The amplification and sequencing of TRB gene libraries followed the protocol as previously described (25). The sequences were mapped to human reference sequences as described in detail previously (25, 26). CMV-specific cells demonstrate clonal expansion. However, TCR diversity is similar between CD85j+ and CD85j? compartments, suggesting that CD85j does not directly impact the repertoire of antigen-specific cells. Further phenotypic and functional analyses revealed that CD85j CM 346 (Afobazole) identifies a specific subset of CMV-responsive CD8 T cells that coexpress a marker of senescence (CD57) but retain polyfunctional cytokine production and expression of cytotoxic mediators. Blocking CD85j binding enhanced proliferation of CMV-specific CD8 KDELC1 antibody T CM 346 (Afobazole) cells upon antigen stimulation but did not alter polyfunctional cytokine production. Taken together, these data demonstrate that CD85j characterizes a population of senescent, but not exhausted antigen-specific effector CD8 T cells and indicates that CD85j is an important checkpoint regulator controlling expansion of virus-specific T cells during aging. Inhibition of CD85j activity may be a mechanism to promote stronger CD8 T cell effector responses during immune aging. Sequencing Total T cells were isolated by unfavorable selection using human T cell RosetteSep enrichment kit (StemCell Technologies) from platelet donor apheresis lymphocytes of HLA-A2 donors who are CMV seropositive. T cells were stained with CD4, CD8, pp65 HLA-A*0201 tetramer, and CD85j antibodies. CD85j+ and CD85j? pp65-HLA-A*0201 tetramer+ CD8 T cells were sorted using a FACSAria (BD Bioscience) and split into two to four replicates with 4,000C5,000 cells per replicate. Total RNA was extracted from each T cell replicate using RNeasy Plus Micro kit (Qiagen), followed by generation of cDNA using SuperScript VILO grasp mix (Invitrogen). The amplification and sequencing of TRB gene libraries followed the protocol as previously described (25). The sequences were mapped to human reference sequences as described in detail previously (25, 26). Clonotypes were defined as sequences with the same and gene segments and identical CDR3 amino acid sequences. In addition, any clonotype that was only found in one replicate library was filtered out of the analysis. The clonality index for each population was calculated using the lymphclon package (https://arxiv.org/abs/1408.1149) (25). CyTOF PBMCs were left unstimulated or stimulated for 18?h with CMV peptide pools in the presence of brefeldin A and monensin (BD Bioscience). For CMV-specific stimulation, two peptide super pools, each made up of overlapping peptide pools of four different antigens, were used. The immediate early (IE) pool consisted of IE-1, IE-2, US3, and UL36. The late pool consisted of pp65, UL32, UL48AB, and UL55 (gB), based on previously described work (27). Following stimulation, cells were resuspended in CyFACS buffer (1 PBS with 0.1% BSA, 2?mM EDTA, and 0.5% sodium azide) and stained with isotope-tagged antibodies before being acquired around the CyTOF. For a detailed protocol, see http://iti.stanford.edu/himc/protocols.html (CyTOF ICS protocol) and Table S1 in Supplementary Material. Data acquired from CyTOF were initially analyzed using FlowJo v10.1 (FlowJo Inc.). CD3+CD19?CD8+CD4? cells expressing CD107a or one of the following cytokines, IFN, TNF, IL-2, GM-CSF, or MIP1, after stimulation with IE or late pool were considered CMV-responsive CD8 T cells. The CMV-responsive cells for 30 individuals were concatenated, and cluster analysis was performed using X-shift (28). For final clustering, basic phenotypic (CD45RA, CCR7, CD28, CD27, and CD127) and the six preselected response factors were excluded. Blocking Experiments Reagents Peptide pools were purchased from JPT Peptide Technologies. The late antigen pp65 peptide pool was a combination of 138 peptides derived from a peptide scan (15mers with 11 amino-acid overlap) through 65?kDa phosphoprotein (pp65) (Swiss-Prot ID: “type”:”entrez-protein”,”attrs”:”text”:”P06725″,”term_id”:”130714″,”term_text”:”P06725″P06725) of human cytomegalovirus (HHV-5). The immediate early antigen IE-1 peptide pool was a combination of 120 peptides derived from a peptide scan (15mers with 11 amino acid overlap) through 55?kDa immediate early protein 1 (IE-1) (Swiss-Prot ID: “type”:”entrez-protein”,”attrs”:”text”:”P13202″,”term_id”:”138476″,”term_text”:”P13202″P13202) of human cytomegalovirus (HHV-5). PBMC Assays PBMCs were stimulated with pp65 or IE-1 peptide pools in the presence of brefeldin A. Monoclonal IgG2B mouse anti-human CD85j (ILT-2) antibody (R&D Systems) or an isotype control CM 346 (Afobazole) (eBioscience) (5?g/mL) was added prior to stimulation. For cytokine production, cells were stimulated for 13?h. For proliferation, cells were prelabeled with CFSE and stimulated for 7?days. Following stimulation, cells were resuspended in FACS buffer and stained with fluorescently tagged antibodies before being acquired around the flow.