Marilyn R. Kehry

Depletion of B Cells in Murine Lupus: Efficacy and Resistance

In mice, genetic deletion of B cells strongly suppresses systemic autoimmunity, providing a rationale for depleting B cells to treat autoimmunity. In fact, B cell depletion with rituximab is approved for rhematoid arthritis patients, and clinical trials are underway for systemic lupus erythematosus. Yet, basic questions concerning mechanism, pathologic effect, and extent of B cell depletion cannot be easily studied in humans. To better understand how B cell depletion affects autoimmunity, we have generated a transgenic mouse expressing human CD20 on B cells in an autoimmune-prone MRL/MpJ-Faslpr (MRL/lpr) background. Using high doses of a murine anti-human CD20 mAb, we were able to achieve significant depletion of B cells, which in turn markedly ameliorated clinical and histologic disease as well as antinuclear Ab and serum autoantibody levels. However, we also found that B cells were quite refractory to depletion in autoimmune-prone strains compared with nonautoimmune-prone strains. This was true with multiple anti-CD20 Abs, including a new anti-mouse CD20 Ab, and in several different autoimmune-prone strains. Thus, whereas successful B cell depletion is a promising therapy for lupus, at least some patients might be resistant to the therapy as a byproduct of the autoimmune condition itself.

Dendritic cells and B cells maximize mucosal Th1 memory response to herpes simplex virus

Although the importance of cytotoxic T lymphocytes and neutralizing antibodies for antiviral defense is well known, the antiviral mechanism of Th1 remains unclear. We show that Th1 cells mediate noncytolytic antiviral protection independent of direct lysis through local secretion of IFN-γ after herpes simplex virus (HSV) 2 infection. IFN-γ acted on stromal cells, but not on hematopoietic cells, to prevent further viral replication and spread throughout the vaginal mucosa. Importantly, unlike other known Th1 defense mechanisms, this effector function did not require recognition of virally infected cells via MHC class II. Instead, recall Th1 response was elicited by MHC class II+ antigen-presenting cells at the site of infection. Dendritic cells (DCs) were not required and only partially sufficient to induce a recall response from memory Th1 cells. Importantly, DCs and B cells together contributed to restimulating memory CD4 T cells to secrete IFN-γ. In the absence of both DCs and B cells, immunized mice rapidly succumbed to HSV-2 infection and death. Thus, these results revealed a distinct mechanism by which memory Th1 cells mediate noncytolytic IFN-γ–dependent antiviral protection after recognition of processed viral antigens by local DCs and B cells.

Suppression of Proteoglycan-Induced Arthritis by Anti-CD20 B Cell Depletion Therapy Is Mediated by Reduction in Autoantibodies and CD4+ T Cell Reactivity

B cells have been implicated in the pathogenesis of rheumatoid arthritis (RA) since the discovery of RA as an autoimmune disease. There is renewed interest in B cells in RA based on the clinical efficacy of B cell depletion therapy in RA patients. Although, reduced titers of rheumatoid factor and anti-cyclic citrullinated peptide Abs are recorded, the mechanisms that convey clinical improvement are incompletely understood. In the proteoglycan-induced arthritis (PGIA) mouse model of RA, we reported that Ag-specific B cells have two important functions in the development of arthritis. PG-specific B cells are required as autoantibody-producing cells as well as Ag-specific APCs. Herein we report on the effects of anti-CD20 mAb B cell depletion therapy in PGIA. Mice were sensitized to PG and treated with anti-CD20 Ab at a time when PG-specific autoantibodies and T cell activation were evident but before acute arthritis. In mice treated with anti-CD20 mAb, development of arthritis was significantly reduced in comparison to control mAb-treated mice. B cell depletion reduced the PG-specific autoantibody response. Furthermore, there was a significant reduction in the PG-specific CD4+ T cell recall response as well as significantly fewer PG-specific CD4+ T cells producing IFN-γ and IL-17, but not IL-4. The reduction in PG-specific T cells was confirmed by the inability of CD4+ T cells from B cell-depleted mice to adoptively transfer disease into SCID mice. Overall, B cell depletion during PGIA significantly reduced disease and inhibited both autoreactive B cell and T cell function.

B-cell depletion with anti-CD20 ameliorates autoimmune cholangitis but exacerbates colitis in transforming growth factor-β receptor II dominant negative mice

