Murine natural killer T(NKT) cells [correction of natural killer cells] contribute to the granulomatous reaction caused by mycobacterial cell walls

Mice injected with deproteinized cell walls prepared from the strain H37rv of Mycobacterium tuberculosis develop a granuloma-like lesion in which NKT cells are predominant. NKT cells play a primary role in the granulomatous response, because the latter does not occur in Jalpha281(-/-) mice, which miss NKT cells. The glycolipidic fraction of the cell walls is responsible for the recruitment of NKT cells; the recruiting activity is associated with fractions containing phosphatidylinositolmannosides. These results define a powerful experimental set up for studying the in vivo induction of NKT cell responses to microbial components.     

Suppression of natural killer cell-mediated bone marrow cell rejection by CD4+CD25+ regulatory T cells

Naturally occurring CD4(+)CD25(+) T regulatory (Treg) cells have been shown to inhibit adaptive responses by T cells. Natural killer (NK) cells represent an important component of innate immunity in both cancer and infectious disease states. We investigated whether CD4(+)CD25(+) Treg cells could affect NK cell function in vivo by using allogeneic (full H2-disparate) bone marrow (BM) transplantation and the model of hybrid resistance, in which parental marrow grafts are rejected solely by the NK cells of irradiated (BALB/c x C57BL/6) F(1) recipients. We demonstrate that the prior removal of host Treg cells, but not CD8(+) T cells, significantly enhanced NK cell-mediated BM rejection in both models. The inhibitory role of Treg cells on NK cells was confirmed in vivo with adoptive transfer studies in which transferred CD4(+)CD25(+) cells could abrogate NK cell-mediated hybrid resistance. Anti-TGF-beta mAb treatment also increased NK cell-mediated BM graft rejection, suggesting that the NK cell suppression is exerted through TGF-beta. Thus, CD4(+)CD25(+) Treg cells can potently inhibit NK cell function in vivo, and their depletion may have therapeutic ramifications for NK cell function in BM transplantation and cancer therapy.     

The role of natural killer T (NKT) cells in the pathogenesis of type 1 diabetes

In type 1 diabetes, a failure in the regulation of either innate or acquired immunity may be the cause of autoimmune response. A cell population that may have a regulatory role of the immune response are the Natural Killer T (NKT) cells, which are a population expressing T lymphocyte antigen receptor (TCR), and are a common marker for NK cells. A distinctive characteristic in NKT cells is their capacity to produce large amounts of immune-modulating cytokines. A decrease in the number and/or functional incapability of NKT cells is associated with progression of type 1 diabetes and with other self-immune diseases. However, the relevance of such findings is not completely understood. Limitations of the current studies include the existing methods to measure NKT activation and the lack of assessment of the expression of genes affected by NKT action. Nevertheless, the study of NKT cells may be a new clinical approach to detect individuals at risk for having type 1 diabetes. Additional studies are needed to evaluate the clinical value of this new predictive tool.     

Requirement for natural killer T (NKT) cells in the induction of allograft tolerance

In this study, we investigated the role of Valpha14 natural killer T (NKT) cells in transplant immunity. The ability to reject allografts was not significantly different between wild-type (WT) and Valpha14 NKT cell-deficient mice. However, in models in which tolerance was induced against cardiac allografts by blockade of lymphocyte function-associated antigen-1/intercellular adhesion molecule-1 or CD28/B7 interactions, long-term acceptance of the grafts was observed only in WT but not Valpha14 NKT cell-deficient mice. Adoptive transfer with Valpha14 NKT cells restored long-term acceptance of allografts in Valpha14 NKT cell-deficient mice. The critical role of Valpha14 NKT cells to mediate immunosuppression was also observed in vitro in mixed lymphocyte cultures in which lymphocyte function-associated antigen-1/intercellular adhesion molecule-1 or CD28/B7 interactions were blocked. Experiments using IL-4- or IFN-gamma-deficient mice suggested a critical contribution of IFN-gamma to the Valpha14 NKT cell-mediated allograft acceptance in vivo. These results indicate a critical contribution of Valpha14 NKT cells to the induction of allograft tolerance and provide a useful model to investigate the regulatory role of Valpha14 NKT cells in various immune responses.     

