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Complex interactions among multiple cell types contribute to the immunosuppressive milieu of the tumor microenvironment. Using a murine model of adoptive T-cell immunotherapy (ACT) for B16 melanoma, we investigated the impact of tumor infiltrating cells on this complex regulatory network in the tumor. Transgenic pmel-1-specific cytotoxic T lymphocytes (CTLs) were injected intravenously into tumor-bearing mice and could be detected in the tumor as early as on day 1, peaking on day 3. They produced IFN-γ, exerted anti-tumor activity and inhibited tumor growth. However, CTL infiltration into the tumor was accompanied by the accumulation of large numbers of cells, the majority of which were CD11b(+) Gr1(+) myeloid-derived suppressor cells (MDSCs). Notably, CD11b(+) Gr1(int) Ly6G(-) Ly6C(+) monocytic MDSCs outnumbered the CTLs by day 5. They produced nitric oxide, arginase I and reactive oxygen species, and inhibited the proliferation of antigen-specific CD8(+) T cells. The anti-tumor activity of the adoptively-transferred CTLs and the accumulation of MDSCs both depended on IFN-γ production on recognition of tumor antigens by the former. In CCR2(-/-) mice, monocytic MDSCs did not accumulate in the tumor, and inhibition of tumor growth by ACT was improved. Thus, ACT triggered counter-regulatory immunosuppressive mechanism via recruitment of MDSCs. Our results suggest that strategies to regulate the treatment-induced recruitment of these MDSCs would improve the efficacy of immunotherapy.

Background Proinflammatory state has been implicated as a pathogenetic mechanism in the progression of intracranial large artery atherosclerosis (ILA). High levels of inflammatory biomarkers in healthy populations and in patients with acute stroke or acute coronary syndrome are known to be associated with subsequent stroke events. This study investigated the relationship between circulating biomarkers measured early after stroke onset and future ILA progression. Methods In 48 patients with acute ischemic stroke, high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), IL-18, tumor necrosis factor–α, matrix metalloproteinase (MMP)-2 and MMP-9 were measured within 48 hours after onset. Baseline severity and ILA progression were assessed by serial magnetic resonance angiography (MRA). The median follow-up period for MRA was 3.1 years. Hazard ratio (HR) was calculated using the Cox proportional hazard model adjusted for traditional risk factors, and accuracy of predicted ILA progression was analyzed by receiver operating characteristic (ROC) curve analysis. Results ILA progression was observed in 6 of 48 patients (12.5%). After adjusting for age, sex, and presence of hypertension, baseline ILA severity score (HR 2.814; 95% confidence interval [CI] 1.172-6.754) and IL-6 (HR 1.215; 95% CI 1.002-1.473) were significantly associated with ILA progression. Area under the ROC curve (AUC) for prediction of ILA progression by traditional risks, baseline ILA severity score and IL-6, was 0.647. When IL-6 was removed from this model, AUC remained at 0.631. Conclusions In addition to traditional risk factors and baseline radiologic findings, circulating levels of IL-6 measured soon after stroke onset are associated with future ILA progression.

Both innate and adaptive immunity are crucial for cancer immunosurveillance, but precise therapeutic equations to restore immunosurveillance in patients with cancer patients have yet to be developed. In murine models, α-galactosylceramide (α-GalCer)–loaded, tumor antigen–expressing syngeneic or allogeneic cells can act as cellular adjuvants, linking the innate and adaptive immune systems. In the current study, we established human artificial adjuvant vector cells (aAVC) consisting of human HEK293 embryonic kidney cells stably transfected with the natural killer T (NKT) immune cell receptor CD1d, loaded with the CD1d ligand α-GalCer and then transfected with antigen-encoding mRNA. When administered to mice or dogs, these aAVC-activated invariant NKT (iNKT) cells elicited antigen-specific T-cell responses with no adverse events. In parallel experiments, using NOD/SCID/IL-2rγcnull–immunodeficient (hDC-NOG) mouse model, we also showed that the human melanoma antigen, MART-1, expressed by mRNA transfected aAVCs can be cross-presented to antigen-specific T cells by human dendritic cells. Antigen-specific T-cell responses elicited and expanded by aAVCs were verified as functional in tumor immunity. Our results support the clinical development of aAVCs to harness innate and adaptive immunity for effective cancer immunotherapy. Cancer Res; 73(1); 62–73. ©2012 AACR.

An induction of long-term cellular and humoral immunity is for the goal of vaccines, but the combination of antigens and adjuvant remain unclear. Here, we show, using a cellular vaccine carrying foreign protein antigen plus iNKT cell glycolipid antigen, designated as artificial adjuvant vector cells (aAVCs), that mature XCR1− DCs in situ elicit not only ordinal antigen-specific CD4+T cells, but also CD4+ Tfh and germinal center, resulted in inducing long-term antibody production. As a mechanism for leading the long-term antibody production by aAVC, memory CD4+ Tfh cells but not iNKTfh cells played an important role in a Bcl6 dependent manner. To develop it for influenza infection, we established influenza hemagglutinin-carrying aAVC (aAVC-HA) and found that all the mice vaccinated with aAVC-HA were protected from life-threatening influenza infection. Thus, the in vivo DC targeting therapy by aAVC would be useful for protection against viral infection.

Reprogramming of antigen-specific T lymphocytes into induced pluripotent stem cells (iPSCs) and their subsequent re-differentiation has enabled expansion of functional T lymphocytes in vitro, thus opening up new approaches for immunotherapy of cancer and other diseases. In this study, we have established a robust protocol to reprogram human invariant NKT (Vα24

Immunotherapy using dendritic cells (DCs) has the potential to activate both T cells and NK cells. We previously demonstrated the long-lasting antitumor responses by NK cells following immunization with bone marrow-derived DCs. In the current study, we demonstrate that long-term antitumor NK responses require endogenous DCs and a subset of effector memory CD4+ T (CD4+ TEM) cells. One month after DC immunization, injection of a tumor into DC-immunized mice leads to an increase in the expression of CXCL10 by endogenous DCs, thus directing NK cells into the white pulp where the endogenous DCs bridged CD4+ TEM cells and NK cells. In this interaction, CD4+ TEM cells express CD40L, which matures the endogenous DCs, and produce cytokines, such as IL-2, which activates NK cells. These findings suggest that DC vaccination can sustain long-term innate NK cell immunity but requires the participation of the adaptive immune system.

