Simon Yue

Effects of the Administration of High-Dose Interleukin-2 on Immunoregulatory Cell Subsets in Patients with Advanced Melanoma and Renal Cell Cancer

Purpose: High-dose recombinant human interleukin-2 (IL-2) therapy is of clinical benefit in a subset of patients with advanced melanoma and renal cell cancer. Although IL-2 is well known as a T-cell growth factor, its potential in vivo effects on human immunoregulatory cell subsets are largely unexplored. Experimental Design: Here, we studied the effects of high-dose IL-2 therapy on circulating dendritic cell subsets (DC), CD1d-reactive invariant natural killer T cells (iNKT), and CD4+CD25+ regulatory-type T cells. Results: The frequency of both circulating myeloid DC1 and plasmacytoid DC decreased during high-dose IL-2 treatment. Of these, only a significant fraction of myeloid DC expressed CD1d. Although the proportion of Th1-type CD4− iNKT increased, similarly to DC subsets, the total frequency of iNKT decreased during high-dose IL-2 treatment. In contrast, the frequency of CD4+CD25+ T cells, including CD4+Foxp3+ T cells, which have been reported to suppress antitumor immune responses, increased during high-dose IL-2 therapy. However, there was little, if any, change of expression of GITR, CD30, or CTLA-4 on CD4+CD25+ T cells in response to IL-2. Functionally, patient CD25+ T cells at their peak level (immediately after the first cycle of high-dose IL-2) were less suppressive than healthy donor CD25+ T cells and mostly failed to Th2 polarize iNKT. Conclusions: Our data show that there are reciprocal quantitative and qualitative alterations of immunoregulatory cell subsets with opposing functions during treatment with high-dose IL-2, some of which may compromise the establishment of effective antitumor immune responses.

Primary deficiency of microsomal triglyceride transfer protein in human abetalipoproteinemia is associated with loss of CD1 function

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.

The A2aR adenosine receptor controls cytokine production in iNKT cells

The purine nucleoside adenosine is an important anti‐inflammatory molecule, inhibiting a variety of immune cells by adenosine receptor‐mediated mechanisms. Invariant NKT (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‐galactosylceramide 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.

Circulating Myeloid Dendritic Cells of Advanced Cancer Patients Result in Reduced Activation and a Biased Cytokine Profile in Invariant NKT Cells

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.

CD1d activation and blockade: a new antitumor strategy

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.

Adoptive Transfer of Invariant NKT Cells as Immunotherapy for Advanced Melanoma: A Phase I Clinical Trial

Purpose: Invariant NKT cells (iNKT) are innate-like CD1d-restricted T cells with immunoregulatory activity in diseases including cancer. iNKT from advanced cancer patients can have reversible defects including IFNγ production, and iNKT IFNγ production may stratify for survival. Previous clinical trials using iNKT cell activating ligand α-galactosylceramide have shown clinical responses. Therefore, a phase I clinical trial was performed of autologous in vitro expanded iNKT cells in stage IIIB–IV melanoma. Experimental Design: Residual iNKT cells [<0.05% of patient peripheral blood mononuclear cell (PBMC)] were purified from autologous leukapheresis product using an antibody against the iNKT cell receptor linked to magnetic microbeads. iNKT cells were then expanded with CD3 mAb and IL2 in vitro to obtain up to approximately 109 cells. Results: Expanded iNKT cells produced IFNγ, but limited or undetectable IL4 or IL10. Three iNKT infusions each were completed on 9 patients, and produced only grade 1–2 toxicities. The 4th patient onward received systemic GM-CSF with their second and third infusions. Increased numbers of iNKT cells were seen in PBMCs after some infusions, particularly when GM-CSF was also given. IFNγ responses to α-galactosylceramide were increased in PBMCs from some patients after infusions, and delayed-type hypersensitivity responses to Candida increased in 5 of 8 evaluated patients. Three patients have died, three were progression-free at 53, 60, and 65 months, three received further treatment and were alive at 61, 81, and 85 months. There was no clear correlation between outcome and immune parameters. Conclusions: Autologous in vitro expanded iNKT cells are a feasible and safe therapy, producing Th1-like responses with antitumor potential. Clin Cancer Res; 23(14); 3510–9. ©2017 AACR.

