Exposing the oral mucosa to antigen may stimulate immune tolerance. It is unknown whether treatment with oral insulin can induce a tolerogenic immune response in children genetically susceptible to type 1 diabetes. OBJECTIVE To assess the immune responses and adverse events associated with orally administered insulin in autoantibody-negative, genetically at-risk children. DESIGN, SETTING, AND PARTICIPANTS The Pre-POINT study, a double-blind, placebo-controlled, dose-escalation, phase 1/2 clinical pilot study performed between 2009 and 2013 in Germany, Austria, the United States, and the United Kingdom and enrolling 25 islet autoantibody-negative children aged 2 to 7 years with a family history of type 1 diabetes and susceptible human leukocyte antigen class II genotypes. Follow-up was completed in August 2013. INTERVENTIONS Children were randomized to receive oral insulin (n = 15) or placebo (n = 10) once daily for 3 to 18 months. Nine children received insulin with dose escalations from 2.5 to 7.5 mg (n = 3), 2.5 to 22.5 mg (n = 3), or 7.5 to 67.5 mg (n = 3) after 6 months; 6 children only received doses of 22.5 mg (n = 3) or 67.5 mg (n = 3). MAIN OUTCOMES AND MEASURES An immune response to insulin, measured as serum IgG and saliva IgA binding to insulin, and CD4 + T-cell proliferative responses to insulin. RESULTS Increases in IgG binding to insulin, saliva IgA binding to insulin, or CD4 + T-cell proliferative responses to insulin were observed in 2 of 10 (20% [95% CI, 0.1%-45%]) placebo-treated children and in 1 of 6 (16.7% [95% CI, 0.1%-46%]) children treated with 2.5 mg of insulin, 1 of 6 (16.7%[ 95% CI, 0.1%-46%]) treated with 7.5 mg, 2 of 6 (33.3% [95% CI, 0.1%-71%]) treated with 22.5 mg, and 5 of 6 (83.3% [ 95% CI, 53%-99.9%]) treated with 67.5 mg (P = .02). Insulin-responsive T cells displayed regulatory T-cell features after oral insulin treatment. No hypoglycemia, IgE responses to insulin, autoantibodies to glutamic acid decarboxylase or insulinoma-associated antigen 2, or diabetes were observed. Adverse events were reported in 12 insulin-treated children (67 events) and 10 placebo-treated children (35 events). CONCLUSIONS AND RELEVANCE In this pilot study of children at high risk for type 1 diabetes, daily oral administration of 67.5 mg of insulin, compared with placebo, resulted in an immune response without hypoglycemia. These findings support the need for a phase 3 trial to determine whether oral insulin can prevent islet autoimmunity and diabetes in such children.
CD4+CD25+FOXP3+ regulatory T cells (Tregs) control the activation and expansion of alloreactive and autoreactive T cell clones. Because uncontrolled activation and expansion of autoreactive T cells occur in an IL-7–rich environment, we explored the possibility that IL-7 may affect the function of Treg. We show that the functional high-affinity IL-7R is expressed on both naive and memory Tregs, and exposure to IL-7 results in STAT-5 phosphorylation. Naive, but not memory, Tregs proliferated greatly and acquired a memory phenotype in the setting of a suppression assay when IL-7 was present. Importantly, the presence of IL-7 abrogated the capacity of Tregs to suppress proliferation of conventional T cells in response to TCR activators, including alloantigens and autoantigens. Removal of IL-7 restored the suppressive function of Tregs. Preblocking of the IL-7R on the Tregs also restored suppressor function, indicating that IL-7 directly affected Treg function. Thus, prolonged periods of homeostatic expansion can temporarily release natural regulatory brakes on T cells, thereby providing an additional mechanism for activating and expanding alloreactive and autoreactive T cells.
