Supraphysiological FOXP3 expression in human CAR-Tregs results in improved stability, efficacy, and safety of CAR-Treg products for clinical application.

The forkhead family transcription factor (FOXP3) is an essential regulator for the development of regulatory T cells (Tregs) and orchestrates both suppressive function and Treg lineage identity. Stable expression of FOXP3 enables Tregs to maintain immune homeostasis and prevent autoimmunity. However, under pro-inflammatory conditions, FOXP3 expression in Tregs can become unstable, leading to loss of suppressive function and conversion into pathogenic T effector cells. Therefore, the success of adoptive cell therapy with chimeric antigen receptor (CAR) Tregs is highly dependent on the stability of FOXP3 expression to ensure the safety of the cell product. To warrant the stable expression of FOXP3 in CAR-Treg products, we have developed an HLA-A2-specific CAR vector that co-expresses FOXP3. The transduction of isolated human Tregs with the FOXP3-CAR led to an increase in the safety and efficacy of the CAR-Treg product. In a hostile microenvironment, under pro-inflammatory and IL-2-deficient conditions, FOXP3-CAR-Tregs showed a stable expression of FOXP3 compared to Control-CAR-Tregs. Furthermore, additional exogenous expression of FOXP3 did not induce phenotypic alterations and dysfunctions such as cell exhaustion, loss of functional Treg characteristics or abnormal cytokine secretion. In a humanized mouse model, FOXP3-CAR-Tregs displayed an excellent ability to prevent allograft rejection. Furthermore, FOXP3-CAR-Tregs revealed coherent Treg niche-filling capabilities. Overexpression of FOXP3 in CAR-Tregs has thereby the potential to increase the efficacy and reliability of cellular products, promoting their clinical use in organ transplantation and autoimmune diseases.

Treg-specific IL-2 therapy can reestablish intrahepatic immune regulation in autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a chronic autoimmune inflammatory disease that usually requires life-long immunosuppression. Frequent relapses after discontinuation of therapy indicate that intrahepatic immune regulation is not restored by current therapies. As steroid therapy preferentially depletes intrahepatic regulatory T cell (Tregs), immune regulation might be re-established by increasing and functionally strengthening intrahepatic Tregs. In recent clinical trials with low dose IL-2, the Treg compartment was strengthened in autoimmune diseases. Therefore, we tested complexed IL-2/anti-IL-2 to increase the selectivity for Tregs. We used our model of experimental murine AIH (emAIH) and treated the mice with complexed IL-2/anti-Il-2 in the late course of the disease. The mice showed increased intrahepatic and systemic Treg numbers after treatment and a reduction in activated, intrahepatic effector T cells (Teffs). This resulted in a reduction in liver-specific ALT levels and a molecular pattern similar to that of healthy individuals. In conclusion, complexed IL-2/anti-IL-2 restored the balance between Tregs and Teffs within the liver, thereby improving the course of emAIH. Treg-specific IL-2 augmentation offers new hope for reestablishing immune tolerance in patients with AIH.

Lipid nanoparticle-mediated siRNA delivery for safe targeting of human CML in vivo.

Efficient and safe delivery of siRNA in vivo is the biggest roadblock to clinical translation of RNA interference (RNAi)-based therapeutics. To date, lipid nanoparticles (LNPs) have shown efficient delivery of siRNA to the liver; however, delivery to other organs, especially hematopoietic tissues still remains a challenge. We developed DLin-MC3-DMA lipid-based LNP-siRNA formulations for systemic delivery against a driver oncogene to target human chronic myeloid leukemia (CML) cells in vivo. A microfluidic mixing technology was used to obtain reproducible ionizable cationic LNPs loaded with siRNA molecules targeting the BCR-ABL fusion oncogene found in CML. We show a highly efficient and non-toxic delivery of siRNA in vitro and in vivo with nearly 100% uptake of LNP-siRNA formulations in bone marrow of a leukemic model. By targeting the BCR-ABL fusion oncogene, we show a reduction of leukemic burden in our myeloid leukemia mouse model and demonstrate reduced disease burden in mice treated with LNP-BCR-ABL siRNA as compared with LNP-CTRL siRNA. Our study provides proof-of-principle that fusion oncogene specific RNAi therapeutics can be exploited against leukemic cells and promise novel treatment options for leukemia patients.

