BACKGROUND & AIMS:Autoreactive CD4 T cells, which recognize liver self-antigens such as SepSecS, are potentially main drivers of the chronic inflammatory response during autoimmune hepatitis (AIH). Previous studies have revealed SepSecS epitopes often associated with HLA-DRB1∗03 or HLA-DRB1∗04 restriction, two alleles enriched in AIH population. However, human leukocyte antigen (HLA) restriction of numerous SepSecS epitopes remains incomplete, and whether they could be presented by non-HLA-DR molecules remains unclear. Here, we investigated epitope recognition and HLA restriction of SepSecS-specific CD4 T cells from AIH patients. METHODS:We cloned 17 T-cell receptor αβ (TCRαβ) from the most expanded SepSecS-specific CD4 T cells of five AIH patients from our previous studies into a murine hybridoma T-cell line. The epitope reactivity of each TCR cell line was identified via IL-2 secretion following culture with 20-mer overlapping peptides of the SepSecS protein and immortalized B-cell lines. HLA restriction was defined using HLA blocking antibodies. We analyzed 484 SepSecS-specific TCRs from nine AIH patients using GLIPH2 algorithm. CD154 activation-induced marker assay was used to evaluate the SepSecS epitope reactivity in AIH patients (n = 11). RESULTS:Autoreactive TCRs recognized six SepSecS amino acid regions in vitro, with four distinct class II HLA restrictions, including non-HLA-DR restrictions. The GLIPH2 algorithm clustered SepSecS-specific TCRs into six groups. Notably, 6% of the total SepSecS-specific TCR clonotypes shared common CDR3β motifs linked to the recognition of HLA-DPA1∗02:01/HLA-DPB1∗01:01-restricted SepSecS152-179 epitope. Ex vivo peptide stimulation assay revealed that SepSecS152-179 epitope represents 15% to 67% of total SepSecS CD4 T-cell reactivity in HLA-DPA1∗02:01∼HLA-DPB1∗01:01 AIH patients (n = 5). CONCLUSIONS:We highlighted that SepSecS epitopes could be presented by HLA-DR and non-HLA-DR molecules to autoreactive CD4 TCRs with potentially conserved CDR3 motifs for common epitope recognition. IMPACT AND IMPLICATIONS:In AIH, SepSecS is a key autoantigen targeted by the adaptive immune system, but only few T-cell epitopes are defined and associated with a precise HLA restriction. By reconstructing TCRs from the most expanded SepSecS-specific CD4 T cells of AIH patients, we identified new T-cell epitopes and their specific class II HLA restriction, including peptides presented by non-HLA-DR molecules. From a clinical perspective, these findings could be used to develop tools to track the frequency of autoreactive T cells in patients. This work could also pave the way for self-antigen-specific immunotherapies, such as peptide-based desensitization or the development of HLA-multimer technologies to selectively deplete or reprogram pathogenic T cells without inducing systemic immunosuppression.

Autoreactive CD4 T cells, recognizing liver-self-antigens such as SepSecS, are main drivers of the chronic inflammatory response during autoimmune hepatitis (AIH). Previous studies have uncovered immunodominant SepSecS epitopes often associated with HLA-DRB1*03 or HLA-DRB1*04 restriction, two alleles enriched in AIH population. However, HLA restriction of numerous SepSecS epitopes remains incomplete and it is still unclear if immunodominant epitopes could be presented by non-HLA-DR molecules. Here, we investigated epitope recognition of SepSecS-specific TCRs isolated from AIH patients, and their HLA restriction by generating TCR hybridoma cell lines. Seventeen TCRs recognized eight SepSecS epitopes with four distinct HLA restrictions, including a novel HLA-DPA1\*02:01/DPB1\*01:01-restricted SepSecS epitope. TCR clustering analysis using GLIPH2 algorithm suggested that this epitope is recognized by multiple distinct TCRs in HLA-DPA1\*02:01∼HLA-DPB1\*01:01 patients. Our study provides new insights into liver-self-antigen T cell reactivity during AIH, which could offer potential therapeutic strategies by targeting autoreactive CD4 T cells. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-19 CE17-0024, ANR-24 CE15-7123

