Heterogeneity in phenotype and function of CD8+ and CD4/CD8 double‐negative Natural Killer T cell subsets in sooty mangabeys

Background We have recently reported the presence of CD8⁺ and CD4/8 double‐negative (DN) natural killer T (NKT) lymphocytes in sooty mangabeys. To investigate differences in the two NKT cell subsets, we compared the phenotype and function of sooty mangabey CD8⁺ and DN NKT cells. Methods Flow‐sorted NKT lymphocytes from one SIV‐negative sooty mangabey were subjected to limiting dilution cloning. Invariant NKT clones were characterized by flow cytometry and cytokine ELISA. Results The majority of NKT clones displayed an effector memory phenotype and expressed CXCR3 and NKG2D. While CD8⁺ NKT subsets expressed significantly higher levels of granzyme B and perforin and produced more IFN‐γ, the DN NKT subsets secreted significantly more IL‐4, IL‐13, and IL‐10. Conclusions The Th1 and Th2 cytokine bias of CD8⁺ and DN NKT cells, respectively, indicates the presence of functionally heterogeneous populations of NKT cells in sooty mangabeys.     

Induction of Th17 differentiation by corneal epithelial‐derived cytokines

This study was to explore a potential role of epithelium‐derived cytokines in Th17 differentiation. Th17 induction was evaluated by murine CD4⁺ T cells treated with different combinations of five inducing cytokines, or conditioned media of human corneal epithelial cells (HCECs) exposed to a variety of stimuli. Th17 differentiation was determined by measuring Th17 associated molecules, IL‐17A, IL‐17F, IL‐22, CCL‐20, and STAT3 at mRNA and protein levels, and numbers of IL‐17‐producing T cells by real‐time PCR, and cytokine immunobead and ELISPOT assays, respectively. IL‐23 was the strongest inducer for expanding Th17 cells in the presence of TGF‐β1 + IL‐6; and IL‐1β was the strongest Th17 amplifier in the presence of TGF‐β1 + IL‐6 + IL‐23. These inducing cytokines were found to be significantly stimulated in HCECs challenged by hyperosmotic media (450 mOsM), microbial components (polyI:C, flagellin, R837, and other TLR ligands) and TNF‐α. Interestingly, when incubated with conditioned media of HCECs irritated by polyI:C or TNF‐α, CD4⁺ T cells displayed increased mRNA levels of IL‐17A, IL‐17F, IL‐22, CCL‐20, and STAT3, increased IL‐17 protein in the supernatant, and increased numbers of IL‐17‐producing T cells (Th17 cells). These findings demonstrate for the first time that Th17 differentiation can be promoted by cytokines produced by corneal epithelium that are exposed to hyperosmotic, microbial, and inflammatory stimuli. J. Cell. Physiol. 222:95–102, 2010. © 2009 Wiley‐Liss, Inc.     

Proteome profiling of interleukin-12 treated human T helper cells

Selective activation of T helper subsets 1 (Th1) and 2 (Th2) plays a crucial role in different pathological conditions. Th1 cell response is involved in pathogenesis of autoimmune diseases, such as type II diabetes and multiple sclerosis, and Th2 cell response in pathogenesis of allergy and asthma. Cytokine interleukin-12 (IL-12) is one of the key factors in the differentiation of na?ve CD4(+) T cells into Th1 cells. In this study we used 2-DE and MS to find and identify IL-12 regulated proteins in human CD4(+) T cells. In total, 42 protein spots were found to be differentially expressed following IL-12 stimulation, of which 22 were up- and 20 down-regulated. Among the upregulated proteins there are a multifunctional cytokine macrophage migration inhibitory factor and a known IL-12 target gene Programmed cell death 4. Downregulated proteins include p21-activated kinase 2 and its upstream GTPase Cdc42. Compared to previous reports our analysis provides a new view on the IL-12 induced changes on CD4(+) T cells underscoring the importance of creating and combining the data generated at various levels to build a comprehensive view of a given biological process of the cell.     