Bispecific Abs hold great potential for immunotherapy of malignant diseases. Because the first components of this new drug class are now entering clinical trials, all aspects of their mode of action should be well understood. Several studies proved that CD8+ and CD4+ effector T cells can be successfully redirected and activated against tumor cells by bispecific Abs both in vitro and in vivo. To our knowledge, this study provides the first evidence that bispecific Abs can also redirect and activate regulatory T cells against a surface Ag, independently of their TCR specificity. After cross-linking, via a bispecific Ab, redirected regulatory T cells upregulate the activation markers CD69 and CD25, as well as regulatory T cell-associated markers, like CTLA-4 and FOXP3. The activated regulatory T cells secrete the immunosuppressive cytokine IL-10, but, in contrast to CD8+ and CD4+ effector T cells, almost no inflammatory cytokines. In addition, the redirected regulatory T cells are able to suppress effector functions of activated autologous CD4+ T cells both in vitro and in vivo. Therefore, the potential risk for activation of regulatory T cells should be taken into consideration when bispecific Abs are applied for the treatment of malignant diseases. In contrast, an Ag/tissue-specific redirection of regulatory T cells with bispecific Abs holds great potential for the treatment of autoimmune diseases and graft rejection.
Summary Regulatory T cell (Treg) therapy has been exploited in autoimmune disease, solid organ transplantation and in efforts to prevent or treat graft‐versus‐host disease (GVHD). However, our knowledge on the in‐vivo persistence of transfused Treg is limited. Whether Treg transfusion leads to notable changes in the overall Treg repertoire or whether longevity of Treg in the periphery is restricted to certain clones is unknown. Here we use T cell receptor alpha chain sequencing (TCR‐α‐NGS) to monitor changes in the repertoire of Treg upon polyclonal expansion and after subsequent adoptive transfer. We applied TCR‐α‐NGS to samples from two patients with chronic GVHD who received comparable doses of stem cell donor derived expanded Treg. We found that in‐vitro polyclonal expansion led to notable repertoire changes in vitro and that Treg cell therapy altered the peripheral Treg repertoire considerably towards that of the infused cell product, to different degrees, in each patient. Clonal changes in the peripheral blood were transient and correlated well with the clinical parameters. We suggest that T cell clonotype analyses using TCR sequencing should be considered as a means to monitor longevity and fate of adoptively transferred T cells.
Peroxisomal disorders are genetic diseases in which an impairment in one or more peroxisomal function(s) causes clinical and multiple biochemical abnormalities. Early recognition of the major peroxisomal disorders in which functional peroxisomes are virtually absent, leading to a generalised impairment of peroxisomal functions, is of utmost importance, as this will enable the prenatal diagnosis of these severe diseases in future pregnancies. Unfortunately, clinical recognition of these disorders can be difficult because of the aspecific and varying phenotypic presentation. We analysed the clinical characteristics in 40 patients suspected of having a peroxisomal disorder to identify specific clinical criteria for diagnosis. From this study we conclude that the combined presence of at least three major clinical characteristics (present in greater than 75% of the affected patients, including psychomotor retardation, hypotonia, impaired hearing, low/broad nasal bridge, abnormal ERG, hepatomegaly) and one or more minor characteristics (present in 50%-75% of the patients, like large fontanelles, shallow orbital ridges, epicanthus, anteverted nostrils, retinitis pigmentosa) warrants biochemical investigation of peroxisomal functions. Further prospective investigations will have to be done to evaluate these criteria.
Cord blood has been used as a cell source for therapeutic purposes in children with type 1 diabetes and other disorders. Here, we explore the benefits of cord blood as an autologous source of T regulatory cells for immune cell therapy in patients. CD4(+)CD25(+) T regulatory cells were isolated from cord blood and adult peripheral blood of healthy donors and compared during and after expansion in a 14-day protocol incorporating anti-CD3/anti-CD28 beads, and IL-2 with or without rapamycin. Cord blood T regulatory cells were largely naïve (89±7 vs. 31±10% in young adults, p<0.0001), and had higher expansion yields (median 5,968-fold) than adult T regulatory cells (median 516-fold, p=0.001) and adult naïve T regulatory cells (median 820-fold, p=0.003). Rapamycin reduced expansion yields, but was not necessary to obtain pure expanded cord blood T regulatory cells as judged by FOXP3 staining (94±3%), methylation status of FOXP3 (97%), and intracellular effector cytokine staining (< 6%). Expanded adult T regulatory cells were much less pure in the absence of rapamycin (72±19% FOXP3; 76% by methylation status, <13% INF-γ, <16% IL-4, <5% IL-17 positive), but purity was achieved by inclusion of rapamycin during expansion. Despite differences in purity, all preparations of expanded T regulatory from all sources were able to strongly suppress proliferation of T effector cells in vitro. Our findings suggest that cord blood is an excellent source of T regulatory cells for expansion and autologous cell therapy that may be considered as a strategy to prevent immune-mediated destruction of beta cells in type 1 diabetes.
