Murine NKT cells produce Th17 cytokine interleukin-22

Natural killer T (NKT) cells are known to produce Th17 cytokine IL-17 in addition to Th1/2 cytokines. In this study, the ability of NKT cells to produce IL-22, another Th17 cytokine, was examined in mice. When murine spleen cells were stimulated with α-galactosyl ceramide, a ligand for NKT cells, not only Th1/2 cytokines (IFN-γ, IL-4) but Th17 cytokines (IL-17, IL-22) were produced. NKT cells isolated from splenocytes released IL-17 and IL-22 following CD3, CD3/IL-2 or CD3/CD28 stimulation, in which CD3/CD28 costimulation was most effective. Production of IL-17 and IL-22 in CD4+ and CD8+ T cells from splenocytes was little, if any, even after CD3/CD28 costimulation. Treatment with IL-6/TGF-β decreased CD3/CD28-stimulated production of IL-22, but not that of IL-17, in NKT cells. These findings show for the first time that NKT cells are a cell source of IL-22, and that expression of two Th17 cytokines might be regulated in NKT cells by different mechanisms.     

Induction of polyclonal CD8+ T cell activation and effector function by Pertussis toxin

Pertussis toxin (PTX) has pronounced adjuvant activity and strongly enhances innate and adaptive immune responses, including increased antibody production and Th1/Th2 cytokine production. Adjuvant effects of PTX on Th1 and Th2 cells are primarily mediated via CD80/86 costimulation via enhanced expression of these molecules by APCs. However, it has remained unresolved whether PTX modulates the expression of costimulatory and inhibitory molecules on CD4+ and CD8+ T cells. To address this question, we determined the expression kinetics of CD28, CTLA-4, and CD40L on spleen CD4+ and CD8+ T cells after incubation with PTX. The results show that PTX upregulated the expression of CD28 by CD8+ T cells, but not by CD4+ T cells. In contrast, the expression of CTLA-4 and CD40L was not substantially altered on CD4+ or CD8+ T cells. CD28 upregulation by CD8+ T cells was paralleled by upregulation of CD69 and the induction of IFN-γ, Granzyme B (GrB), and IL-17. CD8+ T cell activation and cytokine production could be substantially blocked with anti-CD80 and CD86 antibodies, consistent with CD28 mediated signaling. Treatment of highly purified CD8+ T cells with PTX resulted in upregulation of CD28 and CD69, and production of IFN-γ. Incubation with CD28 mAb further enhanced this effect, suggesting that PTX has direct effects on CD8+ T cells which are enhanced by CD80/86-mediated costimulation provided by APCs.     

New CD1d agonists: synthesis and biological activity of 6″-triazole-substituted α-galactosyl ceramides

Huisgen [3+2] dipolar cycloaddition of 6″-azido-6″-deoxy-α-galactosyl ceramide 11 with a range of alkynes (or a benzyne precursor) yielded a series of triazole-containing α-galactosyl ceramide (α-GalCer) analogues in high yield. These α-GalCer analogues and the precursor azide 11 were tested for their ability to activate iNKT cells and stimulate IL-2 cytokine secretion in vitro, and IFN-γ and IL-4 cytokine secretion in vivo. Some of these analogues, specifically 11, 12b, 12f and 13, were more potent IL-2 stimulators than the prototypical CD1d agonist, α-GalCer 1. In terms of any cytokine bias, most of the triazole-containing analogues exhibited a small Th2 cytokine-biasing response relative to that shown by α-GalCer 1. In contrast, the cycloaddition precursor, namely azide 11, provided a small Th1 cytokine-biasing response.     

