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Defining the correlates of immune protection conferred by SIVΔnef, the most effective vaccine against SIV challenge, could enable the design of a protective vaccine against HIV infection. Here we provide a comprehensive assessment of immune responses that protect against SIV infection through detailed analyses of cellular and humoral immune responses in the blood and tissues of rhesus macaques vaccinated with SIVΔnef and then vaginally challenged with wild-type SIV. Despite the presence of robust cellular immune responses, animals at 5 weeks after vaccination displayed only transient viral suppression of challenge virus, whereas all macaques challenged at weeks 20 and 40 post-SIVΔnef vaccination were protected, as defined by either apparent sterile protection or significant suppression of viremia in infected animals. Multiple parameters of CD8 T cell function temporally correlated with maturation of protection, including polyfunctionality, phenotypic differentiation, and redistribution to gut and lymphoid tissues. Importantly, we also demonstrate the induction of a tissue-resident memory population of SIV-specific CD8 T cells in the vaginal mucosa, which was dependent on ongoing low-level antigenic stimulation. Moreover, we show that vaginal and serum antibody titers inversely correlated with post-challenge peak viral load, and we correlate the accumulation and affinity maturation of the antibody response to the duration of the vaccination period as well as to the SIVΔnef antigenic load. In conclusion, maturation of SIVΔnef-induced CD8 T cell and antibody responses, both propelled by viral persistence in the gut mucosa and secondary lymphoid tissues, results in protective immune responses that are able to interrupt viral transmission at mucosal portals of entry as well as potential sites of viral dissemination., Author Summary Annually, more than two million people worldwide are infected with HIV, the virus that causes AIDS. Rhesus macaques can be infected with SIV, a close relative and ancestor of HIV, resulting in simian AIDS, recapitulating key aspects of human HIV infection. SIVΔnef, a live attenuated form of SIV, protects rhesus macaques from subsequent challenge with pathogenic SIV and is widely viewed as the most effective SIV vaccine. Here we demonstrate that SIVΔnef persistence during the vaccination period drives both cell-mediated and humoral immune response maturation. During the vaccination period, cell-mediated immune responses elicited by SIVΔnef target more conserved regions of the virus rendering immune escape more difficult. Furthermore, the localization of the cell-mediated immune responses is shifted over time from peripheral blood to sites of viral production that are rich in uninfected SIV target cells, thereby positioning cell-mediated immune responses where they are most needed after wild-type SIV challenge. Similarly, SIVΔnef persistence during the vaccination period also leads to the accumulation and maturation of the humoral immune response. Our findings highlight the unique capacity of persistent vaccines to elicit durable and effective immune responses against wild-type SIV challenge.

Human immunodeficiency virus type 1 (HIV-1) does not replicate in primary cells of New World primates. To better understand this restriction, we expressed owl monkey ( Aotus nancymaae ) CD4 and CXCR4 in the owl monkey kidney cell line, OMK. An HIV-1 variant modified to evade the owl monkey restriction factor TRIM-cyp replicated efficiently in these cells but could not replicate in primary A. nancymaae CD4-positive T cells. To understand this difference, we examined APOBEC3G and tetherin orthologs from OMK cells and primary A. nancymaae cells. We observed that OMK cells expressed substantially lower levels of APOBEC3G than did A. nancymaae cells. A. nancymaae , but not marmoset ( Callithrix jacchus ), APOBEC3G was partially downregulated by HIV-1 vif and reduced but did not abolish HIV-1 replication when stably expressed in OMK cells. The functional difference between A. nancymaae and marmoset APOBEC3Gs mapped to residue 128, previously shown to distinguish African green monkey from human APOBEC3G. We also characterized tetherin orthologs from OMK and A. nancymaae cells. The A. nancymaae tetherin ortholog, but not OMK tetherin, prevented HIV-1 release. Alteration of threonine 181 of OMK tetherin rescued its function and its efficient N glycosylation. All alleles of A otus lemurinus griseimembra examined, but none of A. nancymaae or A otus vociferans , encoded this nonfunctional tetherin ortholog. Our data indicate that HIV-1 replication in owl monkeys is not restricted at entry but can be limited by APOBEC3G and tetherin. Further, A. lemurinus griseimembra does not restrict HIV-1 replication via tetherin, a property likely useful for the study of tetherin-restricted viruses.