Early‐shared Mycobacterium bovis bacillus Calmette–Guérin sub‐strains induce Th1 cytokine production in vivo

Interleukin‐12 is one of the cytokines that induce acquired immunity by progressing the differentiation of T cells. When antigens are presented by APCs, including macrophages and DCs, T cells are activated and produce the Th1 cytokines IL‐2 and IFN‐γ. We have previously reported greater IL‐12 production from macrophages infected with early‐shared BCG sub‐strains (ex. BCG‐Japan, ‐Sweden) than from those infected with late‐shared BCG (ex. BCG‐Pasteur and ‐Connaught) 1 . In this study, we investigated the Th1 cytokine‐inducing activity of splenocytes co‐cultured with BCG‐infected DCs. Early‐shared BCG‐infected DCs produced IL‐12 and TNF‐α? Furthermore, when they were co‐cultured with purified protein derivative‐stimulated DCs, the splenocytes of mice immunized with BCG‐Tokyo/Japan produced more Th1 cytokine than did those of mice immunized with BCG‐Connaught. In conclusion, early‐shared BCG sub‐strains more strongly induce Th1 cytokine production in vivo. This study provides basic information to inform the selection of candidates for primary vaccination.

     

Effect of mesenchymal stem cells on inhibiting airway remodeling and airway inflammation in chronic asthma

Previous studies proved that bone marrow‐derived mesenchymal stem cells (BMSCs) could improve a variety of immune‐mediated disease by its immunomodulatory properties. In this study, we investigated the effect on airway remodeling and airway inflammation by administrating BMSCs in chronic asthmatic mice. Forty‐eight female BALB/c mice were randomly distributed into PBS group, BMSCs treatment group, BMSCs control group, and asthmatic group. The levels of cytokine and immunoglobulin in serum and bronchoalveolar lavage fluid were detected by enzyme‐linked immunosorbent assay. The number of CD4⁺CD25⁺regulatory T cells and morphometric analysis was determined by flow cytometry, hematoxylin‐eosin, immunofluorescence staining, periodic‐acid Schiff, and masson staining, respectively. We found that airway remodeling and airway inflammation were evident in asthmatic mice. Moreover, low level of IL‐12 and high levels of IL‐13, IL‐4, OVA‐specific IgG1, IgE, and IgG2a and the fewer number of CD4⁺CD25⁺regulatory T cells were present in asthmatic group. However, transplantation of BMSCs significantly decreased airway inflammation and airway remodeling and level of IL‐4, OVA‐specific IgE, and OVA‐specific IgG1, but elevated level of IL‐12 and the number of CD4 + CD25 + regulatory T cells in asthma (P < 0.05). However, BMSCs did not contribute to lung regeneration and had no significant effect on levels of IL‐10, IFN‐Y, and IL‐13. In our study, BMSCs engraftment prohibited airway inflammation and airway remodeling in chronic asthmatic group. The beneficial effect of BMSCs might involved the modulation imbalance cytokine toward a new balance Th1–Th2 profiles and up‐regulation of protective CD4 + CD25 + regulatory T cells in asthma, but not contribution to lung regeneration. J. Cell. Biochem. 114: 1595–1605, 2013. © 2013 Wiley Periodicals, Inc.     

Peptidoglycan‐induced T helper 2 immune response in mice involves interleukin‐10 secretion from Langerhans cells

