Flow cytometric detection of perforin in normal human lymphocyte subpopulations defined by expression of activation/differentiation antigens
Introduction
Perforin (Pf) is a 70 kDa glycoprotein stored in cytoplasmic granules of cytotoxic T lymphocytes and NK cells [1]. Monomeric perforin acts as a cytolytic mediator, polymerizing into target cells' membranes to form aggregates and causing death by colloid osmotic lysis. Genes encoding human and mouse perforin were shown to map to chromosome 10 in humans and in mice; similarities in genomic structure and protein sequence suggest analogous mechanisms of gene transcription regulation.
Human peripheral blood CD56+ NK cells and γδ T lymphocytes were shown by immunocytochemistry to express constitutively and store substantial levels of Pf in cytoplasmic granules. Similarly, CD8+CD11b+ suppressor T cells express Pf and exhibit cytolytic potential after in vitro or in vivo activation [2].
We investigated by three-color flow cytometry the expression of Pf in normal CD4+ and CD8+ lymphocytes and in NK cells defined by surface expression of a variety of differentiation/activation markers.
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Lymphocyte membrane phenotype
Triple labeling experiments were performed on EDTA-anticoagulated normal peripheral blood samples (n=10); aliquots of 100 μl were incubated for 30 min at 4°C with pre-titered dilutions of the following fluorescein isothyocyanate (FITC)-, phycoerythrin (PE)- or peridinin chlorophyll protein (PerCP)-conjugated monoclonal antibodies (mAb): CD45 (2D1 clone, IgG1), CD14 (MØP9 clone, IgG2b), CD4 (SK3 clone, IgG1), CD3 (SK7 clone, IgG1), CD8 (SK1 clone, IgG1), CD25 (2A3 clone, IgG1), HLA-DR (L243
Expression of Pf in normal CD3+ lymphocytes
On average, 22.3±5.7% normal peripheral blood lymphocytes identified by means of expression of CD3 were stained with anti-Pf mAb; a negligible subset (<1%) of CD3+HLA−DR+, CD3+CD69+, CD3+CD25+ and CD3+CD95+ lymphocytes were Pf+, indicating that the expression of activation/differentiation related markers and Pf identifies different cellular subsets (Table 1). In vitro short-term PHA stimulation caused an increase of Pf+ cells, which comprised up to 30±7% of phenotypically activated CD69+
Discussion
Perforin (Pf) is a killer-cell specific cytolytic mediator produced by cytotoxic lymphocytes and stored in cytoplasmic granules; mRNA encoding Pf and granzyme A or B, a family of synergistically acting cytolytic serine-proteinases, can be detected in CD8+ T cells infiltrating heart and kidney allografts during rejection and in myocardium cells in post-viral myocarditis, supporting the concept that Pf plays a role in vivo in disease immunopathogenesis 1, 6. In particular, Pf-dependent cytotoxic
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Functional impact of A91V mutation of the PRF1 perforin gene
2013, Human ImmunologyCitation Excerpt :Both parents were carriers and furthermore, the mother presented the A91V variant in trans (Fig. 1). Perforin expression is mainly confined to NK cells, as well as CD8+, CD56+, TcRγδ+ and some activated CD4+ T cells [20–22]. An analysis of NK and T cells from the asymptomatic A91V/G149S compound heterozygous revealed decreased perforin expression levels as compared to a healthy control (Fig. 2); as expected, perforin was undetectable in cells from the (G149S/G149S) patient.
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