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The liver displays a strong capacity to induce tolerance toward hepatic antigens. However, hepatic tolerance can be overcome with the development of local autoimmune diseases such as autoimmune hepatitis (AIH). This chronic inflammatory disorder leads to a progressive destruction of liver parenchyma if non-treated. Although the CD4+ T cell response seems a key player of this immune disorder, the dynamics and biology of emerging liver antigen-specific CD4+ T cells are poorly described. Here, we developed a new murine model which mimics hepatic autoreactivity allowing the study and monitoring of antigen-specific CD4+ T cells from their emergence to local immune response. We show that the induction of the expression of an antigen in the liver in non-inflammatory condition leads to antigen tolerance. In inflammatory condition, using viral vector transduction, we observe the development of a complete adaptive immune response concomitant with the antigen expression in the liver. The presence of antigen-specific CD4+ T cells in the liver is associated to transient hepatic damages. Interestingly, the neo-antigen expression by hepatocytes after peripheral immunisation induces the recruitment of antigen-specific CD4+ T cells and hepatic damages. These data demonstrate that the recruitment of antigen-specific CD4+ T cells in the liver is conditioned by an immune coordination between surface antigen expression by hepatocytes and peripheral immune response and mimics the first step of a local autoreactive process. In the long-term, we observe that the hepatic environment has the capacity to control the local, but not the systemic, antigen-specific CD4+ T cells. Additional immune events might be involved in the long-term chronic immune reactivity in the liver, following the first steps described in this study.Key pointsAntigen expression in the liver in non-inflammatory condition leads to antigen toleranceAntigen expression in the liver in immunization condition (concomitant or pre-existing) is sufficient to induce liver recruitment of antigen-specific CD4+ T cells and hepatic damagesThis model mimics the first step of an autoreactive process against a liver antigenIn the long-term, the hepatic environment induces a local tolerance toward the antigen expression and the clearance of liver-infiltrating, but not peripheral, antigen-specific CD4+ T cellsThis new murine model can be of interest for the analysis of the immunomodulatory pathways implicated in liver tolerization of autoreactive CD4+ T cells and identification of potential extrinsic factors implicated in an acute-to-chronic transition as observed during autoimmune hepatitis

International audience; Background & aims: In most autoimmune disorders, crosstalk of B cells and CD4 T cells results in the accumulation of autoantibodies. In autoimmune hepatitis (AIH), the presence of anti-soluble liver antigen (SLA) autoantibodies is associated with reduced overall survival, but the associated autoreactive CD4 T cells have not yet been characterised. Herein, we isolated and deeply characterised SLA-specific CD4 T cells in patients with AIH.Methods: We used brief ex vivo restimulation with overlapping SLA peptides to isolate and phenotype circulating SLA-specific CD4 T cells, and integrative single-cell RNA-seq (scRNA-seq) to characterise their transcriptome and T-cell receptor (TCR) repertoire. Autoreactive TCRs were cloned and used to identify dominant SLA-derived epitopes. SLA-specific CD4 T cells were tracked in peripheral blood through TCR sequencing to identify their phenotypic niche. We further characterised disease-associated peripheral blood T cells by high-content flow cytometry in 42 patients with AIH and 17 controls with non-alcoholic steatohepatitis.Results: Autoreactive SLA-specific CD4 T cells were only detected in patients with anti-SLA autoantibodies and had a memory PD-1+CXCR5-CCR6-CD27+ phenotype. ScRNA-seq revealed their pro-inflammatory/B-helper profile. SLA81-100 and SLA177-204 contain dominant T-cell epitopes. Autoreactive TCR clonotypes were predominantly found in the memory PD-1+CXCR5-CD4 T cells, which were significantly increased in the blood of patients with AIH and supported B-cell differentiation through IL-21. Finally, we identified specific T-cell phenotypes linked to disease activity and IgG level during AIH.Conclusions: We provide a deep characterisation of rare circulating autoreactive CD4 T cells and identify their peripheral reservoir in AIH. We also propose a specific phenotype of autoreactive T cells related to AIH disease activity, which will be essential to track, delineate, and potentially target these pathogenic cells.Lay summary: One principal characteristic of autoimmune hepatitis (AIH), like for many other autoimmune diseases, is the accumulation of autoantibodies produced by B lymphocytes following their interaction with autoreactive CD4 T lymphocytes. In this study, we identified and characterised with high resolution these CD4 T cells. This will be essential to track, delineate, and potentially target them during AIH.

