Approaches Towards Better Immunosuppressive Agents.

Several classes of compounds are applied in clinics due to their immunosuppressive properties in transplantology and the treatment of autoimmune diseases. Derivatives of mycophenolic acid, corticosteroids and chemotherapeutics bearing heterocyclic moieties like methotrexate, azathioprine, mizoribine, and ruxolitinib are active substances with investigated mechanisms of action. However, improved synthetic approaches of known drugs and novel derivatives are still being reported to attempt better accessibility and therapeutic properties. In this review article, we present the synthesis of the designed chemical structures based on recent literature reports concerning novel compounds as promising immunosuppressive drugs. Moreover, some of the discussed derivers revealed also other types of activities with prospective medicinal potential.

PD-1 Receptor (+) T cells are associated with the efficacy of the combined treatment with regulatory t cells and rituximab in type 1 diabetes children via regulatory t cells suppressive activity amelioration.

An imbalance between exaggerated autoaggressive T cell responses, primarily CD8 + T cells, and impaired tolerogenic mechanisms underlie the development of type 1 diabetes mellitus. Disease-modifying strategies, particularly immunotherapy focusing on FoxP3 + T regulatory cells (Treg), and B cells facilitating antigen presentation for T cells, show promise. Selective depletion of B cells may be achieved with an anti-CD20 monoclonal antibody (mAb). In a 2-year-long flow cytometry follow-up, involving 32 peripheral blood T and B cell markers across three trial arms (Treg + rituximab N = 12, Treg + placebo N = 13, control N = 11), we observed significant changes. PD-1 receptor (+) CD4 + Treg, CD4 + effector T cells (Teffs), and CD8 + T cell percentages increased in the combined regimen group by the end of follow-up. Conversely, the control group exhibited a notable reduction in PD-1 receptor (+) CD4 + Teff percentages. Considering clinical endpoints, higher PD-1 receptor (+) expression on T cells correlated with positive responses, including a higher mixed meal tolerance test AUC, and reduced daily insulin dosage. PD-1 receptor (+) T cells emerged as a potential therapy outcome biomarker. In vitro validation confirmed that successful Teff suppression was associated with elevated PD-1 receptor (+) Treg levels. These findings support PD-1 receptor (+) T cells as a reliable indicator of treatment with combined immunotherapy consisting of Tregs and anti-CD20 mAb efficacy in type 1 diabetes mellitus.

The Complex Network of Cytokines and Chemokines in Pediatric Patients with Long-Standing Type 1 Diabetes.

Type 1 diabetes (T1D) is a progressive disorder leading to the development of microangiopathies and macroangiopathies. Numerous cytokines and chemokines are involved in the pathogenesis of T1D complications. The study aimed to assess the presence of complications in patients with long-standing T1D and its relationship with serum biomarker concentrations. We examined 52 T1D subjects, with a disease duration ≥4 years and 39 healthy controls. The group of T1D patients was further divided into subgroups based on the duration of the disease (<7 years and ≥7 years) and the metabolic control assessed by the HbAlc level (<8% and ≥8%). We used Luminex Technology to assess a wide range of biomarker concentrations. A 24 h urine test was done to evaluate the rate of albuminuria. Optical coherence tomography (OCT) was conducted to detect early retinopathic changes. Subclinical atherosclerosis was assessed by measuring the carotid intima-media thickness (IMT). T1D patients showed remarkably higher concentrations of EGF, eotaxin/CCL11, MDC/CCL22, sCD40L, TGF-α, and TNF-α. Moreover, we reported statistically significant correlations between cytokines and IMT. Biomarker concentrations depend on numerous factors such as disease duration, metabolic control, and the presence of complications. Although the majority of pediatric T1D patients do not present signs of overt complications, it is indispensable to conduct the screening for angiopathies already in childhood, as its early recognition may attenuate the further progression of complications.

Beyond FOXP3: a 20-year journey unravelling human regulatory T-cell heterogeneity.

