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Neonatal CD4(+) T cells express less CD154 protein and mRNA than adult CD4(+) T cells after activation by calcium ionophore and phorbol ester, but the mechanism for this reduced expression and its relevance to the primary immune response remain unclear. We compared expression of CD154 protein and mRNA and CD154 gene promoter activity by purified naive (CD45RA(high)CD45RO(low)) neonatal and adult CD4(+) T cells after activation by calcium ionophore (ionomycin) and phorbol myristate acetate (PMA) treatment or by engagement of alphabeta TCR-CD3 complex. Substantial and consistent reductions in expression by neonatal cells were found in all cases and were paralleled by decreased CD154-dependent activation of a B cell line. CD69 expression by neonatal CD4(+) T cells after alphabeta TCR-CD3 engagement was also reduced compared to adult cells, which suggested that limitations in activation-induced signaling by neonatal CD4(+) T cells occurred at a point upstream of where the signaling pathways leading to CD154 and CD69 expression diverge. Decreased CD154 expression by neonatal cells after alphabeta TCR-CD3 engagement was paralleled by a lower free intracellular calcium concentration, a key event for CD154 gene transcription. Reduced CD154 promoter activity by neonatal cells persisted when proximal signaling events were bypassed using ionomycin and PMA, suggesting an additional and more distal mechanism for decreased transcription. In contrast, CD154 mRNA stability was similar in neonatal and adult cells after either ionomycin and PMA stimulation or engagement of the alphabeta TCR-CD3 complex. We conclude that decreased CD154 production by neonatal CD4(+) T cells is due to limitations in both proximal and distal activation events, which together ultimately limit CD154 gene transcription.

CD154 (CD40-ligand) has a wide variety of pleiotropic effects throughout the immune system and is critical to both cellular and humoral immunity. Cell surface and soluble CD154 are primarily expressed by activated CD4 T cells. Expression of CD154 is tightly regulated in a time-dependent manner, and, like most T cell-derived cytokines and other members of the tumor necrosis factor (TNF) superfamily, CD154 is largely regulated at the level of gene transcription. Recently, dysregulated expression of CD154 has been noted in a number of autoimmune disorders, including systemic lupus erythematosus (SLE). In addition, abnormal expression of CD154 has been hypothesized to contribute to a wider array of diseases, from atherosclerosis to Alzheimer's disease. Until recently, very little was known about the transcriptional regulation of CD154. We are exploring CD154 regulation in primary human CD4 T cells in hopes of understanding the cis- and trans-regulatory elements that control its expression in the cells that normally express CD154. Ultimately, we hope to be able to correct abnormal expression of CD154 in various disease states and to help design gene therapy vectors for treating CD154-deficient individuals with hyper-IgM syndrome.

The expression of CD154 (CD40 ligand) by activated T lymphocytes plays a central role in humoral and cellular immunity. The fundamental importance of this protein in mounting an immune response has made it an attractive target for immunomodulation. Several studies have demonstrated that CD154 expression is regulated at the level of mRNA turnover in a manner distinct from other cytokine genes. We have purified, sequenced, and characterized the two major proteins that bind the CD154 3' untranslated region (3'UTR) as members of the polypyrimidine tract binding protein (PTB) family. One of these proteins is a previously unreported alternatively spliced PTB isoform, which we call PTB-T. These proteins interact with a polypyrimidine-rich region within the CD154 3'UTR that lacks any known cis-acting instability elements. The polypyrimidine-rich region of the CD154 3'UTR was both necessary and sufficient to mediate changes in reporter gene expression and mRNA accumulation, indicating the presence of a novel cis-acting instability element. The presence of a cis-acting instability element in the polypyrimidine-rich region was confirmed using a tetracycline-responsive reporter gene approach. The function of this cis-acting element appears to be dependent on the relative cytoplasmic levels of PTB and PTB-T. Cotransfection of vectors encoding PTB-T consistently decreased the CD154 3'UTR-dependent luciferase expression. In contrast, transfection of plasmids encoding PTB tended to increase CD154 3'UTR-dependent luciferase expression. Thus, the CD154 3'UTR contains a novel cis-acting element whose function is determined by the binding of PTB and PTB-T. These data identify a specific pathway that regulates CD154 expression that can potentially be selectively targeted for the treatment of autoimmune disease and allograft rejection.

Treatment of chronic arthritis in childhood is progressing quickly. Nonsteroidal antiinflammatory drugs still remain mainstays of treatment, but weekly methotrexate and intra-articular corticosteroid injections have made the biggest impact on disease control. Recently, tumor necrosis factor inhibitors have become available for the treatment of chronic childhood arthritis and have already had a substantial influence on disease activity. Very aggressive measures are still reserved for the sickest children who do not respond to conventional therapy, but the future for children with chronic arthritis looks bright with newer, highly targeted biologic therapies in the pipeline.

NFAT proteins play a key role in the inducible expression of cytokine genes in T lymphocytes. NFATc1 and NFATc2 are the predominant NFAT family members in the peripheral immune system. NFATc2 is found abundantly in the cytoplasm of resting T cells, whereas Nfatc1 expression is induced during T cell activation. To investigate Nfatc1 regulation, we characterized the structure of the murine Nfatc1 gene and its 5'-flanking region. A 290-bp sequence proximal to the transcription start site is highly conserved between mouse and human and possesses both basal and inducible promoter activities. Multiple binding sites for transcription factors were identified within this region, including a consensus NFAT-binding site. This promoter segment was cyclosporin A-sensitive, and mutation of the NFAT site abrogated inducible promoter activity and inhibited formation of an inducible DNA x protein complex containing NFATc2 in primary T cells. Overexpression of NFATc2 increased inducible Nfatc1 promoter activity, whereas this inducibility was attenuated in NFATc2(-/-) splenocytes. This study suggests that pre-existing NFATc2 contributes to the subsequent induction of Nfatc1 during T cell activation.

We observed that the human CD40 ligand (CD40L) gene 5'-flanking region conferred weak promoter activity in activated CD4 T cells, suggesting that additional regions are required for optimal CD40L gene transcription. We therefore examined a 3'-flanking segment of the CD40L gene, which contained a putative NF-kappaB/Rel cis-element, for its ability to enhance CD40L promoter function. This segment augmented CD40L promoter activity in an orientation-independent manner in CD4 T-lineage cells but not in human B cell or monocyte cell lines. Mapping of CD4 T-lineage cell nuclei identified a DNase I-hypersensitive site in the flanking region near the NF-kappaB/Rel sequence, suggesting a transcriptional regulatory role. This was further supported by truncation analysis and site-directed mutagenesis, which indicated that the CD40L 3'-flanking NF-kappaB/Rel cis-element was critical for enhancer function. Electrophoretic mobility shift assays showed that the cis-element preferentially bound the p50 form of the NF-kappaB1 gene contained in human T cell nuclear protein extracts. This binding also appeared to occur in vivo in CD4 T cells based on chromatin immunoprecipitation assays using NF-kappaB p50-specific antiserum. Together, these results suggest that the CD40L gene 3'-flanking region acts as a T cell-specific classical transcriptional enhancer by a NF-kappaB p50-dependent mechanism.