Download Supplemental Information Supplementary Materials and Methods

Supplemental Information
Supplementary Materials and Methods
Antibodies. Purified, biotinylated and directly conjugated antibodies were obtained from BD
Biosciences (anti-B220, CD3, CD4, CD8, CD19, CD24, CD43, IgM, Bcl-2, Ki67), eBioscience
(anti-IL-7R) or Southern Biotech (anti-Bcl-xL). Anti-Bim was purified from supernatants of
Ham151 cells (Oliver et al., 2004) and conjugated to Alexa647 (Invitrogen). Unlabeled
antibodies were obtained from Southern Biotech (anti-Bcl-xL), Cell Signaling Technology (antic-myc) or Abcam (anti-Mcl-1). Fluorescent secondary antibodies used in the Odyssey Infrared
Imaging System (LiCOR) were obtained from Rockland Immunochemicals or Invitrogen.
Flow cytometry. Bone marrow progenitor B cells were characterized based on expression of
surface markers B220, CD43, CD24 and IgM (Hardy et al., 1991): B220+CD43+IgM-CD24–
(pre-pro-B), B220+CD43+IgM-CD24+ (pro-B), B220midCD43-IgM- (pre-B), and B220hiCD43IgM+ (mature B). For analysis of Ki67, cells were stained for IgM and B220, fixed for 10 min in
1% paraformaldehyde at room temperature, permeabilized in 100% ice-cold methanol and
stained for intracellular Ki67. Bcl-2 and Bim expression was measured as previously described
(Osborne et al., 2007). For Bcl-xL and Mcl-1 detection, cells were stained with antibodies to
surface markers, treated with eBioscience Fixation & Permeabilization buffers and antibodies
against Bcl-xL or Mcl-1. Mcl-1 was visualized with donkey anti-rabbit IgG (H+L) PE from
Jackson ImmunoResearch. MFI (mean fluorescence intensity) was calculated by subtracting the
MFI value of the relevant isotype control from the raw MFI value for each sample. Briefly, BM
was isolated, depleted of Lineage positive cells using biotinylated antibodies against Gr-1,
NK1.1, CD3, CD8, Ter119 and IgM and immunomagnetic negative selection (StemCell
Technologies) (similar to protocol published by Li et al., 2010). The recovered cells were labeled
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with streptavidin-FITC, rested for 30 minutes at 37oC, then stimulated with 25 ng/ml IL-7 and
processed as previously described (Osborne et al., 2007) with concomitant anti-pSTAT5 and
anti-B220 labeling. Samples were collected on an LSRII (BD Biosciences), and data analyzed
with FlowJo software (Tree Star).
Apoptosis induction. Splenic lymphocytes were plated in IMDM supplemented with 10% FBS
at a density of 105 cells/ml and treated with 10-7 M dexamethasone (Sigma-Aldrich).
Alternatively, splenocytes were cytokine stripped (Osborne et al., 2007), plated in IMDM +
0.5% BSA with or without hydrogen peroxide. Cells were labeled with antibodies to CD3, CD4,
CD8 and B220 and stained for AnnexinV (eBioscience) and propidium idodide (PI) or 7AAD
according to manufacturer’s instructions and analyzed by flow cytometry.
Western blotting. Samples were lysed in modified RIPA lysis buffer (30 mM Tris-HCl, pH 7.4,
150 mM NaCl, 2 mM EDTA, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS) and processed
as previously described (Osborne et al., 2007). Quantification is based on integrated pixel
intensity from the LiCor Odyssey imager where loading was normalized to total Coomassie stain
of the SDS-PAGE gel (Aldridge et al., 2008; Duthie et al., 2007).
Quantitative RT-PCR. Quantitative PCR of IL-7 transcript levels was performed as previously
described (Abraham et al., 2005) using a BioRad CFX96 Real-Time PCR system.
Tumor cell in vivo transfer. Murine RMA (a Rauscher virus induced T-cell lymphoma line
from C57BL/6 (Karre et al., 1986), kindly provided by Dr. Hung-Sia Teh, UBC) and EL4
(C57BL/6 chemically induced lymphoma, ATCC) cells were grown in RPMI supplemented with
10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate and 50 µM -mercaptoethanol. Cells
were washed in PBS and 105 RMA cells or PBS were injected sub-cutaneously into the right
flank of mice.
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Supplemental Material – References
Abraham N, Ma MC, Snow JW, Miners MJ, Herndier BG, Goldsmith MA. (2005).