【目的】粥腫形成に炎症機転が推測されている．本研究は急性期血中炎症性バイオマーカーと脳梗塞亜病型の関連を検討した．【方法】発症48時間以内の脳梗塞患者142例を対象とし，入院時interleukin (IL)-6，IL-10，IL-18，matrix metalloproteinase (MMP)-2，MMP-9，高感度CRPを測定，TOAST分類での亜病型，頭蓋内外動脈硬化との関連をロジスティック回帰分析で検討した．【結果】心原性脳塞栓症では梗塞体積（OR 1.006，95%CI 1.001-1.010）と，MMP-2（OR 1.003，95%CI 1.000-1.005）が有意に関連した．脳動脈狭窄が高度なほどmaxIMT，高感度CRP，IL-6が有意に高値を示した．【結論】急性期血中MMP-2は，他の亜病型に比べ心原性脳塞栓症で高値であり，IL-6，高感度CRPは脳動脈狭窄度と関連する．

Invariant natural killer T (iNKT) cells possess potent antitumor effects after activation with a specific glycolipid antigen, α-galactosylceramide (αGalCer). A phase I–II clinical study of αGalCer-pulsed dendritic cells (DC) to activate endogenous iNKT cells was previously performed in patients with non-small-cell lung cancer (NSCLC). In this clinical trial, the patients with increased interferon-γ (IFN-γ) production (>two-fold) in PBMC after the DC treatment (good responder group) experienced a prolonged overall survival time in comparison with the poor responder group. We extended the previous study and performed a microarray-based gene expression analysis using peripheral blood CD56+ cells and CD56−CD3+ T cells from patients enrolled in the above-mentioned clinical study. We sought to identify any biomarkers associated with the immune responses in this immunotherapy trial. Six patient samples corresponding to three subjects in the good responder group and three subjects in the poor responder group were included in the microarray analysis. Genes differentially expressed between pre-treatment and post-treatment samples were selected for analysis. Subsequently, genes that were only expressed in the good responder group or poor responder group were chosen. After these procedures, four selected genes were quantified by reverse transcriptase–polymerase chain reaction in another eight patient samples, and two genes, LTB4DH and DPYSL3, were confirmed to be candidate genes for the predictor of a good immune response. The expression profile of these two genes may be associated with the responsiveness of IFN-γ production after αGalCer-pulsed DC treatment. (Cancer Sci 2010; 101: 2333–2340)

Establishment of a system with efficient generation of natural killer T (NKT) cells from embryonic stem (ES) cells would enable us to identify the cells with NKT-cell potential and obtain NKT cells with desired function. Here, using cloned ES (NKT-ES) cells generated by the transfer of nuclei from mature NKT cells, we have established a culture system that preferentially developed functional NKT cells and also identified early NKT progenitors, which first appeared on day 11 as a c-kit+ population in the cocultures on OP9 cells with expression of Notch ligand, delta-like1 (OP9/Dll-1) and became c-kitlo/− on day 14. Interestingly, in the presence of Notch signals, NKT-ES cells differentiated only to thymic CD44lo CD24hi NKT cells producing mainly interleukin-4 (IL-4), whereas NKT cells resembling CD44hi CD24lo liver NKT cells producing mainly interferon γ (IFN-γ) and exhibiting strong adjuvant activity in vivo were developed in the switch culture starting at day 14 in the absence of Notch. The cloned ES culture system offers a new opportunity for the elucidation of the molecular events on NKT-cell development and for the establishment of NKT-cell therapy.

Because of their unique role in linking the innate and adaptive immune systems, dendritic cells (DCs) have been a logical focus for novel immunotherapies. However, strategies employing active immunization with ex vivo generated and antigen-pulsed DCs have shown limited efficacy in clinical trials. These past approaches did not take into account the complex interactions between cells of the innate immune system and DCs during DC maturation, antigen processing, and presentation to naïve T cells. By better understanding the natural sequence of events occurring in vivo during an effective immune response, we can tailor antitumor immunotherapeutic strategies to augment aspects of this response from the activation of innate immune cells to antigen uptake and DC maturation to priming of naïve T cells and, ultimately, to the establishment of antitumor immunity. Current DC vaccination strategies utilize a number of methods to recapitulate the cascade of events that culminate in a protective antitumor immune response.

The maturation of dendritic cells (DCs) in situ by danger signals plays a central role in linking innate and adaptive immunity. We previously demonstrated that the activation of invariant natural killer T (iNKT) cells by administration of α-galactosylceramide (α-GalCer)–loaded tumor cells can act as a cellular adjuvant through the DC maturation. In the current study, we used allogeneic fibroblasts loaded with α-GalCer and transfected with antigen-encoding mRNA, thus combining the adjuvant effects of iNKT-cell activation with delivery of antigen to DCs in vivo. We found that these cells produce antigen protein and activate NK and iNKT cells. When injected into major histocompatibility complex (MHC)–mismatched mice, they elicited antigen-specific T-cell responses and provided tumor protection, suggesting that these immune responses depend on host DCs. In addition, antigen-expressing fibroblasts loaded with α-GalCer lead to a more potent T-cell response than those expressing NK cell ligands. Thus, glycolipid-loaded, mRNA-transfected allogeneic fibroblasts act as cellular vectors to provide iNKT-cell activation, leading to DC maturation and T-cell immunity. By harnessing the innate immune system and generating an adaptive immune response to a variety of antigens, this unique tool could prove clinically beneficial in the development of immunotherapies against malignant and infectious diseases.

