In Vitro Induction of Specific Unresponsiveness of Immunologically Reactive, Normal Bone Marrow Cells

Abstract

Normal rabbit bone marrow cells have been studied according to their immunological reactivity in vitro. The test system involved stimulation by antigen after the subsequent stimulation into cellular proliferation by measuring the uptake of tritium-labeled thymidine. Specific separation of immunological reactivity was obtained by filtration of cells through antigen-coated bead columns. All experimental evidence supported the view that this separation was due to the existence of preformed antibody molecules on the outer cell surface of the antigen-recognizing cells. The response to antigenic stimulation was shown to be strictly dose related and, using supraoptimal concentrations of one antigen, no increased DNA synthesis was recorded. That this state of unresponsiveness represented a state of immunological paralysis was indicated by the normal response of these cells to stimulation by a second antigen in optimal concentration. Thus both methods, cell separation on antigen-coated columns or induction of specific unresponsiveness by antigen in vitro, can produce a cell population specifically devoid of cells reactive against a given antigen.

Lymphocyte Proliferation in Vitro Induced by Hapten Autologous Protein Conjugates

“…A major reason for the increased relative DNA synthetic responses was the frequent drastic reduction in background DNA synthesis of the passed cells. The exact reason for this reduction is unknown but would seem linked with the column separation as such, since normal serum columns will also have this impact (28). However, the present results indicate that purified, immune guinea pig T lymphocytes are quite capable of proliferating in vitro upon antigen stimulation in agreement with the findings of others (16,17).…”

“…0.5 ml of Hyamine was added to each tube and kept over-night at room temperature in complete darkness. The contents of the tubes were then transferred to counting vials containing 10 ml of scintillation fluid (Packard Instrument Inc. Downers Grove, Ill.), cooled to 4°C in the dark and analyzed for radioactivity (see reference 28).…”

Separation of Normal and Immune Lymphoid Cells by Antigen-Coated Columns

“…This was shown by tile selective retention of such cells when being filtered through antigen-coated bead columns in the way previously demonstrated for immune cells (11) and for normal bone marrow cells (12,27,28). The column fractionation could be shown to produce a cellular population being very similar in immunological reactivity to one being induced to become immunologically tolerant by "classical" means (23).…”

“…These experiments failed to enrich anti-NIP-reactive cells and we consider them as providing further information on the mode of action of antigen-coated columns when retaining antigen-reactive cells. It has previously been found that mere changes in the flow rate of filtered cells through a normal serum-coated column will significantly alter the percentage of cells retained for nonserological reasons and, with a sufficiently reduced rate, very few cells will pass through (11,12). Preliminary experiments on the actual profile of antigen-reactive cells coming out at the bottom of the columns strongly suggest that if, eg.…”

“…The increase of the number of reactive cells in such systems after immunization (4)(5)(6)(7)(8)(9)(10) and the specific inhibition of the reactions by blocking antigen (7,8) indicate that lymphoidal cells can display antibody activity on their surfaces. Direct demonstration that these surface receptors could be linked to potential immune capacity of the cells carrying the surface antibodies has come from experiments where filtration of immunologically competent cells through antigen-coated columns was carried out (11,12). In such experiments, specific elimination of the antigen-reactive cells directed against the antigen used for coating the column was obtained as indicated by the reactivity of the passed cells.…”