Background & AimsIn most autoimmune disorders, crosstalk of B cells and CD4 T cells results in the accumulation of autoantibodies. In autoimmune hepatitis (AIH), the presence of anti-Soluble Liver Antigen (SLA or SepSecs) autoantibodies is associated with significantly reduced overall survival, but the associated autoreactive CD4 T cells have not been characterized yet. Here we isolated and deeply characterized SLA-specific CD4 T cells in AIH patients.MethodsWe used brief ex vivo restimulation with overlapping SLA-derived peptides to isolate and phenotype circulating SLA-specific CD4 T cells, and integrative single-cell RNA-seq (scRNA-seq) to characterize their transcriptome and TCR repertoire in n=5 AIH patients. SLA-specific CD4 T cells were tracked in peripheral blood through TCR sequencing, to identify their phenotypic niche. We further characterized disease-associated peripheral blood T cells by high content flow cytometry in an additional cohort of n=46 AIH patients and n=18 non-alcoholic steatohepatitis (NASH) controls.ResultsAutoreactive SLA-specific CD4 T cells were only detected in patients with anti-SLA autoantibodies and had a memory PD-1+CXCR5−CCR6−CD27+ phenotype. ScRNA-seq revealed their pro-inflammatory/B-Helper profile (IL21, IFNG, TIGIT, CTLA4, NR3C1, CD109, KLRB1 and CLEC2D). Autoreactive TCR clonotypes were restricted to the memory PD-1+CXCR5− CD4 T cells. This subset was significantly increased in the blood of AIH patients and supported B cell differentiation through IL-21. Finally, we identified a specific phenotype (PD-1+CD38+CD27+CD127−CXCR5−) of CD4 T cells linked to disease activity and IgG response during AIH.ConclusionsThis work provides for the first time a deep characterization of rare circulating autoreactive CD4 T cells and the identification of their peripheral reservoir in AIH. We also propose a generic phenotype of pathogenic CD4 T cells related to AIH disease activity.

International audience; Xenocell therapy from neonate or adult pig pancreatic islets is one of the most promising alternatives to allograft in type 1 diabetes for addressing organ shortage. In humans, however, natural and elicited anti-bodies specific for pig xenoantigens, α-(1,3)-galactose (GAL) and N-glycolylneuraminic acid (Neu5Gc), are likely to significantly contribute to xenoislet rejection. We obtained double-knockout (DKO) pigs lacking GAL and Neu5Gc. Because Neu5Gc 2/2 mice exhibit glycemic dys-regulations and pancreatic b-cell dysfunctions, we evaluated islet function and glucose metabolism regulation in DKO pigs. Isolation of islets from neonate piglets yielded identical islet equivalent quantities to quantities obtained from control wild-type pigs. In contrast to wild-type islets, DKO islets did not induce anti-Neu5Gc antibody when grafted in cytidine monophosphate-N-acetylneuraminic acid hydroxylase KO mice and exhibited in vitro normal insulin secretion stimulated by glucose and theophylline. Adult DKO pancreata showed no histological abnormalities , and immunostaining of insulin and glucagon was similar to that from wild-type pancreata. Blood glucose, insulin, C-peptide, the insulin-to-glucagon ratio, and HOMA-insulin resistance in fasted adult DKO pigs and blood glucose and C-peptide changes after intravenous glucose or insulin administration were similar to wild-type pigs. This first evaluation of glucose homeostasis in DKO pigs for two major xenoantigens paves the way to their use in (pre)clinical studies.

Immunotherapy is a promising strategy against hepatocellular carcinoma (HCC). We assessed the therapeutic effects of stimulating CD137, a member of the TNF receptor family, with agonistic monoclonal antibodies (mAb). Agonistic anti-CD137 mAb treatment was tested on two in situ models of HCC in immunocompetent mice. We also studied the mediators involved at different time points. In an orthotopic HCC the treatment consistently leads to complete tumor regression in 40-60% of animals. The protection is long lasting in the animals responding to the treatment, which can reject a second tumor challenge more than 3 months after treatment and eradication of the first malignancy. The main mediators of the effect are T lymphocytes and NK cells, demonstrated through depletion experiments. In addition, adoptive transfer of splenocytes prepared from anti-CD137 mAb-treated and -cured mice to naive mice allowed them to, in turn, reject the tumor. The efficacy of anti-CD137 mAb treatment is associated with early, sustained recruitment of iNOS-positive macrophages within tumor nodules. Moreover, in the absence of treatment, tumor development is accompanied by infiltration by myeloid derived suppressor cells (MDSC) and regulatory T lymphocytes. In mice responding to the anti-CD137 mAb treatment, this infiltration is very limited, and a combination treatment with a depletion of MDSC leads to the recovery of 80% of the mice. These results demonstrate that agonistic anti-CD137 mAb is a promising therapeutic strategy for anti-tumor immunity stimulation against HCC.