In autoimmune hepatitis (AIH), hepatocellular damage is linked to an accumulation of autoreactive T cells in the liver of patients, but how these cells emerge in the tissue remains unclear. Here we used a mouse model based on recombination-dependent inducible expression of influenza A hemagglutinin (HA) by hepatocytes to investigate initiation of liver antigen-specific response. Our study revealed that peripheral immunization, unlike inflammatory triggers, is essential to initiate an immune response against a liver antigen. We showed that liver T cell reactivity after peripheral immunization is marked by PD-1 and TIGIT co-expression and that the frequency of PD-1+ TIGIT+ HLA-DR+ CD38+ CD8 T cells in the blood of AIH patients is associated with liver injury. Our findings suggest a potential influence of the peripheral immunization for the liver-antigen-specific responses during AIH. Liver tissue-activated T cells probably recirculate during active phase of the disease, unveiling potential immunomarkers to monitor disease activity. Highlights In brief Guinebretière et al. demonstrate that after peripheral immunization, liver-antigen-specific T cell accumulation in the tissue is marked by local PD-1/TIGIT co-expression, a phenotype shared with liver and circulating T cell subsets enriched in active autoimmune hepatitis (AIH) patients. These findings suggest the influence of peripheral immunization on the initiation of AIH and provide potential immunomarker of AIH activity. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-19 CE17-0024, ANR-24 CE15-7123

Autoimmune liver diseases (AILD) involve dysregulated CD4 T cell responses against liver self-antigens, but how these autoreactive T cells relate to liver tissue pathology remains unclear. Here we perform single-cell transcriptomic and T cell receptor analyses of circulating, self-antigen-specific CD4 T cells from patients with AILD and identify a subset of liver-autoreactive CD4 T cells with a distinct B-helper transcriptional profile characterized by PD-1, TIGIT and HLA-DR expression. These cells share clonal relationships with expanded intrahepatic T cells and exhibit transcriptional signatures overlapping with tissue-resident T cells in chronically inflamed environments. Using a mouse model, we demonstrate that, following antigen recognition in the liver, CD4 T cells acquire an exhausted phenotype, play a crucial role in liver damage, and are controlled by immune checkpoint pathways. Our findings thus suggest that circulating autoreactive CD4 T cells in AILD are imprinted by chronic antigen exposure to promote liver inflammation, thereby serving as a potential target for developing biomarkers and therapies for AILD.

Autoimmune liver diseases (AILD) are immune-mediated disorders in which CD4 T cells play a central role. However, the link between circulating self-antigen-specific CD4 T cells and the targeted tissue has not been extensively studied in AILD. We hypothesized that circulating autoreactive CD4 T cells were clonally and functionally related to dominant intra-hepatic pathogenic CD4 T cell clones. Single cell transcriptomic analysis of circulating self-antigen-specific CD4 T cells revealed a specific B-helper and immuno-exhausted transcriptional profile, which was conserved for different autoantigens, but distinct from several other types of foreign antigen specificities. In the blood, the dominant hepatic CD4 T cell clones had a similar transcriptomic signature and were enriched in the PD-1+ TIGIT+ HLA-DR+ CD4 T cell subset. In a mouse model, antigen-specific CD4 T cells acquired the immuno-exhausted transcriptional profile when they accumulated in the liver after local antigen reactivity. Locally, immune checkpoint molecules controlled the response of antigen-specific CD4 T cells responsible for liver damage. Our study reveals the origin and biology of liver-derived autoreactive CD4 T cells in the blood of AILD patients that are imprinted by the liver environment, and suggest a dysregulation of the immune checkpoint molecules pathways. Our study enables tracking and isolating circulating autoreactive CD4 T cells for future diagnostic and therapeutic purposes.### Competing Interest StatementThe authors have declared no competing interest.

