Abstract

African green monkeys (genus Chlorocebus) can be infected with species-specific simian immunodeficiency virus (SIVagm) but do not develop AIDS. These natural hosts of SIV, like sooty mangabeys, maintain high levels of SIV replication but have evolved to avoid immunodeficiency. Elucidating the mechanisms that allow natural hosts to coexist with SIV without overt disease may provide crucial information for understanding AIDS pathogenesis. Here we show that many CD4⁺ T cells from African green monkeys downregulate CD4 in vivo as they enter the memory pool; that downregulation of CD4 by memory T cells is independent of SIV infection; that the CD4^- memory T cells maintain functions that are normally attributed to CD4⁺ T cells, including production of interleukin-2 (IL-2), production of IL-17, expression of forkhead box P3 and expression of CD40 ligand; that loss of CD4 expression protects these T cells from infection by SIVagm in vivo; and that these CD4^- T cells can maintain major histocompatibility complex class II restriction. These data show that the absence of SIV-induced disease progression in natural host species may be partially explained by preservation of a subset of T cells that maintain CD4⁺ T cell function while being resistant to SIV infection in vivo.

Introduction

SIV belongs to the group of lentiviruses that infect nonhuman primates. The lentiviruses that cause immunodeficiencies in humans and Asian macaques originated from cross-species transmission of viruses that naturally infect nonhuman primates in Africa¹. Like HIV-1 and HIV-2, all known SIV subtypes use CD4 as a receptor and either CCR5, CXCR4, or, as for the red capped mangabey virus, SIVrcm, CCR2^{2, 3, 4} as co-receptors. Moreover, both SIV infection of Asian macaques and HIV-1 infection of humans result in chronic infection, and the majority of infected individuals progress to AIDS.

In contrast, after SIV infection, natural hosts generally do not progress to AIDS. Because natural hosts of SIV have coevolved with the virus to avoid disease progression, dissecting the mechanisms underlying the nonprogressive nature of natural SIV infection will lead to a better understanding of the aspects of HIV infection responsible for the progressive nature of the disease in humans^{5, 6, 7}. Natural hosts do not avoid disease progression by immunological control of the virus, as SIV-infected natural hosts maintain high levels of viremia^{8, 9, 10, 11, 12}. Moreover, experimental depletion of CD8⁺ T cells does not affect plasma viremia¹³, and natural hosts do not show superior cellular control of viremia compared to HIV-infected humans or SIV-infected rhesus macaques¹⁴. The lentiviruses that infect natural hosts can be pathogenic. SIV infections of African green monkeys and sooty mangabeys have been correlated with short life spans of infected cells in vivo^{15, 16, 17}. Moreover, SIVagm, which naturally infects African green monkeys, can be used to infect pigtail macaques, who subsequently manifest simian AIDS^{18, 19}. Isolates of the sooty mangabey virus, SIVsmm, can also cause progressive infection in rhesus macaques^{20, 21, 22, 23}. One fundamental difference between progressive SIV and HIV infections and nonprogressive SIV infection is the absence of immune activation, associated with disease progression in HIV-infected individuals²⁴, during the chronic phase of infection in natural hosts^{9, 11, 25, 26, 27, 28}.

Previous studies performed in African green monkeys have reported very low frequencies of CD4⁺ T cells²⁸. African green monkeys, however, remain disease free despite having low numbers of CD4⁺ T cells. In two reports, the investigators found low frequencies of CD4⁺ T cells and high frequencies of CD8^dim T cells in healthy adult African green monkeys^{29, 30}. They also found that the CD8^dim T cells could induce antibody production from B cells in vitro and suggested that the CD8^dim T cells might act as substitutes for the CD4⁺ T cells²⁹. Here we have studied the frequencies, functionalities and in vivo infection frequencies of lymphocyte subsets from 36 African green monkeys: ten naturally infected, eight experimentally infected, eleven uninfected adults and seven uninfected juveniles (Table 1). Our data describe a mechanism by which African green monkeys are able to survive SIVagm infection without succumbing to AIDS.

Table 1: Clinical information for African green monkeys

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Results

Inverse correlation between CD4⁺ and CD4^-CD8^dim T cells

Loss of CD4⁺ T cells is a hallmark of progression to AIDS in humans and Asian macaques. We therefore compared the frequencies of CD4⁺ T cells in SIVagm-infected and uninfected vervet African green monkeys (one of four subspecies of African green monkeys), HIV-uninfected humans, SIV-uninfected rhesus macaques and SIVsmm-infected and uninfected sooty mangabeys. We observed three distinct T cell populations in African green monkeys, on the basis of expression of CD4 and CD8: CD4⁺ T cells, CD4^-CD8^dim T cells and CD4^-CD8^bright T cells (Fig. 1a). We further analyzed the phenotypes of each subset on the basis of expression of CD28 and CD95 (Fig. 1b). The CD4⁺ and CD4^-CD8^bright T cells consisted of both memory and naive subsets (Fig. 1b). However, the CD4^-CD8^dim T cells consisted predominantly of memory T cells (Fig. 1b). Moreover, adult African green monkeys seemed to have unexpectedly low frequencies of CD4⁺ T cells and low CD4⁺ T cell counts (Table 1). There were significantly lower frequencies of CD4⁺ T cells in both SIVagm-infected and uninfected African green monkeys compared to SIVsmm-infected or uninfected sooty mangabeys, SIV-uninfected rhesus macaques or HIV-1–uninfected humans (Fig. 1c). In addition, the decrease in CD4⁺ T cells was accompanied by an increase in the frequency of CD4^-CD8^dim T cells (Fig. 1a). Further phenotypic analysis showed that this CD4^-CD8^dim T cell population lacked expression of CD8 (Fig. 1d). In addition, the frequency of activated Ki67⁺ cells was significantly lower among CD4^-CD8^dim T cells compared to either the CD4⁺ or CD4^-CD8^bright subsets, suggesting that the high frequency of CD4^-CD8^dim T cells was not due to preferential proliferation of CD4^-CD8^dim T cells in vivo (Fig. 1e).

