Separation of T cell subpopulations capable of DNA synthesis, lymphotoxin release, and regulation of antigen and phytohemagglutinin responses on the basis of density and adherence properties.

Durkin, Helen G.; Bash, Jerry A.; Waksman, Byron H.

doi:10.1073/pnas.72.12.5090

Cited by 17 publications

(8 citation statements)

References 17 publications

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“…Since it is quite possible that in certain immunodeficiencies or in immune reactions a lack of correlation between the proliferative and transcriptional response of lymphocytes exists (16,17), the present method might be of unique value in such situations. Likewise, the method may be helpful in recognizing lymphocyte differentiation into blastoid compared to plasmocytoid cells (18,19).…”

Section: Resultsmentioning

confidence: 99%

Lymphocyte stimulation: a rapid multiparameter analysis.

Darżynkiewicz¹,

Traganos²,

Sharpless³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

315

215

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ABSIRACTWe propose a simple cytofluorometric assay of lymphocyte stimulation. It was developed from earlier efforts in this laboratory to differentially stain DNA and RNA in fixed cells by use of a metachromatic dye, acridine orange (4), and applied to distinguish stimulated from nonstimulated lymphocytes (5). Under appropriate conditions, acridine orange preferentially stains nucleic acids, among other cellular polyanions. The dye intercalates into the double helix as a monomer that fluoresces green (530 nm) (6), while electrostatic dye binding to the phosphates of single-stranded nucleic acids involves dye aggregation ("stacking") and dye-dye interactions that result in red fluorescence (640 nm) (7). To obtain differential stainability of DNA compared to RNA in situ, it is necessary to selectively denature any double-stranded RNA prior to staining (4, 5), because otherwise the binding of acridine orange to double-stranded RNA is indistinguishable from binding to native DNA (4). This is achieved by treatment of cells with the chelating agent EDTA, which is known to unfold ribosomes (8). MATERIALS AND METHODSHuman mononuclear cells were isolated from peripheral blood of normal volunteers by density centrifugation of Ficoll-Hypaque. The lymphocytes were separated from monocytes, and were cultured in the absence or presence of phytohemagglutinin, as described before (9 (10).Observation of cells under phase microscopy reveals that Triton X-100 does not lyse the cells; the cell membrane remains preserved, and the mitotic cells remain intact after the treatment with detergent at that low pH. Inspection of cells stained with.acridine orange under ultraviolet fluorescence microscopy indicates that the green fluorescence is localized exclusively in the cell nucleus, while the red fluorescence is confined to the cytoplasm and nucleoli. RESULTS AND DISCUSSIONThe present method involves initial treatment of unfixed cells with chelating agents (citrate, EDTA) in the presence of the nonionic detergent Triton X-100, at pH 3.0, followed by staining with acridine orange at pH 3.8. Control experiments on cells first treated with DNase or RNase (Table 1) reveal that the detergent treatment makes cells permeable not only to the dye but also to nucleases, and that at least 88% of the green fluorescence (Fmo) is the result of acridine orange interaction with cellular DNA, while at least 87% of the red fluorescence (F>600) of stimulated cells is due to RNA. This relatively good specificity in differential staining of DNA and RNA by acridine orange may be due to the following: (i) Exposure of permeable cells to chelating agents and the dye, as in the case of fixed cells (4, 5) or isolated ribosomes (8), denatures double-stranded RNA in situ. (i)0 Cell staining is done at pH 3.8, i.e., near optimal for the discrimination of DNA from RNA (11). In fact, morphological recognition of stimulated lymphocytes, based on RNA stainability with acridine orange, was shown to be optimal at pH 3.8 (12). (iii) The cells are stained in the presence of...

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Section: Resultsmentioning

confidence: 99%

Lymphocyte stimulation: a rapid multiparameter analysis.

Darżynkiewicz¹,

Traganos²,

Sharpless³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

315

215

View full text Add to dashboard Cite

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“…The functioning of T lymphocytes in regulation of antibody responses has been recognized as both helping and suppressing the responsive bone marrow-derived lymphocyte (B cell) I (reviewed in 1). With regard to the termination of the response, many suppressor systems have been described, some nonspecific, spontaneous (2)(3)(4), or induced (5)(6)(7), some specific in their induction and nonspecific in their activity (8)(9)(10), or some specific both in their induction and in their effector phases (11)(12)(13).…”

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confidence: 99%

Changes in suppressor mechanisms during postnatal development in mice.

