Attenuation of invariant natural killer T-cell anergy induction through intradermal delivery of alpha-galactosylceramide.

CD1d restricted, alpha-galactosylceramide (alphaGC) responsive invariant (i)NKT cells positively regulate immune responses. Both intravenous and intradermal administered alphaGC are known to activate iNKT cells. iNKT cells become unresponsive to a second intravenous alphaGC injection, whereas no data are available regarding potential anergy upon intradermal administration. Here, comparative analysis of two intradermal versus two intravenous injections in mice demonstrated that iNKT cell anergy was prevented by intradermal injection and when combined with a vaccine, superior tumor protection afforded by intradermally administered alphaGC. Moreover, human skin dendritic cells (DC) took up intradermally injected alphaGC and activated iNKT cells upon migration, while iNKT cells in human skin-draining lymph nodes expanded in response to alphaGC presented either by exogenously added DC or by CD1d positive antigen presenting cells in the lymph nodes. In conclusion, glycolipids such as alphaGC may greatly improve the efficacy of skin immunization strategies, targeting cutaneous and lymph node DC.

Generation of antitumor invariant natural killer T cell lines in multiple myeloma and promotion of their functions via lenalidomide: a strategy for immunotherapy.

PURPOSE: CD1d-restricted invariant natural killer T (iNKT) cells are important immunoregulatory cells in antitumor immune responses. However, the quantitative and qualitative defects of iNKT cells in advanced multiple myeloma hamper their antitumor effects. Therefore, the development of functional iNKT cells may provide a novel strategy for the immunotherapy in multiple myeloma. EXPERIMENTAL DESIGN: We activated and expanded iNKT cells from multiple myeloma patients with alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells, characterized their antitumor effects by the cytokine production profile and cytotoxicity against multiple myeloma cells, and explored the effects of immunomodulatory drug lenalidomide on these iNKT cells. We also investigated the expression of CD1d by primary multiple myeloma cells and its function to activate iNKT cells. RESULTS: We established highly purified functional iNKT cell lines from newly diagnosed and advanced multiple myeloma patients. These CD1d-restricted iNKT cell lines produced high level of antitumor Th1 cytokine in response to alpha-GalCer-pulsed primary multiple myeloma cells, CD1d-transfected MM1S cell line, and dendritic cells. Moreover, iNKT cell lines displayed strong cytotoxicity against alpha-GalCer-pulsed primary multiple myeloma cells. Importantly, lenalidomide further augmented the Th1 polarization by iNKT cell lines via increased Th1 cytokine production and reduced Th2 cytokine production. We also showed that CD1d was expressed in primary multiple myeloma cells at mRNA and protein levels from the majority of multiple myeloma patients, but not in normal plasma cells and multiple myeloma cell lines, and CD1d(+) primary multiple myeloma cells presented antigens to activate iNKT cell lines. CONCLUSIONS: Taken together, our results provide the preclinical evidence for the iNKT cell-mediated immunotherapy and a rationale for their use in combination with lenalidomide in multiple myeloma treatment.

Invariant natural killer T cells and immunotherapy of cancer.

Invariant CD1d restricted natural killer T (iNKT) cells are regulatory cells that express a canonical TCR-Valpha-chain (Valpha24.Jalpha18 in humans and Valpha14.Jalpha18 in mice) which recognizes glycolipid antigens presented by the monomorphic CD1d molecule. They can secrete a wide variety of both pro-inflammatory and anti-inflammatory cytokines very swiftly upon their activation. Evidence for the significance of iNKT cells in human cancer has been ambiguous. Still, the (pre-)clinical findings reviewed here, provide evidence for a distinct contribution of iNKT cells to natural anti-tumor immune responses in humans. Furthermore, clinical phase I studies that are discussed here have revealed that the infusion of cancer patients with ligand-loaded dendritic cells or cultured iNKT cells is well tolerated. We thus underscore the potential of iNKT cell based immunotherapy in conjunction with established modalities such as surgery and radiotherapy, as adjuvant therapy against carcinomas.

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-beta, but it was further reduced by the neutralization of IL-10 in vitro. Additional experiments revealed discordant roles for TGF-beta and IL-10 on human iNKT cells, because TGF-beta suppressed iNKT cell activation and proliferation and IFN-gamma 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.

Chronically stimulated mouse invariant NKT cell lines have a preserved capacity to enhance protection against experimental tumor metastases.

