AUTOIMMUNE, CHOLESTATIC AND BILIARY DISEASE

Depletion of B Cells Induces Remission of Autoimmune Hepatitis in Mice Through Reduced Antigen Presentation and Help to T Cells Kathie Beland,1 Gabriel Marceau,1 Agathe Labardy,1 Sara Bourbonnais,1 and Fernando Alvarez1,2,3 Autoimmune hepatitis (AIH) is known as a T cell–mediated disease. However, AIH patients refractory to conventional treatment have been successfully treated with antiCD20-mediated B-cell depletion. The aim of this project was to understand the immunological changes underlying the AIH remission caused by B-cell depletion in an experimental model of AIH. C57BL/6 AIH mice, xenoimmunized with DNA coding for human liver antigens, were treated with a single dose of depleting mouse anti-CD20 antibody at the peak of liver inflammation. Liver inflammation, alanine aminotransferase levels, chemokine (C-X-C) ligand 10 expression, and circulating B-cell, autoantibody, and total immunoglobulin G levels were monitored following depletion. T-cell and B-cell phenotype and function were characterized. Administration of a single dose of anti-CD20 resulted in a drastic reduction of liver inflammation accompanied by a significant reduction of alanine aminotransferase levels and of proinflammatory chemokine (C-X-C) ligand 10 expression. The treatment did not result in significant changes in total immunoglobulin G levels or autoantibodies. There were significantly more naive and less antigen-experienced CD41 and CD81 T cells, and T-cell proliferation was significantly reduced following anti-CD20 treatment. B cells served as antigen-presenting cells to CD41 T cells. Anti-CD20 treatment also led to a profound reduction of T follicular helper cells. Conclusion: B cells play an active role in the pathogenesis of AIH in antigen presentation processes and the modulation of T-cell functions and influence the T follicular helper–cell population; this active role of B cells could explain the success of B-cell depletion for remission of AIH despite its classification as a T cell–mediated autoimmune liver disease. (HEPATOLOGY 2015;62:1511-1523)

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utoimmune hepatitis (AIH) is a disease of unknown etiology characterized by a loss of tolerance against liver autoantigens resulting in the progressive destruction of the hepatic parenchyma. AIH has been classified as a T cell–mediated autoimmune disease, with an important contribution of CD41 T helper 1 cells to its pathogenesis.1,2 Recent clinical observations have blurred the classification of autoimmune diseases as either B cell–mediated or T cell– mediated.3 It is now believed that most autoimmune

diseases, like immune responses to pathogens, involve both B cells and T cells in their pathogenesis.4 Numerous observations suggest that B cells are involved in AIH pathogenesis. A distinct feature of AIH compared to other liver diseases is that immunoglobulin-secreting plasma cells are abundantly present in liver inflammatory infiltrates, along with CD41 and CD81 T cells.2 AIH follows a chronic but fluctuating course, and the activation of B cells results in the characteristic hyper–immunoglobulin G (IgG) and

Abbreviations: AIH, autoimmune hepatitis; ALT, alanine aminotransferase; APC, antigen-presenting cell; CXCL10, chemokine (C-X-C) ligand 10; CXCR, chemokine (C-X-C motif) receptor; FTCD, formiminotransferase cyclodeaminase (mFTCD, murine FTCD); IFN, interferon; IgG, immunoglobulin G; IL, interleukin; MHC, major histocompatibility complex; PBS, phosphate-buffered saline; SEM, standard error of the mean; Tfh, follicular T helper (cell); TNF, tumor necrosis factor; Treg, regulatory T cell. From the 1Division of Gastroenterology, Hepatology and Nutrition, CHU Sainte-Justine, Montreal, QC, Canada; 2Microbiology and Immunology Department and 3Department of Pediatrics, University of Montreal, Montreal, QC, Canada Received December 12, 2014; accepted July 13, 2015. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.27991/suppinfo. Supported by the Canadian Liver Foundation (grant 2012, to F.A.). 1511

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circulating autoantibodies, a hallmark of AIH.1 Although not believed to be pathogenic,1 autoantibodies are a key aspect of AIH and have enabled the classification of the disease into two types. In type 2 AIH, autoantibodies share the same autoantigen as T cells with overlapping B-cell and T-cell epitopes5 and are directed against autoantigens mainly expressed in the liver, i.e., cytochrome P450 2D6 and formiminotransferase cyclodeaminase (FTCD).6,7 Another interesting observation is that titers of specific autoantibodies and levels of total IgG in sera of AIH patients correlate with disease activity.8 Recently, two studies9,10 reporting few AIH patients refractory to conventional treatment have described successful rescue therapy through B-cell depletion with rituximab, an anti-CD20 monoclonal antibody. Both studies described biochemical improvement, prednisone reduction, and/or weaning and complete remission with sustained B-cell depletion.9,10 Moreover, this remission is accompanied by a reduction of chemokine (C-X-C) ligand 10 (CXCL10, also interferon-c [IFN-c]–inducible protein 10), a biomarker of hepatic inflammation and fibrosis.11 These and other clinical studies in several autoimmune diseases highlight a role for B cells in the perpetuation of organ-specific inflammation and the usefulness of B cell–targeted therapy in autoimmune diseases classically believed to be T cell–mediated.3 However, how B-cell depletion can lead to remission of these T cell–mediated disease is unknown. B cells could be involved in the onset and perpetuation of many autoimmune diseases in several ways. Beyond their classical role as (auto-)antibody-secreting cells, B cells can be potent antigen-presenting cells (APCs) and lead to the activation of autoreactive T cells, perpetuating inflammation.4 They can modulate inflammation through the secretion of anti-inflammatory or proinflammatory cytokines such as interleukin-10 (IL10), IFN-c, and tumor necrosis factor-a (TNF-a).12 In addition, they can influence secondary lymphoid organ structure,4 impact antigen presentation,13 and influence follicular T helper (Tfh) cells.14 The broad range of immune functions that B cells display could explain how B-cell depletion can be effective in T cell–driven autoimmune diseases such as AIH.

