Human immunodeficiency virus type 1 envelope epitope-specific CD4(+) T lymphocytes in simian/human immunodeficiency virus-infected and vaccinated rhesus monkeys detected using a peptide-major histocompatibility complex class II tetramer

Abstract

A tetrameric recombinant major histocompatibility complex (MHC) class II-peptide complex was used to quantitate human immunodeficiency virus type 1 (HIV-1) envelope (Env)-specific CD4(+) T cells in vaccinated and in simian/human immunodeficiency virus (SHIV)-infected rhesus monkeys. A rhesus monkey MHC class II DR molecule, Mamu-DR*W201, and an HIV-1 Env peptide (p46) were employed to construct this tetrameric complex. A p46-specific proliferative response was seen in sorted, tetramer-binding, but not nonbinding, CD4(+) T cells, directly demonstrating that this response was mediated by the epitope-specific lymphocytes. Although staining of whole blood from 10 SHIV-infected Mamu-DR*W201(+) rhesus monkeys failed to demonstrate tetramer-binding CD4(+) T cells (<0.02%), p46-stimulated peripheral blood mononuclear cells (PBMCs) from 9 of these 10 monkeys had detectable p46 tetramer-binding cells, comprising 0.5 to 15.2% of the CD4(+) T cells. p46-stimulated PBMCs from 7 of 10 Mamu-DR*W201(+) monkeys vaccinated with a recombinant canarypox virus-HIV-1 env construct also demonstrated p46 tetramer-binding cells, comprising 0.9 to 7.2% of the CD4(+) T cells. Thus, Env p46-specific CD4(+) T cells can be detected by tetrameric Mamu-DR*W201-p46 complex staining of PBMCs in both SHIV-infected and vaccinated rhesus monkeys. These epitope-specific cell populations appear to be present in peripheral blood at a very low frequency.

FIG. 1

Identification of Mamu-DRB*W201 alleles by PCR with allele-specific primers. Shown is the gel electrophoresis profile of PCR products amplified with allele-specific primers from DNA extracted from PBMCs of various rhesus monkeys (A to G). DRB, PCR products amplified by primers specific for a conserved region of the Mamu-DRB sequences; DRB*W201, PCR product amplified by Mamu-DRB*W201 allele-specific primers; −, Mamu-DR*W201 was not amplified from DNA; +, Mamu-DR*W201 was amplified from DNA; M, DNA standard (HaeIII digest of φ174 DNA).

FIG. 2

Tetrameric Mamu-DR*W201–p46 complex binds specifically to a p46-specific CD4⁺ T-cell line generated from PBMCs of a Mamu-DR*W201⁺ rhesus monkey. The p46 tetramer was used to stain two HIV-1 Env-specific T-cell lines generated from PBMCs of the same rhesus monkey, one specific for p46 and the other specific for the p14 Env peptide. Flow-cytometric analysis was performed on gated CD4⁺ CD3⁺ T cells.

FIG. 3

Kinetics of in vitro expansion of tetrameric Mamu-DR*W201–p46 complex-binding CD4⁺ T cells in p46-stimulated PBMCs of SHIV-infected Mamu-DR*W201⁺ rhesus monkeys. PBMCs of two SHIV-infected monkeys (L23 and H318) were stained at different times after beginning p46 stimulation in vitro. Flow-cytometric analysis was performed on gated CD3⁺ T cells stained with PE-coupled tetrameric Mamu-DR*W201–p46 complex and fluorescein isothiocyanate-coupled anti-CD4 antibody. Whole-blood staining was performed on day 0. The indicated percentages are the proportions of CD4⁺ T cells that bound tetramer.

FIG. 4

Tetrameric Mamu-DR*W201–p46 complex binds specifically to p46-stimulated CD4⁺ T cells from PBMCs of SHIV-infected Mamu-DR*W201⁺ rhesus monkeys. p46-stimulated PBMCs of three groups of monkeys (three monkeys per group) were assessed: SHIV-positive Mamu-DR*W201⁺ monkeys, SHIV-positive Mamu-DR*W201-negative monkeys, and SHIV-negative Mamu-DR*W201⁺ monkeys. PBMCs were stimulated in vitro with p46 in IL-2-containing medium for 11 days, and flow-cytometric analysis was performed on gated CD3⁺ cells stained with PE-coupled tetrameric Mamu-DR*W201–p46 complex and fluorescein isothiocyanate-coupled anti-CD4 antibody. The indicated percentages are the proportions of CD4⁺ T cells that bound tetramer.

FIG. 5

p46-stimulated T-cell proliferation is mediated by tetrameric Mamu-DR*W201–p46 complex-binding CD4⁺ T lymphocytes. In vitro p46-stimulated PBMCs from the SHIV-infected Mamu-DR*W201⁺ rhesus monkey L23 were stained with PE-coupled tetrameric Mamu-DR*W201–p46 complex. CD4-positive, Mamu-DR*W201–p46 complex-positive cells and CD4-positive, Mamu-DR*W201–p46 complex-negative cells were sorted by flow cytometry and expanded by concanavalin A stimulation for 10 days with irradiated PBMCs in IL-2-containing medium. The cells were again stained and analyzed by flow cytometry, and the p46-specific proliferative response of each cell population was assessed. SI, stimulation index.

FIG. 6

Peptide epitope-specific CD4⁺ T cells detected by tetramer binding in peptide-stimulated PBMCs of SHIV-infected rhesus monkeys and recombinant canarypox virus-vaccinated monkeys. p46-specific CD4⁺ T cells among in vitro p46-stimulated PBMCs from SHIV-infected Mamu-DR*W201⁺ monkeys (A) and recombinant canarypox virus-vaccinated Mamu-DR*W201⁺ rhesus monkeys (B) were identified by their binding to tetrameric Mamu-DR*W201–p46 complex. p46-stimulated PBMCs from 10 infected and 10 vaccinated rhesus monkeys were stained with PE-coupled tetrameric Mamu-DR*W201–p46 complex. Flow-cytometric analysis was performed on gated CD4⁺ CD3⁺ T cells.