Implications of IL-21 in solid tumor therapy.

Cancer, the most deadly disease, is known as a recent dilemma worldwide. Presently different treatments are used for curing cancers, especially solid cancers. Because of the immune-enhancing functions of cytokine, IL-21 as a cytokine may have new possibilities to manipulate the immune system in disease conditions, as it stimulates NK and CTL functions and drives IgG antibody production. Indeed, IL-21 has been revealed to elicit antitumor-immune responses in several tumor models. Combining IL-21 with other agents, which target tumor cells, immune-regulatory circuits, or other immune-enhancing molecules enhances this activity. The exciting breakthrough in the results obtained in pre-clinical situations has led to the early outset of present developing clinical trials in cancer patients. In the paper, we have reviewed the function of IL-21 in solid tumor immunotherapy.

C-type lectin receptor agonists elicit functional IL21-expressing Tfh cells and induce primary B cell responses in neonates.

Introduction: C-type lectin receptor (CLR) agonists emerged as superior inducers of primary B cell responses in early life compared with Toll-like receptor (TLR) agonists, while both types of adjuvants are potent in adults. Methods: Here, we explored the mechanisms accounting for the differences in neonatal adjuvanticity between a CLR-based (CAF®01) and a TLR4-based (GLA-SE) adjuvant administered with influenza hemagglutinin (HA) in neonatal mice, by using transcriptomics and systems biology analyses. Results: On day 7 after immunization, HA/CAF01 increased IL6 and IL21 levels in the draining lymph nodes, while HA/GLA-SE increased IL10. CAF01 induced mixed Th1/Th17 neonatal responses while T cell responses induced by GLA-SE had a more pronounced Th2-profile. Only CAF01 induced T follicular helper (Tfh) cells expressing high levels of IL21 similar to levels induced in adult mice, which is essential for germinal center (GC) formation. Accordingly, only CAF01- induced neonatal Tfh cells activated adoptively transferred hen egg lysozyme (HEL)-specific B cells to form HEL+ GC B cells in neonatal mice upon vaccination with HEL-OVA. Discussion: Collectively, the data show that CLR-based adjuvants are promising neonatal and infant adjuvants due to their ability to harness Tfh responses in early life.

Interleukins 4 and 21 Protect Anti-IgM Induced Cell Death in Ramos B Cells: Implication for Autoimmune Diseases.

Interleukins 4 (IL-4) and 21 (IL-21) belong to the common gamma chain cytokine family which are highly involved in the progression of autoimmune diseases. While IL-4 is well known to be involved in the suppression of apoptosis of autoreactive B cells, the role played by IL-21 remains unclear. In the current study, we activated the human Burkitt's lymphoma Ramos B cells with anti-IgM to mimic B cell hyperactivation observed in patients of autoimmune diseases. Consistent with other reported findings, anti-IgM led to the downregulation of proteins involved in B cell survival and proliferation, as well as the activation of caspase 3 activity and DNA damage, resulting in apoptotic cell death after 48-hour treatment. Although both IL-4 and IL-21 reversed anti-IgM-induced apoptosis and cell cycle arrest, they did so via different mechanisms: while IL-4 could directly suppress anti-IgM-induced caspase 3 activation and marker indicative of DNA damage, IL-21 could induce B cell proliferation in the presence of anti-IgM. Importantly, IL-21 also suppressed activation induced cell death in human primary B cells. Pre-treatment with clinically validated JAK inhibitors completely reversed the effects of IL-4 and IL-21 to rescue anti-IgM induced cell death and DNA damage. The results indicate the underlying mechanisms of how IL-4 and IL-21 differentially promote survival of hyperactivated B cells and provide hints to treat autoimmune diseases.

Altered T Follicular Helper Cell Subsets and Function in Chronic Lymphocytic Leukemia.