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The aim of this study was to assess the outcome of Bcell depletion in an animal model of AIH and the immunological changes taking place following this intervention. Herein, we report that a single dose of anti-CD20 B-cell depleting antibodies was effective at inducing remission of liver inflammation in a mouse model of type 2 AIH. This anti-CD20–induced remission was driven by reduced autoantigen presentation to T cells. leading to lowered T-cell activation, reduced autoantigen-specific proliferation, and cytotoxic activity.

Materials and Methods Experimental Type 2 AIH and Anti-CD20 Antibody Treatment. Experimental AIH was induced in mice by xenoimmunization as described.15 DNAinjected mice were treated with a single intravenous injection of 250 lg of anti-CD20 antibodies (clone 5D2 IgG2a, kindly provided by Genentech, San Francisco, CA) or 100 lL of saline buffer (AIH controls) 7 months postxenoimmunization, when AIH is fully active in this mouse model.15 A second group of mice were treated at 5.5 months postxenoimmunization, with two dose of anti-CD20 antibody 4 weeks apart, to test whether anti-CD20 treatment can prevent AIH. Unless otherwise mentioned, mice were sacrificed 4 weeks after the last anti-CD20 treatment, by injection of sodium pentobarbital followed by bleeding through cardiac puncture. All experiments were carried out following protocols approved by the institutional committee for animal care (CHU Sainte-Justine) following guidelines published by the Canadian Council on Animal Care. Serum Alanine Aminotransferase Activity. Serum alanine aminotransferase (ALT) levels were measured in a Beckman-Synchron CX9 apparatus, from blood taken 2 days before and 4 weeks after (at sacrifice) the last anti-CD20 treatment. Histopathology. Liver sections were formalin-fixed, paraffin-embedded, stained with hematoxylin–phloxin– safran, and assessed blindly by two observers using the Ishak modified histological activity index scoring system.16 Data are expressed as the mean score of inflammation 6 standard error of the mean (SEM). Spleen architecture integrity following anti-CD20 treatment

Address reprint requests to: Fernando Alvarez, M.D., Division of Gastroenterology, Hepatology and Nutrition, CHU Sainte-Justine, 3175 C^ ote Ste-Catherine, Montr eal, QC, Canada, H3T 1C5. E-mail: [email protected]; tel: 11-514-345-4626; fax: 11-514-345-4999. C 2015 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27991 Potential conflict of interest: Nothing to report.

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was also assessed by hematoxylin–phloxin–safran coloration. Autoantibodies and Total Immunoglobulin Assays. Autoantibodies against murine FTCD (mFTCD) were measured by enzyme-linked immunosorbent assay using purified pMal-mFTCD fusion protein as described.17 Anti-mFTCD titers were log-transformed before statistical tests to ensure normal distribution. Total immunoglobulin levels were assessed by enzyme-linked immunosorbent assay by coating mouse sera (serial dilution from 1:1000 to 1:128,000) in 0.1 mM NaHCO3 pH 8 solution overnight at 48C. Plates were then washed and incubated with antimouse IgG conjugated with alkaline phosphatase (Jackson Laboratories, Bar Harbor, ME) at a 1:500 dilution. Plates were revealed as described.17 A standard curve was done using serial dilution of purified IgG (Sigma-Aldrich, St. Louis, MO) spanning 0.8-10 lg. Immunoglobulin concentration in mouse sera was calculated based on the standard curve and expressed in milligrams per milliliter. Lymphocyte Isolation and Purification. Lymphocytes were isolated from liver (liver infiltrating lymphocytes), blood, and spleen as described.17 For lymphoproliferation assays using CD191 B cells as APCs, CD191 B cells were purified and T cells were enriched from splenocytes using the RoboSep Mouse CD19 Positive Selection kit and the RoboSep Mouse T cell Enrichment kit following the manufacturer’s instructions (StemCell Technologies, Vancouver, Canada). Flow-Cytometric Analysis. The following antibodies/reagents were used for flow-cytometric analyses: antimouse CD45 (clone 30-F11), antimouse CD19 (6D5), antimouse CD3 (145-2C11), antimouse CD8 (53-6.7), antimouse CD4 (RM4.5), anti-CD62L (MEL-14), anti-CD44 (IM7), anti-B220 (RA3-6B2), anti-TNF-a (MP6-XT22), anti-IFN-c (XMG1.2), anti-IL-10 (JES516E3), anti-CD25 (PC61.5), anti-FoxP3 (FJK-16s), anti–B- and T-lymphocyte attenuator (8F4), anti-PD1 (J43), anti-granzyme B (NGZB), anti-CD107a (eBio1D4B), anti-lag3 (C9B7W), anti–major histocompatibility complex class II (MHCII; M5/114.15.2), streptavidin-phycoerythrin (all from eBioscience, San Diego, CA); anti-CD49b (DX5), anti-CD138 (281-2), anti-CD80 (16-10A1), anti-CD11b (M1/70) (Biolegend, San Diego, CA); anti–chemokine (C-X-C motif ) receptor 5 (CXCR5) biotin (2G8), anti-PD1 (J43) and 7-amino-actinomycin D (BD Biosciences, Canada). Acquisitions were performed on a Fortessa flow cytometer (BD Biosciences), and analysis used Flowjo software (Tree Star, Ashland, OR). Immunofluorescence. Cryopreserved liver sections fixed in methanol (7 lm) were blocked with bovine