Activation of invariant natural killer T cells by synthetic glycolipid ligands suppresses autoantibody-induced arthritis

OBJECTIVE: Stimulation of invariant natural killer T (iNKT) cells with SGL-S23, a novel synthetic glycolipid analog of alpha-galactosylceramide with an elongated sphingosine chain, has been shown to strongly suppress K/BxN serum transfer arthritis. This study was designed to evaluate the protective effects of SGL-S23 in an effector phase of arthritis. METHODS: To induce arthritis, C57BL/6 mice were injected with 150 mul of serum from K/BxN mice (KRN TCR-transgenic mice crossed with nonobese diabetic mice). Subsequently, synthetic glycolipid ligands were administered intraperitoneally twice, either 3 times starting on day 0 (the day of K/BxN serum injection) or twice starting on day 3. Neutralizing antibody against interferon-gamma (IFNgamma) interleukin-4 (IL-4), IL-10, or transforming growth factor beta was administered 4 hours before injection of SGL-S23. Recombinant IFNgamma was administered subcutaneously every day. The severity of arthritis was monitored using a macroscopic scoring system. Cytokine production and plasma histamine levels were measured by enzyme-linked immunosorbent assay. RESULTS: SGL-S23 strongly suppressed K/BxN serum transfer arthritis by inhibiting inflammatory cell infiltration and subsequent destruction of cartilage and bone. The inhibitory effect mediated by SGL-S23 was abolished by neutralization of IFNgamma. Systemic administration of IFNgamma prevented the development of inflammatory arthritis. Histamine release was suppressed by administration of SGL-S23 or IFNgamma. Degranulated mast cells in the synovium were significantly reduced in SGL-S23-treated mice, suggesting that suppression of mast cell activation contributed to the inhibition of arthritis. CONCLUSION: These findings suggest that activation of iNKT cells with glycolipid ligands holds promise with regard to the treatment of autoimmune diseases such as rheumatoid arthritis. SGL-S23 has clinical benefit over alpha-galactosylceramide since it induces a weaker cytokine production response in iNKT cells, therefore reducing potential side effects caused by excessive cytokine release.     

Expansion of natural killer cell receptor (CD94/NKG2A)-expressing cytolytic CD8 T cells and CD4+CD25+ regulatory T cells from the same cord blood unit

OBJECTIVE: Cord blood contains a significant number of precursor cells that differentiate to cytotoxic effector cells and immunoregulatory cells. We tried to expand inhibitory natural killer cell receptor CD94-expressing CD8 T cells with cytolytic activity and CD4(+)CD25(+) regulatory T cells from the same cord cell unit. METHODS: Cytotoxic CD94-expressing CD8 T cells were expanded from CD4-depleted cord blood using an immobilized anti-CD3 monoclonal antibody and a cytokine and also CD4(+)CD25(+) regulatory T cells were expanded from a CD4-enriched fraction derived from the same cord blood unit using anti-CD3/CD28 monoclonal antibody-coated Dynabeads and cytokines. RESULTS: We were able to obtain a more than 1000-fold expansion of CD94-expressing CD8 T cells and a more than 50-fold expansion of CD4(+)CD25(+) cells from the same cord blood unit. These expanded CD4(+)CD25(+) cells expressed FoxP3 mRNA at a level about 100-fold higher than that in isolated CD25(-) cells and could suppress allogeneic mixed lymphocyte culture by >80% (effector cells: CD4(+)CD25(+) cells = 2:1). Cytolytic activities of purified CD94-expressing cells detected by a 4-hour (51)Cr release assay against K562 were >60%. Coculture of CD94-expressing cells with expanded CD4(+)CD25(+) cells did not have any effect on cytolytic activities of purified CD94-expressing cells against K562 cells. CONCLUSION: These expanded cytolytic CD94-expressing CD8 cells might be able to induce a graft-vs-leukemia effect without enhancing graft-vs-host disease, and CD4(+)CD25(+) cells might be able to suppress allogeneic responses, including graft-vs-host disease and graft rejection after cord blood transplantation.     

