INTRODUCTION {#S1}
============

Simian immunodeficiency viruses (SIV) belong to the group of lentiviruses that infect non-human primates (NHP). The lentiviruses that cause immunodeficiencies in humans and Asian macaques originated from cross-species transmission of viruses that naturally infect nonhuman primates in Africa[@R1]. Like HIV-1 and HIV-2, all known SIV subtypes use CD4 as a receptor and either CCR5, CXCR4, or, as for SIVrcm, CCR2[@R2]--[@R4] as a co-receptor. 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. Since natural hosts of SIV have co-evolved 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[@R5]--[@R7]. Natural hosts do not avoid disease progression by immunological control of the virus since SIV-infected natural hosts maintain high levels of viremia[@R8]--[@R12]. Moreover, experimental depletion of CD8^+^ T cells does not affect plasma viremia[@R13] and natural hosts do not exhibit superior cellular control of viremia compared to HIV-infected humans or SIV-infected rhesus macaques(RM)[@R14]. The lentiviruses that infect natural hosts can be pathogenic. SIV infections of AGM and Sooty mangabeys (SM) have been correlated with short life spans of infected cells *in vivo*[@R15]--[@R17]. Moreover, SIVagm, which naturally infects African green monkeys (AGM), can be used to infect pigtail macaques who subsequently manifest simian AIDS[@R18],[@R19]. Isolates of SIVsmm can also cause progressive infection in RM[@R20]--[@R23]. One fundamental difference between progressive SIV/HIV infection and nonprogressive SIV infection is the absence of immune activation, which is associated with disease progression in HIV-infected individuals[@R24], during the chronic phase of infection in natural hosts[@R9],[@R11],[@R25]--[@R28].

Previous studies performed using AGM have reported very low frequencies of CD4^+^ T cells[@R28]. AGM, however, remain disease free despite having low numbers of CD4^+^ T cells. In two reports, Murayama *et al*, described low frequencies of CD4^+^ T cells and high frequencies of CD8^dim^ T-cells in healthy adult AGM[@R29],[@R30]. They also found the CD8^dim^ T cells could induce antibody production from B-cells *in vitro* and suggested that the CD8^dim^ T-cells might supplement for the lack of CD4^+^ T-cells[@R29]. Here we have studied the frequencies, functionalities and *in vivo* infection frequencies of lymphocyte subsets from 36 AGM, 10 naturally infected, 8 experimentally infected, 11 uninfected adults and 7 uninfected juveniles ([Table 1](#T1){ref-type="table"}). Our data describe a mechanism by which AGM are able to survive SIVagm infection without succumbing to AIDS.

RESULTS {#S2}
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Decreased frequencies of CD4^+^ T-cells correlate with increased frequencies of CD4^−^CD8α^dim^ T-cells {#S3}
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Loss of CD4^+^ T-cells is a hallmark of progression to AIDS in humans and Asian macaques. We therefore compared the frequency of CD4^+^ T-cells in SIVagm-infected and uninfected vervet AGM (one of four subspecies of AGM), HIV-uninfected humans, SIV-uninfected RM and SIVsmm-infected and uninfected SM. We observed three distinct T-cell populations in AGM based upon expression of CD4 and CD8α: CD4^+^ T-cells, CD4^−^CD8α^dim^ T-cells and CD4^−^CD8α^bright^ T-cells ([Fig. 1a](#F1){ref-type="fig"}). We analyzed further the phenotypes of each subset based upon expression of CD28 and CD95 ([Fig. 1b](#F1){ref-type="fig"}). The CD4^+^ and CD4^−^CD8α^bright^ T-cells consisted of both memory and naive subsets ([Fig. 1b](#F1){ref-type="fig"}). However, the CD4^−^CD8α^dim^ T-cells consisted predominantly of memory T-cells ([Fig. 1b](#F1){ref-type="fig"}). Moreover, adult AGM appeared to have surprisingly low frequencies of CD4^+^ T-cells and low CD4^+^ T-cell counts([Table 1](#T1){ref-type="table"}). There were significantly lower frequencies of CD4^+^ T-cells in both SIVagm-infected and uninfected AGM compared to SIVsmm-infected or uninfected SM, SIV-uninfected RM or HIV-1-uninfected humans ([Fig. 1c](#F1){ref-type="fig"}). In addition, the decrease in CD4^+^ T-cells was accompanied by an increase in the frequency of CD4^−^CD8α^dim^ T-cells ([Fig. 1a](#F1){ref-type="fig"}). Further phenotypic analysis showed that this CD4^−^CD8α^dim^ T-cell population lacked expression of CD8β ([Fig. 1d](#F1){ref-type="fig"}). In addition, the frequency of Ki67^+^ cells was significantly among CD4^−^CD8α^dim^ T-cells compared to either the CD4^+^ or CD4^−^CD8α^bright^ subsets, suggesting that the high frequency of CD4^−^CD8α^dim^ T-cells was not due to preferential proliferation ofCD4^−^CD8α^dim^ T-cells *in vivo* **(**[Fig. 1e](#F1){ref-type="fig"}).