Relapse after immunosuppression (IS) treatment withdrawal is frequent in patients with Autoimmune Hepatitis (AIH), and non-invasive biomarkers predictive of this risk are lacking. We assessed the frequency of circulating T cell subsets as potential biomarkers of disease activity and predictor of the risk of relapse after IS withdrawal. Serum levels of the cytokine B-cell Activating Factor (BAFF) were also investigated. Blood samples from 58 patients with active AIH, 56 AIH patients in remission, and 31 patients with NASH were analyzed. The frequency of activated CD4+ T peripheral helper (TPH) cells (CD4+CD45RA-CXCR5-PD1+CD38+) and of activated CD8+ T cells (CD8+CD45RA-PD1+CD38+) were assessed by flow cytometry. BAFF levels were determined by ELISA. Activated TPH and CD8+ T cell frequencies were significantly increased in patients with active AIH compared to remission AIH or NASH (TPH: 0.88% of total CD3+ vs. 0.42% and 0.39% respectively, p < 0.0001; CD8+ subset: 1.42% vs. 0.09% and 0.11% p < 0.0001). Among patients in remission undergoing treatment withdrawal (n = 18), those with increased frequencies of activated TPH (> 0.5% of total CD3+) and/or activated CD8+ T cells (> 0.18% total CD3+) had a higher risk of relapse (80% vs. 15% after 2 years, p = 0.0071). High BAFF serum concentration (> 213pg/ml) was also associated to a higher risk of relapse (57% vs. 11%, p = 0.0452). In conclusion, high frequency of activated TPH and of activated CD8+, as well as high levels of BAFF, before IS discontinuation, were significantly associated to a greater risk of relapse during the first two years. Thus, they represent promising biomarkers to provide personalized clinical follow-up for patients with AIH.

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 points Antigen expression in the liver in non-inflammatory condition leads to antigen tolerance Antigen 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 damages This model mimics the first step of an autoreactive process against a liver antigen In 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 cells This 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

permettant la réparation tissulaire après une lésion (Figure 1).Ils ouvrent ainsi la voie à une recherche de nouveaux traitements contre des maladies caractérisées par une inflammation excessive ou chronique, telles que le sepsis ou les myopathies dégénératives.‡ Tissue repair: Key role of annexin A1 in the control of inflammatory response LIENS D'INTÉRÊTLes auteurs déclarent n'avoir aucun lien d'intérêt concernant les données publiées dans cet article.

Perfusion of convalescent plasma (CP) has demonstrated a potential to improve the pneumonia induced by SARS-CoV-2, but procurement and standardization of CP are barriers to its wide usage. Many monoclonal antibodies (mAbs) have been developed but appear insufficient to neutralize SARS-CoV-2 unless two or three of them are being combined. Therefore, heterologous polyclonal antibodies of animal origin, that have been used for decades to fight against infectious agents might represent a highly efficient alternative to the use of CP or mAbs in COVID-19 by targeting multiple antigen epitopes. However, conventional heterologous polyclonal antibodies trigger human natural xenogeneic antibody responses particularly directed against animal-type carbohydrate epitopes, mainly the N-glycolyl form of the neuraminic acid (Neu5Gc) and the Gal α1,3-galactose (αGal), ultimately forming immune complexes and potentially leading to serum sickness or allergy. To circumvent these drawbacks, we engineered animals lacking the genes coding for the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) and α1,3-galactosyl-transferase (GGTA1) enzymes to produce glyco-humanized polyclonal antibodies (GH-pAb) lacking Neu5Gc and α-Gal epitopes. We found that pig IgG Fc domains fail to interact with human Fc receptors and thereby should confer the safety advantage to avoiding macrophage dependent exacerbated inflammatory responses, a drawback possibly associated with antibody responses against SARS-CoV-2 or to avoiding a possible antibody-dependent enhancement (ADE). Therefore, we immunized CMAH/GGTA1 double knockout (DKO) pigs with the SARS-CoV-2 spike receptor-binding domain (RBD) to elicit neutralizing antibodies. Animals rapidly developed a hyperimmune response with anti-SARS-CoV-2 end-titers binding dilutions over one to a million and end-titers neutralizing dilutions of 1:10,000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV-19, neutralized Spike/angiotensin converting enzyme-2 (ACE-2) interaction at a concentration < 1μg/mL and inhibited infection of human cells by SARS-CoV-2 in cytopathic assays. These data and the accumulating safety advantages of using glyco-humanized swine antibodies in humans warranted clinical assessment of XAV-19 to fight against COVID-19.