Calkins¹,

Stutman²

1978

The Journal of Experimental Medicine

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The activity of suppressor cells from spleens of mice of varying ages was assessed by their addition to cultures of normal or SRBC immune spleen cells together with a challenge of SRBC. 1-wk and adult spleen cells were highly suppressive of the secondary in vitro antibody response to SRBC. 3-wk spleen cells were less active in suppressing this response. The nature of the suppression and the character of the suppressor cells changed in this period. Whereas adult spleen cells demonstrated specificity, 1-wk cells nonspecifically suppressed all responses tested. Further, unlike adult suppressor cells (which are Thy.1.2 positive), 1-wk suppressor cells are insensitive to anti-Thy.1.2 treatment in this system. Both cells are nonadherent to glass beads and nylon wool and are undetectable in the normal thymus.

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“…This characterization of PHA-reactive cells is consistent with the experiments reported here in which they were shown by 'suicide' with '%€'HA t o be distinct from both helper T cells which are Ly-1+, 2-[ 321 and suppressor T cells which are Ly-1-, 2+ and Ia+ [33] (Table 3). Furthermore, PHA-reactive cells do not appear to include antigen-reactive T cells [34]. Although these experiments fail to demonstrate the normal function of PHA-reactive cells, a clue may be found in the recent finding of a role for Ly-1+,2+, 3+ T cells as amplifiers for both helper and suppressor T cells [35], and Feldmann (personal communication).…”

Section: Discussionmentioning

confidence: 93%

Identification of T cell subpopulations binding phytohemagglutinin: functional characteristics

Callard

Basten

1978

Eur J Immunol

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Lymphocytes binding 1251-labeled phytohemagglutinin ( i251-PHA) were directly quantitated by autoradiography. The distribution of grain counts proved t o be trimodal indicating the presence of three distinct subpopulations of PHA-binding cells. Low intensity binders (median grain count of 8-12 grains/cell) predominated in spleen and lymph node, included B and T cells, and responded to the mitogen, lipopolysaccharide (LPS). High intensity binders (median grain count greater than 60 grains/cell) occurred at low frequency in every lymphoid organ, were T cells, and included mixed lymphocyte culture (MLC)-reactive but not PHA-reactive cells as judged by preferential sensitivity to suicide with lz5I-PHA. Medium intensity binders (median grain count of 40-45 grains/cell) predominated in the thymus, formed a subpopulation in the spleen and lymph nodes of normal mice, but were absent from bone marrow of normal mice and the spleen of nude mice. In addition, their numbers were markedly reduced following adult thymectomy and cortisone treatment indicating that they were most likely T cells of recent thymic origin.PHA-reactive cells were considered t o belong to the medium intensity PHAbinding subpopulation. This conclusion was based on the following: first, medium intensity binders, like PHA-reactive cells, were T cells; second, they were contained in the same subpopulation of T cells as PHA-reactive cells. That is, they were of recent thymic origin (T, cells) according to studies of tissue distribution, cortisone sensitivity and effect of adult thymectomy; third, 'suicide' of the response t o PHA as well as to allogeneic cells but not LPS was achieved by prior incubation of cells with high doses of soluble '2SI-PHA, whereas lower doses resulted only in abrogation of the MLC response. In other words, PHA-reactive cells resided in the medium, rather than high intensity subpopulation of binding cells. By analogy with suicide of antigen-binding cells, this observation implies that PHA binds preferentially to those cells responding to it, and confirms the validity of using mitogen activation as a model for antigen-induced triggering. On the other hand, binding of PHA t o a subpopulation of T cells casts doubt on the value of the PHA response as a measure of overall T cell function. Support for this conclusion came from the failure of PHA suicide to abrogate effector T cell activities such as help and suppression.

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Separation of T cell subpopulations capable of DNA synthesis, lymphotoxin release, and regulation of antigen and phytohemagglutinin responses on the basis of density and adherence properties.

Cited by 17 publications

References 17 publications

Lymphocyte stimulation: a rapid multiparameter analysis.

Lymphocyte stimulation: a rapid multiparameter analysis.

Changes in suppressor mechanisms during postnatal development in mice.

Identification of T cell subpopulations binding phytohemagglutinin: functional characteristics

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