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.

Author Summary

Several African nonhuman primate species such as sooty mangabeys are naturally infected with SIV and maintain high levels of viral replication without developing AIDS. SIV-infected natural hosts do not show evidence of increased chronic immune activation, a feature that distinguishes them from AIDS-susceptible SIV-infected Asian macaques. In this study we compared natural killer T (NKT) lymphocytes, a unique subset of innate T lymphocytes with anti-inflammatory properties, in AIDS-resistant and AIDS-susceptible hosts. Sooty mangabey NKT cells retained normal functionality following SIV infection and were more potent than macaque NKT cells in their ability to produce interferon-γ and secrete anti-inflammatory cytokines. In contrast, NKT cells of SIV-infected macaques were markedly hypo-functional with regards to secretion of anti-inflammatory and effector cytokines and showed an association between loss of CD4+ NKT cells and increased immune activation. These findings suggest that dysfunctional NKT cells may promote increased immune activation in AIDS-susceptible hosts while intact effector and anti-inflammatory NKT cells could help to prevent immunodeficiency and increased immune activation in natural hosts.

Background

We have recently reported the presence of CD8+ and CD4/8 double negative (DN) Natural Killer T (NKT) lymphocytes in sooty mangabeys. To investigate differences in the two NKT cell subsets, we compared the phenotype and function of sooty mangabey CD8+ and DN NKT cells.

Methods

Flow sorted NKT lymphocytes from one SIV-negative sooty mangabey were subjected to limiting dilution cloning. Invariant NKT clones were characterized by flow cytometry and cytokine-ELISA.

Results

The majority of NKT clones displayed an effector memory phenotype and expressed CXCR3 and NKG2D. While CD8+ NKT subsets expressed significantly higher levels of granzyme B and perforin and produced more IFN-γ, the DN NKT subsets secreted significantly more IL-4, IL-13, and IL-10.

Conclusions

The Th1 and Th2 cytokine bias of CD8+ and DN NKT cells respectively indicates the presence of functionally heterogeneous populations of NKT cells in sooty mangabeys.

The purine nucleoside adenosine is an important anti-inflammatory molecule, inhibiting a variety of immune cells by adenosine receptor-mediated mechanisms. Invariant natural killer T (iNKT) cells recognize glycolipids presented on CD1d molecules and produce vigorous amounts of cytokines upon activation, hence regulating immune reactions. The mechanisms polarizing their cytokine pattern are elusive. Previous studies demonstrated that adenosine can suppress IFN-γ production by iNKT cells.

We describe the expression of all four known adenosine receptors A1R, A2aR, A2bR, and A3R, on mouse iNKT cells. We show that IL-4 production in primary mouse iNKT cells and a human iNKT line is efficiently inhibited by A2aR blockade with an inverse relation to IL-4. These data are supported by A2aR-deficient mice, which exhibit largely decreased levels of IL-4, IL-10 and TGF-β concomitantly with an increase of IFN-γ upon α-GalCer administration in vivo. While A2aR inhibits other lymphocyte populations, A2aR is required for the secretion of IL-4 and IL-10 by iNKT cells. These data suggest adenosine:A2aR-mediated mechanisms can control the cytokine secretion pattern of iNKT cells.

CD1d-restricted ‘NKT’ rapidly stimulate innate and adaptive immunity through production of Th1 and/or Th2 cytokines and induction of CD1d+ antigen-presenting cell (APC) maturation. However, therapeutic exploitation of NKT has been hampered by their paucity and defects in human disease. NKT:APC interactions can be modeled by direct stimulation of human APC through CD1d in vitro. We have now found that direct ligation with multiple CD1d mAbs also stimulated bioactive IL-12 release from CD1d+ but not CD1d KO murine splenocytes in vitro. Moreover, all CD1d mAbs tested also induced IL-12 as well as both IFN-γ and IFN-α in vivo from CD1d+ but not CD1d-deficient recipients. Unlike IFN-γ, CD1d-induced IFN-α was at least partially dependent on invariant NKT. Optimal resistance to infection with picornavirus encephalomyocarditis virus (EMCV) is known to require CD1d-dependent APC IL-12-induced IFN-γ as well as IFN-α. CD1d ligation in vivo enhanced systemic IL-12, IFN-γ, and IFN-α, and was protective against infection by EMCV, suggesting an alternative interpretation for previous results involving CD1d ‘blocking’ in other systems. Such protective responses, including elevations in Th1 cytokines, were also seen with CD1d FAb’2s in vivo, while an IgM mAb (with presumably minimal tissue penetration) was comparably effective at protection in vivo as well as cytokine induction both in vivo and in vitro. Although presumably acting immediately ‘downstream’, CD1d mAbs were protective later during infection than iNKT agonist α-galactosylceramide. These data indicate that NKT can be bypassed with CD1d-mediated induction of robust Th1 immunity, which may have therapeutic potential both directly and as adjuvant.