Follicular helper T cells (TFH) have specialized properties in promoting normal B cell activation but their role in chronic lymphocytic leukemia (CLL) is unknown. We find that TFH cells are elevated in CLL patients and are phenotypically abnormal, expressing higher levels of PD-1, TIGIT, CD40L, IFNγ and IL-21, and exhibiting abnormal composition of TFH1, TFH2 and TFH17 subsets. Frequencies of CD4-positive T cells expressing TFH1 markers and IL-21 were positively correlated with patient lymphocyte counts and RAI stage, suggesting that accumulation of abnormal TFH cells is concomitant with expansion of the leukemic B cell clone. Treatment with ibrutinib led to normalization of TFH frequencies and phenotype. TFH cells identified in CLL bone marrow display elevated expression of several functional markers compared to blood TFH cells. CLL T cell-B cell co-culture experiments revealed a correlation of patient TFH frequencies with functional ability of their CD4-positive T cells to promote CLL proliferation. Conversely, CLL cells can preferentially activate the TFH cell subset in co-culture. Together our results indicate that CLL development is associated with expansion of abnormal TFH populations that produce elevated levels of cytokines and costimulatory molecules which may help support CLL proliferation.

Inflammatory versus Anti-inflammatory Profiles in Major Depressive Disorders-The Role of IL-17, IL-21, IL-23, IL-35 and Foxp3.

BACKGROUND: The authors of this research study intended to verify whether there are any changes in gene expression in depressed patients without coexisting inflammatory diseases for selected immune-inflammatory factors that are particularly important in autoimmune disease pathogenesis (IL-17, IL-21, IL-23, IL-35, Foxp3). METHODS: The study was carried out on a group of 190 patients with depression and 100 healthy volunteers. The severity of depressive symptoms was assessed using the Hamilton Depression Scale. RT-PCR was used to evaluate mRNA expression and ELISA was used to measure protein expression of these genes. RESULTS: The level of gene expression for IL-17, IL-21, IL-23, and IL-35 was substantially higher in the group of patients with depression compared to the control group. The mean mRNA expression of Foxp3 was considerably reduced in patients suffering from depressive disorders. There was a statistically significant correlation between the number of hospitalizations and the expression of specific inflammatory factors. CONCLUSIONS: Expression of specific inflammatory genes may be a factor in the etiopathogenesis of depressive disorders. The duration of the disease seems to be more important for the expression of the genes in question than the severity of depression. These cytokines may affect the metabolism of neurotransmitters and neuroendocrine functions in the brain as well as be a marker and a new potential therapeutic target for recurrent depressive disorders.

IL-21+ CD4+ T helper cells co-expressing IFN-γ and TNF-α accumulate in the joints of antinuclear antibody positive patients with juvenile idiopathic arthritis.

Juvenile Idiopathic Arthritis (JIA) is currently classified into seven subgroups. Recently, antinuclear antibody (ANA) positive JIA patients were suggested to encompass a clinically homogenous new subgroup. CD4+ T helper (Th) cells play an essential role in JIA pathogenesis. Herein, we analyzed cytokine expression in synovial fluid (SF) CD4+ Th cells of JIA patients by using flow cytometry and compared cytokine patterns between JIA subgroups. We could show increased frequencies of IL-21 expressing CD4+ Th cells in the joints of ANA+ Oligo-/Poly-JIA patients, which co-expressed the Th-1 cytokines IFN-γ/TNF-α. In contrast, frequencies of IL-17 expressing cells were lowest in the joints of ANA+ Oligo-/Poly-JIA but enriched in that of ERA-JIA patients. This is the first description of a diverse SF Th cell cytokine pattern in different JIA subgroups. Additionally, we could define IL-21 as an effector cytokine expressed in SF Th cell in a significant proportion of ANA+ JIA patients.

Serum Ghrelin Levels in Saudi Obese Asthmatic School-Children-Correlation with Interleukin-4, Interleukin-5, and Interleukin-21.

Ghrelin is a peptide hormone with direct or indirect effects on obesity and asthma. More data are required to understand the effect of ghrelin on the control and pathogenesis of these diseases. The aim of this study was to evaluate ghrelin levels in selected groups of children to identify the association between serum ghrelin, obesity, and the severity of asthma. The study included 401 school children selected from the Najran area and grouped into non-obese asthmatics, obese asthmatics, obese non-asthmatics and controls (non-obese non-asthmatics). Blood levels of ghrelin, interleukin (IL)-4, IL-5 and IL-21 were determined by ELISA. The mean ghrelin values were insignificantly increased in obese children compared with non-obese children. The highest blood ghrelin values were in the non-obese asthmatic group. Serum ghrelin, IL-4 and IL-21 levels were significantly increased in asthmatic children compared with non-asthmatic children (p < 0.05), and there were significant positive correlations between ghrelin and IL-4, IL-5, and IL-21 in asthmatic children. Furthermore, ghrelin, IL-4, and IL-21 levels were significantly higher in uncontrolled asthmatics compared with controlled-asthmatic children (p < 0.05). Asthma was the only significant risk factor for high ghrelin values. This study provides evidence supporting the anti-inflammatory role of ghrelin in the pathogenesis of asthma. Asthma might be considered as an important determinant of high ghrelin values in children.

