Analysis of Gag-specific Cytotoxic T Lymphocytes in Simian Immunodeficiency Virus–infected Rhesus Monkeys by Cell Staining with a Tetrameric Major Histocompatibility Complex Class I–Peptide Complex

Abstract

A tetrameric recombinant major histocompatibility complex (MHC) class I–peptide complex was used as a staining reagent in flow cytometric analyses to quantitate and define the phenotype of Gag-specific cytotoxic T lymphocytes (CTLs) in the peripheral blood of simian immunodeficiency virus macaque (SIVmac)-infected rhesus monkeys. The heavy chain of the rhesus monkey MHC class I molecule Mamu-A*01 and β2-microglobulin were refolded in the presence of an SIVmac Gag synthetic peptide (p11C, C–M) representing the optimal nine–amino acid peptide of Mamu-A*01–restricted predominant CTL epitope to create a tetrameric Mamu-A*01/p11C, C–M complex. Tetrameric Mamu-A*01/p11C, C–M complex bound to T cells of SIVmac-infected, Mamu-A*01+, but not uninfected, Mamu-A*01+, or infected, Mamu-A*01− rhesus monkeys. Specific staining of peripheral blood mononuclear cells (PBMC) from SIVmac-infected, Mamu-A*01+ rhesus monkeys was only found in the cluster of differentiation (CD)8α/β+ T lymphocyte subset and the percentage of CD8α/β+ T cells in the peripheral blood of four SIVmac-infected, Mamu-A*01+ rhesus monkeys staining with this complex ranged from 0.7 to 10.3%. Importantly, functional SIVmac Gag p11C-specific CTL activity was seen in sorted and expanded tetrameric Mamu-A*01/p11C, C–M complex–binding, but not nonbinding, CD8α/β+ T cells. Furthermore, the percentage of CD8α/β+ T cells binding this tetrameric Mamu-A*01/p11C, C–M complex correlated well with p11C-specific cytotoxic activity as measured in both bulk and limiting dilution effector frequency assays. Finally, phenotypic characterization of the cells binding this tetrameric complex indicated that this lymphocyte population is heterogeneous. These studies indicate the power of this approach for examining virus-specific CTLs in in vivo settings.

Construction of Soluble Mamu-B*1703, a Class I Major Histocompatibility Complex of Chinese Rhesus Macaques, Monomer and Tetramer Loaded with a Simian Immunodeficiency Virus Peptide

“…In the present study, we used human  2 m in place of monkey  2 m. Our results showed that the Mamu-B*1703 heavy chain could be refolded with IW9 in the presence of human  2 m. This is consistent with the previous publication (20), which also reported that the monkey MHC I heavy chain could refolded with human  2 m in construction of tetramers. The reason may be due to the high homology between human and monkey  2 m (92% identity).…”

Interleukin-18 gene transfer increases antitumor effects of suicide gene therapy through efficient induction of antitumor immunity

“…The reason might be that LDA analysis depends on cell proliferation. 26 Peptide/MHC-I tetramers were intensively used for the staining of virus-specific T cells. Study with tetrameric complexes of HLA-A2/HIV gag or pol showed that 2% of CD8 + T cells were antigen specific.…”

“…7 For persistent HIV infection, the range is commonly 0.1-2% of CD8 + T cells. In persistent simian immunodeficiency virus infection in macaques, Kuroda et al 26 showed that epitope-specific T cells can account for as much as 10% of CD8 + T cells in the blood. Callan et al 27 have shown that between 7 and 44% of blood CD8 + T cells are EBV-specific in acute infection and the total number of CD8 + T cells is greatly increased, almost entirely by antigen-specific cells.…”

Interleukin-15 but Not Interleukin-7 Abrogates Vaccine-Induced Decrease in Virus Level in Simian Immunodeficiency Virusmac251-Infected Macaques

“…For the physical detection of SIV-specific CD8 ϩ T cells, staining was performed with pretitered, allophycocyanin-conjugated (Molecular Probes) Gag 181-189 CM9 (p11C) (CTPYDINQM)-Mamu-A*01 tetrameric complexes for 30 min at room temperature (51). One hundred thousand events were collected in the lymphocyte region (R1) and analyzed with CellQuest software and PAINT-A-GATE (BD Biosciences).…”