We vaccinated mice with DC loaded with or without invariant NKT-cell ligand alpha-galactosylceramide and evaluated long-term resistance against tumor challenge. When mice had been given either DC or DC/galactosylceramide and were challenged with tumor cells even 6-12 months later, both NK and NKT cells were quickly activated to express CD69 and produce IFN-gamma. The NK cells could resist a challenge with several different tumors in vivo. The activated NK and NKT cells could be depleted with anti-NK1.1 treatment. In spite of this, the activated cells recovered, indicating that tumor-responsive NK and NKT cells were being generated continuously as a result of vaccination with DC and were not true memory cells. The NK and NKT antitumor response in DC-vaccinated mice depended on CD4(+) T cells, but neither CD8(+)T cells nor CD4(+)CD25(+) regulatory T cells. However, both vaccine DC and host DC were required for the development of long-term, tumor reactive innate immunity. These results indicate that DC therapy in mice induces long-lasting innate NK- and NKT-cell activation through a pathway that requires host DC and CD4(+) T cells and that the continued generation of active NK cells resists the establishment of metastases in vivo.

The observation that the glycolipid alpha-galactosylceramide (alpha-GalCer) is a potent stimulator of natural killer T (NKT) cells has provided an important means for investigating NKT cell biology. alpha-GalCer is presented on CD1d to the invariant NKT receptor, leading to interleukin-12 (IL-12) production by dendritic cells (DCs) and to NK cell activation. We review our research on the tumor-protective properties of alpha-GalCer, particularly the major role played by DCs. We compared administration of alpha-GalCer on mature DCs with soluble glycolipid and found that DCs induced more prolonged interferon-gamma (IFN-gamma) production by NKT cells and better protection against B16 melanoma. Human alpha-GalCer-loaded DCs also expanded NKT cell numbers in cancer patients. alpha-GalCer-activated NKT cells were then found to induce DC maturation in vivo. The maturing DCs produced IL-12, upregulated co-stimulatory molecules, and induced adaptive immunity to captured cellular antigens, including prolonged, combined CD4(+)/CD8(+) T-cell immunity to dying tumor cells. Surprisingly, co-stimulator-poor tumor cells, if directly loaded with alpha-GalCer ('tumor/Gal') and injected intravenously, also induced strong NKT- and NK-cell responses. The latter killed the tumor/Gal, which were subsequently cross presented by CD1d on DCs to elicit DC maturation and prolonged adaptive T-cell immunity, which lasted 6-12 months. These findings help explain tumor protection via alpha-GalCer and urge development of the DC-NKT axis to provide innate and adaptive immunity to human cancers.

We report a mechanism to induce combined and long-lived CD4(+) and CD8(+) T cell immunity to several mouse tumors. Surprisingly, the initial source of antigen is a single low dose of tumor cells loaded with alpha-galactosylceramide (alpha-GalCer) glycolipid (tumor/Gal) but lacking co-stimulatory molecules. After tumor/Gal injection intravenously (i.v.), innate NKT and NK cells reject the tumor cells, some of which are taken up by dendritic cells (DCs). The DCs in turn cross-present glycolipid on CD1d molecules to NKT cells and undergo maturation. For B16 melanoma cells loaded with alpha-GalCer (B16/Gal), interferon gamma-producing CD8(+) T cells develop toward several melanoma peptides, again after a single low i.v. dose of B16/Gal. In all four poorly immunogenic tumors tested, a single dose of tumor/Gal i.v. allows mice to become resistant to tumors given subcutaneously. Resistance requires CD4(+) and CD8(+) cells, as well as DCs, and persists for 6-12 mo. Therefore, several immunogenic features of DCs are engaged by the CD1d-mediated cross-presentation of glycolipid-loaded tumor cells, leading to particularly strong and long-lived adaptive immunity.

Here, we report a case of Cronkhite-Canada syndrome in a patient with schizophrenia. A 64-year-old man, who had been diagnosed as having a schizophrenic disorder at the age of 30, presented with alopecia, atrophic nail changes, hyperpigmentation of the skin, and inflammatory polyposis of the stomach and colon. Endoscopic ultrasonography of the stomach and colon revealed diffuse mucosal thickening with small hypoechoic areas, corresponding to edema of the lamina propria. After treatment with parenteral hyperalimentation and tranexamic acid, his physical findings and polyposis gradually improved. This is the first report of Cronkhite-Canada syndrome in a patient with schizophrenia.

Persistent complex bereavement disorder (PCBD) was proposed as a bereavement-related clinical category in the Diagnostic and Statistical Manual of Mental Disorders, 5th edition, which included the disorder among conditions for further study. This is an independent clinical category in which intense yearning for the deceased continues for at least 12 months. However, the diagnostic features are still inconclusive. We suggest a variation of PCBD for making category from our clinical experiences.We presented two representative case studies in which grief caused by bereavement was observed as the root of the pathological condition. We examined the disorder's pathological conditions, diagnoses, and appropriate treatments based on the cases we experienced.Both cases involved elderly women who lost their spouse through illness and experienced prolonged grief for an extended period, resulting in hospital admission. Based on the two cases, we believe that PCBD can also include a psychotic type with hallucinations as a major symptom. While studying PCBD, we took into account specific cultural characteristics of Japanese people and their present day social environment.Such cases would be suggestive when determining the PCBD clinical category in the future. PCBD is considered to be clinically very useful, especially in an extremely aged society as seen in developed countries, including Japan.

An ideal cancer vaccine would inhibit tumor cells by inducing both innate and adaptive immunity, allowing for specific and long-term cancer immunosurveillance.

NKT cells play a role in immunological regulation of certain diseases, and their frequency and/or function may be related to disease prognosis. However, it is often difficult to evaluate NKT cell function in patients with malignancies due to reduced numbers of NKT cells as well as the dysfunction of the APCs used as stimulators. We found that NKT cell function could not be evaluated by conventional ELISPOT assays, confirming the impaired function of APCs in chronic myelogenous leukemia (CML)-chronic phase patients. To overcome this problem, we have established a sensitive assay using murine dendritic cells to evaluate the function of small numbers of human NKT cells independent of autologous APCs. We found that imatinib-treated CML-chronic phase patients showing a complete cytogenetic response had NKT cells capable of producing IFN-γ, whereas NKT cells from patients who were only partially responsive to imatinib treatment did not produce IFN-γ. Functional NKT cells found in imatinib-treated, CML-complete cytogenetic response patients may offer the promise of effective immunotherapy with ex vivo-generated α-galactosylceramide-pulsed dendritic cells. This new approach should be available for evaluating the functions of NKT cells and APCs in cancer patients.