T Follicular Helper Cell Subsets and the Associated Cytokine IL-21 in the Pathogenesis and Therapy of Asthma.

For many decades, T helper 2 (TH2) cells have been considered to predominantly regulate the pathogenic manifestations of allergic asthma, such as IgE-mediated sensitization, airway hyperresponsiveness, and eosinophil infiltration. However, recent discoveries have significantly shifted our understanding of asthma from a simple TH2 cell-dependent disease to a heterogeneous disease regulated by multiple T cell subsets, including T follicular helper (TFH) cells. TFH cells, which are a specialized cell population that provides help to B cells, have attracted intensive attention in the past decade because of their crucial role in regulating antibody response in a broad range of diseases. In particular, TFH cells are essential for IgE antibody class-switching. In this review, we summarize the recent progress regarding the role of TFH cells and their signature cytokine interleukin (IL)-21 in asthma from mouse studies and clinical reports. We further discuss future therapeutic strategies to treat asthma by targeting TFH cells and IL-21.

MiR302c, Sp1, and NFATc2 regulate interleukin-21 expression in human CD4+CD45RO+ T lymphocytes.

Interleukin-21 (IL-21) is a cytokine with potent regulatory effects on different immune cells. Recently, IL-21 has been contemplated for use in the treatment of cancers. However, the molecular mechanisms regulating human IL-21 gene expression has not yet been described. In this study, we initially studied the promoter region and identified the transcription start site. We thereafter described the essential region upstream of the transcription start site and showed the in vivo binding of NFATc2 and SP1 transcription factors to this region, in addition to their positive role in IL-21 expression. We also studied the role of microRNAs (miRNAs) in regulating IL-21 expression. We, thus, established the miRNA profile of CD4+CD45RO+ versus CD4+CD45RA+ isolated from healthy volunteers and identified a signature composed of 12 differentially expressed miRNAs. We showed that miR-302c is able to negatively regulate IL-21 expression by binding directly to its target site in the 3'-untranslated region. Moreover, after using fresh human CD4-positive T cells, we observed the high acetylation level of histone H4, an observation well in line with the already described high expression of IL-21 in CD4+CD45RO+ versus CD4+CD45RA+ T cells. Altogether, our data identified different molecular mechanisms regulating IL-21 expression.

The pathogenesis of thyroid autoimmune diseases: New T lymphocytes - Cytokines circuits beyond the Th1-Th2 paradigm.

Autoimmune thyroid disease (AITD) is one of the most common organ-specific autoimmune disorders. It mainly manifests as Hashimoto's thyroiditis (HT) and Graves' disease (GD). HT is characteristic of hypothyroidism resulting from the destruction of the thyroid while GD is characteristic of hyperthyroidism due to excessive production of thyroid hormone induced by thyrotropin receptor-specific stimulatory autoantibodies. T lymphocytes and their secretory cytokines play indispensable roles in modulating immune responses, but their roles are often complex and full of interactions among distinct components of the immune system. Dysfunction of these T cells or aberrant expressions of these cytokines can cause the breakdown of immune tolerance and result in aberrant immune responses during the development of AITDs. This review summarizes recently identified T subsets and related cytokines and their roles in the pathogenesis of AITDs with the hope to provide a better understanding of the precise roles of notably identified T subsets in AITDs and facilitate the discovery of functional molecules or novel immune therapeutic targets for AITDs.

