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. 2011 Jun 26;17(7):830-6.
doi: 10.1038/nm.2395.

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

Mirko Paiardini  1 , Barbara Cervasi, Elane Reyes-Aviles, Luca Micci, Alexandra M Ortiz, Ann Chahroudi, Carol Vinton, Shari N Gordon, Steven E Bosinger, Nicholas Francella, Paul L Hallberg, Elizabeth Cramer, Timothy Schlub, Ming Liang Chan, Nadeene E Riddick, Ronald G Collman, Cristian Apetrei, Ivona Pandrea, James Else, Jan Munch, Frank Kirchhoff, Miles P Davenport, Jason M Brenchley, Guido Silvestri
Affiliations

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

Mirko Paiardini et al. Nat Med. .

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.

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Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

Figure 1
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 (TN), central memory (TCM) and effector memory (TEM) subsets of CD4+ and CD8+ T-cells of 18 SIV uninfected sooty mangabeys (SMs) and 30 SIV uninfected rhesus macaques (RMs). (a) TN, TCM, and TEM 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 TCM and TEM 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 ( formula image). 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
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 ( formula image) 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
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 ( formula image) 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 SIVmac239 infection of five RMs and (ii) non-progressive experimental SIVsmm 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
Figure 4. Lower fraction of CCR5+ cells after in vitro activation of sorted CD4+ TCM of SMs as compared to RMs
The fractions of sorted CD4+ TCM and TEM that express CCR5 were longitudinally determined after in vitro mitogen activation in eight SIV-uninfected SMs (■) and eight SIV-uninfected RMs ( formula image) TCM (CD95+CD62L+) and TEM (CD95+CD62L−) cells that were sorted and stimulated with ConA/IL-2. The graphs show the fraction of CD4+CCR5+ TEM (a) and TCM (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
Figure 5. CD4+ TCM of SMs are relatively resistant to SIV infection in vivo
The fraction of SIV-infected CD4+ (white boxes), CD4+ TCM (light gray) and TEM (dark gray) cells, determined measuring by q-PCR the number of SIVgag DNA copies for cell equivalent, was determined in 18 naturally SIVsmm infected SMs and 7 experimentally SIVmac239 infected RMs. As showed in the graph, the fraction of SIV-infected CD4+ TCM 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+ TEM. Of note, in RMs the fraction of SIV-infected cells was significantly higher (p=0.03) in CD4+ TCM than TEM.
Figure 6
Figure 6. CD4+ TCM of SMs are relatively resistant to SIV infection in vitro.
(a–d) CD4+ TCM and TEM purified from six SMs and eleven RMs were infected in vitro with SIVsmm M949 (six SMs and five RMs), or with SIVmac (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+ TCM than CD4+ TEM 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+ TCM where similar to those in CD4+ TEM following infection with SIVmac (b) or higher than CD4+ TEM following infection with SIVsmm (c). As a result, the TCM vs TEM 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 SIVsmm, and the fraction of infected SM CD4+ TCM and TEM that express GFP was determined by multiparametric flow cytometry at day 4 post-infection. Figure 6e shows flow plots of GFP expression in CD4+ TCM and TEM in two representative animals. In SMs the fraction of GFP+ cells (f) is approximately 1 log lower in CD4+ TCM than CD4+ TEM (p=0.0006).
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