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1.
Front Immunol ; 12: 661580, 2021.
Article in English | MEDLINE | ID: mdl-33897706

ABSTRACT

Donor-specific antibodies (DSAs) play a key role in chronic kidney allograft injury. Follicular T helper (Tfh) cells trigger the humoral alloimmune response and promote DSA generation, while T-follicular regulatory (Tfr) cells inhibit antibody production by suppressing Tfh and B cells. Interleukin (IL)-21 exerts a distinct effect on Tfh and Tfr. Here, we studied whether blocking IL-21R with anti-IL-21R monoclonal antibody (αIL-21R) changes the Tfh/Tfr balance and inhibits DSA generation. First, we investigated the impact of αIL-21R on CD4+ T cell proliferation and apoptosis. The results showed that αIL-21R did not have cytotoxic effects on CD4+ T cells. Next, we examined Tfh and regulatory T cells (Tregs) in an in vitro conditioned culture model. Naïve CD4+ T cells were isolated from 3-month-old C57BL/6 mice and cultured in Tfh differentiation inducing conditions in presence of αIL-21R or isotype IgG and differentiation was evaluated by CXCR5 expression, a key Tfh marker. αIL-21R significantly inhibited Tfh differentiation. In contrast, under Treg differentiation conditions, FOXP3 expression was inhibited by IL-21. Notably, αIL-21R rescued IL-21-inhibited Treg differentiation. For in vivo investigation, a fully mismatched skin transplantation model was utilized to trigger the humoral alloimmune response. Consistently, flow cytometry revealed a reduced Tfh/Tfr ratio in recipients treated with αIL-21R. Germinal center response was evaluated by flow cytometry and lectin histochemistry. We observed that αIL-21R significantly inhibited germinal center reaction. Most importantly, DSA levels after transplantation were significantly inhibited by αIL-21R at different time points. In summary, our results demonstrate that αIL-21R shifts the Tfh/Tfr balance toward DSA inhibition. Therefore, αIL-21R may be a useful therapeutic agent to prevent chronic antibody mediated rejection after organ transplantation.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies/immunology , Receptors, Interleukin-21/antagonists & inhibitors , Skin Transplantation/methods , T Follicular Helper Cells/immunology , Tissue Donors , Animals , Antibodies, Monoclonal/immunology , Apoptosis/drug effects , Apoptosis/immunology , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cell Proliferation/drug effects , Cells, Cultured , Germinal Center/cytology , Germinal Center/drug effects , Germinal Center/immunology , Male , Mice, Inbred BALB C , Mice, Inbred C57BL , Receptors, Interleukin-21/immunology , Receptors, Interleukin-21/metabolism , T Follicular Helper Cells/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism
2.
Int J Immunopathol Pharmacol ; 32: 2058738418781634, 2018.
Article in English | MEDLINE | ID: mdl-30103640

ABSTRACT

Oxymatrine (OMT) as a type of alkaloids collected from Sophora flavescens Ait exerts some biological functions including anticancer properties. Here, we investigated the therapeutic effects of OMT in gastric cancer cells (HGC 27 and AGS). As a result, the exposure of gastric cancer (GC) cells to OMT contributed to the suppression of cell proliferation and invasion. Interleukin 21 receptor (IL-21R) was identified to be differentially expressed between OMT treatment group (4 mg/mL) and control group (0 mg/mL), and knockdown of IL-21R repressed cell proliferation and invasion via inactivation of the JAK2/STAT3 pathway. The rescue experiment showed that IL-21R overexpression attenuated the anti-tumor effects of OMT through activation of the JAK2/STAT3 pathway. Moreover, the expression of IL-21R was significantly upregulated in GC samples compared with the adjacent normal tissues and associated with overall survival (OS) and tumor recurrence of GC patients. Taken together, in this study, we evaluated the anti-tumor effects of OMT on GC by investigating proliferation and invasion ability changes, and our findings show that OMT exhibits effects via regulation of JAK/STAT signaling pathway. Through the mechanism study, we may enlighten the potential therapeutic target for treatment of GC.