CD1d is expressed on APCs and presents glycolipids to CD1d-restricted NKT cells. For the first time, we demonstrate the ability of anti-CD1d mAbs to inhibit the growth of different CD1d-negative experimental carcinomas in mice. Anti-CD1d mAbs systemically activated CD1d+ APC, as measured by production of IFN-γ and IL-12. Tumor growth inhibition was found to be completely dependent on IFN-γ and IL-12 and variably dependent on CD8+ T cells and NK cells, depending upon the tumor model examined. Anti-CD1d mAb induced greater CD8+ T cell-dependent tumor suppression where regulatory CD1d-restricted type II NKT cells have been implicated, and were less effective in a NK cell-dependent manner against tumors where T regulatory cells were immunosuppressive. The ability of anti-CD1d mAbs to coincidently activate CD1d+ APCs to release IL-12 and inhibit CD1d-restricted type II NKT cells makes CD1d an exciting new target for immunotherapy of cancer based on tumor immunoregulation.

CD1d-restricted invariant NKT (iNKT) cells play important regulatory roles in various immune responses, including antitumor immune responses. Previous studies have demonstrated quantitative and qualitative defects in iNKT cells of cancer patients, and these defects are clinically relevant as they are associated with poor prognosis. In this study we demonstrate that defects in the iNKT cell population can, at least in part, be attributed to defective interactions between iNKT cells and CD1d-expressing circulating myeloid dendritic cells (mDC), as mDC of patients with advanced melanoma and renal cell cancer reduced the activation and Th1 cytokine production of healthy donor-derived iNKT cells. Interestingly, this reduced activation of iNKT cells was restricted to patients with low circulating iNKT cell numbers and could be reversed by IL-12 and in part by the neutralization of TGF-β, but it was further reduced by the neutralization of IL-10 in vitro. Additional experiments revealed discordant roles for TGF-β and IL-10 on human iNKT cells, because TGF-β suppressed iNKT cell activation and proliferation and IFN-γ production while IL-10 was identified as a cytokine involved in stimulating the activation and expansion of iNKT cells that could subsequently suppress NK cell and T cell responses.

Abetalipoproteinemia (ABL) is a rare Mendelian disorder of lipid metabolism due to genetic deficiency in microsomal triglyceride transfer protein (MTP). It is associated with defects in MTP-mediated lipid transfer onto apolipoprotein B (APOB) and impaired secretion of APOB-containing lipoproteins. Recently, MTP was shown to regulate the CD1 family of lipid antigen-presenting molecules, but little is known about immune function in ABL patients. Here, we have shown that ABL is characterized by immune defects affecting presentation of self and microbial lipid antigens by group 1 (CD1a, CD1b, CD1c) and group 2 (CD1d) CD1 molecules. In dendritic cells isolated from ABL patients, MTP deficiency was associated with increased proteasomal degradation of group 1 CD1 molecules. Although CD1d escaped degradation, it was unable to load antigens and exhibited functional defects similar to those affecting the group 1 CD1 molecules. The reduction in CD1 function resulted in impaired activation of CD1-restricted T and invariant natural killer T (iNKT) cells and reduced numbers and phenotypic alterations of iNKT cells consistent with central and peripheral CD1 defects in vivo. These data highlight MTP as a unique regulator of human metabolic and immune pathways and reveal that ABL is not only a disorder of lipid metabolism but also an immune disease involving CD1.

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.

OBJECTIVE

Bone marrow (BM) Th1 populations can contribute to graft-versus-leukemia (GvL) responses. G/GM-CSF-mobilized peripheral blood progenitor cells (PBPC) have become widely accepted alternatives to BM transplantation (BMT). T cells co-expressing NK proteins (NKT) include a CD1d-reactive subset which influence immunity by rapidly producing large amounts of Th1 and/or Th2 cytokines dependent upon microenvironment and disease. There are two types of CD1d-reactive NKT. “iNKT” express a semi-invariant TCR-α. Other “non-invariant” CD1d-reactive NKT from BM and liver produce large amounts of IL-4 or IFN-γ respectively, and within the intestine can be biased in either direction. Recent data suggests that NKT might contribute to clinical benefits of PBPC.

METHODS

To address these issues, we phenotypically and functionally studied PBPC NKT.

RESULTS

Similarly to BM, NKT-like cells were common in allogeneic and autologous PBPC, there were relatively few classical iNKT, but high CD1d-reactivity concentrated in NKT fractions. Significantly, PBPC CD1d-reactive cells were relatively Th1-biased and their presence was associated with better prognosis. G-CSF treatment of BM to yield PBPC in vivo as well as in vitro Th2-polarizes conventional T cells and iNKT. However, G-CSF treatment of BM in vitro produced Th1-biased NKT, providing a mechanism for opposite polarization of NKT from BM versus PBPC.

CONCLUSIONS

These results suggest distinct Th1 CD1d-reactive NKT cells could stimulate anti-tumor responses from those previously described, which can suppress GvHD.