Role of Valpha 14 NKT cells in the development of impaired liver regeneration in vivo

Although we have previously demonstrated that IL-12 stimulation increases the number of hepatic natural killer (NK) T (NKT) cells and enhances liver injury during the early phase of liver regeneration, the role of NKT cells has remained unknown. We therefore evaluated the influence of NKT cells activated by IL-12 or by alpha-galactosylceramide (alpha-GalCer) on murine liver regeneration using Valpha 14 NKT knockout (Jalpha 281(-/-)) mice. Levels of serum alanine aminotransferase (sALT) 24 hours after partial hepatectomy were enhanced in Jalpha 281(+/+) but not in Jalpha 281(-/-) mice by both procedures. Hepatic NKT cells expressed considerably more interferon (IFN) gamma and tumor necrosis factor alpha (TNF-alpha) messenger RNA (mRNA) after stimulation with both factors in Jalpha 281(+/+) mice. Either anti-IFN-gamma or TNF-alpha antibody inhibited the enhancement of liver injury. Furthermore, recombinant TNF-alpha injection similarly caused injury in hepatectomized livers of both Jalpha 281(+/+) and Jalpha 281(-/-) mice; indeed, adoptively transferred TNF-alpha(+/+) NKT cells enhanced liver injury after hepatectomy in TNF-alpha knockout mice. TNF receptor expressions on hepatocytes increased and peaked 24 hours after partial hepatectomy. In conclusion, simultaneous TNF-alpha synthesis and high levels of TNF receptor expression on hepatocytes cause severe liver damage by activated NKT cells during liver regeneration.     

Valpha24+ NKT cells are decreased in elderly humans.

Natural killer T (NKT) cells represent a novel cell lineage characterized by the restricted expression of an invariant TCRalpha chain encoded by Valpha24/JalphaQ gene segments in humans and Valpha14/Jalpha281+ in mice. Different aspects of the immune response are severely affected by age. Thus, we have studied the effect of aging on NKT cells from healthy elderly individuals. Our results demonstrated a decreased percentage of CD3+Valpha24+ cells in peripheral blood from elderly donors, whereas mainstream T lymphocytes showed an age-associated decrease in the expression of CD28, the vast majority of CD3+Valpha24+ cells from old individuals were CD28+. A significant increase in the percentage of Valpha24+ cells with the CD4-CD8+ phenotype was also found in the elderly, indicating a redistribution of Valpha24+ subsets according to the CD4/CD8 phenotype. Given the important immunoregulatory role of these cells, the decrease of NKT cells will contribute to the deleterious immune response in the elderly.     

Recovery of Valpha24+ NKT cells after hematopoietic stem cell transplantation

Human Valpha24+ natural killer T (NKT) cells have an invariant T-cell receptor-alpha chain and are activated in a CD1d-restricted manner. Valpha24+ NKT cells are thought to regulate immune responses and to play important roles in the induction of allograft tolerance. In this report, we analyzed the recovery of Valpha24+ NKT cells after hematopoietic stem cell transplantation and its correlation with graft-versus-host disease (GVHD). Patients who received a dose-reduced conditioning regimen, antithymocyte globulin- or CAMPATH-1H-containing conditioning regimen were excluded. NKT cells were reconstituted within 1 month after transplantation in peripheral blood stem cell transplantation recipients, while their numbers remained low for more than 1 year in bone marrow transplantation (BMT) recipients. The number of Valpha24+ NKT cells in BMT recipients with acute GVHD was lower than that in patients without acute GVHD, and both the CD4+ and CD4- Valpha24+ NKT subsets were significantly reduced. With regard to chronic GVHD, BMT recipients with extensive GVHD had significantly fewer Valpha24+ NKT cells than other patients. Furthermore, the number of CD4+ Valpha24+ NKT cells was also significantly reduced in patients with chronic extensive GVHD. Our results raise the possibility that the number of Valpha24+ NKT cells could be related to the development of GVHD.     