Natural killer (NK) cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. We previously reported that the binding of a common MHC class I molecule in the rhesus macaque, Mamu-A1*002, to the inhibitory receptor Mamu-KIR3DL05 is stabilized by certain simian immunodeficiency virus (SIV) peptides, but not by others. Here we investigated the functional implications of these interactions by testing SIV peptides bound by Mamu-A1*002 for the ability to modulate Mamu-KIR3DL05+ NK cell responses. Twenty-eight of 75 SIV peptides bound by Mamu-A1*002 suppressed the cytolytic activity of primary Mamu-KIR3DL05+ NK cells, including three immunodominant CD8+ T cell epitopes previously shown to stabilize Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. Substitutions at C-terminal positions changed inhibitory peptides into disinhibitory peptides, and vice versa, without altering binding to Mamu-A1*002. The functional effects of these peptide variants on NK cell responses also corresponded to their effects on Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. In assays with mixtures of inhibitory and disinhibitory peptides, low concentrations of inhibitory peptides dominated to suppress NK cell responses. Consistent with the inhibition of Mamu-KIR3DL05+ NK cells by viral epitopes presented by Mamu-A1*002, SIV replication was significantly higher in Mamu-A1*002+ CD4+ lymphocytes co-cultured with Mamu-KIR3DL05+ NK cells than with Mamu-KIR3DL05- NK cells. These results demonstrate that viral peptides can differentially affect NK cell responses by modulating MHC class I interactions with inhibitory KIRs, and provide a mechanism by which immunodeficiency viruses may evade NK cell responses., Author Summary Natural killer (NK) cells recognize and kill infected cells without prior antigenic stimulation, and thus provide an important early defense against virus infection. NK cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. Inhibitory KIRs normally suppress NK cell responses through interactions with their MHC class I ligands on the surface of healthy cells. However, when these interactions are perturbed, this inhibition is lost resulting in NK cell activation and killing of the target cell. We investigated the functional implications of simian immunodeficiency virus (SIV) peptides bound by a common MHC class I molecule in the rhesus macaque that stabilize or disrupt binding to an inhibitory KIR. Whereas SIV peptides that stabilized KIR-MHC class I binding suppressed NK cell activation, peptides that disrupted this interaction did not and resulted in NK cell lysis. These findings demonstrate that viral peptides can modulate NK cell responses through KIR-MHC class I interactions, and are consistent with the possibility that human and simian immunodeficiency viruses may acquire changes in epitopes that increase the binding of MHC class I ligands to inhibitory KIRs as a mechanism to suppress NK cell responses.

The onset of protective immunity against pathogenic SIV challenge in SIVΔnef-vaccinated macaques is delayed for 15-20 weeks, a process that is related to qualitative changes in CD8+ T cell responses induced by SIVΔnef. As a novel approach to characterize cell differentiation following vaccination, we used multi-target qPCR to measure transcription factor expression in naïve and memory subsets of CD8++ T cells, and in SIV-specific CD8+ T cells obtained from SIVΔnef-vaccinated or wild type SIVmac239-infected macaques. Unsupervised clustering of expression profiles organized naïve and memory CD8+ T cells into groups concordant with cell surface phenotype. Transcription factor expression patterns in SIV-specific CD8+ T cells in SIVΔnef-vaccinated animals were distinct from those observed in purified CD8+ T cell subsets obtained from naïve animals, and were intermediate to expression profiles of purified central memory and effector memory T cells. Expression of transcription factors elicited by SIVΔnef vaccination also varied over time: cells obtained at later time points, temporally associated with greater protection, appeared more central-memory like than cells obtained at earlier time points, which appeared more effector memory-like. Expression of transcription factors associated with effector differentiation, such as ID2 and RUNX3, were decreased over time, while expression of transcription factors associated with quiescence or memory differentiation, such as TCF7, BCOR and EOMES, increased. CD8+ T cells specific for a more conserved epitope expressed higher levels of TBX21 and BATF, and appeared more effector-like than cells specific for an escaped epitope, consistent with continued activation by replicating vaccine virus. These data suggest transcription factor expression profiling is a novel method that can provide additional data complementary to the analysis of memory cell differentiation based on classical phenotypic markers. Additionally, these data support the hypothesis that ongoing stimulation by SIVΔnef promotes a distinct protective balance of CD8+ T cell differentiation and activation states., Author Summary The live attenuated vaccine SIVΔnef can induce robust CD8+ T cell- mediated protection against infection with pathogenic SIV in macaques. Thus, there is substantial interest in characterizing these immune responses to inform HIV vaccine design. Animals challenged at 15–20 weeks post vaccination exhibit robust protection, whereas animals challenged at 5 weeks post-vaccination manifest little protection. Since the frequency of SIV-specific T cells decreases from week 5 to week 20, it is likely that the quality of the response to challenge changes as virus-specific cells differentiate. We applied a novel approach of transcription factor expression profiling to characterize the differences in SIV-specific cell function and phenotype at more protected and less protected time points. Using unsupervised clustering methods informed by expression profiles assessed in purified CD8+ T cell subsets, we show that SIV-specific cells display expression profiles different than any purified CD8+ T cell subset, and intermediate to sorted effector memory and central memory subsets. SIV-specific cells overall appear more effector memory-like at week 5 post-vaccination, and more central memory-like at week 20 post-vaccination. Distinct profiles of CD8+ T cells specific for different SIV epitopes having different immune escape kinetics suggests maturation is regulated by ongoing low-level replication of vaccine virus.