Figure 1: Phenotypic analysis of T cell populations in vervet African green monkeys.

(a) Phenotype of T cells in adult African green monkeys, as determined by flow cytometry. (b) Phenotype of individual subsets of T cells in adult African green monkeys, as determined by flow cytometry. (c) Comparison of percentages of CD3⁺ T cells that express CD4 in peripheral blood in SIV⁺ and SIV^- adult African green monkeys (AGMs), adult SIV⁺ and SIV^- sooty mangabeys (SMs), adult SIV^- rhesus macaques (RMs) and HIV^- adult humans. (d) Characterization of CD4^-CD8^dim T cells as CD8^- by analysis of CD8 expression. Cells were gated on live CD3⁺ small lymphocytes and analyzed for both CD4 and CD8 expression. (e) Ki67 expression by various subsets of memory T cells. (f) Negative correlation between the frequencies of CD4⁺ T-cell and CD4^-CD8^dim T cells in adult AGMs. (g) Correlation of the frequency of CD4⁺ T cells and the frequency of CD4^-CD8^bright T cells in adult African AGMs. A Mann-Whitney U test was performed for c. A Spearman rank correlation was calculated for f and g. Lines in c and e represent median values, and percentages in a and b represent frequencies of boxed cells.

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The increased frequency of CD4^-CD8^dim T cells could be a reflection of a mathematical shift in the percentages of T cell populations due to the loss of CD4⁺ T cells. However, we found that there was a significant negative correlation between the decrease in CD4⁺ T cells and the increase in CD4^-CD8^dim T cells (Fig. 1f), but not between the CD4^-CD8^bright T cells and CD4⁺ T cells (Fig. 1g). Therefore, the increase in the CD4^-CD8^dim T cell population was directly related to the decrease in CD4⁺ T cells, suggesting that some CD4^-CD8^dim T cells might develop from CD4⁺ T cells.

Some CD4^-CD8^dim T cells develop from CD4⁺ memory T cells

Given that the frequencies of the CD4^-CD8^dim T cells negatively correlated with the frequencies of CD4⁺ T cells (Fig. 1f), we hypothesized that some of the CD4^-CD8^dim T cells might have developed from CD4⁺ T cells. Phenotypic analysis of the CD4⁺ T cells from SIVagm-infected and uninfected African green monkeys illustrated that the CD95⁺ memory CD4⁺ T cells downregulated CD4 (Fig. 2a) and upregulated CD8 (Fig. 2b) compared to naive CD4⁺ T cells. We also performed fluorescence-minus-one analysis for CD8 to confirm that the CD4⁺ T cells expressed CD8 (Fig. 2c). The median fluorescent intensity (MFI) of CD4 was significantly lower in the memory subset of CD4⁺ T cells compared to naive CD4⁺ T cells (Fig. 2d), whereas the MFI of CD8 was significantly higher in the memory CD4⁺ T cells over naive CD4⁺ T cells (Fig. 2e). Therefore, we hypothesized that upon stimulation CD4⁺ T cells are able to transition to become CD4^-CD8^dim T cells.

Figure 2: CD8 and CD4 expression by naive and memory CD4⁺ T cells.

(a) Representative staining of CD4⁺ memory and naïve T cells for CD4, as determined by flow cytometry. (b) Representative staining of CD4⁺ memory and naive T cells for CD8, as determined by flow cytometry. (c) Fluorescence-minus-one (FMO) analysis by flow cytometry for all markers except CD8. (d) Median fluorescence intensity for CD4 expression in naive and memory CD4⁺ T cells. (e) MFI for CD8 expression in naive and memory CD4⁺ T cells. A Wilcoxon matched-pairs test was performed for d and e.

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To study downregulation of CD4 in vitro, we CFSE-labeled and mitogenically stimulated peripheral blood mononucelear cells (PBMCs) from several African green monkeys and, for comparison, from pigtail macaques. We then studied expression of CD4 and proliferation by the cells by flow cytometry after 5, 7 and 9 d in culture (Fig. 3a,b and Supplementary Fig. 1). We found that, whereas African green monkey CD4⁺ T cells lost CD4 expression, CD4⁺ T cells from pigtail macaques maintained CD4 expression with cell division (Fig. 3a). It is possible that the CD4 downregulation in African green monkeys was transient; however, we did not see upregulation of CD4 by day 9 (Supplementary Fig. 1). To confirm this finding, we also sorted naive CD4⁺ T cells (>99% pure) before stimulation and found that after stimulation they became CD4^- (Supplementary Fig. 1).