The increased frequency of CD4^−^CD8α^dim^ T-cells could be a reflection of a mathematical shift in 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^−^CD8α^dim^ T-cells ([Fig. 1f](#F1){ref-type="fig"}), but not for the CD4^−^CD8α^bright^ T-cells ([Fig. 1g](#F1){ref-type="fig"}). Therefore, the increase in the CD4^−^CD8α^dim^ T-cell population was directly related to the decrease in CD4^+^ T-cells, suggesting that someCD4^−^CD8α^dim^ T-cells might develop from CD4^+^ T-cells.

Some CD4^−^CD8α^dim^ T-cells develop from CD4^+^ memory T-cells {#S4}
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Since the frequencies of the CD4^−^CD8α^dim^ T-cells negatively correlated with the frequencies of CD4^+^ T-cells ([Fig. 1f](#F1){ref-type="fig"}), we hypothesized that some of the CD4^−^CD8α^dim^ T-cells may have developed from CD4^+^ T-cells. Phenotypic analysis of the CD4^+^ T-cells from SIVagm-infected and uninfected AGM illustrated that the CD95^+^ memory CD4^+^ T-cells down-regulated CD4 ([Fig. 2a](#F2){ref-type="fig"}) and up-regulated CD8α ([Fig. 2b](#F2){ref-type="fig"}) compared to naive CD4^+^ T-cells. Fluorescence minus one analysis for CD8α was also performed to confirm that the CD4^+^ T-cells expressed CD8α ([Fig 2c](#F2){ref-type="fig"}). 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](#F2){ref-type="fig"}) while the MFI of CD8α was significantly increased in the memory CD4^+^ T-cells over naive ([Fig. 2e](#F2){ref-type="fig"}). Therefore, we hypothesized that upon stimulation CD4^+^ T-cells could transition to become CD4^−^CD8α^dim^ T-cells.

To study down-regulation of CD4 *in vitro*, we CFSE labeled and mitogenically stimulated peripheral blood mononucelear cells (PBMC) from several AGM and, for comparison, pigtail macaques. We then studied expression of CD4 and proliferation by flow cytometry after 5, 7, and 9 days in culture ([Fig. 3a--b](#F3){ref-type="fig"} and [Supplemental Fig. 1](#SD1){ref-type="supplementary-material"}). We found that while AGM CD4^+^ T-cells lost CD4 expression, CD4^+^ T-cells from pigtail macaques maintained CD4 expression with cell division ([Fig. 3a](#F3){ref-type="fig"}). It is a possibility that the CD4 down-regulation in AGM was transient, however, we did not see up-regulation of CD4 by day 9 ([Supplemental Fig. 1](#SD1){ref-type="supplementary-material"}). To confirm this finding, we also sorted naive CD4^+^ T-cells (\>99% pure) prior to stimulation and found that after stimulation they became CD4^−^([Supplemental Fig. 1](#SD1){ref-type="supplementary-material"}).

Analysis for CD4 mRNA within sorted CD4^+^, CD4^−^CD8α^dim^, CD4^−^CD8α^bright^ T-cells and monocytes revealed that CD4 mRNA was only detected within CD4^+^ T-cells ([Fig. 3c](#F3){ref-type="fig"}). Taken together, these data suggested that AGM memory CD4^+^ T-cells could become CD4^−^CD8α^dim^ T-cells. If our hypothesis was correct, AGM 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 ofCD4^+^ T-cells ([Fig. 3d](#F3){ref-type="fig"}). Hence as AGM accumulate memory CD4^+^ T-cells, the overall frequency of CD4^+^ T-cells decreases.

