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Whole blood was collected in sodium heparinized vacutainer tubes and processed within 24 hours of collection. Some samples were shipped in a temperature controlled shipping container (23 C) over night from Colorado or Florida prior to processing without any detectable loss of viability or expansion capacity. Whole blood was diluted with an equal volume of Hank's Buffered Salt Solution (HBSS) and subsequently underlayed in each tube with 10 ml Ficoll-hypaque solution (Amersham/GE Healthcare, Piscataway, NJ). The PBMC layer was collected following density gradient centrifugation (900 x g for 20 min at 23Â°C, without brake) and subsequently washed twice in 10 ml HBSS at 420 x g for 5 min at 23Â°C. Cells were resuspended in phenol red-free HBSS at a final concentration of 1 x 108/ml.
Isolation of Tregs by FACS.
Tregs were isolated by FACS according to the selection markers of CD4, CD25, and CD127. Setup controls (5 x 105 cells in 5 Î¼l HBSS) were stained for 30 min at 4Â°C with 2.5 Î¼l of each single color compensation antibody (CD4-PerCP (clone SK3), CD127-PE (hIL-7R-M21), CD25-APC (2A3). For a limited number of experiments, CD45RA-FITC (L48), and CD45RO-PE.Cy5 (UCHL1) (in conjunction with CD4-Pacific Blue) were used to isolate naÃ¯ve and memory Treg subsets, respectively. All antibodies for cell isolation were provided by BD Biosciences (San Jose, CA) and BD PharMingen (San Diego, CA). Compensation controls were washed in 2 ml phenol red-free HBSS and resuspended in 500 Î¼l phenol red-free HBSS for instrument setup.
All remaining cells were stained for collection by adding fluorescent labeled antibodies at the following concentrations: 1 Î¼l/106 cells for CD4-PerCP and CD127-PE, and 0.7 Î¼l/106 cells of CD25-APC. Cells were stained for 30 min at 4Â°C, washed in 12 ml of phenol red-free HBSS and resuspended at a final concentration of 25-30 x 106 cells/ml in phenol red-free HBSS containing 10% human heat-inactivated pooled AB serum (Valley Biomedical, Winchester, VA) for sorting. CD4+CD127lo/-CD25+ and CD4+CD127lo/- T cells were sorted using aseptic technique at a cGMP-level clean room facility with a sorting rate of 15-20,000 events per second. The sorted populations were collected into 3 ml of 4Â°C X-VIVO 15 media (Lonza, Walkersville, MD) containing 10% human heat-inactivated pooled AB serum.
In vitro expansion procedure.
Following FACS isolation, cells were centrifuged at 420 x g for 5 min at 4oC to remove residual sheath fluid and collection media. Cells were resuspended in 1 ml X-VIVO 15 media (Lonza) containing 10% serum and plated at 2.5 x 105 Tregs per well in a 24-well plate (Costar; Cambridge, MA). Human serum often varies in composition. To test the requirement of serum for expansion, additional experiments were conducted in a defined serum-free media formulation (OpTmizer T-Cell Expansion Media, Invitrogen). This analysis indicated continued Treg expansion in serum-free media compared to X-VIVO with 10% human serum (data not shown).
Tregs were activated with Dynabeads ClinExVivo anti-CD3/anti-CD28 coated microbeads (Invitrogen; Carlsbad, CA) at a 1:1 bead to cell ratio. CD4+CD127lo/- cells received Rapamycin (Wyeth, Madison, NJ) immediately to a final concentration of 100 ng/ml and maintained (assuming consumption) until day 7 of culture. On day 2, the culture volume was doubled and IL-2 (Proleukin; Chiron Therapeutics, Emeryville, CA) was added to a final concentration of 300 U/ml. Cells were resuspended, counted and fresh media and IL-2 was added on days 2, 5, 7, 9, and 12 and maintained at 300U/ml assuming consumption. On day 9, cells were restimulated with fresh anti-CD3/anti-CD28 coated beads at a 1:1 bead to cell ratio. Culture guidelines including cell numbers, volumes, and respective culture flasks are outlined below. For cultures containing intermediate cell numbers, a combination of vessels was used.
