Suppression of autoreactive T and B lymphocytes by anti-annexin A1 antibody in a humanized NSG murine model of systemic lupus erythematosus.

Systemic lupus erythematosus is a chronic inflammatory disease which involves multiple organs. Self-specific B and T cells play a main role in the pathogenesis of lupus and have been defined as a logical target for selective therapy. The protein annexin A1 (ANX A1) is a modulator of the immune system involving many cell types. An abnormal expression of ANX A1 was found on activated B and T cells during autoimmunity, suggesting its importance as a potential therapeutic target. We hypothesize that it may be possible to down-regulate the activity of autoreactive T and B cells from lupus patients in a humanized immunodeficient mouse model by treating them with an antibody against ANX A1. When cultured in the presence of anti-ANX A1, peripheral blood mononuclear cells (PBMC) from lupus patients showed a decreased number of immunoglobulin (Ig)G anti-dsDNA antibody-secreting plasma cells, decreased T cell proliferation and expression of activation markers and increased B and T cell apoptosis. We employed a humanized model of SLE by transferring PBMCs from lupus patients to immunodeficient non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. The humanized animals presented autoantibodies, proteinuria and immunoglobulin deposition in the renal glomeruli. Treatment of these NOD-SCID mice with an anti-ANX A1 antibody prevented appearance of anti-DNA antibodies and proteinuria, while the phosphate-buffered saline (PBS)-injected animals had high levels after the transfer. The treatment reduced the levels of autoantibodies to several autoantigens, lupus-associated cytokines and disease symptoms.

Protein-engineered molecules carrying GAD65 epitopes and targeting CD35 selectively down-modulate disease-associated human B lymphocytes.

Type 1 diabetes mellitus is an autoimmune metabolic disorder characterized by chronic hyperglycemia, the presence of autoreactive T and B cells and autoantibodies against self-antigens. A membrane-bound enzyme on the pancreatic beta-cells, glutamic acid decarboxylase 65 (GAD65), is one of the main autoantigens in type 1 diabetes. Autoantibodies against GAD65 are potentially involved in beta-cell destruction and decline of pancreatic functions. The human complement receptor type 1 (CD35) on B and T lymphocytes has a suppressive activity on these cells. We hypothesized that it may be possible to eliminate GAD65-specific B cells from type 1 diabetes patients by using chimeric molecules, containing an anti-CD35 antibody, coupled to peptides resembling GAD65 B/T epitopes. These molecules are expected to selectively bind the anti-GAD65 specific B cells by the co-cross-linking of the immunoglobulin receptor and CD35 and to deliver a suppressive signal. Two synthetic peptides derived from GAD65 protein (GAD65 epitopes) and anti-CD35 monoclonal antibody were used for the construction of two chimeras. The immunomodulatory activity of the engineered antibodies was tested in vitro using peripheral blood mononuclear cells (PBMCs) from type 1 diabetes patients. A reduction in the number of anti-GAD65 IgG antibody-secreting plasma cells and increased percentage of apoptotic B lymphocytes was observed after treatment of these PBMCs with the engineered antibodies. The constructed chimeric molecules are able to selectively modulate the activity of GAD65-specific B lymphocytes and the production of anti-GAD65 IgG autoantibodies by co-cross-linking of the inhibitory CD35 and the B cell antigen receptor (BCR). This treatment presents a possible way to alter the autoimmune nature of these cells.

Target silencing of disease-associated B-lymphocytes by chimeric molecules in SCID model of pristane-induced autoimmunity.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the generation of autoantibodies against a diverse array of self-antigens. The B cells producing immunoglobulin G (IgG) antibodies to double-stranded DNA appear to play a main role in the disease progression. Their specific elimination is a reasonable mechanism for effective therapy of SLE. The presently used approaches for silencing autoreactive disease-associated B cells are nonspecific and more precise therapies are needed. We have previously constructed a chimeric protein molecule consisting of several DNA-mimotope peptides coupled to a rat monoclonal anti-mouse CD32 (FcγRIIb) antibody. The mineral oil pristane induces a lupus-like syndrome in non-autoimmune mice leading to the development of glomerulonephritis and lupus-associated autoantibodies. In the present paper, using a pristane-induced autoimmune model in SCID mice, we analyzed the ability of the chimeric antibody to suppress selectively the autoreactive B lymphocytes by cross-linking B-cell surface immunoglobulin receptors with the inhibitory IgG FcγRIIb receptors. Treatment with DNA-like chimeric molecules inhibited B- and T-cell proliferation, restricted the number of anti-DNA antibody-producing cells and suppressed the generation of IgG anti-DNA antibodies. In contrast, phosphate buffered saline (PBS)-injected control mice experienced an increase of disease-associated antibody levels and developed glomerulonephritis similar to pristane-treated donor Balb/c mice.

Inflammation-induced enhancement of IgG immunoreactivity.

Natural polyreactive IgG antibodies are found in the sera of all healthy individuals. The in vitro exposure of pooled human IgG to protein-destabilizing chemical or physical factors has been previously shown to result in the exposure of their "hidden" polyspecificity. We hypothesize that such an enhancement of their pre-existing immunoreactivity may occur in vivo in the aggressive microenvironment of inflammation sites. An increase in the antigen binding intensity as well as of the number of recognized antigens was observed in the sera of IgG-infused immunodeficient SCID mice with induced local inflammation. The expansion of the IgG pathogen-binding repertoire may have important biological consequences.

IgM-enriched human intravenous immunoglobulin suppresses T lymphocyte functions in vitro and delays the activation of T lymphocytes in hu-SCID mice.

Previous studies of an experimental human immunoglobulin preparation for intravenous use, containing normal pooled IgM (IVIgM), have shown its beneficial therapeutic effect in experimental autoimmune diseases. The mechanisms of its immunomodulatory activity remain however, poorly understood. In the experiments reported here, IVIgM inhibited the proliferation of various autonomously growing human lymphoid cell lines in vitro, as well as of MLR- and of PHA-stimulated human T-lymphocytes. These effects of IVIgM were observed at non-apoptotic concentrations and were stronger on a molar basis than those of normal pooled IgG for intravenous use (IVIg). Both preparations, when administered to SCID mice, repopulated with human peripheral blood mononuclear cells, delayed the expression of the early activation marker CD69 on both human CD4+ and CD8+ T-lymphocytes, activated by the mouse antigenic environment. The data obtained show that normal pooled human IgM exerts a powerful antiproliferative effect on T-cells that is qualitatively similar but quantitatively superior to that of therapeutic IVIg. Our results suggest that infusions with IVIgM might have a significant beneficial immunomodulating activity in patients with selected autoimmune diseases.