REDUCED EXPRESSION OF DISTINCT T-CELL CD MOLECULES BY COLLAGENASE DNASE TREATMENT

DNase/collagenase treatments are widely used to obtain single-cell sus pensions of tumour cells and tumour-infiltrating T lymphocytes (TIL) f rom solid tumours. Since the functional integrity of such cells has be en questioned, we have studied whether treatments with commonly used p reparations of these enzymes could affect the expression of lymphocyte surface molecules and lymphocyte proliferative responsiveness. With p eripheral-blood-derived T cells as a model, flow-cytometric analysis r evealed strongly reduced expression of distinct CD molecules for each enzyme, notably CD2, CD4, CD8 and CD44 for DNase, and CD4, CD14, CD16, and CD56 for collagenase. The effects were found to be due to proteas e contaminations present in all but the purest enzyme preparations tes ted. Addition of serum or trypsin inhibitor abolished the effects. Sin ce serum-free media are widely used to expand tumour-infiltrating T ce lls for clinical therapeutic use, data from early phenotypic analyses can be strongly misleading. Even after an 18-h rest period following t he enzyme treatments, re-expression of the affected membrane markers w as still far from complete. On the other hand, despite strongly reduce d expression of CD2 molecules on the lymphocyte membrane, anti-CD2-ind uced proliferation was not affected, showing the redundancy of this si gnal molecule. Since other important T cell activation molecules (TCR, CD3, CD28) were not affected by enzymatic treatment, the use of expen sive, highly purified collagenase/DNase preparations does not seem to be mandatory in clinical studies with expanded TIL.