Low levels of SIV infection in sooty mangabey central memory CD⁴⁺ T cells are associated with limited CCR5 expression

Abstract

Naturally simian immunodeficiency virus (SIV)-infected sooty mangabeys do not progress to AIDS despite high-level virus replication. We previously showed that the fraction of CD4(+)CCR5(+) T cells is lower in sooty mangabeys compared to humans and macaques. Here we found that, after in vitro stimulation, sooty mangabey CD4(+) T cells fail to upregulate CCR5 and that this phenomenon is more pronounced in CD4(+) central memory T cells (T(CM) cells). CD4(+) T cell activation was similarly uncoupled from CCR5 expression in sooty mangabeys in vivo during acute SIV infection and the homeostatic proliferation that follows antibody-mediated CD4(+) T cell depletion. Sooty mangabey CD4(+) T(CM) cells that express low amounts of CCR5 showed reduced susceptibility to SIV infection both in vivo and in vitro when compared to CD4(+) T(CM) cells of rhesus macaques. These data suggest that low CCR5 expression on sooty mangabey CD4(+) T cells favors the preservation of CD4(+) T cell homeostasis and promotes an AIDS-free status by protecting CD4(+) T(CM) cells from direct virus infection.

Figure 1. The fraction of CCR5+ cells ex vivo is significantly lower in all subsets of CD4+ T-cells of SMs as compared to RMs

The fraction of CCR5+ cells was determined in the naive (T_N), central memory (T_CM) and effector memory (T_EM) subsets of CD4+ and CD8+ T-cells of 18 SIV uninfected sooty mangabeys (SMs) and 30 SIV uninfected rhesus macaques (RMs). (a) T_N, T_CM, and T_EM cells were defined based on the expression of the surface markers CD28, CD95, and CD62L, as showed for CD4+ T-cells in a representative SM. (b) Staining of CCR5 on T_CM and T_EM CD4+ and CD8+ cells in a representative SM and a representative RM. (c) Fraction of CD4+ (left graph) or CD8+ (right graph) T-cells that express CCR5 in 18 uninfected SMs (■) and 30 uninfected RMs ( ). Statistical analyses were performed to compare, in SMs versus RMs, the fraction of CCR5+ cells within each CD4+ and CD8+ T-cell subset.

Figure 2. CCR5 expression upon in vitro activation and proliferation is significantly lower in CD4+ T-cells of SMs than RMs

Fractions of CD4+CCR5+ T-cells were determined in PBMCs from uninfected SMs (■) and RMs ( ) after in vitro stimulations. Fraction of CD4+Ki-67+ (a) and CD4+CCR5+ (b) T-cells following stimulation with ConA/IL-2. Asterisks indicate time points when the fraction of CD4+CCR5+ T-cells was significantly lower in SMs than RMs (p values are detailed in the Result section). (c) Representative dot plots showing the fraction of CD4+CCR5+ T-cells post-stimulation in RMs (top) and SMs (bottom). (d) Flow cytometry dot plots showing Ki-67/CCR5 double staining in a representative RM and SM at 120 hours post stimulation with ConA/IL-2. (e) PBMCs isolated from RMs and SMs were labeled with CFSE prior to mitogen stimulation; levels of CCR5 were analyzed on cells expressing various levels of CFSE dilution at 120 hours post-stimulation. (f) and (g) PBMCs isolated from SMs and RMs were stimulated with recombinant IL-7, and the fraction of CD4+Ki-67+ (f) and CD4+CCR5+ (g) T-cells determined following stimulation. Asterisks indicate time points where, in RMs, the IL-7-induced increase in CD4+CCR5+ T-cells (as compared to baseline) was statistically significant (p values are detailed in the Result section). (h) In a subset of animals, levels of CCR5 mRNA (expressed as CCR5/GAPDH ratio) were determined on purified CD4+ T-cells at 0, 24, 72 and 120 hours post-stimulation with ConA/IL-2. Statistical analyses were performed to compare CCR5/GAPDH ratio between SMs and RMs.

