Quantitation of T cell depletion by limiting dilution analysis

We evaluated a culture method for enumeration of residual T cells remaining in marrow after treatment with antibody and complement or with immunotoxin.

Marrow cells were cultured at limiting dilutions with phytohemagglutinin in the presence of Epstein Barr virus transformed human lymphoblastoid cells, and supernates were tested three days later for IL-2 by a cell proliferation assay. This method provides a simple, reliable, objective and rapid enumeration of T cells in marrow before and after treatment.

Approximately 6% of untreated marrow mononuclear cells can produce IL-2 in such clonal cultures. Treatment with antibodies and complement under conditions identical to those used for our previous clinical trials produced a 3.7 log depletion of IL-2 precursors, whereas treatment with a ricin A chain anti-CD3 immunotoxin produced a 3.0 log depletion.

Clinical correlations are in progress for assessing whether T cell depletion evaluated with the present method equals previous techniques. The extreme depletion of T cells accomplished by these methods may partly account for the high graft failure rate seen in our clinical trials.