Factors affecting long-term efficacy of T regulatory cell-based therapy in type 1 diabetes
- Natalia Marek-Trzonkowska†1,
- Małgorzata Myśliwiec†2,
- Dorota Iwaszkiewicz-Grześ3,
- Mateusz Gliwiński3,
- Ilona Derkowska2,
- Magdalena Żalińska2,
- Maciej Zieliński3,
- Marcelina Grabowska3,
- Hanna Zielińska3,
- Karolina Piekarska1,
- Anna Jaźwińska-Curyłło4,
- Radosław Owczuk5,
- Agnieszka Szadkowska6,
- Krystyna Wyka6,
- Piotr Witkowski7,
- Wojciech Młynarski6,
- Przemysława Jarosz-Chobot8,
- Artur Bossowski9,
- Janusz Siebert1 and
- Piotr Trzonkowski3Email authorView ORCID ID profile
© The Author(s) 2016
Received: 17 May 2016
Accepted: 22 November 2016
Published: 1 December 2016
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.
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.
The disease progressed and all patients were insulin-dependent 2 years after inclusion. The β-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.
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
KeywordsDiabetes type 1 Children T regulatory cells Immunotherapy
Type 1 diabetes (T1DM) is an emerging medical problem, since there is no causal treatment and patients inevitably develop full onset of the disease, e.g., in Poland the consequent morbidity doubles every 10 years . The majority of patients are children and the initial manifestation can often be severe, including deep ketoacidosis or coma. It is, therefore, important to investigate novel treatments, aiming at early intervention, while a significant mass of β-cells is still present and can be preserved.
A common consensus exists that the disease develops as a result of the attack of autoaggressive T-cells that infiltrate pancreatic islets and destroy insulin-producing β-cells . This autoaggression is usually unleashed when suppressive subsets, such as CD3+CD4+FoxP3+ T regulatory cells (Tregs), are somehow impaired . Indeed, the adoptive transfer of Tregs was confirmed in animal models as an effective way to stop or delay the progression of the disease . Translational studies in humans seem to confirm this observation, however, the disease still progresses in patients treated with Tregs preparation. It is, therefore, necessary to identify the factors that influence the efficacy of this therapy in the clinical setting.
Starting from 2009, therapy using T regulatory cells moved to the clinical stage and its efficacy is currently being assessed in various conditions, including T1DM . Our group performed several such studies, including that for the treatment of T1DM [5–8]. In this paper, apart from the clinical background, we will present some immunity studies in order to identify the factors that possibly influence the efficacy of the adoptive transfer of Tregs in T1DM.
Protocol and treatment
This was an open-labeled study conducted according to the Declaration of Helsinki principles and was approved by the Ethics Committee of the Medical University of Gdańsk, Poland (NKEBN/8/2010 with amendments). The trial was registered at the Current Controlled Trials database: http://www.controlled-trials.com/ISRCTN06128462 (Additional file 1). Written informed consent was received from parents of all the participants and from the patients themselves, if above 16-years of age.
Clinical characteristics of the patients
Treated (n = 12)
Not treated (n = 10)
Age (years) [median; min–max]
BMI [median; min–max]
Polydipsia at diagnosis (number of patients)
Polyuria at diagnosis (number of patients)
Loss of weight at diagnosis (number of patients)
pH at diagnosis (capillary blood) [median; min–max]
pO2 at diagnosis (capillary blood—mmHg) [median; min–max]
pCO2 at diagnosis (capillary blood—mmHg) [median; min–max]
HCO3 at diagnosis (capillary blood—mmHg) [median; min–max]
Acid/base balance at diagnosis (BE—mEq/l) [median; min–max]
Sat02 at diagnosis (capillary blood—%) [median; min–max]
Anti-GAD65 antibody (number of patients)
ICA (number of patients)
IAA (number of patients)
Tregs were isolated from the patients’ peripheral blood with a GMP-compliant FACS sorter (Influx; BDBiosciences, USA). The purity of Tregs after sorting was ≈98% (range 97–100%). The expansion was performed under GMP conditions and according to our previously described protocol using anti-CD3/anti-CD28 beads, interleukin 2 (IL-2) and autologous serum. The final product on release kept the FoxP3 expression above 90% [median (min.–max) = 91% (90–97)] .
