Proinsulin multi-peptide immunotherapy induces antigen-specific regulatory T cells and limits autoimmunity in a humanized model

Peptide immunotherapy (PIT) is a targeted therapeutic approach, involving administration of disease-associated peptides, with the aim of restoring antigen-specific immunological tolerance without generalized immunosuppression. In type 1 diabetes, proinsulin is a primary antigen targeted by the autoimmune response, and is therefore a strong candidate for exploitation via PIT in this setting. To elucidate the optimal conditions for proinsulin-based PIT and explore mechanisms of action, we developed a preclinical model of proinsulin autoimmunity in a humanized HLA-DRB1*0401 transgenic HLA-DR4 Tg mouse. Once proinsulin-specific tolerance is broken, HLA-DR4 Tg mice develop autoinflammatory responses, including proinsulin-specific T cell proliferation, interferon (IFN)-γ and autoantibody production. These are preventable and quenchable by pre- and post-induction treatment, respectively, using intradermal proinsulin-PIT injections. Intradermal proinsulin-PIT enhances proliferation of regulatory [forkhead box protein 3 (FoxP3⁺)CD25^high] CD4 T cells, including those capable of proinsulin-specific regulation, suggesting this as its main mode of action. In contrast, peptide delivered intradermally on the surface of vitamin D3-modulated (tolerogenic) dendritic cells, controls autoimmunity in association with proinsulin-specific IL-10 production, but no change in regulatory CD4 T cells. These studies define a humanized, translational model for in vivo optimization of PIT to control autoimmunity in type 1 diabetes and indicate that dominant mechanisms of action differ according to mode of peptide delivery.     

A novel mechanism of regulatory T cell-mediated down-regulation of autoimmunity

We have established a novel CD4 and CD8 double-positive CD25+ T regulatory (Treg) clone, MT-5B, from lymph nodes of type 1 diabetes prone non-obese diabetic (NOD) mice immunized with CFA. CFA has previously been shown to prevent the onset of diabetes by inducing Treg cells. In vitro, clone MT-5B was anergic to a panel of antigen stimulations and exerted an immunosuppressive effect in antigen-non-specific and cell contact-independent manners. In vivo, clone MT-5B blocked the adoptive transfer of diabetes. Proteomics and immunoadsorption studies identified the suppressive proteins secreted by clone MT-5B as granzyme B (GrB) and perforin (PFN). GrB-mediated immune suppression was PFN dependent. Removal of GrB or PFN from the culture supernatant (SN) of MT-5B cells or pre-incubation of MT-5B cells with ethyleneglycol-bis(aminoethylether)-tetraacetic acid which blocks PFN activity reduced the immunosuppressive effect in vitro. Pre-incubation of diabetogenic splenocytes from NOD mice with MT-5B SN impaired their ability to transfer disease by inducing T cell apoptosis, and removal of GrB from MT-5B SN by immunoadsorption decreased the effector function of MT-5B SN on diabetogenic splenocytes. Immunization of NOD mice with CFA increased the expression of GrB+ CD4 T cells, indicating that these cells are present in vivo. In conclusion, we describe a novel mechanism of cell contact-independent immune suppression in which Treg cells maintain immune homeostasis by secreting GrB/PFN.     

Regulatory T cells in skin

Foxp3⁺ CD4⁺ regulatory T (Treg) cells are a subset of immune cells that function to regulate tissue inflammation. Skin is one of the largest organs and is home to a large proportion of the body's Treg cells. However, relative to other tissues (such as the spleen and gastrointestinal tract) the function of Treg cells in skin is less well defined. Here, we review our understanding of how Treg cells migrate to skin and the cellular and molecular pathways required for their maintenance in this tissue. In addition, we outline what is known about the specialized functions of Treg cells in skin. Namely, the orchestration of stem cell‐mediated hair follicle regeneration, augmentation of wound healing, and promoting adaptive immune tolerance to skin commensal microbes. A comprehensive understanding of the biology of skin Treg cells may lead to novel therapeutic approaches that preferentially target these cells to treat cutaneous autoimmunity, skin cancers and disorders of skin regeneration.     

