Article Figures & Data
Figures
- FIGURE 1.
LCK-B-myb transgene construct. A 2.4-kb (▦) human B-myb cDNA was inserted at the BamHI site downstream of the 3.2-kb LCK proximal promoter (□). Introns, exons, and polyadenylation sites (▪) of the human growth hormone gene constitute the 3′-untranslated region.
- FIGURE 2.
B-myb expression in transgenic LCK-B-myb mice. A, Human B-myb mRNA expression was analyzed (Northern blot) in the indicated organs from wt and the indicated lines of LCK-B-myb tg mice. GAPDH mRNA was measured to control for RNA loading. B, RT-PCR analysis of human (top) and murine (bottom) B-myb mRNA from thymus and spleen of wt and LCK-B-myb tg mice. β-Actin transcripts were amplified for normalization. Oligonucleotide primers are described in Materials and Methods. C, RT-PCR analysis of human B-myb mRNA from the indicated cell types of wt and LCK-B-myb tg mice. T, B, and NK cells were isolated from the spleen. RT-PCR products were obtained using human B-myb-specific oligonucleotide primers and were hybridized to a 32P end-labeled human B-myb-specific oligo probe. β-Actin transcripts were amplified for normalization. D, Western blot analysis of total thymus and spleen extracts from non-tg wt and LCK-B-myb tg mice (5986). Analysis was performed on an equivalent number of cells using polyclonal rabbit anti-B-myb Ab (Lewis). A mouse anti-heat shock protein 90 (HSP90) mAb was used to control for protein loading.
- FIGURE 3.
Lymphocyte subsets in spleen and thymus. Lymphocyte subsets were analyzed in thymus (left) and spleen (right) from wt and tg, 5986, mice. CD4, CD8, and CD3 expressions were detected by three-color immunofluorescence as described in Materials and Methods, using PE-conjugated anti-CD4, FITC-conjugated anti-CD8, and CyChrome-conjugated anti-CD3. Lymphocytes were gated on the basis of light scatter characteristics. Correlate measurements of FITC (x-axis) and PE (y-axis) were performed on gated CyChrome anti-CD3+ cells and are displayed as density plots on 4-decade log10 scales. Based on negative controls with irrelevant Ab (not shown), the plots were divided into quadrants in which <0.5% control cells were included. The percentage of positive cells is reported for each quadrant. Top left, PE only positive cells; top right, double-positive cells; bottom right, FITC only positive cells. The bottom panels in each section are histograms of cells reacting with CyChrome anti-CD3 Ab in the total population. The percentage of positive cells is indicated. x-axis, Fluorescence intensity; y-axis, relative cell number. Results are from one experiment using one mouse and are representative of 5 wt and 20 tg mice analyzed.
- FIGURE 4.
B, T, NK, and NKT subsets in spleen. Lymphocyte subsets were analyzed in spleen from wt and tg, 5986, mice. B220, CD3, and Pan-NK expressions were detected by three-color immunofluorescence as described in Fig. 3, using PE-conjugated anti-B220, FITC-conjugated anti-Pan-NK, and CyChrome-conjugated anti-CD3. The bottom panels in each section indicate cells reacting with FITC anti-Pan-NK mAb within gated CD3+/B220− and CD3−/B220− lymphocytes. The percentage of positive cells within the gated population is indicated. x-axis, Fluorescence intensity; y-axis, relative cell number. Results are from one experiment using one mouse and are representative of five wt and 20 tg mice analyzed.
- FIGURE 5.
Viability of thymus and spleen cells from tg LCK-B-myb mice. Thymocytes and splenocytes from wt and LCK-B-myb tg mice were cultured in RPMI 1640 without the addition of growth factors or cytokines. The percentage of apoptotic cells was determined by DNA content analysis of PI-stained nuclei at 0, 6, 12, and 24 h. Bars and error bars are the mean ± SD of the results from three independent experiments.
- FIGURE 6.
FasL expression in tg LCK-B-myb mice. A, Western blot analysis of total thymus and spleen extracts from wt and tg LCK-B-myb mice. Analysis was performed on an equivalent number of cells using polyclonal rabbit anti-FasL Ab for detection. The 35/43-kDa band corresponds to full-length FasL; the 26-kDa band corresponds to truncated, soluble FasL (sFasL). Mouse anti-heat shock protein 90 (HSP90) mAb was used to control for protein loading. B, Semiquantitative RT-PCR analysis of FasL mRNA from thymus and spleen of wt and tg LCK-B-myb mice. RT-PCR products obtained using FasL-specific oligonucleotide primers were hybridized to a 32P-end-labeled FasL-specific oligo probe. The expected 522-bp RT-PCR product corresponding to FasL is indicated. The RT-PCR reaction was performed with 22 cycles; oligonucleotide primers and oligo probe are detailed in Materials and Methods. The results are from one experiment representative of three independent ones performed. β-Actin transcripts were amplified for normalization. C, Semiquantitative RT-PCR analysis of FasL mRNA from NK and T cells purified to homogeneity from spleen of wt and tg LCK-B-myb mice. RT-PCR analysis was performed as described in B.
- FIGURE 7.
IFN-γ production in transgenic LCK-B-myb mice. A, IFN-γ ELISPOT assay with splenic lymphocytes from wt and tg LCK-B-myb mice. Assays were performed 14 days after priming i.p. with influenza A PR/8/34 virus (107 PFU/mouse). Unprimed mice and uninfected L-Kd cells were used as controls. The number of spontaneous (left) and A PR/8/34-specific (right) IFN-γ-producing cells/106 splenocytes is shown. Bars and error bars are the mean ± SD of three independent experiments. B, Western blot analysis of total spleen extracts from wt and LCK-B-myb tg, 5986 and 6085, mice. Analysis was performed with an equivalent number of splenocytes using rat anti-IFN-γ mAb. The 20-kDa band corresponding to IFN-γ is indicated. Mouse anti-heat shock protein 90 (HSP90) mAb was used to control for protein loading.
- FIGURE 8.
T and NK cell-mediated cytotoxicity. A, Cytotoxicity assays were performed in complete medium (CM) with or without added 1 mM EGTA and 2 mM MgCl2 and Fas/Fc chimera or a control fusion protein (TAT-HA mutant BAD), as indicated, with cells from wt and tg mice. Jurkat cells (104/well) were used as targets in 6-h 51Cr release assays. Effector cells were splenic lymphocytes. B, Yac-1 cells (104/well) were used as targets in 4-h 51Cr release assays. Effector cells were splenic lymphocytes. C and D, Jurkat cells (2.5 × 103/well) were used as targets in 6-h 51Cr release assays. Effector cells in C were NK cells purified to homogeneity from spleen by cell sorting. Effector cells in D were T cells purified by panning (negative selection). Results are from one experiment using one mouse and are representative of three independent ones with each cell type. x-axis, E:T cell ratio; y-axis, percentage of specific 51Cr release.
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