Age-associated accumulation of CMV-specific CD8+ T cells expressing the inhibitory killer cell lectin-like receptor G1 (KLRG1)

Large clonal expansions of peripheral CD8+ T cells carrying receptors for single epitopes of CMV and EBV are common in the elderly and may be associated with an immune risk phenotype predicting mortality. Here we show that the frequency of CD8+ T cells expressing the inhibitory killer cell lectin-like receptor G1 (KLRG1), a marker of cells unable to undergo further clonal expansion, was markedly elevated in CD8+ T cells from old donors. Moreover, tetramer staining revealed that the elevated frequency of CMV-specific CD8+ T cells in the elderly was due to an accumulation of cells bearing this dominant negative receptor. The fraction of CMV-specific T cells able to secrete interferon-gamma after specific antigenic stimulation was significantly lower in the elderly than in the young, although the total number of functional cells was comparable. Therefore, the majority of the clonally expanded virus-specific CD8+ cells in the elderly was dysfunctional. Thus, T cell responses are altered in the aged by an accumulation of replicatively senescent dysfunctional T cells carrying receptors for persistent herpes viruses. The presence of clonal expansions of such virus-specific cells may shrink the available repertoire for other antigens and contribute to the increased incidence of infectious disease in the elderly.     

CD3+CD56+自然杀伤T细胞在系统性红斑狼疮中的检测及意义

目的 检测系统性红斑狼疮(SLE)患者外周血中自然杀伤T细胞(NKT)的表达,探讨其与SLE疾病活动的关系.方法 入选确诊的SLE患者30例及健康对照30名,采用流式细胞仪检测外周血CD3+D56+NKT细胞的表达,分析NKT细胞的表达与SLE疾病活动评分和免疫学指标的相关性,采用两样本均数的t检验和Pearson相关分析进行统计学处理.结果与健康对照(4.16±0.22)%相比,SLE患者外周血中CD3+CD56+比例(2.53±0.33)%显著降低,NKT值与血清IgG水平、抗双链DNA抗体定量及SLE疾病活动指数(SLEDAI)评分呈负相关(r值=-0.595,-0.408,-0.637;P均<0.05).结论 SLE患者外周血中NKT细胞表达下降,其下降与SLE的疾病活动存在相关性.     

In vitro differentiation of natural killer T cells from human cord blood CD34+ cells

Natural killer T (NKT) cells are involved in innate immune defence and also in the regulation of adaptive immune responses. However, the development of NKT cells in vitro has not been fully characterized and culture conditions have not been fully optimized. In the present study, we found that an NKT cell fraction developed during the in vitro culture of cord blood (CB) CD34+ cells, and this was subsequently characterized both phenotypically and morphologically. CD34+ cells purified from 10 human CB were cultured in the presence of several cytokines and analysed by flow cytometry, light microscopy and electron microscopy. The NKT cell fraction, defined phenotypically (CD3+CD16+CD56+CD94+) as expressing the invariant T-cell receptor Valpha24 and Vbeta11, appeared in the CD56hi fractions. Intracytoplasmic staining demonstrated that interferon-gamma and interleukin 4 (IL-4) were detected in the CD56hi fractions. IL-15 was essential and, in combination with either flt3-ligand (FL) or stem cell factor (SCF), was sufficient to induce the development of NKT cells. The phenotype of the NKT cell fraction was CD45RO+CD45RA- and CD4+CD8alpha+. Morphologically, they were very large, with either round or oval nuclei, moderately condensed chromatins, voluminous weakly basophilic cytoplasm and various cytoplasmic granules such as dense core granules, multivesicular bodies, and intermediate form granules. When CD34+ cells purified from bone marrow (BM) were compared with those from CB, the latter were consistently more efficient at generating CD56hi NKT cell fractions. In conclusion, IL-15 in combination with FL and/or SCF can induce the differentiation of NKT cells from human CB CD34+ cells.     

Natural killer T (NKT) cells and their role in antitumor immunity

Natural killer T (NKT) cells have become a major focus for those who study the innate immune response to tumors and infectious diseases, as well as autoimmunity. These novel T lymphocytes produce both Th1 and Th2 cytokines, recognize phospholipid and glycolipid antigens presented by CD1 molecules in a similar manner as peptides are recognized by cytotoxic T lymphocytes (CTL), and kill tumor cell targets by a perforin-dependent mechanism like NK cells and CTL. These ascribed functions thus demonstrate that NKT cells are a unique cytotoxic effector cell subpopulation with a kaleidoscope of activities. Because they can mediate antitumor effects in vivo with or without the collaboration of NK cells, the study of NKT cells in antitumor immunity may lead to novel treatments based on the ability to manipulate the generation and/or activity of these multifunctional lymphocytes.     

