Exploring the Utility of NK Cells in COVID-19.

Coronavirus disease 2019 (COVID-19) can manifest as acute respiratory distress syndrome and is associated with substantial morbidity and mortality. Extensive data now indicate that immune responses to SARS-CoV-2 infection determine the COVID-19 disease course. A wide range of immunomodulatory agents have been tested for the treatment of COVID-19. Natural killer (NK) cells play an important role in antiviral innate immunity, and anti-SARS-CoV-2 activity and antifibrotic activity are particularly critical for COVID-19 control. Notably, SARS-CoV-2 clearance rate, antibody response, and disease progression in COVID-19 correlate with NK cell status, and NK cell dysfunction is linked with increased SARS-CoV-2 susceptibility. Thus, NK cells function as the key element in the switch from effective to harmful immune responses in COVID-19. However, dysregulation of NK cells has been observed in COVID-19 patients, exhibiting depletion and dysfunction, which correlate with COVID-19 severity; this dysregulation perhaps contributes to disease progression. Given these findings, NK-cell-based therapies with anti-SARS-CoV-2 activity, antifibrotic activity, and strong safety profiles for cancers may encourage the rapid application of functional NK cells as a potential therapeutic strategy to eliminate SARS-CoV-2-infected cells at an early stage, facilitate immune-immune cell interactions, and favor inflammatory processes that prevent and/or reverse over-inflammation and inhibit fibrosis progression, thereby helping in the fight against COVID-19. However, our understanding of the role of NK cells in COVID-19 remains incomplete, and further research on the involvement of NK cells in the pathogenesis of COVID-19 is needed. The rationale of NK-cell-based therapies for COVID-19 has to be based on the timing of therapeutic interventions and disease severity, which may be determined by the balance between beneficial antiviral and potential detrimental pathologic actions. NK cells would be more effective early in SARS-CoV-2 infection and prevent the progression of COVID-19. Immunomodulation by NK cells towards regulatory functions could be useful as an adjunct therapy to prevent the progression of COVID-19.

Immunosurveillance of Cancer and Viral Infections with Regard to Alterations of Human NK Cells Originating from Lifestyle and Aging.

Natural killer (NK) cells are cytotoxic immune cells with an innate capacity for eliminating cancer cells and virus- infected cells. NK cells are critical effector cells in the immunosurveillance of cancer and viral infections. Patients with low NK cell activity or NK cell deficiencies are predisposed to increased risks of cancer and severe viral infections. However, functional alterations of human NK cells are associated with lifestyles and aging. Personal lifestyles, such as cigarette smoking, alcohol consumption, stress, obesity, and aging are correlated with NK cell dysfunction, whereas adequate sleep, moderate exercise, forest bathing, and listening to music are associated with functional healthy NK cells. Therefore, adherence to a healthy lifestyle is essential and will be favorable for immunosurveillance of cancer and viral infections with healthy NK cells.

Effective induction of melanoma-antigen-specific CD8+ T cells via Vγ9γδT cell expansion by CD56(high+) Interferon-α-induced dendritic cells.