The initial idea of a distinct group of T-cells responsible for suppressing immune responses was first postulated half a century ago. However, it is only in the last three decades that we have identified what we now term regulatory T-cells (Tregs), and subsequently elucidated and crystallized our understanding of them. Human Tregs have emerged as essential to immune tolerance and the prevention of autoimmune diseases and are typically contemporaneously characterized by their CD3+CD4+CD25high CD127lowFOXP3+ phenotype. It is important to note that FOXP3+ Tregs exhibit substantial diversity in their origin, phenotypic characteristics, and function. Identifying reliable markers is crucial to the accurate identification, quantification, and assessment of Tregs in health and disease, as well as the enrichment and expansion of viable cells for adoptive cell therapy. In our comprehensive review, we address the contributions of various markers identified in the last two decades since the master transcriptional factor FOXP3 was identified in establishing and enriching purity, lineage stability, tissue homing and suppressive proficiency in CD4+ Tregs. Additionally, our review delves into recent breakthroughs in innovative Treg-based therapies, underscoring the significance of distinct markers in their therapeutic utilization. Understanding Treg subsets holds the key to effectively harnessing human Tregs for immunotherapeutic approaches.

Novel amides of mycophenolic acid and some heterocyclic derivatives as immunosuppressive agents.

The group of 18 new amide derivatives of mycophenolic acid (MPA) and selected heterocyclic amines was synthesised as potential immunosuppressive agents functioning as inosine-5'-monophosphate dehydrogenase (IMPDH) uncompetitive inhibitors. The synthesis of 14 of them employed uronium-type activating system (TBTU/HOBt/DIPEA) while 4 of them concerned phosphonic acid anhydride method (T3P/Py) facilitating amides to be obtained in moderate to excellent yields without the need of phenolic group protection. Most of optimised protocols did not require complicated reaction work-ups, including chromatographic, solvent-consuming methods. The biological activity assay was performed on the T-Jurkat cell line and peripheral mononuclear blood cells (PBMCs) which are both dedicated for antiproliferative activity determination. Each of designed derivatives was characterised by reduced cytotoxicity and benzoxazole analogue (A2) revealed the most promising activity. Subsequently, an observed structure-activity relationship was discussed.

Combined therapy with CD4+ CD25highCD127- T regulatory cells and anti-CD20 antibody in recent-onset type 1 diabetes is superior to monotherapy: Randomized phase I/II trial.

AIMS: Monotherapy with autologous expanded CD4+ CD25high CD127- T regulatory cells (Tregs) or rituximab has been documented to slow disease progression in patients with recent-onset type 1 diabetes mellitus (T1DM). Whether a combined therapy including both drugs would further benefit this patient population is unknown. MATERIALS AND METHODS: We conducted a three-arms clinical trial to explore the efficacy and safety of the combined treatment with Tregs and rituximab in paediatric patients with T1DM. The patients were allocated to three groups: Tregs only (n = 13), Tregs + rituximab (n = 12) and control (n = 11). The key primary efficacy analyses were C-peptide levels (mixed meal tolerance test) and the proportion of patients in remission at 12 and 24 months. RESULTS: At month 24, as compared with the control, both treatment groups remained superior in the area under the curve of C-peptide mixed meal tolerance test, whereas in the analysis of all visits only the combined therapy improved area under the curve at 12 and 24 months. The proportion of patients in remission was significantly higher in the combined group than in the control group at 3, 6, 9 and 21 months but not at 18 and 24 months. There was no significant difference between the Tregs only group and control group. Adverse events occurred in 80% patients, mostly in the combined group and Tregs only group. No adverse events led to the withdrawal of the intervention or death. All comparisons were performed with alpha level of 5%. CONCLUSIONS: Over 2 years, combined therapy with Tregs and rituximab was consistently superior to monotherapy in delaying T1DM progression in terms of C-peptide levels and the maintenance of remission.

Regulatory T Cells-Related Genes Are under DNA Methylation Influence.

Regulatory T cells (Tregs) exert a highly suppressive function in the immune system. Disturbances in their function predispose an individual to autoimmune dysregulation, with a predominance of the pro-inflammatory environment. Besides Foxp3, which is a master regulator of these cells, other genes (e.g., Il2ra, Ctla4, Tnfrsf18, Ikzf2, and Ikzf4) are also involved in Tregs development and function. Multidimensional Tregs suppression is determined by factors that are believed to be crucial in the action of Tregs-related genes. Among them, epigenetic changes, such as DNA methylation, tend to be widely studied over the past few years. DNA methylation acts as a repressive mark, leading to diminished gene expression. Given the role of increased CpG methylation upon Tregs imprinting and functional stability, alterations in the methylation pattern can cause an imbalance in the immune response. Due to the fact that epigenetic changes can be reversible, so-called epigenetic modifiers are broadly used in order to improve Tregs performance. In this review, we place emphasis on the role of DNA methylation of the genes that are key regulators of Tregs function. We also discuss disease settings that have an impact on the methylation status of Tregs and systematize the usefulness of epigenetic drugs as factors able to influence Tregs functions.

