Upregulated TCRζ enhances interleukin-2 production in T-cells from patients with CML

Abstract

T-cell immunodeficiency is a common feature in patients with chronic myeloid leukemia (CML), and deficiency in CD3 levels was detected in T cells from these patients, which may represent a characteristic that is related to a lower T cell activation. In this study, we explored the possibility that forced TCRζ gene expression may upreg-u-late T cell receptor (TCR) signaling activation and reverse interleukin-2 (IL-2) production in T cells from patients with CML. A recombinant eukaryotic vector expressing TCRζ was transfected into T cells by nucleofection. Phosphorylated TCRζ, phosphorylated NF-κB, and the IL-2 level in TCRζ-transfected CD3+T cells that were activated with anti-CD3 and anti-CD28 antibodies were measured by Western blot and enzyme-linked immunosorbent assay (ELISA). Significantly increased TCRζ levels were found in TCRζ-transfected CD3+T cells. After CD3 and CD28 antibody stimulation, a significantly higher phosphorylated TCRζ chain level was demonstrated, and an increased IL-2 production in TCRζ-upregulated T cells was associated with the increased expression of the phosphorylated NF-κB. In conclusion, TCRζ gene transfection could restore TCRζ chain deficiency and enhance IL-2 production in T cells from patients with CML. It is possible that TCRζ chain reconstitution in leukemia-specific, clonally expanded T cells will effectively increase their activation of antileukemia cytotoxicity.

FIG. 1.

Immunofluorescence detection (200×) of enhanced green fluorescent protein (EGFP) expression to measure the TCRζ-IRES2-EGFP transfection efficiency in K293 (A) and Jurkat cells (B) and Western blot analysis of TCRζ protein expression in K293 (C, 1–2) and Jurkat cells (C, 3–4) 24 h post-transfection. Color images available online at www.liebertpub.com/dna

FIG. 2.

The TCRζ expression level in peripheral blood mononuclear cells (PBMCs) from patients with chronic myeloid leukemia (CML) as determined by flow cytometry (FCM) analysis. (A) Comparison of the mean fluorescence intensity (MFI) of TCRζ in PBMCs between healthy individuals (HI) (17.2±5.7, n=10) and patients with CML (8.5±4.9, n=12), (B) Example of the shift in the MFI of TCRζ in PBMCs from a patient with CML relative to the MFI (PE-A) of TCRζ in PBMCs of a healthy individual. PE-A=fluorescence intensity of PE. Color images available online at www.liebertpub.com/dna

FIG. 3.

The transfection efficiencies of TCRζ-IRES2-EGFP or IRES2-EGFP transfected CD3+T cells as determined by fluorescence microscopy (A) and FCM detection (B) 18 h post-transfection. FITC-A=the fluorescence intensity of FITC. Color images available online at www.liebertpub.com/dna

FIG. 4.

The expression levels of TCRζ and phosphorylated TCRζ as determined by Western blot analysis. (A) Western blot results, P1–P4: CML samples, HI: healthy individual control, (B) Comparison of the expression levels of TCRζ and phosphorylated TCRζ between the TCRζ-IRES2-EGFP and IRES2-EGFP transfected CD3+T cell groups (n=4).

FIG. 5.

The interleukin-2 (IL-2) levels in TCRζ-IRES2-EGFP or IRES2-EGFP transfected CD3+T cell groups (n=4).

FIG. 6.

The expression level of phosphorylated NF-κB by Western blot analysis. (A) Western blot results, P1–P4: CML samples, HI: healthy individual control, (B) Comparison of the expression levels of phosphorylated NF-κB between the TCRζ-IRES2-EGFP and IRES2-EGFP transfected CD3+T cell groups (n=4).