Effect and mechanism of mesenchymal stem cell derived from different sources on inhibiting follicular helper T cells / 器官移植

Objective To investigate the inhibitory effect and underlying mechanism of mesenchymal stem cell (MSC) derived from different sources on follicular helper T cell (Tfh cell). Methods Umbilical cord-derived MSC (UC MSC), bone marrow-derived MSC (BM MSC) and fat-derived MSC (Fat MSC) were co-cultured with peripheral blood mononuclear cell (PBMC) for 48 h. A control group was established. Flow cytometry was adopted to calculate the proportion of Tfh cells among the lymphocytes in four groups. The content of interleukin (IL)-21 in the supernatant was detected by enzyme-linked immune absorbent assay (ELISA) in four groups. BM MSC was co-cultured with PBMC, and supplemented with indoleamine 2,3-dioxygenase (IDO) inhibitor 1-methyl tryptophan (1-MT), IL-10 antibody, human leukocyte antigen (HLA)-G antibody in the 1-MT group, IL-10 inhibition group, HLA-G inhibition group and BM MSC group without addition of other substances. After 48 h culture, flow cytometry was used to detect the percentage of Tfh cells among lymphocytes. Results Flow cytometry demonstrated that compared with the control group, the proportion of Tfh cells in the BM MSC group was significantly decreased (P<0.05). Compared with the BM MSC group, the percentage of Tfh cells in the UC MSC and Fat MSC groups was significantly higher (both P<0.05). ELISA revealed that compared with the control group, the IL-21 content in the BM MSC group was significantly decreased (P<0.05). Compared with the BM MSC group, the IL-21 contents were considerably higher in the UC MSC and Fat MSC groups (both P<0.05). The analysis of underlying mechanism revealed that the proportions of Tfh cells in the 1-MT, IL-10 inhibition and the HLA-G inhibition groups were (1.75±0.07)%, (1.31±0.09)% and (1.50±0.03)%, respectively, which were significantly higher than (1.03±0.43)% in the BM MSC group (all P<0.05). Conclusions BM MSC exerts the highest inhibitory effect upon the differentiation of Tfh cell and IL-21. The mechanism underlying suppressing the differentiation of Tfh cells differentiation is probably correlated to promoting the secretion of IDO.

Expression of IL-21/BCL-6/Blimp-1 in echinococcosis / 中国免疫学杂志

Objective:To study the expression of IL-21/BCL-6/Blimp-1 in CE patients and discusse the mechanism of the pathogenesis of the echinococcosis.Methods:27 patients and 30 health persons were collected from the first affiliated hospital of Xinjiang medical university in the same period.IL-21 was detected by ELISA and the expression of IL-21/BCL-6 /Blimp-1 mRNA was detected by Real-time fluorescence quantitative PCR (qRT-PCR) in CE patients.At the same time,17 patients were followed up in the group of patients,and the expression of IL-21/BCL-6/Blimp-1 was detected before and after treatment.Results:(1) The results of PCR showed that the levels of IL-21/BCL-6 mRNA were significantly increased in the peripheral blood mononuclear cells of the CE patients compared with the healthy control group (P<0.05).The expression of IL-21/BCL-6 /Blimp-1 mRNA in patients before the treatment was higher than that of patients after treatment(P<0.05).(2)The level of IL-21 in peripheral blood of CE patients was sig-nificantly higher than that in the healthy control group and basically returned to normal after the cure (P<0.05).IL-21 was positively correlated with BCL-6(r=0.733, P<0.01).Conclusion:BCL-6 and Blimp-1 May promote the human immune system to resist parasitic infection in the course of the development of the disease.IL-21, BCL-6 and Blimp-1 are significantly reduced after effective treatment,indicating that these factors are involved in the immune mechanism of the development of the disease.

Increased IL-21/IL-21R expression and its proinflammatory effects in autoimmune thyroid disease.