Antigenic Challenge Influences Epigenetic Changes in Antigen-Specific T Regulatory Cells.

Background: Human regulatory T cells (Tregs) are the fundamental component of the immune system imposing immune tolerance via control of effector T cells (Teffs). Ongoing attempts to improve Tregs function have led to the creation of a protocol that produces antigen-specific Tregs, when polyclonal Tregs are stimulated with monocytes loaded with antigens specific for type 1 diabetes. Nevertheless, the efficiency of the suppression exerted by the produced Tregs depended on the antigen with the best results when insulin ß chain peptide 9-23 was used. Here, we examined epigenetic modifications, which could influence these functional differences. Methods: The analysis was pefromed in the sorted specific (SPEC, proliferating) and unspecific (UNSPEC, non-proliferating) subsets of Tregs and Teffs generated by the stimulation with monocytes loaded with either whole insulin (INS) or insulin ß chain peptide 9-23 (B:9-23) or polyclonal cells stimulated with anti-CD3/anti-CD28 beads (POLY). A relative expression of crucial Tregs genes was determined by qRT-PCR. The Treg-specific demethylated region (TSDR) in FoxP3 gene methylation levels were assessed by Quantitative Methylation Specific PCR (qMSP). ELISA was used to measure genomic DNA methylation and histone H3 post-translational modifications (PTMs). Results: Tregs SPECB:9-23 was the only subset expressing all assessed genes necessary for regulatory function with the highest level of expression among all analyzed conditions. The methylation of global DNA as well as TSDR were significantly lower in Tregs SPECB:9-23 than in Tregs SPECINS. When compared to Teffs, Tregs were characterized by a relatively lower level of PTMs but it varied in respective Tregs/Teffs pairs. Importantly, whenever the difference in PTM within Tregs/Teffs pair was significant, it was always low in one subset from the pair and high in the other. It was always low in Tregs SPECINS and high in Teffs SPECINS, while it was high in Tregs UNSPECINS and low in Teffs UNSPECINS. There were no differences in Tregs/Teffs SPECB:9-23 pair and the level of modifications was low in Tregs UNSPECB:9-23 and high in Teffs UNSPECB:9-23. The regions of PTMs in which differences were significant overlapped only partially between particular Tregs/Teffs pairs. Conclusions: Whole insulin and insulin ß chain peptide 9-23 affected epigenetic changes in CD4+ T cells differently, when presented by monocytes. The peptide preferably favored specific Tregs, while whole insulin activated both Tregs and Teffs.

Administration of CD4+CD25highCD127-FoxP3+ Regulatory T Cells for Relapsing-Remitting Multiple Sclerosis: A Phase 1 Study.

BACKGROUND: Multiple sclerosis (MS) is an immune-mediated disease in which autoimmune T conventional (Tconv) cells break the blood-brain barrier and destroy neurons of the central nervous system. It is hypothesized that CD4+CD25highCD127-FoxP3+ T regulatory (Treg) cells may inhibit this destruction through suppressive activity exerted on Tconv cells. METHODS: We present the results of a phase 1b/2a, open-label, two-arm clinical trial in 14 patients treated with autologous Treg cells for relapsing-remitting MS. The patients received either expanded ex vivo Treg cells intravenously (intravenous [IV] group, n = 11; dose 40 × 106 Treg cells/kg of body weight) or freshly isolated Treg cells intrathecally (intrathecal [IT] group, n = 3; dose 1.0 × 106 Treg cells). Importantly, patients were not treated with any other disease-modifying drugs for at least 6 months before the recruitment and during the follow-up. RESULTS: No severe adverse events were observed. Self-assessed quality of life (EuroQol-5 Dimensions [EQ-5D] form) did not change and did not differ significantly between the groups. A total of 12 relapses were noted in five intravenously treated patients, who had from one to three attacks per year. Three out of ten participants who completed the trial in the IV group deteriorated more than 1 point on the Expanded Disability Status Scale (EDSS) during the follow-up. At the same time, no patients in the IT group experienced a relapse or such a deterioration in the EDSS. No significant differences were found in the Multiple Sclerosis Functional Composite (MSFC) scale in both the IV and IT groups. Magnetic resonance imaging (MRI) scans revealed a significantly lower change in the T2 lesion volume in the IT group compared to the IV group. The increase in the number of new T2 lesions during the follow-up was significant for the IV group only. There were no significant changes in the level of Treg cells or Tconv cells in the peripheral blood throughout the follow-up or between the groups. Interestingly, Treg cells in all patients consisted of two different phenotypes: peripheral Treg cells Helios(-) (≈ 20%) and thymic Treg cells Helios(+) (≈ 80%). The analysis of the cytokine pattern revealed higher levels of transforming growth factor-α and proinflammatory factors MCP3, CXCL8, and IL-1RA in the IT group compared with the IV group. CONCLUSIONS: No serious adverse events were reported in the 14 patients with MS treated with Treg cells in this study. The results suggest that IT administration is more promising than IV administration. Because of the low number of patients recruited, the statistical results may be underpowered and further studies are necessary to reach conclusions on efficacy and safety. TRIAL REGISTRATION: EudraCT: 2014-004320-22; registered 18 November 2014.

