet (BioLegend catalog 644817; clone 4B10), RORγt (BD Biosciences catalog 562684; clone Q31-378), and Foxp3 (Invitrogen 48-5773-82; clone FJK-16s). In some experiments, donor CD45.1 cells and recipient CD45.2 cells were identified by staining with antibodies against mouse CD45.1 (eBioscience catalog 12-0453-81; clone A20) and CD45.2 (BD Pharmingen catalog 553772; clone 104). Cells were evaluated using an LSR II flow cytometer (BD Biosciences), and data were analyzed using FlowJo software, version 9.6 (Tree Star).

B6 mice were immunized i.p. with 50 μg FL9-68/IFA or PBS/IFA at days 0 and 7 followed by a BALB/C → B6 skin transplant at day 10. FL9-68/IFA or PBS/IFA immunization was repeated on days 10, 13, and 16. An additional group of hosts were treated with 30 μg of α-Ly49F Ab (Novus Biologicals, clone HBF-719) on days of immunization to deplete CD8 Tregs. At day 27, fully vascularized BALB/c hearts were transplanted into the abdominal cavity of B6 mice using microsurgical techniques, as previously described (43 (link)). Heart graft survival was determined by monitoring palpable heart beating. At day 16 after skin sensitization, levels of FL9 T cells, Tfh, GC B, and plasma cells in dLNs were analyzed by flow cytometry. Serum was collected from the heart graft recipient B6 mice that were either immunized with FL9-68/adjuvant or adjuvant alone at day 16. Serially diluted serum was incubated with 1 × 10⁶ donor splenocytes in total volume 100 μL PBS for 30 minutes followed by detection of surface-bound antibodies on CD4 cells using anti-CD4 (Biolegend, Clone RM4-5) and anti-mouse IgG1 antibodies (BD Biosciences, Clone A85-1). Histological analysis of heart grafts was performed by InvivoEx company using anti-C4d Ab (Hycult Biotech) and Vector Blue Alkaline Phosphatase Substrate Kit (Vector Laboratories).