Hepatic T Cell Tolerance Induction in An Inflammatory Environment.

For the development of autoimmune hepatitis (AIH), genetic predisposition and environmental triggers are of major importance. Although experimental AIH can be induced in genetically susceptible mice, the low precursor frequency of autoreactive T cells hampers a deeper analysis of liver-specific T cells. Here, we established a system where the model antigen hemagglutinin (HA) is expressed exclusively in hepatocytes of Rosa26-HA mice following administration of a replication deficient adenovirus expressing Cre recombinase (Ad-Cre). Under these conditions, hepatocytes mimic the generation of altered-self neoantigens. To follow autoreactive T cells during AIH, we adoptively transferred HA--specific Cl4-TCR and 6.5-TCR T cells into Ad-Cre infected -Rosa26-HA mice. Alternatively, Rosa26-HA mice have been crossed with TCR transgenic mice that were infected with Ad-Cre to break hepatic tolerance and induce the expression of the HA antigen as a hepatic self-antigen. Surprisingly, neither adoptive transfer nor a very high precursor frequency of autoreactive T cells was able to break tolerance in the context of adenoviral infection. The low proliferation of the antigen experienced autoreactive T cells despite the presence of the autoantigen and inflammation points to anergy as a potential tolerance mechanism. This model underscores the crucial importance of genetic susceptibility to break tolerance against hepatic autoantigens.

The influence of genetic predisposition and autoimmune hepatitis inducing antigens in disease development.

Autoimmune hepatitis (AIH) is defined as a chronic liver inflammation with loss of tolerance against hepatocytes. The etiology and pathophysiology of AIH are still poorly understood because reliable animal models are limited. Therefore, we recently introduced a model of experimental murine AIH by a self-limited adenoviral infection with the AIH type 2 antigen formiminotransferase cyclodeaminase (FTCD). We could demonstrate that break of humoral tolerance towards liver specific autoantigens like FTCD and cytochrome P450 2D6 (CYP2D6) is not dependent on the genetic background. However, the development of AIH in autoantibody positive animals is determined by genetic background genes. We could also show that the break of humoral tolerance is necessary but not sufficient for the development of AIH. In contrast the break of tolerance against the ubiquitously expressed nuclear antigens (ANAs) is strictly dependent on genetic predisposition. Priming with the UGA suppressor tRNA-associated protein (soluble liver antigen; SLA) is a strong inducer of ANA reactivity, but not sufficient to cause AIH development thereby questioning the importance of anti-SLA immune response as an important driver in AIH. Monogenetic mutations such as Aire-deficiency can cause AIH in otherwise genetically resistant strains. CONCLUSION: The results have important implications for our understanding of the pathophysiology of AIH development and for the interpretation of humoral antibody responses in AIH.

Preferential accumulation of T helper cells but not cytotoxic T cells characterizes benign subclinical rejection of human liver allografts.

Subclinical rejection (SCR) is a common event in protocol biopsies after liver transplantation (LT). So far the interpretation of the underlying histological changes and clinical significance is limited. Previous studies were restricted to SCR manifestations within the first weeks after transplantation with limited follow-up. We analyzed clinical data from our prospective protocol biopsy program and found late SCR (at least 3 months after transplantation) to be a common event (41/94 patients). SCR manifested much later than acute cellular rejection (ACR). In the second year after transplantation, the SCR incidence in protocol biopsies reached a plateau of approximately 25% and remained at this level until the latest observed manifestations more than 5 years after transplantation. During a median follow-up of 32 months after SCR, no acute or chronic rejection, relevant graft fibrosis, graft loss, or liver-related death occurred even without specific therapy for SCR. Immunophenotyping of liver biopsies during SCR showed that similar to ACR, the composition of intrahepatic T cells depended on the severity of histological rejection. However, SCR showed a different pattern of infiltrating T cells with a stronger accumulation of CD4(+) cells, an increasing CD4(+) /CD8(+) ratio, and an increasing CD4(+) forkhead box P3 (FOXP3)(+) regulatory T cell (Treg)/CD8(+) ratio, which was not seen in ACR. These intrahepatic T cell patterns were not reflected in the peripheral blood. In conclusion, late SCR after LT has a good clinical prognosis, and it seems safe to leave it untreated. This benign clinical course compared to ACR is associated with intrahepatic T cell infiltration patterns showing less cytotoxic T cells and more CD4(+) FOXP3(+) Tregs. Liver Transplantation 22 943-955 2016 AASLD.