Low expression of CD39+/CD45RA+ on regulatory T cells (Treg) cells in type 1 diabetic children in contrast to high expression of CD101+/CD129+ on Treg cells in children with coeliac disease

Type 1 diabetes (T1D) and coeliac disease are both characterized by an autoimmune feature. As T1D and coeliac disease share the same risk genes, patients risk subsequently developing the other disease. This study aimed to investigate the expression of T helper (Th), T cytotoxic (Tc) and regulatory T cells (T_reg) in T1D and/or coeliac disease children in comparison to healthy children. Subgroups of T cells (Th : CD4⁺ or Tc : CD8⁺); naive (CD27⁺CD28⁺CD45RA⁺CCR7⁺), central memory (CD27⁺CD28⁺CD45RA⁻CCR7⁺), effector memory (early differentiated; CD27⁺CD28⁺CD45RA⁻CCR7⁻ and late differentiated; CD27⁻CD28⁻CD45RA⁻CCR7⁻), terminally differentiated effector cells (TEMRA; CD27⁻CD28⁻CD45RA⁺CCR7⁻) and T_reg (CD4⁺CD25⁺FOXP3⁺CD127⁻) cells, and their expression of CD39, CD45RA, CD101 and CD129, were studied by flow cytometry in T1D and/or coeliac disease children or without any of these diseases (reference group). Children diagnosed with both T1D and coeliac disease showed a higher percentage of TEMRA CD4⁺ cells (P < 0·05), but lower percentages of both early and late effector memory CD8⁺ cells (P < 0·05) compared to references. Children with exclusively T1D had lower median fluorescence intensity (MFI) of forkhead box protein 3 (FoxP3) (P < 0·05) and also a lower percentage of CD39⁺ and CD45RA⁺ within the T_reg population (CD4⁺CD25⁺FOXP3⁺CD127⁻) (P < 0·05). Children with exclusively coeliac disease had a higher MFI of CD101 (P < 0·01), as well as a higher percentage of CD129⁺ (P < 0·05), in the CD4⁺CD25^hi lymphocyte population, compared to references. In conclusion, children with combined T1D and coeliac disease have a higher percentage of differentiated CD4⁺ cells compared to CD8⁺ cells. T1D children show signs of low CD39⁺/CD45RA⁺ T_reg cells that may indicate loss of suppressive function. Conversely, children with coeliac disease show signs of CD101⁺/CD129⁺ T_reg cells that may indicate suppressor activity.     

Increased prevalence of autoimmunity in Turner syndrome – influence of age

Individuals with Turner syndrome (TS) are prone to develop autoimmune conditions such as coeliac disease (CD), thyroiditis and type 1 diabetes (T1DM). The objective of the present study was to examine TS of various karyotypes for autoantibodies and corresponding diseases. This was investigated in a prospective cross‐sectional study of Danish TS patients (n = 107, median age 36·7 years, range: 6–60 years). A medical history was recorded and a blood sample was analysed for autoantibodies against gliadin, transglutaminase, adrenal cortex, intrinsic factor, anti‐thyroid peroxidase (anti‐TPO) and glutamic‐acid‐decarboxylase 65 (GAD‐65). Autoantibodies were present in 58% (n = 61) of all patients, whereof 18% (11) had autoantibodies targeting more than one organ. Patients with autoantibodies were significantly older than those without (P = 0·001). Anti‐TPO was present in 45% (48) of patients, of whom 33% (16) were hypothyroid. Overall, 18% (19) presented with CD autoantibodies, of whom 26% (five) had CD. Anti‐TPO and CD autoantibodies co‐existed in 9% (10). Immunoglobulin A deficiency was found in 3% (three) of patients, who all had CD autoantibodies without disease. Among four patients with anti‐GAD‐65 none had T1DM, but two were classified as having T2DM. One patient had adrenocortical autoantibodies but not adrenal failure. Autoantibodies against intrinsic factor were absent. Anti‐GAD‐65 was increased in isochromosomal karyotypes (3/23 versus 1/84, P = 0·008) with no other association found between autoantibodies and karyotype. In conclusion, TS girls and women face a high prevalence of autoimmunity and associated disease with a preponderance towards hypothyroidism and CD. Thus, health care providers dealing with this patient group should be observant and test liberally for these conditions even before clinical symptoms emerge.     