Haploinsufficiency identifies STAT5 as a modifier of IL-7-induced lymphomas. Oncogene 24:
5252-5257.
Aldridge GM, Podrebarac DM, Greenough WT, Weiler IJ. (2008). The use of total protein stains
as loading controls: An alternative to high-abundance single protein controls in semi-quantitative
immunoblotting. J Neurosci Methods 172: 250-254.
Duthie KA, Osborne LC, Foster LJ, Abraham N. (2007). Proteomic analysis of IL-7 induced
signaling effectors show selective changes in IL-7Ralpha 449F knock-in T cell progenitors. Mol
Cell Proteomics 6: 1700-1710.
Hardy RR, Carmack CE, Shinton SA, Kemp JD, Hayakawa K. (1991). Resolution and
characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J Exp Med
173: 1213-1225.
Karre K, Ljunggren HG, Piontek G, Kiessling R. (1986). Selective rejection of H-2-deficient
lymphoma variants suggests alternative immune defence strategy. Nature 319: 675-678.
Li LX, Goetz CA, Katerndahl CDS, Sakaguchi N, Farrar MA. (2010). A Flt3-and ras-dependent
pathway primes B cell development by inducing a state of IL-7 responsiveness. The Journal of
Immunology 184: 1728-1736.
Oliver PM, Wang M, Zhu Y, White J, Kappler J, Marrack P. (2004). Loss of bim allows
precursor B cell survival but not precursor B cell differentiation in the absence of interleukin 7. J
Exp Med 200: 1179-1187.
Osborne LC, Dhanji S, Snow JW, Priatel JJ, Ma MC, Miners MJ et al. (2007). Impaired CD8 T
cell memory and CD4 T cell primary responses in IL-7R alpha mutant mice. J Exp Med 204:
619-631.
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Supplementary Table 1. Phenotypes of Tg IL-7; IL-7R+/+ tumors. Thymomas and
peripheral lymphoid tumors were phenotyped by flow cytometry using antibodies against CD3,
CD4, CD8 and B220 or CD19. Tumors were designated to be of a certain phenotype when the
population was greater than 60% of the total lymphocyte population.
Genotype
Tg IL-7;
IL-7R+/+
Mouse
ID
3141
3168
3170
Tumor phenotype
Cell type
Affected
Organ
pLNs
Thymus
pLNs
Rate of
occurrence
3/7 (43%)
CD4+ CD8+
DP
2796
3138
CD8+
CD8 SP
Thymus
Spleen
2/7 (29%)
2828
CD8+ and B220+
Mixed CD8 Thymus
SP and B
1/7 (14%)
2797
B220+
B cell
1/7 (14%)
Axial LN
Supplementary Table 2. Phenotypes of Eµ Myc; IL-7R+/+ and Eµ Myc; IL-7R449F
tumors. Lymphoma samples from Eµ Myc; IL-7R+/+ and Eµ Myc; IL-7R449F animals were
phenotyped by flow cytometry using a combination of B220 or CD19, CD43, IgM and CD24.
Tumors were designated to be of a certain phenotype when the population was greater than 60%
of the total lymphocyte population.
Genotype
Eµ Myc;
IL-7R+/+
Eµ Myc;
IL-7R449F
Mouse Tumor phenotype
ID
521
B220+CD43+CD24+
and
B220+CD43-CD24+
Cell type
Mixed
Pro/Pre
Rate of
occurrence
1/8 (12%)
620
930
B220+CD43-CD24+
Pre
2/8 (25%)
506
507
829
968
972
B220+CD43-IgM+
Mature
5/8 (63%)
634
B220+CD43+CD24+
Pro
1/6 (17%)
633
1201
B220+CD43-CD24+
Pre
2/6 (33%)
620
1202
B220+CD43-IgM+
Mature
2/6 (33%)
678
CD4+CD19mid
Aberrant
1/6 (17%)
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Supplemental Figure 1: IL-7R Y449 is required for Tg IL-7 mediated lymphomagenesis.
(a) H & E staining of spleens from age-matched wt, Tg IL-7; IL-7R+/+, IL-7R449F, and Tg IL7; IL-7R449F mice. Representative samples are shown: wt (n=4), Tg IL-7; IL-7R+/+ (n=6), IL7R449F (n=4), Tg IL-7; IL-7R449F (n=6). Original magnification x10. WP (white pulp), RP (red
pulp). (b) Quantitative RT-PCR of absolute levels of cDNA generated from Tg IL-7; IL-7R+/+
(n = 3) and Tg IL-7; IL-7R449F (n = 5) thymi shows that there is no significant difference in the
expression of the IL-7 transgene between the two strains (P=0.66).