Figure 3: CD4^-CD8^dim T cells can develop from memory CD4⁺ T cells.

(a) Flow cytometric analysis of CD4 downregulation by AGM PBMCs after 5 d of stimulation in vitro with SEB. (b) Flow cytometric analysis of CD4 expression by stimulated PBMCs from pigtail macaques in vitro after 5 d of stimulation with SEB. (c) CD4 mRNA expression in CD14⁺, CD4^-CD8^dim, CD4^-CD8^bright or CD4⁺ lymphocyte subsets of an adult African AGM. Bp, base pairs. (d) Negative correlation between the frequency of memory CD4⁺ T cells and the frequency of the total CD4⁺ T cells in adult AGMs. (e) Comparison of the percentage CD4⁺ T cells in peripheral blood in SIV⁺ and SIV^- adult and SIV^- juvenile AGMs. A Spearman rank correlation was calculated for d. A Mann-Whitney U test was performed for e. Horizontal lines in e depict the median value.

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Analysis for CD4 messenger RNA within sorted CD4⁺, CD4^-CD8^dim or CD4^-CD8^bright T cells and monocytes revealed that CD4 mRNA was only detectable within CD4⁺ T cells (Fig. 3c). Taken together, these data suggested that African green monkey memory CD4⁺ T cells can become CD4^-CD8^dim T cells. If our hypothesis was correct, African green monkeys would have concomitant decreases in the overall frequencies of total CD4⁺ T cells with increases in memory CD4⁺ T cells. Indeed, we found a significant negative correlation between the frequencies of memory CD4⁺ T cells and the total frequency of CD4⁺ T cells (Fig. 3d). Hence, as African green monkeys accumulate memory CD4⁺ T cells, the overall frequency of CD4⁺ T cells decreases.

We reasoned that if some CD4⁺ T cells become CD4^-CD8^dim T cells upon stimulation into the memory pool, then African green monkeys with very little antigenic experience should have frequencies of CD4⁺ T cells comparable to those of healthy rhesus macaques and humans (Fig. 1c). Therefore, we measured the frequencies of T cell subsets from PBMCs obtained ex vivo directly from juvenile African green monkeys (all fewer than 2 years old). In a total of seven juvenile African green monkeys, the median frequency of CD4⁺ T cells was 59% (39.6–64.4%, Fig. 3e). These frequencies were comparable with those from nonimmunocompromised rhesus macaques and humans and were higher than the frequencies of CD4⁺ T cells in adult African green monkeys (P<0.0006, Fig. 3e). Furthermore, after we vaccinated the juvenile African green monkeys with standard influenza vaccine, we observed an influenza-specific T cell response from the CD4^-CD8^dim T cells to the major histocompatibility complex class II (MHC-II)-restricted antigen (Supplementary Fig. 2c). Taken together, these observations strongly suggest that many of the CD4^-CD8^dim T cells developed from memory CD4⁺ T cells.

Although we do not know the ages of many of the African green monkeys, as they were imported from Tanzania, in the few monkeys for whom we do have this information, there was no correlation between age and the frequency of CD4⁺ T cells (data not shown). However, it is likely that age may be a factor in the accumulation of CD4^-CD8^dim T cells, as older monkeys are exposed to more antigens in vivo.

Function and SIVagm infection of CD4^-CD8^dim T cells in vivo

To elucidate the functions of the CD4^-CD8^dim T cells, we obtained PBMCs from SIVagm-infected and uninfected adult African green monkeys. As it seemed apparent that many of the CD4^-CD8^dim T cells developed from CD4⁺ T cells, we examined these T cells for functions generally attributed to CD4⁺ T cells. We found that upon stimulation with staphylococcal enterotoxin B (SEB), the CD4^-CD8^dim T cells could produce IL-2 and IL-17 (Supplementary Fig. 2 and Fig. 4a) and expressed CD40 ligand (CD40L) (Fig. 4a and Supplementary Fig. 2). CD40L is typically expressed by activated CD4⁺ T cells, resulting in enhancement of antigen presentation and induction of B cell class switching³¹. Additionally, a portion of the CD4^-CD8^dim T cells expressed the transcription factor forkhead box P3 (FoxP3), thought to be predominantly expressed by regulatory CD4⁺ T cells (Fig. 4a and Supplementary Fig. 2)³². Hence, the CD4^-CD8^dim T cell subset includes T cells that perform functions normally restricted to CD4⁺ T cells. Although the overall frequency of memory CD4⁺ T cells that could perform each function was significantly higher compared to the CD4^-CD8^dim T cells (Fig. 4a), the total frequency of CD4^-CD8^dim T cells was significantly higher than the overall frequency of CD4⁺ T cells (Fig. 1a). We therefore calculated the relative number of cytokine-expressing, CD40L⁺ and FoxP3⁺ T cells for each T cell subset (Fig. 4b). For IL-2, IL-17 and FoxP3, the relative number of CD4^-CD8^dim T cells was significantly larger than the numbers of the CD4⁺ and CD4^-CD8^bright T-cell subsets (Fig. 4b). The relative number of CD40L⁺CD4^-CD8^dim T cells was equal to the number of CD40L⁺CD4⁺ T cells (Fig. 4b). These observations suggest that there are actually greater numbers of CD4^-CD8^dim T cells performing CD4⁺ T cell functions compared to classical CD4⁺ T cells.

