Lymphocyte surface markers in sheep blood and lymph

Lymphocytes from sheep blood and lymph were analysed for the percentages of cells which had receptors for Fc mu, Fc gamma or C', which had surface immunoglobulin (sIg) or which could form rosettes with sheep red blood cells in the presence of saline (E) or 14% Ficoll-saline (Efic). Compared with lymphocytes prepared by treating blood with carbonyl iron followed by glycerol lysis of the red cells, lymphocytes prepared by Ficoll-Paque flotation were enriched for B-cells (sIg+), depleted of T-cells (Efic+) and retained a high proportion of phagocytic cells. Efferent popliteal lymph contained almost twice the percentage of Efic+ cells as did blood prepared by glycerol lysis, but only half the percentage of sIg+ cells. An unmarked or 'null' cell population was defined by subtracting the percentage of Efic+ and sIg+ cells from 100%. In glycerol lysed blood 39.0% of the cells were 'null' compared with 18.6% in lymph. Fractionation of lymphocytes on Percoll discontinuous density gradients allowed some separation of T, B and 'null' cells from each other. With blood lymphocytes, 'null' cells were concentrated in the least dense region (1.01 g/ml), B-cells in the region of intermediate density (1.05-1.07 g/ml) and T-cells in the most dense regions (1.08-1.09 g/ml). Separation of blood and lymph cells on nylon wool columns and anti-immunoglobulin affinity plates showed that 'null' cells were relatively non-adherent and that there was a small population of C' receptor-bearing lymphocytes which were not sIg+. Phytohaemagglutin (PHA) stimulation of lymphocytes prepared from blood, using Ficoll-Paque or glycerol lysis and from lymph, showed that all three preparations had similar dose-response curves irrespective of their T:B cell ratios. Interestingly, the 'null' cell enriched fraction from Percoll gradients also responded well to PHA. It was concluded that sheep blood contains three major populations, T, B and 'null' and that T-cells (Efic+) are relatively concentrated in efferent popliteal lymph compared to blood. Some separation of these three cell types could be obtained using Percoll density gradients, nylon wool columns or anti-immunoglobulin affinity plates. Comparing the PHA responsiveness of lymphocytes from different layers of the Percoll gradients, it was concluded that the sheep blood 'null' cells should be considered to be part of the T-cell population.