Tipping the balance in autoimmunity: are regulatory t cells the cause, the cure, or both?

Abstract Regulatory T cells (Tregs) are a specialized subgroup of T-cell lymphocytes that is crucial for maintaining immune homeostasis and preventing excessive immune responses. Depending on their differentiation route, Tregs can be subdivided into thymically derived Tregs (tTregs) and peripherally induced Tregs (pTregs), which originate from conventional T cells after extrathymic differentiation at peripheral sites. Although the regulatory attributes of tTregs and pTregs partially overlap, their modes of action, protein expression profiles, and functional stability exhibit specific characteristics unique to each subset. Over the last few years, our knowledge of Treg differentiation, maturation, plasticity, and correlations between their phenotypes and functions has increased. Genetic and functional studies in patients with numeric and functional Treg deficiencies have contributed to our mechanistic understanding of immune dysregulation and autoimmune pathologies. This review provides an overview of our current knowledge of Treg biology, discusses monogenetic Treg pathologies and explores the role of Tregs in various other autoimmune disorders. Additionally, we discuss novel approaches that explore Tregs as targets or agents of innovative treatment options

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

BackgroundRegulatory 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. MethodsTherefore, our aim was to enhance the function and stability of transferred CAR-Tregs via expression of membrane-associated IL-2 (mbIL-2). ResultsmbIL-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. DiscussionThe 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 Prior to Experimental Induction of Autoimmune Hepatitis Promotes More Severe Hepatic Inflammation, Production of IL-17 and Apoptosis

Autoimmune hepatitis (AIH) is detected at a late stage in the course of the disease. Therefore, induction and etiology are largely unclear. It is controversial if the induction of autoimmunity occurs in the liver or in the spleen. In our experimental murine AIH model, the induction of autoimmunity did not occur in the spleen. Instead, a protective role of the spleen could be more likely. Therefore, we splenectomized mice followed by induction of experimental murine AIH. Splenectomized mice presented more severe portal inflammation. Furthermore, these mice had more IL-17, IL-23 receptor (IL-23R) and caspase 3 (casp3) and a decreased amount of erythropoietin in serum, while intrahepatic T cell compartments were unaffected. These results indicate that the spleen is not necessary for induction of AIH, and splenectomy disrupts the ability to immune regulate the intensity of hepatic inflammation, production of IL-17 and apoptosis

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 β-amyloid (Aβ) 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 β-amyloid. Results: The β-amyloid-specific CARs obtained were stimulated by both recombinant and membrane-bound Aβ 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

Hyperferritinemia and hypergammaglobulinemia predict the treatment response to standard therapy in autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a chronic hepatitis with an increasing incidence. The majority of patients require life-long immunosuppression and incomplete treatment response is associated with a disease progression. An abnormal iron homeostasis or hyperferritinemia is associated with worse outcome in other chronic liver diseases and after liver transplantation. We assessed the capacity of baseline parameters including the iron status to predict the treatment response upon standard therapy in 109 patients with untreated AIH type 1 (AIH-1) in a retrospective single center study. Thereby, a hyperferritinemia (> 2.09 times upper limit of normal; Odds ratio (OR) = 8.82; 95% confidence interval (CI): 2.25-34.52) and lower immunoglobulins (<1.89 times upper limit of normal; OR = 6.78; CI: 1.87-24.59) at baseline were independently associated with the achievement of complete biochemical remission upon standard therapy. The predictive value increased when both variables were combined to a single treatment response score, when the cohort was randomly split into a training (area under the curve (AUC) = 0.749; CI 0.635-0.863) and internal validation cohort (AUC = 0.741; CI 0.558-0.924). Patients with a low treatment response score (<1) had significantly higher cumulative remission rates in the training (p<0.001) and the validation cohort (p = 0.024). The baseline hyperferritinemia was accompanied by a high serum iron, elevated transferrin saturations and mild hepatic iron depositions in the majority of patients. However, the abnormal iron status was quickly reversible under therapy. Mechanistically, the iron parameters were not stringently related to a hepatocellular damage. Ferritin rather seems deregulated from the master regulator hepcidin, which was down regulated, potentially mediated by the elevated hepatocyte growth factor. In conclusion, baseline levels of serum ferritin and immunoglobulins, which are part of the diagnostic work-up of AIH, can be used to predict the treatment response upon standard therapy in AIH-1, although confirmation from larger multicenter studies is pending

The cyclin E regulator cullin 3 prevents mouse hepatic progenitor cells from becoming tumor-initiating cells

Cyclin E is often overexpressed in cancer tissue, leading to genetic instability and aneuploidy. Cullin 3 (Cul3) is a component of the BTB-Cul3-Rbx1 (BCR) ubiquitin ligase that is involved in the turnover of cyclin E. Here we show that liver-specific ablation of Cul3 in mice results in the persistence and massive expansion of hepatic progenitor cells. Upon induction of differentiation, Cul3-deficient progenitor cells underwent substantial DNA damage in vivo and in vitro, thereby triggering the activation of a cellular senescence response that selectively blocked the expansion of the differentiated offspring. Positive selection of undifferentiated progenitor cells required the expression of the tumor suppressor protein p53. Simultaneous loss of Cul3 and p53 in hepatic progenitors turned these cells into highly malignant tumor-initiating cells that formed largely undifferentiated tumors in nude mice. In addition, loss of Cul3 and p53 led to the formation of primary hepatocellular carcinomas. Importantly, loss of Cul3 expression was also detected in a large series of human liver cancers and correlated directly with tumor de-differentiation. The expression of Cul3 during hepatic differentiation therefore safeguards against the formation of progenitor cells that carry a great potential for transformation into tumor-initiating cells