Dendritic cells (DCs) can be differentiated from CD14+ monocytes in the presence of interferon-α (IFNα) and granulocyte/macrophage-colony stimulating factor (GM-CSF) in vitro and are known as IFN-DCs. Circulating blood CD56+ cells expressing high levels of CD14, HLA-DR and CD86 have been shown to spontaneously differentiate into DC-like cells in vitro after their isolation from blood. We show here that IFN-DCs expressing high levels of CD56 (hereafter, CD56(high+) IFN-DCs) can be differentiated in vitro from monocytes obtained as adherent cells from healthy donors and patients with metastatic melanoma. These cells expressed high levels of CD14, HLA-DR and CD86 and possessed many pseudopodia. These CD56(high+) IFN-DCs may be an in vitro counterpart of the circulating CD56+ CD14+ CD86+ HLA-DR+ cells in blood. Conventional mature DCs differentiated from monocytes as adherent cells in the presence of GM-CSF, IL-4 and TNF-α (hereafter, mIL-4DCs) did not express CD56 or CD14. In contrast to mIL-4DCs, the CD56(high+) IFN-DCs exhibited a stronger capacity to stimulate autologous CD56+ Vγ9γδT cells highly producing IFNγ in the presence of zoledronate and IL-2. The CD56(high+) IFN-DCs possessing HLA-A*0201 effectively induced Mart-1-modified melanoma peptide (A27L)-specific CD8+ T cells through preferential expansion of CD56+ Vγ9γδT cells in the presence of A27L, zoledronate and IL-2. Vaccination with CD56(high+) IFN-DCs copulsed with tumor antigens and zoledronate may orchestrate the induction of various CD56+ immune cells possessing high effector functions, resulting in strong immunological responses against tumor cells. This study may be relevant to the design of future clinical trials of CD56(high+) IFN-DCs-based immunotherapies for patients with melanoma.

Large-scale expansion of γδ T cells and peptide-specific cytotoxic T cells using zoledronate for adoptive immunotherapy.

Specific cellular immunotherapy for cancer requires efficient generation and expansion of cytotoxic T lymphocytes (CTLs) that recognize tumor-associated antigens. However, it is difficult to isolate and expand functionally active T-cells ex vivo. In this study, we investigated the efficacy of a new method to induce expansion of antigen-specific CTLs for adoptive immunotherapy. We used tumor-associated antigen glypican-3 (GPC3)-derived peptide and cytomegalovirus (CMV)-derived peptide as antigens. Treatment of human peripheral blood mononuclear cells (PBMCs) with zoledronate is a method that enables large-scale γδ T-cell expansion. To induce expansion of γδ T cells and antigen-specific CTLs, the PBMCs of healthy volunteers or patients vaccinated with GPC3 peptide were cultured with both peptide and zoledronate for 14 days. The expansion of γδ T cells and peptide-specific CTLs from a few PBMCs using zoledronate yields cell numbers sufficient for adoptive transfer. The rate of increase of GPC3­specific CTLs was approximately 24- to 170,000-fold. These CD8(+) cells, including CTLs, showed GPC3-specific cytotoxicity against SK-Hep-1/hGPC3 and T2 pulsed with GPC3 peptide, but not against SK-Hep-1/vec and T2 pulsed with human immunodeficiency virus peptide. On the other hand, CD8(-) cells, including γδ T cells, showed cytotoxicity against SK-Hep-1/hGPC3 and SK-Hep-1/vec, but did not show GPC3 specificity. Furthermore, adoptive cell transfer of CD8(+) cells, CD8(-) cells, and total cells after expansion significantly inhibited tumor growth in an NOD/SCID mouse model. This study indicates that simultaneous expansion of γδ T cells and peptide-specific CTLs using zoledronate is useful for adoptive immunotherapy.

Ex vivo-expanded natural killer cells kill cancer cells more effectively than ex vivo-expanded γδ T cells or αß T cells.

Adoptive immunotherapy of cancer is evolving with the development of novel technologies for generating a large number of activated killer cells such as natural killer (NK) cells, γδ T cells, and αß T cells. We have recently established large-scale culture methods to generate activated NK cells from human peripheral blood, and demonstrated that expanded NK cells have higher cytotoxicity against cancer cells than freshly isolated NK cells. In this study, we compared cultured NK cells with cultured γδ T and αß T cells that were prepared by conventional culture methods regarding the expression of cytotoxic molecules and cytotoxicity against cancer cells. Natural cytotoxicity receptors such as NKp30, NKp44 and NKp46, and perforin were expressed most exclusively on NK cells. Granzyme A, NKG2D, and interferon-γ were dominantly expressed in NK cells and γδ T cells but not in αß T cells. Consistent with the expression profiles of the cytotoxic molecules, cultured NK cells from both healthy volunteers and cancer patients demonstrated significantly higher cytotoxicity against cancer cell lines, including MHC class I-positive cell lines, compared with cultured γδ T cells and cultured αß T cells. Additionally, NK cells, unlike γδ T cells or αß T cells, expressed high levels of CD16, and showed augmented cytotoxicity when co-administered with an anti-CD20 monoclonal antibody drug, rituximab. These results suggest the excellent efficacy of expanded NK cells for cancer treatment.