T-bet concomitantly controls migration, survival, and effector functions during the development of Valpha14i NKT cells

Valpha14i natural killer T (NKT)-cell function has been implicated in a number of disease conditions. The molecular events that drive Valpha14i NKT-cell development remain elusive. We recently showed that T-bet is required for the terminal maturation of these cells. Here we identify some of the genetic targets of T-bet during Valpha14i NKT-cell lineage development. Microarray gene-expression analyses on developing Valpha14i NKT cells were performed and provide a molecular framework to study these maturation events. In vitro ectopic expression of T-bet in immature Valpha14i NKT cells, which do not yet express T-bet, was sufficient to promote Valpha14i NKT-cell maturation, driving the expression of multiple genes, including those that participate in migration, survival, and effector functions. By regulating the expression of T-helper 1 (Th1)-associated cytokines, chemokines, chemokine receptors, and molecules involved in cytolysis, T-bet defines the unique lineage attributes of mature Valpha14i NKT cells and acts to link these attributes to a developmental process.     

The T cell antigen receptor expressed by Valpha14i NKT cells has a unique mode of glycosphingolipid antigen recognition

Natural killer (NK) T cells with an invariant Valpha14 rearrangement (Valpha14i) are the largest population of lipid antigen-specific T lymphocytes identified in animals. They react to the glycolipid alpha-galactosyl ceramide (alpha-GalCer) presented by CD1d, and they may have important regulatory functions. It was previously shown that the Valpha14i T cell antigen receptor (TCR) has a high affinity for the alpha-GalCer/CD1d complex, driven by a long half-life (t(1/2)). Although this result could have reflected the unique attributes of alpha-GalCer, using several related glycolipid compounds, we show here that the threshold for full activation of Valpha14i NKT cells by these glycosphingolipids requires a relatively high-affinity TCR interaction with a long t(1/2). Furthermore, our data are consistent with the view that the mechanism of recognition of these compounds presented by CD1d to the Valpha14i NKT cell TCR is likely to fit a lock-and-key model. Overall, these findings emphasize the distinct properties of glycosphingolipid antigen recognition by Valpha14i NKT cells.     

Human peripheral blood Valpha24+ Vbeta11+ NKT cells expand following administration of alpha-galactosylceramide-pulsed dendritic cells

BACKGROUND AND OBJECTIVES: We have undertaken the first clinical trial involving the administration of alpha-GalactosylCeramine (alpha-GalCer)-pulsed dendritic cells (DCs) to human subjects, to determine safety, optimal dose, optimal administration route and immunological effects. MATERIALS AND METHODS: Subjects (n = 4) with metastatic malignancy received two infusions of alpha-GalCer-pulsed DCs intravenously, and two infusions intradermally. The percentages of Valpha24 Vbeta11 NKT cells in peripheral blood (PB) were determined by three-colour flow cytometry and the PB NKT cell numbers were calculated using the total number of PB lymphocytes/ml determined by automated full-blood counts. RESULTS: No serious treatment related adverse events were observed during the study period. Administration of alpha-GalCer-pulsed DCs in vivo can significantly (P < 0.03) increase PB Valpha24+ Vbeta11+ NKT cell numbers above pretreatment baseline levels after the transient fall in the NKT numbers within 48 h. CONCLUSIONS: Administration of alpha-GalCer-pulsed DCs is well tolerated, modulates PB Valpha24+ Vbeta11+ NKT cells and may have a role in the therapy of malignancies sensitive to activities of Valpha24+ Vbeta11+ NKT cells, or for autoimmune diseases.     

