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Technical Advances |
-Secreting T-Cell Populations in Rejecting Murine Cardiac Allografts
From the Department of Pathology,*
Immunology Division,
and Department of Medicine,§
Brigham and
Women's Hospital, Boston, Massachusetts, the Department of
Surgery,
Osaka University Medical School,
Osaka, and Department of Cardiothoracic
Surgery,
Tokyo Medical and Dental University,
Tokyo, Japan
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)-
, 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- 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-, 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.
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