The dose-escalation scheme of Tregs administration was: 10 × 106 of Tregs/kg b.w. in a single infusion (three patients), 20 × 106 of Tregs/kg b.w. in a single infusion (three patients), and a total of 30 × 106 of Tregs/kg b.w. in two infusions (six patients), with the second dose being administered 6–9 months after the first one. Two patients were lost to follow-up at +6 and +9 months, while ten patients completed the trial.
The primary endpoints of the trial were safety and remission defined as daily dose of insulin (DDI) ≤0.5 UI/kg b.w. and fasting C-peptide levels >0.5 ng/mL 1 year after recruitment. Secondary endpoints included the immune background of the patients treated with the preparation of Tregs.
Metabolic and immune responses
Fasting C-peptide levels, fasting glucose, HbA1c and T1DM autoantibody [glutamic acid decarboxylase autoantibody (anti-GAD65), insulin autoantibody (IAA), insulin antigen 2 antibody (IA2) and zinc transporter 8 autoantibody (anti-ZnT8)] levels were measured during a 24-month-long follow-up at different time points, as previously described [7, 8]. The mixed meal tolerance test (MMTT) was performed according to standard criteria on the day of the 24th month of follow-up .
Immune phenotyping was performed using a seven-color panel: CD3/CD4/CD25/CD127/CD45RA/CD62L/FoxP3. Two phenotypes of Tregs were analyzed: CD3+CD4+FoxP3+ and CD3+CD4+CD25highCD127− T-cells. The gate CD25highCD127− from the later population was also used to assess the content of FoxP3+ T-cells in order to estimate an overlap between the two phenotypes of Tregs. Finally, CD3+CD4+FoxP3+ T-cells were subdivided into naive CD3+CD4+FoxP3+CD62L+CD45RA+ (Tn) Tregs, central memory, CD3+CD4+FoxP3+CD62L+CD45RA− (Tcm) Tregs, and effector memory, CD3+CD4+FoxP3+CD62L−CD45RA− (Tem) Tregs .
The following anti-human monoclonal antibodies were used in this procedure (fluorochrome/class/clone): anti-CD3 (PacificBlue/IgG1/UCHT1), anti-CD4 (PerCP/IgG1/RPA-T4), anti-CD25 (PE/IgG1/M-A251), anti-CD127 (FITC/IgG1/hIL-7R-M21) and, anti-CD45RA (PE-Cy7/IgG1/L48). All of the antibodies were purchased from BDBiosciences, Poland. Anti-CD62L (APC-Cy7/IgG1/3B5) was supplied by Invitrogen, USA, and the FoxP3 staining kit by eBioscience, USA.
Serum levels of IFNγ, VEGF, TNFα, IL1, IL2, IL4, IL6, IL8, IL10 and, IL12 were measured with the Luminex Bead based Multiplex Assay (ebioscience, USA), while BAFF, TGFβ, and, IL17 were measured with Quantikine High Sensitivity ELISA kit (R&D Systems, USA). All assays were performed according to the manufacturers’ instructions.
IL2 dependency tests
Samples of cells from Tregs expansions administered to the patients and autologous CD3+CD4+CD127+ conventional/effector T-cells (Teffs) expanded along with Tregs were cultured for additional 8 days after the release of Treg products to the clinic. Tregs and Teffs were cultured separately or co-cultured in a 1:1 ratio, 1 × 106 cells/well in 24-well plates, in a 5% CO2 atmosphere, at 37.0 °C, in the following concentrations of IL2: 0.0, 10.0, 100.0 and 1000 UI/mL. Survival of the cells was measured every day by flow cytometry using 7-aminoactinomycin D staining (7-AAD, Via-probe BDBiosciences, Poland).
