Virus-specific T cell responses in macaques acutely infected with SHIV(sf162p3)

Abstract

CD4(+) T helper and CD8(+) cytotoxic T lymphocyte responses are believed to play an important role in the control of primary HIV and SIV infection. However, the role of these cells in macaques acutely infected with SHIV(sf162p3) has not been well characterized. In this study, ten adult rhesus macaques were intravaginally infected with SHIV(sf162p3), and antigen-specific cytokine responses to SHIV-Tat, Nef, Gag and Env peptide pools were examined through 70 days post inoculation (p.i.) using ELISPOT and/or cytokine flow cytometry (CFC). Peak plasma viral replication occurred between 14 and 21 days p.i. followed by low to undetectable plasma viremia by 70 days of infection in most macaques. Although some animals had strong virus-specific cellular immune responses, many had weak or minimal responses that did not correlate with the post peak decline in plasma viremia.

Figure 1

Plasma viral RNA levels from all SHIV_sf162p3 inoculated animals. Ten animals became infected while three (T887, EV63 and AA41) did not. Data shown is from the day of inoculation (day 0) through 84 days post inoculation (p.i.). Note that viral loads were undetectable in 9 out of 10 animals by 84 days p.i.

Figure 2

Absolute counts (top) and percentages (bottom) of CD4 and CD8 lymphocytes in peripheral blood of SHIV_sf162p3 infected (A & B) (n=10) and uninfected (C & D) (n=3) macaques. Data presented are means ± standard errors of CD4 and CD8 T cells at different time points of infection.

Figure 3

Absolute counts of naïve (CD28⁺CD95⁻), central memory (CD28⁺CD95⁺) and effector memory (CD28⁻CD95⁺) CD3⁺ CD4⁺ T cells in peripheral blood of SHIV_sf162p3 infected (A) and uninfected (B) macaques after inoculation. Six-color flow cytometry was performed to define the cell populations as described in Table 1.

Figure 4

(A). T cell proliferation assessed by %Ki67 expression is shown for CD4⁺ and CD8⁺ T cells. Both CD4⁺ and CD8⁺ T cell proliferation increased following SHIV_sf162p3 infection between 14 to 35 days post inoculation, but returned to baseline levels thereafter. (B). Representative expression of CD95 and Ki67 in CD4⁺ and CD8⁺ T cells in blood from a SHIV_sf162p3 infected macaque (DT54). Percentages of Ki67⁺ CD95⁺ T cells in either CD4⁺ or CD8⁺ T cells are shown in the box of each dot plot. Six-color flow cytometry staining was performed to define proliferating T cells as described in Table 1.

Figure 5

Intracellular cytokine flow cytometry for IFN-γ and TNF-α responses from a representative SHIV_sf162p3 infected macaque (L274). PBMC were unstimulated (medium control), or stimulated for 6 h with different peptide pools at day 56 post inoculation. Cells were gated first on lymphocytes followed by CD3⁺ and then on CD8⁺ T cells. The percentages of IFN-γ and/or TNF-α positive cells are shown in each quadrant. Bold numbers represent positive responses. Four color CFC staining was performed to define antigen specific cytokine responses as described in Table 1.

Figure 6

Plasma viral loads (line graphs) compared with SHIV-peptide specific T cell responses in seven SHIV_sf162p3 infected macaques following intravaginal infection. ELISPOT responses (IFN-γ-SFC/1×10⁶ cells) are shown on the left after stimulation with Tat, Gag and Env peptide stimulation (symbols). CFC data showing percentages of different antigen specific TNF-α⁺ T cells responding to peptide pools are shown in the right panels. Although all peptides were tested at each time point, only positive responses are shown. Note that while some animals (AK46, EV50) have persistent cellular responses to multiple peptides, other animals (DT91) have minimal responses, yet most animals have marked reductions in viral loads by 21 days of infection, regardless of the frequency or magnitude of cytokine responses tested.

Figure 7

Plasma viral loads in relation to SHIV-specific T cell responses in three SHIV_sf162p3 infected macaques after intravaginal infection. Panels were generated using CFC data and show plasma viral load (lines) compared with IFN-γ and TNF-α⁺ T cell responses after Tat, Nef, Gag and Env peptide stimulation (symbols). Although all peptides were tested at each time point, only positive responses are shown.