We reasoned that if some CD4^+^ T-cells were becoming CD4^−^CD8α^dim^ T-cells upon stimulation into the memory pool, then animals with very little antigenic experience should have frequencies of CD4^+^ T-cells comparable to those of healthy RM and humans ([Fig. 1c](#F1){ref-type="fig"}). Therefore, we measured the frequencies of T-cell subsets from PBMC obtained directly *ex vivo* from juvenile AGM (all less than 2 years old). In a total of seven juvenile AGM, the median frequency of CD4^+^ T-cells was 59% (39.6%--64.4%, [Fig. 3e](#F3){ref-type="fig"}). These frequencies were comparable with those from non-immunocompromised RM and humans and were higher than the frequencies of CD4^+^ T-cells in adult AGM (*P*\<0.0006). Furthermore, after we vaccinated the juvenile AGM with the standard influenza vaccine we observed an influenza-specific T-cell response from the CD4^−^CD8α^dim^ T-cells to the MHC-II restricted antigen ([Supplemental Fig. 2c](#SD2){ref-type="supplementary-material"}). Taken together, these observations strongly suggest that many of the CD4^−^CD8α^dim^ T-cells developed from memory CD4^+^ T-cells.

While we do not know the ages of many of the AGM, as they were imported from Tanzania, in the few animals from which 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 accumulation of CD4^−^CD8α^dim^ T-cells as older animals are exposed to more antigens *in vivo*.

CD4^−^CD8α^dim^ T-cells preserve CD4^+^ T-cell function while evading SIV infection {#S5}
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To elucidate the functions of the CD4^−^CD8α^dim^ T-cells, we obtained PBMC from SIVagm-infected and uninfected adult AGM. Since it seemed apparent that many of the CD4^−^CD8α^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^−^CD8α^dim^ T-cells could produceIL-2 and IL-17 ([Supplemental Fig. 2](#SD2){ref-type="supplementary-material"} and [Fig. 4a](#F4){ref-type="fig"}) and express CD40 ligand (CD40L) ([Supplemental Fig. 2](#SD2){ref-type="supplementary-material"} and [Fig. 4a](#F4){ref-type="fig"}). CD40L is typically expressed by activated CD4^+^ T-cells and results in enhancement of antigen presentation and induces B-cell class switching[@R31]. Additionally, a portion of the CD4^−^CD8α^dim^ T-cells expressed the transcription factor FoxP3, thought to be predominantly expressed by regulatory CD4^+^ T-cells ([Supplemental Fig. 2](#SD2){ref-type="supplementary-material"} and [Fig. 4a](#F4){ref-type="fig"})[@R32]. Hence the CD4^−^CD8α^dim^ T-cell subset included T-cells that performed functions normally restricted to CD4^+^ T-cells. While the overall frequencies of memory CD4^+^ T-cells that could perform each function was significantly higher compared to the CD4^−^CD8α^dim^ T-cells([Fig. 4a](#F4){ref-type="fig"}), the total frequency of CD4^−^CD8α^dim^ T-cells was significantly greater than the overall frequency of CD4^+^ T-cells. We therefore calculated the relative number of cytokine^+^, CD40L^+^, and FoxP3^+^ T-cells for each T-cell subset ([Fig. 4b](#F4){ref-type="fig"}). For IL-2, IL-17, and FoxP3 the relative number of CD4^−^CD8α^dim^ T-cells was significantly greater than those of the CD4^+^ and CD4^−^CD8α^bright^ T-cell subsets. The relative number of CD40L^+^ CD4^−^CD8α^dim^ T-cells was equal to that of CD4^+^ T-cells. These observations suggest that there were actually greater numbers of T-cells performing CD4^+^ T-cell functions without actually expressing CD4.

CD4^+^ T-cells are restricted by MHC-II, while CD8^+^ T-cells are restricted to MHC-I. Therefore, to test further our hypothesis that the CD4^−^CD8α^dim^ T-cells were acting as CD4^+^ T-cells, we screened PBMC from adult AGM for T-cell responses to a cytomegalovirus (CMV) whole antigen preparation. Presentation of this antigen requires processing through MHC-II[@R33]. We found one AGM that had T-cells responsive to CMV (AG731) and the entire CMV-specific T-cell response was restricted to CD4^−^CD8α^dim^ T-cells([Fig. 4c](#F4){ref-type="fig"} and [Supplemental Fig. 2b,c](#SD2){ref-type="supplementary-material"}). To confirm that these CMV-specific CD4^−^CD8α^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 anti-MHC class-II antibody decreased the frequency of responding CMV-specific CD4^−^CD8α^dim^ T-cells by more than two-thirds, while blocking MHC-I had virtually no effect on the ability of CMV-specific CD4^−^CD8α^dim^ T-cells to respond ([Fig. 4c](#F4){ref-type="fig"}). Hence CD4^−^CD8α^dim^ T-cells can be restricted by MHC-II in AGM.