The treatment of primary biliary cirrhosis (PBC) with conventional immunosuppressive drugs has been relatively disappointing and there have been few efforts in defining a role for the newer biological agents useful in rheumatoid arthritis and other systemic autoimmune diseases. In this study we took advantage of transforming growth factor‐β (TGF‐β) receptor II dominant negative (dnTGF‐βRII) mice, a mouse model of autoimmune cholangitis, to address the therapeutic efficacy of B‐cell depletion using anti‐CD20. Mice were treated at either 4‐6 weeks of age or beginning at 20‐22 weeks of age with intraperitoneal injections of anti‐CD20 every 2 weeks. We quantitated B‐cell levels in all mice as well as antimitochondrial antibodies (AMA), serum and hepatic levels of proinflammatory cytokines, and histopathology of liver and colon. In mice whose treatment was initiated at 4‐6 weeks of age, anti‐CD20 therapy demonstrated a significantly lower incidence of liver inflammation associated with reduced numbers of activated hepatic CD8+ T cells. However, colon inflammation was exacerbated. In contrast, in mice treated at 20‐22 weeks of age, anti‐CD20 therapy had relatively little effect on either liver or colon disease. As expected, all treated animals had reduced levels of B cells, absence of AMA, and increased levels in sera of tumor necrosis factor alpha (TNF‐α), interleukin 6 (IL‐6), and chemokine (C‐C motif) ligand (CCL2) (monocyte chemoattractant protein 1 [MCP‐1]). Conclusion: These data suggest potential usage of anti‐CD20 in early PBC resistant to other modalities, but raise a cautionary note regarding the use of anti‐CD20 in inflammatory bowel disease. (HEPATOLOGY 2009.)

Prolonged effects of short‐term anti‐CD20 B cell depletion therapy in murine systemic lupus erythematosus

OBJECTIVE Although B cells are implicated in the pathogenesis of systemic lupus erythematosus, the role of B cell depletion (BCD) as a treatment is controversial, given the variable benefit in human disease. This study was undertaken to test the effects of BCD therapy in a murine lupus model to better understand the mechanisms, heterogeneity, and effects on disease outcomes. METHODS (NZB x NZW)F(1) female mice with varying degrees of disease severity were treated with an anti-mouse CD20 (anti-mCD20) antibody (IgG2a), BR3-Fc fusion protein (for BAFF blockade), or control anti-human CD20 monoclonal antibody (approximately 10 mg/kg each). Tissue samples were harvested and analyzed by flow cytometry. The development and extent of nephritis were assessed by monitoring proteinuria (using a urine dipstick) and by immunohistochemical analysis of the kidneys. Serum immunoglobulin levels were measured by enzyme-linked immunosorbent assay. RESULTS After a single injection of anti-mCD20, BCD was more efficient in the peripheral blood, lymph nodes, and spleen compared with the bone marrow and peritoneum of normal mice as well as younger mice with lupus. Since depletion of the marginal zone and peritoneal B cells was incomplete and variable, particularly in older mice with established nephritis, a strategy of sequential weekly dosing was subsequently used, which improved the extent of depletion. BAFF blockade further enhanced depletion in the spleen and lymph nodes. Early BCD therapy delayed disease onset, whereas BCD therapy in mice with advanced disease reduced the progression of nephritis. These effects were long-lasting, even after B cell reconstitution occurred, and were associated with a reduction in T cell activation but no significant change in autoantibody production. CONCLUSION The lasting benefit of a short course of BCD therapy in lupus-prone mice with an intact immune system and established disease highlights the validity of this treatment approach.

Humoral Immunity to Vimentin Is Associated with Cardiac Allograft Injury in Nonhuman Primates

Immunity to autologous protein has not previously been described following nonhuman primate cardiac transplant. Native hearts and cardiac allografts from cynomolgus monkeys were assessed by immunohistology for vimentin, a highly conserved intermediate filament protein. IgM and IgG to vimentin were measured in serial sera from untreated (n = 4) or cyclosporine (CsA)‐treated (n = 8, 2 with ATG) cardiac allograft recipients, and in groups treated with anti‐CD154 antibody with (n = 6) or without ATG (n = 28). IgM or IgG reactive with vimentin was elaborated within 30 days with unmodified acute rejection (3/4) or in CsA‐treated animals (5/6). CD154 blockade did not prevent anti‐vimentin IgM (14/28) but tended to delay the IgG response during therapy (anti‐CD154: 8/28, p = 0.10 vs. CsA; anti‐CD154+ATG: 2/6). CAV and alloantibody were seen in 25 of 26 animals with grafts surviving over 30 days, including seven animals without increasing anti‐vimentin antibody. Anti‐vimentin antibodies and vascular complement deposition were found in rejected hearts. Acute and chronic alloimmunity disrupt modulation of autoreactivity to vimentin through pathways, which are resistant to CsA, but may be partially regulated by CD154.

