A lack of evidence for down-modulation of CD3 zeta expression in colorectal carcinoma and pregnancy using multiple detection methods

Abstract

Loss of the T cell receptor-associated CD3 zeta chain has been proposed as a possible mechanism of the acquired immunosuppression in both tumour-bearing hosts, and in symptomatic patients with HIV infection. However, other reports suggest that the zeta-chain loss may in part be caused by protease activity of contaminating phagocytes ex vivo. Using flow cytometry and Western blot analysis on highly purified T cells, and ensuring adequate addition of protease inhibitors, we have studied the expression of CD3zeta on peripheral blood T cells from patients with colorectal carcinoma, and compared these with normal controls, and pregnant donors, as a further example of an immunocompromised state. Immunohistochemistry was performed on tumour sections from patients with colorectal carcinoma to measure CD3zeta expression in tumour infiltrating T cells, and compared with normal mucosa and tonsil. Using these three approaches, our data provide no evidence for downregulation of CD3zeta chain expression either in colorectal carcinoma or pregnancy and suggest that this explanation is unlikely to fully account for the reduced T cell function associated with these conditions in all patients.

Fig. 1

Example of flow cytometric analysis of CD3ζ expression of PBL from (a) healthy control (male, 41 years) compared with (b) a patient with colorectal carcinoma Dukes B/C (male, 51 years).

Fig. 2

Levels of CD3ζ and ε in purified CD3 purified cells from the peripheral blood of patients with colorectal carcinoma (T1–T3, all Dukes C) and healthy controls by Western blot (C1–C4). Samples were loaded at 10 μg of total protein per lane.

Fig. 3

CD3ζ chain expression in colorectal carcinoma (Dukes stage B) compared with nonmalignant tonsil. Staining was performed on sequential tissue sections at the dilutions shown and visualized using alkaline phosphatase. An appropriate murine IgG antibody was included as a negative control.

Fig. 4

Image analysis of CD3ζ expression in colorectal carcinoma (Dukes stage B) compared with nonmalignant tonsil. Staining was peformed on sequential tissue sections at the dilutions shown and visualized using alkaline phosphatase, without counterstain. The sections were colour (green channel) imaged on a Leica Q600 Image Analyser with a × 10 objective lens. The mean intensity of the staining is measured in arbitrary units. There was no difference in CD3ζ staining intensity observed between the tumour and tonsil sections at a range of antibody dilutions.

Fig. 5

CD3ζ and ε-chain expression in colorectal carcinoma (Dukes stage B) compared with nonmalignant colon and tonsil. Staining was performed on sequential tissue sections at the optimal dilutions shown and visualized using alkaline phosphatase. An appropriate murine IgG antibody was included as a negative control.

Fig. 6

Levels of CD3ζ and ε in CD3 purified cells from the peripheral blood of pregnant (P1–P2) and nonpregnant female donors (C1–C3) detected by Western blot. Samples were loaded at 10 μg of total protein per lane.