ABSTRACT.Purpose: To investigate the cytokine composition and anti-inflammatory effects of allogeneic serum preparations for improved use as serum eye drops.
Adoptive transfer of autologous polyclonal regulatory T cells (Tregs) is a promising
option for reducing graft rejection in allogeneic transplantation. To gain therapeutic
levels of Tregs there is a need to expand obtained cells ex vivo, usually in the presence
of the mTOR inhibitor Rapamycin due to its ability to suppress proliferation of non-Treg T
cells, thus promoting a purer Treg yield. Azithromycin is a bacteriostatic macrolide with
mTOR inhibitory activity that has been shown to exert immunomodulatory effects on several
types of immune cells. In this study we investigated the effects of Azithromycin, compared
with Rapamycin, on Treg phenotype, growth, and function when expanding bulk, naïve, and
memory Tregs. Furthermore, the intracellular concentration of Rapamycin in CD4+ T cells as
well as in the culture medium was measured for up to 48 h after supplemented. Treg
phenotype was assessed by flow cytometry and Treg function was measured as inhibition of
responder T-cell expansion in a suppression assay. The concentration of Rapamycin was
quantified with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS).
Azithromycin and Rapamycin both promoted a FoxP3-positive Treg phenotype in bulk Tregs,
while Rapamycin also increased FoxP3 and FoxP3+Helios positivity in naïve and memory
Tregs. Furthermore, Rapamycin inhibited the expansion of naïve Tregs, but also increased
their suppressive effect. Rapamycin was quickly degraded in 37°C medium, yet was retained
intracellularly. While both compounds may benefit expansion of FoxP3+ Tregs in vitro,
further studies elucidating the effects of Azithromycin treatment on Tregs are needed to
determine its potential use.
With the increasing interest in clinical trials with regulatory T cells (Tregs), immunological profiling of prospective target groups and standardized procedures for Treg isolation are needed. In this study, flow cytometry was used to assess peripheral blood lymphocyte profiles of young healthy individuals and patients undergoing haemodialysis treatment. Tregs obtained from the former may be used in haematopoietic stem cell transplantation and Tregs from the latter in the prevention of kidney transplant rejection. FOXP3 mRNA expression with accompanying isoform distribution was also assessed by the quantitative reverse transcriptase polymerase chain reaction. Flow-cytometric gating strategies were systematically analysed to optimize the isolation of Tregs. Our findings showed an overall similar immunological profile of both cohorts in spite of great differences in both age and health. Analysis of flow-cytometric gating techniques highlighted the importance of gating for both CD25high and CD127low expression in the isolation of FOXP3-positive cells. This study provides additional insight into the immunological profile of young healthy individuals and uraemic patients as well as in-depth analysis of flow-cytometric gating strategies for Treg isolation, supporting the development of Treg therapy using cells from healthy donors and uraemic patients.
Allogeneic islet transplantation in type 1 diabetes requires lifelong immunosuppression to prevent graft rejection. This medication can cause adverse effects and increases the susceptibility for infections and malignancies. Adoptive therapies with regulatory T cells (Tregs) have shown promise in reducing the need for immunosuppression in human transplantation settings but have previously not been evaluated in islet transplantation. In this study, five patients with type 1 diabetes undergoing intraportal allogeneic islet transplantation were co-infused with polyclonal autologous Tregs under a standard immunosuppressive regimen. Patients underwent leaukapheresis from which Tregs were purified by magnetic-activated cell sorting (MACS) and cryopreserved until transplantation. Dose ranges of 0.14-1.27 9 10 6 T cells per kilo bodyweight were transplanted. No negative effects were seen related to the Treg infusion, regardless of cell dose. Only minor complications related to the immunosuppressive drugs were reported. This first-in-man study of autologous Treg infusion in allogenic pancreatic islet transplantation shows that the treatment is safe and feasible. Based on these results, future efficacy studies will be developed under the label of advanced therapeutic medical products (ATMP), using modified or expanded Tregs with the aim of minimizing the need for chronic immunosuppressive medication in islet transplantation.
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