Data were computed with the software Statistica 10.0 (Statsoft, Poland). As indicated by the distribution of the variables non-parametric tests were used. The analysis was performed using Kruskal–Wallis ANOVA (KW), the U-Mann–Whitney test (MW), Wilcoxon test, and Spearman’s rank correlation. The p value was considered statistically significant when <0.05 (Additional file 2).
β-cell function was best preserved in patients treated with two doses of Tregs, slightly less preserved in those treated with one dose and the least preserved in untreated controls. The criteria of remission were achieved at the 24th month of follow-up in 3 out of 6 patients treated with two doses of Tregs, 1 out of 6 patients treated with a single dose of Tregs, while no remission occurred in the control group. There were no additional adverse effects, as compared to previous reports [7, 8].
A significant difference was also seen when daily insulin requirements were analyzed. When compared to untreated controls, both groups treated with Tregs required significantly lower doses of insulin throughout the study (KW p < 0.05). The group treated with two Tregs doses required the lowest doses of exogenous insulin, which maintained the levels of metabolic parameters, such as HbA1c and fasting glucose under control, notably at 2 years post inclusion (KW p < 0.05 at the 4th and the 24 month of follow-up for fasting glucose). Importantly, two patients in the group treated with two doses were insulin independent more than 1 year after infusion.
Tregs levels in vivo
Tregs subsets in vivo
Immune markers of disease progression in peripheral blood
The control untreated group was characterized by a significant shift of Treg subsets during the study (Fig. 4). Compared to the baseline, there was an increase in the level of Tcm Tregs at the expense of naïve Tregs at the 24th month of follow-up. Similarly, but not as significant, changes were seen in patients treated with a single dose of Tregs. These differences were the least noticeable in the group treated with two doses of Tregs (Wilcoxon test: no Tregs p < 0.05, treated groups p > 0.05). Among the groups, the patients treated with two doses of Tregs preserved the proportions of Treg subsets at the 24th month, having the highest level of Tn Tregs and the lowest level of Tem Tregs (KW p < 0.05) (Fig. 4b).
In the control untreated group, the levels of TNFα, IL1, and IL6 were higher at the 12 month than at the start of the study (Wilcoxon test: p < 0.05). Unfortunately, we have no data for these cytokines from the 24th month of follow-up. Apart from IL2, no significant changes were found in serum levels of other cytokines measured.
Tregs and IL2
Dependency of Tregs on IL2 found in vitro was further confirmed by the levels of this cytokine in the sera of patients. Shortly after Tregs infusions, notably in the group treated with two doses of Tregs, concentrations of IL2 transiently decreased, possibly due to the cytokine being metabolized by the infused Tregs (Wilcoxon test: first dose p > 0.05, second dose p < 0.05) (Fig. 7b).
Cellular therapy with autologous expanded Tregs seemed to delay the progression of T1DM but its efficacy could be limited by several factors. As the treatment was introduced after the initial onset of T1DM, probably only a small fraction of β-cells was still preserved in the body and could be protected from autoimmune attack, which was a major limitation of the success of the treatment. The advanced stage of the disease also affected the Tregs. The analysis of this population suggested that it was substantially evolving with the progression of the disease, and it was, therefore, beneficial to use autologous Tregs as early as possible in order to obtain an optimal preparation of Tregs for clinical applications. An additional dose of Tregs later in the study improved the results, but the effects were limited. The disease not only modified Tregs, but also the cytokine milieu towards stronger proinflammatory activity, by increasing the levels of IL6 and, to a lesser extent, TNFα and, IL1. Finally, as confirmed in vitro, survival of the expanded Tregs was dependent on IL2 and the interaction with other lymphocytes.
