Identification of dominant optimal HLA-B60- and HLA-B61-restricted cytotoxic T-lymphocyte (CTL) epitopes: rapid characterization of CTL responses by enzyme-linked immunospot assay

Abstract

Human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T-lymphocyte (CTL) responses play a major role in the antiviral immune response, but the relative contribution of CTL responses restricted by different HLA class I molecules is less well defined. HLA-B60 or the related allele B61 is expressed in 10 to 20% of Caucasoid populations and is even more highly prevalent in Asian populations, but yet no CTL epitopes restricted by these alleles have been defined. Here we report the definition of five novel HLA-B60-restricted HIV-1-specific CTL epitopes, using peripheral blood mononuclear cells in enzyme-linked immunospot (Elispot) assays and using CTL clones and lines in cytolytic assays. The dominant HLA-B60-restricted epitope, Nef peptide KEKGGLEGL, was targeted by all eight subjects with B60 and also by both subjects with B61 studied. This study additionally establishes the utility of the Elispot assay as a more rapid and efficient method of defining novel CTL epitopes. This approach will help to define new CTL epitopes that may play an important role in the immune control of HIV-1.

FIG. 1

Definition of an HLA-B60-restricted novel epitope in Nef. The subject studied was 166j (HLA A3/− B14/60 Cw3/8). (a) Recognition of 3 of 20 20-mer overlapping peptides spanning Nef in the Elispot assay. Frequency of responses is expressed as SFC per million PBMC. (b) CD8⁺ T-cell dependence of the response toward Nef-10 demonstrated in the Elispot assay following CD4-CD8 enrichment or depletion. Frequency of IFN-γ-producing cells is expressed as SFC per million input cells. (c) Titration curves using PBMC in an Elispot assay mixture incubated with serial dilutions of peptides as shown. (d) Titration curves using CTL specific clones in a standard chromium release assay; the peptides (and symbols) are the same as shown in panel c. (e) Titration curves using the CTL clones employed in panel d but in an Elispot assay, incubated with the same peptides as shown in panels c and d. (f) Determination of HLA restriction using CTL clones in an Elispot assay (hatched bars) and in a chromium release assay (solid bars). Targets either were pulsed with no peptide (−) or were pulsed with peptide KEKGGLEGL (+). The HLA class I types of the targets used were A2/3, B7/60, and Cw3/7; A3/−, B7/−, and Cw7/−; A28/29, B14/44, and Cw5/8; and A34/68, B57/71, and Cw3/7 (matching HLA class I alleles are shown in boldface).

FIG. 2

Definition of novel HLA-B60-restricted CTL epitopes in p17^Gag (a to c) and RT (d to f). The subject studied was 166j (HLA A3/− B14/60 Cw3/8). (a and d) Titration curves of truncated peptides using PBMC in an Elispot assay. (b and e) Titration curves using peptides in panels a and d, respectively, in chromium release assays using peptide-specific CTL clones and autologous B-cell line targets. (c and f) Titration curves using peptides in panels a and d, respectively, incubated in Elispot assays with the respective CTL clones.

FIG. 3

Recognition of novel HLA-B60-restricted CTL epitopes in p24^Gag and gp41^Env. The subject studied was 161j (HLA A2/3 B7/60 Cw3/7). (a) Incubation of PBMC with peptide truncations of p24^Gag peptide SEGATPQDL in an Elispot assay as shown. (b) Incubation of PBMC with peptide truncations of gp41 peptide QELKNSAVSL in an Elispot assay as shown.

FIG. 4

Contribution of responses toward HLA-B60-restricted CTL epitope peptides in p17^Gag (IEIKTDKEAL), p24^Gag (SEGATPQDL), and p24^Nef (KEKGGLEGL) compared to total CTL activity detected toward epitopes within these proteins for six subjects. The method used was as illustrated in Fig. 1.