Decrease of intrathyroidal CD161+Valpha24+Vbeta11+ NKT cells in Graves' disease

To clarify changes in the intrathyroidal natural killer T (NKT) cell subset, which prevents autoimmunity in patients with Graves' disease (GD), we examined intrathyroidal and peripheral lymphocytes in 11 patients with GD and peripheral lymphocytes in nine healthy volunteers using three-color flow cytometry. The proportion of CD161 (+) T cell receptor Valpha24 (+) Vbeta11 (+) cells, which represent the NKT cell subset, was lower in the thyroid of patients with GD than in the peripheral blood of the same patients and in the peripheral blood of healthy subjects. These results indicate that the proportion of intrathyroidal NKT cells is decreased in patients with GD and that this decrease may contribute to incomplete regulation of autoreactive T cells in GD.     

alpha -galactosylceramide-activated Valpha 14 natural killer T cells mediate protection against murine malaria

Natural killer T (NKT) cells are a unique population of lymphocytes that coexpress a semiinvariant T cell and natural killer cell receptors, which are particularly abundant in the liver. To investigate the possible effect of these cells on the development of the liver stages of malaria parasites, a glycolipid, alpha-galactosylceramide (alpha-GalCer), known to selectively activate Valpha14 NKT cells in the context of CD1d molecules, was administered to sporozoite-inoculated mice. The administration of alpha-GalCer resulted in rapid, strong antimalaria activity, inhibiting the development of the intrahepatocytic stages of the rodent malaria parasites Plasmodium yoelii and Plasmodium berghei. The antimalaria activity mediated by alpha-GalCer is stage-specific, since the course of blood-stage-induced infection was not inhibited by administration of this glycolipid. Furthermore, it was determined that IFN-gamma is essential for the antimalaria activity mediated by the glycolipid. Taken together, our results provide the clear evidence that NKT cells can mediate protection against an intracellular microbial infection.     

The Pten/PI3K pathway governs the homeostasis of Valpha14iNKT cells

The tumor suppressor PTEN is mutated in many human cancers. We previously used the Cre-loxP system to generate mice (LckCrePten mice) with a Pten mutation in T-lineage cells. Here we describe the phenotype of Pten-deficient Valpha14iNKT cells. A failure in the development of Valpha14iNKT cells occurs in the LckCrePten thymus between stage 2 (CD44(high)NK1.1(-)) and stage 3 (CD44(high)NK1.1(+)), resulting in decreased numbers of peripheral Valpha14iNKT cells. In vitro, Pten-deficient Valpha14iNKT cells show reduced proliferation and cytokine secretion in response to alphaGalCer stimulation but enhanced inhibitory Ly49 receptor expression. Following interaction with dendritic cells (DCs) loaded with alphaGalCer, Pten-deficient Valpha14iNKT cells demonstrate activation of PI3K. Indeed, the effects of the Pten mutation require intact function of the PI3K subunits p110gamma and p110delta. In vivo, LckCrePten mice display reduced serum IFNgamma after alphaGalCer administration. Importantly, Valpha14iNKT cell-mediated protection against the metastasis of melanoma cells to the lung was impaired in the absence of Pten. Thus, the Pten/PI3K pathway is indispensable for the homeostasis and antitumor surveillance function of Valpha14iNKT cells.     

Therapeutic activation of Valpha24+Vbeta11+ NKT cells in human subjects results in highly coordinated secondary activation of acquired and innate immunity

Human Valpha24+Vbeta11+ natural killer T (NKT) cells are a distinct CD1d-restricted lymphoid subset specifically and potently activated by alpha-galactosylceramide (alpha-GalCer) (KRN7000) presented by CD1d on antigen-presenting cells. Preclinical models show that activation of Valpha24+Vbeta11+ NKT cells induces effective antitumor immune responses and potentially important secondary immune effects, including activation of conventional T cells and NK cells. We describe the first clinical trial of cancer immune therapy with alpha-GalCer-pulsed CD1d-expressing dendritic cells. The results show that this therapy has substantial, rapid, and highly reproducible specific effects on Valpha24+Vbeta11+ NKT cells and provide the first human in vivo evidence that Valpha24+Vbeta11+ NKT cell stimulation leads to activation of both innate and acquired immunity, resulting in modulation of NK, T-, and B-cell numbers and increased serum interferon-gamma. We present the first clinical evidence that Valpha24+Vbeta11+ NKT cell memory produces faster, more vigorous secondary immune responses by innate and acquired immunity upon restimulation.     