GC–olfactometry was used to characterize the specific aroma of a pentane extract of snake fruit (Salacca edulis Reinw cv. Pondoh). Ten compounds, including two carboxylic acids, six methyl esters, an alcohol and a furaneol (2,5-dimethyl-4-hydroxy-3(2H)-furanone), were detected as the most characteristic odorants. Methyl esters exhibited particularly sweet and fruity odour characteristics in snake fruit at more mature stages, whereas carboxylic acids exhibited the sweaty odour that tends to prevent non-native consumers from liking the fruit. A headspace of snake fruit at different stages of maturation was subjected to analysis using two analytical systems an electronic nose equipped with 18 sensors and fingerprint mass spectrometry. Three sensors and five ions derived from esters were chosen as fingerprint parameters to discriminate between maturation levels. Both systems appeared to discriminate efficiently between the fruits at different maturation stages. Copyright © 2003 John Wiley & Sons, Ltd.

Here we show that patients with myelodysplastic syndromes (MDS) have a severe deficiency of glycolipid reactive Valpha24+/Vbeta11+ natural killer T (NKT) cells, but not NK cells or CD4+ or CD8+ T cells. Neither the blood nor marrow of MDS patients had detectable interferon-gamma-producing NKT cells in response to the NKT ligand, alpha-galactosyl ceramide, although influenza-virus-specific effector T-cell function was preserved. This severe and selective deficiency of an important immune regulatory cell may contribute to the pathogenesis of MDS.

The maturation of dendritic cells (DCs) allows these antigen-presenting cells to initiate immunity. We pursued this concept in situ by studying the adjuvant action of alpha-galactosylceramide (alphaGalCer) in mice. A single i.v. injection of glycolipid induced the full maturation of splenic DCs, beginning within 4 h. Maturation was manifest by marked increases in costimulator and major histocompatibility complex class II expression, interferon (IFN)-gamma production, and stimulation of the mixed leukocyte reaction. These changes were not induced directly by alphaGalCer but required natural killer T (NKT) cells acting independently of the MyD88 adaptor protein. To establish that DC maturation was responsible for the adjuvant role of alphaGalCer, mice were given alphaGalCer together with soluble or cell-associated ovalbumin antigen. Th1 type CD4+ and CD8+ T cell responses developed, and the mice became resistant to challenge with ovalbumin-expressing tumor. DCs from mice given ovalbumin plus adjuvant, but not the non-DCs, stimulated ovalbumin-specific proliferative responses and importantly, induced antigen-specific, IFN-gamma producing, CD4+ and CD8+ T cells upon transfer into naive animals. In the latter instance, immune priming did not require further exposure to ovalbumin, alphaGalCer, NKT, or NK cells. Therefore a single dose of alphaGalCer i.v. rapidly stimulates the full maturation of DCs in situ, and this accounts for the induction of combined Th1 CD4+ and CD8+ T cell immunity to a coadministered protein.

We studied the function of antitumor T and natural killer T (NKT) cells from the blood and tumor bed in 23 patients with premalignant gammopathy, nonprogressive myeloma, or progressive multiple myeloma. We show that antitumor killer T cells can be detected in patients with both progressive or nonprogressive myeloma. V alpha 24+V beta 11+ invariant NKT cells are detectable in the blood and tumor bed of all cohorts. However, freshly isolated NKT cells from both the blood and tumor bed of patients with progressive disease, but not nonprogressive myeloma or premalignant gammopathy, have a marked deficiency of ligand-dependent interferon-gamma production. This functional defect can be overcome in vitro using dendritic cells pulsed with the NKT ligand, alpha-galactosylceramide (alpha-GalCer). Fresh myeloma cells express CD1d, and can be efficiently killed by autologous NKT cells. We hypothesize that presentation of tumor derived glycolipids by myeloma cells leads to NKT dysfunction in vivo. These data demonstrate that clinical progression in patients with monoclonal gammopathies is associated with an acquired but potentially reversible defect in NKT cell function and support the possibility that these innate lymphocytes play a role in controlling the malignant growth of this incurable B cell tumor in patients.

Human CD1d-restricted natural killer T (NKT) cells, which are postulated to regulate the immune response in several clinical settings, can be activated by alpha-galactosylceramide (alpha-GalCer) presented by CD1d molecules on antigen presenting cells (APCs). Simple methods to quantify NKT function in fresh blood will greatly benefit studies targeting NKT cells in humans. Here we show that freshly isolated human NKT cells can be readily quantified by an enzyme-linked immunospot (ELISPOT) assay and have a Th1 profile (secreting interferon-gamma, but not IL-4), after stimulation using alpha-GalCer loaded APCs. Using this assay, we also evaluated APC requirements for human NKT cell activation in fresh blood. Monocyte-derived dendritic cells (DCs) are more effective than monocytes/macrophages for detecting and activating NKT cells in fresh blood, with mature alpha-GalCer pulsed DCs being optimal. DCs are also efficient APCs for expanding NKT cells in culture and generating NKT cell lines. NKT cells expanded with DCs were functional, secreting both IFN-gamma and IL-4, and killing NKT-sensitive targets. Optimal activation of these lines was seen using mature DCs loaded with 10-100 ng/ml of alpha-GalCer. DCs matured with several different stimuli were effective. These data help to establish the conditions for loading DCs with alpha-GalCer for immune therapeutic targeting of NKT cells, and provide a new simple assay to monitor NKT function in humans.

Dendritic cells (DCs) play a central role in immune regulation. Some leukemic cells are argued to be malignant counterparts of DC because of their ability to differentiate into leukemic DC. We characterize DC-like leukemia homogenously expressing CD11c+CD86+ in acute myelogenous leukemia patients. They express the Wilms’ tumor-1 antigen and common DC phenotypes (i.e., fascin+, CD83+, and DR+) directly. Purified leukemic cells produce interleukin-12 (IL-12) simultaneously with Fas ligand (FasL) and IL-6, which may suppress T cell-mediated immunity. These cells can elicit strong allogeneic T cell responses as well as induce tumor-specific CD8+ cytotoxic T cells, suggesting that they effectively present tumor-associated antigens. In contrast, they drive primary T cells toward apoptosis mediated in a tumor-specific way by a Fas-FasL interaction. Taken together, DC-like leukemia uniquely influences immune surveillance in contadictory ways, some of which may be involved in the mechanism of immune escape.

