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Marcelo J. Kuroda^*, Jörn E. Schmitz^*, Dan H. Barouch^*, Abie Craiu^*, Todd M. Allen^,, Alessandro Sette^||, David I. Watkins^,, Meryl A. Forman^¶, and Norman L. Letvin^*

From the ^* Harvard Medical School, Division of Viral Pathogenesis, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215; Wisconsin Regional Primate Research Center and Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin 53715; ^|| Eppimune, San Diego, California 92121; and the ^¶ Coulter Corporation, Miami, Florida 33116

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)8/β⁺ T lymphocyte subset and the percentage of CD8/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, CD8/β⁺ T cells. Furthermore, the percentage of CD8/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.

Abbreviations used: β₂m, β₂-microglobulin; 1-D IEF, one-dimensional isoelectric focusing; APC, allophycocyanin; B-LCL, B lymphoblastoid cell line; CD, cluster of differentiation; LDA, limiting dilution assay; mac, macaque; p11C, 12–amino acid fragment of SIVmac 251 Gag (amino acids 179–190); p11C, C–M, 9–amino acid fragment of SIVmac 251 Gag (amino acids 181–189); pCTL, precursor frequency of CTLs; SIV, simian immunodeficiency virus.

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