Pro5 mhc class 1 pentamers

Similar to the T_reg Assay, after washing two times with HBSS, isolated T cells were directly combined with PPI-treated BMDCs at a ratio of 4:1 (T cells to BMDCs) and incubated for 72 h at 37°C and 5% CO₂. In the OVA-specific assay, cells were washed two times with a stain solution containing DPBS +1% BSA (10 mg/mL) + 0.1 mM EDTA before being stained with 50 μL staining solution +5 μL ProT2 MHC Class II tetramers +10 μL Pro5 MHC Class I pentamers (ProImmune) per sample at 37°C and 5% CO₂ for 30 min. Without washing, each sample was stained with 2 μL mouse CD16/CD32 monoclonal antibodies on ice for 30 min. Then each sample was stained with 60 μL antibody cocktails containing surface markers of CD8, CD3, CD69, CD19, CD127, CD4, CD25, CD27, CD44, CTLA-4 (CD152), CD28, PD-1 (CD279), and CD45 in the fridge for 1 h. The cells were then washed one time with stain solution, used Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), and followed standard protocols to stain Foxp3. After washing two times with stain solution, each sample was resuspended in 200 μL stain solution and tested by flow cytometry using BDFACS Symphony A5. The unspecific assay was identical to the OVA-specific assay, except that tetramers and pentamers were not stained. Samples were compensated based on single staining. All raw data were analyzed by FlowJo.

All fluorescently tagged antibodies, αCD28/CD3 antibodies, ELISA kits, RBC lysis buffer, Foxp3/Transcription Factor Staining Buffer Set, EDTA, and Cell Proliferation Dye eFluor^™ 670 were purchased from Invitrogen. All inhibitors (modulators) were purchased from MedChemExpress. Bovine serum albumin (BSA) was purchased from VWR Life Science. HBSS, DPBS, PBS, DMEM, RPMI 1640, AIM-V medium, FBS, HI-FBS, HEPES, and non-essential amino acid solutions were purchased from Gibco. RAW-Dual cells, RAW Blue cells, QB buffer and reagent, EndoFit Ovalbumin (OVA), ODN 1826 (CpG), and all other TLR agonists were purchased from InvivoGen. Spleen Dissociation Medium and EasySep Mouse T-Cell Isolation Kit were purchased from STEMCELL Technologies. β-Mercaptoethanol was purchased from MP Biomedicals. Cell Activation Cocktail (without Brefeldin A), Recombinant Mouse GM-CSF (carrier-free) (20 ng/mL), and LEGENDplex MU Th1/Th2 Panel (8-plex) Kit were purchased from BioLegend. ProT2 MHC Class II tetramers and Pro5 MHC Class I pentamers were purchased from ProImmune. All plates, unless noted otherwise, were purchased from Thermo Fisher Scientific.

Splenic mononuclear cell suspensions were obtained from 10 Mamu-A*01⁺ RM (Macaca mulatta) infected with either SIVmac239, SIVsmE543, or SIVsmE660 and with established SIV infection or AIDS. Macaques were classified as viremic (>1000 copies/mL; n = 6) or controller (<1000 copies/mL; n = 4) based on plasma RNA VLs (Supplemental Table 2). Cryopreserved samples were homogenized as described previously (83 (link)). Tissue homogenates were washed twice in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin (HyClone via GE Healthcare Life Sciences). SIV-specific CD8⁺ T cells were identified using Mamu-A*01-CTPYDINQM Gag_181–189 (CM9) Pro5 MHC Class I Pentamers (ProImmune). Cells were stained with the viability marker LIVE/DEAD Aqua (Thermo Fisher Scientific), and surface antigens were subsequently assessed with the fluorochrome-conjugated antibodies listed in Supplemental Table 3. For detection of intracellular proteins, surface antigen–labeled cells were fixed and permeabilized with the eBioscience Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) and incubated with the fluorochrome-labeled antibodies listed in Supplemental Table 3. All macaque samples were evaluated in a single experiment.