Subject(s)
Alkaloids/pharmacology , Antineoplastic Agents/pharmacology , Janus Kinase 2/metabolism , Quinolizines/pharmacology , Receptors, Interleukin-21/antagonists & inhibitors , STAT3 Transcription Factor/metabolism , Stomach Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Receptors, Interleukin-21/genetics , Receptors, Interleukin-21/metabolism , Signal Transduction/drug effects , Stomach Neoplasms/drug therapy
3.
Trans Am Clin Climatol Assoc ; 127: 224-234, 2016.
Article in English | MEDLINE | ID: mdl-28066055

ABSTRACT

Atherosclerosis is the leading cause of morbidity and mortality in the Western world. Peripheral artery disease (PAD) has been less studied then coronary artery disease but is nearly as common. PAD impairs blood flow to the leg(s) and causes functional impairment, leg pain, and amputation. The last drug approved for PAD was in 1999. Blood flow to leg proceeds through one major artery and in PAD total occlusions in the course of that vessel are common. Thus, the extent of new blood vessel growth determines a patients' clinical course. Promoting the growth of new blood vessels (therapeutic angiogenesis) was a major goal of therapy. Results from studies using cytokine growth factors have shown disappointing results. Using clinical and preclinical studies, our laboratory has identified several novel therapeutic approaches. One, a modulator of innate immunity, will be reviewed as an approach that has the potential to create new therapies for PAD.


Subject(s)
Immunologic Factors/pharmacology , Peripheral Arterial Disease/therapy , Angiogenesis Inducing Agents/pharmacology , Animals , Humans , Immunity, Innate , Receptors, Interleukin-21/antagonists & inhibitors
4.
Arthritis Rheumatol ; 67(10): 2723-31, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26097207

ABSTRACT

OBJECTIVE: Systemic lupus erythematosus (SLE) is a complex autoimmune disease that is driven in part by chronic B and T lymphocyte hyperresponsiveness to self antigens. A deficiency of interleukin-21 (IL-21) or IL-21 receptor (IL-21R) in mice dramatically reduces inflammation and B and T cell activation in models of autoimmunity, including SLE. However, whether IL-21 is essential for the maintenance and amplification of preestablished inflammation has not been widely examined in various animal models. The purpose of this study was to examine the impact of novel mouse IL-21R neutralizing antibodies on recall responses to antigen challenge and on disease progression in the (NZB × NZW)F1 (NZB/NZW) mouse model of SLE. METHODS: Humoral and cellular immune responses to immunization with sheep red blood cells (SRBCs) were measured in mice dosed with IL-21R blocking antibodies. Progression of nephritis and markers of immune activation was monitored in NZB/NZW mice following different anti-IL-21R treatment regimens. RESULTS: IL-21R blockade specifically inhibited secondary IgG responses to SRBC immunization. In NZB/NZW mice, IL-21R blockade completely inhibited the onset of nephritis, which was associated with dramatic reductions in splenomegaly and in B cell and T cell activation. When administered to mice with preexisting disease, anti-IL-21R antibody halted the disease progression and mortality and reversed the nephritis in a subset of mice. Furthermore, treatment cessation was not followed by rapid reemergence of disease. CONCLUSION: Our results highlight the importance of IL-21 in promoting humoral recall responses and in sustaining autoimmune inflammation.


Subject(s)
Antibodies, Blocking/therapeutic use , Disease Models, Animal , Disease Progression , Immunity, Humoral/immunology , Lupus Erythematosus, Systemic/drug therapy , Lupus Erythematosus, Systemic/immunology , Receptors, Interleukin-21/antagonists & inhibitors , Animals , Antibodies, Blocking/immunology , Antibodies, Blocking/pharmacology , Autoimmunity/immunology , B-Lymphocytes/drug effects , B-Lymphocytes/pathology , Female , Immunity, Humoral/drug effects , Immunoglobulin G/blood , Lupus Erythematosus, Systemic/pathology , Mice , Mice, Inbred NZB , Receptors, Interleukin-21/drug effects , Receptors, Interleukin-21/immunology , Sheep/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/pathology , Treatment Outcome
5.
J Immunol Methods ; 417: 60-66, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25523924

