Paucity of CD4+ Natural Killer T (NKT) Lymphocytes in Sooty Mangabeys Is Associated with Lack of NKT Cell Depletion after SIV Infection

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 α-galactosylceramide loaded CD1d tetramers co-staining with Vα24-positive invariant NKT lymphocytes were detected at frequencies ≥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 α-galactosylceramide loaded on human CD1d molecules, sooty mangabey NKT lymphocytes underwent degranulation and secreted IFN-γ, TNF-α, 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.     

Loss of Effector and Anti-Inflammatory Natural Killer T Lymphocyte Function in Pathogenic Simian Immunodeficiency Virus Infection

Chronic immune activation is a key determinant of AIDS progression in HIV-infected humans and simian immunodeficiency virus (SIV)-infected macaques but is singularly absent in SIV-infected natural hosts. To investigate whether natural killer T (NKT) lymphocytes contribute to the differential modulation of immune activation in AIDS-susceptible and AIDS-resistant hosts, we compared NKT function in macaques and sooty mangabeys in the absence and presence of SIV infection. Cynomolgus macaques had significantly higher frequencies of circulating invariant NKT lymphocytes compared to both rhesus macaques and AIDS-resistant sooty mangabeys. Despite this difference, mangabey NKT lymphocytes were functionally distinct from both macaque species in their ability to secrete significantly more IFN-γ, IL-13, and IL-17 in response to CD1d/α-galactosylceramide stimulation. While NKT number and function remained intact in SIV-infected mangabeys, there was a profound reduction in NKT activation-induced, but not mitogen-induced, secretion of IFN-γ, IL-2, IL-10, and TGF-β in SIV-infected macaques. SIV-infected macaques also showed a selective decline in CD4⁺ NKT lymphocytes which correlated significantly with an increase in circulating activated memory CD4⁺ T lymphocytes. Macaques with lower pre-infection NKT frequencies showed a significantly greater CD4⁺ T lymphocyte decline post SIV infection. The disparate effect of SIV infection on NKT function in mangabeys and macaques could be a manifestation of their differential susceptibility to AIDS. Alternately, these data also raise the possibility that loss of anti-inflammatory NKT function promotes chronic immune activation in pathogenic SIV infection, while intact NKT function helps to protect natural hosts from developing immunodeficiency and aberrant immune activation.     

Diverse Host Responses and Outcomes following Simian Immunodeficiency Virus SIVmac239 Infection in Sooty Mangabeys and Rhesus Macaques

Sooty mangabeys naturally infected with simian immunodeficiency virus (SIV) do not develop immunodeficiency despite the presence of viral loads of 10⁵ to 10⁷ RNA copies/ml. To investigate the basis of apathogenic SIV infection in sooty mangabeys, three sooty mangabeys and three rhesus macaques were inoculated intravenously with SIVmac239 and evaluated longitudinally for 1 year. SIVmac239 infection of sooty mangabeys resulted in 2- to 4-log-lower viral loads than in macaques and did not reproduce the high viral loads observed in natural SIVsmm infection. During acute SIV infection, polyclonal cytotoxic T-lymphocyte (CTL) activity coincident with decline in peak plasma viremia was observed in both macaques and mangabeys; 8 to 20 weeks later, CTL activity declined in the macaques but was sustained and broadly directed in the mangabeys. Neutralizing antibodies to SIVmac239 were detected in the macaques but not the mangabeys. Differences in expression of CD38 on CD8⁺ T lymphocytes or in the percentage of naive phenotype T cells expressing CD45RA and CD62L-selection did not correlate with development of AIDS in rhesus macaques. In macaques, the proportion of CD4⁺ T lymphocytes expressing CD25 declined during SIV infection, while in mangabeys, CD25-expressing CD4⁺ T lymphocytes increased. Longitudinal evaluation of cytokine secretion by flow cytometric analysis of unstimulated lymphocytes revealed elevation of interleukin-2 and gamma interferon in a macaque and only interleukin-10 in a concurrently infected mangabey during acute SIV infection. Differences in host responses following experimental SIVmac239 infection may be associated with the divergent outcome in sooty mangabeys and rhesus macaques.     