Natural killer T (NKT) lymphocytes mediate a rapid reaction to the glycolipid drug alpha-galactosylceramide (alpha GalCer), which triggers release of large amounts of cytokines into the serum within 12 h, starting with interleukin 4 (IL-4). When alpha GalCer is administered to mice on dendritic cells (DCs) instead, the response is more prolonged (>4 days) and marked by a large expansion in IFN-gamma-producing NKT cells as well as greater resistance to metastases of the B16 melanoma. Nevertheless, DCs from mice given free alpha GalCer are able to induce strong IFN-gamma-producing NKT responses when transferred to naive mice, but not when transferred to alpha GalCer-treated recipients. In the latter, the NKT cells are energized and can respond to glycolipid only in the presence of supplemental IL-2. Therefore, when alpha GalCer is selectively targeted to DCs, mice develop a stronger, more prolonged and effector type of NKT response, but this response can be blocked by the induction of anergy after presentation of alpha GalCer on other cells.

Immunological memory has been regarded as a unique feature of the adaptive immune response mediated in an antigen-specific manner by T and B lymphocytes. However, natural killer (NK) cells and γδT cells, which traditionally are classified as innate immune cells, have been shown in recent studies to have hallmark features of memory cells. Invariant NKT cell (iNKT cell)-mediated antitumor effects indicate that iNKT cells are activated in vivo by vaccination with iNKT cell ligand-loaded CD1d(+) cells, but not by vaccination with unbound NKT cell ligand. In such models, it previously was thought that the numbers of IFN-γ-producing cells in the spleen returned to the basal level around 1 wk after the vaccination. In the current study, we demonstrate the surprising presence of effector memory-like iNKT cells in the lung. We found long-term antitumor activity in the lungs of mice was enhanced after vaccination with iNKT cell ligand-loaded dendritic cells. Further analyses showed that the KLRG1(+) (Killer cell lectin-like receptor subfamily G, member 1-positive) iNKT cells coexpressing CD49d and granzyme A persisted for several months and displayed a potent secondary response to cognate antigen. Finally, analyses of CDR3β by RNA deep sequencing demonstrated that some particular KLRG1(+) iNKT-cell clones accumulated, suggesting the selection of certain T-cell receptor repertoires by an antigen. The current findings identifying effector memory-like KLRG1(+) iNKT cells in the lung could result in a paradigm shift regarding the basis of newly developed extrathymic iNKT cells and could contribute to the future development of antitumor immunotherapy by uniquely energizing iNKT cells.

The Lck protein (p56(lck)), a src family tyrosine kinase that is essential for T cell development and function, is aberrantly expressed in metastatic colon cancers. p56(lck) seems to facilitate the malignant transformation of epithelial cells through initiation of anchorage-independent proliferation. We demonstrate that the lck gene encodes antigenic epitopes recognized by the HLA class I-restricted and tumor-specific CTL of metastatic cancer patients. Lck peptides augmented CTL activity in peripheral blood mononuclear cells (PBMC) of colon and other epithelial cancer patients with distant metastases, but not those without distant metastases. CTL precursors recognizing the Lck peptide were identified in freshly prepared PBMC of patients with distant metastases, and their frequency was significantly augmented by stimulation with the peptide. Thus, Lck peptides could be useful in developing a specific immunotherapy for cancer patients with distant metastases.

The macrolide lactone, tacrolimus (FK506), is utilized in bone marrow transplantation (BMT) to prevent graft-versus-host disease (GVHD). In the current study, we evaluated the ability of FK506 to modify the function of dendritic cells (DCs) derived from CD34+ hematopoietic progenitor cells (HPCs). Comparable to DCs obtained in the absence of FK506, DCs cultured in the presence of FK506 (FK-DCs) had higher expression of CD1a+ and formed a greater number of DC colonies. Despite the same expression of costimulatory molecules, FK-DCs displayed a reduced capacity to stimulate an allogeneic T cell response, and showed significantly lower interleukin (IL)-12 production. While normal DCs pulsed with the exogenous antigen, keyhole limpet hemocyanin (KLH) induced specific Th1-like interferon-γ(IFN-γ) producing CD4+ T cell line, FK-DCs induced Th2-like interleukin-4 (IL-4) producing CD4+ T cell line. These data demonstrate the ability of FK506 to induce Th2-promoting function in developing DCs.

Human natural killer T (NKT) cells are characterized by their expression of an invariant T cell antigen receptor α chain variable region encoded by a Vα24Jα18 rearrangement. These NKT cells recognize α-galactosylceramide (α-GalCer) in conjunction with the MHC class I-like CD1d molecule and bridge the innate and acquired immune systems to mediate efficient and augmented immune responses. A prime example of one such function is adjuvant activity: NKT cells augment anti-tumor responses because they can rapidly produce large amounts of IFN-γ, which acts on NK cells to eliminate MHC negative tumors and also on CD8 cytotoxic T cells to kill MHC positive tumors. Thus, upon administration of α-GalCer-pulsed DCs, both MHC negative and positive tumor cells can be effectively eliminated, resulting in complete tumor eradication without tumor recurrence. Clinical trials have been completed in a cohort of 17 patients with advanced non-small cell lung cancers and 10 cases of head and neck tumors. Sixty percent of advanced lung cancer patients with high IFN-γ production had significantly prolonged median survival times of 29.3 months with only the primary treatment. In the case of head and neck tumors, 10 patients who completed the trial all had stable disease or partial responses 5 weeks after the combination therapy of α-GalCer-DCs and activated NKT cells. We now focus on two potential powerful treatment options for the future. One is to establish artificial adjuvant vector cells containing tumor mRNA and α-GalCer/CD1d. This stimulates host NKT cells followed by DC maturation and NK cell activation but also induces tumor-specific long-term memory CD8 killer T cell responses, suppressing tumor metastasis even 1 year after the initial single injection. The other approach is to establish induced pluripotent stem (iPS) cells that can generate unlimited numbers of NKT cells with adjuvant activity. Such iPS-derived NKT cells produce IFN-γ in vitro and in vivo upon stimulation with α-GalCer/DCs, and mediated adjuvant effects, suppressing tumor growth in vivo.