Autoimmune hepatitis in a murine autoimmune polyendocrine syndrome type 1 model is directed against multiple autoantigens.

UNLABELLED: Autoimmune polyendocrine syndrome type 1 (APS-1) is caused by mutations of the autoimmune regulator (AIRE) gene. Mouse studies have shown that this results in defective negative selection of T cells and defective early seeding of peripheral organs with regulatory T cells (Tregs). Aire deficiency in humans and mice manifests as spontaneous autoimmunity against multiple organs, and 20% of patients develop an autoimmune hepatitis (AIH). To study AIH in APS-1, we generated a murine model of human AIH on a BALB/c mouse background, in which Aire is truncated at exon 2. A subgroup of 24% of mice is affected by AIH, characterized by lymphoplasmacytic and periportal hepatic infiltrates, autoantibodies, elevated aminotransferases, and a chronic and progressive course of disease. Disease manifestation was dependent on specific Aire mutations and the genetic background of the mice. Though intrahepatic Treg numbers were increased and hyperproliferative, the intrahepatic CD4/CD8 ratio was decreased. The targets of the adaptive autoimmune response were polyspecific and not focussed on essential autoantigens, as described for other APS-1-related autoimmune diseases. The AIH could be treated with prednisolone or adoptive transfer of polyspecific Tregs. CONCLUSION: Development of AIH in APS-1 is dependent on specific Aire mutations and genetic background genes. Autoimmune response is polyspecific and can be controlled by steroids or transfer with Tregs. This might enable new treatment options for patients with AIH.

Isolation of human antigen-specific regulatory T cells with high suppressive function.

Adoptive transfer of regulatory T (Treg) cells could be an alternative to chronic immunosuppression for prevention of allogeneic graft rejection. While polyspecific Treg cells can prevent immune responses under lymphopenic conditions, Ag-specific Treg cells are needed to treat autoimmunity and graft rejection. Yet, reliable markers for Ag-specific Treg cells are missing. We report that latency-associated peptide (LAP) and glycoprotein A repetitions predominant (GARP) can identify human Ag-specific Treg cells. In addition, we show that the depletion of CD154(+) cells from LAP(+) or GARP(+) Treg cells increases the Treg-cell purity to over 90%, as assessed by epigenetic analysis. These Ag-specific Treg cells can be isolated magnetically and might contribute to the development of GMP-based protocols. In addition, Ag-specific Treg cells are functionally far superior to CD4(+) CD25(high) or CD4(+) CD25(high) CD127(low) Treg cells in vitro and in preventing strong alloreactions in humanized mice. They could, therefore, have a high therapeutic potential for the control of alloimmune, autoimmune, and allergic immune responses in patients.

Intrahepatic regulatory T cells in autoimmune hepatitis are associated with treatment response and depleted with current therapies.

