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 IFN-gamma production, and iNKT IFN-gamma production may stratify for survival. Previous clinical trials using iNKT cell activating ligand alpha-galactosylceramide have shown responses. Therefore, a phase 1 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 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 IL-2 in vitro to obtain up to ~109 cells. Results Expanded iNKT cells produced IFN-gamma, but limited or undetectable IL-4 or IL-10. 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 PBMC after some infusions, particularly when GM-CSF was also given. IFN-gamma responses to alpha-galactosylceramide were increased in PBMC from some patients after infusions, and DTH responses to Candida increased in 5/8 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 anti-tumor potential.

Natural killer T cells (NKT cells) expressing a semiinvariant CD1d-reactive T cell receptor (invariant NKT, iNKT) can be rapidly activated by monocytes or immature dendritic cells (iDCs) bearing aCD1d-presented glycolipid antigen and can in turn stimulate these myeloid cells to mature and produce IL-12. Previous studies have shown that iNKT-produced IFN␥ and CD40 ligand contribute to this dendritic cell maturation. This study demonstrates that CD1d ligation alone, in the absence of iNKT, could rapidly (within 24 h) stimulate production of bioactive IL-12p70 by CD1d ؉ human peripheral blood monocytes as well as iDCs. IFN␥ alone had no effect, but it markedly enhanced CD1d-stimulated IL-12 production. Monocyte differentiation, as assessed by CD40 and CD1a upregulation, was also accelerated by CD1d stimulation, consistent with this representing a physiological response. CD1d ligation on the human monocytic cell line THP-1 similarly specifically stimulated IL-12 production. Biochemical studies showed that IL-12 release correlated with rapid phosphorylation of I B, a critical step in NF-B activation. Selective NF-B inhibition blocked this CD1d-stimulated IL-12 production. Finally, CD1d ligation could also enhance IL-12 production in the presence of suboptimal LPS or CD40 stimulation. These findings demonstrate an innate immune signaling function for CD1d and provide a mechanism for the rapid activation of monocytes and iDCs by CD1d-reactive T cells.antigen-presenting cell ͉ innate immunity ͉ natural killer T cell C D1d is a nonpolymorphic MHC class I-like protein constitutively expressed by antigen-presenting cells (APC) and by some epithelia, whereas CD1a-c are induced upon dendritic cell (DC) maturation. CD1d is recognized by a subpopulation of T cells, many of which express markers typical of natural killer cells and have been termed natural killer T cells (NKT cells) (1-3). A major fraction of CD1d-restricted T cells recognize CD1d through an invariant T cell receptor (TCR)␣ chain (V␣24-J␣18 in humans). CD1d-reactive NKT cells using this invariant TCR (iNKT) are largely primed in vivo for the rapid production of T helper 1 and T helper 2 cytokines (IFN␥ and IL-4) and have regulatory functions in innate and adaptive immune responses (3, 4). A glycolipid antigen isolated from marine sponge (␣-galactosylceramide, ␣galcer) is recognized by iNKT in the context of CD1d (5-8). In vivo, ␣galcer causes activation of iNKT, rapid cytokine production, and subsequent systemic activation of innate and adaptive immune cells (9, 10).Data from many groups indicate that iNKT functions can be mediated through interactions with CD1d ϩ APC including . Murine studies have shown that iNKT activation can be enhanced by B7 ligation of CD28 and that IL-12 produced by DCs can further enhance iNKT IFN␥ production (11,12,20). Conversely, murine and human studies have shown that iNKT can stimulate the maturation of ␣galcer-pulsed DCs (11,12,18,20). This DC stimulation appears to be mediated by CD40 ligand on the iNKT through ligation of CD40 ...

PurposeTo 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.MethodsUsing 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.ResultsHIV 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.ConclusionNovel 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.