Complex interactions among multiple cell types contribute to the immunosuppressive milieu of the tumor microenvironment. Using a murine model of adoptive T-cell immunotherapy (ACT) for B16 melanoma, we investigated the impact of tumor infiltrating cells on this complex regulatory network in the tumor. Transgenic pmel-1-specific cytotoxic T lymphocytes (CTLs) were injected intravenously into tumor-bearing mice and could be detected in the tumor as early as on day 1, peaking on day 3. They produced IFN-γ, exerted anti-tumor activity and inhibited tumor growth. However, CTL infiltration into the tumor was accompanied by the accumulation of large numbers of cells, the majority of which were CD11b(+) Gr1(+) myeloid-derived suppressor cells (MDSCs). Notably, CD11b(+) Gr1(int) Ly6G(-) Ly6C(+) monocytic MDSCs outnumbered the CTLs by day 5. They produced nitric oxide, arginase I and reactive oxygen species, and inhibited the proliferation of antigen-specific CD8(+) T cells. The anti-tumor activity of the adoptively-transferred CTLs and the accumulation of MDSCs both depended on IFN-γ production on recognition of tumor antigens by the former. In CCR2(-/-) mice, monocytic MDSCs did not accumulate in the tumor, and inhibition of tumor growth by ACT was improved. Thus, ACT triggered counter-regulatory immunosuppressive mechanism via recruitment of MDSCs. Our results suggest that strategies to regulate the treatment-induced recruitment of these MDSCs would improve the efficacy of immunotherapy.

A key goal of vaccine immunotherapy is the generation of long-term memory CD8+ T cells capable of mediating immune surveillance. We discovered a novel intercellular pathway governing the development of potent memory CD8+ T cell responses against cell-associated Ags that is mediated through cross-presentation by XCR1+ dendritic cells (DCs). Generation of CD8+ memory T cells against tumor cells pulsed with an invariant NKT cell ligand depended on cross-talk between XCR1+ and plasmacytoid DCs that was regulated by IFN-α/IFN-αR signals. IFN-α production by plasmacytoid DCs was stimulated by an OX40 signal from the invariant NKT cells, as well as an HMGB1 signal from the dying tumor cells. These findings reveal a previously unknown pathway of intercellular collaboration for the generation of tumor-specific CD8+ memory T cells that can be exploited for strategic vaccination in the setting of tumor immunotherapy.

Summary Antigen-specific T cells represent a potential therapeutic avenue for a variety of conditions, but current approaches for generating such cells for therapeutic purposes are limited. In this study, we established iPSCs from mature cytotoxic T cells specific for the melanoma epitope MART-1. When cocultured with OP9/DLL1 cells, these iPSCs efficiently generated TCRβ + CD4 + CD8 + double positive (DP) cells expressing a T cell receptor (TCR) specific for the MART-1 epitope. Stimulation of these DP cells with anti-CD3 antibody generated a large number of CD8 + T cells, and more than 90% of the resulting cells were specific for the original MART-1 epitope. Stimulation of the CD8 + T cells with MART-1 antigen-presenting cells led to the secretion of IFNγ, demonstrating their specific reactivity. The present study therefore illustrates an approach for cloning and expanding functional antigen-specific CD8 + T cells that might be applicable in cell-based therapy of cancer.

We prospectively studied the effects of early statin treatment on stroke-induced changes in the levels of inflammatory biomarkers. Patients admitted within 48 hours after the onset of ischemic stroke were enrolled. They were divided into 2 groups according to their lipid profiles and history of statin treatment. In patients who had received statin treatment prior to admission and those who had abnormal lipid profiles on admission, daily treatment with 10 mg atorvastatin was initiated within 48 hours after the onset of stroke (Statin group; n = 45). In patients who had normal lipid profiles on admission, statin was not administered for at least 2 weeks after admission (Non-Statin group; n = 101). The serum concentrations of interleukin (IL)-6, IL-10, IL-18, matrix metalloproteinase (MMP)-2, MMP-9, and high sensitive C-reactive protein were measured on days 1, 3, 7, and 14. In percentage changes in serially measured circulating IL-6 levels, a significant interaction between group and repeated measures (group X time factor) was demonstrated (p = 0.047). Frequency of neurological deterioration episodes (NIHSS score > or = 2) during 14 days after admission was lower in the Statin group than in the Non-Statin group, however the difference did not reach statistically significant level (7.9% vs 20.2%, p = 0.118). The initiation of usual dose of atorvastatin early after the onset of ischemic stroke significantly decreased the elevation of IL-6 and may protect against the early neurological deterioration. Circulating levels of IL-6 may be one of the candidates for monitoring the acute effects of statin. Further studies wherein IL-6 levels are monitored in larger samples would be feasible for investigating the effect of early treatment with usual dose of atorvastatin on the functional outcome.

Intracerebroventricular (ICV) administration of gonadotropin-releasing hormone II (GnRH II), which plays a crucial role in the regulation of reproduction in vertebrates, markedly reduces food intake in goldfish. However, the neurochemical pathways involved in the anorexigenic action of GnRH II and its interaction with other neuropeptides have not yet been identified. Alpha-melanocyte-stimulating hormone (α-MSH), corticotropin-releasing hormone (CRH) and CRH-related peptides play a major role in feeding control as potent anorexigenic neuropeptides in goldfish. However, our previous study has indicated that the GnRH II-induced anorexigenic action is not blocked by treatment with melanocortin 4 receptor (MC4R) and CRH receptor antagonists. Therefore, in the present study, we further examined whether the anorexigenic effects of α-MSH and CRH in goldfish could be mediated through the GnRH receptor neuronal pathway. ICV injection of the MC4R agonist, melanotan II (80 pmol/g body weight; BW), significantly reduced food intake, and its anorexigenic effect was suppressed by ICV pre-administration of the GnRH type I receptor antagonist, antide (100 pmol/gBW). The CRH-induced (50 pmol/gBW) anorexigenic action was also blocked by treatment with antide. ICV injection of CRH (50 pmol/gBW) induced a significant increase of the GnRH II mRNA level in the hypothalamus, while ICV injection of melanotan II (80 pmol/gBW) had no effect on the level of GnRH II mRNA. These results indicate that, in goldfish, the anorexigenic actions of α-MSH and CRH are mediated through the GnRH type I receptor-signaling pathway, and that the GnRH II system regulates feeding behavior.