Epigenetic Regulation of Tumor Necrosis Factor α (TNFα) Release in Human Macrophages by HIV-1 Single-stranded RNA (ssRNA) Is Dependent on TLR8 Signaling

Background: TLR7/TLR8 recognize viral ssRNA, but in human macrophages recognition of HIV-1 ssRNA is not known. Results: In human macrophages, HIV-1 ssRNA promotes TNFα release and requires endocytosis through TLR8, MyD88, and histone modification. Conclusion: TLR8 recognizes HIV-1 ssRNA and promotes TNFα release through chromatin remodeling. Significance: Targeting macrophage TLR8 signaling may serve as a novel therapeutic strategy to modify HIV infection. Human macrophages at mucosal sites are essential targets for acute HIV infection. During the chronic phase of infection, they are persistent reservoirs for the AIDS virus. HIV virions gain entry into macrophages following ligation of surface CD4-CCR5 co-receptors, which leads to the release of two copies of HIV ssRNA. These events lead to reverse transcription and viral replication initiation. Toll-like receptors TLR7 and TLR8 recognize specific intracellular viral ssRNA sequences, but in human alveolar macrophages, their individual roles in TLR-mediated HIV ssRNA recognition are unclear. In the current study, HIV-1 ssRNA induced TNFα release in a dose-dependent manner in adherent human macrophages expressing both intracellular TLR7 and TLR8. This response was reduced by inhibiting either endocytosis (50 μm dynasore) or endosomal acidification (1 μg/ml chloroquine). Either MYD88 or TLR8 gene knockdown with relevant siRNA reduced HIV-1 ssRNA-mediated TNFα release, but silencing TLR7 had no effect on this response. Furthermore, HIV-1 ssRNA induced histone 4 acetylation at the TNFα promoter as well as trimethylation of histone 3 at lysine 4, whereas TLR8 gene knockdown reduced these effects. Taken together in human macrophages, TLR8 binds and internalizes HIV ssRNA, leading to endosomal acidification, chromatin remodeling, and increases in TNFα release. Drugs targeting macrophage TLR8-linked signaling pathways may modulate the innate immune response to acute HIV infection by reducing viral replication.

Novel HIV-1 MiRNAs Stimulate TNFα Release in Human Macrophages via TLR8 Signaling Pathway

Purpose To determine whether HIV-1 produces microRNAs and elucidate whether these miRNAs can induce inflammatory response in macrophages (independent of the conventional miRNA function in RNA interference) leading to chronic immune activation. Methods Using sensitive quantitative Real Time RT-PCR and sequencing, we detected novel HIV-derived miRNAs in the sera of HIV+ persons, and associated with exosomes. Release of TNFα by macrophages challenged with HIV miRNAs was measured by ELISA. Results HIV infection of primary alveolar macrophages produced elevated levels of viral microRNAs vmiR88, vmiR99 and vmiR-TAR in cell extracts and in exosome preparations from conditioned medium. Furthermore, these miRNAs were also detected in exosome fraction of sera from HIV-infected persons. Importantly, vmiR88 and vmiR99 (but not vmiR-TAR) stimulated human macrophage TNFα release, which is dependent on macrophage TLR8 expression. These data support a potential role for HIV-derived vmiRNAs released from infected macrophages as contributing to chronic immune activation in HIV-infected persons, and may represent a novel therapeutic target to limit AIDS pathogenesis. Conclusion Novel HIV vmiR88 and vmiR99 are present in the systemic circulation of HIV+ persons and could exhibit biological function (independent of gene silencing) as ligands for TLR8 signaling that promote macrophage TNFα release, and may contribute to chronic immune activation. Targeting novel HIV-derived miRNAs may represent a therapeutic strategy to limit chronic immune activation and AIDS progression.

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.

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.