Use of human CD3 monoclonal antibody for accurate CD4+ and CD8+ lymphocyte determinations in macaques: phenotypic characterization of the CD3- CD8+ cell subset

Macaque monkeys are frequently used in models for studies of infectious diseases, immunity, transplantation and vaccine development. Such use is largely due to the conservation of functionally important cell surface molecules and the phylogenetic proximity of their immune systems to that of humans. Some monoclonal antibodies (mAb) raised against human leukocyte antigens can be utilized in the monkey. Until recently, many primate centers have utilized the CD2 monoclonal antibody to enumerate T lymphocytes. We have evaluated the anti-human CD3 mAb in macaques and sooty mangabeys. Using this monoclonal antibody, pigtailed macaques were found to have a much higher proportion of CD2+ CD3- CD8+ cells as compared with rhesus macaques and sooty mangabeys. Such cells comprised approximately one-half of all CD8+ cells in the pigtailed macaque, but only one-quarter of CD8+ cells in the rhesus, and one-fifth in the sooty mangabey. Use of the CD2 monoclonal antibody as the T-cell marker resulted in underestimating CD4/CD8 ratios compared with using the CD3 mAb in pigtailed macaques. Phenotypic characterization of this subset of CD3- CD8+ cells indicated that they are CD16+, CD45RA+, CD11b+, CD69+ and CD28-. This would indicate that these cells represent an activated natural killer cell subset.     

Flow microfluorometric analysis of peripheral blood mononuclear cells from nonhuman primates: Correlation of phenotype with immune function

A panel of monoclonal antibody reagents has been identified that can be used for routine monitoring of subsets of peripheral blood mononuclear cells (PBMC) from Macaca mulatta (rhesus macaques), Macaca nemestrina (pig-tailed macaques), and Cercocebus atys (sooty mangabeys). The procedure uses fluorescein and phycoerythrin conjugates of the monoclonal antibodies in appropriate combinations, so that two-color microfluorometric analyses can be readily performed on as little as 1.2 ml of EDTA blood. PBMC from a total of 20 normal adult rhesus macaques, 21 normal adult pig-tailed macaques, 4 SIV^? sooty mangabeys, and 16 SIV⁺ adult sooty mangabeys were analyzed with the panel of monoclonal reagents and flow microfluorometry. The mean frequency, absolute numbers, and range for each subset in these nonhuman primate species are described. Sooty mangabeys appeared markedly different from the other two primate species. The PBMCs from the mangabeys had a higher mean frequency and absolute number of total T cells, Leu-3a⁺/18^? T cells, suppressor (Leu?2a⁺) T cells, which were HLA-DR⁺, and IL-2R⁺ cells. Functional helper, suppressor, natural killer (NK), lymphokine activated killer (LAK), and antigen-presenting cell studies were also performed to correlate phenotype with immune function. Data indicate that Leu?3a⁺ T cells (CD4⁺) and Leu?2a⁺ T cells (CD8⁺) in these primate species represent human equivalents of helper and suppressor T cells, respectively. NK and LAK effector cells in the rhesus and pig-tailed macaques appear to be predominantly Leu?19⁺. In contrast, Leu?2a⁺ cells appear to be the predominant NK and LAK effector cell in sooty mangabeys. These data provide a basis for routine evaluation of lymphocyte subsets in these nonhuman primate species, and provide a means to correlate phenotype with immune function.     