Desmethyl homologs of dimethylallyl diphosphate and of isopentenyl diphosphate were examined for the reactivity as substrates for the thermostable farnesyl diphosphate synthase of Bacillus stearothermophilus. E-But-2-enyl diphosphate was not accepted as a substrate at all, but but-3-enyl diphosphate reacted with dimethylallyl- and geranyl diphosphates to give norgeranyl- and norfarnesyl diphosphates, respectively, which lack methyl groups at the 3-positions.

NKT cells are characterized by their production of both T(h)1 and T(h)2 cytokines immediately after stimulation with alpha-galactosylceramide (alpha-GalCer), which is composed of alpha-galactopyranose linked to ceramide (itself composed of sphingosine and fatty-acyl chains); the chain length of the ceramide varies and this affects the ability of alpha-GalCer to stimulate cytokine production. However, the contribution of its galactopyranose sugar moiety remains unclear. We synthesized alpha-carba-GalCer, which has an alpha-linked carba-galactosyl moiety; here, the 5a'-oxygen atom of the D-galactopyranose ring of alpha-GalCer is replaced by a methylene group. The alpha-carba-GalCer was more stable and showed higher affinity to the NKT receptor. It thus enhanced and prolonged production of IL-12 and IFN-gamma compared with alpha-GalCer, resulting in augmented NKT cell-mediated adjuvant effects in vivo. The alpha-carba-GalCer, which has an ether linkage, was more resistant to degradation by liver microsomes than was alpha-GalCer, which has an acetal bond. Modulation of the sugar moiety in glycolipids might therefore provide optimal therapeutic reagents for protective immune responses against tumor or pathogens.

Invariant NKT cells (NKT) cells become activated after stimulation with antigen-presenting cells (APCs) loaded with the NKT cell ligand, alpha-galactosylceramide (alpha-GalCer). In this study, we investigated whether human APCs loaded with alpha-GalCer have the ability to activate NKT cells in mice. We found that human dendritic cells (DCs) loaded with alpha-GalCer (hDC/Gal) and injected into C57BL/6 mice stimulated the secretion of IFN-gamma by activated murine NKT cells. Furthermore, the number of transferred hDC/Gal correlated with the number of recovered IFN-gamma-producing spleen cells, indicating that the capacity of APCs to load alpha-GalCer can be measured by IFN-gamma release in an ELISPOT assay. Finally, alpha-GalCer-loaded human leukemic cell lines and primary leukemic cells injected into C57BL/6 mice also had the capacity to stimulate murine NKT cells in vivo. These results indicate that in vivo murine NKT cell responses can be used to quantitate the alpha-GalCer-loading capacity of human APCs. This method could be utilized to develop future immunotherapies in which NKT cells are targeted for activation.

Dendritic cells (DCs) work as a natural adjuvant to elicit T cell immunity. Though DCs have been widely used in immunotherapy, little is known about their number and function in patients with cancer or autoimmune disease. In recent studies, antigen has been targeted to DCs through DC-specific receptors, such as DEC205, the mannose receptor and dying cell receptors. However, antigen captured by DCs in the absence of danger signals induces tolerance. Therefore, the duration and/or magnitude of danger signals plays a crucial role in generating an immunogeneic response. Various danger signals, i.e., pathogen-associated molecular pattern (PAMP), damage-associated molecular pattern (DAMP) and the activation of innate lymphocytes, serve as maturation signals for DCs. An immunotherapeutic approach which delivers both maturation signals and antigen to DCs would link the innate and adaptive arms of the immune system for a more effective and global immune response. It is therefore crucial to determine optimal conditions for antigen delivery to DCs in an environment suited to maximally stimulate the immune system.

Dendritic cells (DCs) loaded with α-galactosylceramide (α-GalCer) are known to be active APCs for the stimulation of innate NKT and NK cell responses in vivo. In this study, we evaluated the capacity of non-DCs to present α-GalCer in vitro and in vivo, particularly tumor cells loaded with α-GalCer (tumor/Gal). Even though the tumor cells lacked expression of CD40, CD80, and CD86 costimulatory molecules, the i.v. injection of tumor/Gal resulted in IFN-γ secretion by NKT and NK cells. These innate responses to tumor/Gal, including the induction of IL-12p70, were comparable to or better than α-GalCer-loaded DCs. B16 melanoma cells that were stably transduced to express higher levels of CD1d showed an increased capacity relative to wild-type B16 cells to present α-GalCer in vivo. Three different tumor cell lines, when loaded with α-GalCer, failed to establish tumors upon i.v. injection, and the mice survived for at least 6 mo. The resistance against tumor cells was independent of CD4 and CD8 T cells but dependent upon NKT and NK cells. Mice were protected from the development of metastases if the administration of live B16 tumor cells was followed 3 h or 3 days later by the injection of CD1dhigh-α-GalCer-loaded B16 tumor cells with or without irradiation. Taken together, these results indicate that tumor/Gal are effective APCs for innate NKT and NK cell responses, and that these innate immune responses are able to resist the establishment of metastases in vivo.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population with the ability to suppress immune responses and are currently classified into three distinct MDSC subsets: monocytic, granulocytic and non-monocytic, and non-granulocytic MDSCs. Although NK cells provide an important first-line defense against newly transformed cancer cells, it is unknown whether NK cells can regulate MDSC populations in the context of cancer. In this study, we initially found that the frequency of MDSCs in non-Hodgkin lymphoma (NHL) patients was increased and inversely correlated with that of NK cells, but not that of T cells. To investigate the regulation of MDSC subsets by NK cells, we used an EL4 murine lymphoma model and found the non-monocytic and non-granulocytic MDSC subset, i.e., Gr1+CD11b+Ly6GmedLy6Cmed MDSC, is increased after NK cell depletion. The MDSC population that expresses MHC class II, CD80, CD124, and CCR2 is regulated mainly by CD27+CD11b+NK cells. In addition, this MDSC subset produces some immunosuppressive cytokines, including IL-10 but not nitric oxide (NO) or arginase. We also examined two subsets of MDSCs (CD14+HLA-DR− and CD14− HLA-DR− MDSC) in NHL patients and found that higher IL-10-producing CD14+HLA-DR−MDSC subset can be seen in lymphoma patients with reduced NK cell frequency in peripheral blood. Our analyses of MDSCs in this study may enable a better understanding of how MDSCs manipulate the tumor microenvironment and are regulated by NK cells in patients with lymphoma.