A low-grade increase of serum pancreatic exocrine enzyme levels by dipeptidyl peptidase-4 inhibitor in patients with type 2 diabetes.

A potential adverse effect of dipeptidyl peptidase-4 inhibitors (DPP-4i) on the pancreas remains controversial. We evaluated the DPP-4i effects on pancreatic amylase and lipase activity in patients with type 2 diabetes. These enzymes were slightly but significantly increased, suggesting DPP-4i cause a low-grade inflammatory change in the exocrine pancreas.

Clinical scale electroloading of mature dendritic cells with melanoma whole tumor cell lysate is superior to conventional lysate co-incubation in triggering robust in vitro expansion of functional antigen-specific CTL.

Recent commercial approval of cancer vaccine, demonstrating statistically significant improvement in overall survival of prostate cancer patients has spurred renewed interest in active immunotherapies; specifically, strategies that lead to enhanced biological activity and robust efficacy for dendritic cell vaccines. A simple, widely used approach to generating multivalent cancer vaccines is to load tumor whole cell lysates into dendritic cells (DCs). Current DC vaccine manufacturing processes require co-incubation of tumor lysate antigens with immature DCs and their subsequent maturation. We compared electroloading of tumor cell lysates directly into mature DCs with the traditional method of lysate co-incubation with immature DCs. Electroloaded mature DCs were more potent in vitro, as judged by their ability to elicit significantly (p < 0.05) greater expansion of peptide antigen-specific CD8(+) T cells, than either lysate-electroloaded immature DCs or lysate-co-incubated immature DCs, both of which must be subsequently matured. Expanded CD8(+) T cells were functional as judged by their ability to produce IFN-γ upon antigen-specific re-stimulation. The electroloading technology used herein is an automated, scalable, functionally closed cGMP-compliant manufacturing technology supported by a Master File at CBER, FDA and represents an opportunity for translation of enhanced potency DC vaccines at clinical/commercial scale.

Synergistic cytotoxicity of ex vivo expanded natural killer cells in combination with monoclonal antibody drugs against cancer cells.

The adoptive transfer of highly cytotoxic natural killer (NK) cells is an emerging tool for cancer immunotherapy. Antibody-dependent cellular cytotoxicity (ADCC) has recently been identified as one of the critical factors for the clinical efficacy of anticancer antibodies, in which NK cells are the major effectors of ADCC. NK cells were expanded from PBMC by a feeder-cell-free expansion method. NK cell expansion efficiency was evaluated within a period of 21 days. The kinetics of NK cell expansion and the expression of activating and inhibitory receptors on NK cells were monitored. NK cells producing IFN-γ and TNF-α were detected by intracellular cytokine staining. The cytotoxicity of expanded NK cells against various cancer cells was compared with that of freshly isolated NK cells. The ADCC functions of expanded NK cells in combination with rituximab against CD20+ lymphoma cell lines were evaluated. Our method efficiently expanded NK cells ex vivo, which showed a much higher activity to induce the expression of activating receptors and to produce IFN-γ and TNF-α as well as cytotoxicity against various cancer cell lines including CD133+ primary cancer cells than freshly isolated NK cells. We observed a synergistic cytotoxicity of our expanded NK cells against CD20+ B lymphoma cell lines as well as higher IFN-γ and TNF-α production when combined with rituximab. Our results suggest that the adoptive transfer of a large number of ex vivo expanded NK cells, particularly in combination with monoclonal antibody drugs, is a useful tool for cancer immunotherapy.