T cell exclusion causes resistance to cancer immunotherapies via immune checkpoint blockade (ICB). Myeloid cells contribute to resistance by expressing signal regulatory protein-α (SIRPα), an inhibitory membrane receptor that interacts with ubiquitous receptor CD47 to control macrophage phagocytosis in the tumor microenvironment. Although CD47/SIRPα-targeting drugs have been assessed in preclinical models, the therapeutic benefit of selectively blocking SIRPα, and not SIRPγ/CD47, in humans remains unknown. We report a potent synergy between selective SIRPα blockade and ICB in increasing memory T cell responses and reverting exclusion in syngeneic and orthotopic tumor models. Selective SIRPα blockade stimulated tumor nest T cell recruitment by restoring murine and human macrophage chemokine secretion and increased anti-tumor T cell responses by promoting tumor-antigen crosspresentation by dendritic cells. However, nonselective SIRPα/SIRPγ blockade targeting CD47 impaired human T cell activation, proliferation, and endothelial transmigration. Selective SIRPα inhibition opens an attractive avenue to overcoming ICB resistance in patients with elevated myeloid cell infiltration in solid tumors.

The success of inducing human pluripotent stem cells (hIPSC) offers new opportunities for cell-based therapy. Since B cells exert roles as effector and as regulator of immune responses in different clinical settings, we were interested in generating B cells from hIPSC. We differentiated human embryonic stem cells (hESC) and hIPSC into B cells onto OP9 and MS-5 stromal cells successively. We overcame issues in generating CD34(+)CD43(+) hematopoietic progenitors with appropriate cytokine conditions and emphasized the difficulties to generate proper hematopoietic progenitors. We highlight CD31(int)CD45(int) phenotype as a possible marker of hematopoietic progenitors suitable for B cell differentiation. Defining precisely proper lymphoid progenitors will improve the study of their lineage commitment and the signals needed during the in vitro process.

Granzyme B-expressing B cells have been shown to be an important regulatory B cell subset in humans. However, it is unclear which subpopulations of B cells express GZMB under normal conditions and which protocols effectively induce ex vivo expansion of GZMB+ B cells. We found that in the peripheral blood of normal individuals, plasmablasts were the major B cell subpopulation that expressed GZMB. However, when using an in vitro plasmablast differentiation protocol, we obtained only 2% GZMB+ B cells. Nevertheless, using an expansion mixture containing IL-21, anti-BCR, CpG oligodeoxynucleotide, CD40L, and IL-2, we were able to obtain more than 90% GZMB+ B cells after 3 d culture. GZMB+ B cells obtained through this protocol suppressed the proliferation of autologous and allogenic CD4+CD25- effector T cells. The suppressive effect of GZMB+ B cells was partially GZMB dependent and totally contact dependent but was not associated with an increase in effector T cell apoptosis or uptake of GZMB by effector T cells. Interestingly, we showed that GZMB produced by B cells promoted GZMB+ B cell proliferation in ERK1/2-dependent manner, facilitating GZMB+ B cell expansion. However, GZMB+ B cells tended to undergo apoptosis after prolonged stimulation, which may be considered a negative feedback mechanism to limit their uncontrolled expansion. Finally, we found that expanded GZMB+ B cells exhibited a regulatory phenotype and were enriched in CD307bhi, CD258hiCD72hi, and CD21loPD-1hi B cell subpopulations. Our study, to our knowledge, provides new insight into biology of GZMB+ B cells and an efficient method to expand GZMB+ B cells for future cell therapy applications.

