Interferon-γ-Secreting T-Cell Populations in Rejecting Murine Cardiac Allografts

• Published in: The American journal of pathology Vol. 153; no. 5; pp. 1383 - 1392
• Main Authors: Stinn, Jennifer L., Taylor, Marta K., Becker, Gerold, Nagano, Hiroaki, Hasegawa, Satoru, Furakawa, Yutaka, Shimizu, Koichi, Libby, Peter, Mitchell, Richard N.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.11.1998
• ISSN: 0002-9440; 1525-2191
• DOI: 10.1016/S0002-9440(10)65725-2

Interplay between T-helper-1 (Th1) and T-helper-2 (Th2) cells is considered important in the development of acute allograft rejection and many other immune-mediated disease processes. Existing methods for evaluating expression of Th1 and Th2 cytokines, including reverse transcriptase polymerase chain reaction (RT-PCR), RNase protection assay (RPA), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) all have limitations; alternate techniques to quantify cell populations expressing specific cytokine proteins, generate statistically analyzable data, and allow simultaneous identification of cytokine-secreting cell type are needed. To this end, we adapted a flow cytometric technique for intracellular cytokine immunofluorescence staining for use with cells isolated from solid tissue. To demonstrate the utility of the method, we determined the number of CD4 + and CD8 + cells secreting the prototypical Th1 and Th2 cytokines, interferon (IFN)-γ, and interleukin (IL)-4 in acutely rejecting murine cardiac allografts. We also measured the cytokine production via ELISA, RPA, and semiquantitative competitive RT-PCR. The number of CD4 + cells producing IFN-γ increased as rejection proceeded, in agreement with previous data; we detected no IL-4 production at any time, although relatively low numbers of IL-10-producing cells were identified. In addition, a high percentage of CD8 + cells, which outnumber CD4 + cells at day 6 after transplant, also produce IFN-γ, suggesting that cytotoxic lymphocytes contribute significantly to the local cytokine milieu. This new application of intracellular cytokine staining provides a powerful methodology for studying transplantation immunology. The method may also be easily adapted to the study of other immune-mediated processes.