The K1 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) efficiently transduces extracellular signals to elicit cellular activation events through its cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM). In addition, the extracellular domain of K1 demonstrates regional homology with the immunoglobulin (Ig) family and contains conserved regions (C1 and C2) and variable regions (V1 and V2). To generate mouse monoclonal antibodies directed against the KSHV K1 protein, BALB/c mice were primed and given boosters with K1 protein purified from mammalian cells. Twenty-eight hybridomas were tested for reactivity with K1 protein by enzyme-linked immunosorbent assay, immunofluorescence, flow cytometry, immunohistochemistry, and immunoblotting. Deletion mutants of the K1 extracellular domain were used to map the epitope of each antibody. All antibodies were directed to the Ig, C1, and C2 regions of K1. Furthermore, antibody recognition of a short sequence (amino acids 92 to 125) of the C2 region overlapping with the Ig region of K1 efficiently induced intracellular free calcium mobilization; antibody recognition of the other regions of K1 did not. The efficient signal transduction of K1 induced by antibody stimulation required both the ITAM sequence of the cytoplasmic domain and the normal structure of the extracellular domain. Finally, immunological assays showed that K1 was expressed during the early lytic cycle of viral replication in primary effusion lymphoma cells. K1 was readily detected in multicentric Castleman's disease tissues, whereas it was not detected in Kaposi's sarcoma lesions, suggesting that K1 is preferentially expressed in lymphoid cells. Thus, these results indicate that the conserved regions, particularly the Ig and C2 regions, of the K1 extracellular domain are exposed on the outer surface and play an important role in K1 structure and signal transduction, whereas the variable regions of K1 appear to be away from the surface.

Plasmacytoid dendritic cells (pDCs), a primary source of interferon α (IFN-α), provide a first line of innate immune defense against human immunodeficiency virus infection. However, their kinetics and functions during acute infection are poorly understood. In mucosal tissues of normal rhesus macaques, we found CD4(+) pDCs to be the subset responsible for most IFN-α and tumor necrosis factor α (TNF-α) production in response to Toll-like receptor (TLR) 7/8 stimulation, compared with relatively anergic CD4(-) pDCs. During acute simian immunodeficiency virus (SIV) infection, gut homing was imprinted on pDCs in the bone marrow, resulting in a decline in pDCs from circulation and secondary lymphoid tissues. Although the accumulated pDCs in the gut mucosae had robust cytokine responses to TLR7/8 stimulation in vitro, pDC gut migration occurred after infection and detection of SIV in plasma. Our data suggest that innate pDC responses do not control initial SIV seeding and dissemination but instead may contribute to ongoing immune activation in the gut.