The time of recruitment to the treatment was an obvious medical limitation of the trial. Current diagnostics identify T1DM patients relatively late, when only 10–30% of the islets are still functional, so only this small fraction can be spared from the disease . Routine markers of autoimmunity in T1DM, such as autoantibodies, may identify patients in danger of disease development. However, their accuracy in prediction of the onset—notably when titers are low or some autoantibodies are not detectable yet—is not sufficient to identify early stage T1DM . It is therefore imperative to develop credible criteria of imminent T1DM, ideally before clinical manifestation, while there is still a substantial mass of β-cells. An enrichment of the routine algorithm of T1DM diagnosis with HLA haplotyping, non-HLA polymorphisms, and an assessment of islet-autoreactive T-cells or circulating DNA of the islets may allow the identification of patients in a very early stage of the disease [13–15]. The administration of Tregs to such patients would definitely improve the success of the therapy.
The need for early intervention is also justified by changes in the Treg population during T1DM progression. We have found that two phenotypes of Tregs, that is, CD3+CD4+FoxP3+ and CD3+CD4+CD25highCD127−, overlap, but diverge with time. It implies problems with acquiring Tregs for clinical expansion in the more advanced stage of the disease. It can be performed with either worse purity of putative Tregs or with pure Tregs at the expense of the number of sorted cells. In both cases, this can affect the quality of the preparation that is administered to the patient. Additional proof of the change in the population of Tregs in T1DM was a shifting proportion of naïve and memory Tregs as the disease progressed, notably in the untreated controls. In these subjects, there was a continuous shrinkage of the Tn compartment at the expense of more differentiated memory subsets. Our previous studies suggested that inflammation associated with long-lasting T1DM was responsible for such an effect on Tregs. These cells were characterized by decreased expression of FoxP3 and CD62L under proinflammatory conditions, and this effect could be reverted using anti-inflammatory agents [16–19]. It was intriguing that, in the current study, gradual increase in the level of proinflammatory cytokines was seen early, starting from the clinical onset of T1DM. In addition, it was the only examined immune phenomenon that could not be stopped completely by the adoptive transfer of Tregs.
Interestingly, a swap between naïve and memory Tregs could be also seen in interventional groups immediately after infusion of expanded Tregs. However, it would always revert to the baseline: 2 years after Tregs infusion naïve and memory proportions were close to those observed at the beginning of the study. The patients administered with two doses of Tregs preserved proportions closest to the baseline ratio. This temporary change was probably mostly caused by the content of the administered expanded Tregs, which are usually Tcm FoxP3high Tregs . The fact that they reverted to the baseline ratio in treated subjects suggests a possibility that the level and proportions of Tregs, like other lymphocytes, are homeostatically regulated to some ‘baseline’ levels. Indeed, in our study, the Tcm/Tn proportion of Tregs reverted along with the numbers of total Tregs. Interestingly, the increase in the percentage of Tregs after infusions did not clearly correlate with the dose of administered Tregs, but with C-peptide levels, as shown in our previous report . In addition, the increased level of Tregs, followed by higher C-peptide levels, could be seen after each infusion of the cells. Hence, the number of doses might be an independent factor affecting the efficacy of the treatment, equally important as the cumulative number of Tregs administered. If repetitive doses of Tregs can maintain an increased level of Tregs for a longer period, they can also maintain the C-peptide levels, which was observed in this study in patients treated with two doses of Tregs. The hypothesis on homeostatic regulation is therefore important for the treatment regimen, as splitting the therapy to repetitive smaller doses of Tregs could keep an increased level of Tregs in peripheral blood longer than a single high dose. The assumption on the Tregs homeostat should be completed with the effect of proinflammatory cytokines in T1DM. This proinflammatory activity probably exerts significant influence, as seen in untreated controls, whose sera maintained high levels of proinflammatory cytokines, as well as their Tregs populations were shifted towards the memory subsets in a sustained way.