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serum albumin and with avidin/biotin solutions (DakoCytomation, Denmark) and then incubated with antiCD19 fluorescein isothiocyanate (1D3, 1:100) and biotin-coupled antimouse CD8 or CD4 (53-6.7 and GK1.5, 1:100) (eBiosciences). Slides were then incubated with streptavidin-Cy3 (1:1000) (Jackson Laboratories). Slides were mounted with ProLong gold antifade reagent with 40 ,6-diamidino-2-phenylindole (Invitrogen, CA). CXCL10 Expression Analysis. Total RNA from liver was prepared using the RNeasy kit (Qiagen, Santa Clarita, CA). A DNA digestion was performed on each sample with RNAse-Free DNAse (Qiagen). Expression levels were measured using specific primers for CXCL10 with the One-Step reverse-transcription polymerase chain reaction kit (Qiagen) as described.18 Murine b-actin was used as internal reference. Fluorescence-Based Lymphoproliferation Assay. A carboxyfluorescein diacetate succinimidyl ester–based proliferation assay was performed as described.19 Cells were plated in complete Roswell Park Memorial Institute 1640 medium (10% fetal calf serum, 20 U/mL murine recombinant IL-2 [Invitrogen], 5 lM 2-mercaptoethanol) with 20 lg of purified mFTCD-maltose binding protein in phosphate-buffered saline (PBS) and cultured for 72 hours. Cells were collected; labeled with antimouse CD8 APC, anti-CD19 phycoerythrin, and anti-CD4 eFluor 780 (eBioscience); and stained for viability (7-amino-actinomycin D; BD Biosciences). Alternatively, 100,000 enriched T cells from xenoimmunized mice were combined in wells with 100,000 cells from CD191 or CD19– cell fractions isolated from xenoimmunized mice. Control wells contained 20 lg of purified maltose binding protein. Degranulation and Cytokine Secretion Assays. Splenocytes or liver lymphocytes from anti-CD20– treated mice or from xenoimmunized control mice were incubated for 5 hours in complete Roswell Park Memorial Institute 1640 medium (10% fetal calf serum, 20 U/mL murine recombinant IL-2 [Invitrogen], 5 lM 2mercaptoethanol), supplemented with 50 ng/mL phorbol myristate acetate, 0.5 lg/mL ionomycin, 5 lg/mL brefeldin A, 20 lg/mL monensin A (Sigma-Aldrich), and 0.2 lg anti-CD107a APC (eBioscience). Cells were harvested, washed, and then surface-labeled with antiCD45, anti-CD62L, anti-CD8, and anti-CD4. Cells were then permeabilized with fix/perm buffer (eBiosciences) and then stained with anti-IFN-c, anti-TNF-a, and anti–granzyme B. Alternatively, isolated cells from AIH xenoimmunized mice or control mice (female C57BL/6 nonvaccinated) were incubated in Roswell Park Memorial Institute 1640 medium containing 10%

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fetal calf serum, 20 U/mL murine recombinant IL-2 (Invitrogen), 5 lM 2-mercaptoethanol, 50 ng/mL phorbol myristate acetate, 0.5 lg/mL ionomycin, 10 lg/mL brefeldin A, and 10 lg/mL lipopolysaccharide (SigmaAldrich) for 5 hours. Cells were harvested and stained as described above using the following antibodies: antiCD45, anti-CD19, anti-IFN-c, anti-TNF-a, anti-IL10, and anti-B220. Statistical Analysis. All statistical analyses were performed using GraphPad Prism 4. Differences were considered significant at P < 0.05. Only two-tailed analyses were performed. The statistical tests used are mentioned in the figure legends. Unless mentioned otherwise, all graphs represent means and SEM.