Figure 4: CD4^-CD8^dim T cells can preserve CD4⁺ T-cell function.

(a) Comparison of the frequency of memory CD4⁺, CD4^-CD8^dim and CD4^-CD8^bright T cells for IL-2 and IL-17 production and FoxP3 and CD40L expression. (b) Comparison of the relative numbers of memory CD4⁺, CD4^-CD8^dim and CD4^-CD8^bright T cells producing IL-2 and IL-17 and expressing FoxP3 and CD40L. (c) Responses of CD4^-CD8^dim T cells to CMV whole antigen (ag) in the presence or absence of blocking antibodies to MHC-II or MHC-I. IFN-, interferon-. Percentages represent frequencies of boxed cells. A Wilcoxon matched-pairs test was performed for a and b.

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CD4⁺ T cells are restricted by MHC-II, whereas CD8⁺ T cells are restricted by MHC-I. Therefore, to test further our hypothesis that the CD4^-CD8^dim T cells were acting as CD4⁺ T cells, we screened PBMCs from adult African green monkeys for T cell responses to a cytomegalovirus (CMV) whole-antigen preparation. Presentation of this antigen requires processing through MHC-II³³. We found one African green monkey that had T cells responsive to CMV (AG731) whose entire CMV-specific T cell response was restricted to CD4^-CD8^dim T cells (Fig. 4c and Supplementary Fig. 2b,c). To confirm that these CMV-specific CD4^-CD8^dim T cells were restricted by MHC-II, we stimulated the T cells in the presence of blocking antibodies specific to either MHC-II or MHC-I and measured production of cytokines. Blocking with MHC-II–specific antibody decreased the frequency of responding CMV-specific CD4^-CD8^dim T cells by more than two thirds, whereas blocking MHC-I had virtually no effect on the ability of CMV-specific CD4^-CD8^dim T cells to respond (Fig. 4c). Hence, CD4^-CD8^dim T cells can be restricted by MHC-II in African green monkeys.

Vervets are one of four subspecies of African green monkey, along with sabeus, tantalus and grivets. To determine whether the phenomena that we observed are specific to vervet African green monkeys, we examined T cell populations in six adult sabeus African green monkeys. Indeed, we found CD4^-CD8^dim T cells among their T cell populations (Supplementary Fig. 3). Similar to the case in vervets, the CD4^-CD8^dim T cells from sabeus were able to perform CD4⁺ T cell functions (Supplementary Fig. 3). Confirmation of this phenomenon occurring in a second subspecies of African green monkeys suggests that downregulation of CD4 may be characteristic of all African green monkeys.

Since the CD4^-CD8^dim T cells have many functional characteristics of CD4⁺ T cells, we next determined which lymphocytes were infected by SIVagm in vivo. We used flow cytometry to sort individual subsets of lymphocytes from 18 SIVagm-infected African green monkeys and measured the in vivo infection frequency by quantitative real-time PCR for SIVagm DNA. We sorted naive and memory CD4⁺ T cells, memory CD4^-CD8^dim T cells, memory CD4^-CD8^bright T cells, memory CD4^-CD8^- T cells and monocytes. We found that memory CD4⁺ T cells were the primary targets for SIVagm in vivo (Fig. 5). Consistent with the infection frequency patterns of HIV³⁴, we also found that SIVagm could infect naive CD4⁺ T cells, but memory CD4⁺ T cells were preferentially infected (Fig. 5). Notably, CD4^-CD8^dim T cells, which we have shown can develop from CD4⁺ T cells and maintain functions of CD4⁺ T cells, were only very rarely, if ever, infected by SIVagm in vivo (Fig. 5). In 78% of the SIVagm-infected African green monkeys, we detected no viral DNA within the CD4^-CD8^dim T cell subset (Fig. 5). Cell numbers were often limiting, and it is conceivable that, in some cases, we did not detect any viral DNA as a result of the small number of sorted cells in each PCR. Therefore, in samples in which we did not detect viral DNA, we reported values calculated as one-half of the lower limit of detection; these values are based on the number of sorted cells (Fig. 5). In the few (22%) African green monkeys in whom we did detect viral DNA within the CD4^-CD8^dim T cell subset, the infection frequencies were very low (<0.01%). Taken together, these data suggest that CD4^-CD8^dim T cells preserve CD4⁺ T cell function while evading SIV infection in vivo, and, in turn, these findings suggest a mechanism by which African green monkeys are able remain disease free despite SIV infection.

Figure 5: Infection frequencies of lymphocyte subsets.

Infection frequencies of sorted lymphocyte subsets from SIVagm-infected adult AGMs, as determined by quantitative PCR for viral DNA. White circles represent cells with detectable viral DNA, and black circles represent an undetectable infection frequency reported as one-half the lower limit of detection on the basis of the number of cells within each PCR reaction. A Wilcoxon matched pairs test was performed for this analysis.