Comparison of clinical and immunological effects of intravenous and intradermal administration of α-galactosylceramide (KRN7000)-pulsed dendritic cells.

PURPOSE: Human Vα24+Vß11+ natural killer T-cells (NKT cells) have antitumor activity via direct cytotoxicity and by induction of antitumor actions of T and NK cells. Activation of NKT cells is crucial for their antitumor activity and is induced by α-galactosylceramide (α-GalCer, KRN7000) presented by CD1d on dendritic cells (DC). We conducted a phase I clinical trial of therapy with α-GalCer-pulsed DC to determine safety, tolerability, immune effects and an optimal dose, and administration route. EXPERIMENTAL DESIGN: Twelve subjects (3 cohorts) with metastatic malignancy received 4 treatments of α-GalCer-pulsed DC, 2 treatments intravenously (IV), and 2 treatments intradermally (ID). Each successive cohort received a log higher cell dose. Clinical and immunological outcomes were evaluated, including secondary effects on NK and T cells. RESULTS: Substantial effects on peripheral blood NKT cells were observed but were greater following IV treatment. Secondary immune effects including activation of T and NK cells, increases in T- and NK-cell cytoplasmic interferon-γ, and increases in serum interferon-γ levels were seen after IV but not after ID treatment. Therapy was well tolerated, but 9 of 12 subjects had tumor flares with clinical findings consistent with transient tumor inflammation. Disease response (minor) or stabilization of disease progressing up to enrollment was observed in 6 of the 12 subjects. Stabilization of previously progressive disease lasted for at least one year in three subjects. CONCLUSION: We conclude that therapy with α-GalCer-pulsed DC induced clinically beneficial immune responses that are highly dependent on cell dose and administration route.

Clinical and immunological evaluation of zoledronate-activated Vgamma9gammadelta T-cell-based immunotherapy for patients with multiple myeloma.

OBJECTIVE: To evaluate the potential anti-tumor activity of zoledronate-activated Vgamma9gammadelta T cells in vivo, we initiated a pilot study involving administration of zoledronate-activated Vgamma9gammadelta T lymphocyte-activated killer (LAK) cells to patients with multiple myeloma. MATERIALS AND METHODS: Subjects (n = 6) received four intravenous infusions at 2-week intervals of zoledronate-activated Vgamma9gammadelta T LAK cells generated from the culture of peripheral blood mononuclear cells (PBMCs) in the presence of zoledronate and interleukin-2. If the M-protein level in the patient's serum remained at baseline following four intravenous infusions, the patient underwent four more treatments at 4-week intervals. Subjects (n = 6) received a median of 0.99 x 10(9) Vgamma9gammadelta T LAK cells per infusion. RESULTS: No serious treatment-related adverse effects were observed during the study period. The percentage of Vgamma9gammadelta T cells in PBMCs and absolute numbers of Vgamma9gammadelta T cells in peripheral blood, particularly those of CD45RA(-)CD27(-) effector memory (TEM) Vgamma9gammadelta T-cell subsets increased in all the patients. Percentages of Vgamma9gammadelta T cells and TEM Vgamma9gammadelta T cells in bone marrow also increased in all the patients so far tested. M-protein levels in the serum remained at baseline in four of six patients and increased in two of six patients. Soluble major histocompatibility complex class I chain-related antigen A was detected only in the serum of patients whose M-protein level increased. CONCLUSION: Administration of zoledronate-activated Vgamma9gammadelta T LAK cells is a safe and promising immunotherapy approach for treatment of patients with multiple myeloma.

A combination therapy of gemcitabine with immunotherapy for patients with inoperable locally advanced pancreatic cancer.

