Coating human pancreatic islets with CD4(+)CD25(high)CD127(-) regulatory T cells as a novel approach for the local immunoprotection.

OBJECTIVES: To develop a novel approach for local immunoprotection using CD4(+)CD25(high)CD127(-) T regulatory cells (Tregs) attached to the surface of the islets before transplantation. BACKGROUND: Tregs expanded ex vivo can control allo and autoreactivity, therefore, Treg-based therapy may offer more effective protection for transplanted islets from immunologic attack than currently used immunosuppression. Local application of Tregs can make such therapy more clinically feasible and efficient. METHODS: Human islets were isolated and coated with allogeneic ex vivo expanded Tregs using biotin-poly(ethylene glycol)-N-hydroxysuccinimide ester (biotin-PEG-NHS) and streptavidin as binding molecules. RESULTS: Coating pancreatic islets with Tregs did not affect islet viability (>90% fluorescein diacetate/propidium iodide) or the insulin secretion profile in dynamic islet perifusion assays. After in vitro incubation with allogeneic T effector cells, Treg-coated islets revealed preserved function with higher insulin secretion compared with controls-native islets, coated islets with T effector cells or when Tregs were added to the culture, but not attached to islets (P < 0.05). In addition, the Enzyme-linked immunosorbent spot (ELISPOT) assay revealed suppression of interferon (IFN)-γ secretion, when T effector cells were challenged with Treg-coated islets comparing to controls (99 ± 7 vs 151 ± 8 dots, respectively; P < 0.01). CONCLUSIONS: We demonstrated, for the first time, the ability to bind immune regulatory cells to target cells with preservation of their viability and function and protective activity against immune attack. If successfully tested in an animal model, local delivery of immunoprotective Tregs on the surface of transplanted pancreatic islets may be an alternative or improvement to the currently used immunosuppression.

Homeostatic proliferation of NK cells: friend or foe in cellular immunotherapy?

NK cells are an important tool in cellular immunotherapy owing to their role in infections and antitumor immunity. Until recently, these cells have been thought to be short-lived cytotoxic effectors that are cleared from the body soon after resolution of an immune response. In the commented study, Sun et al. confirmed that, similar to T cells, NK cells sensed the space in the immune system and underwent homeostatic proliferation in order to provide necessary protection to the body. Moreover, homeostatically driven NK cells persisted in the tissues for a long time without loss of activity. These findings have important consequences for immunotherapy, suggesting that the mechanisms of homeostatic expansion can be deployed in order to expand NK cells for therapeutic purposes in vivo. As homeostatically driven NK cells are long-lived effectors, such therapies can exert prolonged effects for the immunity of the patients.

Anti-TNF rescue CD4+Foxp3+ regulatory T cells in patients with type 1 diabetes from effects mediated by TNF.

The presence of low-grade chronic inflammation is a known feature of long standing diabetes type 1. The association between serum level of several markers of inflammation and severity of DM1 was proven. Serum concentrations of TNF were reported to be elevated in diabetic patients, especially those who developed diabetic complications. Lately, it has been also shown that TNF may impair the subset of naturally arising regulatory T cells, which control autoimmunity. The presented study, for the first time, shows the regulatory T cells in the context of an inflammatory environment that is present in patients with type 1 diabetes. It indicates that TNF reduces the number and frequency of regulatory CD4(+)Foxp3(+) T cells in children with diabetes type 1 and that in vitro treatment with anti-TNF antibody seems to rescue this cell subset from its defective effects.

Decreased angiogenin concentration in vitreous and serum in proliferative diabetic retinopathy.

Diabetic retinopathy is the most common cause of vision loss in young adults in developed countries. The disease therapy with anti-vascular endothelial growth factor (VEGF) agents gives some positive results, but is associated with retinal ischemia and vasoconstriction. Therefore, determination of factors involved in the physiological and pathological angiogenesis in the diabetic eye is of great importance for understanding of the pathogenesis of diabetic retinopathy and its effective treatment. Previously, we found that diabetic patients were characterized by increased serum concentration of VEGF, but decreased levels of other proangiogenic factor-angiogenin. The involvement of VEGF in pathogenesis of diabetic retinopathy is well established, but there is lack of data regarding angiogenin in retinopathy. Therefore, in the present study we measured angiogenin concentration in vitreous and serum samples of the patients with type 1 diabetes to determine its role in diabetic retinopathy. In addition, in each time, we compared the level of angiogenin with level of VEGF as a known factor involved in the pathogenesis of the disease. Angiogenin was found to be significantly more abundant in serum than in vitreous in both diabetic groups. In addition, patients with retinopathy had twofold lower vitreous angiogenin levels than diabetic individuals without complications. On the contrary, vitreous concentration of VEGF was dramatically increased only in participants with retinopathy. Patients without diabetic complications had significantly lower VEGF levels in vitreous than in serum and were characterized by high local and systemic concentration of angiogenin. These data suggest a local imbalance between two proangiogenic factors-VEGF and angiogenin in retinopathy. Low vitreous concentration of angiogenin in diabetic patients suggests that this factor is not responsible for pathological neovascularization in diabetic eye. Further studies will elucidate if angiogenin can be used to improve the insufficient angiogenesis in diabetes and prevent retinal ischemia after retinopathy treatment with anti-VEGF agents.