Cytomegalovirus (CMV) is a herpesvirus establishing life-long persistence after primary infection. CMV infection can cause severe manifestations in immunocompromised subjects and fetuses but is usually asymptomatic in immunocompetent hosts. Data from several studies support an important role for CD4+ T lymphocytes in the control of human CMV infection. Early appearance of functional CMV-specific CD4+ T cells is associated with asymptomatic infections after kidney transplantation [1] and with a lower risk of viremia after hematopoietic stem cell transplantation [2]. In chronic infection, CMV-specific CD4+ T cells acquire direct antiviral functions and are pivotal for optimal B and CD8+ T-lymphocyte responses [3]. CMV-specific CD4+ T cells produce multiple cytokines, including interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), and interleukin 2 (IL-2), have potent proliferative capacity and can display major histocompatibility complex class II–restricted cytotoxicity [4, 5]. Asymptomatic primary CMV infection in immunocompetent hosts, particularly children, often leads to virus shedding in bodily secretions for a prolonged duration, suggesting incomplete immune control [6, 7]. Following primary infection, CMV-specific CD4+ T cells emerge rapidly but acquire the capacity to proliferate in vitro in response to viral antigens only several months after seroconversion [1, 7, 8]. While the delayed acquisition of functional CD4+ T-lymphocyte responses in primary infection is likely to contribute to the incomplete control of CMV replication, the etiology of functional CD4+ T-lymphocyte exhaustion remains uncertain. We recently observed that the slow acquisition of proliferative responses in primary CMV infection is associated with a reduced capacity to produce cytokines, including IL-2, IFN-γ, and TNF-α [9]. Clinical studies suggest that this functional impairment is a risk factor for mother-to-fetus transmission of CMV and for CMV retinitis in human immunodeficiency virus (HIV)–infected patients [10–13]. Thus, development of therapeutic strategies to prevent the clinical complications associated with primary CMV infection will require a better understanding of the mechanisms compromising its immune control. Rhesus macaques are currently the best available animal model of human CMV infection [14]. CMV infection is widely prevalent among captive group-housed rhesus macaques, and rhesus CMV shares close genetic similarity to human CMV [15–17]. Similar to humans, rhesus CMV infection is asymptomatic in immunocompetent macaques, and CMV reactivation-associated disease manifests only in the setting of immunosuppression [18–20]. In contrast to chronic infection, there are limited data on the virologic and immunologic events in naturally acquired primary rhesus CMV infection. We and others have shown that maternal antibodies to CMV wane around 1 year of age and that seroconversion following natural infection occurs soon thereafter [15, 18]. Furthermore, hand-reared newborn macaques separated at birth from their mothers remain CMV seronegative after loss of maternal CMV antibodies [18], suggesting that natural CMV acquisition occurs in the postnatal period. In this study, we investigated the relationship between viral load and CMV-specific CD4+ T-lymphocyte responses at different time points following natural CMV acquisition in rhesus macaques.

Genetic modification of hematopoietic stem cells often results in the expression of foreign proteins in pluripotent progenitor cells and their progeny. However, the potential for products of foreign genes introduced into hematopoietic stem cells to induce host immune responses is not well understood. Gene marking and induction of immune responses to enhanced green fluorescent protein (eGFP) were examined in rhesus macaques that underwent nonmyeloablative irradiation followed by infusions of CD34+ bone marrow cells transduced with a retroviral vector expressing eGFP. CD34+ cells were obtained from untreated animals or from animals treated with recombinant human granulocyte colony-stimulating factor (G-CSF) alone or G-CSF and recombinant human stem cell factor. Levels of eGFP-expressing cells detected by flow cytometry peaked at 0.1% to 0.5% of all leukocytes 1 to 4 weeks after transplantation. Proviral DNA was detected in 0% to 17% of bone marrow–derived colony-forming units at periods of 5 to 18 weeks after transplantation. However, 5 of 6 animals studied demonstrated a vigorous eGFP-specific cytotoxic T lymphocyte (CTL) response that was associated with a loss of genetically modified cells in peripheral blood, as demonstrated by both flow cytometry and polymerase chain reaction. The eGFP-specific CTL responses were MHC-restricted, mediated by CD8+lymphocytes, and directed against multiple epitopes. eGFP-specific CTLs were able to efficiently lyse autologous CD34+ cells expressing eGFP. Antibody responses to eGFP were detected in 3 of 6 animals. These data document the potential for foreign proteins expressed in CD34+ hematopoietic cells and their progeny to induce antibody and CTL responses in the setting of a clinically applicable transplantation protocol.

Background Although vaccination with live attenuated SIV is the most effective means of inducing protection against lentiviruses, the immunologic mechanisms responsible remain unclear. Previous studies have yielded conflicting data regarding the role of adaptive immune responses in mediating protection, suggesting that innate immune responses, including natural killer (NK) cells, may play a role.

