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 OutcomeABSTRACT
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 therapyABSTRACT
Interleukin-21 receptor (IL-21R) is widely expressed in lymphocytes, and plays an important role in immunological cell proliferation and cytokine production. The present study aims to express a recombinant extracellular domain of human IL-21R (rhIL-21R-ECD) with high yield, and to screen the anti-IL-21R single-chain variable fragments (scFvs) from a synthetic human phage display library. The rhIL-21R-ECD, being expressed mainly as insoluble inclusion bodies in Escherichia coli BL21 (DE3), was purified and refolded. ELISA analysis showed that the refolded rhIL-21R-ECD bound to its ligand IL-21 in a concentration-dependent manner. Using a phage display technique, anti-IL-21R scFvs were screened from a naïve human phage display library by biopanning. After four rounds of panning, positive clones were isolated, sequenced, and characterized. The clone with highest activity was designated as C2. Flow cytometry analysis showed that the scFv C2 could recognize IL-21R on Jurkat cells. Furthermore, proliferation assay revealed a concentration-dependent inhibitory effect of C2 on the Jurkat cell, with fifty percent inhibitory concentration (IC(50)) of 78 nM. A human scFv antibody C2 with a high binding specificity to IL-21R was isolated and characterized. The antibody showed a concentration-dependent inhibitory effect on Jurkat cell proliferation.