BACKGROUND & AIMS: Autoimmune hepatitis (AIH) is a chronic autoimmune liver disease usually requiring life-long immunosuppression. The mechanisms for disease initiation and chronicity are largely unknown. A contribution of deficient regulatory T cells (Tregs) in the blood was controversially discussed recently. So far investigations in the target organ have been limited to single parameter analysis in untreated AIH. METHODS: We retrospectively analysed the pattern of liver infiltrating T, B and regulatory T cells quantitatively with simultaneous multicolour immunofluorescence before (n=45) and under (n=31) therapy in adult AIH type 1 (AIH-1) patients. RESULTS: Intrahepatic CD4(+) cells dominate over CD8(+) at diagnosis, but with increasing disease activity the CD4(+)/CD8(+) ratio approached one. While there is no change of Tregs in the blood, they are enriched with effector T cells (Teffs) within the liver of patients with untreated AIH-1 with a constant Treg/Teff ratio. Even more importantly, immunosuppression mostly with steroids and azathioprine caused a disproportional loss of intrahepatic Tregs. Patients reaching biochemical remission had higher intrahepatic Treg/Teff and Treg/B cell ratios compared to patients failing to reach remission. In vitro proliferation of Tregs seemed to be more suppressed by prednisolone than expansion of Teffs. Furthermore, intraportal B cells correlated with serum IgG suggesting an autochthonous intrahepatic IgG production. CONCLUSIONS: Intrahepatic Tregs are rather enriched than numerically deficient in untreated AIH-1. The disproportional decrease of intrahepatic Tregs during therapy might explain high relapse rates after discontinuation of immunosuppression. Thus, future therapies increasing intrahepatic immunoregulation might be better suited for long-term control of AIH.

Genetic predisposition and environmental danger signals initiate chronic autoimmune hepatitis driven by CD4+ T cells.

UNLABELLED: Autoimmune hepatitis (AIH) is defined as a chronic liver disease with loss of tolerance against liver tissue eventually leading to cirrhosis if left untreated. 80%-90% of patients can be treated with a life-long immunosuppression. Unfortunately, there are strong drug-related side effects and steroid-refractory patients. Therefore, there is a need for a model system to investigate the complex immunopathogenesis of this chronic disease and subsequently to develop new therapeutic interventions. We developed a new model of experimental murine AIH (emAIH) by a self-limited adenoviral infection with the hepatic autoantigen formiminotransferase cyclodeaminase (FTCD). After an initial transient hepatitis there was a chronic evolving AIH, finally leading to portal and lobular fibrosis. We could show that the genetic predisposition provided by the NOD background was essential for creating a fertile field for the development of liver-specific autoimmunity. However, a strong environmental trigger was additionally necessary to initiate the disease. Besides the break of humoral tolerance, T-cell tolerance against hepatic self-antigens was also broken and CD4(+) T cells were identified as essential drivers of the disease. As the disease was successfully treated with prednisolone and budesonide, the model will be helpful to develop and test new therapeutic interventions. CONCLUSION: We developed a new murine AIH model closely resembling AIH in patients that explains the mechanisms of AIH pathophysiology. In addition, emAIH provides options to test therapeutic alternatives for patients not achieving remission, with reduced side effects of chronic nonspecific immunosuppression.

Graft-Specific Regulatory T Cells for Long-Lasting, Local Tolerance Induction.

BACKGROUND: Solid organ transplantation is hindered by immune-mediated chronic graft dysfunction and the side effects of immunosuppressive therapy. Regulatory T cells (Tregs) are crucial for modulating immune responses post-transplantation; however, the transfer of polyspecific Tregs alone is insufficient to induce allotolerance in rodent models. METHODS: To enhance the efficacy of adoptive Treg therapy, we investigated different immune interventions in the recipients. By utilizing an immunogenic skin transplant model and existing transplantation medicine reagents, we facilitated the clinical translation of our findings. Specifically, antigen-specific Tregs were used. RESULTS: Our study demonstrated that combining the available induction therapies with drug-induced T-cell proliferation due to lymphopenia effectively increased the Treg/T effector ratios. This results in significant Treg accumulation within the graft, leading to long-term tolerance after the transfer of antigen-specific Tregs. Importantly, all the animals achieved operational tolerance, which boosted the presence of adoptively transferred Tregs within the graft. CONCLUSIONS: This protocol offers a means to establish tolerance by utilizing antigen-specific Tregs. These results have promising implications for future trials involving adoptive Treg therapy in organ transplantation.