SIV infection of macaques is the most widely employed model for preclinical AIDS vaccine and pathogenesis research. In macaques, high-titer virus-specific antibodies are induced by infection, and antibody responses can drive evolution of viral escape variants. However, neutralizing antibodies (Nabs) induced in response to SIVmac239 and SIVmac251 infection or immunization are generally undetectable or of low titer, and the identification and cloning of potent Nabs from SIVmac-infected macaques remains elusive. Based on recent advances in labeling HIV-specific B lymphocytes [1–3], we have generated recombinant, secreted, soluble SIVmac envelope (Env) proteins (gp120 and gp140) for detection and quantification of SIVmac Env-specific B lymphocytes. In contrast to HIV-1, we found that soluble SIVmac239 gp140 retains the ability to form stable oligomers without the necessity for introducing additional, stabilizing modifications. Soluble oligomeric gp140 reacted with rhesus anti-SIV Env-specific monoclonal antibodies (MAbs), and was used to deplete Env-specific antibodies with SIV neutralization capability from plasma taken from a rhesus macaque immunized with live attenuated SIVmac239∆nef. Soluble gp120 and gp140 bound to SIV-specific immortalized B cells, and to SIV Env-specific B lymphocytes in peripheral blood of immunized animals. These reagents will be useful for analyzing development of Env-specific B cell responses in preclinical studies using SIV-infected or vaccinated rhesus macaques.

In light of findings demonstrating that the macaque TRIM5alpha protein inhibits infection of cells by human immunodeficiency virus (HIV)-1, simian immunodeficiency virus (SIV)-based lentiviral vectors may have distinct advantages over HIV-1 vectors for the transduction of macaque hematopoietic stem cells. We evaluated the ability of an SIV vector (VRX859) encoding an antisense SIV envelope sequence and enhanced green fluorescent protein (GFP) to inhibit viral replication and to transduce rhesus CD34(+) lymphoid progenitor cells. After infection with homologous SIV strains, CD4(+) cell lines transduced with VRX859 exhibited more than 600-fold inhibition of viral replication compared with control cells. Less inhibition was observed with the divergent SIV strain SIVsmE660. Partial inhibition of a chimeric simian-human immunodeficiency virus, which contains an HIV-1 envelope in an SIV backbone, was observed, suggesting that the SIV vector also contributes to viral inhibition independent of the antisense envelope inhibitor. Transduction of rhesus CD34(+) cells with VRX859 at various multiplicities of infection resulted in transduction efficiencies comparable to those obtained with the HIV vector VRX494. However, when we evaluated transduction of rhesus T lymphocyte progenitors by examining GFP expression in CD4(+) T cells derived from transduced CD34(+) cells, we observed more efficient transduction with the SIV-based vector. GFP(+)CD4(+) T cells derived from VRX859-transduced CD34(+) cells strongly inhibited SIVmac239 replication as compared with control CD4(+) T cells. The ability of this SIV-based vector to mediate potent inhibition of SIV replication, coupled with its efficient transduction of rhesus hematopoietic progenitor cells, make it an important candidate for proof-of-principle experiments of stem cell gene therapy in the SIV-macaque model.

