Impairment of organ-specific T cell negative selection by diabetes susceptibility genes: genomic analysis by mRNA profiling

Abstract

Background: T cells in the thymus undergo opposing positive and negative selection processes so that the only T cells entering circulation are those bearing a T cell receptor (TCR) with a low affinity for self. The mechanism differentiating negative from positive selection is poorly understood, despite the fact that inherited defects in negative selection underlie organ-specific autoimmune disease in AIRE-deficient people and the non-obese diabetic (NOD) mouse strain

Results: Here we use homogeneous populations of T cells undergoing either positive or negative selection in vivo together with genome-wide transcription profiling on microarrays to identify the gene expression differences underlying negative selection to an Aire-dependent organ-specific antigen, including the upregulation of a genomic cluster in the cytogenetic band 2F. Analysis of defective negative selection in the autoimmune-prone NOD strain demonstrates a global impairment in the induction of the negative selection response gene set, but little difference in positive selection response genes. Combining expression differences with genetic linkage data, we identify differentially expressed candidate genes, including Bim, Bnip3, Smox, Pdrg1, Id1, Pdcd1, Ly6c, Pdia3, Trim30 and Trim12.

Conclusion: The data provide a molecular map of the negative selection response in vivo and, by analysis of deviations from this pathway in the autoimmune susceptible NOD strain, suggest that susceptibility arises from small expression differences in genes acting at multiple points in the pathway between the TCR and cell death.

Figure 1

Global gene expression differences induced upon positive or negative selection on the B10^k and NOD^k genetic backgrounds. The global difference in gene expression between conditions was calculated as a Euclidean distance, taking into account the number of genes with differential expression, and the scale of differential expression, for each replicate chip. Euclidean distances are represented as the distance between conditions for both the B10^k and NOD^k genetic backgrounds, with the average positions of groups located at the apexes of the triangles. Dotted lines correspond to the Euclidean distance between B10^k and NOD^k genetic backgrounds, for the same population.

Figure 2

Patterns of gene expression changes induced by positive and negative selection in the B10^k and NOD^k strains. Analysis of Affymetrix GeneChip data segregated those probesets that were significantly changed upon positive and/or negative selection (p < 0.005) into patterns A to F based on logical sets of significant contrasts (p < 0.05), where patterns are defined by the relative expression during pre-selection (PreS, left), positive selection (S+, middle) and negative selection (S-, right). The number of probesets falling into each pattern in B10^k and NOD^k mice is listed on the expression pattern. Assignment of probesets to patterns was based only on direction of change, thus the graphical depictions do not represent the degree of change.

Figure 3

Gene expression changes localized to cytogenetic band 2F. Cytogenetic band 2F was analyzed for the relative expression values of Affymetrix annotated genes. The log₂ ratio of each gene (diamonds), plotted by genomic position, is displayed for: (a) B10^k PreS compared to B10^k S+; (b) B10^k S- compared to B10^k S+; and (c) B10^k S- compared to NOD^k S-. Comparisons of gene expression changes under these conditions were made by plotting the log₂ ratios for all genes within cytogenetic band 2F against each other (diamonds) and calculating the r² value. The distance from the origin thus reflects the degree of expression change. (d) Ratio of gene expression in B10^k PreS compared to B10^k S+ (y-axis) versus the ratio of expression in B10^k S- compared to B10^k S+ (x-axis). (e) Ratio of gene expression in B10^k S- compared to NOD^k S- (y-axis) versus B10^k S- compared to B10^k S+ (x-axis). In each case the probeset measuring Bim expression is indicated with a red diamond. All probesets with a relative change greater than +1 or less than -1 are shaded grey and annotated.

Figure 4

Graphical representation of expression changes between the B10^k and NOD^k strains. Probesets falling in specific B10^k clusters were assessed for the ratio of expression during negative selection in NOD^k:B10^k mice. A value of 1 represents equal expression during negative selection, <1 represents lower expression in NOD^k thymocytes than B10^k thymocytes during negative selection, and >1 represents higher expression in NOD^k thymocytes than B10^k thymocytes during negative selection. (a) Probeset patterns from the B10^k analysis increased (D, E, L) or decreased (J, K, F) specifically during negative selection trended towards showing that the same probeset in NOD^k thymocytes had a lower/higher expression, respectively. (b) The probeset pattern from the B10^k analysis increased during maturation (A) showed roughly the same level of expression during negative selection in NOD^k thymocytes (divided into random groups for clarity).

Figure 5

Regulation of surface CD3 levels on 3A9 TCR transgenic thymocytes on the B10^k and NOD^k genetic backgrounds. 3A9 TCR transgenic mice on the B10^k and NOD^k backgrounds were assessed for CD3 surface levels by flow cytometry, at both the DP and SP 1G12⁺ developmental stages. (a) Representative histograms for CD3 expression on DP cells and SP 1G12⁺ cells. B10^k mice are shown in grey, NOD^k mice in white. (b) The mean fluorescence intensity (normalized to B10^k SP 1G12⁺ thymocytes) and standard error is shown with B10^k in black and NOD^k in white (n = 7 for B10 values, 17 for NOD values). Significance of differences between B10^k and NOD^k groups of the same genotype are indicated by t-test p values about the group. t-tests comparing SP 1G12⁺ thymocyte expression were tested using a two sided t-test of the hypothesis that NOD^k expression levels are different to '100'.

Figure 6

Grouping of key gene changes between NOD^k and B10^k thymocytes. Analysis of Affymetrix GeneChip data segregated those probesets that were significantly modified in a 2 × 2 × 2 F-test (p < 0.005) into groups 1 to 6 based on logical sets of significant contrasts (p < 0.05). The diagrams show relative changes for the mean gene expression in the pre-selection (PreS), positive selection (S+) and negative selection (S-) conditions. The number of probesets falling into each group is listed on the group pattern. B10^k expression changes are shown in red, NOD^k expression changes are shown in blue.

Figure 7

Expression of candidate genes for the D7mit101-linked defective thymic deletion allele. Individual values of biological replicates from the Affymetrix 430A microarray chips are indicated in the biological groups of 'pre-selection' (PreS), 'positive selection' (S+) and 'negative selection' (S-). Values are represented in black (B10^k) and white (NOD^k) boxes.

Figure 8

Expression of candidate genes for D15mit229-linked defective thymic deletion. Individual values of biological replicates from the Affymetrix 430A microarray chips are indicated in the biological groups of 'pre-selection' (PreS), 'positive selection' (S+) and 'negative selection' (S-). Values are represented in black (B10^k) and white (NOD^k) boxes.

Figure 9

Expression of candidate genes for D2mit490-linked defective thymic deletion. Individual values of biological replicates from the Affymetrix 430A microarray chips are indicated in the biological groups of 'pre-selection' (PreS), 'positive selection' (S+) and 'negative selection' (S-). Values are represented in black (B10^k) and white (NOD^k) boxes.

Figure 10

Expression of candidates for the defective thymic deletion allele linked to D1mit181. Individual values of biological replicates from the Affymetrix 430A microarray chips are indicated in the biological groups of 'pre-selection' (PreS), 'positive selection' (S+) and 'negative selection' (S-). Values are represented in black (B10^k) and white (NOD^k) boxes.