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. 2002 Sep 3;99(18):11796-801.
doi: 10.1073/pnas.092284399. Epub 2002 Aug 23.

Genomic expression programs and the integration of the CD28 costimulatory signal in T cell activation

Maximilian Diehn  1 , Ash A Alizadeh, Oliver J Rando, Chih Long Liu, Kryn Stankunas, David Botstein, Gerald R Crabtree, Patrick O Brown
Affiliations

Genomic expression programs and the integration of the CD28 costimulatory signal in T cell activation

Maximilian Diehn et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A 2002 Nov 12;99(23):15245

Abstract

Optimal activation of T cells requires effective occupancy of both the antigen-specific T cell receptor and a second coreceptor such as CD28. We used cDNA microarrays to characterize the genomic expression program in human peripheral T cells responding to stimulation of these receptors. We found that CD28 agonists alone elicited few, but reproducible, changes in gene expression, whereas CD3 agonists elicited a multifaceted temporally choreographed gene expression program. The principal effect of simultaneous engagement of CD28 was to increase the amplitude of the CD3 transcriptional response. The induced genes whose expression was most enhanced by costimulation were significantly enriched for known targets of nuclear factor of activated T cells (NFAT) transcription factors. This enhancement was nearly abolished by blocking the nuclear translocation of NFATc by using the calcineurin inhibitor FK506. CD28 signaling promoted phosphorylation, and thus inactivation, of the NFAT nuclear export kinase glycogen synthase kinase-3 (GSK3), coincident with enhanced dephosphorylation of NFATc proteins. These results provide a detailed picture of the transcriptional program of T cell activation and suggest that enhancement of transcriptional activation by NFAT, through inhibition of its nuclear export, plays a key role in mediating the CD28 costimulatory signal.

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Figures

Figure 1
Figure 1
Gene expression responses of T cells to diverse stimuli mimicking antigen receptor stimulation. Peripheral blood T cells were subjected to six distinct treatments in parallel time series and harvested at seven time intervals (0, 1, 2, 6, 12, 24, and 48 h). Treatments included: no stimulus (mock treated); stimulation with beads coated with αCD3, αCD28, or both; or treatment with 5 μg/ml of phytohemagglutinin or ionomycin [1 μM]/PMA [25 ng/ml]. Array elements that were induced or repressed more than 3-fold compared with baseline on at least two microarrays were included (4,359 cDNA elements representing 2,926 genes). The data are displayed as a self-organizing map that temporally orders the matrix of gene expression data where rows represent genes (unique cDNA elements), and columns represent experimental samples. Colored pixels capture the magnitude of the response for any gene. Shades of red and green represent induction and repression, respectively, relative to the prestimulation specimen (t = 0). Black pixels reflect no change from baseline and gray pixels represent missing data. Supplemental data and enhanced versions of the figures, including searchable clusters and raw microarray data, can be found at our web site (http://genome-www.stanford.edu/costimulation).
Figure 2
Figure 2
Gene expression responses to CD28 ligands. (A) Genes with detectable responses to monostimulation by CD28 ligands. Geometric means are shown for genes represented by multiple cDNA elements (indicated by asterisks and number of averaged elements). (B) Aggregate enhancement of gene expression responses to αCD3 stimulation by costimulation with αCD28. The abscissa represents the difference in the response between monostimulation with αCD3 and costimulation with αCD3/αCD28 coated beads using a customized distance metric. Representative genes from both ends of the distribution are shown, exemplifying genes whose responses to αCD3 were enhanced or diminished by αCD28. Genes from the bottom 5 and top 10 percentiles of the distribution are depicted in C (348 elements), and D (538 elements), respectively. (E) Example genes from c, plotted as line graphs. Depicted data points represent the geometric mean of independent cDNA elements, with error bars indicating the corresponding standard error. (F) Comparison of IL-2 mRNA and protein levels.
Figure 3
Figure 3
FK506 inhibition of gene expression enhancement by CD28. (A) Global overview of T cells stimulated with either αCD3 beads, αCD3/αCD28 costimulatory beads alone, or the latter in combination with FK506. T cells were isolated and stimulated as described in Fig. 1. Cells treated with FK506 were pretreated with 24 nM FK506 for 60 min before addition of costimulatory beads. (B) Distribution of the magnitudes of expression changes. The plot shows the fraction of genes passing a given minimum fold change at two or more time points in the different stimulations. (C) FK506 inhibits aggregate enhancement of transcription by CD28. The customized distance metric from Fig. 2B was used to determine CD28 enhancement of CD3-dependent transcription in the absence or presence of FK506.
Figure 4
Figure 4
Enhancement of NFAT activation by CD28 engagement and GSK3 inactivation. (A) Simultaneous engagement of CD28 enhances CD3-dependent dephosphorylation of NFATc2. The phosphorylation state of the NFATc2 bands is as indicated. αHsp90 antibody was used as a loading control. (B) Simultaneous engagement of CD28 enhances CD3-dependent phosphorylation of GSK3. Cells were treated as above. Westerns were probed with antibody to GSK3β as well as an antibody specific for the serine 9 phosphorylated form of GSK3β. Some lots of phospho-GSK3 antibody detect phosphorylated GSK3α as well, and similar results were seen for both isoforms (C and Fig. 12). (C) CD28 engagement alone promotes GSK3 phosphorylation. Cells were isolated as above, and treated for the indicated times with αCD28 beads after pretreatment with the indicated drugs for 15 min. CD28, no inhibitor; LY, 10 μM LY294002; PD, 50 μM PD98059. Westerns were probed with antibody specific for phosphorylated GSK3α. HSP90 was used as a loading control. (D) GSK3 inhibition by lithium enhances proliferation of T cells treated with αCD3 beads. Proliferation assays were preformed as described above. Mock, no stimulus; I+P, 1 μM ionomycin and 25 ng/ml PMA; CD3, αCD3 coated beads with or without 10 mM lithium; CD3/CD28, αCD3/αCD28 costimulation beads with or without 24 nM FK506.
Figure 5
Figure 5
A model of T cell costimulation. Illustrated are the dominant signaling pathways that likely characterize and distinguish TCR stimulation in the absence (A) or presence (B) of CD28-mediated signals. The thickness of each arrow reflects the relative strength of the transduced signal.
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