The time is crucial for ex vivo expansion of T regulatory cells for therapy.

Ex vivo expanded CD4(+)CD25(high)CD127(-) T regulatory cells (Tregs) are recognized as a promising candidate for immunosuppressive therapy in humans. However, due to the plasticity of Tregs lineage and artificial environment present during ex vivo expansion, Tregs easily lose suppressive activity. Here, we followed expanding CD4(+)CD25(high)CD127(-) Tregs and their naive (CD45RA(+)) and memory-like (CD45RA(-)) subsets in order to establish the best conditions of the expansion. We found that, regardless of the phenotype sorted, expanding Tregs were undergoing changes resembling homeostatic proliferation and transformed into effector memory-like cells which produced not only suppressive interleukin-10 (IL-10) but also IL-6, IL-17, and interferon-γ (IFN-γ). With the time ex vivo, Tregs were losing the expression of FoxP3 and suppressive activity both when stimulated and when at rest. The only variable that helped preserve suppressive abilities of Tregs was the limitation of the time of ex vivo cultures to 2 weeks only. According to our study, the highest number of highly suppressive Tregs could be yielded with CD4(+)CD25(high)CD127(-) Tregs cultured no longer than 2 weeks. Thorough quality check, preferentially with the assessment of FoxP3 expression and IFN-γ suppression assay, should be applied to assess suppressive activity of the cells.

Increased spontaneous production of VEGF by CD4+ T cells in type 1 diabetes.

In the present study we report that CD4(+) T cells from patients with type 1 diabetes produce significantly higher amounts of VEGF than respective cells from the healthy individuals. Among CD4(+) T cells memory subsets were the main source of VEGF. In addition, memory CD4(+) T subsets were the most numerous in patients with diabetic retinopathy (DR). DR was also characterized by significant increase of VEGF concentration in serum and culture supernatants. Hence, these data indicate that there is a sustained spontaneous production of VEGF by CD4(+) T cells in type 1 diabetes, which is additionally exacerbated in DR. In our opinion alterations in the proportions of CD4(+) T cell subsets and their VEGF production may be a useful tool for early assessment of the risk of DR onset and progression.

Glycemic control influences serum angiogenin concentrations in patients with type 2 diabetes.

OBJECTIVE: Because diabetes is the most frequent factor responsible for microvascular and macrovascular disease, we investigated angiogenin serum levels within the diabetic patient group. RESEARCH DESIGN AND METHODS: We investigated 49 patients who met the criteria to be in the diabetic group. Forty nondiabetic patients were included in the control group. We set A1C <7% as well-controlled diabetes. Serum angiogenin level was measured using the enzyme-linked immunosorbent assay method. RESULTS: Serum angiogenin levels of poorly controlled patients with type 2 diabetes were significantly lower than those of group with well-controlled diabetes (361.23 +/- 126.03 ng/ml vs. 446.37 +/- 134.10 ng/ml; P = 0.001). Moreover, they were characterized by a significantly longer duration of the disease (P = 0.006), higher BMI (P = 0.0003), and higher systolic blood pressure (P = 0.01). Levels of total cholesterol, triglycerides, LDL, and HDL were not significantly different in both groups. CONCLUSIONS: Patients with poorly controlled type 2 diabetes (A1C >7%) have lower angiogenin levels than patients with well-controlled diabetes.

Membrane potential of CD4+ T cells is a subset specific feature that depends on the direct cell-to-cell contacts with monocytes.

CD4(+) T cells can be divided into three subsets: naive (Tn), central memory (Tcm), and effector memory (Tem) lymphocytes. These subpopulations differ in phenotype, migratory capacity, pattern of secreted cytokines, and activation threshold. T-cell activation is associated with changes in membrane potential, which provide an electrical driving force for calcium entry into the cytosol. These phenomena were shown to precede lymphocyte proliferation, cytokine synthesis, migration, and apoptosis. Hence the aim of the study was the analysis of these early activation events in the subsets of CD4(+) T cells. We measured the membrane potential and intracellular calcium concentration ([Ca(2+)](i)) in CD4(+) Tn, Tcm, and Tem cells as well as the dependency of these parameters in CD4(+) T cells on their cell-to-cell contacts with other leukocyte subsets. The data indicate that membrane potential of CD4(+) T cells is a subset specific feature maintained by direct contact with monocytes. In addition, monocytes were found to control Ca(2+) influx in CD4(+) T cells.

First-in-man clinical results of the treatment of patients with graft versus host disease with human ex vivo expanded CD4+CD25+CD127- T regulatory cells.

Here, we describe a procedure and first-in-man clinical effects of adoptive transfer of ex vivo expanded CD4+CD25+CD127- T regulatory cells (Tregs) in the treatment of graft versus host disease (GvHD). The cells were sorted from buffy coats taken from two family donors, expanded ex vivo and transferred to respective recipients who suffered from either acute or chronic GvHD. The therapy allowed for significant alleviation of the symptoms and reduction of pharmacologic immunosuppression in the case of chronic GvHD, while in the case of grade IV acute GvHD it only transiently improved the condition, for the longest time within all immunosuppressants used nonetheless.