ABSTRACT

Interleukin (IL)-21 is crucial for the regulation of lymphocytes and is implicated in autoimmune and other diseases. The relevance of being able to measure human IL-21 prompted us to develop ELISA and ELISpot assays for analysis of IL-21 levels and IL-21-producing cells, respectively. Monoclonal antibodies (mAbs) to IL-21 were made and ELISA and ELISpot assays were developed. The selected detection mAb also neutralized IL-21-mediated activation of human cells. Peripheral blood mononuclear cells (PBMCs) from healthy donors (n=24) were stimulated polyclonally (phytohemagglutinin; PHA) or with antigen (Candida albicans extract and tetanus toxoid). Using ELISpot, high numbers of IL-21-producing cells were detected after PHA activation; lower but positive responses to antigen were seen in approximately 50% of the donors. In contrast, the ELISA detected IL-21 in supernatants from PHA-activated cells but not from antigen-stimulated cells. When analyzing IL-17A in parallel, PHA and antigens induced detectable responses in ELISpot as well as in ELISA. Hypothesizing that the lack of detectable IL-21 levels after antigenic stimulation was due to a combination of low frequencies of IL-21-secreting cells and consumption of IL-21 by cellular receptors during cell culture, PBMCs (n=18) were stimulated in the presence of the neutralizing detection mAb. When preventing IL-21 from interacting with its receptor, increased IL-21 levels were found by ELISA after PHA activation and IL-21 could also be measured after antigen stimulation. ELISpot results were unaffected by the addition of the neutralizing mAb. In conclusion, IL-21 secreted by low frequencies of antigen-specific ex vivo-stimulated PBMC can be difficult to detect by ELISA but prevention of IL-21 interaction with its receptor leads to detectable IL-21 levels. In ELISpot, where the cytokine is captured by mAbs on a solid phase immediately upon secretion, blocking the receptor interaction does not affect the detection of IL-21-secreting cells.


Subject(s)
Enzyme-Linked Immunosorbent Assay , Enzyme-Linked Immunospot Assay , Interleukins/analysis , Interleukins/immunology , Receptors, Interleukin-21/antagonists & inhibitors , T-Lymphocytes, Helper-Inducer/immunology , Antibodies, Monoclonal/immunology , Candida albicans/immunology , Cell Line , HEK293 Cells , Humans , Interleukin-17/immunology , Interleukin-17/metabolism , Leukocytes, Mononuclear/immunology , Phytohemagglutinins/immunology , Tetanus Toxoid/immunology
6.
J Invest Dermatol ; 134(10): 2639-2647, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24756111

ABSTRACT

Sézary syndrome is an aggressive cutaneous T-cell lymphoma. The malignant cells (Sézary cells) are present in skin, lymph nodes, and blood, and express constitutively activated signal transducer and activator of transcription (STAT)3. STAT3 can be activated by IL-21 in vitro and the IL-21 gene itself is a STAT3 target gene, thereby creating an autocrine positive feedback loop that might serve as a therapeutic target. Sézary cells underwent apoptosis when incubated with Stattic, a selective STAT3 inhibitor. STAT3 activation in Sézary cells did not affect expression of the supposed anti-apoptotic STAT3 target genes BCL2, BCL-xL, and SURVIVIN, whereas expression of (proto)oncogenes miR-21, TWIST1, MYC, and PIM1 was significantly increased. CD3/CD28-mediated activation of Sézary cells induced IL-21 expression, accompanied by STAT3 activation and increased proliferation. Blocking IL-21 in CD3/CD28-activated cells had no effects, whereas Stattic abrogated IL-21 expression and cell proliferation. Thus, specific inhibition of STAT3 is highly efficient in the induction of apoptosis of Sézary cells, likely mediated via the regulation of (proto)oncogenes. In contrast, blocking IL-21 alone seems insufficient to affect STAT3 activation, cell proliferation, or apoptosis. These data provide further insights into the pathogenic role of STAT3 in Sézary syndrome and strengthen the notion that STAT3 represents a promising therapeutic target in this disease.


Subject(s)
Interleukins/physiology , STAT3 Transcription Factor/physiology , Sezary Syndrome/physiopathology , Signal Transduction/physiology , Skin Neoplasms/physiopathology , Aged , Aged, 80 and over , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Cyclic S-Oxides/pharmacology , Female , Humans , Male , Middle Aged , Receptors, Interleukin-21/antagonists & inhibitors , Receptors, Interleukin-21/drug effects , Receptors, Interleukin-21/physiology , Recombinant Fusion Proteins/pharmacology , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/drug effects , Sezary Syndrome/drug therapy , Signal Transduction/drug effects , Skin Neoplasms/drug therapy
7.
J Clin Pharmacol ; 54(1): 14-22, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23913720

ABSTRACT

Safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of ATR-107, a fully human monoclonal anti-IL-21 receptor (IL-21R) antibody, administered as ascending single doses, subcutaneously or intravenously, was evaluated in a placebo-controlled, double-blind trial in healthy subjects. The dose levels were 3-300 mg by SC and 30-120 mg by IV. The most important adverse events were hypersensitivity reactions occurring in three out of six subjects in 300 mg SC cohort and considered as dose limiting toxicity. More than 75% of the subjects who received ATR-107 developed anti-drug antibodies (ADAs), which had no discernible impact on PK or safety. The PK of ATR-107 appeared to be dose -proportional. T1/2 was shorter than typical therapeutic antibodies. Bioavailability of ATR-107 was about 30%. IL-21R occupancy was measured in circulating B cells in the 60 and 120 mg IV cohort. The data indicated that single dose of ATR-107 was able to maximally occupy IL-21Rs through at least Day 42. Further escalation in the FIH study was halted partially due to the high rates of ADA formation. In conclusion, ATR-107 had a prolonged PD effect measured by IL-21R occupancy; was highly immunogenic after single dose administration and had PK properties with rapid clearance and low bioavailability.