Decrease in phenotypic regulatory T cells in subsets of patients with common variable immunodeficiency

Common variable immunodeficiencies (CVID) are a heterogeneous group of antibody deficiency disorders complicated by autoimmune, lymphoproliferative and/or granulomatous manifestations, suggesting variations in immunoregulation. We sought to quantify regulatory CD4 T cells (T_reg cells) in the blood of CVID patients and to correlate the frequency with clinical manifestations and classification subgroups. Blood samples from 99 CVID patients in Freiburg, London and Sydney, who had been phenotyped clinically and stratified according to their memory B cell phenotype (Freiburg and Paris classification schemes), were analysed for the proportion of T_reg cells, defined either as CD25⁺/forkhead box P3 (FoxP3)⁺, CD25⁺/CD127^low/FoxP3⁺ or CD25⁺/CD127^low CD4⁺ T cells, and results compared with 49 healthy controls. Irrespective of the phenotype used to define them, there was a significant decrease in the T_reg cell proportion in patients with granulomatous disease and immune cytopenias. This allowed the definition of a subgroup of CVID patients with abnormally low T_reg cells, which had a higher rate of these two manifestations as well as autoimmune disease in general. There was also a significant reduction in the proportion of T_reg cells in the Freiburg group Ia compared with other CVID patients and controls, but there were no differences between the Paris groups. The reduction in T_reg cells in subsets of CVID patients may be relevant to their clinical manifestations, and may contribute to our understanding of the pathogenesis of CVID complications.     

Altered regulatory T cell phenotype in latent autoimmune diabetes of the adults (LADA)

Latent autoimmune diabetes of the adults (LADA) accounts for up to 12% of all patients with diabetes. Initially the disease resembles type 2 diabetes (T2D); however, the typical presence of β cell autoantibodies indicates an autoimmune basis of LADA. While dysfunctional regulatory T cells (T_regs) have been implicated in autoimmune diabetes, these cells have been scarcely studied in LADA. The aim of this study was to investigate the frequency and phenotype of circulating T_regs in LADA patients early during disease progression. Flow cytometric analysis was performed on whole blood and peripheral mononuclear cells (PBMC) from patients diagnosed with LADA prior to insulin deficiency (n = 39) and from healthy volunteers (n = 20). Overall, we found the frequency and activation status of peripheral putative T_regs to be altered in LADA patients compared to healthy controls. While total T cells and CD4⁺ T cells expressing high levels of CD25 (CD4⁺CD25^hi) were unchanged, the frequency and total numbers of CD4⁺ T cells expressing an intermediate level of CD25 (CD4⁺CD25^int) were decreased in LADA patients. Interestingly, the expression of the T_reg‐specific marker forkhead box protein 3 (FoxP3), as well as the activation and memory makers CD69, cytotoxic T lymphocyte associated antigen 4 (CTLA‐4), CCR4 and CD45RO were increased in CD4⁺CD25⁺ T cells of the patients. Our data depict phenotypical changes in T cells of LADA patients that may reflect a derangement in peripheral immune regulation contributing to the slow process leading to insulin‐dependent diabetes in these patients.     

Diminished regulatory T cells in cutaneous lesions of thymoma-associated multi-organ autoimmunity: a newly described paraneoplastic autoimmune disorder with fatal clinical course