SUPPLEMENTAL FIG. 1. 3D FACS plot of Teff and Treg populations. Cells were gated on Lymphocytes (FSC vs. SSC), then CD4+ cells (CD4 vs. SSC), and shown as CD127-PE (x axis), vs. CD25-APC (y axis) vs. FOXP3-Alexa 488 (z axis) using CXP Data Acquisition & Analysis software, version 2.0 from Beckman Coulter, Inc. Plot shows CD4+CD127hi Teff (A, Isotype control) and (B, FOXP3 stained cells) or CD4+CD127lo/-CD25+ Tregs (C, Isotype control) and (D, FOXP3 stained cells). See also Supplemental videos for animated images of these plots.
SUPPLEMENTAL FIG. 2. Rapamycin delays cell cycle progression of CD4+CD127lo/- T cells during in vitro expansion. A: For days 1-7, plot shows dilution of CFSE labeled cells cultured with IL-2 alone (left plot) or cells cultured with IL-2 and Rapamycin (right plot). B: Cells were relabeled with CFSE and restimulated with anti-CD3/anti-CD28 beads on day 9 of expansion. Plots shows dilution of CFSE labeled cells as described above for days 9-14 of culture, with IL-2 only (left plot) or IL-2 and Rapamycin (right plot).
SUPPLEMENTAL FIG. 3. FOXP3 expression in expanded Tregs is upregulated following reactivation. A: CD4+CD127lo/-CD25+ Tregs were harvested following 14 days of culture and stained for intracellular FOXP3 expression immediately (solid light gray histogram), or following 5 h of reactivation with either beads (solid gray histogram) or PMA/ionomycin (solid dark gray histogram). Dashed histograms represent matched isotype controls. B: The suppressive capacity of these cells following each activation condition was tested by their capacity to suppress proliferation of responder T cells as measured by the incorporation of 3H-thymidine. Graph shows data from triplicate co-cultures at the indicated ratios of Tregs to responders, with responders alone in black bar.
SUPPLEMENTAL FIG. 4. Expression of phenotypic markers on expanded human Tregs. Cells were gated on FOXP3+ T cells and analyzed for the expression of MHC II molecule HLA-DR, CTLA-4, and TGF-ï¢1-LAP by FACS analysis. Shown is one representative example. Histogram overlays indicate isotype control background staining (dashed lines) as well as marker expression (solid lines).
SUPPLEMENTAL FIG. 5. Production of IFN-Î³ from expanded FOXP3+ Treg populations. A: Plot shows IFN-Î³ production from expanded CD4+CD127lo/-CD25+ T cells reactivated with PMA/ionomycin (6.0% FOXP3+IFN-Î³+, upper-right hand quadrant). B: IFN-Î³ could also be detected upon reactivation of expanded cells with anti-CD3 and anti-CD28 coated beads, as well as from (C) expanded cord blood Tregs. To further isolate the source of IFN-Î³ producing cells, CD4+CD127lo/-CD25+ Tregs were sorted into naÃ¯ve (D, CD45RA+) and memory (E, CD45RO+) fractions prior to expansion. FOXP3+ NaÃ¯ve Tregs produce less IFN-Î³ compared to memory Tregs (2.1% vs. 5.3%, respectively). Data shown are representative examples of at least three independent experiments.
Supplemental Methods Section. Culture conditions for isolation and in vitro expansion of Tregs.
SUPPLEMENTAL TABLE 1. Cytokine production profiles from expanded Tregs. Seventeen subjects were analyzed with the mean + standard deviation reported in pg/ml (upper un-shaded rows) and in pM (lower shaded rows). Instances where cytokine was below the limit of detection were reported as no detection (ND). TGF-ï¢ levels were below the limit of detection prior to acid activation.
SUPPLEMENTAL TABLE 2. IFN-ï§ production following ï¡CD3/ï¡CD28 beads or PMA/Ionomycin activation of freshly isolated or expanded CD4+CD127lo/-CD25+ Tregs. Data are indicated as a percent of IFN-ï§+/- within the FOXP3+ subset.