Patients with atopic dermatitis (AD) have superficial skin colonization with Staphylococcus aureus and an increased number of T helper (Th)2 cells in their peripheral blood. The purpose of this study was to clarify the involvement of interleukin (IL)‐10 secretion from Langerhans cells (LCs) in staphylococcal peptidoglycan (PEG)‐induced Th2 immune responses in mice. Mice were primed with LCs pulsed with PEG (or LPS) and ovalbumin (OVA) and then given a booster OVA injection 2 days later in the hind footpad. Five days after the OVA injection, cytokine responses in the draining popliteal lymph nodes were investigated by RT‐PCR and ELISA. Production of both IL‐10 and IL‐12 by cultured LCs was detected by ELISA. Administration of PEG‐ or LPS‐stimulated LCs into the hind footpads of the mice induced Th2‐prone and Th1‐prone immune responses, respectively, as represented by expression of IL‐4 and interferon ‐γ . In vitro experiments showed that PEG induced greater production of IL‐12 p40 from LCs than did LPS, whereas LPS induced greater production of IL‐12 p70 from LCs than did PEG. Furthermore, it was found that PEG‐stimulated LCs induced greater production of IL‐10 than did LPS‐stimulated LCs, and that neutralization of IL‐10 augmented IL‐12 p70 production and inhibited Th2 development by PEG‐stimulated LCs. These results suggest that PEG can induce Th2 development through down‐regulation of IL‐12 p70 production by LCs in an IL‐10 production‐dependent manner and would explain the role of S. aureus colonization in patients with AD.     

Quantitative proteomics reveals GIMAP family proteins 1 and 4 to be differentially regulated during human T helper cell differentiation

T helper (Th) cells differentiate into functionally distinct effector cell subsets of which Th1 and Th2 cells are best characterized. Besides T cell receptor signaling, IL-12-induced STAT4 and T-bet- and IL-4-induced STAT6 and GATA3 signaling pathways are the major players regulating the Th1 and Th2 differentiation process, respectively. However, there are likely to be other yet unknown factors or pathways involved. In this study we used quantitative proteomics exploiting cleavable ICAT labeling and LC-MS/MS to identify IL-4-regulated proteins from the microsomal fractions of CD4(+) cells extracted from umbilical cord blood. We were able to identify 557 proteins of which 304 were also quantified. This study resulted in the identification of the down-regulation of small GTPases GIMAP1 and GIMAP4 by IL-4 during Th2 differentiation. We also showed that both GIMAP1 and GIMAP4 genes are up-regulated by IL-12 and other Th1 differentiation-inducing cytokines in cells induced to differentiate toward Th1 lineage and down-regulated by IL-4 in cells induced to Th2. Our results indicate that the GIMAP (GTPase of the immunity-associated protein) family of proteins is differentially regulated during Th cell differentiation.     

Localized interleukin‐12 delivery for immunotherapy of solid tumours

Interleukin (IL)‐12 is the key cytokine in the initiation of a Th1 response and has shown promise as an anti‐cancer agent; however, clinical trials involving IL‐12 have been unsuccessful due to toxic side‐effects. To address this issue, lentiviral vectors were used to transduce tumour cell lines that were injected as an autologous tumour cell vaccine. The focus of the current study was to test the efficacy of this approach in a solid tumour model. SCCVII cells that were transduced to produce IL‐12 at different concentrations were then isolated. Subcutaneous injection of parental SCCVII cells results in tumour development, while a mixture of IL‐12‐producing and non‐producing cells results in tumour clearance. Interestingly, when comparing mice injected a mixture of SCCVII and either high IL‐12‐producing tumour cells or low IL‐12‐producing tumour cells, we observed that mixtures containing small amounts of high producing cells lead to tumour clearance, whereas mixtures containing large amounts of low producing cells fail to elicit protection, despite the production of equal amounts of total IL‐12 in both mixtures. Furthermore, immunizing mice with IL‐12‐producing cells leads to the establishment of both local and systemic immunity against challenge with SCCVII. Using depletion antibodies, it was shown that both CD4⁺ and CD8⁺ cells are crucial for therapy. Lastly, we have established cell clones of other solid tumour cell lines (RM‐1, LLC1 and moto1.1) that produce IL‐12. Our results show that the delivery of IL‐12 by cancer cells is an effective route for immune activation.     

