Figure 1.
Activity of Ox40-cre in regulatory T cells.
A. Thymic and lymph node expression of YFP in Ox40-cre/YFP mice (i.e., Ox40-cre mice carrying a ROSA26-loxP-STOP-loxP-YFP allele). The contour plots show that YFP expression is predominantly found on CD4+CD8− cells in both tissues (as determined by flow cytometry). FoxP3+ cells are enriched 30–40 fold in the YFP+CD4+CD8−population compared to YFP− cells. B. Penetrance of Ox40-cre recombination in CD4+CD8−CD25+ cells in the lymph nodes and thymus. The histograms show the frequency of YFP expression in regulatory phenotype cells in both tissues of Ox40-cre/YFP mice. C. Developmental stage-specific activity of Ox40-cre in the thymus. The plots show YFP expression is predominantly found in cells that are CD4+CD8− and is barely detectable at the CD4+CD8+ stage.
Figure 2.
Ox40-cre-mediated inactivation of p56Lck in T cells.
A. Selective inactivation of p56Lck in CD4+ T cells in lymph nodes of Ox40-cre mice carrying a conditional null allele of the Lck gene. The histograms show intracellular p56Lck detected with the 1F6 monoclonal antibody in B cells, CD4+ and CD8+ T cells in mice of the indicated genotypes. B. Intracellular p56Lck (as in A.) in T cells of the indicated phenotypes (regulatory, memory and naïve) from Lck/Fyn mice. C. Increased frequency of Treg cells that had not undergone Ox40-cre-mediated recombination in mice carrying the conditional null Lck allele compared to mice carrying the ROSA26-YFP allele. The graph shows the frequencies of YFP− or p56Lck-positive (1F6+) CD25+ Treg cells in individual mice of the indicated genotypes.
Figure 3.
p56Lck-deficient regulatory T cells.
A. Flow cytometry data showing frequencies of Treg cells in mice of the indicated genotypes in the thymus, spleen and lymph nodes. B. Frequencies of thymic and peripheral Treg cells in individual mice of the indicated genotypes (bottom panel). The top panel shows total cell recovery from each of the tissues in the same mice. Asterisks identify differences (compared to control) that were statistically significant by ANOVA (p<0.05).
Figure 4.
Retention of FoxP3 but abrogation of in vitro suppression in Lck-mutant regulatory cells.
A. Real-time PCR analysis of FoxP3 expression in CD25+CD4+ T cells from mice of the indicated genotypes. Expression values were calculated relative to HPRT as an internal standard using the ΔΔCt method [76], and with normalization to the expression value obtained for the CD25− sample from control mice. B. The graphs show 3H-Thymidine uptake in cultures comprised of CD25− and CD25+ T cells at the stated ratios from mice of the indicated genotypes. Lymph node cells from mice of the indicated genotypes were flow-sorted then stimulated with soluble anti-CD3 and anti-CD28 in the presence of irradiated wild-type splenic accessory cells.
Figure 5.
Phenotype of p56Lck-deficient T cells.
Flow cytometry data showing expression of the indicated molecules on CD25+ or CD25− lymph node CD4+ T cells from Lck/Fyn mice. Cells were permeabilized and stained with the 1F6 antibody to discriminate cells that had undergone Ox40-cre-mediated inactivation of the Lck gene from those that had not (open versus solid curve respectively).
Figure 6.
Gene expression in Lck-mutant regulatory T cells.
A. The graphs show normalized log2 ratios of hybridization signals (Lck/Control) versus average hybridization intensity for all spots on the microarrays. Ratios corresponding to greater than 1.5-fold changes in spot intensity (dotted lines) are colored black instead of grey, and the numbers of these are indicated at the top and bottom right of the graphs. RNA was purified from flow-sorted Treg cells (CD4+CD25+) labeled differentially according to its origin (Lck mutant vs. control) and hybridized to microarrays spotted with 70-mer oligos from the MEEBO collection as described in Methods and Materials. B. Bivariate plot of Lck/Control spot ratios in memory versus regulatory T cells. Numbers of spots with vectors greater than 1.5 (colored black instead of grey) in the indicated parts of the four quadrants are shown. C. Venn diagram showing overlap between genes that are differentially expressed in control versus Lck memory and/or Treg cells and the Treg signature gene set identified by Hill et al. [40].
Figure 7.
Phenotype and localization of Lck-mutant T cells.
A. Flow cytometry validation of selected gene expression differences identified by microarray analysis. The overlayed histograms show expression of the indicated molecules in CD25+ (top) or CD25− (bottom) T cells that retain (filled curve) or lack (open curve) expression of functional p56Lck taken from the lymph nodes of Lck mice. B. Expression of Ly-6C and CD103 on cells from mice of the indicated genotypes (top panel) and in cells that retain or lack expression of p56Lck from Lck/Fyn mice (bottom panel). C. Frequencies of regulatory (CD25+) or memory (CD25−CD44hi) phenotype cells in the indicated locations in Lck, Lck/Fyn or Ox40-cre/YFP mice. Horizontal bars represent means of the combined Lck and Lck/Fyn data for each location. D. Frequencies of cells with recombined alleles resulting in inactivation of p56Lck or acquisition of YFP in the same mice and populations studied in C.