PURPOSE OF REVIEW:Autoimmune hepatitis (AIH) is a chronic disease characterized by a lymphocyte infiltrate in the liver. For decades, nonspecific immunosuppression has been used to limit chronic liver inflammation. The high risk of relapse, the treatments side effects, and the significant number of refractory patients are the main clinical issues that require efforts to understand AIH immune mechanisms.RECENT FINDINGS:The balance between regulatory CD4 T cells, known to control autoimmunity, and effector CD4 T cells, that recognize liver self-antigens and mediate the liver inflammation, appears central in AIH immune mechanisms. Recent advances in the identification of pathogenic auto-reactive CD4 T cells, and of new mechanisms of immune regulatory defects in AIH patients, give new insights into the pathophysiology of this disease.SUMMARY:In this review, we propose an overview of the central role of CD4 T cells (both regulatory and pathogenic) in mechanisms of AIH, with a focus on recent advances regarding defective regulatory mechanisms and immune profile of auto-reactive CD4 T cells. These findings may have implication for the orientation of new therapeutic strategies to treat AIH, such as regulatory T-cell infusion or targeting B cells and cytokines released by pathogenic CD4 T cells.

The development of innovative immune cell therapies relies on efficient cell tracking strategies. For this, multiscale fluorescence-based analyses of transferred cells into the host with complementary techniques, including flow cytometry for high-throughput cell analysis and two-photon microscopy for deep tissue imaging would be highly beneficial. Ideally, cells should be labelled with a single fluorescent probe combining all the properties required for these different techniques. Due to the intrinsic autofluorescence of most tissues and especially the liver, far-red emission is also an important asset. However, the development of far-red emitting probes suitable for two-photon microscopy and compatible with clearing methods to track labelled immune cells in thick samples, remains challenging. A newly-designed water-soluble far-red emitting polymer probe, 19K-6H, with a large Stokes shift, was thus evaluated for the tracking of primary immune CD8 T cells. These cells, prepared from mouse spleen, were efficiently labelled with the 19K-6H probe, which was internalized via endocytosis and was highly biocompatible at concentrations up to 20 μM. Labelled primary CD8 T cells were detectable in culture by both confocal and two-photon microscopy as well as flow cytometry, even after 3 days of active proliferation. Finally, 19K-6H-labelled primary CD8 T cells were injected to mice in a classical model of immune mediated hepatitis. The efficient tracking of the transferred cells in the liver by flow cytometry (on purified non-parenchymal cells) and by two-photon microscopy on 800 μm thick cleared sections, demonstrated the versatility of the 19K-6H probe.

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 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. Methods We 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. 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 ( 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. Conclusions This 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.

Two well‐characterized carbohydrate epitopes are absent in humans but present in other mammals. These are galactose‐α1,3‐galactose (αGal) and N ‐glycolylneuraminic acid (Neu5Gc) which are introduced by the activities of two enzymes including α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP‐Neu5Gc hydroxylase (encoded by the CMAH gene) that are inactive in humans but present in cattle. Hence, bovine‐derived products are antigenic in humans who receive bioprosthetic heart valves (BHVs) or those that suffer from red meat syndrome. Using programmable nucleases, we disrupted (knockout, KO) GGTA1 and CMAH genes encoding for the enzymes that catalyse the synthesis of αGal and Neu5Gc, respectively, in both male and female bovine fibroblasts. The KO in clonally selected fibroblasts was detected by polymerase chain reaction (PCR) and confirmed by Sanger sequencing. Selected fibroblasts colonies were used for somatic cell nuclear transfer (SCNT) to produce cloned embryos that were implanted in surrogate recipient heifers. Fifty‐three embryos were implanted in 33 recipients heifers; 3 pregnancies were carried to term and delivered 3 live calves. Primary cell cultures were established from the 3 calves and following molecular analyses confirmed the genetic deletions. FACS analysis showed the double‐KO phenotype for both antigens confirming the mutated genotypes. Availability of such cattle double‐KO model lacking both αGal and Neu5Gc offers a unique opportunity to study the functionality of BHV manufactured with tissues of potentially lower immunogenicity, as well as a possible new clinical approaches to help patients with red meat allergy syndrome due to the presence of these xenoantigens in the diet.