Supplemental Figure 2: Increased Bcl-2 and Bcl-xL expression in T lymphocytes as a result
of chronic IL-7 exposure is IL-7R Y449-dependent. (a) Intracellular flow cytometry
histograms showing expression of anti-apoptotic proteins Bcl-2 in DP (top panels) and CD8 SP
(bottom panels) thymocytes of age-matched wt, IL-7R449F, Tg IL-7; IL-7R+/+ (pre-disease)
and Tg IL-7; IL-7R449F splenic CD8 SP T cells. Data is representative of 4 independent
experiments, n=3 for each genotype per experiment. Grey histogram, isotype control; blue
histogram, anti-Bcl-2 fluorescence. Average MFI values are indicated in the top corners, dashed
vertical line represents the MFI for wt samples as a reference point. (b) Quantification of
intracellular levels of Bcl-2, Bcl-xL, Mcl-1 and Bim in splenic CD8 SP T cells of wt, IL-7R449F,
pre-disease Tg IL-7; IL-7R+/+ and Tg IL-7; IL-7R449F counterparts, n=3 or 4 for each
genotype. MFI of the isotype control was subtracted from the MFI of each sample to correct for
non-specific antibody binding. *, P<0.05, represents that Tg IL-7; IL-7R+/+ samples are
statistically different from all other genotypes. NS represents that Tg IL-7; IL-7R+/+ samples
are not statistically different from other genotypes. (c) Expression of Bcl-xL and Mcl-1 in Tg IL7; IL-7R+/+ thymomas and control thymi. Equal amounts of whole thymus lysate from healthy
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controls and Tg IL-7; IL-7R+/+ thymomas were subjected to SDS-PAGE and immunoblots
probed for expression of Bcl-xL and Mcl-1 using the Odyssey infra red imager (LiCor).
Representative results are shown; numbers below the blots indicate quantification of Bcl-xL and
Mcl-1 band intensity (in pixels/mm2, normalized to total protein loaded) from 3 mice of each
genotype. The protein gel was Coomassie stained and intensity (in pixels/mm2) of total protein in
each lane was quantified as loading control. (d) Splenic lymphocytes of age-matched, pre-disease
wt, IL-7R449F, Tg IL-7; IL-7R+/+ and Tg IL-7; IL-7R449F mice were cytokine stripped and
plated in IMDM + 0.5% BSA for 24 hours with the indicated concentrations of hydrogen
peroxide or vehicle control (IMDM). Viable (AnnexinV-7AAD-) CD8 SP T cells were quantified
after treatment. Comparison of untreated samples (black bars) allows comparison of cell viability
after cytokine withdrawal, *, P<0.05. Data is representative of 2 independent experiments with
2-3 mice of each genotype.
Supplemental Figure 3
(a) Coomassie stained protein gel was used to normalize for protein loading of samples in Figure
3B. Intensity (in pixels/mm2) of total protein in each lane was quantified as loading control.
Supplemental Figure 4: Mature B cells from wt and IL-7R449F mice lack surface
expression of IL-7R. Splenic B220+ CD43- B cells of age-matched wt and IL-7R449F mice
were analyzed for expression of IL-7R. Filled histogram, isotype control. Black trace, anti-IL7R fluorescence.
Supplemental Figure 5: IL-7R Y449 is essential for IL-7 mediated activation of STAT5 in
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bone marrow progenitor B cells. Cytometry plots of representative data showing
phosphorylation of STAT5 in Lineage- B220+ BM B cells in response to IL-7 in mice bearing a
wt copy of the IL-7R, but not in IL-7R449F or Eµ-myc; IL-7R449F mice.
Supplemental Figure 6: PI3 kinase p110 activity is non-essential for Tg IL-7 mediated
lymphomagenesis. PI3 kinase p110D910A mice, which express a catalytically inactive version of
the p110 subunit, were crossed to Tg IL-7 mice to generate Tg IL-7; p110+/+, Tg IL-7;
p110+/D910A, Tg IL-7; p110D910A animals. Survival analysis of these mice showed that tumor
progression and morbidity were unaffected by loss of signals initiated by PI3 kinase p110.
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