This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Mukherji, B. Articles by Nathanson, L. Search for Related Content PubMed PubMed Citation Articles by Mukherji, B. Articles by Nathanson, L.

Variables and Specificity of in Vitro Lymphocyte-mediated Cytotoxicity in Human Melanoma¹

Tufts University School of Medicine and New England Medical Center Hospitals, Boston, Massachusetts 02111

In vitro cell-mediated cytotoxicity (CMC) assays have been carried out in human melanoma system with blood effector lymphocytes on [³H]proline-labeled target cells in a 48-hr microcytotoxicity technique. Three lymphocyte purification procedures (Ficoll:Hypaque gradient, plasma gel sedimentation followed by nylon column incubation, and plasma gel sedimentation followed by separation with nylon powder and glass beads) are compared in parallel experiments for characteristic effector cell composition and cytotoxic potential against target cells of dissimilar histology. The cytotoxicity is defined by the loss of target cell ³H cpm as measured by residual target cell ³H cpm in individual microwell following incubation with lymphocytes. Target cell ³H cpm loss by test lymphocytes is compared with target cell ³H cpm loss by several age and sex matched control lymphocytes (from normal donors and unrelated cancer patients); further comparison between the various control lymphocytes is also made in each assay. As control for target cells, autologous fibroblasts and homologous tumor cells of dissimilar histology are always included in each assay. Specific cytotoxicity is defined as statistically significant and selective destruction of only melanoma cells by the test lymphocytes as compared to the control lymphocytes. Significant but nonselective destruction of 2 or more target cells of unrelated histology is regarded as nonspecific cytotoxicity, while no destruction of any target cells signifies no cytotoxicity.

The Ficoll:Hypaque preparations consistently exhibit the highest nonlymphocytic cell contamination (8 to 16%); the nonlymphocytic cells are, almost exclusively, monocytes. They also produce relatively high percentage of thymus independent (B) cells (8 to 15%). The ultimate cell composition of the 2 plasma gel-nylon preparations is essentially identical. In either plasma gel-nylon preparations, the nonlymphocytic contamination is minimal (0 to 4%) and thymus-dependent (T) cell percentage is considerably higher (92 to 99%) with none or few B cells. Nylon column fractionation of Ficoll:Hypaque preparation exhibits the same characteristics of any nylon preparation. Results of the CMC assays indicate that the control lymphocytes often produce variable base-line cytotoxicity. Interpretation of cytotoxicity by test lymphocytes, therefore, varies since specificity of cytotoxicity by test lymphocytes is determined in relation to control lymphocytes. Dramatic reversal of interpretation is often seen in the same assay when comparison is made between test lymphocyte cytotoxicity and different control lymphocyte cytotoxicity. Target cells of dissimilar histology exhibit significant variation in sensitivity to a given lymphocyte preparation even under identical experimental conditions. Furthermore, interpretation of specificity may vary when comparison is made between different target cells incorporated in the assay.

These results do not argue against demonstration of tumor-related specificity in in vitro CMC assay in human tumor system(s). Despite variabilities in the cytotoxic behavior of different effector cell preparations and in the sensitivity of different target cell types to a given effector cell preparation, tumor-specific cytotoxicity by autologous and/or allogeneic lymphocytes agaisnt tumor cells of similar histology might be demonstrated through large scale in vitro CMC assay and by appropriate statistical analysis. Results of this study, however, suggest that biological significance of such "specific," perhaps more appropriately, "selective," cytotoxicity should be drawn in context of these possible variables in the assay.

¹ This work was supported in part by NIH Cancer Research Center Grant CA-12924-03 and General Research Support Grant, New England Medical Center Hospitals RR05598-09.

² To whom reprint request should be sent, at Tufts University School of Medicine and New England Medical Center Hospitals, 171 Harrison Avenue, Boston, Mass. 02111.

Received 5/23/75. Accepted 9/ 4/75.