Role of γδ T cells in α-galactosylceramide-mediated immunity

Attempts to harness mouse type I NKT cells in different therapeutic settings including cancer, infection, and autoimmunity have proven fruitful using the CD1d-binding glycolipid α-galactosylceramide (α-GalCer). In these different models, the effects of α-GalCer mainly relied on the establishment of a type I NKT cell-dependent immune cascade involving dendritic cell, NK cell, B cell, or conventional CD4(+) and CD8(+) T cell activation/regulation as well as immunomodulatory cytokine production. In this study, we showed that γδ T cells, another population of innate-like T lymphocytes, displayed a phenotype of activated cells (cytokine production and cytotoxic properties) and were required to achieve an optimal α-GalCer-induced immune response. Using gene-targeted mice and recombinant cytokines, a critical need for IL-12 and IL-18 has been shown in the α-GalCer-induced IFN-γ production by γδ T cells. Moreover, this cytokine production occurred downstream of type I NKT cell response, suggesting their bystander effect on γδ T cells. In line with this, γδ T cells failed to directly recognize the CD1d/α-GalCer complex. We also provided evidence that γδ T cells increase their cytotoxic properties after α-GalCer injection, resulting in an increase in killing of tumor cell targets. Moreover, using cancer models, we demonstrated that γδ T cells were required for an optimal α-GalCer-mediated anti-tumor activity. Finally, we reported that immunization of wild-type mice with α-GalCer enhanced the adaptive immune response elicited by OVA, and this effect was strongly mediated by γδ T cells. We conclude that γδ T cells amplify the innate and acquired response to α-GalCer, with possibly important outcomes for the therapeutic effects of this compound.     

Synthesis and biological activities of amino acids functionalized α-GalCer analogues

Invariant natural killer T-cells (iNKT-cells) are promising targets for manipulating the immune system, which can rapidly release a large amount of Th1 and Th2 cytokines upon the engagement of their T cell receptor with glycolipid antigens presented by CD1d. In this paper, we wish to report a novel series of α-GalCer analogues which were synthesized by incorporation of l-amino acid methyl esters in the C-6′ position of glycolipid. The evaluation of these synthetic analogues for their capacities to stimulate iNKT-cells into producing Th1 and Th2 cytokines both in vitro and in vivo indicated that they were potent CD1d ligands and could stimulate murine spleen cells into a higher release of the Th1 cytokine IFN-γ in vitro. In vivo, Gly-α-GalCer (1) and Lys-α-GalCer (3) showed more Th1-biased responses than α-GalCer, especially analogue 3 showed the highest selectivity for IFN-γ production (IFN-γ/IL-4 = 5.32) compared with α-GalCer (IFN-γ/IL-4 = 2.5) in vivo. These novel α-GalCer analogues might be used as efficient X-ray crystallographic probes to reveal the relationship between glycolipids and CD1d proteins in α-GalCer/CD1d complexes and pave the way for developing new potent immunostimulating agents.     

B cells as a therapeutic target for IFN-β in relapsing-remitting multiple sclerosis

IFN-β-1b is a first-line immunomodulatory therapy for relapsing-remitting multiple sclerosis (RR MS). However, its effects on B cells have not been characterized. In vitro studies of B cells derived from RR MS patients revealed that IFN-β-1b decreases B cells' stimulatory capacity, as detected by inhibition of the Ag-specific T cell proliferative response upon Ag presentation by IFN-β-1b-treated B cells. Our study has identified that IFN-β-1b inhibited B cells' stimulatory capacity in RR MS patients and healthy controls through the suppression of CD40 and CD80 expression, whereas the MHC class I and II expression was not changed. IFN-β-1b in vitro treatment inhibited B cell secretion of IL-1β and IL-23 and induced IL-12 and IL-27. Supernatants transferred from IFN-β-1b-treated B cells inhibited Th17 cell differentiation, as they suppressed gene expression of the retinoic acid-related orphan nuclear hormone receptor C and IL-17A and secretion of IL-17A. In addition, IFN-β-1b induced B cells' IL-10 secretion, which may mediate their regulatory effect. Studies of B cells derived from RR MS patients treated with recombinant s.c. injected IFN-β-1b revealed that they induced a significantly lower proliferative response in allogenic MLR than the B cells from untreated patients. Further confirming the IFN-β-1b in vitro-induced changes in B cell cytokine secretion, B cells derived from the IFN-β-1b-treated patients secreted significantly lower levels of IL-1β and IL-23 and higher levels of IL-12 and IL-27 in comparison with the B cells derived from untreated patients. We conclude that IFN-β-1b exerts its therapeutic effects in part by targeting B cells' functions that contribute to the autoimmune pathogenesis of RR MS.     

