Autologous regulatory T cells in clinical intraportal allogenic pancreatic islet transplantation.

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 × 106 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.

Comparing the Effects of the mTOR Inhibitors Azithromycin and Rapamycin on In Vitro Expanded Regulatory T Cells.

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.

Improving the anti-inflammatory effect of serum eye drops using allogeneic serum permissive for regulatory T cell induction.

PURPOSE: To investigate the cytokine composition and anti-inflammatory effects of allogeneic serum preparations for improved use as serum eye drops. METHODS: Serum of 15 healthy blood donors was extensively screened for cytokines, including IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-15, 1L-17A, E and F, IL-21, IL-22, IL-23, IL-27, IL-28A, IL-31, IL-33, granulocyte macrophage colony-stimulating factor (GM-CSF), chemokine ligand 20 (CCL20), tumour necrosis factor (TNF)-α and TNF-ß, interferon (IFN)-γ and transforming growth factor (TGF)-ß. The levels of cytokines were assessed before and after heat-induced inactivation. Individual serum preparations were tested for their anti-inflammatory effect using an in vitro test to differentiate effector T lymphocytes into anti-inflammatory regulatory T cells. RESULTS: The anti-inflammatory cytokine TGF-ß was readily detected in the serum of all blood donors and was only modestly affected by heat-induced inactivation. Serum containing high amounts of TGF-ß was more effective at inducing anti-inflammatory regulatory T cells. The serum of one healthy blood donor displayed high levels of inflammatory cytokines. CONCLUSION: We propose that serum used as eye drops is screened for its cytokine content, making it possible to correlate the composition to the clinical outcome. Based on the findings in this study, tailored serum eye drops produced from allogeneic donors may provide increased anti-inflammatory effects. This may be superior to autologous serum eye drops, which in many cases are retrieved from patients with inflammatory diseases.