Th-1-type cytotoxic CD8+ T-lymphocyte responses to simian immunodeficiency virus (SIV) are a consistent feature of natural SIV infection in sooty mangabeys

Sooty mangabeys are a natural host of simian immunodeficiency virus (SIV) that remain asymptomatic and do not exhibit increased immune activation or increased T-lymphocyte turnover despite sustained high levels of SIV viremia. In this study we asked whether an altered immune response to SIV contributes to the lack of immunopathology in sooty mangabeys as opposed to species with pathogenic lentivirus infection. SIV-specific cellular immune responses were investigated in a cohort of 25 sooty mangabeys with natural SIV infection. Gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay responses targeting a median of four SIV proteins were detected in all 25 mangabeys and were comparable in magnitude to those of 13 rhesus macaques infected with SIVmac251 for more than 6 months. As with rhesus macaques, Th2 ELISPOT responses to SIV were absent or >10-fold lower than the IFN-gamma ELISPOT response to the same SIV protein. The SIV-specific ELISPOT response was predominantly mediated by CD8+ T lymphocytes; the frequency of circulating SIV-specific CD8+ T lymphocytes ranged between 0.11% and 3.26% in 13 mangabeys. Functionally, the SIV-specific CD8+ T lymphocytes were cytotoxic; secreted IFN-gamma, tumor necrosis factor alpha, and macrophage inflammatory protein 1beta; and had an activated effector phenotype. Although there was a trend toward higher frequencies of SIV-specific CD8+ T lymphocytes in mangabeys with lower viral loads, a significant inverse correlation between SIV viremia and SIV-specific cellular immunity was not detected. The consistent detection of Th1-type SIV-specific cellular immune responses in naturally infected sooty mangabeys suggests that immune attenuation is neither a feature of nor a requirement for maintenance of nonpathogenic SIV infection in its natural host.     

Gamma/Delta T-cell functional responses differ after pathogenic human immunodeficiency virus and nonpathogenic simian immunodeficiency virus infections

The objective of this study was to functionally assess gamma/delta (γδ) T cells following pathogenic human immunodeficiency virus (HIV) infection of humans and nonpathogenic simian immunodeficiency virus (SIV) infection of sooty mangabeys. γδ T cells were obtained from peripheral blood samples from patients and sooty mangabeys that exhibited either a CD4-healthy (>200 CD4⁺ T cells/μl blood) or CD4-low (<200 CD4 cells/μl blood) phenotype. Cytokine flow cytometry was utilized to assess production of Th1 cytokines tumor necrosis factor alpha and gamma interferon following ex vivo stimulation with either phorbol myristate acetate/ionomycin or the Vδ2 γδ T-cell receptor agonist isopentenyl pyrophosphate. Sooty mangabeys were observed to have higher percentages of γδ T cells in their peripheral blood than humans did. Following stimulation, γδ T cells from SIV-positive (SIV⁺) mangabeys maintained or increased their ability to express the Th1 cytokines regardless of CD4⁺ T-cell levels. In contrast, HIV-positive (HIV⁺) patients exhibited a decreased percentage of γδ T cells expressing Th1 cytokines following stimulation. This dysfunction is primarily within the Vδ2⁺ γδ T-cell subset which incurred both a decreased overall level in the blood and a reduced Th1 cytokine production. Patients treated with highly active antiretroviral therapy exhibited a partial restoration in their γδ T-cell Th1 cytokine response that was intermediate between the responses of the uninfected and HIV⁺ patients. The SIV⁺ sooty mangabey natural hosts, which do not proceed to clinical AIDS, provide evidence that γδ T-cell dysfunction occurs in HIV⁺ patients and may contribute to HIV disease progression.     

Semiquantitative analysis of Th1 and Th2 cytokine expression in CD3+, CD4+, and CD8+renal-cell-carcinoma-infiltrating lymphocytes

