T-cell responses can be mapped using matrix pools of HERV-K(HML-2) Gag- and Env-derived peptides. (A and B) Peripheral blood mononuclear cells (PBMC) from HIV-1-infected subject 125 were screened by IFN-γ ELISPOT using peptide matrix pools. Results are shown in mean spot forming units (SFU)/million PBMC. Tests were performed in duplicate, and error bars are standard deviations. Background levels were established by measuring responsiveness to 0.5% DMSO, and a pool of CMV pp65 peptides was included as a positive control. The horizontal line depicts the cutoff, with a positive response based on both (i) >3× background and (ii) >50 SFU/million PBMC after background subtraction. (A) The response to HERV-K(HML-2) Env peptide matrix pools 7 and 26 maps to HERV-K(HML-2) Env pep124. (B) The response to HERV-K(HML-2) Gag peptide matrix pools 9 and 21 maps to pep100, while the response to HERV-K(HML-2) Gag peptide matrix pools 4 and 23 maps to pep121. (C) IFN-γ ELISPOT results with 200,000 PBMC from subject 125/well are shown, confirming the response to the HERV-K(HML-2) Env pep124 15mer peptide common to matrix pools 7 and 23. The responses to these matrix pools are also shown in parallel. HK, HERV-K(HML-2).

CD8⁺ T-cell clones identified by responses to HERV-K(HML-2) Env pep124, Gag pep100, and Gag pep121 are specific for an HIV-1 Gag peptide contaminant. (A) CD8⁺ T-cell clones responsive to the original batches of HERV-K(HML-2) peptides were obtained by IFN-γ capture, followed by two rounds of limiting dilution cloning. IFN-γ ELISPOT results are shown, indicating that these T-cell clones, presumably of 3 separate specificities, in fact respond to all three original HERV-K(HML-2) peptides while failing to respond to newly synthesized batches of peptides with the same sequences. A CMV pp65-specific CD8⁺ T-cell clone did not respond to the original HERV-K(HML-2)-specific peptides, ruling out a nonspecific effect. All peptides were tested at 100 μg/ml in 0.05% DMSO. HK, HERV-K(HML-2). (B) These CD8⁺ T-cell clones were tested against HIV-1-derived peptide matrix pools to test the hypothesis that they were specific for a contaminating HIV-1 peptide. All three clones responded to matrix pools 5, 20, and 21, mapping to the 15mers AAWDRLHPVHAGPI and DRLHPVHAGPIAPGQ. Results for the clone obtained using HERV-K(HML-2) Gag pep100 are shown. (C) The CD8⁺ T-cell clone from panel B was mixed with autologous B lymphoblastoid cells and either pulsed with peptides or maintained as a no-peptide control. Flow cytometry data, gated on CD8⁺ cells, are shown. PHA, phytohemagglutinin.