Finally, we assessed the effect of IL2 on expanded Tregs. This is the cytokine known to influence the survival and function of Tregs  and it was also used in high concentrations to expand Tregs for clinical application in this study. The production of the clinical preparation of Tregs required high doses of IL2 and its sudden deprivation on release of the product resulted in a fast decline in cell survival. Fortunately, there were two factors that probably protected Tregs viability in vivo. Even small doses of IL2 added to cell culture—equal to the concentrations of IL2 measured in sera of the patients—significantly improved Tregs viability. Furthermore, the addition of other lymphocytes, which probably secreted IL2 or supported Tregs through cell-to-cell interactions, improved Tregs viability. Most importantly, we found a synergic effect of IL2 and co-cultured Teffs on Tregs viability, since the survival of Tregs in co-cultures with Teffs and exogenous IL2 was comparable to the survival of other lymphocytes. In vivo, the concentration of IL2 decreased shortly after the infusion of Tregs in the group treated with two doses of Tregs, which might simply reflect an increased consumption of IL2 by the infused Tregs. The dependence of Tregs on IL2 was tested in several clinical trials, in which IL2 was either given alone or administered together with expanded Tregs. The former has already been tested in T1DM, but appeared to be ineffective . The latter was tested in graft-versus-host disease with some effect; however, although IL2 as a medicinal product is indicated against tumors, the co-administration of IL2 and Tregs was associated with manifestation of malignancy . Paradoxically, this might be the major threat of systemic injections of IL2 with Tregs, since the exogenous cytokine might disturb trafficking of administered Tregs attracted to local inflammation, as in insulitis. It is an important argument as we confirmed that such an accumulation of Tregs in human inflamed tissues exists . Instead of chemotaxis towards inflamed tissues, Tregs injected in combination with exogenous IL2 may impose generalized systemic immunosuppression, facilitating progression of tumors. Hence, such combined interventions should be performed with caution.
Currently, there are two centers that completed their first studies with Tregs in T1DM [7, 8, 24]. Safety was confirmed in both studies; however, the results are from small cohorts and, therefore, should be interpreted with caution. Still, the results can help design new, improved treatment protocols. As our trial included matched untreated controls, some efficacy could be also confirmed when comparing the data of the treated and untreated subjects. More importantly, we have found factors that most probably affect the efficacy of this therapy. Mainly, it is the advanced stage of the disease, which has a negative impact on the changes in the Tregs compartment. Based on our previous work, as well as this study, we assume that the inflammatory milieu characteristic for the progression of diabetes is responsible for these changes. High dependency of Tregs on IL2 may also influence the results, but this seems to be controlled by the IL2 produced in vivo. Early administration and repetitive doses of Tregs seemed to improve the results. Hence, new trials should take into account these observations in order to improve the efficacy of this approach. These factors were considered in the design of our new currently ongoing trial TregVac2.0 registered as EudraCT: 2014-004319-35 , in which two doses of Tregs are administered in a 3-month interval, and patients are recruited in earlier phases of the disease.
glutamic acid decarboxylase autoantibody
zinc transporter 8 autoantibody
B cell activating factor
fluorescence activated cell sorter
good manufacturing practice
islet antigen 2 antibody
mixed meal tolerance test
type 1 diabetes
T central memory
T effector/conventional cells
T effector memory
T regulatory cells
tumour necrosis factor α
vascular endothelial growth factor
NMT contributed to the study design, protocol writing, cell preparation, data collection, analysis, interpretation, and writing and reviewing of the report; MM contributed to the study design, protocol writing, cell preparation, data collection, analysis, interpretation, and writing and reviewing of the report; DIG, MG, contributed to cell separation and data collection; ID, MŻ, MZ, MG, HZ, KP contributed to data collection and interpretation; AJC contributed to data collection; RO contributed to data collection; AS, KW contributed to data collection and interpretation; PW contributed to data analysis, interpretation and reviewed the report; WM contributed to data collection, analysis and interpretation and reviewed the report; PJCh contributed to data collection and interpretation and reviewed the report; AB contributed to data collection and interpretation and reviewed the report; JS contributed to data collection, interpretation and reviewed the report; PT was a supervisor of the study who contributed to the study design, protocol writing, cell preparation, data collection, analysis, interpretation writing and, reviewing of the report. All authors read and approved the final manuscript.