The mRNA expression of Th1 and Th2 cytokines was compared in freshly isolated CD3⁺ tumor-infiltrating lymphocytes (CD3⁺ TIL) and in autologous CD3⁺ peripheral blood lymphocytes (CD3⁺ PBL) obtained simultaneously from 20 patients with renal cell carcinomas (RCC). In addition cytokine expression was compared in CD4⁺ TIL and CD8⁺ TIL from another group of 20 patients with RCC. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation and subsequent selection with anti-CD3, anti-CD4 or anti-CD8 magnetic beads. In these pure lymphocyte preparations the constitutive expression of interleukin-1 (IL-1), IL-2, IL-10, interferon γ (IFN), and tumor necrosis factor α (TNF) was determined by using a polymerase-chain-reaction-assisted mRNA amplification assay. In the CD3⁺ TIL, levels of mRNA for IFN, IL-10, IL-1 and TNF were significantly higher than in the autologous CD3⁺ PBL whereas IL-2 expression was rather low and did not differ in the two populations. Comparison of cytokine mRNA expression in CD4⁺ TIL and simultaneously obtained CD8⁺ TIL revealed a significantly higher expression of IFN in the CD8⁺ cells. These data reflect an in vivo activation of RCC-infiltrating lymphocytes at the mRNA level with respect to the Th1 as well as the Th2 immune response. Th1 activation seems to be most evident in the CD8⁺ TIL. Received: 14 January 1999 / Accepted: 30 April 1999     

Dynamics of T- and B-lymphocyte turnover in a natural host of simian immunodeficiency virus

Increased lymphocyte turnover is a hallmark of pathogenic lentiviral infection. To investigate perturbations in lymphocyte dynamics in natural hosts with nonpathogenic simian immunodeficiency virus (SIV) infection, the nucleoside analog bromodeoxyuridine (BrdU) was administered to six naturally SIV-infected and five SIV-negative sooty mangabeys. As a measure of lymphocyte turnover, we estimated the mean death rate by fitting a mathematical model to the fraction of BrdU-labeled cells during a 2-week labeling and a median 10-week delabeling period. Despite significantly lower total T- and B-lymphocyte counts in SIV-infected sooty mangabeys than in SIV-negative mangabeys, the turnover rate of B lymphocytes and CD4⁺ and CD8⁺ T lymphocytes was not increased in the SIV-infected animals. A small, rapidly proliferating CD45RA⁺ memory subset and a large, slower-proliferating CD45RA⁻ central memory subset of CD4⁺ T lymphocytes identified in the peripheral blood of sooty mangabeys also did not show evidence of increased turnover in the context of SIV infection. Independently of SIV infection, the turnover of CD4⁺ T lymphocytes in sooty mangabeys was significantly higher (P < 0.01) than that of CD8⁺ T lymphocytes, a finding hitherto not reported in rhesus macaques or humans. The absence of aberrant T-lymphocyte turnover along with an inherently high rate of CD4⁺ T-lymphocyte turnover may help to preserve the pool of central memory CD4⁺ T lymphocytes in viremic SIV-infected sooty mangabeys and protect against progression to AIDS.     

Human herpesvirus-6-specific interleukin 10-producing CD4+ T cells suppress the CD4+ T-cell response in infected individuals

Human herpesvirus‐6 (HHV‐6) infection normally persists for the lifetime of the host and may reactivate with immunosuppression. The mechanism behind HHV‐6 latent infection is still not fully understood. In this study, we observed that decreased proliferation of CD4⁺ T cells and PBMCs but not CD8⁺ T cells from HHV‐6‐infected individuals was stimulated with HHV‐6‐infected cell lysates. Moreover, HHV‐6‐stimulated CD4⁺ T cells from HHV‐6‐infected individuals have suppressive activity on naïve CD4⁺ T and CD8⁺ T cells from HHV‐6‐uninfected individuals. However, no increased proportion of CD4⁺ CD25⁺ Treg cells from HHV‐6‐infected individuals contributed to the suppressive activity of the HHV‐6‐stimulated CD4⁺ T cells from HHV‐6‐infected individuals. Transwell experiments, ELISA and anti‐IL‐10 antibody blocking experiment demonstrated that IL‐10 may be the suppressive cytokine required for suppressive activity of CD4⁺ T cells from HHV‐6‐infected individuals. Results of intracellular interleukin (IL)‐10 and IL‐4 further implicated the HHV‐6‐speciflc IL‐10‐producing CD4⁺ T cells in the suppressive activity of CD4⁺ T cells from HHV‐6‐infected individuals. Results of intracellular interferon (IFN)‐γ demonstrated a decreased frequency of HHV‐6‐speciflc IFN‐γ‐producing CD4⁺ T, but not CD8⁺ T cells in HHV‐6‐infected individuals, indicating that it was the CD4⁺ Th1 responses in HHV‐6‐infected individuals that were selectively impaired. Our findings indicated that HHV‐6‐specific IL‐10‐producing CD4⁺ T cells from HHV‐6‐infected individuals possess T regulatory type 1 cell activity: immunosuppression, high levels of IL‐10 production, with a few cells expressing IFN‐γ, but none expressing IL‐4. These cells may play an important role in latent HHV‐6 infection.     

