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Single-cell suspensions of C57BL/6J splenocytes were incubated with CD5 beads (Miltenyi Biotech) and T cells were positively selected by magnetically activated cell sorting (MACS). 50x10 6 or greater cells were incubated with CFSE at a final concentration of 2.5 µm in PBS at 37 C for 25 minutes, washed three times, and 10x10 6 labeled T cells were infused intravenously via the tail vein of lethally irradiated recipient mice. C57BL/6J CD45.1 + mice received a split dose irradiation of 1100 cgy, C3FeB6F1/J mice received a split dose irradiation of 1300 cgy, and BALB/c mice received a split dose irradiation of 850 cgy as described above. Assessment of GVHD Survival was monitored daily, and mice were individually scored weekly for weight loss, posture, activity level, fur ruffling, and skin lesions on a scale from 0 to 2 as described previously 8. A clinical GVHD score was generated by adding these five criteria. Anti-CD3 and CD28 stimulation 96 well plates were coated with anti-cd3 antibody (clone 145-2C11, 0.5 µg / ml) and anti-cd28 antibody (clone 37.51, 0.5 µg / ml) overnight at 4 degrees centigrade and washed 3 times with ice-cold PBS. 10 5 RBC lysed B6 splenocytes per well were added together with varying concentrations of SL327 as described in the legend for Figure 2. Cells were incubated for 24 hours at 37 degrees C and then pulsed with 1 µci of 3 H thymidine. Thymidine incorporation was measured at 18 hours after pulsing.
Mixed leukocyte reactions RBC-lysed splenocytes from B6 and BALB/c cells were irradiated with 2000 cgy from a 60 Co source and used as stimulators. RBC-lysed non-irradiated B6 splenocytes were used as responders. 10 5 stimulators and 10 5 responders were mixed in 96 well plates together with varying concentrations of SL327, cucurbitacin I or cucurbitacin E as described in the legends for Figures 4 and 5, and incubated at 37 degrees C. On day 4 cells were pulsed with 1 µci of 3 H thymidine, and incorporation was then measured at 18 hours after pulsing. Experiments with the adoptive transfer of splenocytes Whole B6 spleens were dissociated into single cell suspensions and incubated for 1 hour at 37 degrees centigrade with 5 nm cucurbitacin E or DMSO control. Cells were then counted, washed twice, and adoptively transferred into lethally irradiated BALB/c mice (850 cgy, split dose) via tail-vein infusion. Recipients were sacrificed at 24 hours and spleens removed for analysis by flow cytometry. Surface and intracellular staining, data acquisition and analysis Cells were fixed with 2% paraformaldehyde in PBS for 10 minutes at 37 C, pelleted by centrifugation (350 x g for 5 minutes), resuspended in 90% methanol while vortexing, and incubated on ice for 30 minutes. Cells were then washed three times with PBS, and incubated for 10 minutes at 4 C with CD16/CD32 FCR block (clone 2.4G2). Subsequently, cells were incubated in 50 µl of staining medium (PBS with 0.05% sodium azide, 0.5% bovine serum albumin) for 15-20 minutes at 4 C with cell-surface and
intracellular antibodies. For some experiments, samples were then washed with staining medium and incubated for 15 minutes at 4 C with the appropriate secondary antibodies for detection of unconjugated primary antibodies. Stained cells were analyzed on a BD FACSCalibur, LSR I or LSR II flow cytometer (Becton-Dickinson, San Jose, CA). Data was analyzed with FlowJo software v8.6 (Tree Star Inc., San Carlos, CA, USA), Microsoft Excel v2004 (Seattle, WA, USA), and Perl v5.8 (The Perl Foundation, Ann Arbor, MI, USA). Graphics for color maps were generated with Perl scripts developed in house and displayed with Mathematica v6.0 (Wolfram Research, Champaign, IL, USA). Antibodies and reagents The following