Baboon CD8 T cells suppress SIVmac infection in CD4 T cells through contact-dependent production of MIP-1α, MIP-1β, and RANTES

Abstract

Simian immunodeficiency virus (SIV) infection in rhesus macaques is often characterized by high viremia and CD4 T cell depletion. By contrast, SIV infection in African nonhuman primate natural hosts is typically nonpathogenic despite active viral replication. Baboons are abundant in Africa and have a geographical distribution that overlaps with natural hosts, but they do not harbor SIVs. Previous work has demonstrated baboons are resistant to chronic SIV infection and/or disease in vivo but the underlying mechanisms remain unknown. Using in vitro SIVmac infections, we sought to identify SIV restriction factors in baboons by comparing observations to the pathogenic rhesus macaque model. SIVmac replicated in baboon PBMC but had delayed kinetics compared to rhesus PBMC. However, SIVmac replication in baboon and rhesus isolated CD4 cells were similar to the kinetics seen for rhesus PBMC, demonstrating intracellular restriction factors do not play a strong role in baboon inhibition of SIVmac replication. Here, we show CD8 T cells contribute to the innate SIV-suppressive activity seen in naïve baboon PBMC. As one mechanism of restriction, we identified higher production of MIP-1α, MIP-1β, and RANTES by baboon PBMC. Contact between CD4 and CD8 T cells resulted in maximum production of these chemokines and suppression of viral replication, whereas neutralization of CCR5-binding chemokines in baboon PBMC increased viral loads. Our studies indicate baboon natural restriction of SIVmac replication is largely dependent on CD4-extrinsinc mechanisms mediated, in part, by CD8 T cells.

Keywords: AIDS; Baboon; CCR5; Chemokine; Cytokine.

FIG 1

SIVmac251 growth is restricted in baboon PBMC. PBMC were isolated from whole blood of rhesus macaques (open circles) or baboons (solid circles) then cultured for 48 hr in (A) RPMI-10 or (B) RPMI-10 containing PHA. PBMC were then infected with SIVmac251 at an M.O.I. of 0.01. Viral loads were quantified by measuring p27 in the supernatant by Luminex. In both panels, each curve represents one animal (n = 10). Repeated measures two-way ANOVA was used for statistical analysis (*P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001).

FIG 2

Baboon and rhesus macaque PBMC have similar levels of SIV target cells and entry receptors. PBMC were isolated from whole blood of rhesus macaques (open circles, n = 9) and baboons (solid circles, n = 8). PBMC were phenotyped by flow cytometry following isolation (Fresh) and after 48 hr in culture with RPMI-10 (RPMI) or RPMI-10 containing PHA. A) Percentage of CD4⁺ T cells within lymphocytes and percentage of CCR5⁺ cells within CD4 ⁺ T cells. B) Median fluorescence intensity (MFI) of CD4 and CCR5 expression on CD4 T cells. In all panels, lines indicate the median and each point represents one animal. Mann-Whitney tests were used for statistical analysis (***P ≤ 0.001). n.s.: not significant.

FIG 3

The levels of SIVmac251 binding, intracellular replication, and egress are similar between baboon and rhesus macaque isolated CD4 cells. CD4 cells were sorted from baboon (dark bars) or rhesus macaque (light bars) PBMC and cultured for 48 hr before infection. Cells were infected with SIVmac251 at an M.O.I. of 1; viral binding was synchronized by keeping cells at 4°C for 1 hr. (A) Cells were washed twice before RNA extraction; SIV RNA was measured by real-time RT-PCR, using GAPDH mRNA to normalize RNA input and for relative quantification (2 ^Ct) (n = 5). (B-C) After incubation at 4°C, cells were placed at 37°C for 1 hr to allow entry. A protease inhibitor, darunavir, was added to cultures to restrict the virus to a single replication cycle. Cells were lysed at indicated time points and SIV DNA was measured by real-time PCR using Oncostatin-M gene to normalize DNA input (n = 3). (D) Supernatant was harvested at 48 h.p.i. and SIV RNA was quantified by real-time RT-PCR (n = 5). In all panels, bars represent the mean ± SD. n.s.: not significant.

