In an in vitro cytotoxicity assay sellckchem using a Hodgkin lymphoma cell that we have previously observed to be susceptible to Treg mediated killing, Tregs expanded in the pres ence of rapamycin had a significant decrease in cytotoxic ity as compared to Tregs expanded without rapamycin. The finding that rapamycin suppresses granzyme B expres sion may indicate that rapamycin expanded Tregs utilize suppressive Inhibitors,Modulators,Libraries pathways other than granzyme B to mediate their effects on bystander lymphocytes under physiologic conditions. Methods Flow cytometry The following antibodies were used for the flow cytomet ric evaluation of T cell subsets in this study anti CD4, anti CD25, anti CD127, anti FOXP3, Inhibitors,Modulators,Libraries all Inhibitors,Modulators,Libraries from Bio legend Inc. Anti granzyme B was from eBioscience.

Inhibitors,Modulators,Libraries For the car boxy fluorescein diacetate, succinimidyl ester based proliferation studies only, anti FOXP3 was purchased from BD Biosciences. Cells were permeabilized using a commercially avail able fixation and permeabilization kit per the manufacturers instructions. Pro pidium iodide and annexin V PE were from Pharmin gen. CFSE was used at a final concentration of 1 M. Cells were labeled for 5 minutes in phosphate buffered saline contain ing CFSE at 37 C then placed in pre warmed media for 10 minutes, washed two times and plated as indicated in the figures. Flow cytometry was performed on a FACSCanto II flow cytometer using FACSDiva soft ware except for cell sorting which was performed on a FACSVantage flow cytometer. Final data analysis was performed using FloJo software.

The statistical sig nificance of differences in granzyme B expression and Foxp3 expression between subsets was assessed using the Students t test. Regulatory T cell isolation enrichment Institutional review board approval was obtained from the University of Utah Health Sciences Center Inhibitors,Modulators,Libraries to obtain peripheral blood selleck chemical from normal adult volunteers. All blood donors provided informed consent prior to blood collection. Blood was collected by peripheral veni puncture in ethylenediaminetetra acetic acid coated vacutainer tubes. Periph eral blood mononuclear cells were isolated from normal donors by density gradient centrifugation using Ficol Paque density medium according to the manufacturers recommendations. Subsequently, CD4, CD25 Tregs were enriched using a magnetic bead based kit according to the manufacturers instructions. This tech nique routinely resulted in samples that were enriched in regulatory T cells to a purity of approximately 60 70% or more based on FOXP3 staining by flow cytometry. How ever, higher purity Tregs were required for the cytotoxicity assays. For the 6 hour cytotoxicity assays, peripheral blood nTregs were isolated based on flow cytometric sort ing of the CD4, CD25bright T cell fraction.