After 7 days of incubation, cells were harvested, stained with appropriately labeled mAbs against CD4 (clone RM4-5), CD8 (clone 53-6
After 7 days of incubation, cells were harvested, stained with appropriately labeled mAbs against CD4 (clone RM4-5), CD8 (clone 53-6.7), CD3 (clone 145-2C11), B220 (clone RA3-6B2), Gr1 (clone RB6-8C5), CD11c (clone HL3) all from BD Biosciences, NK1.1 (clone PK136, Sony) and CD11b (clone M1-70), ckit (CD117, clone 2B-8), Sca1(anti-Ly6A/E, clone D7) and PDCA-1 (clone eBio927) from eBioscience, and analyzed by circulation cytometry for lineage determination. Isolation of Immune Cells from your Spinal Cord Spinal cord isolated from control and MPP-recipient mice were incubated for 30?min in digestion buffer of DNAse and Liberase (27 WU/ml) in PBS 1 at 37C, mixing every 5?min. cutoff is at 0.05. Enrichments in GO (Gene Ontology) terms as well as gene symbols are provided for each significant GO category. Table is usually sorted by decreasing enrichment. Table_1.xlsx (3.3M) GUID:?6F134FF2-41C9-4C48-8B20-63B967E3F17A Supplementary Table 2: Functional annotations of the genes in K-means cluster c8, over-expressed in Treg cultured 4 days PTP1B-IN-8 in presence of MPP Treg alone. FDR (false discovery rate obtained by bootstrapping 50 occasions) cutoff is at 0.05. Enrichments in GO (Gene Ontology) terms as well as gene symbols are provided for each significant GO category. Table is usually sorted by decreasing enrichment. Table_1.xlsx (3.3M) GUID:?6F134FF2-41C9-4C48-8B20-63B967E3F17A Supplementary Table 3: Statistics obtained after supervised differential gene expression analyses. Moderated t-test on linear modeling with empirical Bayes was applied on Treg alone vs. Treg+MPP. FDR: False discovery rate by Benjamini-Hochberg. Fold switch is usually indicated in simple and centered on zero. Table_1.xlsx (3.3M) GUID:?6F134FF2-41C9-4C48-8B20-63B967E3F17A Data Availability StatementThe datasets generated and analyzed for this study can be found in the GEO repository https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE155148. All other data are available upon reasonable request. Abstract Achieving immunoregulation growth of Foxp3+ regulatory CD4+ T cells (Treg) remains challenging. We have shown that mobilization confers to multipotent hematopoietic progenitors (MPPs) the capacity to enhance Treg proliferation. Transcriptomic analysis of Tregs co-cultured with PTP1B-IN-8 MPPs revealed enhanced expression of genes stabilizing the suppressive function of Tregs as well as the activation of IL-1Cdriven pathways. Adoptive transfer of only 25,000 MPPs effectively reduced the development of experimental autoimmune encephalomyelitis (EAE), a pre-clinical model for multiple sclerosis (MS). Production of the pathogenic PTP1B-IN-8 cytokines IL-17 and GM-CSF by spinal cord-derived CD4+ T-cells in MPP-protected recipients was reduced while Treg growth was enhanced. Treg depletion once protection by MPPs was established, brought on disease relapse to the same level as in EAE mice without MPP injection. The key role of IL-1 was further confirmed by the lack of protection against EAE in recipients of IL-1Cdeficient MPPs. Mobilized MPPs may thus be worth considering for cell therapy of MS either per se or for enrichment of HSC grafts in autologous bone marrow transplantation already implemented in patients with severe refractory multiple sclerosis. with IL-2, rapamycin, activation with anti-CD3/CD28 mAb-coated beads (9, 10) or with minute foreign antigen doses (11), for subsequent administration to patients with autoimmune diseases. Notably, Treg cell therapy may require billions of cells (12). Promoting the growth of Tregs directly thus represents a therapeutic strategy well worth of interest. Interleukin-2 (IL-2) at low dose has been demonstrated to expand preferentially Treg and numerous trials in a host of clinical settings are underway (13). However, it remains interesting to develop alternative strategies PTP1B-IN-8 susceptible to confer Ilf3 highly selective growth of Treg with no growth of effector T-cells. We therefore investigated whether adoptive transfer of mobilized MPP could be used to protect against EAE by selectively promoting Treg growth. We herein statement that MPP promote Treg proliferation and survival both and growth of functional Treg can be efficiently induced by MPP, and MPP-based cell therapy could symbolize a therapeutic strategy against autoimmune diseases either per se or as a an enrichment of autologous HSCT, already implemented in patients with severe multiple sclerosis (1C5). Materials and Methods Mice Wild type C57BL/6J, C57BL/6 Foxp3-GFP-KI, and C57BL/6 IL-1?/? (obtained from CDTA, Orlans, France) mice were bred in our animal facility under specific pathogen-free conditions. Live animal experiments were conducted according to the EU Directive 2010/63/EU for animal experiments under an animal study proposal approved by the Paris Descartes University or college Ethical Committee for Animal Experimentation and the French Ministry of Research and Higher Education, number 3846-2015070622031545v4. EAE Induction Active EAE was induced in 10- to 12-week-old female mice by s.c. immunization at two sites, upper back and lower back, with 200 g myelin oligodendrocyte glycoprotein (MOG)35C55 peptide (MEVGWYRSPFSRVVHLYRNGK) emulsified in CFA made up of 400 g heat-killed Mycobacterium tuberculosis H37Ra (Hooke Laboratories, Lawrence, MA, USA), on day 0. Additionally, mice received 300 ng pertussis toxin (Hooke Laboratories, Lawrence, MA, USA) i.p. in 0.1 ml/mouse on days 0 and 1. Clinical indicators of EAE were assessed daily with a.