Crosstalk between decidual NK and CD14+ myelomonocytic cells results in induction of Tregs and immunosuppression

Regulatory T cells (Tregs) are thought to play a major role in pregnancy by inhibiting the maternal immune system and preventing fetal rejection. In decidual tissues, NK cells (dNK) reside in close contact with particular myelomonocytic CD14⁺ (dCD14⁺) cells. Here we show that the interaction between dNK and dCD14⁺ cells results in induction of Tregs. The interaction is mediated by soluble factors as shown by transwell experiments, and the prominent role of IFN-γ is revealed by the effect of a neutralizing monoclonal antibody. Following interaction with dNK cells, dCD14⁺ cells express indoleamine 2,3-dioxygenase (IDO), which, in turn, induces Tregs. Notably, unlike peripheral blood NK (pNK) cells, dNK cells are resistant to inhibition by the IDO metabolite L-kynurenine. “Conditioned” dCD14⁺ cells also may induce Tregs through transforming growth factor-β (TGF-β) production or CTLA-4–mediated interactions, as indicated by the effect of specific neutralizing Abs. Remarkably, only the interaction between dNK and dCD14⁺ cells results in Treg induction, whereas other coculture combinations involving either NK or CD14⁺ cells isolated from peripheral blood are ineffective. Our study provides interesting clues to understanding how the crosstalk between decidual NK and CD14⁺ cells may initiate a process that leads to Treg induction and immunosuppression. Along this line, it is conceivable that an impaired function of these cells may result in pregnancy failure.     

The roles of intrahepatic Valpha14(+) NK1.1(+) T cells for liver injury induced by Salmonella infection in mice.

To investigate the roles of intrahepatic T cells in liver injury after Salmonella infection, we examined serum alanine transaminase (ALT), histopathology, and bacterial numbers in liver after infection with Salmonella choleraesuis strain 31N-1 in mice genetically lacking TCRalpha beta+, CD4(+), CD8(+), or NK1.1(+)T cells with C57BL/6 background. In control (+/+) mice, serum ALT reached a peak level by day 7 after an intraperitoneal inoculation of 2 x 10(6) CFU Salmonella choleraesuis 31N-1. In TCR-beta-/- mice, liver injury, as assessed by serum ALT level and histological examination, was significantly suppressed on day 7 after Salmonella infection but the numbers of bacteria in liver did not differ from those in normal mice, suggesting that alpha beta T cells are responsible for liver injury induced by Salmonella infection. To further determine which subsets in alpha beta T cells are important for the liver injury, we compared serum ALT level in mice genetically lacking CD4, CD8, beta2-microglobulin (beta2m, IAbeta, or Jalpha281 after Salmonella infection. In CD4(-/-) mice, serum ALT was significantly lower in comparison with control mice, but there was no difference in serum ALT levels in CD8(-/-) and IAbeta-/- mice from that in control mice. Notably, serum ALT levels and pathological lesions in liver were significantly decreased in beta2m-/- or Jalpha281(-/-) mice, which lacked in NK1.1(+) T cells bearing TCR Valpha14-Jalpha281 specific for beta2m-associated CD1d, following Salmonella infection. Taken together, it is suggested that alpha beta T cells bearing NK1.1 and CD4 may be main effector cells for liver injury after Salmonella infection.     

Contractile activity of human decidual stromal cells

We previously demonstrated that human decidual stromal cells (DSC), the main cellular component of the decidua, are similar in antigen phenotype and structure to myofibroblasts, cells with contractile activity. In this work we isolated and maintained DSC in fibroblast medium, in which these cells show a stable phenotype similar to that of DSC in vivo. Flow cytometric observations showed that most DSC expressed alpha-smooth muscle (alpha-SM) actin, an intermediate filament that is considered a marker of myofibroblasts and is responsible for the contractile activity of these cells. alpha-SM actin mRNA was detected by RT-PCR in these cells. The contractile activity of DSC was determined by the gel contraction assay; we found that TGF beta 1 and platelet-derived-growth factor, cytokines that are known to be inducers of myofibroblast contractility, also induced contractility of DSC. IL-2, a Th1 cytokine-related with spontaneous abortion, also activated DSC contractility. Our results confirmed that DSC are phenotypically and functionally related with myofibroblast.     