Rapid G0/1 transition and cell cycle progression in CD8+ T cells compared to CD4+ T cells following in vitro stimulation

T‐cell population consists of two major subsets, CD4⁺ T cells and CD8⁺ T cells, which can be distinguished by the expression of CD4 or CD8 molecules, respectively. Although they play quite different roles in the immune system, many of their basic cellular processes such as proliferation following stimulation are presumably common. In this study, we have carefully analyzed time–course of G0/1 transition as well as cell cycle progression in the two subsets of quiescent T‐cell population following in vitro growth stimulation. We found that CD8⁺ T cells promote G0/1 transition more rapidly and drive their cell cycle progression faster compared to CD4⁺ T cells. In addition, expression of CD25 and effects of its blockade revealed that IL‐2 is implicated in the rapid progression, but not the earlier G0/1 transition, of CD8⁺ T cells.     

Acute SIV Infection in Sooty Mangabey Monkeys Is Characterized by Rapid Virus Clearance from Lymph Nodes and Absence of Productive Infection in Germinal Centers

Lymphoid tissue immunopathology is a characteristic feature of chronic HIV/SIV infection in AIDS-susceptible species, but is absent in SIV-infected natural hosts. To investigate factors contributing to this difference, we compared germinal center development and SIV RNA distribution in peripheral lymph nodes during primary SIV infection of the natural host sooty mangabey and the non-natural host pig-tailed macaque. Although SIV-infected cells were detected in the lymph node of both species at two weeks post infection, they were confined to the lymph node paracortex in immune-competent mangabeys but were seen in both the paracortex and the germinal center of SIV-infected macaques. By six weeks post infection, SIV-infected cells were no longer detected in the lymph node of sooty mangabeys. The difference in localization and rate of disappearance of SIV-infected cells between the two species was associated with trapping of cell-free virus on follicular dendritic cells and higher numbers of germinal center CD4⁺ T lymphocytes in macaques post SIV infection. Our data suggests that fundamental differences in the germinal center microenvironment prevent productive SIV infection within the lymph node germinal centers of natural hosts contributing to sustained immune competency.     

Helminth Infections Coincident with Active Pulmonary Tuberculosis Inhibit Mono- and Multifunctional CD4+ and CD8+ T Cell Responses in a Process Dependent on IL-10

