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. 2001 Nov;104(3):269-77.
doi: 10.1046/j.1365-2567.2001.01323.x.

Reduced T-cell Receptor CD3zeta-chain Protein and Sustained CD3epsilon Expression at the Site of Mycobacterial Infection

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Free PMC article

Reduced T-cell Receptor CD3zeta-chain Protein and Sustained CD3epsilon Expression at the Site of Mycobacterial Infection

U Seitzer et al. Immunology. .
Free PMC article

Abstract

Control of mycobacterial infection by the cellular immune system relies both on antigen-presenting cells and on T lymphocytes. The quality of an effective cellular immune response is dependent on functional signal transduction residing in the cytoplasmic tails of the T-cell receptor CD3 components. In order to investigate potential effects of mycobacteria on T-cell receptor signalling, we examined the protein expression of T-cell signal transduction molecules (CD3zeta, ZAP-70, p59fyn, p56lck). In Western blots of peripheral blood mononuclear cells of Mycobacterium tuberculosis infected patients, only the CD3zeta-chain showed a marked reduction in protein expression. To investigate the situation in situ, immunoenzymatic and immunofluorescence stainings for CD3epsilon and CD3zeta expression were performed on sections of normal lymphoid tissue, M. leprae infected and sarcoid tissue. CD3epsilon and CD3zeta expression were similar with respect to intensity, localization and the number of cells stained in normal lymphoid tissue and in sarcoid granulomas. In contrast, the granulomas of M. leprae infected tissues showed a significantly reduced expression of CD3zeta compared to CD3epsilon. Using double immunofluorescence analysis, virtually no CD3zeta expression could be detected in comparison to the CD3epsilon expression in the lesions. Apparently, mycobacteria are capable of significantly reducing CD3zeta-chain expression, which may be restored by cytokines. IL-2-enhanced zeta-chain expression and T-cell effector functions, defined by interferon-gamma release, in M. tuberculosis-specific and human leucocyte antigen-DR restricted CD4+ T cells isolated from granuloma lesions from patients with pulmonary tuberculosis. Because CD3zeta is essential for CD3 signalling and for eliciting T-cell effector functions, reduced CD3zeta protein expression could result in altered signal transduction and inefficient T-cell effector functions. Alternatively, reduced CD3zeta-chain expression may protect T cells from repetitive TCR stimulation associated with anergy or apoptosis.

Figures

Figure 1
Figure 1
Reduced CD3ζ-chain expression in peripheral PBMCs from patients with tuberculosis. Freshly isolated PBMCs of five healthy donors and five patients with tuberculosis were adjusted to 2 × 106 CD3ε positive staining cells as determined by flow cytometry before Western blot analysis. Detection of individual T-cell signalling molecules was performed with specific mAbs as described in the material and methods section. (a) Results obtained from PBMC of five different age-matched healthy donors (lanes 1–5); (b) results of five different tuberculosis patients (lanes 1–5). The expression level of ZAP-70, p56lck and p59fyn appeared to be similar in both groups. In contrast, CD3ζ protein expression was significantly reduced in patients with tuberculosis. The additional band in p56lck or p59fyn was observed in some samples obtained from patients with tuberculosis. The nature of this double band has not yet been defined, it may be associated with the activation status of peripheral T cells from patients with tuberculosis: Upon activation, lck or fyn are enriched in membrane lipid rafts, phosphorylate TCR invariant chains and associate with ZAP-70. These different configurations of lck or fyn (e.g. non-associated or associated with ZAP-70) may provide a reason for a different behaviour in SDS–PAGE analysis and also in Western blot.
Figure 2
Figure 2
Reduced in situ CD3ζ expression in tissues with mycobacterial infection. Serial sections of a hyperplastic tonsil (a, b), a skin biopsy of a lepromatous leprosy patient (c, d) and a lymph node biopsy of a sarcoidosis patient (e, f) were stained for the presence of CD3ε (a, c, e) and CD3ζ (b, d, f) with the APAAP technique. In hyperplastic tonsil, the expression of CD3ε (a) and CD3ζ (b) was localized to the same region, expressed by an equal number of cells, and was of similar intensity. In the representative example of M. leprae-infected tissue, CD3ε (c) expression was distributed uniformly throughout the granuloma, whereas CD3ζ (d) expression was greatly reduced and limited to a few isolated cells throughout the granuloma. In granulomatous tissue of a sarcoid lymph node, CD3ε expression (e) was detected as intense staining of cells localized around epithelioid cells in the granuloma. CD3ζ staining (f) was not as intense as CD3ε staining and appeared not to stain as many cells but was localized to the same areas in the tissue.
Figure 3
Figure 3
Double immunofluorescence analysis of CD3ε and CD3ζ expression in situ. Hyperplastic tonsil (a, b, c), a tuberculoid leprosy skin biopsy (d, e, f) and a sarcoid lymphnode (g, h, i) were stained for CD3ε (green fluorescence, a, d, g), CD3ζ (red fluorescence, b, e, h) and double exposed for colocalization of both CD3ε and CD3ζ (yellow fluorescence, c, f, i). In hyperplastic tonsil, CD3ζ expression colocalized 100% with CD3ε expression. In the tissue area chosen close to endothelium in this example, several cells exhibited CD3ε positivity and CD3ζ negativity. In an example of tuberculoid leprosy, CD3ε positive cells were detected throughout the granuloma (d) whereas CD3ζ expression was barely visible (e), resulting in no detection of CD3ε CD3ζ double positive cells (no yellow fluorescence (f). The sarcoid lymph node lesion exhibited CD3ε (g) and CD3ζ (h) expression to approximately the same extent. All CD3ζ cells were CD3ε positive (yellow fluorescence) and only a minor number of cells were CD3ε positive CD3ζ negative (i, green fluorescence).
Figure 4
Figure 4
Enhanced TCR ζ−chain expression in M. tuberculosis granuloma-associated CD4+ T lymphocytes induced by IL-2. GAL were freshly isolated from pulmonary lesions, as described in detail in the Materials and Methods section, cultured for 3 days in the presence or absence of IL-2 (1000 IU/ml), gated on CD4+ T cells and stained for TCR ζ chain expression by flow cytometry using two different mAb as indicated. Differences in mean fluorescence channel (m.f.c.) intensity. CD4 + T-cells correspond to effector cells described in Table 1. ss=side scatter.

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