In pre-clinical models, CD1d restricted invariant Natural Killer T (iNKT) cells play a pivotal role in natural anti-tumor immune responses, mainly by trans-activating cells of both the innate and adaptive arms via swift and potent cytokine secretion. We have previously reported that patients with a severely reduced circulating iNKT cell pool have a poor clinical response to radio therapy of head and neck squamous cell carcinoma. Therefore, these patients might benefit from an immunotherapeutic approach aimed at the increase of circulating levels of iNKT cells. Furthermore, we have generated both human and mouse iNKT cell lines, and demonstrated that they had retained the capacity to release both Th1 and Th2 type cytokines even after long-term in vitro expansion using alpha-galactosylceramide (alphaGalCer) pulsed dendritic cells (DC). Here, we establish, in a pre-clinical tumor model that the large scale long lived polyclonal iNKT cell lines we generated have a preserved capacity to evoke an in vivo cytokine storm upon adoptive transfer, independently of supplemental alphaGalCer administration. This results in an augmented NK cell mediated protection against B16.F10 experimental lung metastases in vivo. These findings underscore the potential of autologous adoptive transfer of ex vivo expanded iNKT cells as a strategy to enhance immunotherapeutic modalities for the treatment of cancer patients.

Exploiting regulatory T-cell populations for the immunotherapy of cancer.

Tumor escape from the immune system hampers effective immunotherapy for cancer. Recent evidence indicates that in cancer patients the altered activities of 2 innatelike ("natural") immunoregulatory T-cell populations could prevent the establishment of effective antitumor immune responses. These are CD4+ CD25+ (natural) regulatory T cells and invariant natural killer T cells, which normally play critical roles in orchestrating immune responses to self and nonself. Therapeutic modulation of these regulatory T-cell populations is clinically feasible and will potentially allow the induction of effective antitumor immune responses when combined with currently available immunotherapeutic strategies.

Randomized placebo controlled phase I/II trial of alpha-galactosylceramide for the treatment of chronic hepatitis C.

BACKGROUND/AIMS: The glycosphingolipid alpha-galactosylceramide has been shown to activate invariant natural killer T cells when presented in the context of CD1d and induces powerful antiviral immune responses via the production of inflammatory cytokines. The aim of this study was to investigate the safety and the antiviral activity of alpha-galactosylceramide as a novel class of treatment for chronic hepatitis C patients. METHODS: International multicenter dose-escalating randomized placebo-controlled phase I/II trial. RESULTS: Forty patients were allocated to a dose of 0.1 microg/kg (n=9), 1 microg/kg (n=9), 10 microg/kg (n=11) or to placebo (n=11). alpha-Galactosylceramide was well tolerated and no patients were withdrawn due to side effects. Although most patients showed a decrease in invariant natural killer T cells after administration, no clinically relevant suppression of viral replication was observed. Only one patient, a previous non-responder to peginterferon and ribavirin with high baseline invariant natural killer T cell levels, showed profound signs of immune activation, accompanied by a transient 1.3 log decrease in HCV-RNA and a concomitant increase in ALT after the first administration. CONCLUSIONS: alpha-Galactosylceramide used as monotherapy for interferon-refractory patients in doses of 0.1-10 microg/kg is safe and it exerts moderate immunomodulatory effects. However, in its current form it has no significant effect on HCV-RNA levels.

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.

Generation and sustained expansion of mouse spleen invariant NKT cell lines with preserved cytokine releasing capacity.

Invariant Natural Killer T (iNKT) cells are CD1d restricted innate lymphoid cells with an invariant T cell receptor (TCR) alpha chain gene rearrangement (Valpha24-Jalpha18 in human and Valpha14-Jalpha18 in mouse). iNKT cells play a pivotal role in anti-tumor immune responses via cytokine mediated transactivation of various cells which mediate innate and adaptive immune responses. Here we describe, to our knowledge for the first time, the generation of long-term mouse spleen derived iNKT cell lines. We found that dendritic cells (DC) derived from the D1 line, but not Mf4/4 macrophages, loaded with the artificial iNKT cell ligand alpha-Galactosylceramide (alphaGalCer) could be employed to expand iNKT cells in vitro. Furthermore, exogenously added IL-7, but not IL-2 or IL-15 had a pronounced additive effect on iNKT cell expansion. Using this method up to 10(8) iNKT cells could be obtained from one spleen within 12 to 14 weeks, and cell lines could be continued for up to 24 months. Importantly, the iNKT cell lines had retained the capacity to swiftly secrete substantial amounts of both T helper (Th) 1 and Th2 cytokines upon activation. In conclusion we have generated iNKT cell lines with high yields that can be maintained for up to 24 months, by repeated stimulation using alpha-GalCer loaded D1.DC and IL-7. These in vitro expanded iNKT cells preserved the capacity to swiftly produce both Th1 and Th2 type cytokines and are currently being utilized in pre-clinical adoptive transfer models to identify and optimize the characteristics of therapeutically effective iNKT cells in an anti-tumor setting.

Hepatitis C virus (HCV)-specific CD8+ cells produce transforming growth factor beta that can suppress HCV-specific T-cell responses.

