This study was performed under a protocol approved by the Institutional Animal Care and Use Committee of Akita University Graduate School of Medication and Faculty of Medicine

This study was performed under a protocol approved by the Institutional Animal Care and Use Committee of Akita University Graduate School of Medication and Faculty of Medicine. == Experimental protocol == An optimized dose of the humanized anti-hCD20 monoclonal antibody TKM-011 (250 g in 250 L of PBS) or 250 L of PBS alone was administered intraperitoneally to hCD20- and hFcR-expressing IL-10-GFP dnTGF-RII mice using a 26-gauge needle every week starting at 46 weeks of age. of liver memory CD8+T cells and B cells (r= 0.6423,p= 0.0054) were apparent. Accompanying Candesartan cilexetil (Atacand) these changes was a dramatic reduction in anti-mitochondrial antibodies (AMAs), interleukin (IL)-12p40 and IL-5, and elevated levels of the anti-inflammatory chemokine CXCL1/KC. In mice that developed ADAs, clinical improvements were less pronounced. Sustained treatment with B cell-targeted therapies may broadly inhibit effector pathways in PBC, but may need to be administered early in the natural history of PBC. Keywords:anti-drug antibodies (ADAs), anti-mitochondrial antibodies (AMAs), B cell depletion therapy, human anti-chimeric antibodies (HACAs), humanized anti-human CD20 antibody, mouse anti-human antibodies (MAHAs), primary biliary cholangitis (PBC) == Introduction == The destruction of biliary epithelial cells (BECs) in patients with primary biliary cholangitis (PBC) is at least partially secondary to development of autoreactive CD8+T cells (13). In addition, there is evidence that B cells and serum anti-mitochondrial antibodies (AMAs) exacerbate biliary pathology through their effects on apoptotic biliary cells as Candesartan cilexetil (Atacand) well as through B-cell regulatory mechanisms; inflammatory liver infiltrates include B-cell foci (4). Although there is no direct correlation between AMA titer and disease severity, a variety of data support a role of B cells in the immunopathology of PBC (510). For example, dimeric IgA-AMA complexes facilitate induction of BEC apoptosis (8) and AMAs enhance cross-presentation and generation of pyruvate dehydrogenase complex-E2 (PDC-E2)-specific cytotoxic T cell responses in the presence of PDC-E2-pulsed antigen-presenting cells (5). In combination with AMAs, unmodified PDC-E2 localized in apoptotic BECs facilitates production of proinflammatory cytokines from monocyte-derived macrophages in PBC (6,7). These results have led us to postulate that the cellular responses involved in loss of tolerance and biliary pathology include contributions from both B and T cells and that biliary destruction is an orchestrated multi-lineage response (11). We have previously reported the use of dominant negative TGF- receptor II (dnTGF-RII) mice as a model for human PBC (12). The expression of dnTGF-RII is limited to selected cell lineages including CD4+, CD8+, and CD1d-restricted natural killer T (NKT) cells; B cell function is normal in this model (12,13). We previously demonstrated that treatment of juvenile mice with a monoclonal anti-mouse CD20 antibody was effective in preventing PBC, but that responses were reduced when treatment was initiated in older mice (14). This may reflect a limited contribution by B cells in the pathology of advanced bile duct damage during human PBC (15). There is considerable interest in expanding B cell-targeted therapies for human autoimmune diseases (1627). A chimeric antibody against human CD20 (hCD20), rituximab, showed limited clinical efficacy in human PBC (2830). One explanation for its limited efficacy was the appearance of anti-drug antibodies (ADAs; more specifically, human anti-chimeric antibodies, HACAs) as observed in other human autoimmune diseases (3137). Moreover, HACAs have been associated with adverse events such as serum sickness (3840). In this study, we studied a novel humanized IgG1 antibody against hCD20 that is less immunogenic compared with chimeric antibodies, enabling intermittent administration with much lower risk of anti-humanized antibody development during treatment of human PBC [Figure1A; (41)]. This antibody (TKM-011, formerly called BM-ca) has significant direct cytotoxic activity as well as antibody-dependent cell-mediated cytotoxicity (ADCC) activity, similar to rituximab and ofatumumab (4244). To better reflect human immunobiology, we generated dnTGF-RII mice expressing hCD20 as well as human Fc receptors (hFcRs) (Figure1B). After treating these animals with the humanized anti-hCD20 antibody, CDF TKM-011, we observed a significant reduction in portal inflammation, decreased liver-infiltrating Candesartan cilexetil (Atacand) mononuclear cells and a reduction in Candesartan cilexetil (Atacand) liver CD8+T cells. Importantly, direct correlations were apparent between the numbers of liver non-B cells and B cells and between the numbers of liver memory CD8+T cells and B cells. These effects were less pronounced in mice that developed mouse anti-humanized antibodies (MAHAs). These data suggest that the presence.