MAPKAP kinase MK2 maintains self-renewal capacity of haematopoietic stem cells.

The structurally related MAPK-activated protein kinases (MAPKAPKs or MKs) MK2, MK3 and MK5 are involved in multiple cellular functions, including cell-cycle control and cellular differentiation. Here, we show that after deregulation of cell-cycle progression, haematopoietic stem cells (HSCs) in MK2-deficient mice are reduced in number and show an impaired ability for competitive repopulation in vivo. To understand the underlying molecular mechanism, we dissected the role of MK2 in association with the polycomb group complex (PcG) and generated a MK2 mutant, which is no longer able to bind to PcG. The reduced ability for repopulation is rescued by re-introduction of MK2, but not by the Edr2-non-binding mutant of MK2. Thus, MK2 emerges as a regulator of HSC homeostasis, which could act through chromatin remodelling by the PcG complex.

PD-1/CTLA-4 Blockade Leads to Expansion of CD8+PD-1int TILs and Results in Tumor Remission in Experimental Liver Cancer.

Background: Checkpoint inhibitors act on exhausted CD8+ T cells and restore their effector function in chronic infections and cancer. The underlying mechanisms of action appear to differ between different types of cancer and are not yet fully understood. Methods: Here, we established a new orthotopic HCC model to study the effects of checkpoint blockade on exhausted CD8+ tumor-infiltrating lymphocytes (TILs). The tumors expressed endogenous levels of HA, which allowed the study of tumor-specific T cells. Results: The induced tumors developed an immune-resistant TME in which few T cells were found. The few recovered CD8+ TILs were mostly terminally exhausted and expressed high levels of PD-1. PD-1/CTLA-4 blockade resulted in a strong increase in the number of CD8+ TILs expressing intermediate amounts of PD-1, also called progenitor-exhausted CD8+ TILs, while terminally exhausted CD8+ TILs were almost absent in the tumors of treated mice. Although transferred naïve tumor-specific T cells did not expand in the tumors of untreated mice, they expanded strongly after treatment and generated progenitor-exhausted but not terminally exhausted CD8+ TILs. Unexpectedly, progenitor-exhausted CD8+ TILs mediated the antitumor response after treatment with minimal changes in their transcriptional profile. Conclusion: In our model, few doses of checkpoint inhibitors during the priming of transferred CD8+ tumor-specific T cells were sufficient to induce tumor remission. Therefore, PD-1/CTLA-4 blockade has an ameliorative effect on the expansion of recently primed CD8+ T cells while preventing their development into terminally exhausted CD8+ TILs in the TME. This finding could have important implications for future T-cell therapies.

Development of Beta-Amyloid-Specific CAR-Tregs for the Treatment of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease that remains uncured. Its pathogenesis is characterized by the formation of ß-amyloid (Aß) plaques. The use of antigen-specific regulatory T cells (Tregs) through adoptive transfer has shown promise for the treatment of many inflammatory diseases, although the effectiveness of polyspecific Tregs is limited. Obtaining a sufficient number of antigen-specific Tregs from patients remains challenging. AIMS AND METHODS: To address this problem, we used an antibody-like single-chain variable fragment from a phage library and subsequently generated a chimeric antigen receptor (CAR) targeting ß-amyloid. RESULTS: The ß-amyloid-specific CARs obtained were stimulated by both recombinant and membrane-bound Aß isolated from the murine brain. The generated CAR-Tregs showed a normal Treg phenotype, were antigen-specific activatable, and had suppressive capacity. CONCLUSION: This study highlights the potential of CAR technology to generate antigen-specific Tregs and presents novel approaches for developing functional CARs.

Generation of Chimeric Antigen Receptors against Tetraspanin 7.