Results B Cells in the AIH Experimental Model Secrete Proinflammatory Cytokines. In order to understand if B cells play an active role in AIH pathogenesis in our mouse model of type 2 AIH, we first investigated the Bcell secretion profile in xenoimmunized mice during active AIH (8 months after vaccination). A significantly higher proportion of splenic B cells in AIH mice produced IFN-c compared to control C57BL/6 unimmunized mice (Fig. 1A, left panel; P 5 0.0004, n 5 10). In addition, the levels of expression of IFN-c were higher in B cells isolated from both the spleen and liver of AIH mice (Fig. 1A, right panel; P < 0.0001, n 5 10). More polyfunctional B cells, producing both INF-c and TNFa, were present in the spleen and liver of xenoimmunized mice (Fig. 1B; P 5 0.009 and P 5 0.04, respectively, n 5 10) compared to unimmunized mice. In contrast, a lower proportion of splenic B cells from AIH mice (although not statistically significant) produced anti-inflammatory IL-10 cytokine (Fig. 1C; P 5 0.09, n 5 10). In addition, splenic B cells isolated from xenoimmunized mice proliferated significantly more in response to the mFTCD autoantigen compared to C57Bl/6 control mice (Fig. 1D; P 5 0.003, n 5 11). Finally, B cells were found in liver inflammatory foci of AIH mice along with CD41 and CD81 T cells (Fig. 1E). Altogether, these results suggest that B-cell activation and their secretion of proinflammatory cytokines contribute to AIH pathogenesis. Anti-CD20 Depletion Causes Transient Lymphopenia and Affects Mildly Spleen Architecture. Knowing that B cells were proinflammatory and autoreactive, we assessed the consequences of B-cell depletion in our mouse model of AIH. A single dose of anti-CD20 antibody resulted in almost complete elimination of B cells (CD451CD191), followed by a progressive reconstitu-

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tion over a 40-day period after treatment (Supporting Fig. S1A,B, n 5 8-36). Plasma cell (CD1381B2201) proportions were not modified in the spleen and liver of treated animals (Supporting Fig. S1C, n 5 8). The induced depletion led to spleen (P < 0.0001) and liver (P 5 0.017) lymphopenia 4 weeks after anti-CD20 injection (Supporting Fig. S1D, n 5 33), but the CD41/CD81 T-cell ratio remained unchanged in peripheral blood mononuclear cells, spleen, and liver (Supporting Fig. S1E, n 5 8-34). Despite the antiCD20–induced lymphopenia, the spleen architecture was only mildly affected. Anti-CD20 Treatment Prevents Disease Onset and Leads to Remission. A single injection of anti-CD20 antibody was able to induce remission when administered during active AIH (7 months postxenoimmunization). Indeed, a drastic reduction of liver inflammation was observed (Fig. 2A,B; P < 0.0001, n 5 35). This was accompanied by a significant reduction in ALT levels (Fig. 2C; P 5 0.0084, n 5 34) in treated mice compared to controls (AIH untreated mice). Disease remission was accompanied by a significant reduction of CXCL10 and CXCL9 expression in the liver, proinflammatory chemokines associated with liver inflammation (Fig. 2D; P 5 0.0036 and 0.024, respectively, n 5 21). In contrast, a single-dose anti-CD20 treatment did not result in significant change in either total IgG or autoantibody levels (anti-mFTCD) (Fig. 2E,F; n 5 8-45). B-cell depletion was also investigated as a preventive treatment before the onset of liver inflammation, 5.5 months after xenoimmunization. Depletion of B cells was maintained until sacrifice (2 injections over 6 weeks). This treatment prevented the development of AIH as indicated by the status of liver inflammation (Supporting Fig. S2A,B; P 5 0.042, n 5 9) and ALT levels (Supporting Fig. S2C; P 5 0.035, n 5 8). Effect of Anti-CD20 Treatment on Regulatory Lymphocyte Populations. Regulatory cell populations, such as regulatory T cells (Tregs) or Tr1 cells, could be instrumental in the control of liver inflammation and autoimmunity. We thus analyzed the proportion of CD41CD251FoxP31 Tregs in the spleen and liver of anti-CD20–treated mice. No differences in the proportion of Tregs were observed following remission of AIH in both the spleen and liver (Fig. 3A; P 5 0.71 and P 5 0.88, respectively, n 5 13). Similarly, the absolute numbers of Tregs in treated mice were not different in the spleen and liver, despite lymphopenia (Fig. 3B; P 5 0.09 and P 5 0.2, respectively, n 5 13). Splenic regulatory Tr1 cell (CD41CD49b1Lag31) proportions were also not significantly modified by antiCD20 treatment (Fig. 3C; P 5 0.23, n 5 8), but a trend

