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Case Reports
. 2001 Jun;103(2):164-71.
doi: 10.1046/j.1365-2567.2001.01246.x.

Specific immunoglobulin E responses in ZAP-70-deficient patients are mediated by Syk-dependent T-cell receptor signalling

S Toyabe  1 , A Watanabe, W Harada, T Karasawa, M Uchiyama
Affiliations
Case Reports

Specific immunoglobulin E responses in ZAP-70-deficient patients are mediated by Syk-dependent T-cell receptor signalling

S Toyabe et al. Immunology. 2001 Jun.

Abstract

ZAP-70 deficiency is a rare primary immunodeficiency characterized by the absence of peripheral CD8+ T cells and defects in T-cell receptor (TCR) signalling. T cells in ZAP-70-deficient patients are assumed to have no helper functions for B-cell immunoglobulin synthesis, whereas the patients rarely have antigen-specific antibodies. We experienced a ZAP-70-deficient patient, who had immunoglobulin E (IgE) antibodies specific to food allergens, and we investigated the mechanisms of switching to IgE in the patient. Peripheral blood mononuclear cells from the patient did not proliferate upon stimulation with the antigens but produced distinct levels of interleukin-4 (IL-4). Cell sorting analysis indicated that the cells that produced IL-4 in response to the antigens were enriched in CD4+ T cells. Purified CD4+ T cells from the patient produced IL-4 and expressed CD40L upon stimulation with anti-CD3. Moreover, CD4+ T cells pretreated with anti-CD3 induced mature epsilon transcript on naive B cells. Since the results indicated that there remained sufficient T-cell receptor (TCR)-signalling in the patient's T cells to exert antigen-specific IgE switching on B cells, we next investigated the expression of the ZAP-70-homologous kinase Syk. Syk was present in high levels in patient's CD4+ T cells and was tyrosine-phosphorylated after TCR stimulation. Inhibition of Syk by piceatannol resulted in decreased production of IL-4 and expression of CD40L on patient's CD4+ T cells. Moreover, Syk was expressed on all human T-cell leukaemia virus (HTLV-1)-transformed T-cell lines derived from peripheral blood of the patient, whereas it was low or undetectable in control lines. It was therefore concluded that specific IgE responses in the patient were most likely to be mediated by Syk-dependent TCR-signalling.

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Figures

Figure 1
Figure 1
Results of flow cytometry of the patient's PBMNC. PBMNC of the patient were stained with FITC-or PE-conjugated anti-CD3, anti-CD4, and anti-CD8 mAb and then analysed using FACScan.
Figure 2
Figure 2
ZAP-70 and Syk expressions on CD4+ T cells. CD4+ T cells from the patient, her parents, and a normal control were purified from PBMNC using MACS. Cell lysates of the CD4+ T cells (2 × 106 each) were analysed by SDS–PAGE and were subjected to anti-ZAP-70 and anti-Syk blots.
Figure 3
Figure 3
IL-4 mRNA expressions on various cell populations of the patient's PBMNC. Cells positive for CD3, CD4, CD20, CD56, or FcεRI were purified using MACS. Each cell population was stimulated with (+) or without (–) OVA (25 µg/ml) for 48 hr. Total RNA was extracted from 1 × 106 cells and subjected to RT–PCR analysis for IL-4 and β-actin mRNA expression.
Figure 4
Figure 4
CD40L expression on TCR-stimulated CD4+ T cells of the patient. Purified CD4+ T cells were stimulated with biotinylated anti-CD3 plus streptavidin for 3 hr with or without 10 µm piceatannol. Cells were stained with PE-conjugated anti-CD40L antibody and were analysed using FACScan. The clear histogram indicated CD40L expression before stimulation, whereas the shaded histogram corresponded to that after stimulation.
Figure 5
Figure 5
Induction of germline and mature ε transcripts on naive B cells by the patient's T cells. CD4+ T cells were prestimulated with biotinylated anti-CD3 plus streptavidin for 24 hr. Purified IgD+ naive B cells were cultured with IL-4, anti-CD40, or the preactivated CD4+ T cells for 10 days. Total RNA was extracted from the cells and subjected to RT–PCR for the expression of germline (Iε-Cε) and mature (VH-Cε) ε transcripts.
Figure 6
Figure 6
Syk expression on patient's CD4+ T cells that express high levels of CD40L. Purified CD4+ T cells of the patient were stimulated with biotinylated anti-CD3 plus streptavidin for 3 hr. The cells were stained with PE-conjugated anti-CD40L antibody and were sorted into CD40L+ (CD40L high) and CD40L– (CD40L low) populations using FACS Vantage. Cell lysates of the sorted cells (2 × 106 each) were analysed by SDS–PAGE and were subjected to anti-Syk blots.
Figure 7
Figure 7
Expression and tyrosine phosphorylation of Syk, ZAP-70, and TCR ζ chain in the patient's CD4+ T cells. CD4+ T cells obtained from the patient and normal control were cultured with (+) or without (–) biotinylated anti-CD3 plus streptavidin for 2 min. The cells (2 × 106 each) were lysed in 1% NP-40 lysis buffer, and immunoprecipitated with anti-Syk, anti-ZAP-70, and anti-TCR ζ antibodies. Immunopreciptates were resolved on 10% SDS–PAGE and immunoblotted with anti-Syk, anti-ZAP-70, and anti-TCR ζ, and anti-phosphotyrosine (P-Tyr.) antibodies.
Figure 8
Figure 8
Syk expression on T-cell lines. HTLV-1-transformed T-cell lines were established from the patient and normal control. Cell lysates of T-cell lines (2 × 106 each) obtained from the patient (lanes 6–9) and control (lanes 1–5) were analysed by SDS–PAGE and were subjected to anti-Syk blot.
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