Enumeration and isolation of human T and B lymphocytes by rosette formation with antibody-coated erythrocytes

Abstract

Rosette techniques are presented for the enumeration and separation of both Ig+ T- and Ig- T+ human lymphocytes. In order to enumerate Ig+ cells, the direct immunocytoadhesion technique was employed using human erythrocytes (E) coated with purified anti-kappa or anti-lambda light chain antibodies. Specificity of these rosettes was shown with chronic lymphocytic leukaemias of either the kappa or lambda type. T+ cells were enumerated by a new indirect rosette technique in which the lymphocytes were initially treated with rabbit anti-human thymus cell antiserum followed by direct rosetting with human E coated with purified anti-rabbit light chain antibody. For normal individuals, 24-32% Ig+ T- cells and 65-71% Ig- T+ cells were found among the lymphocytes of peripheral blood as well as tonsils with these rosette methods. The Ficoll-Hypaque method was used to obtain purified Ig- T+ and Ig+ T- cells by removing rosetted Ig+ cells or T+ cells, respectively. The purity of the Ig- T+ cells was indicated by greater than 99% indirect rosetting of cells sensitized with anti-human thymus cell antibody (Ab) and by less than 1% direct rosetting with anti-kappa Ab-E+ anti-lambda Ab-E. The purity of the Ig+ T- cells obtained was indicated by 92-96% direct rosetting with anti-kappa Ab-E+anti-lambda Ab-E and by less than 1% indirect rosetting with anti-human thymus cell antibody. A small percentage of Ig- T- 'null' cells could not be identified by either reagent. Thus, essentially pure Ig- T+ and Ig+ T- cells were readily and efficiently isolated by 'negative selection' thereby lessening the possibility of functional changes that may develop by more extensive manipulation of lymphocytes.