Hepatitis C virus (HCV)-specific T-cell responses are rarely detected in peripheral blood, especially in the presence of human immunodeficiency virus (HIV) coinfection. Based on recent evidence that T-regulatory cells may be increased in chronic HCV, we hypothesized that functional blockade of regulatory cells could raise HCV-specific responses and might be differentially regulated in the setting of HIV coinfection. Three groups of subjects were studied: HCV monoinfected, HCV-HIV coinfected, and healthy controls. Frequencies of peripheral T cells specific for peptides derived from HCV core, HIV type 1 p24, and recall antigens were analyzed by gamma interferon (IFN-gamma) enzyme-linked immuno-spot assay. HCV-specific T-cell responses were very weak in groups with HCV and HCV-HIV infections. Addition of blocking antibodies against transforming growth factor beta1 (TGF-beta1), -2, and -3 and interleukin-10 specifically increased the HCV-specific T-cell responses in both infected groups; however, this increase was attenuated in the group with HCV-HIV coinfection compared to HCV infection alone. No increase in recall antigen- or HIV-specific responses was observed. Flow cytometric sorter analysis demonstrated that regulatory-associated cytokines were produced by HCV-specific CD3(+)CD8(+)CD25(-) cells. Enhancement of the IFN-gamma effect was observed for both CD4 and CD8 T cells and was mediated primarily by TGF-beta1, -2, and -3 neutralization. In conclusion, blockade of TGF-beta secretion could enhance peripheral HCV-specific T-cell responses even in the presence of HIV coinfection.

Low levels of circulating invariant natural killer T cells predict poor clinical outcome in patients with head and neck squamous cell carcinoma.

PURPOSE: Evading antitumor immune responses is an important aspect of the pathogenesis of head and neck squamous cell carcinoma (HNSCC). Invariant CD1d-restricted natural killer T (iNKT) cells play an allegedly pivotal role in such responses via transactivation of immune effector cells. It has been reported that iNKT cells are reduced in peripheral blood of cancer patients compared with healthy controls. Here, we investigated whether the extent of this deficiency affected disease outcome in HNSCC patients. PATIENTS AND METHODS: In a prospective study, circulating iNKT cell numbers were evaluated in 47 patients before radiotherapy. Patients were stratified in three groups based on iNKT cell levels, and clinical data were obtained during a median follow-up period of 31 months. RESULTS: A small, compared with an intermediate or large, circulating iNKT cell fraction was significantly associated with decreased 3-year overall survival rate (39% v 75% and 92%, respectively), disease-specific survival rate (43% v 87% and 92%, respectively), and locoregional control rate (31% v 74% and 92%, respectively) in HNSCC patients. Cox regression revealed that the iNKT cell level, as well as clinical T stage, was an independent prognostic parameter even after correction for the confounding effect of age. CONCLUSION: A severe circulating iNKT cell deficiency was related to poor clinical outcome in HNSCC patients, suggesting their critical contribution to antitumor immune responses. Furthermore, screening for iNKT cell levels may be useful for determining which patients can benefit from immunotherapeutic adjuvant therapies aimed at reconstitution of the circulating iNKT cell pool.

IPEX as a result of mutations in FOXP3.

Immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a rare disorder caused by mutations in the FOXP3 gene that result in the defective development of CD4+CD25+ regulatory T cells which constitute an important T cell subset involved in immune homeostasis and protection against autoimmunity. Their deficiency is the hallmark of IPEX and leads to severe autoimmune phenomena including autoimmune enteropathy, dermatitis, thyroiditis, and type 1 diabetes, frequently resulting in death within the first 2 years of life. Apart from its clinical implications, IPEX illustrates the importance of immunoregulatory cells such as CD4+CD25+ regulatory T cells.

Cutting edge: Rapid recovery of NKT cells upon institution of highly active antiretroviral therapy for HIV-1 infection.

CD1d-restricted NKT cells play important regulatory roles in various immune responses and are rapidly and selectively depleted upon infection with HIV-1. The cause of this selective depletion is incompletely understood, although it is in part due to the high susceptibility of CD4+ NKT cells to direct infection and subsequent cell death by HIV-1. Here, we demonstrate that highly active antiretroviral therapy (HAART) results in the rapid recovery of predominantly CD4(-) NKT cells with kinetics that are strikingly similar to those of mainstream T cells. As it is well known that the early recovery of mainstream T cells in response to HAART is due to their redistribution from tissues to the circulation, our data suggest that the selective depletion of circulating NKT cells is likely due to a combination of cell death and tissue sequestration and indicates that HAART can improve immune functions by reconstituting both conventional T cells and immunoregulatory NKT cells.

Peripheral blood IFN-gamma-secreting Valpha24+Vbeta11+ NKT cell numbers are decreased in cancer patients independent of tumor type or tumor load.