Distinct and overlapping effector functions of expanded human CD4+, CD8α+ and CD4-CD8α- invariant natural killer T cells

CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4(+), CD8α(+) and CD4(-)CD8α(-) double-negative (DN) subsets. CD4(+) iNKT cells expanded more readily than CD8α(+) and DN iNKT cells upon mitogen stimulation. CD8α(+) and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d(+) cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4(+) and CD8α(+) fractions, respectively. Only CD4(+) iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α(+), DN or CD4(+) iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.     

乳杆菌对牛乳β-乳球蛋白致敏小鼠淋巴细胞分泌Th1/Th2型细胞因子和抗体的体外影响

为了分析乳杆菌对致敏小鼠脾淋巴细胞分泌Th1/Th2细胞因子及抗体的体外影响,用牛乳β-乳球蛋白腹腔注射BALB/c小鼠建立过敏症模型,造模成功后,分离致敏小鼠的脾淋巴细胞并与4种活/死乳杆菌(107 CFU/mL)体外共同孵育,ELISA法检测细胞上清液中细胞因子(IL-12、IFN-γ和IL-4)和抗体(总IgE、β-Lg特异性IgE和总IgG)含量。4种活/死乳杆菌均可体外调节致敏小鼠脾淋巴细胞分泌细胞因子和抗体的水平,特别是热致死的发酵乳杆菌和嗜酸乳杆菌可提高淋巴细胞IL-12和IFN-γ的分泌,抑制IL-4的分泌,使其IFN-γ/IL-4比值(代表Th1/Th2细胞平衡)高于活菌,与空白对照组比较差异显著(P<0.05)。同时,这两株热致死菌还可显著下调细胞上清液中总IgE、特异性IgE和总IgG抗体的浓度(P<0.05)。试验结果表明乳杆菌可提高牛乳β-乳球蛋白致敏小鼠脾淋巴细胞的IFN-γ/IL-4比值,进而纠正Th2占优势的Th1/Th2失衡,下调抗体分泌量,且具有菌株特异性。     

Expansion and CD2/CD3/CD28 stimulation enhance Th2 cytokine secretion of human invariant NKT cells with retained anti-tumor cytotoxicity

Background aimsKey obstacles in human iNKT cell translational research and immunotherapy include the lack of robust protocols for dependable expansion of human iNKT cells and the paucity of data on phenotypes in post-expanded cells.MethodsWe delineate expansion methods using interleukin (IL)-2, IL-7 and allogeneic feeder cells and anti-CD2/CD3/CD28 stimulation by which to dependably augment Th2 polarization and direct cytotoxicity of human peripheral blood CD3⁺Vα24⁺Vβ11⁺ iNKT cells.ResultsGene and protein expression profiling demonstrated augmented Th2 cytokine secretion (IL-4, IL-5, IL-13) in expanded iNKT cells stimulated with anti-CD2/CD3/CD28 antibodies. Cytotoxic effector molecules including granzyme B were increased in expanded iNKT cells after CD2/CD3/CD28 stimulation. Direct cytotoxicity assays using unstimulated expanded iNKT cell effectors revealed α-galactosyl ceramide (α-GalCer)-dependent killing of the T-ALL cell line Jurkat. Moreover, CD2/CD3/CD28 stimulation of expanded iNKT cells augmented their (α-GalCer-independent) killing of Jurkat cells. Co-culture of expanded iNKT cells with stimulated responder cells confirmed contact-dependent inhibition of activated CD4⁺ and CD8⁺ responder T cells.DiscussionThese data establish a robust protocol to expand and novel pathways to enhance Th2 cytokine secretion and direct cytotoxicity in human iNKT cells, findings with direct implications for autoimmunity, vaccine augmentation and anti-infective immunity, cancer immunotherapy and transplantation.     