Role of NKT cells and alpha-galactosyl ceramide

Alfa-Galactosyl Ceramide was isolated from Ocean sponge which has antitumor effect against several tumors in in vivo animal model with no cytotoxicity. KRN7000(KRN) is the most potent alpha-Galactosyl Ceramide modified from the one isolated from Ocean sponge. KRN is also active against metastatic tumors through the activation ofanimal immune system. Research efforts in learning the mechanism of action, we found the important role of dendritic cells(DC) and NKT cells. NKT cells was first characterized in 1988 which is overlap some part with NK cells and T-Cells and majority is different from NK and T. KRN is active through the activation of DC and NKT in giving antigen specific immune stimulation in animal. This antigen specific stimulation is memorized by immune system and can reject second tumor challenge. KRN is not active in nude mice and NKT deficient animal. NKT cells level in blood is lower in patients with autoimmune disease, cancer, HIV positive or aplastic anemia. NKT rapidly releases IL-4 and IFN-gamma at high level when activated. NKT is CD1d and TCR restricted. NKT plays important role in autoimmune disease such as Type 1 Diabetes, Scleroderma and Systemic Lupus Erythematosus, infections such as Mycobacteria, Listeria and Malaria, GVHD control and tumor rejection. NKT acts as double edge sword, aggressive and suppressive ways. KRN can prevent the onset of Type 1 Diabetes, inhibit replication of hepatitis virus B in liver and suppress malaria replication in activating NKT cells. KRN can activate NKT through DC and activated NKT activates NK, T and macrophage. KRN also expands NKT cells and expanded NKT has full function. Although the exact role of DC and NKT is not clear, KRN clinical study results in conjunction with DC and NKT cell activation are expected.     

Induction of differentiation of pre-NKT cells to mature Vα14 NKT cells by granulocyte/macrophage colony-stimulating factor

Valpha14 NKT cells express an invariant antigen receptor encoded by Valpha14 and Jalpha281 gene segments as well as natural killer (NK) markers, including NK1.1. Here, we describe a precursor population of NKT cells (pre-NKT) that expresses NK1.1, T cell antigen receptor beta, pTalpha, and RAG1/2 but not Valpha14 and surface CD3epsilon. Such pre-NKT cells were differentiated successfully in vitro into mature CD3epsilon+ Valpha14(+) NKT cells by IL-15 and granulocyte/macrophage colony-stimulating factor (GM-CSF) in conjunction with stroma cells. Interestingly, only GM-CSF without stroma cells induced the Valpha14-Jalpha281 gene rearrangement in the pre-NKT cells. This also was confirmed by the findings that the number of mature Valpha14 NKT cells and the frequency of Valpha14-Jalpha281 rearrangements were decreased significantly in the mice lacking a GM-CSF receptor component, common beta-chain. These results suggest a crucial role of GM-CSF in the development of Valpha14 NKT cells in vivo.     

Estrogen and progesterone receptors in the decidual tissue of women administered prostaglandins and experiencing spontaneous abortion

A study was made of the content of estrogen and progesterone receptors (ER and PR, respectively) in the cytosol and nuclei of the decidual tissue of women in induced and spontaneous abortion at 7-12 week pregnancy. Abortion was done surgically (control group) and in preliminary cervical dilatation with the help of intravaginal suppositories with a synthetic prostaglandin analog of E1-16.16-dimethyltrans-delta 2-PGE1 (ONO-802) group or laminaria. The level of ER in the cytosol and nuclei of the decidual tissue of the control group was twice as low as that of PR, and the ER/PR ratio in cell fractions as well as in general cell receptors was 0.58 +/- 0.09, 0.42 +/- 0.07 and 0.56 +/- 0.07, respectively. The suppositories with ONO-802 caused a statistically significant decrease in the PR content in each of the cell fractions and cytoplasmic ER resulting in an increase in the ER/PR ratio to 0.87 +/- 0.06 (P less than 0.05) in the cytosol, 0.09 +/- 0.24 (P less than 0.05) in the nuclei, and 0.86 +/- 0.8 (P less than 0.05) in the total cell. A decrease in the PR level in both cell fractions and the ER level in the fraction of the nuclei with an acute increase in the content of cytoplasmic ER was marked in spontaneous abortion. The ER/PR ratio also rose to ER and was 1.98 +/- +/- 0.2 (P less than 0.001) in the cytosol and 1.02 +/- 0.16 (P less than 0.05) in the total cell. It was concluded that an absolute and/or relative decrease in the PR level plays an important role in mechanism of abortion.     

