T cell depletion of bone marrow for clinical marrow allografting: optimalization of conditions for depletion by anti-CD2 and anti-CD8 monoclonal antibodies with rabbit complement, and for detection of residual T cell content

Normal bone marrow obtained at harvest for bone marrow transplantation was passed over a Ficoll density gradient to obtain the mononuclear cell fraction. These cells were incubated with anti-T cell monoclonal antibodies (MAb) anti-HuLym-1 and anti-HuLym-8 plus rabbit complement, washed, and the degree of T cell depletion was assessed by culture in phytohemagglutinin, by flow cytometry and by limiting dilution analysis. The optimal protocol for T cell depletion was found to be a 2-stage incubation with 2 cycles of complement treatment, using a bone marrow cell concentration as low as possible. The precise conditions of complement dilution and incubation temperature depended on the batch of complement used, and had to be assessed for each batch. Of the 3 methods used for assessing T cell contamination, culture in PHA was found to be inappropriate due to large variation in background proliferation of treated and untreated bone marrow cells. Flow cytometry was accurate for residual T cell content of greater than 1% (corresponding to a 90-95% depletion of T cells) but could not detect lower levels of T cell contamination. The limit dilution assay was found to be by far the most sensitive, being capable of detecting a T cell contamination of 1 in 10,000 cells. When combined with flow cytometry on untreated marrow for calculation of the cloning efficiency, it gave very accurate determinations of residual T cell content of marrow.