Interferon-gamma-secreting T-cell populations in rejecting murine cardiac allografts: assessment by flow cytometry

Am J Pathol. 1998 Nov;153(5):1383-92. 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)-gamma, 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-gamma 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-gamma, 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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • CD8-Positive T-Lymphocytes / drug effects
  • CD8-Positive T-Lymphocytes / immunology
  • Cell Separation / methods*
  • Cell Survival / drug effects
  • Cells, Cultured
  • Coloring Agents
  • Flow Cytometry / methods*
  • Graft Rejection / immunology*
  • Graft Rejection / pathology
  • Heart Transplantation / immunology*
  • Heart Transplantation / pathology
  • Interferon-gamma / metabolism*
  • Interleukin-4 / metabolism
  • Ionomycin / pharmacology
  • Ionophores / pharmacology
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • T-Lymphocytes / drug effects
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism*
  • Tetradecanoylphorbol Acetate / pharmacology
  • Th1 Cells / drug effects
  • Th1 Cells / immunology
  • Th2 Cells / drug effects
  • Th2 Cells / immunology
  • Transplantation, Homologous


  • Coloring Agents
  • Ionophores
  • Interleukin-4
  • Ionomycin
  • Interferon-gamma
  • Tetradecanoylphorbol Acetate