Vervets are one of four subspecies of AGM along with sabeus, tantalus and grivets. To determine whether the phenomena which we observed were specific to vervet AGM, we examined T-cell populations in 6 adult sabeus AGM. Indeed, we found the presence of CD4^−^CD8α^dim^ T-cells. Similar to the vervets, the CD4^−^CD8α^dim^ T-cells from sabeus were able to perform CD4 functions ([Supplementary Fig. 3](#SD3){ref-type="supplementary-material"}). Confirmation of this phenomenon occurring in a second subspecies of AGM suggest that down-regulation of CD4 is characteristic of all AGM.

Since the CD4^−^CD8α^dim^ T-cells have many functional characteristics of CD4^+^ T-cells, wenext determined which lymphocytes were infected by SIVagm *in vivo*. We flow cytometrically sorted individual subsets of lymphocytes from eighteen SIVagm-infected AGM 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^−^CD8α^dim^ T-cells, memory CD4^−^CD8α^bright^ T-cells, memory CD4^−^CD8α^−^ T-cells and monocytes. We found that memory CD4^+^ T-cells were the primary target for SIVagm *in vivo* ([Fig. 5](#F5){ref-type="fig"}). Consistent with infection frequency patterns of HIV[@R34], we also found that SIVagm could infect naive CD4^+^ T-cells, but memory CD4^+^ T-cells were preferentially infected. Importantly, CD4^−^CD8α^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](#F5){ref-type="fig"}). In 78% of the SIVagm-infected AGM we detected no viral DNA within the CD4^−^CD8α^dim^ T-cell subset. Cell numbers were often limiting and it was conceivable that, in some cases, we did not detect any viral DNA due to a small number of sorted cells in each PCR. In samples in which no viral DNA was detected, we reported values calculated as 1/2 the lower limit of detection (closed circles), these values are based upon the number of sorted cells. In the few (22%) AGM where we did detect viral DNA within the CD4^−^CD8α^dim^ T-cell subset the infection frequencies were very low (\<0.01%). Taken together, these data suggest that CD4^−^CD8α^dim^ T-cells preserve CD4^+^ T-cell function while evading SIV infection *in vivo* and in turn, these findings suggest a mechanism by which AGM are able remain disease free despite SIV infection.

DISCUSSION {#S6}
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We have shown that AGM, regardless of infection with SIVagm, have decreased numbers of total CD4^+^ T-cells that correlates with an increased population of CD4^−^CD8α^dim^ T-cells. We demonstrated that some of these CD4^−^CD8α^dim^ T-cells developed from memory CD4^+^ T-cells. This conclusion is strengthened by the observation that juvenile AGM, who have very low frequencies of memory T-cells *in vivo*, have high frequencies of CD4^+^ T-cells and low frequencies of CD4^−^CD8α^dim^ T-cells and that *in vitro* stimulation of naive CD4^+^ T-cells results in down-regulation of CD4 and up-regulation of CD8α. In addition, we have demonstrated that in adult AGM some of these CD4^−^CD8α^dim^ T-cells, upon stimulation, exhibit functions generally attributed to CD4^+^ T-cells. The CD4^−^CD8α^dim^ T-cells can be restricted by MHC-II. Despite the fact many of the T-cells in this population likely developed from CD4^+^ T-cells and maintain the ability to perform functions attributed to CD4^+^ T-cells, they are able to evade infection by SIVagm. These data provide a mechanism by which AGM are able to survive chronic SIVagm infection without progression to simian AIDS.

Our proposed mechanism underlying the nonpathogenic nature of SIVagm infection could contribute to the lack of immune activation seen in the natural hosts of SIV. Indeed, preservation of CD4^+^ T-cell function may well contribute to the lack of immune activation in AGM and SM[@R28],[@R35],[@R36]. Additionally, there is a significantly lower frequency of CD4^+^CCR5^+^ T-cells in natural hosts of SIV when compared to non-natural hosts[@R16],[@R37], suggesting that the lack of CCR5 expression results in decreased homing to sites of inflammation, thereby preventing activation and inflammation. This decrease in activated T-cells could reduce the number targets for SIV. Taken together, these data may suggest that in natural hosts, the virus may be preferentially targeting macrophages. However, we have found no SIVagm DNA within highly purified monocytes, consistent with previous reports that SIVagm preferentially replicates in lymphocytes during chronic infection[@R16],[@R28]. Also, previous comparative studies between pathogenic HIV infection of humans or SIV infection of Asian macaques and SIV infection of SM or AGM have shown that while both SM and AGM lose CD4^+^ T-cells from the gastrointestinal tract during the acute phase of infection, these animals do not manifest immune activation[@R11],[@R28],[@R36]. Microbial translocation, which causes immune activation in the chronic phase of HIV and SIV infection of Asian macaques[@R38]--[@R45], does not occur in the chronic phase of SIV infection of either AGM or sooty mangabeys[@R28],[@R42]. The functionality of mucosal CD4^−^CD8α^dim^ T-cells, which is present at high frequencies in AGM[@R28], should be assessed for a role in preventing microbial translocation and immune activation in SIVagm-infected AGM.

