Multiple differences in gene expression in regulatory Valpha 24Jalpha Q T cells from identical twins discordant for type I diabetes - PubMed (original) (raw)

Multiple differences in gene expression in regulatory Valpha 24Jalpha Q T cells from identical twins discordant for type I diabetes

S B Wilson et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Quantitative and qualitative defects in CD1d-restricted T cells have been demonstrated in human and murine autoimmune diseases. To investigate the transcriptional consequences of T cell receptor activation in human Valpha24JalphaQ T cell clones, DNA microarrays were used to quantitate changes in mRNA levels after anti-CD3 stimulation of clones derived from identical twins discordant for type 1 diabetes and IL-4 secretion. Activation resulted in significant modulation of 226 transcripts in the IL-4 secreting clone and 86 in the IL-4-null clone. Only 28 of these genes were in common. The differences observed suggest both ineffective differentiation of diabetic Valpha24JalphaQ T cells and a role for invariant T cells in the recruitment and activation of cells from the myeloid lineage.

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Figures

Figure 1

Figure 1

Discordant expression of PI3-kinase-regulated events differentiates IL-4+ from IL-4-null Vα24JαQ T cell clones. (A) Vα24JαQ T cell clones GW4 (nondiabetic) and ME10 (diabetic) were activated with plate-bound anti-CD3 or Ig control. Levels of secreted IL-4 and IFN-γ were assayed by ELISA. The concentrations of inhibitors used were 10 nM wortmannin (wort.); 10 μM LY294002 (LY); 50 μM PD98059 (PD), a mitogen-activated protein kinase kinase inhibitor; and 50 μM SB203580 (SB), a p38 kinase inhibitor. Data points were collected in triplicate, and the Fig. is representative of four independent experiments. The concentrations of phorbol ester and calcium ionophore used were 1 ng/ml PMA and 1 μg/ml ionomycin (Iono). Cyclosporin A (CsA) was used at 5 ng/ml. (B) Vα24JαQ T cell clones GW4 (IL-4+) and ME10 (IL-4-null), 1 × 106 cells each, were loaded with Indo-1 (10 μM) for 45 min, then stimulated with 10 μg/ml anti-CD3 (open arrowhead) and analyzed on a Cytomation MoFlo instrument. At the end of the experiment, ionomycin was added to a final concentration of 1 μg/ml (filled arrowhead) to determine maximal flux. The ratio of Indo-1 fluorescence 410/490 nm (410, Ca2+-bound; 490, Ca2+-free) after stimulation in a representative pair of clones is pictured. Control experiments showed no differences in calcium flux in response to thapsigargin treatment (data not shown).

Figure 2

Figure 2

The fraction of genes in Vα24JαQ T cell clones altered by anti-CD3 treatments. Graphical representation of genes differentially expressed in natural killer T cell clones derived from a diabetic/nondiabetic twin pair. Clones ME10 (blue; IL-4-null) and GW4 (red; IL-4+) were treated with control IgG (designated R for Resting) or anti-CD3 (designated A for Activated) for 4 h, after which RNA was isolated and analyzed on Genechips monitoring the expression of 6,800 human genes from the Unigene collection. Genes whose expression was modulated at least 2-fold in either clone were chosen for clustering analysis with the Self-Organizing Map algorithm (15). This method was used to cluster genes into six distinct groups, based on differential expression patterns between ME10 and GW4, independent of expression magnitude. The first group displays the six patterns represented when all genes meeting the 2-fold change criterion are used. The other groupings reveal the differential expression patterns of selected gene functional classes. Individual genes of each functional class falling into the different clusters are identified in Table 1.

Figure 3

Figure 3

Changes of expression on genes for cytokines and chemokines. Vα24JαQ T cell clone GW4 (IL-4+) is compared with ME10 (IL-4-null). For each individual transcript, the anti-CD3-induced hybridization intensity is shown. RNA was isolated, amplified, and hybridized to Genechips, displaying probes for 250 genes of immunological interest. This chip is custom designed for quantitative analysis by increasing the number of probes for the detection of each specific transcript. In the graph, the position of the black dot represents hybridization intensity for each gene before anti-CD3 stimulation, and a line is drawn to the position of hybridization intensity after stimulation. Genes that were called significantly different by a gene expression algorithm, and that changed by at least 2-fold, are indicated by a bold line. Upward- or downward-pointing arrowheads indicate increases or decreases in gene expression in the stimulated cells relative to the unstimulated cells. A, the transcript was not detected; TNF, tumor necrosis factor; LTN, lymphotactin; MIP, macrophage inflammatory protein; GM-CSF, granulocyte–macrophage colony-stimulating factor; M-CSF, macrophage colony-stimulating factor.

Figure 4

Figure 4

A model for identified transcripts whose discordant expression is in accord with observed cell phenotypes. Pictured in cartoon format are genes whose transcripts have clearly defined cellular roles and whose discordant expression between GW4 and ME10 correlated with the established phenotypic differences. The genes are related by either being downstream of PI3-kinase, particularly several genes that regulate cell survival, or are directly required for calcium flux and calcium-regulated gene transcription. The proteins shaded various hues of blue represent genes expressed at significantly greater levels in GW4 (IL-4+) when compared with ME10 (IL-4-null). Conversely, those colored red were overexpressed in ME10 relative to GW4. Average copies of mRNA molecules per million for genes that were significantly altered by anti-CD3 treatment for GW4 (resting, activated) and ME10 (resting, activated), respectively, were: Itk (49, 115) and (25, 19); GATA3 (13, 26) and (8, 11); Jun-B (, 118) and (14, 30); Jun-D (107, 291) and (68, 130); NFAT4 (33, 35) and (64, 26); STAT4 (36, 29) and (76, 36); 14–3-3 (, 45) and (83, 41); Bcl-XL (, 50) and (6, 7); and IAP (, 211) and (, 41). Selected genes whose expression was constitutive but discordantly expressed for GW4 vs. MW10 (GW4, ME10) were NKR-P1A (, 459) and STAT1 (, 79).

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