Antigen-reactive regulatory T cells can be expanded in vitro with monocytes and anti-CD28 and anti-CD154 antibodies.

BACKGROUND: In recent years, therapies with CD4+CD25highFoxP3+ regulatory T cells (Tregs) have been successfully tested in many clinical trials. The important issue regarding the use of this treatment in autoimmune conditions remains the specificity toward particular antigen, as because of epitope spread, there are usually multiple causative autoantigens to be regulated in such conditions. METHODS: Here we show a method of generation of Tregs enriched with antigen-reactive clones that potentially covers the majority of such autoantigens. In our research, Tregs were expanded with anti-CD28 and anti-CD154 antibodies and autologous monocytes and loaded with a model peptide, such as whole insulin or insulin ß chain peptide 9-23. The cells were then sorted into cells recognizing the presented antigen. The reactivity was verified with functional assays in which Tregs suppressed proliferation or interferon gamma production of autologous effector T cells (polyclonal and antigen-specific) used as responders challenged with the model peptide. Finally, we analyzed clonotype distribution and TRAV gene usage in the specific Tregs. RESULTS: Altogether, the applied technique had a good yield and allowed us to obtain a Treg product enriched with a specific subset, as confirmed in the functional tests. The product consisted of many clones; nevertheless, the content of these clones was different from that found in polyclonal or unspecific Tregs. CONCLUSIONS: The presented technique might be used to generate populations of Tregs enriched with cells reactive to any given peptide, which can be used as a cellular therapy medicinal product in antigen-targeted therapies.

GPA33: A Marker to Identify Stable Human Regulatory T Cells.

FOXP3-expressing regulatory T (Treg) cells safeguard immunological tolerance. Treg cells can be generated during thymic development (called thymic Treg [tTreg] cells) or derived from mature conventional CD4+ T cells that underwent TGF-ß-mediated conversion in the periphery (called peripheral Treg [pTreg] cells). Murine studies have shown that tTreg cells exhibit strong lineage fidelity, whereas pTreg cells can revert into conventional CD4+ T cells. Their stronger lineage commitment makes tTreg cells the safest cells to use in adoptive cell therapy, increasingly used to treat autoimmune and inflammatory disorders. Markers to distinguish human tTreg cells from pTreg cells have, however, not been found. Based on combined proteomic and transcriptomic approaches, we report that the Ig superfamily protein GPA33 is expressed on a subset of human Treg cells. GPA33 is acquired late during tTreg cell development but is not expressed on TGF-ß-induced Treg cells. GPA33 identifies Treg cells in human blood that lack the ability to produce effector cytokines (IL-2, IFN-γ, IL-17), regardless of differentiation stage. GPA33high Treg cells universally express the transcription factor Helios that preferentially marks tTreg cells and can robustly and stably be expanded in vitro even without rapamycin. Expanded GPA33high Treg cells are suppressive, unable to produce proinflammatory cytokines, and exhibit the epigenetic modifications of the FOXP3 gene enhancer CNS2, necessary for indelible expression of this critical transcription factor. Our findings thus suggest that GPA33 identifies human tTreg cells and provide a strategy to isolate such cells for safer and more efficacious adoptive cell therapy.

Proinsulin-specific T regulatory cells may control immune responses in type 1 diabetes: implications for adoptive therapy.