α-Galactosylceramide (α-GalCer) is the prototype compound for studying the presentation of glycolipids on CD1d molecules to natural killer T (NKT) lymphocytes. A single i.v. dose of glycolipid triggers a cascade of events involving the production of several cytokines over the course of a day, a short-lived activation of NKT and natural killer (NK) cells, and a more prolonged adaptive T cell immune response if certain antigens are given together with α-GalCer. We find that a recently described analogue, α-C-galactosylceramide (α-C-GalCer), more potently induces these innate and adaptive immune responses in mice. α-C-GalCer acts as a more effective trigger for IL-12 and IFN-γ production, although it minimally elicits IL-4 and TNF-α release into the serum. Also, α-C-GalCer better mobilizes NKT and natural killer cells to resist B16 melanoma. To help understand these effects, we find that α-C-GalCer binds more stably to dendritic cells than α-GalCer and that dendritic cells loaded with α-C-GalCer induce larger and more long lasting NKT cell responses in vivo . When glycolipid is targeted to dendritic cells in spleen together with antigens in dying cells, such as irradiated tumor cells, α-C-GalCer is active as an adjuvant for T cell-mediated immunity at lower doses, just 20 ng per mouse, where it is also able to up-regulate the required CD40L costimulatory molecule on NKT cells. Therefore, α-C-GalCer represents a glycolipid that binds more stably to dendritic cells and acts as a more effective link between innate and adaptive immunity in vivo .

An 88-year old Japanese female with pure red cell aplasia was treated safely and effectively by a combination of thymectomy, cyclosporin A, and erythropoietin. The thymoma was histologically classified as lymphocytic type or cortical type, which are uncommon in cases of a thymoma accompanied by pure red cell aplasia. Immunohistochemical analysis of the thymoma and bone marrow revealed a predominance of CD8(+) cells. Thymectomy alone was ineffective, but cyclosporin A treatment subsequent to thymectomy was safe and effective and resulted in the disappearance of a Vbeta12 bearing T-cell clone in the bone marrow. Additional treatment with erythropoietin enhanced the effects of cyclosporin A and restored the patient's hemoglobin to normal levels. The beneficial effect of cyclosporin A may be attributed not to a broad immunomodulatory effect, but to a local effect on a limited T-cell subset.

Electrochemically generated π-neutral radicals of ZnII- and FeII-protoverdohaem dimethylesters are concluded to be the intermediate species in the process of the biliverdin formation.

Here we show that patients with myelodysplastic syndromes (MDS) have a severe deficiency of glycolipid reactive Valpha24+/Vbeta11+ natural killer T (NKT) cells, but not NK cells or CD4+ or CD8+ T cells. Neither the blood nor marrow of MDS patients had detectable interferon-gamma-producing NKT cells in response to the NKT ligand, alpha-galactosyl ceramide, although influenza-virus-specific effector T-cell function was preserved. This severe and selective deficiency of an important immune regulatory cell may contribute to the pathogenesis of MDS.

Natural killer T (NKT) lymphocytes mediate a rapid reaction to the glycolipid drug alpha-galactosylceramide (alpha GalCer), which triggers release of large amounts of cytokines into the serum within 12 h, starting with interleukin 4 (IL-4). When alpha GalCer is administered to mice on dendritic cells (DCs) instead, the response is more prolonged (4 days) and marked by a large expansion in IFN-gamma-producing NKT cells as well as greater resistance to metastases of the B16 melanoma. Nevertheless, DCs from mice given free alpha GalCer are able to induce strong IFN-gamma-producing NKT responses when transferred to naïve mice, but not when transferred to alpha GalCer-treated recipients. In the latter, the NKT cells are energized and can respond to glycolipid only in the presence of supplemental IL-2. Therefore, when alpha GalCer is selectively targeted to DCs, mice develop a stronger, more prolonged and effector type of NKT response, but this response can be blocked by the induction of anergy after presentation of alpha GalCer on other cells.

Interleukin-10 (IL-10) is a multifunctional cytokine that can exert suppressive and stimulatory effects on T cells. It was investigated whether IL-10 could serve as an immunostimulant for specific CD8(+) cytotoxic T cell (CTL) in vivo after vaccination and, if so, under what conditions. In tumor prevention models, administration of IL-10 before, or soon after, peptide-pulsed primary dendritic cell immunization resulted in immune suppression and enhanced tumor progression. Injection of IL-10, however, just after a booster vaccine significantly enhanced antitumor immunity and vaccine efficacy. Analysis of spleen cells derived from these latter animals 3 weeks after IL-10 treatment revealed that the number of CD8(+) CD44(hi) CD122(+) T cells had increased and that antigen-specific proliferation in vitro was enhanced. Although cytotoxicity assays did not support differences between the various treatment groups, 2 more sensitive assays measuring antigen-specific interferon-gamma production at the single-cell level demonstrated increases in the number of antigen-specific responder T cells in animals in the vaccine/IL-10 treatment group. Thus, IL-10 may maintain the number of antitumor CD8(+) T cells. In adoptive transfer studies, the ability of IL-10 to maintain CTL function could be enhanced by the depletion of CD4(+) T cells. This suggests that IL-10 mediates contrasting effects on both CD4(+) and CD8(+) T cells that result in either immune dampening or immune potentiation in situ, respectively. Appreciation of this dichotomy in IL-10 immunobiology may allow for the design of more effective cancer vaccines designed to activate and maintain specific CD8(+) T-cell effector function in situ.