Distinct APCs explain the cytokine bias of α-galactosylceramide variants in vivo

α-Galactosylceramide represents a new class of vaccine adjuvants and immunomodulators that stimulate NKT cells to secrete Th1 and Th2 cytokines. Synthetic variants with short or unsaturated acyl chains exhibit a striking Th2 bias in vivo but no evidence of defect in TCR signaling or stimulation of NKT cells in vitro. Using cd1d1(fl/fl) mice, we demonstrated that distinct APC types explained the cytokine bias in vivo. Whereas NKT stimulation by α-Galactosylceramide required CD1d expression by dendritic cells (DCs), presentation of the Th2 variants was promiscuous and unaffected by DC-specific ablation of CD1d. This DC-independent stimulation failed to activate the feedback loop between DC IL-12 and NK cell IFN-γ, explaining the Th2 bias. Conversely, forced presentation of the Th2 variants by DC induced high IL-12. Thus, lipid structural variations that do not alter TCR recognition can activate distinct Th1 or Th2 cellular networks by changing APC targeting in vivo.     

Effects of Citrus unshiu Powder on the Cytokine Balance in Peripheral Blood Mononuclear Cells of Patients with Seasonal Allergic Rhinitis to Pollen

We evaluated the effects of a 50% methanol extract of Citrus unshiu powder (MEC) on cytokines in peripheral blood mononuclear cells (PBMCs) obtained from patients with seasonal allergic rhinitis to cedar pollen. The levels of cytokines, such as TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-12 (p70), IL-13, and GM-CSF, produced by pollen-stimulated PBMC were measured. We found that MEC suppressed pollen-induced TNF-α release and increased IFN-γ release from PBMCs. The results suggest that Citrus unshiu powder has an immunomodulatory effect in vitro and that its use could improve seasonal allergic rhinitis symptoms.     

The Characteristics of Th1/Th2 Cytokine Receptors on Monocytes in Untreated Patients of Long Term Nonprogressor or Chronic HIV Infection

Monocytes/macrophages play crucial roles in immunity to microorganisms and are one of the important targets for human immunodeficiency virus (HIV) infection. The phenotypes and function of monocytes in HIV-infected patients were poorly determined. We herein detected the expression of Th1/Th2 cytokine receptors on monocyte subsets in the untreated HIV-infected patients of either long term nonprogressor (LTNP) or chronic infection (CHI). CD14+CD16- monocytes were significantly increased and CD14+CD16+ monocytes were reduced in patients of LTNP or CHI compared with healthy control. IL-6R expression on CD14+CD16- monocytes were decreased in patients of LTNP or CHI, whereas IL-4R and IL-10R expression on both CD14+CD16- and CD14+CD16+ monocyte subsets were increased in patients with LTNP or CHI, as determined by flow cytometry and real time PCR assays. The decreased IL-6R expression and enhanced IL-4R and IL-10R expression were also observed on CD4+ T cells of these patients, indicating that these changes in monocytes are not cell-specific. CD14+CD16- monocytes of HIV-infected patients produced less TNF-α and IL-1β but identical levels of IL-6, and IL-12 as the control after IFN-γ/LPS stimulation. However, in the presence of IL-4 or IL10, CD14+CD16- monocytes of HIV-infected patients produced more TNF-α, IL-6, IL-12 or Il-1β after IFN-γ/LPS stimulation than the healthy control, supporting the impaired IL-4R and IL-10R signal pathways in patients with LTNP and CHI. Therefore, our present study offered the basic information for the Th1/Th2 cytokine receptor expression and function on monocyte subsets in untreated HIV-infected individuals.     

应用体内外实验筛选可降低牛乳β-乳球蛋白过敏的乳杆菌

【目的】通过体内外实验评估5种乳杆菌缓解牛乳β-乳球蛋白(BLG)过敏的作用,为今后筛选具有抗过敏活性的乳杆菌提供参考。【方法】首先体外分析5种活的/热致死的乳杆菌促进小鼠原代淋巴细胞分泌细胞因子(CK)IFN-γ和IL-4的水平,随后应用小鼠BLG过敏模型评估这5种乳杆菌抑制过敏的能力。将实验动物随机分为空白组、BLG致敏组和5种活的/热致死乳杆菌组。采用ELISA法检测各组小鼠淋巴细胞分泌Thl/Th2型CK的水平,并测定小鼠血清中总IgE和BLG特异性IgE的含量。【结果】在体外可促进淋巴细胞分泌IFN-γ、抑制IL-4,使其IFN-γ/IL-4比值(代表Thl/Th2细胞平衡)显著高于正常对照组(P<0.05)的乳杆菌,在体内实验中也能有效提高致敏小鼠淋巴细胞的IFN-γ/IL-4分泌率,并显著降低致敏小鼠血清中总IgE和BLG特异性IgE的水平(P<0.05)。相反,在体外的IFN-γ/IL-4比值较低的乳杆菌,不能缓解特异性IgE抗体介导的食物过敏反应。【结论】基于乳杆菌体外刺激小鼠原代淋巴细胞分泌Th1/Th2型CK的结果,可以预测菌株在体内具有可通过纠正Th2占优势的Th1/Th2细胞失衡,下调抗体分泌量,缓解小鼠BLG过敏症状的能力。     