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C O L O R Fig. 1. B cells have a proinflammatory profile in the type 2 AIH experimental mouse model. (A) A significantly higher proportion of B cells (CD451CD191 cells) in the spleen of AIH experimental model animals (8 months postxenoimmunization) produced INF-c (left panel, ***P 5 0.0004), and these cells produced significantly more IFN-c in both the spleen and the liver (right panel, ***P < 0.0001) than C57BL/6 littermates. (B) A higher proportion of B cells producing both IFN-c and TNF-a was found in the spleen (**P 5 0.009) and the liver (*P 5 0.04) of mice suffering from AIH than unvaccinated mice (C57BL/6). (C) Proportions of IL-10-producing B cells tended to be higher in the spleen of unvaccinated mice (C57BL/6) than in the experimental AIH model (P 5 0.09). Unpaired t tests, n 5 10, means 6 SEM are depicted. (D) B cells (CD191) from AIH mice spleen proliferated significantly more than those isolated from control C57BL/6 mice to autoantigen mFTCD (P 5 0.0033, n 5 11). (E) B cells (CD19-fluorescein isothiocyanate, shown in green) are found in liver infiltrates along with CD81 (a) and CD41 T cells (b) (red) of AIH experimental mice. Nuclei are colored blue by 40 ,6-diamidino-2-phenylindole staining. Magnification 1003. Abbreviations: DAPI, 40 ,6-diamidino-2-phenylindole; NS, nonsignificant.

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C O L O R Fig. 2. Anti-CD20 administration leads to histological and clinical remission of AIH. (A) Typical histological findings of mice treated or not with anti-CD20 at 7 months postvaccination and sacrificed 4 weeks after. Magnified pictures (1003) of liver section stained with hematoxylin– phloxin–safran. (B) Ishak modified histological activity index of mice treated or not with a single dose of anti-CD20 (***P < 0.0001, unpaired t test, n 5 35). (C) ALT values 4 weeks after anti-CD20 treatment or control (**P 5 0.0084, Mann-Whitney test, n 5 34). (D) CXCL10 (IP10; left) and CXCL9 (Mig; right) relative expression levels in liver 4 weeks after anti-CD20 treatment (or PBS) in AIH experimental model (unpaired t test, CXCL10 **P 5 0.0036, CXCL9 *P 5 0.024, n 5 21). (E) Total serum IgG levels in treated and untreated mice following a single dose of anti-CD20 (or PBS) over 3 months (nonsignificant, P 5 0.70, n 5 16 for days 0, 16, and 28 and n 5 8 for days 43, 60, 75, and 90). (F) Anti-mFTCD autoantibodies titers in anti-CD20–treated and untreated mice through time from xenovaccination to 1 month posttreatment (logtransformed, nonsignificant, P 5 0.26, n 5 27 at month 1; n 5 28 for months 2-3; n 5 31 for month 4; n 5 30 for month 5; n 5 34 for month 6; n 5 46 for month 7, n 5 47 for month 8). Dotted line indicates anti-CD20 (or PBS) treatment. Means 6 SEM are depicted.

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Fig. 3. Treg, Tr1, and B10 cells are not affected by anti-CD20 treatment. (A) The Treg (CD41CD251FoxP31) proportion was not significantly changed in the spleen (nonsignificant, P 5 0.71 unpaired t test, n 5 13) and the liver (nonsignificant, P 5 0.88 unpaired t test, n 5 13) following anti-CD20 treatment. Similarly, absolute number of Tregs was not different in the spleen (B) (nonsignificant, P 5 0.09 unpaired t test, n 5 13) and the liver (nonsignificant, P 5 0.2 unpaired t test, n 5 13). Means 6 SEM are depicted. (C) Tr1 (CD41CD49b1Lag31) proportions, in the spleen and the liver, were not significantly modified following anti-CD20–induced remission (P 5 0.23 and P 5 0.06, respectively, n 5 10). (D) B10 cell (CD451CD191IL101) proportions in the spleen and the liver were not modified following anti-CD20 treatment (unpaired t test, P 5 0.36 and P 5 0.49, n 5 8). Means 6 SEM are depicted. Abbreviation: NS, nonsignificant.

to a lower proportion of Tr1 was observed in the liver of anti-CD20–treated AIH mice (P 5 0.06). Regulatory B10 cell (CD451CD191IL101) proportions in the spleen and liver were not modified following anti-CD20 treatment (Fig. 3D; P 5 0.36 and P 5 0.49, n 5 8). Tfh Cell Proportion Is Reduced Following AntiCD20 Treatment. Tfh cells are in close contact with B cells in germinal centers of secondary lymphoid organs. Their role in AIH is not well known, but we observed that in our AIH model mice had significantly more Tfh cells (CD41CXCR51PD11/-) in the spleen than C57BL/6 unvaccinated mice (Fig. 4A,B; P < 0.05,

n 5 17). Interestingly, anti-CD20 treatment reduced significantly the CD41CXCR51 population in the spleen to levels similar to those in control mice (Fig. 4A; P < 0.05). Similarly, CD41CXCR51PD11 Tfh cells were significantly reduced to control levels following anti-CD20 B-cell depletion (Fig. 4B; P < 0.01). Accordingly, B follicles observed in the spleen of B cell– depleted mice were smaller and slightly less organized in tree-like structures (data not shown). Anti-CD20 Treatment Reduced T-Cell Activation and Cytotoxic Activity. Because AIH is believed to be driven by T cells,1,2 the effect of B-cell depletion on