phosphorylation-specific Abs from BD Pharmingen (San Jose, CA, USA) were used: Mouse IgG 1, κ Isotype Control (clone MOPC-21), pan-pp38 MAPK (T180/Y182, clone 30 or 36), perk1/2 (pt202/py204, clone 20A), pstat1 (Y701, clone 14 or 4a), pstat3 (Y705, clone 4), pstat3 (S727, clone 49), pstat4 (Y693, clone 38), and pstat5a (Y694, clone 47). Pan-pSAPK/JNK (T183/Y185, clone G9) was obtained from Cell Signaling Tech (Danvers, MA, USA). These antibodies were conjugated to Alexa- 647 dye. The following unconjugated phosphospecific Abs from Cell Signaling were used: praf (S259), pmek1/2 (S217/S221, polyclonal), ps6 Ribosomal Protein (S235/S236, polyclonal), pakt (T308, clone 244F9), pakt (S73, clone 193H12), and cleaved caspase- 3 (D175, clone 5A1). These antibodies were detected with goat anti-rabbit IgG (H+L) Alexa-647 from Invitrogen (Carlsbad, CA, USA). Anti-phosphatidylinositol 4,5-
diphosphate (clone 2C11) and polyclonal goat anti-mouse IgM Alexa-647 were obtained from Invitrogen. Recombinant murine cytokines including IL-2, IL-7, IL-12, and IL-15 were purchased from PeproTech (Rocky Hill, NJ, USA). Recombinant interferon γ was purchased from BD Biosciences. Cucurbitacin I and cucurbitacin E were purchased from Tocris (Ellisville, MO). SL327 was obtained from Sigma-Aldrich (St. Louis, MO). In vitro stimulation of T cells with IL-2, IL-7, IL-12 and IL-15 Spleens from C57BL/6J (H-2 b ) mice between the ages of 6-8 weeks were removed aseptically. T cells were purified by CD5+ MACS as described above for CFSE-labeling, and resuspended in media (RPMI 1640 supplemented with 1 percent penicillin/streptomycin, L-glutamine, and 10% fetal calf serum) at 10 7 cells/ml. IL-2 (50 U/mL), IL-7 (50 ng/ml), IL-12 (5 ng/ml), and IL-15 (50 ng/ml) or PBS were added, and the samples were incubated for 1 hour at 37ºC. In vitro stimulation of splenocytes with CD3-coated beads Hamster anti-cd3ε (145-2C11) was generated by our monoclonal core facility. Latexsulfate beads were purchased from Invitrogen (Carlsbad, CA, USA). Beads were washed in PBS, and incubated with anti-hamster antibody (Jackson Immunoresearch) for 30 minutes at room temperature, washed, and incubated with anti-cd3e for 30 minutes in PBS and washed before use.
Calculations and generation of color maps All flow cytometric data was visually inspected as univariate or bivariate plots as appropriate. To generate color-maps, median fluorescence intensities were used to summarize the absolute amount of signal in a given population. Fluoresence-minus-one (FMO) isotype-control stained samples with identical gating for populations of interest were used as a reference for background signal. The amount of signal above background was calculated as 100% x [MFI population MFI Isotype ] / MFI Isotype, and the mean of multiple replicates obtained. The log 10 of the ratio of percentages of signals above background for two populations of interest to be compared were then scaled by a constant factor, and the resulting number between [-1, 1] was converted to red (positive numbers) or blue (negative numbers) colors by mapping to blue-black-red colors and manually edited for display. Univarite population comparison metric In Figure 1A, we used the T(X) statistical measure for assessing univariate population distribution differences as developed by Mario Roederer 9. This measure is implanted as a part of the FlowJo analysis software. T(X) is a statistic which not only provides an indication of the probability with which two distributions are different, but simultaneously provides a metric by which multiple distributions can be ranked. A value of T(X) > 4 implies a p<0.01 that the two populations being measured would be identical (i.e. 99 percent confidence that the two populations are in fact different).