Exchange of Cytosolic Content between T Cells and Tumor Cells Activates CD4 T Cells and Impedes Cancer Growth

<div><p>Background</p><p>T cells are known to participate in the response to tumor cells and react with cytotoxicity and cytokine release. At the same time tumors established versatile mechanisms for silencing the immune responses. The interplay is far from being completely understood. In this study we show contacts between tumor cells and lymphocytes revealing novel characteristics in the interaction of T cells and cancer cells in a way not previously described.</p> <p>Methods/ Findings</p><p>Experiments are based on the usage of a hydrophilic fluorescent dye that occurs free in the cytosol and thus transfer of fluorescent cytosol from one cell to the other can be observed using flow cytometry. Tumor cells from cell lines of different origin or primary hepatocellular carcinoma (HCC) cells were incubated with lymphocytes from human and mice. This exposure provoked a contact dependent uptake of tumor derived cytosol by lymphocytes – even in CD4⁺ T cells and murine B cells – which could not be detected after incubation of lymphocytes with healthy cells. The interaction was a direct one, not requiring the presence of accessory cells, but independent of cytotoxicity and TCR engagement.</p> <p>Electron microscopy disclosed 100-200nm large gaps in the cell membranes of connected cells which separated viable and revealed astonishing outcome. While the lymphocytes were induced to proliferate in a long term fashion, the tumor cells underwent a temporary break in cell division. The <i>in vitro</i> results were confirmed <i>in vivo</i> using a murine acute lymphoblastic leukemia (ALL) model. The arrest of tumor proliferation resulted in a significant prolonged survival of challenged mice. </p> <p>Conclusions</p><p>The reported cell-cell contacts reveal new characteristics i.e. the enabling of cytosol flow between the cells including biological active proteins that influence the cell cycle and biological behaviour of the recipient cells. This adds a completely new aspect in tumor induced immunology.</p> </div

Unique properties of thymic antigen-presenting cells promote epigenetic imprinting of alloantigen-specific regulatory T cells.

Regulatory T cells (Tregs) are potential immunotherapeutic candidates to induce transplantation tolerance. However, stability of Tregs still remains contentious and may potentially restrict their clinical use. Recent work suggested that epigenetic imprinting of Foxp3 and other Treg-specific signature genes is crucial for stabilization of immunosuppressive properties of Foxp3+ Tregs, and that these events are initiated already during early stages of thymic Treg development. However, the mechanisms governing this process remain largely unknown. Here we demonstrate that thymic antigen-presenting cells (APCs), including thymic dendritic cells (t-DCs) and medullary thymic epithelial cells (mTECs), can induce a more pronounced demethylation of Foxp3 and other Treg-specific epigenetic signature genes in developing Tregs when compared to splenic DCs (sp-DCs). Transcriptomic profiling of APCs revealed differential expression of secreted factors and costimulatory molecules, however neither addition of conditioned media nor interference with costimulatory signals affected Foxp3 induction by thymic APCs in vitro. Importantly, when tested in vivo both mTEC- and t-DC-generated alloantigen-specific Tregs displayed significantly higher efficacy in prolonging skin allograft acceptance when compared to Tregs generated by sp-DCs. Our results draw attention to unique properties of thymic APCs in initiating commitment towards stable and functional Tregs, a finding that could be highly beneficial in clinical immunotherapy

Exchange of cytosol is mediated by contacts between purified CD4⁺ T cells and tumor cells and associated with transfer of enzymes of high molecular weight.

<div><p>(A)2x10⁶ human PBMCs or purified CD4⁺ T cells were incubated with 2x10⁶ CMFDA-labeled L23 cells for 4 h, subsequently stained for CD3, CD4 and CD8 and analyzed in flow cytometry, respectively. The exposure of lymphocytes to L23 cells led to an inclusion of fluorescence in numerous T cells. </p> <p>(B)Summary of four independent experiments performed as described above. The frequency of green fluorescent T cells is related to bulk population of PBMCs. </p> <p>(C)PBMCs were pre-incubated with 5mM strontium chloride to induce degranulation before the exposure to L23 cells. Adpation of fluorescence was assessed in CD4⁺ and CD8⁺ T cells.</p> <p>(D)2x10⁶ purified T cells and 2x10⁶ L23 were incubated for 4 h together or separated by transwells (TW), the labeled L23 cells at the bottom of a 96well round-bottom plate and T cells placed in the transwells.</p> <p>(E)Purified CD4⁺ T cells were incubated with L23 cells for 4 h, fixed and prepared for electron microscopy (EM). Scanning (i) and transmission EM (ii-iv) revealed intense contacts. In transmission EM flow of cytosol was visualized by the electron dense cytosol of lymphocytes which distributed from the site of contacts in the tumor cell (arrows in (ii) and (iii)). In the highest magnification gaps in the cell membranes could be detected (arrows in iv). The gaps dehisced length up to 200nm allowing free flow of cytosol and soluble components but not of cellular structures e.g. mitochondria. However, the aggregation of mitochondria in the area of contact formation might be an indication of energy consuming processes.</p> <p>(F)CD4⁺ T cells were sorted after incubation with CMFDA-labeled luciferase-transgeneic L23 cells in green-fluorescent and non-fluorescent T cells. 5x10⁴ separated T cells were subsequently incubated in medium supplemented with 100µg/ml luciferin in 96well round-bottom plates. Luminescence was assessed after 30 min incubation using IVIS analysis. 5x10⁴ transgeneic L23 cells served as control of luminescence intensity.</p> <p>Results are shown as representative pictures and scatter-grams or summarizing 4 independent experiments as mean ± SEM.</p></div