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Note: Supplementary information is available on the Nature Medicine website.

Acknowledgments

These studies were supported by the intramural National Institute of Allergy and Infectious Diseases, US National Institutes of Health program and by R01 AI064066 (I.P.), R01 AI065325 (C.A.) and RR-00168 (Tulane National Primate Center). We would like to thank the Bad Boys of Cleveland, D. Douek (Vaccine Research Center, National Institute of Allergy and Infectious Diseases, US National Institutes of Health) and D. Price (Cardiff University) for helpful discussions. We are grateful to J.E. Schmitz and R. Zahn (Harvard University) for the kind donation of microbeads coated with antibody to CD3 and CD28 for stimulation of T cells from nonhuman primates. We also appreciate the technical advice of B. Lafont and G. Mettler.

Author Contributions

C.M.B., L.D.H., N.R.K., S.W., J.M. and J.M.B. performed experiments and analyzed the data. S.G., C.A., I.P. and V.M.H. provided specimens and analyzed data. All authors contributed to the project's planning and writing of the manuscript. J.M.B. supervised the project.

Received 5 December 2008; Accepted 28 April 2009; Published online 14 June 2009.

References

1. Hahn, B.H., Shaw, G.M., De Cock, K.M. & Sharp, P.M. AIDS as a zoonosis: scientific and public health implications. Science 287, 607–614 (2000). | Article | PubMed | ISI | ChemPort |
2. Ling, B., Veazey, R.S. & Marx, P.A. Nonpathogenic CCR2-tropic SIVrcm after serial passage and its effect on SIVmac infection of Indian rhesus macaques. Virology 379, 38–44 (2008). | Article | PubMed | ChemPort |
3. Zhang, Y. et al. Use of inhibitors to evaluate coreceptor usage by simian and simian/human immunodeficiency viruses and human immunodeficiency virus type 2 in primary cells. J. Virol. 74, 6893–6910 (2000). | Article | PubMed | ISI | ChemPort |
4. Beer, B.E. et al. Characterization of novel simian immunodeficiency viruses from red-capped mangabeys from Nigeria (SIVrcmNG409 and -NG411). J. Virol. 75, 12014–12027 (2001). | Article | PubMed | ChemPort |
5. Pandrea, I., Sodora, D.L., Silvestri, G. & Apetrei, C. Into the wild: simian immunodeficiency virus (SIV) infection in natural hosts. Trends Immunol. 29, 419–428 (2008). | Article | PubMed | ChemPort |
6. Silvestri, G., Paiardini, M., Pandrea, I., Lederman, M.M. & Sodora, D.L. Understanding the benign nature of SIV infection in natural hosts. J. Clin. Invest. 117, 3148–3154 (2007). | Article | PubMed | ChemPort |
7. Hirsch, V.M. What can natural infection of African monkeys with simian immunodeficiency virus tell us about the pathogenesis of AIDS? AIDS Rev. 6, 40–53 (2004). | PubMed |
8. Goldstein, S. et al. Comparison of simian immunodeficiency virus SIVagmVer replication and CD4⁺ T cell dynamics in vervet and sabaeus African green monkeys. J. Virol. 80, 4868–4877 (2006). | Article | PubMed | ChemPort |
9. Pandrea, I. et al. Simian immunodeficiency virus SIVagm.sab infection of Caribbean African green monkeys: a new model for the study of SIV pathogenesis in natural hosts. J. Virol. 80, 4858–4867 (2006). | Article | PubMed | ChemPort |
10. Goldstein, S. et al. Wide range of viral load in healthy African green monkeys naturally infected with simian immunodeficiency virus. J. Virol. 74, 11744–11753 (2000). | Article | PubMed | ChemPort |
11. Silvestri, G. et al. Nonpathogenic SIV infection of sooty mangabeys is characterized by limited bystander immunopathology despite chronic high-level viremia. Immunity 18, 441–452 (2003). | Article | PubMed | ISI | ChemPort |
12. Pandrea, I. et al. Simian immunodeficiency viruses replication dynamics in African non-human primate hosts: common patterns and species-specific differences. J. Med. Primatol. 35, 194–201 (2006). | Article | PubMed
13. Barry, A.P. et al. Depletion of CD8⁺ cells in sooty mangabey monkeys naturally infected with simian immunodeficiency virus reveals limited role for immune control of virus replication in a natural host species. J. Immunol. 178, 8002–8012 (2007). | PubMed | ChemPort |
14. Dunham, R. et al. The AIDS resistance of naturally SIV-infected sooty mangabeys is independent of cellular immunity to the virus. Blood 108, 209–217 (2006). | Article | PubMed | ISI | ChemPort |