Thymoma-associated multi-organ autoimmunity is a rare, autoimmune disease that causes colitis, liver dysfunction and cutaneous graft-versus-host (GVH)-like skin damage. This paraneoplastic autoimmune disorder may be due to inadequate T cell selection in the tumour environment of the thymus. Although sporadic case reports have revealed its clinical features, little is known about its pathological mechanism. By comparing the skin-infiltrating T cell subsets with those of GVH disease (GVHD) and other inflammatory skin diseases, we sought to elucidate the pathological mechanism of thymoma-associated multi-organ autoimmunity. Histopathological and immunohistochemical analysis of skin biopsies was performed for three patients with thymoma-associated multi-organ autoimmunity. Histopathological findings of thymoma-associated multi-organ autoimmunity were indistinguishable from those of patients with acute GVHD, although the aetiologies of these diseases are completely different. The frequency of regulatory T cells (T(regs)) is reduced in cutaneous lesions and CD8+ cytotoxic T lymphocytes that massively infiltrate into the epidermis of patients with thymoma-associated multi-organ autoimmunity. Additionally, the ratio of T helper type 17 (Th17) cells to CD4+ cells in patients with thymoma-associated multi-organ autoimmunity and acute GVHD was higher than that in healthy controls, but similar to that in psoriasis vulgaris patients. Similarity of the skin-infiltrating T cell subsets with those of acute GVHD suggested that skin damage in patients with thymoma-associated multi-organ autoimmunity might be induced by self-reactive cytotoxic T lymphocytes under the diminished suppressive capacity of T(regs).     

Induction of self-antigen-specific Foxp3+ regulatory T cells in the periphery by lymphodepletion treatment with anti-mouse thymocyte globulin in mice

Lymphodepletion therapies are increasingly tested for controlling immune damage. One appealing premise for such a therapy is that it may 'reboot' the immune system and restore immune tolerance. However, the tolerogenic potential of lymphodepletion therapies remains controversial. The debate is exemplified by conflicting evidence from the studies of anti-thymocyte globulin (ATG), a prototype of immunodepleting drugs, in particular on whether it induces CD4(+) CD25(+) Foxp3(+) regulatory T (Treg) cells. To understand the impact of ATG on T cells at a clonal level in vivo, we studied the effect of anti-mouse thymocyte globulin (mATG) in a reductionist model in which the T-lymphocyte repertoire consists of a single clone of pathogenic T effector (Teff) cells specific to a physiological self-antigen. The mATG treatment led to peripheral induction of antigen-specific Treg cells from an otherwise monoclonal Teff repertoire, independent of thymic involvement. The de novo induction of Treg cells occurred consistently in local draining lymph nodes, and persistence of induced Treg cells in blood correlated with long-term protection from autoimmune destruction. This study provides in vivo evidence for clonal conversion from a pathogenic self-antigen-specific Teff cell to a Treg cell in the setting of immunodepletion therapies.     

Multi-tasking of helper T cells

CD4 T helper cells (Th) are critical in combating pathogens and maintaining immune homeostasis. Since the establishment of the Th1–Th2 paradigm in the 1980s, many types of specialized Th cells, including Th1, Th2, Th17, Th9, follicular helper T and regulatory T, have been identified. We have become accustomed to the idea that different Th cells are ‘committed’ to their paths but recent emerging evidence suggests that under certain conditions, seemingly committed Th cells possess plasticity and may convert into other types of effector cells. In this review, we will first introduce the major sub‐types of Th cells that are involved in immune regulation. Then, we will describe in detail the inter‐convertibility of Th cells among different sub‐types under in vitro and in vivo conditions. Finally, we will discuss our current understanding of the underlying mechanisms on how a particular type of Th cells may convert into other types of Th cells.     

Mechanisms of autoimmunity in the non‐obese diabetic mouse: effector/regulatory cell equilibrium during peak inflammation

Immune imbalance in autoimmune disorders such as type 1 diabetes may originate from aberrant activities of effector cells or dysfunction of suppressor cells. All possible defective mechanisms have been proposed for diabetes‐prone species: (i) quantitative dominance of diabetogenic cells and decreased numbers of regulatory T cells, (ii) excessive aggression of effectors and defective function of suppressors, (iii) perturbed interaction between effector and suppressor cells, and (iv) variations in sensitivity to negative regulation. The experimental evidence available to date presents conflicting information on these mechanisms, with identification of perturbed equilibrium on the one hand and negation of critical role of each mechanism in propagation of diabetic autoimmunity on the other hand. In our analysis, there is no evidence that inherent abnormalities in numbers and function of effector and suppressor T cells are responsible for the immune imbalance responsible for propagation of type 1 diabetes as a chronic inflammatory process. Possibly, the experimental tools for investigation of these features of immune activity are still underdeveloped and lack sufficient resolution, in the presence of the extensive biological viability and functional versatility of effector and suppressor elements.     