Many other cellular populations, such as NK cells, γδ T-cells and NKT-cells, express the CD8αα homodimer. Indeed, some of the CD4^−^CD8α^dim^ T-cells from AGM express the γδ TCR (data not shown). Moreover, many of the CD28^−^ memory T-cells within the CD4^−^CD8α^dim^ T-cell subset express granzyme B (data not shown), not typically expressed by CD4^+^ T-cells. Therefore, it is clear that not all of the CD4^−^CD8α^dim^ T-cells were originally CD4^+^ T-cells. While AGM have significantly higher frequencies of CD4^−^CD8α^dim^ T-cells compared to humans, Bolassel *et al*, recently showed that slow progressing HIV-1-infected individuals have a significantly higher frequency of CD4^−^CD8α^dim^ T-cells compared to chronically HIV-infected individuals[@R46]. This finding suggests a similar phenomenon to what we describe here could slow disease progression in HIV-infected humans.

While CD4^−^CD8α^dim^ T-cells have previously been studied, it has not been demonstrated that memory CD4^+^ T-cells down-regulate CD4 and up-regulate CD8α. How these T-cells are able to perform functions without the CD4 molecule is unclear. Importantly, Rahemtulla *et al*, demonstrated normal development and function of lymphoid organs, B cells and CD8^+^ T-cells in CD4 knockout mice. However, antibody production, IL-2 secretion, and MHC-II restricted responses were significantly abrogated[@R47]. The authors subsequently showed that CD4^−^CD8^−^ T-cells in CD4 knockout mice could partially compensate for the lack of CD4 T-cells[@R48], similar to what we observe in AGM. CD4 has been shown to be important for initiating the downstream kinase signaling that results in T-cell activation[@R49]. It is possible that in AGM CD4^−^CD8α^dim^ T-cells sufficient lck phosphorylation occurs, or alternative signals exist, but the actual mechanism(s) underlying the switch from CD4^+^ to CD4^−^CD8α^dim^ remains unclear. Indeed, previous studies have reported genetic differences between certain regulatory elements from AGM compared to other primates[@R50]. One possibility is that a change may occur in the methylation states of the enhancer and silencer regions in the genome[@R51]. Also, differential expression of the MAZR protein, a protein suppressor of the CD8α enhancer region[@R52], may also exist.

It is not unprecedented that changes in the frequencies of T-cells within individual subsets occur in lentiviral infections. For example, while SM generally maintain healthy CD4^+^ T-cell counts during chronic SIVsmm infection, infection with a dual tropic (CXCR4/CCR5) strain of SIVsmm resulted in the loss of CD4^+^ T-cells, but preservation CD4^−^CD8^−^ T-cells[@R53]. Moreover, one report identified several naturally SIV-infected SM with very low frequencies of CD4^+^ T-cells[@R26]. The functions and ontogeny of T-cells in these SIVsmm-infected SM infected are currently under investigation but could represent a similar phenomenon.

Natural hosts have co-evolved with SIV to avoid disease progression, though the mechanisms by which this occurs may diverge since most SM maintain healthy frequencies of CD4^+^ T-cells. In AGM, we show that this co-evolution with SIVagm has occurred, in part, by the development of CD4^−^CD8α^dim^ T-cells from memory CD4^+^ T-cells. Additionally, African lions, who remain disease free after infection with feline immunodeficiency virus, maintain a high frequency of CD8β^dull^ T-cells regardless CD4^+^ T-cell loss[@R54]. Hence, down-regulation of CD4 may be associated with lack of disease progression in multiple immunodeficiency lentiviral infections.

We have identified and characterized a mechanism by which AGM are able to survive chronic SIVagm infection. In non-natural hosts of immunodeficiency lentiviruses depletion of CD4^+^ T-cells leads to AIDS. However, SIVagm-infected AGM maintain immune responses, remain healthy, and live normal life spans. We provide evidence for the conversion of CD4^+^ T-cells to CD4^−^CD8α^dim^ T-cells, which likely plays a key role in the lack of clinical signs of AIDS in AGM. Once the mechanism by which the CD4^+^ T-cells are able to convert to CD4^−^CD8α^dim^ T-cells has been understood, interventions aimed at mimicking this phenomenon could be developed for preventative and therapeutic trials.