Induction of donor-specific immune tolerance is a good alternative to chronic life-long immunosuppression for transplant patients. Donor major histocompatibility complex (MHC) molecules represent the main targets of the allogeneic immune response of transplant recipients. Liver targeted gene transfer with viral vectors induces tolerance toward the encoded antigen. The aim of this work was to determine whether alloantigen gene transfer to hepatocytes induces tolerance and promotes graft acceptance.C57BL/6 (H-2b) mice were treated with adeno-associated viral (AAV) vector targeting the expression of the MHC class I molecule H-2KAAV H-2KAAV-mediated long-term expression of a single MHC class I molecule in the liver induces the generation of a subset of allo-specific CD8The liver has a special immune system, biased toward tolerance. In this study, we investigated the possibility of harnessing this property of the liver to induce tolerance to an allogeneic transplantation. We demonstrate for the first time that the in vivo gene transfer of an allogeneic antigen with an adeno-associated viral vector to mouse hepatocytes induces the expansion of a population of CD8

For the first time, the N-glycosylation patterns of immunoglobulin G (IgGs) isolated from the serum of two varieties of knockout pigs (lacking N-glycolylneuraminic acid (Neu5Gc) and/or α 1,3 galactose) were examined for the presence of potential glycan xenoantigens and compared to N-glycosylation patterns obtained for wild-type (WT) pig IgGs. Glycopeptide analysis was chosen over glycan release, as protein-A eluates from pig serum may contain IgA and IgM as shown previously. The experiments focused on the analysis of tryptic glycopeptides EEQFNSTYR and AEQFNSTYR from IgGs, and excluded IgA and IgM, in which N-glycosylated peptides have different sequences and masses. WT pig IgG glycopeptides showed the presence of N-glycolylneuraminic acid (Neu5Gc) and absence of N-acetylneuraminic acid (Neu5Ac). Released glycans from the protein-A eluate, however, showed the presence of both types of sialic acids, allowing Neu5Ac to be attributed to IgA and/or IgM. The WT IgG samples also showed the presence of glycans that could by composition have been α-galactosylated, but treatments with α- and β-galactosidases produced inconclusive results as to the linkage nature of the terminal Gal residues. Single knockout (α-Gal transferase) pig IgG was shown to contain Neu5Gc residues, and there was a definite absence of α-Gal. Double knockout pigs (DKO for α-Gal transferase and cytidine monophosphate-A-acetylneuraminic acid hydroxylase (CMAH)) showed the definite absence of α-Gal and Neu5Gc. Instead of the latter, Neu5Ac residues were observed. Further investigation into the sialylation patterns of WT and DKO pig IgGs consisted of esterifying the glycopeptides to allow the detection and differentiation of α-2,3 and α-2,6 sialic acid-galactose linkages. Fucosylation levels were also compared between IgG species.

International audience; Polyclonal xenogenic IgGs, although having been used in the prevention and cure of severe infectious diseases, are highly immunogenic, which may restrict their usage in new applications such as Ebola hemorrhagic fever. IgG glycans display powerful xenogeneic antigens in humans, for example α1-3 Galactose and the glycolyl form of neuraminic acid Neu5Gc, and IgGs deprived of these key sugar epitopes may represent an advantage for passive immunotherapy. In this paper, we explored whether low immunogenicity IgGs had a protective effect on a guinea pig model of Ebola virus (EBOV) infection. For this purpose, a double knock-out pig lacking α1-3 Galactose and Neu5Gc was immunized against virus-like particles displaying surface EBOV glycoprotein GP. Following purification from serum, hyper-immune polyclonal IgGs were obtained, exhibiting an anti-EBOV GP titer of 1:100,000 and a virus neutralizing titer of 1:100. Guinea pigs were injected intramuscularly with purified IgGs on day 0 and day 3 post-EBOV infection. Compared to control animals treated with IgGs from non-immunized double KO pigs, the anti-EBOV IgGs-treated animals exhibited a significantly prolonged survival and a decreased virus load in blood on day 3. The data obtained indicated that IgGs lacking α1-3 Galactose and Neu5Gc, two highly immunogenic epitopes in humans, have a protective effect upon EBOV infection.