Figure 3. The fraction of CD4+CCR5+ T-cells upon in vivo activation is significantly lower in SMs as compared to RMs

We determined the fraction of CD4+CCR5+ T-cells from SMs (■) and RMs ( ) in two in vivo experimental conditions associated with activation of the CD4+ T-cell compartment, i.e., acute SIV-infection and Ab-mediated CD4+ T-cell depletion. Fraction (a) and fold change vs pre-infection, i.e. day 0 (b) of CD4+CCR5+ T-cells at different time points during (i) pathogenic SIV_mac239 infection of five RMs and (ii) non-progressive experimental SIV_smm infection of four SMs. SIV-induced CD4+ T-cell activation is associated with an increased fraction of CD4+CCR5+ T-cells in RMs, but not in SMs, with a statistically significant difference in the area under the curve (AUC). Fraction (c) and fold change vs pre-depletion, i.e. day -14 (d) of CD4+CCR5+ T-cells at different time points following Ab-mediated CD4+ T-cell depletion in three uninfected RMs and three uninfected SMs. The AUC of the fraction of CD4+CCR5+ T-cells is significantly higher in RMs than SMs. The dotted lines in (c,d) indicate the 10-day period in which the anti-CD4 antibody was administered.

Figure 4. Lower fraction of CCR5+ cells after in vitro activation of sorted CD4+ T_CM of SMs as compared to RMs

The fractions of sorted CD4+ T_CM and T_EM that express CCR5 were longitudinally determined after in vitro mitogen activation in eight SIV-uninfected SMs (■) and eight SIV-uninfected RMs ( ) T_CM (CD95+CD62L+) and T_EM (CD95+CD62L−) cells that were sorted and stimulated with ConA/IL-2. The graphs show the fraction of CD4+CCR5+ T_EM (a) and T_CM (b) cells at different time points following stimulation. Asterisks indicate time points where values are significantly higher in RMs as compared to SMs.

Figure 5. CD4+ T_CM of SMs are relatively resistant to SIV infection in vivo

The fraction of SIV-infected CD4+ (white boxes), CD4+ T_CM (light gray) and T_EM (dark gray) cells, determined measuring by q-PCR the number of SIVgag DNA copies for cell equivalent, was determined in 18 naturally SIV_smm infected SMs and 7 experimentally SIV_mac239 infected RMs. As showed in the graph, the fraction of SIV-infected CD4+ T_CM was significantly lower in SMs than in RMs (p=0.0008), while no significant difference was observed between the two species with respect to the level of SIV-infected CD4+ T_EM. Of note, in RMs the fraction of SIV-infected cells was significantly higher (p=0.03) in CD4+ T_CM than T_EM.

Figure 6. CD4+ T_CM of SMs are relatively resistant to SIV infection in vitro.

(a–d) CD4+ T_CM and T_EM purified from six SMs and eleven RMs were infected in vitro with SIVsmm M949 (six SMs and five RMs), or with SIV_mac (six RMs). The levels of SIV replication were determined by measuring p27 in the supernatants. In SMs (a) the levels of SIV replication were significantly lower in CD4+ T_CM than CD4+ T_EM at days 6, 9, 12, and 15 post-infection (as indicated by asterisks; p values are detailed in the Result section), while in RMs the levels of SIV replication in CD4+ T_CM where similar to those in CD4+ T_EM following infection with SIVmac (b) or higher than CD4+ T_EM following infection with SIVsmm (c). As a result, the T_CM vs T_EM ratios of levels of SIV replication were significantly higher (as indicated by asterisks; p values are detailed in the Result section) in RMs than SMs at all tested time points (d). (e, f) Unfractionated PBMCs from eight SMs were infected with replication competent eGFP-expressing clones of SIV_smm, and the fraction of infected SM CD4+ T_CM and T_EM that express GFP was determined by multiparametric flow cytometry at day 4 post-infection. Figure 6e shows flow plots of GFP expression in CD4+ T_CM and T_EM in two representative animals. In SMs the fraction of GFP+ cells (f) is approximately 1 log lower in CD4+ T_CM than CD4+ T_EM (p=0.0006).