OBJECTIVE: Here we looked for possible mechanisms regulating the progression of type 1 diabetes mellitus (T1DM). In this disease, autoaggressive T cells (T conventional cells, Tconvs) not properly controlled by T regulatory cells (Tregs) destroy pancreatic islets. RESEARCH DESIGN AND METHODS: We compared the T-cell compartment of patients with newly diagnosed T1DM (NDT1DM) with long-duration T1DM (LDT1DM) ones. The third group consisted of patients with LDT1DM treated previously with polyclonal Tregs (LDT1DM with Tregs). We have also looked if the differences might be dependent on the antigen specificity of Tregs expanded for clinical use and autologous sentinel Tconvs. RESULTS: Patients with LDT1DM were characterized by T-cell immunosenescence-like changes and expansion of similar vß/T-cell receptor (TCR) clones in Tconvs and Tregs. The treatment with Tregs was associated with some inhibition of these effects. Patients with LDT1DM possessed an increased percentage of various proinsulin-specific T cells but not GAD65-specific ones. The percentages of all antigen-specific subsets were higher in the expansion cultures than in the peripheral blood. The proliferation was more intense in proinsulin-specific Tconvs than in specific Tregs but the levels of some proinsulin-specific Tregs were exceptionally high at baseline and remained higher in the expanded clinical product than the levels of respective Tconvs in sentinel cultures. CONCLUSIONS: T1DM is associated with immunosenescence-like changes and reduced diversity of T-cell clones. Preferential expansion of the same TCR families in both Tconvs and Tregs suggests a common trigger/autoantigen responsible. Interestingly, the therapy with polyclonal Tregs was associated with some inhibition of these effects. Proinsulin-specific Tregs appeared to be dominant in the immune responses in patients with T1DM and probably associated with better control over respective autoimmune Tconvs. TRIAL REGISTRATION NUMBER: EudraCT 2014-004319-35.

Therapy with CD4+CD25+ T regulatory cells - should we be afraid of cancer?

This review focuses on the role of regulatory T cells (Tregs) in the process of carcinogenesis. The controversy of this issue arose due to the increasing therapeutic use of Tregs in humans (inter alia, in the treatment of autoimmune diseases). It is mainly due to potential dangers related to immunosuppressive activity of these cells, especially regarding cancer. The natural function of regulatory T cells (which is the suppression of excessive activity of the immune system) is purportedly linked to an increased risk of cancer initiation. This work brings together and summarizes the most important reports of researchers dealing with this problem and attempts to explain doubts and fears related to Tregs and their uncertain connection with cancer initiation and progression. It is clearly shown that regulatory T cells are associated with acceleration of existing tumors (they are attracted by microenvironments created by cancer cells) but cannot initiate them on their own.

Corrigendum: Monitoring T-Cell Responses in Translational Studies: Optimization of Dye-Based Proliferation Assay for Evaluation of Antigen-Specific Responses.

[This corrects the article on p. 1870 in vol. 8, PMID: 29312346.].

Cell-Based Therapies with T Regulatory Cells.

CD4+CD25highFoxP3+ T regulatory cells (Tregs) are immunodominant suppressors in the immune system. Tregs use various mechanisms to control immune responses. Preclinical data from animal models have confirmed the huge therapeutic potential of Tregs in many immune-mediated diseases. Hence, these cells are now on the road to translation to cell therapy in the clinic as the first clinical trials are accomplished. To date, clinical research has involved mainly hematopoietic stem cell transplantations, solid organ transplantations, and autoimmunity. Despite difficulties with legislation and technical issues, treatment is constantly evolving and may soon represent a valid alternative for patients with diseases that are currently incurable. This review focuses on the basic and clinical experience with Tregs with adoptive transfer of these cells, primarily from clinical trials, as well as on perspectives on clinical use and technical problems with implementing the therapy.

Investigations on the immunosuppressive activity of derivatives of mycophenolic acid in immature dendritic cells.

The main activity of mycophenolic acid 1 (MPA) and its analogs is the inhibition of proliferation of T cells. Here, we hypothesized that MPA and its conjugates inhibits also the activity of antigen-presenting cells (APC) including dendritic cells (DCs). We tested the effect of novel amino acid derivatives of MPA and conjugates of MPA with acridines/acridones on DCs by flow cytometry, ELISA and MLR assay. Both acridines/acridone derivatives could inhibit the maturation of DC, as shown by the decreased expression of B7 family receptors. It was confirmed in the mixed leucocyte reaction (MLR), in which T cells challenged with DCs pretreated with the analogs showed decreased proliferation and reduced cytokine secretion. The most interesting activity in this series of studies, that is, the suppression of CD86 receptor expression, decreased cytokine production and suppressed mixed leucocyte reaction, exhibited (mycophenoyl-N-3-propyl)-9-acridone-4-carboxamide ester 5a and (mycophenoyl-N-5-pentyl)-9-acridone-4-carboxamide ester 5b. These compounds reduced also the secretion of IL-2 and IL-15. In addition, they increased secretion of suppressive IL-10. Equally promising results were obtained for the N-mycophenoyl-D-glutamic acid 4b, which previously gave the highest value of selectivity. Acridone derivatives of MPA are therefore good immunosuppressive drug candidates for further testing.