Adoptive transfer of antigen-specific regulatory T cells (Tregs) has shown promising results in the treatment of autoimmune diseases; however, the use of polyspecific Tregs has limited effects. However, obtaining a sufficient number of antigen-specific Tregs from patients with autoimmune disorders remains challenging. Chimeric antigen receptors (CARs) provide an alternative source of T cells for novel immunotherapies that redirect T cells independently of the MHC. In this study, we aimed to generate antibody-like single-chain variable fragments (scFv) and subsequent CARs against tetraspanin 7 (TSPAN7), a membrane protein highly expressed on the surface of pancreatic beta cells, using phage display technology. We established two methods for generating scFvs against TSPAN7 and other target structures. Moreover, we established novel assays to analyze and quantify their binding abilities. The resulting CARs were functional and activated specifically by the target structure, but could not recognize TSPAN7 on the surface of beta cells. Despite this, this study demonstrates that CAR technology is a powerful tool for generating antigen-specific T cells and provides new approaches for generating functional CARs.

Inflammatory bowel disease and mutations affecting the interleukin-10 receptor.

BACKGROUND: The molecular cause of inflammatory bowel disease is largely unknown. METHODS: We performed genetic-linkage analysis and candidate-gene sequencing on samples from two unrelated consanguineous families with children who were affected by early-onset inflammatory bowel disease. We screened six additional patients with early-onset colitis for mutations in two candidate genes and carried out functional assays in patients' peripheral-blood mononuclear cells. We performed an allogeneic hematopoietic stem-cell transplantation in one patient. RESULTS: In four of nine patients with early-onset colitis, we identified three distinct homozygous mutations in genes IL10RA and IL10RB, encoding the IL10R1 and IL10R2 proteins, respectively, which form a heterotetramer to make up the interleukin-10 receptor. The mutations abrogate interleukin-10-induced signaling, as shown by deficient STAT3 (signal transducer and activator of transcription 3) phosphorylation on stimulation with interleukin-10. Consistent with this observation was the increased secretion of tumor necrosis factor alpha and other proinflammatory cytokines from peripheral-blood mononuclear cells from patients who were deficient in IL10R subunit proteins, suggesting that interleukin-10-dependent "negative feedback" regulation is disrupted in these cells. The allogeneic stem-cell transplantation performed in one patient was successful. CONCLUSIONS: Mutations in genes encoding the IL10R subunit proteins were found in patients with early-onset enterocolitis, involving hyperinflammatory immune responses in the intestine. Allogeneic stem-cell transplantation resulted in disease remission in one patient.

A syndrome with congenital neutropenia and mutations in G6PC3.

BACKGROUND: The main features of severe congenital neutropenia are the onset of severe bacterial infections early in life, a paucity of mature neutrophils, and an increased risk of leukemia. In many patients, the genetic causes of severe congenital neutropenia are unknown. METHODS: We performed genomewide genotyping and linkage analysis on two consanguineous pedigrees with a total of five children affected with severe congenital neutropenia. Candidate genes from the linkage interval were sequenced. Functional assays and reconstitution experiments were carried out. RESULTS: All index patients were susceptible to bacterial infections and had very few mature neutrophils in the bone marrow; structural heart defects, urogenital abnormalities, and venous angiectasia on the trunk and extremities were additional features. Linkage analysis of the two index families yielded a combined multipoint lod score of 5.74 on a linkage interval on chromosome 17q21. Sequencing of G6PC3, the candidate gene encoding glucose-6-phosphatase, catalytic subunit 3, revealed a homozygous missense mutation in exon 6 that abolished the enzymatic activity of glucose-6-phosphatase in all affected children in the two families. The patients' neutrophils and fibroblasts had increased susceptibility to apoptosis. The myeloid cells showed evidence of increased endoplasmic reticulum stress and increased activity of glycogen synthase kinase 3beta (GSK-3beta). We identified seven additional, unrelated patients who had severe congenital neutropenia with syndromic features and distinct biallelic mutations in G6PC3. CONCLUSIONS: Defective function of glucose-6-phosphatase, catalytic subunit 3, underlies a severe congenital neutropenia syndrome associated with cardiac and urogenital malformations.