Neonatal rhinovirus infection induces mucous metaplasia and airways hyperresponsiveness

Recent studies link early rhinovirus (RV) infections to later asthma development. We hypothesized that neonatal RV infection leads to an IL-13-driven asthma-like phenotype in mice. BALB/c mice were inoculated with RV1B or sham on day 7 of life. Viral RNA persisted in the neonatal lung up to 7 d postinfection. Within this time frame, IFN-α, -β, and -γ peaked 1 d postinfection, whereas IFN-λ levels persisted. Next, we examined mice on day 35 of life, 28 d after initial infection. Compared with sham-treated controls, virus-inoculated mice demonstrated airways hyperresponsiveness. Lungs from RV-infected mice showed increases in several immune cell populations, as well as the percentages of CD4-positive T cells expressing IFN-γ and of NKp46/CD335(+), TCR-β(+) cells expressing IL-13. Periodic acid-Schiff and immunohistochemical staining revealed mucous cell metaplasia and muc5AC expression in RV1B- but not sham-inoculated lungs. Mucous metaplasia was accompanied by induction of gob-5, MUC5AC, MUC5B, and IL-13 mRNA. By comparison, adult mice infected with RV1B showed no change in IL-13 expression, mucus production, or airways responsiveness 28 d postinfection. Intraperitoneal administration of anti-IL-13 neutralizing Ab attenuated RV-induced mucous metaplasia and methacholine responses, and IL-4R null mice failed to show RV-induced mucous metaplasia. Finally, neonatal RV increased the inflammatory response to subsequent allergic sensitization and challenge. We conclude that neonatal RV1B infection leads to persistent airways inflammation, mucous metaplasia, and hyperresponsiveness, which are mediated, at least in part, by IL-13.     

Uncarinic acid C plus IFN-γ generates monocyte-derived dendritic cells and induces a potent Th1 polarization with capacity to migrate

Uncarinic acid C (URC) is triterpene isolated from Uncaria rhynchophylla and modulates human DC function in a fashion that favors Th1 cell polarization depending on TLR4 signaling. The induction of dendritic cells (DC) is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. Monocyte-derived DC used as adjuvant cells in cancer immunotherapy and have shown promising results. We studied the effect of interferon’s (IFN-α and IFN-γ) and TNF-α on phenotypic and functional maturation, and cytokine production of URC-primed DC in vitro. Human monocytes were exposed to either URC alone, or in combination with TNF-α, IFN-α or IFN-γ, and thereafter co-cultured with naïve T cells. We found that the expression levels of CD1a, CD83 and HLA-DR on URC-primed DC were influenced by IFN-γ and IFN-γ augmented the T cell stimulatory capacity in allo MLR to URC-primed DC. Moreover, the production of IL-12p70 by URC-primed DC was enhanced by IFN-γ. IL-12p70 production by URC-primed DC alone was influenced following treatment with anti-TLR4 mAb, but not DC differentiated with URC plus IFN-γ. URC plus IFN-γ-primed DC induced a substantial increase in the secretion of IFN-γ by T cells, which is dependent on IL-12 secretion. DC maturated with URC plus IFN-γ had an intermediate migratory capacity towards CCL19 and CCL21. In addition, the expression levels of CCR7 on URC-primed DC were enhanced by IFN-γ. In contrast, surface molecule up-regulation and function of URC-primed DC were slightly enhanced by TNF-α, and IFN-α. These results suggest that the enhancement of Th1 cells polarization to URC-primed DC induced by IFN-γ depends on the activation of IL-12p70 and independent on TLR4. DC differentiated with URC in combination with IFN-γ might be used on DC-based vaccine for cancer immunotherapy.