15. Klatt, N.R. et al. Availability of activated CD4⁺ T cells dictates the level of viremia in naturally SIV-infected sooty mangabeys. J. Clin. Invest. 118, 2039–2049 (2008). | PubMed | ChemPort |
16. Pandrea, I. et al. Paucity of CD4⁺ CCR5⁺ T cells may prevent transmission of simian immunodeficiency virus in natural nonhuman primate hosts by breast-feeding. J. Virol. 82, 5501–5509 (2008). | Article | PubMed | ChemPort |
17. Gordon, S.N. et al. Short-lived infected cells support virus replication in sooty mangabeys naturally infected with simian immunodeficiency virus: implications for AIDS pathogenesis. J. Virol. 82, 3725–3735 (2008). | Article | PubMed | ChemPort |
18. Hirsch, V.M. et al. Induction of AIDS by simian immunodeficiency virus from an African green monkey: species-specific variation in pathogenicity correlates with the extent of in vivo replication. J. Virol. 69, 955–967 (1995). | PubMed | ChemPort |
19. Goldstein, S. et al. Plateau levels of viremia correlate with the degree of CD4⁺-T-cell loss in simian immunodeficiency virus SIVagm-infected pigtailed macaques: variable pathogenicity of natural SIVagm isolates. J. Virol. 79, 5153–5162 (2005). | Article | PubMed | ChemPort |
20. Watson, A. et al. Plasma viremia in macaques infected with simian immunodeficiency virus: plasma viral load early in infection predicts survival. J. Virol. 71, 284–290 (1997). | PubMed | ISI | ChemPort |
21. Hirsch, V. et al. A molecularly cloned, pathogenic, neutralization-resistant simian immunodeficiency virus, SIVsmE543–3. J. Virol. 71, 1608–1620 (1997). | PubMed | ISI | ChemPort |
22. Li, Y. et al. Complete nucleotide sequence, genome organization and biological properties of human immunodeficiency virus type 1 in vivo: evidence for limited defectiveness and complementation. J. Virol. 66, 6587–6600 (1992). | PubMed | ISI | ChemPort |
23. Fultz, P.N., McClure, H.M., Anderson, D.C. & Switzer, W.M. Identification and biologic characterization of an acutely lethal variant of simian immunodeficiency virus from sooty mangabeys (SIV/SMM). AIDS Res. Hum. Retroviruses 5, 397–409 (1989). | Article | PubMed | ISI | ChemPort |
24. Giorgi, J.V. et al. Shorter survival in advanced human immunodeficiency virus type 1 infection is more closely associated with T lymphocyte activation than with plasma virus burden or virus chemokine coreceptor usage. J. Infect. Dis. 179, 859–870 (1999). | Article | PubMed | ISI | ChemPort |
25. Chakrabarti, L.A. et al. Normal T-cell turnover in sooty mangabeys harboring active simian immunodeficiency virus infection. J. Virol. 74, 1209–1223 (2000). | Article | PubMed | ISI | ChemPort |
26. Sumpter, B. et al. Correlates of preserved CD4⁺ T-cell homeostasis during natural, nonpathogenic simian immunodeficiency virus infection of sooty mangabeys: implications for AIDS pathogenesis. J. Immunol. 178, 1680–1691 (2007). | PubMed | ChemPort |
27. Pandrea, I. et al. High levels of SIVmnd-1 replication in chronically infected Mandrillus sphinx. Virology 317, 119–127 (2003). | Article | PubMed | ChemPort |
28. Pandrea, I.V. et al. Acute loss of intestinal CD4⁺ T cells is not predictive of simian immunodeficiency virus virulence. J. Immunol. 179, 3035–3046 (2007). | PubMed | ChemPort |
29. Murayama, Y., Mukai, R., Inoue-Murayama, M. & Yoshikawa, Y. An African green monkey lacking peripheral CD4 lymphocytes that retains helper T-cell activity and coexists with SIVagm. Clin. Exp. Immunol. 117, 504–512 (1999). | Article | PubMed | ChemPort |
30. Murayama, Y. et al. CD4 and CD8 expressions in African green monkey helper T lymphocytes: implication for resistance to SIV infection. Int. Immunol. 9, 843–851 (1997). | Article | PubMed | ChemPort |
31. Banchereau, J. et al. The CD40 antigen and its ligand. Annu. Rev. Immunol. 12, 881–922 (1994). | Article | PubMed | ISI | ChemPort |
32. Karube, K. et al. Expression of FoxP3, a key molecule in CD4CD25 regulatory T cells, in adult T-cell leukaemia/lymphoma cells. Br. J. Haematol. 126, 81–84 (2004). | Article | PubMed | ISI | ChemPort |
33. Pitcher, C.J. et al. HIV-1–specific CD4⁺ T cells are detectable in most individuals with active HIV-1 infection, but decline with prolonged viral suppression. Nat. Med. 5, 518–525 (1999). | Article | PubMed | ISI | ChemPort |