Tissue invasive helminth infections and tuberculosis (TB) are co-endemic in many parts of the world and can trigger immune responses that might antagonize each other. We have previously shown that helminth infections modulate the Th1 and Th17 responses to mycobacterial-antigens in latent TB. To determine whether helminth infections modulate antigen-specific and non-specific immune responses in active pulmonary TB, we examined CD4⁺ and CD8⁺ T cell responses as well as the systemic (plasma) cytokine levels in individuals with pulmonary TB with or without two distinct helminth infections—Wuchereria bancrofti and Strongyloides stercoralis infection. By analyzing the frequencies of Th1 and Th17 CD4⁺ and CD8⁺ T cells and their component subsets (including multifunctional cells), we report a significant diminution in the mycobacterial–specific frequencies of mono- and multi–functional CD4⁺ Th1 and (to a lesser extent) Th17 cells when concomitant filarial or Strongyloides infection occurs. The impairment in CD4⁺ and CD8⁺ T cell cytokine responses was antigen-specific as polyclonal activated T cell frequencies were equivalent irrespective of helminth infection status. This diminution in T cell responses was also reflected in diminished circulating levels of Th1 (IFN-γ, TNF-α and IL-2)- and Th17 (IL-17A and IL-17F)-associated cytokines. Finally, we demonstrate that for the filarial co-infections at least, this diminished frequency of multifunctional CD4⁺ T cell responses was partially dependent on IL-10 as IL-10 blockade significantly increased the frequencies of CD4⁺ Th1 cells. Thus, co-existent helminth infection is associated with an IL-10 mediated (for filarial infection) profound inhibition of antigen-specific CD4⁺ T cell responses as well as protective systemic cytokine responses in active pulmonary TB.     

Phosphorylation and down-regulation of CD4 and CD8 in human CTLs and mouse L cells

The CD4 and CD8 molecules are rapidly phosphorylated following exposure of CD4⁺ or CD8⁺ human cytotoxic T lymphocytes (CTL) clones to B-lymphoblastoid cell lines bearing the relevant target alloantigens. Treatment of CD4⁺ or CD8⁺ CTL clones with phorbol myristate acetate (PMA), phytohemagglutinin, or mitogenic combinations of CD2-specific antibodies also resulted in CD4 or CD8 phosphorylation. Down-regulation of the surface expression of these molecules could be demonstrated in both CD4⁺ and CD8⁺ clones following exposure to the relevant alloantigen or PMA. Parallel experiments were conducted using mouse L cells in which the human CD4 or CD8 antigens were stably expressed. Exposure of these transfectants to PMA induced rapid phosphorylation of the CD4 and CD8 molecules. As in CD4⁺ CTL clones, rapid modulation of the CD4 antigen could be demonstrated in L cells following PMA treatment. In contrast, there was no demonstrable down-regulation of the CD8 antigen in PMA-treated CD8⁺ L cell transfectants. These studies demonstrate a significant differential property of the CD4 and CD8 antigens and suggest that down-regulation of the CD8 antigen may require its expression in a T-cell environment and/or the association of CD8 with the T-cell receptor or other T cell-specific molecules.     

Changes of frequency and expression level of CD161 in CD8+ T cells and natural killer T cells in peripheral blood of patients with systemic lupus erythematosus

Immunologic abnormalities of natural killer (NK) cells and T cells play a role in the pathogenesis of systemic lupus erythematosus (SLE). CD161 is expressed on most of the NK cells and on some T cells. The quantities of CD161-expressing cells and expression levels of CD161 were analyzed in T cells and NK cells from patients with SLE compared with normal controls. The expression of CD161 on NK cells, NKT cells, CD4⁺ T cells, and CD8⁺ T cells in peripheral blood from patients with inactive SLE and active SLE, and from the normal controls group were determined using flow cytometry. The frequency and expression level of CD161 in the lymphocyte subsets and its relationship with the quantity of regulatory T cells, anti-double stranded DNA antibody, and the titer of antinuclear antibody were evaluated. Both the percentages of the CD161⁺ subpopulation and the mean fluorescence intensities (MFIs) of CD161 in CD8⁺ T cells and NKT cells decreased significantly in SLE patients compared with normal controls (P < .001). The CD161 expression in CD8⁺ T cells and NKT cells also decreased in the anti-dsDNA (+) group (P < .05). The counts of Treg cells were lower in SLE patients and were weakly correlated with the percentage of the CD161 subpopulation (r = 0.229, P = .016) and the MFIs of CD161 expression in CD8⁺ T cells (r = .232, P = .014). The frequencies and levels of CD161 expression on CD8⁺ T cells and NKT cells were reduced in SLE patients, suggesting that an abnormality of these cells was related to the pathogenesis of SLE.