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Fig. 4. Tfh cells are reduced after anti-CD20 treatment. (A) Histogram (left) of CD41CXCR51 cells in untreated (unfilled curve) and anti-CD20 treated (gray curve) AIH mice (cells gated on CD451CD41 population). Splenic CD41CXCR51 cells are increased in AIH mice compared to C57BL/6 control (unvaccinated mice, *P < 0.05). Anti-CD20 treatment significantly reduced this population. (B) Tfh cells (CD41CXCR51PD11) are significantly reduced in spleen of AIH mice following anti-CD20 treatment (**P < 0.01). AIH mice also have significantly more Tfh cells in spleen than control C57Bl/6 mice (**P < 0.01). One-way analysis of variance, Tukey’s posttest, n 5 17. Means 6 SEM are depicted.

the CD41 and CD81 T-cell population was characterized in order to understand how B cells influence these effector cells. Significantly more naive CD41 and CD81 T cells (CD44–CD62L1) were found in the spleen of anti-CD20–treated mice (Fig. 5A; P 5 0.036 and P 5 0.0027, respectively, n 5 23). In parallel, there were less antigen-experienced CD41 and CD81 (CD441) found in the spleen of anti-CD20–treated mice (Fig. 5A; P 5 0.015 and P 5 0.072, respectively, n 5 14-23) but not in the liver (Supporting Fig. S3). Interestingly, B- and T-lymphocyte attenuator–positive cells (CD2721, expressed following antigen encounter and in chronic antigen stimulation20) were significantly reduced among CD41 and CD81 isolated from the spleen and liver of treated mice (Fig. 4B; P < 0.001 for all except P < 0.01 for CD81 from liver, n 5 7-21). Specific cytotoxic CD81 T cells are responsible for hepatocyte lysis and parenchyma destruction in AIH, so we studied the effect of B-cell depletion on the cytotoxic capacity of CD81 T cells in our model. In a degranulation assay, CD81 T cells isolated from the spleen of anti-CD20 mice showed less cytotoxic activity as assessed by CD107a and granzyme B expression (Fig.

5C; P 5 0.03 and P 5 0.05, respectively). CD81 T cells isolated from the liver also showed a tendency to reduced degranulation (CD107a1 P 5 0.09 and granzyme-B1 P 5 0.15, n 5 12). Anti-CD20 Depletion Decreases Proliferation of T Cells Through Reduced Autoantigen Presentation. As activation and cytotoxic capacity were modified following B-cell depletion treatment, we then assessed the proliferation capacity of CD41 and CD81 T cells to autoantigen as a readout of autoreactivity following Bcell depletion. Proliferation of liver-infiltrating lymphocytes, CD41 T cells, and CD81 T cells, isolated from anti-CD20–treated mice, proliferated significantly less than those isolated from AIH mice (Fig. 6A; P < 0.001, n 5 7). So, we hypothesized that antigen presentation may be impaired in the absence of B cells. To investigate if B cells (CD191 cells) act as APCs in our AIH model, we performed a proliferation assay by mixing carboxyfluorescein diacetate succinimidyl ester– stained splenocytes or enriched T cells from AIH mice with CD191 or CD19– cells as autoantigen (mFTCD)– presenting cells. As a mirror of what is happening in the liver, splenocytes, and more specifically CD41 T cells,

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Fig. 5. Effect of antiCD20 on T-cell activation status and cytotoxic activity. (A) Proportions of naive CD41 and CD81 T cells (CD44–CD62L1) were significantly higher in the spleen of mice treated with antiCD20 (CD41 P 5 0.036, top left; CD81 P 5 0.0027, bottom left). Proportions of antigen-experienced CD41 and CD81 T cells (CD441) were reduced in anti-CD20– treated mice (CD41 P 5 0.015, top right; CD81 P 5 0.07, bottom right). (B) Proportions of B- and Tlymphocyte attenuator–positive cells were significantly reduced in CD41 and CD81 T cells in both the spleen and the liver of anti-CD20– treated mice (***P < 0.001, **P < 0.01, twoway analysis of variance, Bonferroni post hoc test). (C) Proportion of CD107a1 in CD81CD62L– T cells were reduced in the spleen (*P 5 0.03) and the liver (P 5 0.09) of anti-CD20–treated mice compared to AIH mice. Similarly, proportions of granzyme-b1 in CD81CD62L– cells were lower in spleen (P 5 0.05) and liver (P 5 0.15) of anti-CD20 mice. Means 6 SEM are depicted.

proliferated similarly when CD191 cells or splenocytes served as APCs (Fig. 6B). In contrast, when the CD19– fraction was used as APCs, CD41 proliferation was reduced. As for CD81 T cells, they did not proliferate when B cells were the APCs and proliferated when the CD19– cell fraction served as APCs. Thus, B cells can be efficient APCs to CD41 T cells but not to CD81 T cells in our model of AIH. However, B-cell depletion also impacted CD81 proliferation (Fig. 6A). Thus, we investigated the antigen presentation capacity through the expression of MHC class II and CD80, molecules implicated in antigen presentation.