4041 Hematopoietic stem cell transplantation is a commonly used treatment for various hematological malignancies. However, a life-threatening complication following this therapy is the development of Graft versus Host Disease (GvHD), during which transplanted donor immune cells attack the host and lead to severe inflammatory responses and tissue damage. Given the key role of regulatory T cells (Tregs) in immune homeostasis and peripheral tolerance, the adoptive transfer of these cells may represent a promising therapeutic opportunity for the treatment of GvHD. This approach has been proven successful to enhance graft acceptance and prevent experimental GvHD in several animal models. However, it becomes increasingly evident that antigen-specific Tregs are more efficient than polyclonal Treg populations. The antigen-specificity enables the cells to exert their suppressive effect locally at the appropriate sites of inflammation. Furthermore, the application of antigen-specific Tregs might lower the risk of unfavourable systemic immunosuppression. Nevertheless, one of the main obstacles for their clinical use is the isolation and expansion of therapeutically relevant numbers of antigen-specific Tregs. In light of these arguments, bispecific antibodies (bsAb) could provide a promising tool for a target-dependent tissue specific redirection of polyclonal Tregs. BsAb redirect T cells to target cells by cross-linking their activating CD3 receptor and any chosen antigen on the surface of the target cell. Several studies have proven that CD8+ and CD4+ effector T cells can be successfully activated by bsAb both in vitro and in vivo. However, so far nobody has ever investigated whether Tregs can be redirected with bsAb. Using bsAb against CD3 and two different target antigens we first examined the expression of different activation associated markers on CD4+CD25+ Tregs. We could show that incubation of Tregs with a bsAb in the presence of the respective target antigen leads to the upregulation of CD25, CD69 and LAP (figure 1). Furthermore, we analyzed the cytokine production profile of the bsAb-activated Tregs. Culturing the cells together with target cells and a cross-linking bsAb triggers the release of the immunomodulatory cytokine IL-10. One prerequisite for the treatment of graft rejection and GvHD with bsAb-redirected Tregs is their suppressive efficacy upon antigen-specific activation via bsAb. Therefore we monitored the ability of bsAb-redirected Tregs to inhibit effector mechanisms of autologous T helper cells. Corresponding experiments could demonstrate the striking capacity of redirected Tregs to suppress proliferation, CD25 upregulation and cytokine production of co-cultured effector T cells (figure 2).Figure 1:Expression of different activation associated markers on Tregs incubated without (−) or with a bsAb (+) and the respective target antigen.Figure 1:. Expression of different activation associated markers on Tregs incubated without (−) or with a bsAb (+) and the respective target antigen.Figure 2:A) CFSE-labeled effector T cells (Teff) were cultured together with either unlabeled autologous effector T cells or Tregs at effector:suppressor ratios of 1:1 or 4:1 in the presence (+) or absence (−) of a bsAb and the respective target antigen. B) Cytokine secretion of bsAb-activated Teff in the presence (white bars) or absence (black bars) of autologous bsAb-activated Tregs.Figure 2:. A) CFSE-labeled effector T cells (Teff) were cultured together with either unlabeled autologous effector T cells or Tregs at effector:suppressor ratios of 1:1 or 4:1 in the presence (+) or absence (−) of a bsAb and the respective target antigen. B) Cytokine secretion of bsAb-activated Teff in the presence (white bars) or absence (black bars) of autologous bsAb-activated Tregs. Taken together, we give evidence for the first time that bsAb can redirect Tregs against a surface antigen independently of their T cell receptor specificity. In view of these results, an antigen- and/or site-specific retargeting of Tregs using bsAb may open novel therapeutic approaches for a long-term establishment of tolerance against allogenic transplants and therefore would offer a new treatment option for severe GvHD after allogenic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.
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