IL7‐hCD25 and IL7‐Cre BAC transgenic mouse lines: New tools for analysis of IL‐7 expressing cells

IL‐7 is a cytokine that is required for T‐cell development and homeostasis as well as for lymph node organogenesis. Despite the importance of IL‐7 in the immune system and its potential therapeutic relevance, questions remain regarding the sites of IL‐7 synthesis, specific cell types involved and molecular mechanisms regulating IL‐7 expression. To address these issues, we generated two bacterial artificial chromosome (BAC) transgenic mouse lines in which IL‐7 regulatory elements drive expression of either Cre recombinase or a human CD25 (hCD25) cell surface reporter molecule. Expression of the IL‐7.hCD25 BAC transgene, detected by reactivity with anti‐hCD25 antibody, mimicked endogenous IL‐7 expression. Fetal and adult tissues from crosses between IL‐7.Cre transgenic mice and Rosa26R or R26‐EYFP reporters demonstrated X‐gal or YFP staining in tissues known to express endogenous IL‐7 at some stage during development. These transgenic lines provide novel genetic tools to identify IL‐7 producing cells in various tissues and to manipulate gene expression selectively in IL‐7 expressing cells. genesis 47:281–287, 2009. © 2009 Wiley‐Liss, Inc.     

Effector CD4+ T cell expression signatures and immune-mediated disease associated genes

Genome-wide association studies (GWAS) in immune-mediated diseases have identified over 150 associated genomic loci. Many of these loci play a role in T cell responses, and regulation of T cell differentiation plays a critical role in immune-mediated diseases; however, the relationship between implicated disease loci and T cell differentiation is incompletely understood. To further address this relationship, we examined differential gene expression in na?ve human CD4+ T cells, as well as in in vitro differentiated Th1, memory Th17-negative and Th17-enriched CD4+ T cells subsets using microarray and RNASeq. We observed a marked enrichment for increased expression in memory CD4+ compared to na?ve CD4+ T cells of genes contained among immune-mediated disease loci. Within memory T cells, expression of disease-associated genes was typically increased in Th17-enriched compared to Th17-negative cells. Utilizing RNASeq and promoter methylation studies, we identified a differential regulation pattern for genes solely expressed in Th17 cells (IL17A and CCL20) compared to genes expressed in both Th17 and Th1 cells (IL23R and IL12RB2), where high levels of promoter methylation are correlated to near zero RNASeq levels for IL17A and CCL20. These findings have implications for human Th17 celI plasticity and for the regulation of Th17-Th1 expression signatures. Importantly, utilizing RNASeq we found an abundant isoform of IL23R terminating before the transmembrane domain that was enriched in Th17 cells. In addition to molecular resolution, we find that RNASeq provides significantly improved power to define differential gene expression and identify alternative gene variants relative to microarray analysis. The comprehensive integration of differential gene expression between cell subsets with disease-association signals, and functional pathways provides insight into disease pathogenesis.     

Interleukin‐17 family cytokines in protective immunity against infections: role of hematopoietic cell‐derived and non‐hematopoietic cell‐derived interleukin‐17s

Interleukin‐17 family cytokines, consisting of six members, participate in immune response in infections and autoimmune and inflammatory diseases. The prototype cytokine of the family, IL‐17A, was originally identified from CD4+ T cells which are now termed Th17 cells. Later, IL‐17A‐producing cells were expanded to include various hematopoietic cells, namely CD8+ T cells (Tc17), invariant NKT cells, γδ T cells, non‐T non‐B lymphocytes (termed type 3 innate lymphoid cells) and neutrophils. Some IL‐17 family cytokines other than IL‐17A are also expressed by CD4+ T cells: IL‐17E by Th2 cells and IL‐17F by Th17 cells. IL‐17A and IL‐17F induce expression of pro‐inflammatory cytokines to induce inflammation and anti‐microbial peptides to kill pathogens, whereas IL‐17E induces allergic inflammation. However, the functions of other IL‐17 family cytokines have been unclear. Recent studies have shown that IL‐17B and IL‐17C are expressed by epithelial rather than hematopoietic cells. Interestingly, expression of IL‐17E and IL‐17F by epithelial cells has also been reported and epithelial cell‐derived IL‐17 family cytokines shown to play important roles in immune responses to infections at epithelial sites. In this review, we summarize current information on hematopoietic cell‐derived IL‐17A and non‐hematopoietic cell‐derived IL‐17B, IL‐17C, IL‐17D, IL‐17E and IL‐17F in infections and propose functional differences between these two categories of IL‐17 family cytokines.     