METHODS {#S7}
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Animals {#S8}
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Eighteen SIVagm-infected vervet AGM (*Chlorocebus pygerythrus)*, 11SIVagm-uninfected vervet AGM, 7SIVagm-uninfected juveline (less than 2 years old) vervet AGM, and 12 SIV-negative rhesus macaques (*Macaca mulatta)* were housed at Bioqual (Rockville, MD) and 6SIV-infected sabaeus AGM (*C. sabeus*) were housed at the Tulane National Primate Center (Convington, LA). SIVsmm-infected and uninfected SM were housed at the Yerkes National Primate Center (Atlanta, GA). 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 relevant institutional animal care and use committees. Ten of the SIV^+^ AGM vervets, were infected in the wild and eight were experimentally infected with 50 or 1000 TCID~50~ of SIVagm90 intravenously ([Table 1](#T1){ref-type="table"}). Virus was isolated as previously described[@R10]. AG11 and AG15 are seropositive for SIVagm.

Human Subjects {#S9}
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Five HIV-1 uninfected subjects were recruited at the National Institutes of Health. The subjects all gave informed consent prior to entry into this study and all studies were approved by the institution's IRB.

Flow cytometry {#S10}
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For intracellular cytokine staining (ICS), PBMC were incubated overnight at 37°C with media alone, 1μg of Staphylococcal enterotoxin B (SEB) (Sigma, St. Louis, MO), or 1μg of cytomegalovirus (CMV) whole antigen (Microbix Biosystems, Toronto, Ontario, Canada) in the presence of 0.5μg each of monoclonal antibodies to CD28 (CD28.2, Beckman Coulter, Fullerton, CA) and CD49d (9F10, BD Bioscience, San Jose, CA) and 1 μg/ml Brefeldin A (Sigma, St. Louis, MO). For some experiments PBMC were pretreated for 1 hour at 37°C with antibodies against MHC-I (G46-2.6, BD Bioscience) or MHC-II (TU39, BD Bioscience). After stimulation, cells were washed twice and incubated with Live/Dead fixable aqua dead cell stain (Invitrogen, Carlsbad, CA). Cells were then stained for the surface markers using monoclonal antibodies to CD3 (SP34-2, BD Bioscience), CD4 (L200, BD Bioscience), CD8 (RPA-T8, BD Bioscience), and CD95 (DX2, BD bioscience). Cells were washed and permeabilized with Cytofix/Cytoperm buffer (BD Bioscience). Cells were then intracellularly stained with fluorescent-conjugated monoclonal antibodies to IFNγ (4S.B3, BD Bioscience), IL-17 (eBio64DEC17, eBioscience, San Diego, CA), IL-2 (MQ1-17H12, BD Bioscience), CD40L (TRAP1, BD Bioscience), or Ki67 (B56, BD Bioscience). Cells were incubated at 4°C for 20 minutes. Cells were washed and then fixed with a 1% paraformaldehyde solution (Electron Microscopy Sciences, Hatfield, PA).

For proliferation, PBMC or flow cytometrically sorted naive CD4 T-cells were stained with 0.125 μM carboxyfluorescein succinimidyl ester (CFSE, Invitrogen) and then stimulated with 1μg/mL concanavalin A (Sigma), 1μg/mL staphylococcal enterotoxin B (Sigma), or anti-CD3/anti-CD28 microbeads at a 1:4 cell to bead ratio (generous gift from Joern E. Schmitz and Roland Zahn) for 5, 7, and 9 days. Cells were then labeled with fluorescent antibodies directed towards CD3, CD4, and CD8 (BD Bioscience).

For analysis of FoxP3 expression fresh PBMCs were surface stained and then permeabilized using FoxP3 permeabilization solution (eBioscience). Cells were intracellularly stained for FoxP3 (PCH101, eBioscience). Cells were washed and then fixed using a 1% PFA solution. All flow cytometry samples were run on a FACSAria (BD Bioscience) using FACSDiva software (BDBioscience) and data were analyzed using FlowJo (Tree Star, Ashland, OR).