Expression of these molecules was drastically reduced on both B cells (CD191) and CD11b1 cells (APCs such as monocytes, macrophages, and dendritic cells) following anti-CD20 treatment (Fig. 6C,D; P < 0.0001, n 5 7). Thus, B cells, in addition to being efficient autoantigenpresenting cells to CD41 T cells, influence the capacity of traditional APCs to present antigen to CD81 T cells.

Discussion Anti-CD20 treatment is effective at obtaining a complete remission of liver inflammation in patients with

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Fig. 6. Anti-CD20 depletion reduces proliferation of T cells through reduced antigen expression. (A) Liver infiltrating lymphocytes isolated from anti-CD20–treated mice proliferate significantly less than untreated AIH mice to specific mFTCD autoantigen (***P < 0.0001, two-way analysis of variance, Bonferroni post hoc test, n 5 8). Means 6 SEM are depicted. (B) In a setting where bulk splenocyte or enriched carboxyfluorescein diacetate succinimidyl ester–stained T-cell fractions were mixed with unstained splenocytes, purified CD191 cells or CD19– cells as APCs, bulk splenocytes, and CD41 T cells proliferated well only when mFTCD was presented by B cells (CD191). This was not the case with CD81 cells that had a higher proliferating rate when autoantigen was presented by CD19– APCs. Representative results of six experiments is shown. (C) MHC class II expression, measures by mean fluorescence index, was significantly reduced on B cells (CD191) isolated from the spleen or the liver of anti-CD20–treated mice (***P < 0.0001, n 5 7). (D) Similarly, MHC class II expression was significantly reduced on CD11b1 cells isolated from the spleen or the liver of anti-CD20–treated mice (***P < 0.0001, n 5 7) as well as CD80 expression on CD11b1 cells from the spleen of treated mice (**P 5 0.0065, unpaired t test, n 5 6). Means 6 SEM are depicted. Abbreviation: MFI, mean fluorescence index.

AIH refractory to standard immunosuppression9,10 and in our AIH mouse model, corroborating the notion that T cell–mediated autoimmune diseases can benefit from the depletion of B cells. Anti-CD20 administration prevents the development and induces AIH remission, distinctively showing that B cells play a significant role in both the initiation and the perpetuation of the disease. Presence of B cells, especially plasma cells, in liver infiltrates of AIH patients along with hyper-IgG were reported in the original description of the disease2; hence, the role of B cells in AIH has been long suspected

but never experimentally proven. In the type 2 AIH model, B cells were autoreactive and secreted proinflammatory cytokines, contributing to a proinflammatory liver environment. Our data suggest that B cells also modulate the T-cell compartment through the reduction of autoantigen presentation, resulting in a reduction of activated T cells and proliferation. Changes induced in the T-cell compartment could explain the success of antiCD20 depletion in AIH remission in mice and patients. The CXCL10 serological level, because of its biological role in promoting the recruitment of lymphocytes to

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inflammatory sites, has been suggested as a biomarker of hepatic inflammation and fibrosis.11 In AIH, CXCL10 serum levels have been correlated to ALT levels and their normalization was associated with successful prednisone treatment.11 Herein, we report a significant decrease in CXCL10 levels in the liver of anti-CD20–treated mice, consistent with the observation of three of the six patients described by Burak et al. following rituximab treatment.9 Expression of CXCL9 (another CXCR3 ligand) was also significantly reduced following treatment. Similarly, other proinflammatory chemokines could be modulated by treatment as their expression is related to inflammatory signals. For example, chemokine (C-C motif ) receptor 5 and CXCR6 receptor/ ligands have been described to be involved in liver inflammation,21,22 and their expression would be interesting to measure in AIH remission. This diminution of chemoattractive molecules in the liver could contribute to the remission of inflammation by reducing the number of autoreactive cells present in the organ. Secretion of antibodies is the archetypal role of B cells; therefore, anti-CD20 can induce remission of autoimmune diseases in which autoantibodies participate to their pathogenesis. As such, autoantibody diminution in human CD20 transgenic mice model of inflammatory arthritis was observed following improvement of the disease after rituximab administration.23 In the present study, the improvement of liver inflammation was not accompanied by a reduction in antimFTCD antibodies. This is coherent with the nature of the disease in which autoantibodies are not believed to be pathogenic. It is also consistent with the observation that plasma cells express CD20 at levels four-fold to five-fold less than B cells and are resistant to anti-CD20 treatment.24 Among the suggested mechanisms by which B-cell depletion induces remission in some autoimmune diseases is an increase in Treg cells.12 Moreover, adoptive transfer of Tregs to mice with AIH induces remission of liver inflammation.19 However, in the month following anti-CD20 treatment in the type 2 AIH mouse model, Treg cell, as well as Tr1 and B10 cell, proportions were not modified. This is concordant with what has been described in patients suffering from refractory AIH treated with rituximab9 and in other autoimmune diseases13 in which no increase in Treg cells was observed following anti-CD20 treatment. While a Treg increase could play an important role in rituximab-induced remission of thrombocytopenia or lupus nephritis,3 it does not seem to be the case in AIH. Interactions between B cells and T cells are believed to potentially generate a positive feedback loop that