CD4 engagement by CD1d potentiates activation of CD4+ invariant NKT cells

The CD4 coreceptor is crucial in the activation of major histocompatibility complex (MHC) class II restricted CD4 (+) T lymphocytes by binding the same MHC class as the T-cell receptor (TCR) and by potentiating TCR-dependent signaling. CD4 is also expressed by invariant natural killer T cells (iNKT), which recognize natural and synthetic lipid antigens, such as alpha-galactosyl ceramide (alpha-GalCer), in association with the MHC class I-like CD1d molecule. Human iNKT cells can be divided into 2 major subsets depending on CD4 expression: CD4 (+) iNKT preferentially produce T-helper (Th)0/Th2 cytokines, whereas CD4(-) iNKT cells produce Th1 cytokines after antigenic activation. Cytokines produced by iNKT may have immunomodulatory roles in various physiopathologic contexts, but their mode of regulation by iNKT cells remains ill-defined. Using blocking reagents neutralizing CD4 binding, experimental systems where MHC class II molecules are absent and recombinant alpha-GalCer/CD1d complexes, we show that CD4 potentiates human iNKT cell activation by engaging CD1d molecules. These results indicate that the CD4 coreceptors may contribute to the fine tuning of iNKT cells reactivity.     

Activation of invariant natural killer T cells by synthetic glycolipid ligands suppresses autoantibody‐induced arthritis

Objective

Stimulation of invariant natural killer T (iNKT) cells with SGL‐S23, a novel synthetic glycolipid analog of α‐galactosylceramide with an elongated sphingosine chain, has been shown to strongly suppress K/BxN serum transfer arthritis. This study was designed to evaluate the protective effects of SGL‐S23 in an effector phase of arthritis.

Methods

To induce arthritis, C57BL/6 mice were injected with 150 μl of serum from K/BxN mice (KRN TCR–transgenic mice crossed with nonobese diabetic mice). Subsequently, synthetic glycolipid ligands were administered intraperitoneally twice, either 3 times starting on day 0 (the day of K/BxN serum injection) or twice starting on day 3. Neutralizing antibody against interferon‐γ (IFNγ) interleukin‐4 (IL‐4), IL‐10, or transforming growth factor β was administered 4 hours before injection of SGL‐S23. Recombinant IFNγ was administered subcutaneously every day. The severity of arthritis was monitored using a macroscopic scoring system. Cytokine production and plasma histamine levels were measured by enzyme‐linked immunosorbent assay.

Results

SGL‐S23 strongly suppressed K/BxN serum transfer arthritis by inhibiting inflammatory cell infiltration and subsequent destruction of cartilage and bone. The inhibitory effect mediated by SGL‐S23 was abolished by neutralization of IFNγ. Systemic administration of IFNγ prevented the development of inflammatory arthritis. Histamine release was suppressed by administration of SGL‐S23 or IFNγ. Degranulated mast cells in the synovium were significantly reduced in SGL‐S23–treated mice, suggesting that suppression of mast cell activation contributed to the inhibition of arthritis.

Conclusion

These findings suggest that activation of iNKT cells with glycolipid ligands holds promise with regard to the treatment of autoimmune diseases such as rheumatoid arthritis. SGL‐S23 has clinical benefit over α‐galactosylceramide since it induces a weaker cytokine production response in iNKT cells, therefore reducing potential side effects caused by excessive cytokine release.

     

Depletion of CD25^＋CD4^＋T cells （Tregs） enhances the H BV-specific CD8^＋ T cell response primed by DNA immunization

AIM: Persistent hepatitis B virus (HBV) infection is characterized by a weak CD8+ T cell response to HBV. Immunotherapeutic strategies that overcome tolerance and boost these suboptimal responses may facilitate viral clearance in chronically infected individuals. Therefore, we examined whether CD25+CD4+ regulatory T (Treg) cells might be involved in a inhibition of CD8+T cell priming or in the modulation of the magnitude of the 'peak' antiviral CD8+ T cell response primed by DNA immunization. METHODS: B10.D2 mice were immunized once with plasmid pCMV-S. Mice received 500 μg of anti-CD25 mAb injected intraperitoneally 3 d before DNA immunization to deplete CD25+ cells. Induction of HBV-specific CD8+ T cells in peripheral blood mononuclear cells (PBMCs) was measured by S28-39 peptide loaded DimerX staining and their function was analyzed by intracellular IFN-γ staining. RESULTS: DNA immunization induced HBV-specific CD8+ T cells. At the peak T cell response (d 10), 7.1±2.0% of CD8+ T cells were HBV-specific after DNA immunization, whereas 12.7±3.2% of CD8+ T cells were HBV-specific in Treg-depleted mice, suggesting that DNA immunization induced more antigen-specific CD8+ T cells in the absence of CD25+ Treg cells (n = 6, P<0.05). Similarly, fewer HBV specific memory T cells were detected in the presence of these cells (1.3±0.4%) in comparison to Treg-depleted mice (2.6±0.9%) on d 30 after DNA immunization (n - 6, P<0.01). Both IFN-γ production and the avidity of the HBV-specific CD8+ T cell response to antigen were higher in HBV-specific CD8+ T cells induced in the absence of Treg cells. CONCLUSION: CD25+ Treg cells suppress priming and/or expansion of antigen-specific CD8+ T cells during DNA immunization and the peak CD8+ T cell response is enhanced by depleting this cell population. Furthermore, Treg cells appear to be involved in the contraction phase of the CD8+ T cell response and may affect the quality of memory T cell pools. The elimination of Treg cells or their inhibition may be important in immunotherapeutic strategies to control HBV infection by inducing virus-specific cytotoxic T lymphocyte responses in chronically infected subjects.     

