Use of major histocompatibility complex class I/peptide/beta2M tetramers to quantitate CD8(+) cytotoxic T lymphocytes specific for dominant and nondominant viral epitopes in simian-human immunodeficiency virus-infected rhesus monkeys

Abstract

To evaluate the impact of the diversity of antigen recognition by T lymphocytes on disease pathogenesis, we must be able to identify and analyze simultaneously cytotoxic T-lymphocyte (CTL) responses specific for multiple viral epitopes. Many of the studies of the role of CD8(+) CTLs in AIDS pathogenesis have been done with simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys. These studies have frequently made use of the well-defined SIV Gag CTL epitope p11C,C-M presented to CTL by the HLA-A homologue molecule Mamu-A*01. In the present study we identified and fine mapped two novel Mamu-A*01-restricted CTL epitopes: the SIVmac Pol-derived epitope p68A (STPPLVRLV) and the human immunodeficiency virus type 1 (HIV-1) Env-derived p41A epitope (YAPPISGQI). The frequency of CD8(+) CTLs specific for the p11C,C-M, p68A, and p41A epitopes was quantitated in the same animals with a panel of tetrameric Mamu-A*01/peptide/beta2m complexes. All SHIV-infected Mamu-A*01(+) rhesus monkeys tested had a high frequency of SIVmac Gag-specific CTLs to the p11C,C-M epitope. In contrast, only a fraction of the monkeys tested had detectable CTLs specific for the SIVmac Pol p68A and HIV-1 Env p41A epitopes, and these responses were detected at very low frequencies. Thus, the p11C,C-M-specific CD8(+) CTL response is dominant and the p41A- and p68A-specific CD8(+) CTL responses are nondominant. These results indicate that CD8(+) CTL responses to dominant CTL epitopes can be readily quantitated with the tetramer technology; however, CD8(+) CTL responses to nondominant epitopes, due to the low frequency of these epitope-specific cells, may be difficult to detect and quantitate by this approach.

FIG. 1

Mapping of the optimal Mamu-A*01-restricted HIV-1 Env CTL epitope. (A) PBMC from a SHIV-HXBc2-infected rhesus monkey (L28) were cultured for 10 days with paraformaldehyde-fixed, autologous B-LCL cells infected with a vaccinia virus (vv299) expressing HIV-1 gp160. The effector cells were assayed at an E/T ratio of 10:1 with autologous B-LCL targets cultured overnight with the indicated synthetic peptides. (B) PBMC from a SHIV-HXBc2-infected rhesus monkey (L3) were cultured for 10 days with the HIV-1 gp160 20-amino-acid p41 peptide (GKAMYAPPIEGQIRCSSNIT) at a final concentration of 50 μg/ml. The effector cells were assayed at an E/T ratio of 10:1 with Mamu-A*01⁺ B-LCL targets cultured overnight with the indicated synthetic peptides. (C) Sequences of p41-derived peptides with a proline (P) at position 3.

FIG. 2

Mapping of the optimal Mamu-A*01-restricted SIVmac Pol CTL epitope. (A) PBMC from an SIVmac-infected rhesus monkey (GL9) were cultured for 10 days with 10 μg of the SIVmac Pol 25-amino-acid p68 peptide (WQVTWIPEWDFISTPPLVRLVFNLV) per ml. The effector cells were assayed at an E/T Ratio of 10:1 with autologous B-LCL cells cultured overnight with the indicated synthetic peptides. (B) Sequences of p68-derived peptides with a proline (P) at position 3.

FIG. 3

Folding of soluble Mamu-A*01 and human β2m around SIVmac Pol-derived peptides in vitro. (A) Gel filtration profile of soluble Mamu-A*01 monomers refolded with human β2m and the SIVmac Pol-derived peptide p68A. (B) Gel filtration profile of soluble Mamu-A*01 monomers refolded with human β2m and the SIVmac Pol-derived peptide p68B. (C) Gel filtration profile of soluble Mamu-A*01 monomers refolded with human β2m in the absence of exogenous peptide.

FIG. 4

Tetramer binding to peptide-stimulated PBMC from SHIV- and SIVmac-infected, Mamu-A*01⁺ rhesus monkeys. PBMC from SHIV-89.6-infected monkey 287 and SIVmac-infected monkey 4DK were stimulated in vitro with 1.0 μg of the indicated optimal peptide (p11C, C-M [CTPYDINQM], p68A [STPPLVRLV], or p41A [YAPPISGQI]) per ml for 10 days in rIL-2-containing medium. The PE-coupled tetrameric Mamu-A*01/peptide/β2m complexes were used in combination with anti-CD8α-FITC, anti-CD8αβ-ECD, and anti-CD3-APC to stain 2 × 10⁵ lymphocytes isolated by Ficoll-Hypaque density gradient centrifugation following this in vitro peptide stimulation. The percentage of CD8⁺ T lymphocytes staining positively with the Mamu-A*01/peptide/β2m complex is indicated in the upper right quadrant.

FIG. 5

Tetramer binding to freshly isolated PBMC from SHIV- and SIVmac-infected, Mamu-A*01⁺ rhesus monkeys. Fresh blood (100 μl) from SHIV-HXBc2-infected monkey L3 and SIVmac-infected monkey 4DK were stained with the indicated Mamu-A*01/peptide/β2m complex in combination with anti-CD8α-FITC, anti-CD8αβ-ECD, and anti-CD3-APC. The percentage of CD8⁺ T lymphocytes staining positively with the Mamu-A*01/peptide/β2m complex is indicated in the upper right quadrant.