Subject(s)
Antibodies, Monoclonal/pharmacology , Receptors, Interleukin-21/antagonists & inhibitors , Adolescent , Adult , Antibodies, Monoclonal/adverse effects , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal, Humanized , Biological Availability , Cohort Studies , Dose-Response Relationship, Drug , Double-Blind Method , Electrocardiography/drug effects , Female , Half-Life , Humans , Injections, Intravenous , Injections, Subcutaneous , Male , Middle Aged , Young Adult
8.
J Transl Med ; 8: 51, 2010 May 28.
Article in English | MEDLINE | ID: mdl-20509950

ABSTRACT

BACKGROUND: In preparation for potential clinical development of Ab-01, an antagonistic antibody directed against the IL21R, studies were undertaken to address translational medicine needs that fall into four categories: 1) development of a pharmacodynamic biomarker assay suitable for use in the clinic, 2) demonstration that Ab-01 has the desired biological activity in vitro and in vivo in cynomolgus monkeys, the preferred safety study species, 3) pre-clinical in vivo proof-of-concept that the assay can be used to detect Ab-01 pharmacodynamic (PD) activity in treated subjects, and 4) comprehensive assessment of the agonistic potential of Ab-01 when cross-linked. This report and a recently published companion report address the first three of these needs. The fourth has been addressed in a separate study. METHODS: Genes that change RNA expression upon ex vivo rhIL21 stimulation of whole blood were identified in human and cynomolgus monkey. The inhibitory effects of exogenously added Ab-01 were measured ex vivo in human and monkey, and the in vivo inhibitory effects of Ab-01 treatment were measured in monkey. RESULTS: Stimulation of whole human blood for 2 hours with rhIL21 induced robust increases in RNA expression of 6 genes. This response was blocked by Ab-01, indicating that the assay is suitable for measuring Ab-01 activity in blood. rhIL21 induced expression of a similar set of genes in cynomolgus monkey blood. This response was blocked with Ab-01, thus demonstrating that Ab-01 has the desired activity in the species, and that safety studies done in cynomolgus monkeys are relevant. Proof -of-concept for using this assay system to detect PD activity in vivo was generated by measuring the response in monkey blood to ex vivo rhIL21 stimulation before and 5 minutes following in vivo Ab-01 administration. CONCLUSIONS: A robust PD biomarker assay suitable for clinical use has been developed in human whole blood. The successful adaptation of the assay to cynomolgus monkeys has enabled the demonstration of Ab-01 activity both in vitro and in vivo in monkey, thus validating the use of this species in safety studies and establishing proof-of-concept for using this PD assay system to aid in dose selection in clinical studies.


Subject(s)
Antibodies/pharmacology , Biological Assay/methods , Receptors, Interleukin-21/antagonists & inhibitors , Receptors, Interleukin-21/blood , Animals , Antibodies/immunology , Antibodies/therapeutic use , Biomarkers/blood , Gene Expression Regulation/drug effects , Humans , Interleukins/immunology , Macaca fascicularis/immunology , Receptors, Interleukin-21/immunology , Signal Transduction/drug effects , Time Factors , Titrimetry
9.
MAbs ; 2(3): 335-46, 2010.
Article in English | MEDLINE | ID: mdl-20424514

ABSTRACT

Using phage display, we generated a panel of optimized neutralizing antibodies against the human and mouse receptors for interleukin 21 (IL-21), a cytokine that is implicated in the pathogenesis of many types of autoimmune disease. Two antibodies, Ab-01 and Ab-02, which differed by only four amino acids in V(L) CDR3, showed potent inhibition of human and mouse IL-21R in cell-based assays and were evaluated for their pharmacological and pharmacodynamic properties. Ab-01, but not Ab-02, significantly reduced a biomarker of disease (anti-dsDNA antibodies) and IgG deposits in the kidney in the MRL-Fas(lpr) mouse model of lupus, suggesting that anti-IL-21R antibodies may prove useful in the treatment of lupus. Ab-01 also had a consistently higher exposure (AUC(0-infinity)) than Ab-02 following a single dose in rodents or cynomolgus monkeys (2-3-fold or 4-7-fold, respectively). Our data demonstrate that small differences in CDR3 sequences of optimized antibodies can lead to profound differences in in vitro and in vivo properties, including differences in pharmacological activity and pharmacokinetic profiles. The lack of persistent activity of Ab-02 in the MRL-Fas(lpr) mouse lupus model may have been a consequence of faster elimination, reduced potency in blocking the effects of mouse IL-21R, and more potent/earlier onset of the anti-product response relative to Ab-01.