OBJECTIVES: Dendritic cell (DC) therapy frequently induces a measurable immune response. However clinical responses are seen in a minority of patients, presumably due to insufficient expansion of antigen-specific cytotoxic T lymphocytes (CTLs) capable of eradicating tumor cells. To increase therapeutic efficacy of DC-based vaccination, we have undertaken the first clinical trial involving a combination therapy of gemcitabine (GEM) with immunotherapy for patients with inoperable locally advanced pancreatic cancer. METHODS: Patients (n = 5) received the treatment course, which consisted of intravenous GEM administration at 1000 mg/m (day 1) and the endoscopic ultrasound-guided fine-needle injection of OK432-pulsed DCs into a tumor, followed by intravenous infusion of lymphokine-activated killer cells stimulated with anti-CD3 monoclonal antibody (CD3-LAKs) (day 4), at 2-week intervals. RESULTS: No serious treatment-related adverse events were observed during the study period. Three of the 5 patients demonstrated effective responses to this clinical trial; 1 had partial remission and 2 had long stable disease more than 6 months. In the patient with partial remission, it has been shown that DC-based vaccination combined with GEM administration induces tumor antigen-specific CTLs. CONCLUSION: This combined therapy was considered to be synergistically effective and may have a role in the therapy of pancreatic cancer for inducing tumor antigen-specific CTLs.

V gamma 9 V delta 2 T cell cytotoxicity against tumor cells is enhanced by monoclonal antibody drugs--rituximab and trastuzumab.

V gamma 9 V delta 2 T cells exert potent cytotoxicity toward various tumor cells and adoptive transfer of V gamma 9 V delta 2 T cells is an attractive proposition for cell based immunotherapy. V gamma 9 V delta 2 T cells expanded in the presence of Zoledronate and IL-2 express CD16 (Fc gamma RIII), which raises the possibility that V gamma 9 V delta 2 T cells could be used in conjunction with tumor targeting monoclonal antibody drugs to increase antitumor cytotoxicity by antibody dependent cellular cytotoxicity (ADCC). Cytotoxic activity against CD20-positive B lineage lymphoma or chronic lymphocytic leukemia (CLL) and HER2-positive breast cancer cells was assessed in the presence of rituximab and trastuzumab, respectively. Cytotoxicity of V gamma 9 V delta 2 T cells against CD20-positive targets was higher when used in combination with rituximab. Similarly, V gamma 9 V delta 2 T cells used in combination with trastuzumab resulted in greater cytotoxicity against HER2-positive cells in comparison with either agent alone and this effect was restricted to the CD16(+)V gamma 9 V delta 2 T cell population. Our results show that CD16(+)V gamma 9 V delta 2 T cells recognize monoclonal antibody coated tumor cells via CD16 and exert ADCC similar to that observed with NK cells, even when target cells are relatively resistant to monoclonal antibodies or V gamma 9 V delta 2 T cells alone. Combination therapy involving ex vivo expanded CD16(+)V gamma 9 V delta 2 T cells and monoclonal antibodies may enhance the clinical outcomes for patients treated with monoclonal antibody therapy.

Copulsing tumor antigen-pulsed dendritic cells with zoledronate efficiently enhance the expansion of tumor antigen-specific CD8+ T cells via Vgamma9gammadelta T cell activation.

