Gag-specific CD8+ T lymphocytes recognize infected cells before AIDS-virus integration and viral protein expression

Abstract

CD8(+) T cells are a key focus of vaccine development efforts for HIV. However, there is no clear consensus as to which of the nine HIV proteins should be used for vaccination. The early proteins Tat, Rev, and Nef may be better CD8(+) T cell targets than the late-expressed structural proteins Gag, Pol, and Env. In this study, we show that Gag-specific CD8(+) T cells recognize infected CD4(+) T lymphocytes as early as 2 h postinfection, before proviral DNA integration, viral protein synthesis, and Nef-mediated MHC class I down-regulation. Additionally, the number of Gag epitopes recognized by CD8(+) T cells was significantly associated with lower viremia (p = 0.0017) in SIV-infected rhesus macaques. These results suggest that HIV vaccines should focus CD8(+) T cell responses on Gag.

FIGURE 1

KICS assay schematic. A, PBMC-derived CD4⁺ T cell targets were activated by overnight stimulation with SEB and CD28, CD3, and CD49d Abs. B, Effector cells were generated by three rounds of limiting dilution of a 2-wk in vitro-stimulated CD8⁺ T cell line. C, Activated CD4⁺ target cells are synchronously infected by a 15-min incubation with magnetically activated SIVmac239 (multiplicity of infection ≤1) in the presence of a magnetic field. Spinoculation (41) was used to verify the results of some experiments. Identical results were obtained with both methods (data not shown). D, Synchronously infected CD4⁺ target cells are cocultured with SIV-specific clones for 1.5 h and then for an additional 5 h in the presence of BFA at various times postinfection. ICS is then performed to detect activation of the CD8⁺ T cell clones via staining for IFN-γ and TNF-α.

FIGURE 2

Presentation kinetics of SIV-derived CD8⁺ T cell epitopes. MHC-I-matched (■) and MHC-I-mismatched (large asterisk) CD4⁺ T cell targets were synchronously infected with SIVmac239 and cocultured at an E:T of 1:1 with SIV-specific CD8⁺ T cell clones specific for Gag CM9 (A), Gag GY9 (B), Tat SL8 (C), or Env FW9 (D). Results are shown as a percentage of the maximum cytokine staining of TNF-α and IFN-γ detected during the assay. Representative raw flow data is shown in Fig. 1D. Uninfected, MHC-I-matched targets did not activate the CD8⁺ T cell clones (data not shown). Data shown are mean ± SD for at least three independent experiments.

FIGURE 3

Gag-specific CD8⁺ T cells recognize infected cells by 2 h postinfection. Synchronously infected MHC-I-matched (■) and MHC-I-mismatched (large asterisk) CD4⁺ T cell targets were cocultured at an E:T of 1:1 with SIV-specific CD8⁺ T cell clones specific for Gag CM9 (A), Gag GY9 (B), Tat SL8 (C), or Env FW9 (D). Results are shown as a percentage of the maximum cytokine secretion detected during the assay. For Tat- and Env-specific CD8⁺ T cells, the infected targets used for the first 7 h were used as APCs at 24 h postinfection to obtain maximum responses. Data shown are mean ± SD for at least three independent experiments.

FIGURE 4

Early presentation of Gag-derived CD8⁺ T cell epitopes does not require de novo protein synthesis. CD4⁺ T cell targets (■) were infected with equal amounts of virus, as measured by Gag p27 content, of either AT2-inactivated SIVmac239 or infectious SIVmac239 in the presence of 400 μM Tenofovir to inhibit reverse transcription and then cocultured with CD8⁺ T cells specific for Gag CM9 (A), Gag GY9 (B), Tat SL8 (C), or Env FW9 (D). Cells infected with infectious SIVmac239 were pretreated with 400 μM Tenofovir for at least 2 h before infection and cultured with 400 μM tenofovir throughout the experiment to block reverse transcription and the KICS assay was performed as described. Data shown are mean ± SD for at least three independent experiments.

FIGURE 5

Titration of virus inoculum. Gag CM9-specific (A) or Gag GY9-specific (B) CD8⁺ T cell clones were cocultured with CD4⁺ T cell targets synchronously infected with decreasing amounts of virus, as measured by Gag p27 content, at an E:T of 1:1 (at 6 h postinfection). Positive results are underlined. Dot plots were generated by gating on live lymphocytes and CD8⁺ T cells, and results are shown as percentages of IFN-γ⁺ and/or TNF-α⁺ CD8⁺ T cells. Results are representative of at least two independent experiments.

FIGURE 6

Gag-specific CD8⁺ T cells eliminate infected cells early after infection. A Mamu-A*01⁺, Mamu-A*02⁺, and Mamu-B*17⁺ CD4⁺ target cell line was synchronously infected and cocultured at an E:T of 1:1 with either no CD8⁺ T cells (A), or with SIV-specific CD8⁺ T cell clones specific for Gag GY9 (B), Tat SL8 (C), or Env FW9 (D). E, To ensure that elimination of infected cells was MHC-I-dependent, a Mamu-A*01⁻, Mamu-A*02⁻, and Mamu-B*17⁻ CD4⁺ target cell line was synchronously infected and cocultured for 24 h with the CD8⁺ T cell clones described above. Dot plots were generated by gating on live, CD8⁻ cells. Data are representative of three independent experiments.

FIGURE 7

Multiple Gag-specific, not Env-specific, CD8⁺ T cell responses are linked with lower viremia in SIVmac239-infected macaques. Distribution of viral loads of 24 unvaccinated, SIV-infected rhesus macaques during the postacute phase according to number of responses (Δ, zero to two responses, ▼, three or more responses) against the entire length of Env (A) or Gag (B). Values of p were generated through independent group Student's t tests (n = 24).