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Heterogeneity in P-glycoprotein (multidrug resistance) activity among murine peripheral T cells: correlation with
U Bommhardt1, J C Cerottini, H R MacDonald
1Ludwig Institute for Cancer Research, University of Lausanne, Epalinges, Switzerland.
Abstract:
P-glycoprotein (P-gly) is the transmembrane efflux pump responsible for multidrug resistance in tumor cells. Functional P-gly activity can be conveniently assessed microfluorometrically using the fluorescent dye rhodamine 123 (Rh123), which is an artificial substrate for the P-gly transporter. Here we assess P-gly activity in subsets of mouse peripheral T lymphocytes using the Rh123 efflux assay. Our data indicate that virtually all CD8+ cells extrude Rh123 efficiently, whereas only a subset of CD4+ cells exhibit P-gly activity. Correlation of P-gly activity in CD4+ cells with the expression of a panel of surface markers revealed that cells bearing an "activated/memory" phenotype (CD45RB-, CD44hi, CD62L-, CD25+, CD69+) were exclusively found in the fraction that can extrude Rh123. In contrast "naive" phenotype CD4+ cells (CD45RB+, CD44lo, CD62L+, CD25-, CD69-) could be further subdivided into two major subsets based on P-gly activity. In functional studies of sorted cell populations the Rh123-extruding subset of "naive" CD4+ cells proliferated more strongly and secreted higher levels of interleukin (IL)-2 than its Rh123-retaining counterpart when activated by a variety of polyclonal stimuli. Furthermore, this subset produced detectable levels of interferon (IFN)-gamma upon stimulation but no IL-4 or IL-10. As expected, the Rh123-retaining "naive" subset produced only IL-2 after stimulation, whereas the "memory" subset produced IFN-gamma, IL-4 and IL-10 in addition to low levels of IL-2. Collectively, our data indicate that P-gly activity is a novel parameter that can be used to distinguish a subset of "preactivated" CD4+ cells that would be considered as naive on the basis of their surface phenotype.
Insights
P-glycoprotein (P-gly) activity, measured by rhodamine 123 (Rh123) efflux, distinguishes subsets of T lymphocytes. This efflux pump activity identifies pre-activated CD4+ T cells within the naive population, impacting their immune response.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- P-glycoprotein (P-gly) is a transmembrane efflux pump crucial for multidrug resistance in tumor cells.
- Functional P-gly activity can be assessed using the fluorescent dye rhodamine 123 (Rh123) and microfluorometry.
- T lymphocytes play a central role in adaptive immunity, with distinct subsets mediating different functions.
Purpose of the Study:
- To assess P-glycoprotein (P-gly) activity in subsets of mouse peripheral T lymphocytes.
- To correlate P-gly activity with surface marker expression and functional responses in CD4+ T cells.
- To identify novel parameters for distinguishing T cell subsets, particularly within the naive CD4+ population.
Main Methods:
- Utilized the rhodamine 123 (Rh123) efflux assay to measure P-glycoprotein (P-gly) activity in mouse peripheral T lymphocytes.
- Employed flow cytometry to analyze P-gly activity in conjunction with surface marker expression (e.g., CD45RB, CD44, CD62L, CD25, CD69).
- Performed functional studies on sorted T cell populations, assessing proliferation and cytokine secretion (IL-2, IFN-γ, IL-4, IL-10) upon polyclonal activation.
Main Results:
- All CD8+ T cells efficiently extruded Rh123, indicating high P-gly activity.
- P-gly activity was detected in a subset of CD4+ T cells, correlating with an activated/memory phenotype (CD45RB-, CD44hi, CD62L-, CD25+, CD69+).
- Within the 'naive' CD4+ T cell population, P-gly activity subdivided cells into subsets with differential proliferation and cytokine production (IL-2, IFN-γ).
Conclusions:
- P-glycoprotein (P-gly) activity serves as a functional marker to distinguish T cell subsets.
- P-gly activity identifies a subset of 'preactivated' CD4+ T cells within the conventionally defined naive population.
- This P-gly-defined subset exhibits enhanced proliferative capacity and distinct cytokine profiles, suggesting a role in early immune responses.