34. Brenchley, J.M. et al. T-cell subsets that harbor human immunodeficiency virus (HIV) in vivo: implications for HIV pathogenesis. J. Virol. 78, 1160–1168 (2004). | Article | PubMed | ISI | ChemPort |
35. Brenchley, J.M. et al. Differential T_H17 CD4 T-cell depletion in pathogenic and nonpathogenic lentiviral infections. Blood 112, 2826–2835 (2008). | Article | PubMed | ChemPort |
36. Gordon, S.N. et al. Severe depletion of mucosal CD4⁺ T cells in AIDS-free simian immunodeficiency virus–infected sooty mangabeys. J. Immunol. 179, 3026–3034 (2007). | PubMed | ChemPort |
37. Pandrea, I. et al. Paucity of CD4⁺CCR5⁺ T cells is a typical feature of natural SIV hosts. Blood 109, 1069–1076 (2007). | Article | PubMed | ISI | ChemPort |
38. Hunt, P.W. et al. Relationship between T-cell activation and CD4₊ T-cell count in HIV-seropositive individuals with undetectable plasma HIV RNA levels in the absence of therapy. J. Infect. Dis. 197, 126–133 (2008). | Article | PubMed
39. Jiang, W. et al. Plasma levels of bacterial DNA correlate with immune activation and the magnitude of immune restoration in persons with antiretroviral-treated HIV infection. J. Infect. Dis. 199, 1177–1185 (2009). | Article | PubMed | ChemPort |
40. Papasavvas, E. et al. Delayed loss of control of plasma lipopolysaccharide levels after therapy interruption in chronically HIV-1–infected patients. AIDS 23, 369–375 (2009). | Article | PubMed | ChemPort |
41. Gregson, J.N. et al. Elevated plasma lipopolysaccharide is not sufficient to drive natural killer cell activation in HIV-1–infected individuals. AIDS 23, 29–34 (2009). | Article | PubMed | ChemPort |
42. Brenchley, J.M. et al. Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat. Med. 12, 1365–1371 (2006). | Article | PubMed | ChemPort |
43. Ancuta, P. et al. Microbial translocation is associated with increased monocyte activation and dementia in AIDS patients. PLoS One 3, e2516 (2008). | Article | PubMed | ChemPort |
44. Balagopal, A. et al. Human immunodeficiency virus–related microbial translocation and progression of hepatitis C. Gastroenterology 135, 226–233 (2008). | Article | PubMed | ChemPort |
45. Marchetti, G. et al. Microbial translocation is associated with sustained failure in CD4⁺ T-cell reconstitution in HIV-infected patients on long-term highly active antiretroviral therapy. AIDS 22, 2035–2038 (2008). | Article | PubMed | ChemPort |
46. Boulassel, M.R., Mercier, F., Gilmore, N. & Routy, J.P. Immunophenotypic patterns of CD8⁺ T-cell subsets expressing CD8 and IL-7R in viremic, aviremic and slow progressor HIV-1–infected subjects. Clin. Immunol. 124, 149–157 (2007). | Article | PubMed | ChemPort |
47. Rahemtulla, A. et al. Normal development and function of CD8⁺ cells but markedly decreased helper cell activity in mice lacking CD4. Nature 353, 180–184 (1991). | Article | PubMed | ISI | ChemPort |
48. Rahemtulla, A. et al. Class II major histocompatibility complex–restricted T-cell function in CD4-deficient mice. Eur. J. Immunol. 24, 2213–2218 (1994). | Article | PubMed | ISI | ChemPort |
49. Barber, E.K., Dasgupta, J.D., Schlossman, S.F., Trevillyan, J.M. & Rudd, C.E. The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex. Proc. Natl. Acad. Sci. USA 86, 3277–3281 (1989). | Article | PubMed | ChemPort |
50. Matsunaga, S., Mukai, R., Inoue-Murayama, M., Yoshikawa, Y. & Murayama, Y. Sequence and functional properties of African green monkey CD4 silencer. Immunol. Lett. 75, 47–53 (2000). | Article | PubMed | ChemPort |
51. Kioussis, D. & Ellmeier, W. Chromatin and CD4, CD8A and CD8B gene expression during thymic differentiation. Nat. Rev. Immunol. 2, 909–919 (2002). | Article | PubMed | ISI | ChemPort |
52. Bilic, I. et al. Negative regulation of CD8 expression via Cd8 enhancer–mediated recruitment of the zinc finger protein MAZR. Nat. Immunol. 7, 392–400 (2006). | Article | PubMed | ISI | ChemPort |
53. Milush, J.M. et al. Virally induced CD4⁺ T-cell depletion is not sufficient to induce AIDS in a natural host. J. Immunol. 179, 3047–3056 (2007). | PubMed | ChemPort |
54. Roelke, M.E. et al. T-lymphocyte profiles in FIV-infected wild lions and pumas reveal CD4 depletion. J. Wildl. Dis. 42, 234–248 (2006). | PubMed | ChemPort |
55. Mattapallil, J.J. et al. Massive infection and loss of memory CD4+ T cells in multiple tissues during acute SIV infection. Nature 434, 1093–1097 (2005). | Article | PubMed | ChemPort |

