RNA content in human lymphocyte subpopulations.

Abstract

Human peripheral blood lymphocytes are stained with the metachromatic dye acridine orange and the fluorescence of individual cells is measured by flow cytometry. The relative content of stainable RNA per cell is estimated by comparison with RNase-treated cells. Non-T and T lymphocytes have different mean quantities of RNA per cell, and these classes exhibit different distributions of RNA content. Non-T cells have a unimodal distribution with a sharp peak and exponential distribution towards higher RNA values. T cells have a bimodal distribution with two separate peaks. When T cells having receptors for IgG (T., cells) and IgM (T,, cells) are separated, each of these cell populations displays a unimodal distribution. Of these three lymphocyte subpopulations,T, cells have the lowest content of RNA per cell. Non-T cells have slightly higher RNA content than Ty, and T,, cells have twice as much RNA as T cells. The RNA content, which surely relates to the different functions of these lymphocyte subpopulations, may also be a useful marker for rapidly distinguishing the lymphocyte subpopulations.Although immunologic functions of thymus-derived (T) and bone marrow-derived (B) lymphocytes are very different, their morphologic features are remarkably similar (1, 2). These cell populations are indistinguishable by light microscopy and share a number of cytochemical properties. Differences, however, can be ascertained at the membrane level through analysis of immunologic markers (3,4). For example, human T lymphocytes consist of at least two subpopulations, namely cells having receptors either for IgG (To) or for IgM (T,), as described recently (5,6 More than 98% of the cells were viable as determined by trypan blue dye exclusion. Non-T cells were removed from the interface of the Ficoll/Hypaque gradient. Sheep erythrocytes attached to T cells were lysed by incubation for 6 min at 370 with Tris buffer containing 0.83% ammonium chloride (sample III). The same incubation was done with non-T cells (sample IV). Cells were then washed thrice in saline.T Cell Subpopulations. T cells were resuspended in RPMI-1640 medium containing penicillin (100 units/ml), streptomycin (100 Ag/ml), and 20% fetal calf serum at a concentration of 2 X 106 cells per ml. These cells were cultured at 370 in a humidified incubator with 5% C02/95% air for 20 hr. At the end of the incubation, cells were washed thrice in phosphatebuffered saline and resuspended at a concentration of 4 X 106/ml (sample V). More than 98% of cells were viable as tested by trypan blue dye exclusion. Cells were then assayed for TM, and T7 subpopulations as previously described (12).Antibodies to Ox Erythrocytes. These antibodies were prepared as described elsewhere (13 Table 1). The peak value of non-T cells is slightly higher than that of the first peak of T cells.(17, 18). More detailed description of the instrument and the computer data-handling system is given elsewhere (9,17 (Fig. 1, upper row). Large differences were observed among normal-individuals, expressed...

Relationship between RNA content and progression of lymphocytes through S phase of cell cycle.

“…As was documented before in various cell systems including stimulated lymphocytes (9)(10)(11)(12)17) and confirmed here (Table 1), the F>600 value represents mostly cellular RNA whereas F53o is a measure of the DNA content per cell (9)(10)(11)(12)18 (3)(4)(5). By using flow cytometry, we observe a similar variation; whereas some lymphocytes double their DNA within 5 hr, others replicate DNA at rates up to 5 times slower (Fig.…”

Fluorescence spectra of DNA dyes measured in a flow cytometer

“…The shift may also depend on DNA conformation and solvent polarity (16). Shapiro (14) has suggested that Hoechst, because of its milieu- (1,5,6). dependent fluorescence spectrum, may be used as a probe for chromatin structure.…”

“…Photons absorbed by one dye may be emitted by another if 1 ) the emission spectrum of one of the dyes (the donor) overlaps the excitation spectrum of the other (the acceptor), 2 ) the dyes are in close proximity, and 3) the dye molecules are in a favorable orientation (17). This process is called resonance energy transfer and produces fluorescence with the characteristic spectrum of the acceptor at the cost of the fluorescence intensity of the donor.…”

Isolation and partial chemical characterization of the IgG Fc receptor of human T lymphocytes and production of an antiserum.

“…Ty cells are more sensitive to irradiation and high-dose corticosteroid treatment (9), have diminished RNA content (10), and have reduced surface charge (11) when compared with the majority of T lymphocytes. In addition, at least some Ty cells can suppress immunoglobulin production and can participate in natural killer-cell activity and antibody-dependent, cell-mediated cytotoxicity (12)(13)(14)(15).…”