NKG2D ligand RAE1ε induces generation and enhances the inhibitor function of myeloid‐derived suppressor cells in mice

Expression of surface NKG2D ligands on tumour cells, which activates nature killer (NK) cells and CD8⁺ T cells, is crucial in antitumour immunity. Some types of tumours have evolved mechanisms to suppress NKG2D‐mediated immune cell activation, such as tumour‐derived soluble NKG2D ligands or sustained NKG2D ligands produced by tumours down‐regulate the expression of NKG2D on NK cells and CD8⁺ T cells. Here, we report that surface NKG2D ligand RAE1ε on tumour cells induces CD11b⁺Gr‐1⁺ myeloid‐derived suppressor cell (MDSC) via NKG2D in vitro and in vivo. MDSCs induced by RAE1ε display a robust induction of IL‐10 and arginase, and these MDSCs show greater suppressive activity by inhibiting antigen‐non‐specific CD8⁺ T‐cell proliferation. Consistently, upon adoptive transfer, MDSCs induced by RAE1ε significantly promote CT26 tumour growth in IL‐10‐ and arginase‐dependent manners. RAE1ε moves cytokine balance towards Th2 but not Th1 in vivo. Furthermore, RAE1ε enhances inhibitory function of CT26‐derived MDSCs and promotes IL‐4 rather than IFN‐γ production from CT26‐derived MDSCs through NKG2D in vitro. Our study has demonstrated a novel mechanism for NKG2D ligand⁺ tumour cells escaping from immunosurveillance by facilitating the proliferation and the inhibitory function of MDSCs.     

Dendritic cells engineered to express CD40L continuously produce IL12 and resist negative signals from Tr1/Th3 dominated tumors

TNFα-matured dendritic cells (DCs) pulsed with tumor antigens are being evaluated as cancer vaccines. It has been shown that DCs produce IL12 during a limited time span and subsequently enter a stage of IL12 exhaustion. If DCs are generated ex vivo, the patient could receive IL12-exhausted DCs which may be detrimental for stimulating anti-tumor Th1 responses. Furthermore, many cancer patients exhibit a cytokine profile skewed toward IL10 and TGFβ. This immunological profile, called the Tr1/Th3 response, is associated with the presence of regulatory T-cells. Tr1/Th3 responses potently inhibit DC maturation, thereby regulating Th1 responses. In the present study, we produced genetically engineered DCs that continuously express Th1-related cytokines such as IL12, and resist negative signals from Tr1/Th3-dominated bladder carcinoma cells. Human immature DCs were genetically engineered by adenoviral vectors to express CD40L, or were treated with TNFα as a positive control for maturation. The expression of different Th1/Th3 and inflammatory cytokines was monitored. IL12 and IFNγ were expressed by CD40L-engineered DCs, while TNFα-matured DCs lacked IFNγ and exhibited low IL12 expression. The addition of recombinant IL10 to genetically engineered DCs did not abolish their Th1 profile. Likewise, coculture with tumor cell lines expressing TGFβ with or without recombinant IL10 did not revert to the engineered DCs. We further demonstrate that the resistance of CD40L-expressing DCs to TGFβ and IL10 may be due to decreased levels of TGFβ and IL10 receptors. Thus, CD40L-engineered DCs are robust Th1-promoting ones that are resistant to Tr1/Th3-signaling via IL10 and TGFβ.     