To determine the potential role of interleukin-21 (IL-21) / IL-21 receptor (IL-21R) in the pathogenesis of autoimmune thyroid disease (AITD) mainly known as Graves' disease (GD) and Hashimoto's thyroiditis (HT). IL-21 and IL-21R of peripheral blood samples and/or thyroid tissues from AITD patients and healthy controls were analyzed by ELISA, quantitative real-time polymerase chain reaction (qRT-PCR), flow cytometry and immunohistochemistry. In vitro, the mRNA and protein of inflammatory cytokines of cultured peripheral blood mononuclear cells (PBMCs) upon recombinant human IL-21 (rhIL-21) stimulation were detected. There was an increased serum concentration of IL-21 in untreated GD and HT patients, and IL-21(+)CD3(+)CD8(-)T cells were significantly increased in PBMCs of HT patients compared with healthy volunteers. The IL-21 mRNA expression in PBMCs increased dramatically in GD and HT patients, and marked augmentations of IL-21 and IL-21R mRNA in thyroid tissues of HT patients were observed. Immunohistochemical staining confirmed the expression of IL-21R protein in HT thyroid cells and lymphocytes. In vitro, PBMCs from GD cultured with rhIL-21 induced increased IL-17A but decreased IL-4 production, while from HT stimulated by rhIL-21 induced augmented production of IFN-γ. In conclusion, the expression of IL-21 and IL-21R were up-regulated in AITD and may be involved in the pathogenesis of the disease through augmenting aberrant immune cascade.

Expression and purification of mIL-21-hIgGFc fusion protein in 293E cells and its effects on CD8+T cell phenotype / 中国免疫学杂志

Objective:To express recombinant protein mIL-21-hIgGFc in 293E cells,and investigate its effect on CD8+T cell.Methods:Total RNA was extracted from the mouse spleen cells ,and then IL-21 gene was amplified by RT-PCR and inserted into expression vector PTT3-hIgGFc.PTT3-mIL-21-hIgGFc were transfected into 293E cells by calcium phosphate method.The supernatants were collected at 48 hours and 72 hours and concentrated by MOLLIPORE Labscale TM TFF system ( 5 kD membrane ).The mIL-21-hIgGFc fusion protein was purified with HiTrap TM Protein G column.The protein was quantified by SDS-PAGE and ELISA.The biological activity of the protein was determined by detecting the change of the phenotypes of CD 8+ T cells treated with the protein.Results: The constructed recombinant plasmid PTT 3-mIL-21-Fc was confirmed by sequencing.PTT3-mIL-21-Fc was transfected into 293E cells,mIL-21-Fc protein in culture supernatant was collected after 48 hours and 72 hours.The protein in cell su-pernatant reached a concentration of 787 ng/ml which was determined by ELISA.The protein was purified by Protein G chromatography column.P1A-specific T cells were treated with mIL-21-hIgGFc, and found that the CD44low CD62Lhi CD8+ population increased compared to the control.Conclusion:We built PTT3-mIL-21-hFc recombinant plasmid, expressed mIL21-hFc fusion protein in 293E cells,and purified by Protein G column.By treating mIL-21-hFc ,the antigen-primed CD8+T cells prefer to differentiate into CD44low CD62Lhi CD8+T cells which had been reported as a memory stem phenotype .This protein may be used to improve the effectness of adoptive T cell cancer therapy.

Human IL-21 gene was expressed in Hela cell and it costimulate T cell proliferation in vitro / 中国免疫学杂志

Objective:To construct Interleukin 21 (IL 21) expression plasimd pCDNA3.1 IL21 and express it in Hela cell and analysis its acitivity of costimilating T cell proliferation with anti CD3 monoclonal antibody in vitro.Methods:The CDs of IL 21 was amplified by PCR using the pMD IL21 as templet.The expression plasimd pCDNA3.1 IL21 was constructed by inserting the sequence of coding region of the IL 21 into pCDNA3.1/Hismyc B containing CMV promoter and transfected into Hela cells.The stability expression stain was screened by the condition media containing 400 mg/L G418 and cloned through the limited dilution method.The target protein was purified through Ni 2+ chelating Sepharose Fast Flow.The bioactivity was confirmed by MTT method on costimulating the T cell proliferation with anti CD3.SDS PAGE and Western blot analysis the rhIL 21 expressed.Results:hIL 21 was expressed in Hela cell successfully.SDS PAGE analysis showed the IL 21 fusion protein with Mr 18 000 or so was expressed in supernatant of the cells.The rhIL 21 has significant proliferation effect on mature human T cells in the presence of anti CD3 monoantibody.Conclusion:The rhIL 21 with bioactivity has been obtained,which may help researcher study its new function and effects. [