Faculty Bibliography

Severe Combined Immunodeficiency (SCID) is a primary immunodeficiency affecting T cells, B cells, or both. Whereas the clinical symptoms are uniformly dominated by recurrent infections, the molecular causes for SCID are very heterogeneous. Mutations in cell surface receptors, signal transduction molecules and transcription factors have been described, including the common gamma chain of the IL-2 (and IL-4, IL-7, IL-9 and IL-15) receptors, the kinase JAK-3, the epsilon and gamma chains of CD3, the protein tyrosine kinase ZAP-70, as well as CIITA and RFX5 involved in MHC class II gene expression. In this work we describe two infants with SCID whose T cells display a severe defect in T cell activation and cytokine transcription due to impaired activation of the transcription factor NFAT. We show that this defect in activation is not due to mutations in the NFAT proteins expressed in T cells or the phosphatase calcineurin which regulates the activation of NFAT. However, nuclear import of NFAT in response to T cell activation was severely compromised in the patients' T cells. A modest degree of nuclear translocation of NFAT was achieved in the patients' T cells when nuclear export was inhibited using lithium chloride. This low level of nuclear NFAT in the nucleus was not sufficient to compensate for the defect in cytokine production in the patients' T cells. However, elevated levels of extracellular calcium led to an increase in cytokine gene transcription by the SCID T cells, suggesting that the underlying genetic defect in the patients involved calcium influx or the initiation of calcium signalling.

The expression of cytokine genes and other inducible genes is crucially dependent on the pattern and duration of signal transduction events that activate transcription factor binding to DNA. Two infant patients with SCID and a severe defect in T cell activation displayed an aberrant regulation of the transcription factor NFAT. Whereas the expression levels of the NFAT family members NFAT1, -2, and -4 were normal in the patients' T cells, dephosphorylation and nuclear translocation of these NFAT proteins occurred very transiently and incompletely upon stimulation. Only after inhibition of nuclear export with leptomycin B were we able to demonstrate a modest degree of nuclear translocation in the patients' T cells. This transient activation of NFAT was not sufficient to induce the expression of several cytokines, including IL-2, IL-3, IL-4, and IFN-gamma, whereas mRNA levels for macrophage inflammatory protein-1alpha, GM-CSF, and IL-13 were only moderately reduced. By limiting the time of NFAT activation in normal control cells using the calcineurin inhibitor cyclosporin A, we were able to mimic the cytokine expression pattern in SCID T cells, suggesting that the expression of different cytokine genes is differentially regulated by the duration of NFAT residence in the nucleus.

Peripheral blood lymphocytes (PBL) and alloreactive T cell lines of two male infants born to consanguinous parents and presenting with severe combined immunodeficiency (SCID) showed a pronounced deficiency in T cell activation. Although phenotypically normal, the proliferative response of the childrens' T cells was strongly reduced but could be improved by the addition of interleukin-2 (IL-2). Furthermore both childrens' T cells were unable to produce the cytokines IL-2, interferon-gamma (IFN-gamma), IL-4 and tumor necrosis factor-alpha (TNF-alpha). This multiple cytokine production deficiency could not be restored by IL-2 or co-stimulatory signals provided by antigen-presenting cells (APC). Moreover, mRNA for IL-2 and IFN-gamma could not be detected. In contrast, expression of the activation-dependent cell surface markers CD25 and CD69 was within normal limits. To determine whether the functional defect of the patients' T cells was due to the absence or abnormal binding of transcription factors involved in cytokine gene expression, electrophoretic mobility shift assays were used to examine the DNA binding of AP-1, Oct, CREB, SP1, NF-kappa B and the nuclear factor of activated T cells (NF-AT) to their respective response elements in the promoter of the IL-2 gene. Whereas AP-1, NF-kappa B, Oct, CREB and SP1 displayed normal binding activities in nuclear extracts, the binding of NF-AT to its IL-2 promoter response element was barely detectable both before and after T cell stimulation. Our results strongly suggest that this NF-AT/DNA binding defect is responsible for the multiple cytokine deficiency and the SCID phenotype observed in the two infant brothers.