Natural killer-like nonspecific tumor cell lysis mediated by specific ligand-activated Vα14 NKT cells

We have recently identified α-galactosylceramide (α-GalCer) as a specific ligand for an invariant Vα14/Vβ8.2 T cell receptor exclusively expressed on the majority of Vα14 NKT cells, a novel subset of lymphocytes. Here, we report that α-GalCer selectively activates Vα14 NKT cells resulting in prevention of tumor metastasis. The effector mechanisms of the ligand-activated Vα14 NKT cells seem to be mediated by natural killer (NK)-like nonspecific cytotoxicity. Indeed, the cytotoxic index obtained by α-GalCer-activated Vα14 NKT cells was reduced by the addition of cold target tumor cells or by treatment with concanamycin A, which inhibits activation and secretion of perforin, but not by mAbs against molecules involved in the NKT cell recognition and conventional cytotoxicity, such as CD1d, Vβ8, NK1.1, Ly49C, Fas, or Fas ligand. These results suggest that the ligand-activated Vα14 NKT cells kill tumor cells directly through a CD1d/Vα14 T cell receptor-independent, NK-like mechanism.     

Evidence for the secretion of decidual luteotropin: a prolactin-like hormone produced by rat decidual cells

Rat decidual tissue contains a PRL-like hormone named decidual luteotropin. We have recently revealed some of its physiological and biochemical characteristics. However, because rat decidual tissue contains specific binding sites for PRL, it was important to demonstrate that the hormone found in the tissue is not locally stored and structurally transformed PRL but a hormone actively synthesized by the rat decidual tissue. Decidual explants or decidual cells obtained from day 9 pseudopregnant rats were incubated for different times under either static conditions or continuously perifused with medium at a rate of 1 ml/h. Levels of decidual luteotropin were measured by a specific radioreceptor assay using luteal membranes as source of receptors and [125I]iodo-ovine(o)PRL as a tracer. In the static incubation, no proof of hormone production was obtained; levels of decidual luteotropin in medium and tissue or cells at the end of the incubation were similar to levels found in either cells or tissue before incubation. In sharp contrast, decidual cells perifused with media secreted large amounts of hormone. This may suggest that an inhibitor of decidual luteotropin production was being removed from the culture by the perifusion. For the first 4 h of perifusion, no hormone was produced. However by the fifth hour, cells began to actively release decidual luteotropin. Secretion of the hormone increased with time and reached maximal values between 7-15 h of perifusion. During the 15 h of perifusion, decidual cells released approximately 1000 times more decidual luteotropin than the amount they originally contained. A dose-response increase in hormone secretion was obtained with increased concentrations of decidual cells. The net amount of decidual luteotropin released into the medium over an 18-h period was approximately 6.5 micrograms/30 X 10(6) cells, 3.5 micrograms/10 X 10(6) cells, and 0.5 micrograms/2 X 10(6) cells. A similar profile of decidual luteotropin release was obtained when decidual explants were perifused. However, in contrast to decidual cells which secreted no hormone for the first 4 h of culture, decidual explants immediately began to release decidual luteotropin in the medium. The secretion of decidual luteotropin in vitro was inhibited 75% by the protein synthesis inhibitor, cycloheximide. In summary, results of this investigation demonstrate for the first time that rat decidual cells secrete in vitro a PRL-like hormone, decidual luteotropin.