The response of natural killer T cells to glycolipid antigens is characterized by surface receptor down-modulation and expansion

CD1d-restricted natural killer T (NKT) cells are a subset of regulatory T cells that react with glycolipid antigens. Although preclinical studies have effectively targeted NKT cells for immunotherapy, little is known regarding the early in vivo response of these cells to antigenic stimulation. We have analyzed the early response of NKT cells to glycolipid antigens and bacterial infection by using specific reagents for tracking these cells. Our results demonstrate dramatic in vivo expansion and surface phenotype alterations after NKT cell activation with alpha-galactosylceramide. In addition, we show significant NK1.1 down-modulation on NKT cells in the setting of oral Salmonella infection. Our results indicate that in vivo activation of NKT cells leads to a dynamic response characterized by surface receptor down-modulation and expansion. These findings alter current understanding of NKT cell biology and should aid in the rational design of NKT cell-based immunotherapies.     

Adoptive immunotherapy mediated by ex vivo expanded natural killer T cells against CD1d-expressing lymphoid neoplasms

Background

CD1d is a monomorphic antigen presentation molecule expressed in several hematologic malignancies. Alpha-galactosylceramide (α-GalCer) is a glycolipid that can be presented to cytotoxic CD1d-restricted T cells. These reagents represent a potentially powerful tool for cell mediated immunotherapy.

Design and Methods

We set up an experimental model to evaluate the use of adoptively transferred cytotoxic CD1d-restricted T cells and α-GalCer in the treatment of mice engrafted with CD1d⁺ lymphoid neoplastic cells. To this end the C1R cell line was transfected with CD1c or CD1d molecules. In addition, upon retroviral infection firefly luciferase was expressed on C1R transfected cell lines allowing the evaluation of tumor growth in xenografted immunodeficient NOD/SCID mice.

Results

The C1R-CD1d cell line was highly susceptible to specific CD1d-restricted T cell cytotoxicity in the presence α-GalCer in vitro. After adoptive transfer of CD1d-restricted T cells and α-GalCer to mice engrafted with both C1R-CD1c and C1R-CD1d, a reduction in tumor growth was observed only in CD1d⁺ masses. In addition, CD1d-restricted T-cell treatment plus α-GalCer eradicated small C1R-CD1d⁺ nodules. Immunohistochemical analysis revealed that infiltrating NKT cells were mainly observed in CD1d nodules.

Conclusions

Our results indicate that ex vivo expanded cytotoxic CD1d-restricted T cells and α-GalCer may represent a new immunotherapeutic tool for treatment of CD1d⁺ hematologic malignancies.     

Lysis of lymphoma cells by autologous and allogeneic natural killer cells

Studies were undertaken to determine whether natural killer (NK) cells would lyse autologous and allogeneic lymphoma cells. When large granular lymphocytes, which are known to mediate NK activity, were enriched from peripheral blood and used as effector cells, they lysed autologous lymphoma cells of all of eight patients tested, and those of healthy donors lysed lymphoma cells of all of ten patients tested. The addition of interferon to the culture medium enhanced their cytotoxicity in three of the eight patients in the autologous effector- tumor system and in four of the ten patients in the above allogeneic system. On the basis of the unlabeled target competition test and the decrease in cytotoxicity with anti-NK antibody treatment, NK cells appeared to be the main cytotoxic effector cells for autologous and allogeneic lymphoma cells.