B cell depletion therapy exacerbates murine primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is considered a model autoimmune disease due to the clinical homogeneity of patients and the classic hallmark of antimitochondrial antibodies (AMAs). Indeed, the presence of AMAs represents the most highly directed and specific autoantibody in autoimmune diseases. However, the contribution of B cells to the pathogenesis of PBC is unclear. Therefore, although AMAs appear to interact with the biliary cell apotope and contribute to biliary pathology, there is no correlation of disease severity and titer of AMAs. The recent development of well‐characterized monoclonal antibodies specific for the B cell populations, anti‐CD20 and anti‐CD79, and the development of a well‐defined xenobiotic‐induced model of autoimmune cholangitis prompted us to use these reagents and the model to address the contribution of B cells in the pathogenesis of murine PBC. Prior to the induction of autoimmune cholangitis, mice were treated with either anti‐CD20, anti‐CD79, or isotype‐matched control monoclonal antibody and followed for B cell development, the appearance of AMAs, liver pathology, and cytokine production. Results of the studies reported herein show that the in vivo depletion of B cells using either anti‐CD20 or anti‐CD79 led to the development of a more severe form of cholangitis than observed in control mice, which is in contrast with results from several other autoimmune models that have documented an important therapeutic role of B cell–specific depletion. Anti‐CD20/CD79–treated mice had increased liver T cell infiltrates and higher levels of proinflammatory cytokines. Conclusion: Our results reflect a novel disease‐protective role of B cells in PBC and suggest that B cell depletion therapy in humans with PBC should be approached with caution (HEPATOLOGY 2011:53:527‐535)

Loss of intra-islet CD20 expression may complicate efficacy of B-cell-directed type 1 diabetes therapies.

OBJECTIVE Consistent with studies in NOD mice, early clinical trials addressing whether depletion of B cells by the Rituximab CD20-specific antibody provides an effective means for type 1 diabetes reversal have produced promising results. However, to improve therapeutic efficacy, additional B-cell–depleting agents, as well as attempts seeking diabetes prevention, are being considered. RESEARCH DESIGN AND METHODS Autoantibodies, including those against insulin (IAAs), are used to identify at-risk subjects for inclusion in diabetes prevention trials. Therefore, we tested the ability of anti-CD20 to prevent diabetes in NOD mice when administered either before or after IAA onset. RESULTS The murine CD20-specific 18B12 antibody that like Rituximab, depletes the follicular (FO) but not marginal zone subset of B cells, efficiently inhibited diabetes development in NOD mice in a likely regulatory T-cell–dependent manner only when treatment was initiated before IAA detection. One implication of these results is that the FO subset of B cells preferentially contributes to early diabetes initiation events. However, most important, the inefficient ability of anti-CD20 treatment to exert late-stage diabetes prevention was found to be attributable to downregulation of CD20 expression upon B cell entry into pancreatic islets. CONCLUSIONS These findings provide important guidance for designing strategies targeting B cells as a potential means of diabetes intervention.

B-cell depletion using an anti-CD20 antibody augments antitumor immune responses and immunotherapy in nonhematopoetic murine tumor models.

The role played by B cells in cancer biology is complex and somewhat controversial. Previous studies using genetically engineered mice suggest that B cells may be immunosuppressive and inhibit tumor rejection. However, the effects of B-cell depletion employing an antibody in mice bearing solid tumors has not been tested owing to difficulties in making an effective antimouse CD20 antibody (similar to rituximab). Injection of a newly developed antimouse CD20 antibody was effective in depleting circulating B cells from blood and lymph nodes, although depletion was less complete in the spleen. B-cell depletion slowed the growth of new solid tumors (not expressing CD20) and retarded the growth of established tumors but did not induce tumor regression. However, when the antibody was combined with an active immunotherapy approach using an adenovirus vaccine expressing the human papilloma virus-E7 gene (Ad.E7) in mice bearing TC1 tumors (murine lung cancer cells expressing human papilloma virus-E7), we noted enhanced antitumor effects and increased numbers of tetramer+/CD8+ T cells within the spleens and activated CD8+ T cells within tumors. B-cell depletion using an anti-CD20 antibody was thus effective in retarding tumor growth in multiple solid tumor models and augmenting immunotherapy in a tumor vaccine model. These studies raise the possibility that B-cell depletion may be a useful adjunct in human immunotherapy trials.

B cell depletion inhibits spontaneous autoimmune thyroiditis in NOD.H-2h4 mice.

B cells are important for the development of most autoimmune diseases. B cell depletion immunotherapy has emerged as an effective treatment for several human autoimmune diseases, although it is unclear whether B cells are necessary for disease induction, autoantibody production, or disease progression. To address the role of B cells in a murine model of spontaneous autoimmune thyroiditis (SAT), B cells were depleted from adult NOD.H-2h4 mice using anti-mouse CD20 mAb. Anti-CD20 depleted most B cells in peripheral blood and cervical lymph nodes and 50–80% of splenic B cells. Flow cytometry analysis showed that marginal zone B cells in the spleen were relatively resistant to depletion by anti-CD20, whereas most follicular and transitional (T2) B cells were depleted after anti-CD20 treatment. When anti-CD20 was administered before development of SAT, development of SAT and anti-mouse thyroglobulin autoantibody responses were reduced. Anti-CD20 also reduced SAT severity and inhibited further increases in anti-mouse thyroglobulin autoantibodies when administered to mice that already had autoantibodies and thyroid inflammation. The results suggest that B cells are necessary for initiation as well as progression or maintenance of SAT in NOD.H-2h4 mice.