FIG 4

SIVmac251 growth is similar in baboon and rhesus macaque isolated CD4 cells. (A) CD4 cells were sorted from baboon (closed triangles, n = 10) or rhesus macaque (open triangles, n = 8) PBMC, cultured for 48 hr in RPMI-10, then infected with SIVmac251 at a M.O.I of 0.01. (B-C) Species-matched compilation of viral loads data from infections in PBMC (shown in Fig. 1A) and sorted CD4 cells (shown in Fig. 4A). (D) PBMC and sorted CD4 cells from the same baboon donor (n = 5) were infected with SIVmac251 at a MOI of 0.01. (E) PBMC were isolated from STLV seronegative (n = 5) and seropositive (n = 5) baboon donors of the same age (4 y/o). PBMC were infected with SIVmac251 at a MOI of 0.01. Viral loads were quantified by measuring p27 in the supernatant by Luminex. In all panels, each curve represents one animal. Repeated measures two-way ANOVA was used for statistical analysis (*P ≤ 0.05, ****P ≤ 0.0001).

FIG 5

CD8 T cells suppress SIVmac251 infection in baboon CD4 cells. Sorted CD4 cells were infected with SIVmac251 and cultured in isolation or with the addition of autologous CD8 T and NK cells. (A-B) The same number of SIV-infected CD4 cells from baboons (n = 6) or rhesus macaques ( n = 3) were cultured alone, with CD8 T cells, or with NK cells. (C) CD8 T and NK cells were cultured with infected CD4 cells in contact or separated by a 0.4 μm membrane (Transwell). In all panels, viral loads were quantified by measuring p27 in the supernatant by Luminex. Bar graphs display SIV p27 values normalized to the CD4 alone condition at peak infection (10–14 d.p.i.) (mean ± SEM). One-way ANOVA was used for statistical analysis (*P ≤ 0.05, ****P ≤ 0.0001). n.s.: not significant.

FIG 6

Elevated production of CCR5-binding chemokines contributes to SIVmac251 restriction in baboon PBMC. (A) Identification of MIP-1α, MIP-1β, and RANTES in PBMC supernatants at 3 d.p.i. PBMC were isolated from baboons (dark bars) and rhesus macaques (light bars) and cultured for 48 hr in medium alone (RPMI) or medium containing PHA then infected with SIVmac251at an M.O.I. of 0.01 (n = 7). Mann-Whitney tests were used for statistical analysis. Top and bottom of boxes represent first and third quartile, band is median, whiskers show minimum and maximum values. (B-C) PBMC were infected with SIVmac251 at an M.O.I. of 0.01. Cultures were treated with medium, normal goat serum, or goat polyclonal anti-chemokine neutralizing antibodies. Viral loads were assessed by measuring p27 in the supernatant by Luminex. Bar graphs display p27 MFI values normalized to the untreated condition at peak infection (10 d.p.i.) (mean ± SEM) (RPMI: n = 6, PHA: n = 5). Paired t-test was used for statistical analysis. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). (D-E) Correlation analysis for total CCR5-binding chemokine concentration at 3 d.p.i. versus log of SIV p27 concentrations at 10 d.p.i. in baboon (closed 20 circles, n = 14) or rhesus macaque (open circles, n = 13) PBMC. Line represents linear regression and each point represents a different donor.

FIG 7

CD8 T and NK cells contribute to high levels of CCR5-binding chemokines in baboon cultures. Luminex cytokine assays performed on supernatants obtained from cultures at 3.d.p.i. (A) CD8 T and NK cells were cultured alone, with uninfected or infected CD4 cells in the indicated combinations (n = 4 - 6). (B) Infected CD4 T cells from baboons (n = 6) and rhesus macaques (n = 3) were cultured with autologous CD8 T and NK cells. Graph shows total CCR5-binding chemokine levels. (C) CD8 T and NK cells were cultured with infected CD4 cells separated by a 0.4 μm membrane (Transwell) or in contact (n = 6). For box and whiskers plots, top and bottom of box represent first and third quartile, band is median, whiskers show minimum and maximum values. One-way ANOVA was used for statistical analysis (*P ≤ 0.05).