Specific Fractionation of Immune Cell Populations

Rutishauser, Urs; Millette, Clarke F.; Edelman, Gerald M.

doi:10.1073/pnas.69.6.1596

Cited by 55 publications

(22 citation statements)

References 10 publications

(12 reference statements)

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“…Nylon fibers were strung in dishes, derivatized with hapten-bovine-serum albumin (BSA) conjugates (at 0.25 mg/ml), BSA (5 mg/ml), Limulus hemocyanin (2.5 mg/ml), or concanavalin A (0.25 mg/ml), and incubated with cells in MEM containing 20 (2). With cells from immunized mice, the inhibition increased with inhibitor concentrations from 0.002 to 300 ,ug/ml.…”

Section: Methodsmentioning

confidence: 99%

“…In previous reports, we have described the fractionation of specific antigen-binding cells from lymphoid cell populations of immunized and unimmunized animals by specific attachment to antigen-derivatized fibers (1,2). Although the isolated lymphoid cells all have specificity for the chosen antigens, it is likely that they are heterogeneous in their function and degree of differentiation.…”

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confidence: 99%

“…Out studies (1,2) have indicated that specific antigenbinding cells isolated by fiber fractionation do not include either thymocytes or plaque-forming cells. In the present paper, we provide evidence that antigen-derivatized fibers bind both T and B cells.…”

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confidence: 99%

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Binding of Thymus- and Bone Marrow-Derived Lymphoid Cells to Antigen-Derivatized Fibers

Rutishauser

Edelman

1972

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

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Thymus-derived lymphocytes (T cells) and bone marrow-derived lymphocytes (B cells) from mouse spleens bind specifically to antigen-derivatized nylon fibers. The fiber-bound population consisted of about 60%-70% B cells and 30% T cells as determined by cytotoxicity, fluorescence, and antibody-complement binding assays. Essentially all fiber-bound cells were viable and could be accounted for as T or B cells. Enriched populations of T or B cells could be isolated on the fibers by destruction of one or the other cell type with the appropriate antiserum plus complement. T or B cells could also be fractionated according to their relative affinity (or avidity) for a given antigen.In previous reports, we have described the fractionation of specific antigen-binding cells from lymphoid cell populations of immunized and unimmunized animals by specific attachment to antigen-derivatized fibers (1, 2). Although the isolated lymphoid cells all have specificity for the chosen antigens, it is likely that they are heterogeneous in their function and degree of differentiation. The most conspicuous functional heterogeneity among lymphocytes is reflected by the presence in the population of thymus-derived lymphocytes (T cells) and bone marrow-or bursa-derived lymphocytes (B cells) (3). Additional distinctions must be made between thymocytes and competent T cells. Similarly, B cells include several functional classes, notably antigen-sensitive B cells that can differentiate into secreting cells when triggered by antigen, and the secreting cells or plaque-forming cells themselves.Out studies (1, 2) have indicated that specific antigenbinding cells isolated by fiber fractionation do not include either thymocytes or plaque-forming cells. In the present paper, we provide evidence that antigen-derivatized fibers bind both T and B cells. The cells can be fractionated according to their relative avidities* for antigen and, by the use of appropriate antisera, enriched populations of T or B cells with specificity for a given antigenic determinant can be obtained.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Binding of Thymus- and Bone Marrow-Derived Lymphoid Cells to Antigen-Derivatized Fibers

Rutishauser

Edelman

1972

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

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“…The specific attachment of cells to chemically derivatized fibers (3,4) has been used to fractionate and manipulate normal lymphoid cells for morphological and functional studies (5)(6)(7). In the present paper, the fiber-rosette method used in antigen-binding studies (5) has been modified for the analysis of cell-cell binding induced by the lectin concanavalin A (Con A).…”

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confidence: 99%

“…In the present paper, the fiber-rosette method used in antigen-binding studies (5) has been modified for the analysis of cell-cell binding induced by the lectin concanavalin A (Con A). This system allows the quantitative study of binding between single cells, in which the binding of Con A to a cell and the binding of that cell to another cell can be carried out as successive steps.…”

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confidence: 99%

Receptor Mobility and the Mechanism of Cell-Cell Binding Induced by Concanavalin A

