Analysis of clones of cytotoxic lymphocytes

Abstract

1. ‘Spotaneously’ generated cytotoxic clones were detected when normal spleen cells from CBA, DBA/2 or (CBA x DBA/2)F1 mice were cultured in polyacrylamide cultures vessels without stimulator cells. Cytotoxicity was mediated by T cells and the highest number of clones occurred after 4 days in culture. The spontaneous cytotoxic T cell clones were detected mainly in adult spleen cell cultures. Few spontaneous clones were generated by lymph node, thymus or neonatal spleen cells. 2. The production of spontaneous clones does not increase linearly with the number of cells cultured which is in contrast with the production of ‘stimulated’ clones of cytotoxic lymphocytes in the polyacylamide vessels. At the optimal cell concentration, 1.3 x 10 7 cells per culture, 20 spontaneous clones of CLs lysing P815 mastocytoma cell targets were detected in cultures of (CBA x DBA/2)F1 spleen cells and 14 clones were detected in cultures of CBA or DBA/2 spleen cells. 3. The specificity of the spontaneous clones was examined by dividing each clone into two halves and assaying the half clones against a pair of different target cells. A range of spontaneous CLs of different specifications was produced. Spontaneous clones lysing syngeneic and allogeneic tumour or normal spleen cell blasts, as well as hapten-modified target cells were detected. 4. Individual spontaneous clones of CLs exhibited a high degree of discrimination and were able to differentiate between many pairs of target cells which were syngeneic with respect to each other. 5. When blast cells which had been induced by various mitogens were used as the target cells, the results indicated that spontaneous CL clones could discriminate between subsets of syngeneic lymphocytes which respond to different mitogens. 6. One-way stimulated CL responses were generated using cells from mice which had been treated with cyclophosphamide. Treatment of mice with 200mg/kg cyclophosphamide abolished the ability of the cells to generate spontaneous clones in culture without impairing their ability to stimulate the production of CLs in responder cell populations. 7. In contrast with spontaneous clones which could discriminate between different H2d target cells, CLs produced by CBA spleen cells stimulated with H2d alloantigens were observed not to differentiate between various H2d target cells. The results indication that spontaneous clones of CLs were not a representative sample of the stimulated clones of CLs. 8. When CBA spleen cells were stimulated simultaneously with H2b and H2d alloantigens, separate populations of Cls against the two sets of antigens were produced. Very few cross-reactive clones were detected. 9. When (CBA x C57B1)F1 spleen cells were stimulated with syngeneic F1 cells modified with TNP, clones of CLs lysing TNP modified cells of the F1 and the two parental strains were produced. The frequency of clones produced by F1 spleen cells against F1-TNP, CBA-TNP and C57B1-TNP target cells was 1 per 3.3 x 10 4, 1 per 6.7 x 10 4 and 1 per 10 5 spleen cells respectively. When (CBA x C57B1)F1 cells were cultured with TNP-modified CBA cells, clones of CLs against TNP-modified cells of both the parental strain were produced. CLs which lyse CBA-TNP and CLs which lyse C75B1-TNP targets segregated as two distinct populations with no cross-reactivity.