Polymerase chain reaction {#S11}
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### Quantitative real time PCR {#S12}

Cell populations were sorted flow cytometrically and were lysed using 25 μL of a 1:100 dilution of proteinase K (Roche, Indianapolis, IN) in 10mM Tris buffer. Quantitative PCR was performed using 5 μL of cell lysates per reaction. Reaction conditions were as follows: 95°C holding stage for 5 minutes, and 40 cycles of 95°C for 15 seconds followed by 60°C for 1 minute. The Taq DNA polymerase kit (Invitrogen) was used. The sequence of the forward primer for SIVagm is GTCCAGTCTCAGCATTTACTTG. The reverse primer sequence is CGGGCATTGAGGTTTTTCAC. The probe sequence is CAGATGTTGAAGCTGACCATTTGGG. For cell number quantitation monkey albumin was measured as previously described[@R55]. The PCR machine used was the StepOne Plus (Applied Biosystems, Foster City, CA) and the analysis was performed using StepOne software (Applied Biosystems).

### Reverse transcription PCR {#S13}

Viable cell populations were sorted flow cytometrically and mRNA was isolated using Oligotex Direct mRNA Mini Kit (Qiagen, Valencia, CA) by following the manufacturer's protocol. Following cDNA synthesis with random hexamers and Superscript II RNase H- reverse transcriptase transcripts of CD4 were amplified by PCR using the forward primer TCGGATTGACTGCCAACTCTG and reverse primer AAGGCGAGCGGGAAGGAGAA. Reaction conditions were as follows: 95°C holding stage for 5 minutes, and 40 cycles of 95°C for 15 seconds followed by 60°C for 1 minute. To control for the amount of mRNA, a second PCR was performed using primers for albumin (as above).

Statistics {#S14}
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All statistical analyses were performed using Prism software (GraphPad, La Jolla, CA). Statistically significance was based upon P values less than 0.05.

Supplementary Material {#S15}
======================

These studies were supported by the intramural NIAID, NIH program and by R01 AI064066 (IP), R01 AI065325 (CA) and RR-00168 (TNPRC). The authors would like to thank the Bad Boys of Cleveland for helpful discussions. We are grateful to Drs. Joern Schmitz and Roland Zahn for the kind donation of anti-CD3/CD28 microbeads for stimulation of T-cells from nonhuman primates. We are also appreciative to Dr. Bernard Lafont and Gary Mettler for their technical advice.

![Phenotypic analysis of T-cell populations in vervet African green monkeys\
(**a**) Phenotype of T-cells in adult AGM. (**b**) Phenotype of individual subsets of T-cells in adult AGM. (**c**) Comparison of percent of CD3^+^ T-cells that express CD4 in peripheral blood in SIV^+^ and SIV^−^ adult AGM, adult SIV^+^ and SIV^−^ SM, adult SIV^−^ RM, and HIV^−^ adult humans. (**d**) Characterization of CD4^−^CD8α^dim^ T-cells as CD8αβ^−^ by analysis of CD8β expression. Cells were gated on live CD3^+^ small lymphocytes and analyzed for both CD4 and CD8β expression. (**e**) Ki67 expression by different subsets of memory T-cells. (**f**) Negative correlation between the frequencies of CD4^+^ T-cell and CD4^−^CD8α^dim^ T-cells in adult AGM. (**g**) Correlation of the frequency of CD4^+^ T-cells and the frequency of CD4^−^CD8α^bright^ T-cells in adult AGM. White circles represent SIV-infected AGM and black circles represent uninfected AGM. White diamonds represent SIV-infected SM and black diamonds prepresent uninfected SM. Black triangles represent SIV-uninfected RM and black squares represent HIV-uninfected humans. A Mann-Whitney U test was performed for **c**. A Spearman rank correlation was calculated for **f** and **g**.](nihms113941f1){#F1}

![CD8α and CD4 expression by naive and memory CD4^+^ T-cells\
(**a**) Representative staining of CD4^+^ memory and naive T-cells for CD4. (**b**) Representative staining of CD4^+^ memory and naive T-cells for CD8. (**c**) Fluorescence minus one control lacking antibodies against CD8α. (**d**) Median fluorescence intensity for CD4 expression in naive and memory CD4^+^ T-cells. (**e**) Median fluorescence intensity for CD8 expression in naive and memory CD4^+^ T-cells. A Wilcoxon matched pairs test was performed for **2d--e**.](nihms113941f2){#F2}