In light of recent findings demonstrating that the rhesus TRIM-5a protein specifically binds to HIV-1 Gag and blocks the uncoating of viral particles, SIV-based lentiviral vectors have been proposed as a more appropriate vector than HIV-1 based vectors for the transduction of rhesus macaque hematopoietic stem cells. We evaluated the ability of a SIVmac239-based lentiviral vector (VRX859), which encodes 1275 bp antisense SIV envelope and eGFP transcriptionally regulated from the SIV LTR, to inhibit viral replication in vitro and compared transduction of rhesus CD34+ lymphoid progenitor cells with an HIV-1 based lentiviral vector. Initially, we evaluated inhibition by VRX859 of the homologous SIVmac239; the related SIVmac251; the heterologous SIVsmE660 (|[sim]|80% homologous in env); the chimeric SHIV 89.6P and HIV-1 NL4-3. CEMx174 cells, which support replication of both HIV-1 and SIV isolates, were transduced with VRX859 at MOI of 10 and the eGFP sorted population contained 1 proviral copy per cell. Following infection with SIVmac239 and SIVmac251, CEMx174-VRX859 cells exhibited over 1000- and 650-fold inhibition of viral replication compared to control cells. Inhibition of SIVsmE660 and SHIV 89.6P was weaker, ranging from 3 to 9-fold and 18 to 32-fold. HIV-1 NL4-3 replication in CEMx174-VRX859 cells was similar to control cells. To evaluate transduction of rhesus CD34+ cells and lymphoid progenitor cells, we prestimulated CD34+ cells with Tpo, Flt3L and SCF (50 ng/ml each) for 1 day and exposed cells to VRX859 vector (or the analogous HIV-1 based lentiviral vector VRX494) for 1, 2 or 3 days on Retronectin-coated plates. Transduction of rhesus CD34+ cells with the SIV vector VRX859 over a range of MOIs (3|[ndash]|50 IU per cell) resulted in similar percentages of eGFP expression as observed with the HIV vector VRX494 at similar MOIs. Transduced CD34+ cells were then cultured with rhesus fetal thymus stromal cells to induce T cell differentiation and analyzed by flow for expression of eGFP. eGFP expression in CD4+ T cells derived from CD34+ cells transduced with VRX859 at low MOIs (3|[ndash]|10 IU per cell) was increased from 13- to 2.2- fold compared cells transduced with VRX494. eGFP expression in CD4+ T cells derived from CD34+ cells transduced at higher MOIs (30 and 50 IU per cell) were basically equivalent between VRX859 and VRX494. Rhesus CD4+ T cells expressing eGFP were obtained by cell sorting and expanded with ConA, IL-2 and irradiated human feeders. GFP+ CD4+ T cells derived from VRX859-transduced cells strongly inhibited SIVmac239, SIVsmE660 and SHIV 89.6P replication (up to 1000-fold) as compared to control CD4+ T cells. These results demonstrate superior transduction of rhesus lymphoid progenitor cells with a SIV-based lentiviral vector, particularly at low MOI, and strong inhibition of viral replication, based partially on competition with genomic RNA, in CD4+ T cells derived from transduced CD34+ cells and in a CD4+ cell line. These results support using a SIV-based lentiviral vector in the rhesus macaque model as a valuable preclinical model for stem cell gene therapy for AIDS.

Background Protective immunity against vaginal challenge in SIVΔnef-vaccinated macaques develops at 20 weeks after vaccination, whereas the magnitude of SIV-specific CD8+ T cell responses peaks at 5 weeks. SIV-specific CD8+ T cells phenotypically mature from week 5 to 20, as characterized by upregulation of CCR7 and CD127, suggesting that the quality of the CD8+ T cell response may correlate with protection.

Lack of chronic immune activation in the presence of persistent viremia is a key feature that distinguishes nonpathogenic simian immunodeficiency virus (SIV) infection in natural hosts from pathogenic SIV and HIV infection. To elucidate novel mechanisms downmodulating immune activation in natural hosts of SIV infection, we investigated natural killer T (NKT) lymphocytes in sooty mangabeys. NKT lymphocytes are a potent immunoregulatory arm of the innate immune system that recognize glycolipid antigens presented on the nonpolymorphic MHC-class I-like CD1d molecules. In a cross-sectional analysis of 50 SIV-negative and 50 naturally SIV-infected sooty mangabeys, ligand alpha-galactosylceramide loaded CD1d tetramers co-staining with Valpha24-positive invariant NKT lymphocytes were detected at frequencies >or=0.002% of circulating T lymphocytes in approximately half of the animals. In contrast to published reports in Asian macaques, sooty mangabey NKT lymphocytes consisted of CD8(+) and CD4/CD8 double-negative T lymphocytes that were CXCR3-positive and CCR5-negative suggesting that they trafficked to sites of inflammation without being susceptible to SIV infection. Consistent with these findings, there was no difference in the frequency or phenotype of NKT lymphocytes between SIV-negative and SIV-infected sooty mangabeys. On stimulation with alpha-galactosylceramide loaded on human CD1d molecules, sooty mangabey NKT lymphocytes underwent degranulation and secreted IFN-gamma, TNF-alpha, IL-2, IL-13, and IL-10, indicating the presence of both effector and immunoregulatory functional capabilities. The unique absence of CD4(+) NKT lymphocytes in sooty mangabeys, combined with their IL-10 cytokine-secreting ability and preservation following SIV infection, raises the possibility that NKT lymphocytes might play a role in downmodulating immune activation in SIV-infected sooty mangabeys.