Monitoring T-Cell Responses in Translational Studies: Optimization of Dye-Based Proliferation Assay for Evaluation of Antigen-Specific Responses.

Adoptive therapy with regulatory T cells or tolerance-inducing antigen (Ag)-presenting cells is innovative and promising therapeutic approach to control undesired and harmful activation of the immune system, as observed in autoimmune diseases, solid organ and bone marrow transplantation. One of the critical issues to elucidate the mechanisms responsible for success or failure of these therapies and define the specificity of the therapy is the evaluation of the Ag-specific T-cell responses. Several efforts have been made to develop suitable and reproducible assays. Here, we focus on dye-based proliferation assays. We highlight with practical examples the fundamental issues to take into consideration for implementation of an effective and sensitive dye-based proliferation assay to monitor Ag-specific responses in patients. The most critical points were used to design a road map to set up and analyze the optimal assay to assess Ag-specific T-cell responses in patients undergoing different treatments. This is the first step to optimize monitoring of tolerance induction, allowing comparison of outcomes of different clinical studies. The road map can also be applied to other therapeutic interventions, not limited to tolerance induction therapies, in which Ag-specific T-cell responses are relevant such as vaccination approaches and cancer immunotherapy.

Minimum Information about T Regulatory Cells: A Step toward Reproducibility and Standardization.

Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications.

Factors affecting long-term efficacy of T regulatory cell-based therapy in type 1 diabetes.

BACKGROUND: Recent studies suggest that immunotherapy using T regulatory cells (Tregs) prolongs remission in type 1 diabetes (T1DM). Here, we report factors that possibly affect the efficacy of this treatment. METHODS: The metabolic and immune background of 12 children with recently diagnosed T1DM, as well as that of untreated subjects, during a 2-year follow-up is presented. Patients were treated with up to 30 × 106/kg b.w. of autologous expanded CD3+CD4+CD25highCD127- Tregs. RESULTS: The disease progressed and all patients were insulin-dependent 2 years after inclusion. The ß-cell function measured by c-peptide levels and the use of insulin were the best preserved in patients treated with two doses of Tregs (3/6 in remission), less so after one dose (1/6 in remission) and the worst in untreated controls (no remissions). Increased levels of Tregs could be seen in peripheral blood after their adoptive transfer together with the shift from naïve CD62L+CD45RA+ to memory CD62L+CD45RA- Tregs. Increasing serum levels of proinflammatory cytokines were found: IL6 increased in all subjects, while IL1 and TNFα increased only in untreated group. Therapeutic Tregs were dependent on IL2, and their survival could be improved by other lymphocytes. CONCLUSIONS: The disease progression was associated with changing proportions of naïve and memory Tregs and slowly increasing proinflammatory activity, which was only partially controlled by the administered Tregs. The therapeutic cells were highly dependent on IL2. We conclude that the therapy should be administered at the earliest to protect the highest possible mass of islets and also to utilize the preserved content of Tregs in the earlier phases of T1DM. Trial registration http://www.controlled-trials.com/ISRCTN06128462 ; registered retrospectively.

Synthesis and biological activity of ester derivatives of mycophenolic acid and acridines/acridones as potential immunosuppressive agents.

Improved derivatives of mycophenolic acid (MPA) are necessary to reduce the frequency of adverse effects, this drug exerts in treated patients. In this study, MPA was coupled with N-(ω-hydroxyalkyl)-9-acridone-4-carboxamides or N-(ω-hydroxyalkyl)acridine-4-carboxamides to give respective ester conjugates upon Yamaguchi protocol. This esterification required protection of phenol group in MPA. Designed conjugates revealed higher potency in vitro than parent MPA. Acridine derivatives were more active than acridone analogs and length of the alkyl linker between MPA and heterocyclic units influenced the observed cytotoxicity. Derivatives 2b, 2d, 3a, 3b displayed the most promising immunosuppressive activity.