Subject(s)
Antibodies, Anti-Idiotypic/administration & dosage , Antibodies, Monoclonal/administration & dosage , Antibodies, Neutralizing/administration & dosage , Lupus Erythematosus, Systemic/therapy , Receptors, Interleukin-21/antagonists & inhibitors , Animals , Antibodies, Anti-Idiotypic/immunology , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Neutralizing/immunology , Cells, Cultured , Complementarity Determining Regions/genetics , Disease Models, Animal , Female , Humans , Injections, Intraperitoneal , Injections, Intravenous , Injections, Subcutaneous , Interleukins/immunology , Lupus Erythematosus, Systemic/immunology , Macaca fascicularis , Male , Mice , Mice, Inbred MRL lpr , Mice, Inbred Strains , Rats , Rats, Sprague-Dawley , Receptors, Interleukin-21/immunology
10.
Arthritis Rheum ; 56(4): 1152-63, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17393408

ABSTRACT

OBJECTIVE: Interleukin-21 (IL-21) is a T cell-derived cytokine that modulates T cell, B cell, and natural killer cell responses. In this study, the effects of blocking IL-21 were examined in 2 rodent models of rheumatoid arthritis (RA) to determine whether IL-21 contributes to their pathologic processes. METHODS: DBA/1 mice were immunized with bovine type II collagen and then treated with murine IL-21 receptor Fc fusion protein (IL-21R.Fc), which was initiated after the onset of arthritis symptoms in 10% of the cohort. The mice were assessed 3 times per week for signs of disease, including histologic features as well as serum cytokine, Ig, and cytokine messenger RNA (mRNA) levels in the paws. In a separate experiment, Lewis rats were immunized with Freund's complete adjuvant followed by administration of IL-21R.Fc at the peak of inflammation in the joints. Rats were assessed daily for histologic features and for scoring of arthritis severity. In addition, the effects of IL-21R.Fc on the production of interferon-gamma (IFNgamma) by T cells were examined. RESULTS: Treatment of DBA/1 mice with IL-21R.Fc reduced the clinical and histologic signs of collagen-induced arthritis. Nonspecific IgG1 levels were decreased in response to treatment. The levels of IL-6 mRNA in the paws and the serum IL-6 levels were decreased after treatment with IL-21R.Fc. IFNgamma mRNA levels were increased in the paws, and the addition of IL-21R.Fc to collagen-activated lymph node cultures enhanced the levels of IFNgamma. Collagen-specific spleen cell responses in IL-21R.Fc-treated mice were observed as reduced levels of IFNgamma and increased levels of IL-6. Treatment of Lewis rats with IL-21R.Fc after induction of adjuvant-induced arthritis resulted in reversal of disease signs and improvements in histologic parameters. CONCLUSION: These findings demonstrate a pathogenic role for IL-21 in animal models of RA, and support consideration of IL-21 as a therapeutic target in human RA.


Subject(s)
Arthritis, Experimental/prevention & control , Immunoglobulin Fc Fragments/administration & dosage , Interleukin-21 Receptor alpha Subunit/administration & dosage , Interleukins/antagonists & inhibitors , Receptors, Interleukin-21/antagonists & inhibitors , Recombinant Fusion Proteins/administration & dosage , Animals , Arthritis, Experimental/metabolism , Arthritis, Experimental/pathology , Cells, Cultured , Cytokines/blood , Cytokines/genetics , Dose-Response Relationship, Drug , Gene Expression , Interleukin-21 Receptor alpha Subunit/metabolism , Interleukins/metabolism , Lymph Nodes/drug effects , Lymph Nodes/metabolism , Lymphocyte Activation , Male , Mice , Mice, Inbred DBA , RNA, Messenger/metabolism , Rats , Rats, Inbred Lew , Rats, Sprague-Dawley , Receptors, Interleukin-21/metabolism , Receptors, Tumor Necrosis Factor/administration & dosage , Receptors, Tumor Necrosis Factor/antagonists & inhibitors , Receptors, Tumor Necrosis Factor/genetics , Spleen/drug effects , Spleen/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism
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