Comparison of suppressive function of double\negative regulatory T cells (DN Tregs) grown in the culture media supplemented with interleukin (IL)\2 or IL\2 and IL\7. suppression assay does not impair functionality of double\negative regulatory T cells (DN Tregs). Carboxyfluorescein succinimidyl ester (CFSE)\labelled CD4+ GZD824 cells were stimulated with CD3/CD28 beads cultured with DN Tregs that were expanded in the presence of IL\2 only. The co\culture media during the suppression assay was supplemented with IL\2, IL\2 and IL\7 or devoid of any cytokines. After 4 days, the proliferation of CD4+ responder T cells was determined by CFSE dilution. Data is expressed as mean??standard deviation (s.d.) of three replicate co\cultures. Similar results were obtained with cells from another donor. Fig. S3. Double\negative regulatory T cells (DN Tregs) do not kill autologous CD4+ or CD8+ T cells. After 4 days of suppression assay, CD4+ (a) and CD8+ (b) responder cells were assessed for viability [7\aminoactinomycin D (7\AAD)] and apoptotic markers (annexin V) through flow cytometry. The percentage of viable cells, defined as cells negative for 7\AAD and annexin V, is shown. Bar graphs represent mean??standard deviation (s.d.) from three replicates. Similar results were obtained with DN Tregs from at least four different donors. Fig. S4. Tracking and proliferation of human lymphocytes in non\obese diabetic\expansion of human DN Tregs within 3 weeks with ?97% purity. through direct cell\to\cell contact. to therapeutic numbers. The expanded DN Tregs can suppress proliferation of T and B cells and attenuate GVHD, highlighting the potential clinical use GZD824 of DN Tregs to mitigate GVHD. expansion of a highly pure, functional and stable cellular product 3. In addition to nTregs and Tr1 cells, double\negative (DN) Tregs have been shown to have regulatory properties. DN Tregs express T cell receptor (TCR)\+, are natural killer (NK) lineage marker\negative and lack CD4 and CD8 co\receptors on their cell surface 12. Neither murine 12, 13, 14 nor human 15 DN Tregs express the FoxP3 transcription factor. We and others have demonstrated in various rodent models that DN Tregs were able to induce antigen\specific tolerance to allogeneic skin, heart and pancreas grafts and inhibit various infections and autoimmune diseases 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. Furthermore, DN Tregs were able to inhibit the onset of GVHD while mediating beneficial anti\leukaemia effects 20, 26. Human DN Tregs have also been shown to suppress allogeneic immune responses preclinical studies and, ultimately, clinical use. In this study, we developed a novel protocol that allows for large\scale expansion of highly pure and functional population of human DN Tregs. These was enhanced further by treatment of DN Tregs with rapamycin. These findings emphasize the potential for GZD824 clinical use of DN Tregs poised to broaden T cell\based therapies in treatment of GZD824 GVHD Mouse monoclonal to Calcyclin and prevention of allograft rejection. Materials and methods Cell isolation Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors using Ficoll\Hypaque density gradient centrifugation. DN Tregs were enriched from PBMCs by negative selection using magnetic cell sorting technology (MACS), according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, PBMCs were labelled with fluorescein isothiocyanate (FITC)\conjugated monoclonal antibodies (mAbs) directed against CD4, CD8, CD56 and TCR\, followed by labelling with anti\FITC magnetic beads. CD4+, CD8+ and CD19+ cells were obtained by positive selection using magnetic beads (Miltenyi Biotec). Cell culture Enriched DN Tregs were resuspended in complete RPMI\1640 culture medium supplemented with recombinant human (rh)IL\2 (250 U/ml). To activate DN Tregs, cells were seeded on anti\CD3 mAb [25 g/ml, muromonab\CD3 (OKT3); eBioscience, San Diego, CA, USA] precoated 96\well plates. DN Tregs were restimulated on days 7,.