Background & Aims Induction of donor-specific immune tolerance is a good alternative to chronic life-long immunosuppression for transplant patients. Donor major histocompatibility complex (MHC) molecules represent the main targets of the allogeneic immune response of transplant recipients. Liver targeted gene transfer with viral vectors induces tolerance toward the encoded antigen. The aim of this work was to determine whether alloantigen gene transfer to hepatocytes induces tolerance and promotes graft acceptance. Methods C57BL/6 (H-2b) mice were treated with adeno-associated viral (AAV) vector targeting the expression of the MHC class I molecule H-2K d to hepatocytes, before transplantation with fully allogeneic pancreatic islet from BALB/c mice (H-2d). Results AAV H-2K d treated mice were tolerant to the alloantigen, as demonstrated by its long-term expression by the hepatocytes, even after a highly immunogenic challenge with an adenoviral vector. After chemical induction of diabetes, the AAV treated mice had significantly delayed rejection of fully allogeneic pancreatic islet grafts, with more than 40% of recipients tolerant (>100days). AAV-mediated expression of H-2K d in the liver induced the local expansion of CD8 + T lymphocytes with allo-specific suppressive properties. The adoptive transfer of these liver-generated CD8 + Tregs into naive diabetic mice promoted the long-term survival of allogeneic pancreatic islet grafts. Conclusion AAV-mediated long-term expression of a single MHC class I molecule in the liver induces the generation of a subset of allo-specific CD8 + Treg cells, which promote tolerance toward fully allogeneic graft. Liver gene transfer represents a promising strategy for in vivo induction of donor-specific tolerance. Lay summary The liver has a special immune system, biased toward tolerance. In this study, we investigated the possibility of harnessing this property of the liver to induce tolerance to an allogeneic transplantation. We demonstrate for the first time that the in vivo gene transfer of an allogeneic antigen with an adeno-associated viral vector to mouse hepatocytes induces the expansion of a population of CD8 + regulatory T lymphocytes. These Tregs are then instrumental in preventing the rejection of allogeneic pancreatic islets transplanted in these animals. Allogeneic transplantation is the main treatment for the end-stage diseases of a number of organs. Life-long immunosuppressive treatments are still required to limit graft rejection, and these treatments exhibit serious side effects. Our present findings open a new avenue for promoting allo-specific tolerance via in vivo induction of CD8 + Treg expansion.

Item does not contain fulltext Immunotherapy represents a potential therapeutic option for patients with hepatocellular carcinoma (HCC), especially as secondary treatment to prevent recurrence. It has been shown that a patient's survival is directly correlated to the type and number of tumor-infiltrating immune cells, indicating that immune responses have a direct effect on the clinical course of the disease. We have assessed the potential of immunotherapy against HCC in preclinical models of low tumor burden. An antigen-specific strategy targeting alpha-fetoprotein, and consisting of immunization with a DNA-based synthetic vector (DNAmAFP/704), was tested on an autochthonous model of chemical hepatocarcinogenesis and led to an important (65%) reduction of the tumor burden. A nonspecific approach of CD25(+) T-cell depletion by injection of PC61 antibody was also tested on an orthotopic HCC model and led to a significant protection against tumor development. Antigen-specific immunotherapy and Treg depletion are promising strategies in physiologically relevant HCC preclinical models. Future clinical trials will demonstrate if a combination of Treg depletion with an antigen-specific immunotherapy will also translate into clinical responses in HCC patients. 01 april 2011

Immunotherapy is a promising strategy against hepatocellular carcinoma (HCC). We assessed the therapeutic effects of stimulating CD137, a member of the TNF receptor family, with agonistic monoclonal antibodies (mAb). Agonistic anti-CD137 mAb treatment was tested on two in situ models of HCC in immunocompetent mice. We also studied the mediators involved at different time points. In an orthotopic HCC the treatment consistently leads to complete tumor regression in 40-60% of animals. The protection is long lasting in the animals responding to the treatment, which can reject a second tumor challenge more than 3 months after treatment and eradication of the first malignancy. The main mediators of the effect are T lymphocytes and NK cells, demonstrated through depletion experiments. In addition, adoptive transfer of splenocytes prepared from anti-CD137 mAb-treated and -cured mice to naive mice allowed them to, in turn, reject the tumor. The efficacy of anti-CD137 mAb treatment is associated with early, sustained recruitment of iNOS-positive macrophages within tumor nodules. Moreover, in the absence of treatment, tumor development is accompanied by infiltration by myeloid derived suppressor cells (MDSC) and regulatory T lymphocytes. In mice responding to the anti-CD137 mAb treatment, this infiltration is very limited, and a combination treatment with a depletion of MDSC leads to the recovery of 80% of the mice. These results demonstrate that agonistic anti-CD137 mAb is a promising therapeutic strategy for anti-tumor immunity stimulation against HCC.

Aim. To investigate the ability of recombinant modified vaccinia virus Ankara (rMVA) vector to induce an immune response against a well-tolerated self-antigen.Methods. rMVA vectors expressing different form ofα-fetoprotein (AFP) were produced and characterized. Naïve mice were vaccinated with MVA vectors expressing the AFP antigen in either a secreted, or a membrane-bound, or an intracellular form. The immune response was monitored by an IFNΓ ELISpot assay and antibody detection.Results. Vaccination with the membrane-associated form of AFP induced a stronger CD8+T-cell response compared to the ones obtained with the MVA encoding the secreted or the intracellular forms of AFP. Moreover, the vaccination with the membrane-bound AFP elicited the production of AFP-specific antibodies.Conclusions. The AFP transmembrane form is more immunogenic. Expressing a membrane-bound form in the context of an MVA vaccination could enhance the immunogenicity of a self-antigen.