Membrane-bound IL-2 improves the expansion, survival, and phenotype of CAR Tregs and confers resistance to calcineurin inhibitors.

Background: Regulatory T cells (Tregs) play an important role in the maintenance of immune homeostasis and the establishment of immune tolerance. Since Tregs do not secrete endogenous IL-2, they are especially dependent on external IL-2. IL-2 deficiency leads to lower Treg numbers, instability of the Treg phenotype and loss of immune regulation. After organ transplantation, patients are treated with calcineurin inhibitors (CNIs), which further limits available IL-2. Application of low-dose IL-2 expands Tregs but also activates NK and CD8+ T cells. It was recently shown that graft-specific Tregs recognizing mismatched MHC I molecules via a chimeric antigen receptor were far more potent than polyclonal Tregs in the regulation of immune responses after solid organ transplantation in a humanized mouse model. Methods: Therefore, our aim was to enhance the function and stability of transferred CAR-Tregs via expression of membrane-associated IL-2 (mbIL-2). Results: mbIL-2 promoted higher survival, phenotypic stability, and function among CAR-Tregs than observed in clinical trials. The cells were also more stable under inflammatory conditions. In a preclinical humanized mouse model, we demonstrated that mbIL-2 CAR Tregs survive better in the Treg niche than control CAR Tregs and are even resistant to CNI therapy without affecting other Tregs, thus acting mainly in cis. Discussion: The functional and phenotypic improvements observed after membrane-attached IL-2 expression in CAR-Tregs will be important step for enhancing CAR-Treg therapies currently being tested in clinical trials for use after kidney and liver transplantation as well as in autoimmune diseases.

Splenectomy induces biochemical remission and regeneration in experimental murine autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a chronic immune-mediated inflammatory liver disease. It is known that AIH originates not from the spleen but from the liver itself. Nonetheless, most details of the etiology and pathophysiology are unknown. We induced experimental murine AIH (emAIH) in NOD/Ltj mice by single administration of a replication-deficient adenovirus and performed splenectomy during late-stage disease. Biochemical disease remission occurred, which was characterized by improvement in transaminase levels. The causes of this remission included a shift in the transcriptomic signature of serum proteins toward regeneration. At the cellular level, there was a marked decrease in activated CD8+ T cells and an increase in intrahepatic regulatory T cells (Tregs). Here, intrahepatic Treg numbers correlated with biochemical remission. Notably, an imbalance in the T-cell/B-cell ratio was observed, with a disproportionate increase in total B cells. In summary, intrahepatic increases in Tregs, biochemical remission, and regeneration could be induced by splenectomy in the late stage of emAIH.

The Detrimental Role of Regulatory T Cells in Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is induced by steatosis and metabolic inflammation. While involvement of the innate immune response has been shown, the role of the adaptive immune response in NASH remains controversial. Likewise, the role of regulatory T cells (Treg) in NASH remains unclear although initial clinical trials aim to target these regulatory responses. High-fat high-carbohydrate (HF-HC) diet feeding of NASH-resistant BALB/c mice as well as the corresponding recombination activating 1 (Rag)-deficient strain was used to induce NASH and to study the role of the adaptive immune response. HF-HC diet feeding induced strong activation of intrahepatic T cells in BALB/c mice, suggesting an antigen-driven effect. In contrast, the effects of the absence of the adaptive immune response was notable. NASH in BALB/c Rag1-/- mice was substantially worsened and accompanied by a sharp increase of M1-like macrophage numbers. Furthermore, we found an increase in intrahepatic Treg numbers in NASH, but either adoptive Treg transfer or anti-cluster of differentiation (CD)3 therapy unexpectedly increased steatosis and the alanine aminotransferase level without otherwise affecting NASH. Conclusion: Although intrahepatic T cells were activated and marginally clonally expanded in NASH, these effects were counterbalanced by increased Treg numbers. The ablation of adaptive immunity in murine NASH led to marked aggravation of NASH, suggesting that Tregs are not regulators of metabolic inflammation but rather enhance it.