We thank to Mrs. Anita Dobyszuk, Mrs. Lucyna Szumacher-Sharma and Mrs. Justyna Drabik from Medical University of Gdańsk Medical Centre for their perfect assistance in laboratory and clinical procedures. NMT and PT are members of COST Action BM1305 A FACTT (http://www.afactt.eu) supported by COST (European Cooperation in Science and Technology). COST is part of the EU Framework Programme Horizon 2020.
NMT, MM and PT are co-inventors of patent related to presented content and stakeholders of POLTREG venture. Medical University of Gdańsk received payment for the license to presented content.
Availability of data and materials
All details of the study are presented at http://www.controlled-trials.com/ISRCTN06128462. Further details on the data and materials can be sent by PT, who is the corresponding author.
Ethics approval and consent to participate
The study was conducted according to the Declaration of Helsinki principles and was approved by the Ethics Committee of the Medical University of Gdańsk, Poland (number of approval: NKEBN/8/2010 with amendments). The trial was registered at the Current Controlled Trials database: http://www.controlled-trials.com/ISRCTN06128462 (Additional file 1). Written informed consent was received from parents of all the participants and the patients if above 16-years-old.
Trial supported by National Centre for Research and Development, Poland (grant no STRATEGMED1/233368/1/NCBR/2014, DI-G was supported by the LIDER/160/L-6/14/NCBR/2015), National Science Centre, Poland (Grant No. DEC-2011/01/D/NZ3/00262) and funds of the Polish Ministry of Science and Higher Education (Grant No. IP2011 033771).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Jarosz-Chobot P, Polańska J, Szadkowska A, et al. Rapid increase in the incidence of type 1 diabetes in Polish children from 1989 to 2004, and predictions for 2010 to 2025. Diabetologia. 2011;54:508–15.View ArticlePubMedGoogle Scholar
- Eisenbarth GS. Type I diabetes mellitus. A chronic autoimmune disease. N Engl J Med. 1986;314:1360–8.View ArticlePubMedGoogle Scholar
- Bennett CL, Christie J, Ramsdell F, et al. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet. 2001;27:20–1.View ArticlePubMedGoogle Scholar
- You S, Slehoffer G, Barriot S, Bach JF, Chatenoud L. Unique role of CD4+ CD62L+ regulatory T-cells in the control of autoimmune diabetes in T cell receptor transgenic mice. Proc Natl Acad Sci USA. 2004;101:14580–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Trzonkowski P, Bacchetta R, Battaglia M, et al. Hurdles in therapy with regulatory T-cells. Sci Transl Med. 2015;7(304):304ps18.View ArticlePubMedGoogle Scholar
- Trzonkowski P, Bieniaszewska M, Juścińska J, et al. 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. Clin Immunol. 2009;133:22–6.View ArticlePubMedGoogle Scholar
- Marek-Trzonkowska N, Myśliwiec M, Dobyszuk A, et al. Administration of CD4+ CD25highCD127− regulatory T-cells preserves β-Cell function in type 1 diabetes in children. Diabetes Care. 2012;35:1817–20.View ArticlePubMedPubMed CentralGoogle Scholar
- Marek-Trzonkowska N, Myśliwiec M, Dobyszuk A, et al. Therapy of type 1 diabetes with CD4+ CD25highCD127-regulatory T-cells prolongs survival of pancreatic islets—results of one year follow-up. Clin Immunol. 2014;153:23–30.View ArticlePubMedGoogle Scholar
- Marek N, Bieniaszewska M, Krzystyniak A, et al. The time is crucial for ex vivo expansion of T regulatory cells for therapy. Cell Transpl. 2011;11–12:1747–58.View ArticleGoogle Scholar