Rutishauser

Sachs

1974

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

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The cell-cell binding induced by concanavalin A between single cells has been analyzed by use of cells attached to nylon fibers. Binding of a concanavalin A-coated cell to an untreated cell was found to a high degree between two lymphoma tumor cells, less frequently between a lymphoma cell and a normal lymphocyte, and only rarely between two normal lymphocytes. The binding was inhibited by the presence of a saccharide inhibitor of concanavalin A, but could not be reversed by addition of the inhibitor after the cells had bound to each other. Although nonbinding was obtained when both cells were coated with lectin or fixed with glutaraldehyde, fixation of a cell before coating with concana'valin A enhanced its ability to bind an untreated cell. The results indicate that cell-cell binding induced by concanavalin A requires shortrange lateral movement of cell receptors for the lectin, that only one cell has to have mobile receptors, and that some receptors must be unoccupied by lectin molecules before cell-cell contact. Clustering of the receptors is not necessary and seems to hinder cell-cell binding. It is suggested that the short-range movement is required for alignment of individual receptors so as to form multi-point bridges between two cells by lectin molecules. The bridging is then followed by the formation of irreversible bonds between the cells. The receptors on tumor cells appear to have a greater -ability than receptors on normal cells to align themselves for cell-cell binding.The agglutination of cells by lectins has been used as a probe to study changes in the cell-surface membrane (1, 2). Agglutination is generally determined by adding the lectin to a cell suspension, shaking the suspension, and recording the degree of cell aggregation. Although this agglutination assay is rapid and convenient, it simultaneously measures the binding of lectin to the cells and of several cells to one another.The specific attachment of cells to chemically derivatized fibers (3, 4) has been used to fractionate and manipulate normal lymphoid cells for morphological and functional studies (5-7). In the present paper, the fiber-rosette method used in antigen-binding studies (5) Cells. Normal lymphocytes were obtained from the lymph nodes of 6-to 8-week-old CR/RAR rats or A strain mice.Cells from the two sources contained 70-85% thymus-derived cells and gave the same results in cell-cell binding assays. Lymphocytes were obtained by teasing the cells into phosphate-buffered saline, pH 7.4 (PBS), and then washing the cell three times with PBS. Lymphoma tumor cells were obtained from a Moloney virus-induced lymphoma grown in A strain mice (8). This is a tumor of thymus-derived cells (9). Cells (105) were inoculated intraperitoneally into adult mice, and the cells were harvested 10-14 days later. The cells were washed three times in PBS before they were used.Cell-Cell Binding Assay. Nylon fibers were strung in polyethylene frames, derivatized with concanavalin A (Con A, Miles Yeda) at 0X5 mg/ml, and incubated with cells ...

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Characterization of lymphocyte subpopulations in chronic lymphocytic leukemia

Rowlands

Daniele

Nowell̀

et al. 1974

Cancer

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B cell and T cell characteristics of circulating lymphocytes were studied in 13 patients with chronic lymphocytic leukemia (C.L.L.). Immunofluorescence of surface immunoglobulins and rosette formation using sensitized sheep red blood cells were employed to detect B cells. Rosette formation with untreated sheep red blood cells and in vitro PHA stimulation and cytogenetics were used to study T cells. The findings generally confirm the conclusions of others that C.L.L. usually represents a proliferative disorder of B cells, probably clonal in nature. In 11 of 13 cases the predominant cell surface immunoglobulin was IgM; in 1 case it was IgG, and in 1 case there was only a slight elevation in the incidence of IgM positive cells. In general, EAC rosette studies confirmed the observations made using immunofluorescence, but in one case this correspondence was apparent only after the incidence of EAC rosettes was increased by pretreatment of the C.L.L. cells with 2‐mercaptoethanol. In another case, the incidence of EAC rosette‐forming cells significantly exceeded the incidence of immunofluorescent cells, perhaps suggesting a variable expression of different surface markers on neoplastic cells in C.L.L. E rosette studies and PHA cultures indicated a decreased proportion of T cells in the peripheral blood of these patients, but suggested that the absolute number of T cells was not reduced. Further, kinetic and cytogenetic studies in PHA cultures demonstrated no qualitative abnormality in the circulating T cells in this disease, supporting the view that they represent a normal population diluted in a large pool of neoplastic B cells.

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Specific Fractionation of Immune Cell Populations

Cited by 55 publications

References 10 publications

Binding of Thymus- and Bone Marrow-Derived Lymphoid Cells to Antigen-Derivatized Fibers

Binding of Thymus- and Bone Marrow-Derived Lymphoid Cells to Antigen-Derivatized Fibers

Receptor Mobility and the Mechanism of Cell-Cell Binding Induced by Concanavalin A

Characterization of lymphocyte subpopulations in chronic lymphocytic leukemia

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