We demonstrate that Vgamma9gammadelta T cells activated by zoledronate can link innate and acquired immunity through crosstalk with dendritic cells (DCs) in a way that can amplify activation and proliferation of tumor antigen-specific CD8+ T cells. DCs pulsed with antigen alone or antigen plus zoledronate were used to stimulate the in vitro expansion of antigen-specific CD8+ T cells. MART-1-modified peptide (A27L peptide) and apoptotic HLA-A*0201-positive, MART-1-positive JCOCB tumor cell lines were used as tumor antigen sources. The percentage of A27L-specific CD8+ T cells within the responding lymphocytes on Day 7 when immature DCs (imDCs) were cultured in the presence of A27L peptide and 0.01 microM zoledronate was significantly higher (P=0.002, n=11) than that observed when imDCs were cultured with the lymphocytes in the presence of the A27L peptide alone. This enhancing effect of zoledronate was significantly reduced when gammadelta T cells were depleted from responding lymphocytes (P=0.030, n=5), indicating that the effect is mediated mainly through Vgamma9gammadelta T cells activated by zoledronate-pulsed imDCs. When imDCs copulsed with zoledronate and apoptotic JCOCB tumor cell lines were used, the percentage of A27L-specific CD8+ T cells was higher than that observed using imDCs with the apoptotic JCOCB lines alone, suggesting that zoledronate treatment of imDCs enhances the cross-presentation ability of DCs. These findings suggest a potentially valuable role for Vgamma9gammadelta T cell activation for expanding antigen-specific CD8+T cells using DCs copulsed with tumor antigen and zoledronate in the design of vaccine therapies for malignancy.

Chemotherapy and zoledronate sensitize solid tumour cells to Vgamma9Vdelta2 T cell cytotoxicity.

Combinations of cellular immune-based therapies with chemotherapy and other antitumour agents may be of significant clinical benefit in the treatment of many forms of cancer. Gamma delta (gammadelta) T cells are of particular interest for use in such combined therapies due to their potent antitumour cytotoxicity and relative ease of generation in vitro. Here, we demonstrate high levels of cytotoxicity against solid tumour-derived cell lines with combination treatment utilizing Vgamma9Vdelta2 T cells, chemotherapeutic agents and the bisphosphonate, zoledronate. Pre-treatment with low concentrations of chemotherapeutic agents or zoledronate sensitized tumour cells to rapid killing by Vgamma9Vdelta2 T cells with levels of cytotoxicity approaching 90%. In addition, zoledronate enhanced the chemotherapy-induced sensitization of tumour cells to Vgamma9Vdelta2 T cell cytotoxicity resulting in almost 100% lysis of tumour targets in some cases. Vgamma9Vdelta2 T cell cytotoxicity was mediated by perforin following TCR-dependent and isoprenoid-mediated recognition of tumour cells. Production of IFN-gamma by Vgamma9Vdelta2 T cells was also induced after exposure to sensitized targets. We conclude that administration of Vgamma9Vdelta2 T cells at suitable intervals after chemotherapy and zoledronate may substantially increase antitumour activities in a range of malignancies.

Efficient priming and expansion of antigen-specific CD8+ T cells by a novel cell-based artificial APC.

The ex vivo priming and expansion of human CTL by APC, such as autologous monocyte-derived dendritic cells (DC), has the potential for use in immunotherapy for infectious diseases and cancer. To overcome the difficulty of obtaining sufficient number of autologous DC from patients, we have developed cell-based artificial APC (aAPC), designated Med-APC. These aAPC rapidly activate and expand the corresponding Ag-specific CD8+ T cells when pulsed with CTL epitope peptide(s) as efficiently as mature DC (mDC). We have also shown that Med-APC possess an innate cellular machinery that is sufficient to support the processing of complete Ag into immunodominant peptides, which considerably extends the usefulness of this technology. In addition, we have developed a novel expression vector system that expresses ubiquitinated Ag, resulting in an enhanced APC function of this system. Genetically encoded Ag can be easily introduced into Med-APC by transfection with this vector. Med-APC transfected with ubiquitinated Ag can efficiently expand the corresponding Ag-specific CTL without exogenous peptides. Therefore, Med-APC may have important therapeutic implications for adoptive immunotherapy and can be used for the detection of Ag-specific CTL for immunomonitoring.

Comparative gene expression analysis of NKT cell subpopulations.