- Greenbaum CJ, Mandrup-Poulsen T, McGee PF, Type 1 Diabetes Trial Net Research Group, European C-Peptide Trial Study Group, et al. Mixed-meal tolerance test versus glucagon stimulation test for the assessment of beta-cell function in therapeutic trials in type 1 diabetes. Diabetes Care. 2008;31:1966–71.View ArticlePubMedPubMed CentralGoogle Scholar
- Eisenbarth GS. Type 1 diabetes: molecular, cellular and clinical immunology. Adv Exp Med Biol. 2004;552:306–10.PubMedGoogle Scholar
- Steck AK, Johnson K, Barriga KJ, et al. Age of islet autoantibody appearance and mean levels of insulin, but not GAD or IA-2 autoantibodies, predict age of diagnosis of type 1 diabetes: diabetes autoimmunity study in the young. Diabetes Care. 2011;34:1397–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Akirav EM, Lebastchi J, Galvan EM, et al. Detection of β cell death in diabetes using differentially methylated circulating DNA. Proc Natl Acad Sci USA. 2011;108:19018–23.View ArticlePubMedPubMed CentralGoogle Scholar
- Steck AK, Dong F, Wong R, et al. Improving prediction of type 1 diabetes by testing non-HLA genetic variants in addition to HLA markers. Pediatr Diabetes. 2014;15:355–62.View ArticlePubMedGoogle Scholar
- Peakman M, Tree T, Endl J, et al. Characterization of preparations of GAD65, proinsulin, and the islet tyrosine phosphatase IA-2 for use in detection of autoreactive T-cells in type 1 diabetes: report of phase II of the second international immunology of diabetes society workshop for standardization of T-cell assays in type 1 diabetes. Diabetes. 2001;50:1749–54.View ArticlePubMedGoogle Scholar
- Ryba-Stanisławowska M, Skrzypkowska M, Myśliwska J, Myśliwiec M. The serum IL-6 profile and Treg/Th17 peripheral cell populations in patients with type 1 diabetes. Mediat Inflamm. 2013;2013:205284.Google Scholar
- Ryba M, Rybarczyk-Kapturska K, Zorena K, Myśliwiec M, Myśliwska J. Lower frequency of CD62L(high) and higher frequency of TNFR2(+) Tregs are associated with inflammatory conditions in type 1 diabetic patients. Mediat Inflamm. 2011;2011:645643.View ArticleGoogle Scholar
- Ryba M, Marek N, Hak Ł, et al. Anti-TNF rescue CD4+ Foxp3+ regulatory T-cells in patients with type 1 diabetes from effects mediated by TNF. Cytokine. 2011;55:353–61.View ArticlePubMedGoogle Scholar
- Ryba M, Hak L, Zorena K, Myśliwiec M, Myśliwska J. CD4+ Foxp3+ regulatory T lymphocytes expressing CD62L in patients with long-standing diabetes type 1. Cent Eur J Immunol. 2009;34:90–3.Google Scholar
- Malek TR. The main function of IL-2 is to promote the development of T regulatory cells. J Leukoc Biol. 2003;74:961–5.View ArticlePubMedGoogle Scholar
- Hartemann A, Bensimon G, Payan CA, et al. Low-dose interleukin 2 in patients with type 1 diabetes: a phase 1/2 randomised, double-blind, placebo-controlled trial. Lancet Diabetes Endocrinol. 2013;1:295–305.View ArticlePubMedGoogle Scholar
- Theil A, Tuve S, Oelschlägel U, et al. Adoptive transfer of allogeneic regulatory T-cells into patients with chronic graft-versus-host disease. Cytotherapy. 2015;17:473–86.View ArticlePubMedGoogle Scholar
- Sznurkowska K, Żawrocki A, Sznurkowski J, et al. Peripheral and intestinal T-regulatory cells are upregulated in children with inflammatory bowel disease at onset of disease. Immunol Invest. 2016;19:1–10.Google Scholar
- Bluestone JA, Buckner JH, Fitch M, et al. Type 1 diabetes immunotherapy using polyclonal regulatory T cells. Sci Transl Med. 2015;7(315):315ra189.View ArticlePubMedPubMed CentralGoogle Scholar