RKIP mediates autoimmune inflammation by positively regulating IL‐17R signaling

Th17 cells contribute to the development of autoimmune diseases by secreting interleukin‐17 (IL‐17), which activates its receptor (IL‐17R) that is expressed on epithelial cells, macrophages, microglia, and resident neuroectodermal cells. However, the mechanisms through which IL‐17R‐mediated signaling contributes to the development of autoimmune disease have not been completely elucidated. Here, we demonstrate that Raf‐1 kinase inhibitor protein (RKIP) deficiency in mice ameliorates the symptoms of experimental autoimmune encephalomyelitis (EAE). Adoptive T‐cell‐transfer experiments demonstrate that RKIP plays a predominant role in Th17‐mediated, but not in Th1‐mediated immune responses. RKIP deficiency has no effect on Th17‐cell differentiation ex vivo, nor does it affect Th17‐cell differentiation in EAE mice. However, RKIP significantly promotes IL‐17R‐induced proinflammatory cytokine and chemokine production. Mechanistically, RKIP directly interacts with IL‐17RA and Act1 to promote the formation of an IL‐17R‐Act1 complex, resulting in enhanced MAPK‐ and P65‐mediated NF‐κB activation and downstream cytokine production. Together, these findings indicate that RKIP functions as an essential modulator of the IL‐17R‐Act1 axis in IL‐17R signaling, which promotes IL‐17‐induced inflammation and autoimmune neuroinflammation.     

Th17 cell induction and immune regulatory effects

The T help 1 (Th1) and Th2 cell classification have provided the framework for understanding CD4⁺ T cell biology and the interplay between innate and adaptive immunity for almost two decades. Recent studies have defined a previously unknown arm of the CD4⁺ T cell effector response, the Th17 lineage, which promises to change our understanding of immune regulation, immune pathogenesis and host defense. The factors that specify differentiation of IL‐17 producing effector T cells from naïve T cell precursors are being rapidly discovered and are providing insights into mechanisms by which signals from cells of the innate immune system guide alternative pathways of Th1, Th2, or Th17 development. In this review, we will focus on recent studies that have identified new subsets of Th cells, new insights regarding the induced generation and differentiation mechanisms of Th17 cells and immune regulatory effects. J. Cell. Physiol. 211: 273–278, 2007. © 2007 Wiley‐Liss, Inc.     

MiR‐16 regulates mouse peritoneal macrophage polarization and affects T‐cell activation

MiR‐16 is a tumour suppressor that is down‐regulated in certain human cancers. However, little is known on its activity in other cell types. In this study, we examined the biological significance and underlying mechanisms of miR‐16 on macrophage polarization and subsequent T‐cell activation. Mouse peritoneal macrophages were isolated and induced to undergo either M1 polarization with 100 ng/ml of interferon‐γ and 20 ng/ml of lipopolysaccharide, or M2 polarization with 20 ng/ml of interleukin (IL)‐4. The identity of polarized macrophages was determined by profiling cell‐surface markers by flow cytometry and cytokine production by ELISA. Macrophages were infected with lentivirus‐expressing miR‐16 to assess the effects of miR‐16. Effects on macrophage–T cell interactions were analysed by co‐culturing purified CD4⁺ T cells with miR‐16‐expressing peritoneal macrophages, and measuring activation marker CD69 by flow cytometry and cytokine secretion by ELISA. Bioinformatics analysis was applied to search for potential miR‐16 targets and understand its underlying mechanisms. MiR‐16‐induced M1 differentiation of mouse peritoneal macrophages from either the basal M0‐ or M2‐polarized state is indicated by the significant up‐regulation of M1 marker CD16/32, repression of M2 marker CD206 and Dectin‐1, and increased secretion of M1 cytokine IL‐12 and nitric oxide. Consistently, miR‐16‐expressing macrophages stimulate the activation of purified CD4⁺ T cells. Mechanistically, miR‐16 significantly down‐regulates the expression of PD‐L1, a critical immune suppressor that controls macrophage–T cell interaction and T‐cell activation. MiR‐16 plays an important role in shifting macrophage polarization from M2 to M1 status, and functionally activating CD4⁺ T cells. This effect is potentially mediated through the down‐regulation of immune suppressor PD‐L1.