Natural killer T (NKT) cells are a lymphocyte lineage, which has diverse immune regulatory activities in many disease settings. Most previous studies have investigated the functions of this family of cells as a single entity, but more recent evidence highlights the distinct functional and phenotypic properties of NKT cell subpopulations. It is likely that the diverse functions of NKT cells are regulated and coordinated by these different NKT subsets. Little is known about how NKT subsets differ in their interactions with the host. We have undertaken the first microarray analysis comparing the gene expression profiles of activated human NKT cell subpopulations, including CD8(+) NKT cells, which have often been overlooked. We describe the significant gene expression differences among NKT cell subpopulations and some of the molecules likely to confer their distinct functional roles. Several genes not associated previously with NKT cells were shown to be expressed differentially in specific NKT cell subpopulations. Our findings provide new insights into the NKT cell family, which may direct further research toward better manipulation of NKT cells for therapeutic applications.

Chemotherapy pretreatment sensitizes solid tumor-derived cell lines to V alpha 24+ NKT cell-mediated cytotoxicity.

There is an increasing awareness of the therapeutic potential for combining immune-based therapies with chemotherapy in the treatment of malignant diseases, but few published studies evaluate possible cytotoxic synergies between chemotherapy and cytotoxic immune cells. Human V alpha 24+/V beta 11+ NKT cells are being evaluated for use in cell-based immunotherapy of malignancy because of their immune regulatory functions and potent cytotoxic potential. In this study, we evaluated the cytotoxicity of combinations of chemotherapy and NKT cells to determine whether there is a potential to combine these treatment modalities for human cancer therapy. The cytotoxicity of NKT cells was tested against solid-tumor derived cell lines NCI-H358, DLD-1, HT-29, DU-145, TSU-Pr1 and MDA-MB231, with or without prior treatment of these target cells, with a range of chemotherapy agents. Low concentrations of chemotherapeutic agents led to sensitization of cell lines to NKT-mediated cytotoxicity, with the greatest effect being observed for prostate cancer cells. Synergistic cytotoxicity occurred in an NKT cell in a dose-dependent manner. Chemotherapy agents induced upregulation of cell surface TRAIL-R2 (DR5) and Fas (CD95) expression, increasing the capacity for NKT cells to recognize and kill via TRAIL- and FasL-mediated pathways. We conclude that administration of cytotoxic immune cells after chemotherapy may increase antitumor activities in comparison with the use of either treatment alone.

Analysis of the effect of different NKT cell subpopulations on the activation of CD4 and CD8 T cells, NK cells, and B cells.

OBJECTIVE: NKT cells have diverse immune regulatory functions including activation of cells involved in Th1- and Th2-type immune activities. Most previous studies have investigated the functions of NKT cells as a single family but more recent evidence indicates the distinct functional properties of NKT cell subpopulation. This study aims to determine whether NKT cell subpopulations have different stimulatory activities on other immune cells that may affect the outcome of NKT cell-based immunotherapy. METHODS: NKT cells and NKT cell subpopulations (CD4(+)CD8(-), CD4(-)CD8(+), CD4(-)CD8(-)) were cocultured with PBMC and their activities on immune cells including CD4(+) and CD8(+) T cells, NK cells, and B cells were assessed by flow cytometry. The production of cytokines in culture was measured by enzyme-linked immunsorbent assay. RESULTS: The CD4(+)CD8(-) NKT cells demonstrated substantially greater stimulatory activities on CD4(+) T cells, NK cells, and B cells than other NKT cell subsets. The CD4(-)CD8(+) NKT cells showed the greatest activity on CD8(+) T cells, and were the only NKT cell subset that activated these immune cells. The CD4(-)CD8(-) NKT cells showed moderate stimulatory activity on CD4(+) T cells and the least activity on other immune cells. CONCLUSION: The results here suggest that NKT cell subpopulations differ in their abilities to stimulate other immune cells. This highlights the potential importance of manipulating specific NKT cell subpopulations for particular therapeutic situations and of evaluating subpopulations, rather than NKT cells as a group, during investigation of a possible role of NKT cells in various disease settings.