Online methods

Animals. We housed 18 SIVagm-infected vervet African green monkeys (C. pygerythrus), 11 SIVagm-uninfected vervet African green monkeys, seven SIVagm-uninfected juvenile (fewer than 2 years old) vervet African green monkeys, and 12 SIV-negative rhesus macaques (Macaca mulatta) at Bioqual and six SIV-infected sabaeus African green monkeys (C. sabeus) at the Tulane National Primate Center. We housed SIVsmm-infected and uninfected sooty mangabeys at the Yerkes National Primate Center. All animals were housed in accordance with the National Research Council Guide for the Care and use of Laboratory Animals, and all protocols were approved by the Tulane National Primate Center, Yerkes National Primate Center and National Institute of Allergy and Infectious Diseases Institutional Animal Care and Use committees. Ten of the SIV⁺ African green monkey vervets were infected in the wild, and eight were experimentally infected with 50 or 1,000 TCID₅₀ of SIVagm90 intravenously (Table 1). We isolated virus as previously described¹⁰. AG11 and AG15 are seropositive for SIVagm.

Human subjects. We recruited five HIV-1–uninfected subjects at the National Institutes of Health. The subjects all gave informed consent before entry into this study, and all studies were approved by the Institutional Review Board of the National Institutes of Health.

Flow cytometry. For intracellular cytokine staining, we incubated PBMCs overnight at 37 °C with medium alone, 1 g of SEB) (Sigma) or 1 g of CMV whole antigen (Microbix Biosystems) in the presence of 0.5 g each of monoclonal antibodies to CD28 (CD28.2, Beckman Coulter) and CD49d (9F10, BD Bioscience) and 1 g ml^-1 brefeldin A (Sigma). For some experiments, we pretreated PBMCs for 1 h at 37 °C with antibodies against MHC-I (G46-2.6, BD Bioscience) or MHC-II (TU39, BD Bioscience). After stimulation, we washed the cells twice and incubated them with Live/Dead fixable aqua dead cell stain (Invitrogen). We then stained the cells for surface markers with monoclonal antibodies to CD3 (SP34-2, BD Bioscience), CD4 (L200, BD Bioscience), CD8 (RPA-T8, BD Bioscience) and CD95 (DX2, BD Bioscience). We washed the cells and permeabilized them with Cytofix/Cytoperm buffer (BD Bioscience). We then intracellularly stained the cells with fluorescence-conjugated monoclonal antibodies to interferon- (4S.B3, BD Bioscience), IL-17 (eBio64DEC17, eBioscience), IL-2 (MQ1-17H12, BD Bioscience), CD40L (TRAP1, BD Bioscience) or Ki67 (B56, BD Bioscience) and incubated them at 4 °C for 20 min. We washed and then fixed them with a 1% paraformaldehyde solution (Electron Microscopy Sciences).

For proliferation, we stained PBMCs or flow cytometrically sorted naive CD4⁺ T cells with 0.125 M carboxyfluorescein succinimidyl ester (CFSE, Invitrogen) and then stimulated them with 1 g ml^-1 concanavalin A (Sigma), 1 g ml^-1 staphylococcal enterotoxin B (Sigma) or microbeads coated with antibody to CD3 and CD28 (generous gift from J.E. Schmitz and R. Zahn) at a 1:4 cell-to-bead ratio for 5, 7 and 9 d. We then labeled cells with fluorescent antibodies directed toward CD3, CD4 and CD8 (BD Bioscience).

For analysis of FoxP3 expression, we surface-stained fresh PBMCs and then permeabilized them with FoxP3 permeabilization solution (eBioscience). We stained the cells intracellularly for FoxP3 (PCH101, eBioscience). We then washed them and fixed them with a 1% paraformaldehyde solution. We ran all flow cytometry samples on a FACSAria (BD Bioscience) with FACSDiva software (BD Bioscience) and we analyzed data with FlowJo (Tree Star).

Quantitative real-time PCR. We sorted cell populations by flow cytometry and lysed them with 25 l of a 1 in 100 dilution of proteinase K (Roche) in 10 mM Tris buffer. We performed quantitative PCR with 5 l of cell lysates per reaction. Reaction conditions were as follows: 95 °C holding stage for 5 min and 40 cycles of 95 °C for 15 s followed by 60 °C for 1 min. We used the Taq DNA polymerase kit (Invitrogen). The sequence of the forward primer for SIVagm is 5'-GTCCAGTCTCAGCATTTACTTG-3'. The reverse primer sequence is 5'-CGGGCATTGAGGTTTTTCAC-3'. The probe sequence is 5'-CAGATGTTGAAGCTGACCATTTGGG-3'. For cell number quantification, we measured monkey albumin gene copy number as previously described⁵⁵. We used the StepOne Plus PCR machine (Applied Biosystems), and we performed the analysis with StepOne software (Applied Biosystems).

Reverse-transcription PCR. We sorted viable cell populations by flow cytometry and isolated mRNA with the Oligotex Direct mRNA Mini Kit (Qiagen) following the manufacturer's protocol. After complementary DNA synthesis with random hexamers and Superscript II RNase H, we amplified the reverse transcriptase transcripts of CD4 by PCR with the forward primer 5'-TCGGATTGACTGCCAACTCTG-3' and the reverse primer 5'-AAGGCGAGCGGGAAGGAGAA-3'. Reaction conditions were as follows: 95 °C holding stage for 5 min and 40 cycles of 95 °C for 15 s followed by 60 °C for 1 min. To control for the amount of mRNA, we did a second PCR with primers for albumin (as detailed above).

Statistical analyses. We did all statistical analyses with Prism software (GraphPad). Statistical significance was based on P values <0.05.

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