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amplifies and sustains autoimmunity.25 Anti-CD20 therapy abrogates this B–T cell crosstalk, which could in turn reduce autoantigen presentation, T-cell activation, and the Tfh cell population. Rituximab treatment in lupus nephritis and rheumatoid arthritis patients is associated with a decrease in T helper–cell activation.12 An elegant work studying the effect of anti-CD20 B-cell depletion on CD41 T cells in mice showed that when anti-CD20 was administered following immune challenge, a reduction in activation of CD41 T cells was observed with an increase in CD62L1 cells (naives) and a reduction in CD441 cells (antigen-experienced cells), while CD81 cells were only modestly affected.26 Similar findings were observed in the AIH mouse model following B-cell depletion, where CD41 T-cell activation was more impacted than CD81 T-cell activation. Decrease in activation of CD41 T cells was observed only in the spleen, where B cells are most likely to play their role of APCs/coactivators. In addition, expression of MHC class II and CD80 was significantly reduced on B cells and APCs following treatment, adding to the diminished capacity of antigen presentation, probably as a consequence of reduced liver inflammation, hepatocyte lysis, and autoantigen release. Reduced activation and antigen presentation impact T-cell function such as proliferation, impairing the proinflammatory feedback loop. B cells are efficient at presenting antigen, especially to CD41 T cells; and, accordingly, depletion of B cells has more impact on CD41 than on CD81 T cells.4 Indeed, in a model of Listeria infection, CD41 T cells, but not CD81 T cells, proliferated less in vivo when B cells were depleted.26 This study showed that CD41 T cells’ optimal response needs B-cell and dendritic cell signals.26 This is consistent with our observation that proliferation of CD41 T cells, but not of CD81 T cells, was maintained when CD191 cells were used as APCs and reduced in the absence of CD191 cells. It suggests that B-cell depletion in the AIH mouse model reduced CD41 T-cell expansion and, consequently, reduced their contribution to AIH pathogenesis through impaired help. CD41 T cells are considered to be very important contributors to AIH pathogenesis, and the significant impact observed in CD41 following antiCD20–induced AIH remission highlights their pivotal role. In AIH, an increase in circulating Tfh cells was observed, but their role in the pathogenesis is still unknown.27,28 Such an increase along with an expansion of plasma cells were associated with hypergammaglobulinemia.28 In our model of AIH, Tfh cells were significantly increased in spleen compared to unvaccinated

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mice, adding new evidence that Tfh cells could play a role in AIH pathogenesis. Moreover, Tfh cells were significantly reduced following B-cell depletion, at levels similar to those observed in control mice, possibly contributing to the disease remission. Further studies are needed to understand the specific role of Tfh cells in AIH pathogenesis. Clinical benefit of anti-CD20 treatment has been generally correlated to the extent and duration of B-cell depletion in autoimmune diseases.12 CD20 is expressed on more than 95% of B cells, from immature to memory B cells; but it is not expressed on stem cells or precursor B cells.29 This pattern of expression spares plasma cells, explaining the unchanged immunoglobulin levels following a one-dose treatment, and does not eliminate the entire pool of autoreactive B cells. In our model, relapse of liver inflammation was observed when the B-cell pool was reconstituted (Supporting Fig. S4). Our results on anti-CD20 therapy in AIH experimental model suggest that an amplification loop is temporarily halted but with no immune reeducation or regulation taking place (i.e., Tregs). Translation of these results to patients as well as clinical observations9,10 suggest that B-cell depletion should be prolonged or maintained to achieve long-term remission, while reducing or weaning prednisone and/or azathioprine.9,10 However, the need for maintained B-cell depletion needs to be thoroughly assessed in clinical practice. Moreover, maintenance of B-cell depletion raises the concern of risk of infection. However, rituximab appears to be well tolerated in AIH patients,9 and the rate of severe infection during multiple courses of rituximab is between four and six cases per 100 patients, which is similar to anti-TNF-a therapy.30 AIH pathogenesis remains poorly understood. Studies aiming at understanding the role of different cell populations, such as B cells, through the impact of their depletion could lead to refined treatment strategies. The present study provides additional evidence on the important role of B cells in the pathogenesis of AIH and, along with off-label description of rituximab use in small cohorts of refractory AIH patients,9,10 supports a future clinical trial for treatment of AIH patients with rituximab.

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Author names in bold designate shared co-first authorship.

Supporting Information Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.27991/suppinfo.