In vitro selection of murine antigen-specific T-lymphocytes. I. Description of selection procedure

Abstract

Enrichment of antigen-responsive murine T-lymphocytes was achieved by two in vitro procedures through the preferential adherence of the antigen-specific cells to the antigen-pulsed macrophages and their consequent multiplication. The first procedure involved the addition of column-purified T-enriched lymph node lymphocytes from immunized mice to a monolayer of antigen-pulsed adherent spleen cells from non-primed syngeneic donors. Lymphocytes which failed to adhere to the antigen-pulsed monolayer were removed after 4 h of incubation. The adherent cells were cultured for a week and the lymphocytes obtained after that period from the selection plate were highly responsive to the antigen for which they were selected and for a T-cell mitogen (Con A). On the other hand, these selected cells demonstrated little or no response to other antigens, to which the original donor of the lymphocytes was immune, and to a B-cell mitogen (LPS). The same preferential response to the selecting antigen and T-cell mitogen was obtained in lymphocytes enriched in the alternate procedures on ‘supernatant cultures’. The enriched population from ‘supernatant culture’ was derived from cells that did not adhere to the antigen-pulsed monolayers during 4 h of incubation. The non-adherent lymphocytes which still contained antigen-specific lymphocytes were transferred to a fresh monolayer of antigen-pulsed adherent spleen cells to be grown for a week in culture. The improvement in the response to the selecting antigen and the decreased reaction to other immunizing antigens show that the cells harvested from either ‘selected’ or ‘supernatant’ cultures were enriched for a given antigen — the selecting antigen. In most individual experiments the enrichment was better in the selected cultures. The enrichment procedures were dependent upon effective antigen presentation to the lymphocytes. Spleen cells from mineral oil-injected mice, which are the most effective antigen-presenting cells, formed the most efficient monolayers for the enrichment of the antigen-specific lymphocytes, both in the ‘selected’ and ‘supernatant’ cultures.