Natural killer T (NKT) cells are CD1d-restricted lymphoid cells and are characterized by an invariant T-cell receptor, which in humans consists of a Valpha24 chain paired with a Vbeta11 chain. These cells are known for their rapid production of large amounts of cytokines (e.g., IFN-gamma and IL-4), thereby modulating other cells of the immune system such as T cells, NK cells and dendritic cells. NKT cells have been reported to play important regulatory roles in many immune responses, including antitumor immune responses. Here, we demonstrate an age-dependent decrease in circulating Valpha24(+)Vbeta11(+) NKT cell numbers in both healthy controls and cancer patients and demonstrate that in both groups females have higher NKT cell levels compared to males. In a large group of 120 cancer patients, we show that circulating Valpha24(+)Vbeta11(+) NKT cell numbers are about 50% lower than in age- and gender-matched healthy controls and that this decrease is independent of tumor type or tumor load. This decrease was not restored upon tumor removal by means of surgery or radiotherapy. Even though the percentage of NKT cells that secrete IFN-gamma, as detected by ELISPOT, is normal in cancer patients, the absolute number of circulating IFN-gamma-secreting NKT cells is reduced. Together, our results suggest that the reduced circulating Valpha24(+)Vbeta11(+) NKT cell numbers in cancer patients are not affected by tumor load, but might actually reflect a risk factor for tumor development, e.g., by hampering efficient tumor immunosurveillance.

Circulating Valpha24+Vbeta11+ NKT cell numbers and dendritic cell CD1d expression in hepatitis C virus infected patients.

CD1d-restricted natural killer T (NKT) cells are involved in the regulation of various immune responses, and have been shown to inhibit viral replication in animal hepatitis models when activated by the glycolipid alpha-galactosylceramide (alpha-GalCer, KRN7000). Previous studies have indicated that alpha-GalCer-induced activation of the immune system requires both CD1d expression by antigen-presenting cells as well as (normal) numbers of NKT cells. Discrepancies exist over circulating numbers of human invariant Valpha24+Vbeta11+ NKT cells during hepatitis C virus (HCV) infection. Here, by cross-sectional analysis and longitudinal analysis of patients undergoing effective combination antiviral therapy, we demonstrate that circulating Valpha24+Vbeta11+ NKT cell numbers are not decreased during active HCV infection. Importantly, as we also show that CD1d is expressed at comparable levels by peripheral blood monocytes and CD1c+ myeloid dendritic cells (DC) of healthy individuals and HCV-infected patients, these data indicate that all ingredients for evaluating the antiviral effects of the Valpha24+Vbeta11+ NKT cell ligand alpha-GalCer in HCV-infected patients are present.

The immunoregulatory role of CD1d-restricted natural killer T cells in disease.

Natural killer T (NKT) cells constitute a T cell subpopulation that shares several characteristics with NK cells. NKT cells are characterized by a narrow T cell antigen receptor (TCR) repertoire, recognize glycolipid antigen in the context of the monomorphic CD1d antigen-presenting molecule, and have the unique capacity to rapidly produce large amounts of both T helper (Th) 1 and Th2 cytokines. Important roles of NKT cells have now been demonstrated in the regulation of autoimmune, allergic, antimicrobial, and antitumor immune responses. Here, we review the immunoregulatory role of NKT cells in disease and discuss NKT cell based immunotherapeutic strategies.

Polarization of Valpha24+ Vbeta11+ natural killer T cells of healthy volunteers and cancer patients using alpha-galactosylceramide-loaded and environmentally instructed dendritic cells.

CD1d-restricted natural killer T (NKT) cells play important regulatory roles in various immune responses. NKT cell-derived T helper (Th) 1 cytokines are important in the induction of antitumor immune responses in mice. Because the CD1d-restricted Valpha24(+) Vbeta11(+) NKT cell population in cancer patients is decreased both in size and in its capacity to secrete IFN-gamma, therapeutic strategies based on reconstitution of type 1 polarized Valpha24(+) Vbeta11(+) NKT cells merit additional investigation. Here, we report the simultaneous strong expansion and type 1 polarization of human invariant Valpha24(+) Vbeta11(+) NKT cells using alpha-galactosylceramide-loaded type 1 dendritic cells and interleukin 15. Type 1 polarized Valpha24(+) Vbeta11(+) NKT cells produced high levels of IFN-gamma, tumor necrosis factor alpha, and granulocyte macrophage colony-stimulating factor, and induced strong cytotoxicity in Jurkat cells in an alpha-galactosylceramide-dependent manner. Importantly, the cytokine profile of Valpha24(+) Vbeta11(+) NKT cells that were initially expanded under Th2 polarizing conditions could be reversed to a Th1 cytokine profile, indicating the plasticity of the cytokine profile of the human adult Valpha24(+) Vbeta11(+) NKT cell population.