In vitro growth of cytotoxic human lymphocytes. III. The preparation of lectin-free T cell growth factor (TCGF) and an analysis of its activity

Supernatants containing T cell growth factor (TCGF) that are produced by incubating human peripheral blood lymphocytes with the mitogenic lectin, phytohemagglutinin (PHA) have previously been shown to allow the successful culture of antigen-reactive T cells. The continued presence of PHA in these preparations, however, complicates the analysis of the direct effects of TCGF. In this report we describe in detail methods used to remove >99% of the exogenous mitogen with a 77% yield of TCGF. TCGF activity precipitated between 50 and 75% saturated ammonium sulfate and was freed of 90% of exogenous PHA. Residual PHA was removed by absorption on affinity columns of Sepharose 4B linked to rabbit anti-PHA antibody. Ninety-nine percent of PHA was removed by these techniques based on assays of the mitogenesis of fresh lymphoid cells and the use of tracer quantities of highly purified radiolabeled PHA. This partially purified TCGF (PP-TCGF) supported the continued growth of mitogen or alloactivated lymphoid cells but not fresh resting cells unless high starting numbers of lymphoid cells are used. An increase of up to 500 times the number of specific lytic units/culture was obtained by expanding alloactivated cells in PP-TCGF when compared to the crude TCGF supernatants.