Regulatory T cells in the central nervous system

Regulatory T cells (Tregs) are critical to the human immune system, providing appropriately scaled immune responses and mediating peripheral tolerance. A central role for forkhead box protein 3 (FoxP3)(+) Tregs has been shown in the pathogenesis of mechanistically diverse central nervous system (CNS) diseases from autoimmune diseases such as multiple sclerosis to glioblastomas. Understanding how tumors induce Treg function to escape immune surveillance in marked contrast to autoimmune diseases, where there is loss of Treg function, will provide valuable lessons regarding Treg biology and potential therapeutic targets for CNS diseases.     

调节性T细胞在支气管哮喘中的重要作用

本文论述了调市性T细胞的概念及几种主要的调节性T细胞：Th1、Th2、Th3细胞、TR细胞、CD4^ CD25^ 细胞、NKT细胞的主要特点和研究现状。并论述了这儿种调节性T细胞在支气管哮喘中的重要作用。提出Th1细胞更趋向于一种炎症细胞而非抗炎细胞,而其他各种调节性T细胞可能提供免疫保护及抗炎作用起到抑制哮喘发展的作用,通过控制调节性T细胞的治疗可能成为哮喘治疗的有效手段。     

Systemic lupus erythematosus,regulatory T cells and pregnancy

Systemic lupus erythematosus (SLE) is the most common autoimmune disease affecting women of reproductive age and is associated with poor maternal and fetal outcomes. CD4⁺CD25⁺ Treg cells are a subset of T lymphocytes with potent immunosuppressive activity that play crucial roles in controlling immunological self tolerance. Evidence suggests that they are augmented in pregnancy, especially in the first trimester, suggesting an important role in early placental development. The literature describing Treg cells in SLE is conflicting, but SLE is associated with reduced numbers and functionally defective Treg cells, which may predispose pregnant women with the disease to pregnancy complications. This article discusses the role of Treg cells in SLE and pregnancy, and how these cells may contribute to poor pregnancy outcome in SLE-affected women.     

Regulatory T cells in B-cell-deficient and wild-type mice differ functionally and in expression of cell surface markers

NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) with chronic inflammation of thyroids by T and B cells. B-cell deficient (B^–/–) mice are resistant to SAT but develop SAT if regulatory T (Treg) cells are transiently depleted. We established a transfer model using splenocytes from CD28^–/– B^–/– mice (effector cells and antigen-presenting cells) cultured with or without sorted Treg cells from Foxp3-GFP wild-type (WT) or B^–/– mice. After transfer to mice lacking T cells, mice given Treg cells from B^–/– mice had significantly lower SAT severity scores than mice given Treg cells from WT mice, indicating that Treg cells in B^–/– mice are more effective suppressors of SAT than Treg cells in WT mice. Treg cells from B^–/– mice differ from WT Treg cells in expression of CD27, tumour necrosis factor receptor (TNFR) II p75, and glucocorticoid-induced TNFR-related protein (GITR). After transient depletion using anti-CD25 or diphtheria toxin, the repopulating Treg cells in B^–/– mice lack suppressor function, and expression of CD27, GITR and p75 is like that of WT Treg cells. If B^–/– Treg cells develop with B cells in bone marrow chimeras, their phenotype is like that of WT Treg cells. Addition of B cells to cultures of B^–/– Treg and T effector cells abrogates their suppressive function and their phenotype is like that of WT Treg cells. These results establish for the first time that Treg cells in WT and B^–/– mice differ both functionally and in expression of particular cell surface markers. Both properties are altered after transient depletion and repopulation of B^–/– Treg cells, and by the presence of B cells during Treg cell development or during interaction with effector T cells.