![CD4^−^CD8α^dim^ T-cells can develop from memory CD4^+^ T-cells\
(**a**) Down-regulation of CD4 by stimulated *in vitro* PBMC from AGM after 5 days of stimulation with SEB. (**b**) Maintenance of CD4 expression by stimulated PBMC from pigtail macaques *in vitro* after 5 days of stimulation with SEB. (**c**) CD4 mRNA expression in CD14^+^, CD4^−^CD8α^dim^, CD4^−^CD8α^bright^, or CD4^+^ lymphocyte subsets of an adult AGM. (**d**)Negative correlation between the frequency of memory CD4^+^ T-cells and the frequency of the total CD4^+^ T-cells in adult AGM. (**e**) Comparison of percent CD4^+^ T-cells in peripheral blood in SIV^+^ and SIV^−^ adult and SIV^−^ juvenile AGM. White circles represent SIV-infected AGM and black represent uninfected AGM. A Spearman rank correlation was calculated for **3c**. A Mann Whitney U test was performed for **3e**.](nihms113941f3){#F3}

![CD4^−^CD8α^dim^ T-cells can preserve CD4^+^ T-cell function\
(**a**) Comparison of the frequency of memory CD4^+^, CD4^−^CD8α^dim^, and CD4^−^CD8α^bright^ T-cells performing various functions: IL-2 and IL-17 production, and FoxP3 and CD40L expression. (**b**) Comparison of the relative numbers of memory CD4^+^, CD4^−^CD8α^dim^, and CD4^−^CD8α^bright^ T-cells from different memory T-cell subsets performing various functions: IL-2 and IL-17 production, and FoxP3 and CD40L expression (**c**) Responses of CD4^−^CD8α^dim^ T-cells to CMV whole antigen in the presence and absence blocking antibodies to MHC-II or MHC-I. White circles represent SIV-infected AGM and black represent uninfected AGM. A Wilcoxon matched pairs test was performed for **a--b**.](nihms113941f4){#F4}

![Infection frequency of lymphocyte subsets\
Infection frequency of sorted lymphocyte subsets from SIVagm-infected adult AGM as determined by PCR. White circles represen T-cells with detectable viral DNA and black circles represent an undetectable infection frequency reported as one half the lower limit of detection based on the number of cells within each PCR reaction. A Wilcoxon matched pairs test was performed for this analysis.](nihms113941f5){#F5}

###### 

Clinical information of African green monkeys.

  Animal    Virus Strain          Infection Dose (TCID)   Plasma Viral Load (/mL)   CD4 T-cells (/μl blood)
  --------- --------------------- ----------------------- ------------------------- -------------------------
  AG3       Naturally Infected    NA                      29600                     341
  AG4       Naturally Infected    NA                      19600                     252
  AG5       Naturally Infected    NA                      26000                     63
  AG6       Naturally Infected    NA                      1400                      314
  AG10      Naturally Infected    NA                      8000                      210
  AG11      Naturally Infected    NA                      Undetected                156
  AG12      Naturally Infected    NA                      23600                     60
  AG15      Naturally Infected    NA                      Undetected                495
  AG16      Naturally Infected    NA                      61100                     452
  AG17      Naturally Infected    NA                      148000                    235
  AG7       SIVagm90              1000                    55800                     449
  AG8       SIVagmVer1            50                      1660                      180
  AG13      SIVagm90              1000                    31500                     308
  AG19      SIVagmVer1            50                      44300                     427
  AG22      SIVagm90              1000                    2066100                   185
  AG24      SIVagm90              1000                    11500                     301
  AG302     SIVagm90              1000                    19700                     170
  AG346     SIVagm90              1000                    2000                      17
  AG9       Uninfected            NA                      NA                        289
  AG23      Uninfected            NA                      NA                        821
  AG731     Uninfected            NA                      NA                        129
  AG5339    Uninfected            NA                      NA                        165
  AG5387    Uninfected            NA                      NA                        147
  AG5419A   Uninfected            NA                      NA                        35
  AG5419B   Uninfected            NA                      NA                        67
  AG5431    Uninfected            NA                      NA                        62
  AG5441    Uninfected            NA                      NA                        114
  AG5504    Uninfected            NA                      NA                        55
  AG5506    Uninfected            NA                      NA                        99
  AG25      Uninfected juvenile   NA                      NA                        1625
  AG26      Uninfected juvenile   NA                      NA                        2593
  AG28      Uninfected juvenile   NA                      NA                        2791
  AG30      Uninfected juvenile   NA                      NA                        1531
  AG31      Uninfected juvenile   NA                      NA                        3214
  AG32      Uninfected juvenile   NA                      NA                        2121
  AG33      Uninfected juvenile   NA                      NA                        2798
  HK14      Uninfected sabeus     NA                      NA                        378
