T lymphocytes can mediate lysis of autologous melanoma cells by multiple mechanisms: Evidence with a single T cell clone

Abstract

The specificity analysis of a CD3+, WT31+, CD8+ cytotoxic T lymphocyte (CTL) clone (CTL 49), isolated from peripheral blood lymphocytes of a melanoma patient (no. 665) after mixed lymphocyte culture with an HLA-A2+ allogeneic lymphoblastoid cell line (VSKB-LCL), revealed that CTL 49 could lyse, in addition to HLA-A2+ lines, autologous HLA-A2- melanoma (Me665/2) and K562 targets. Killing of VSKB-LCL, but not of Me665/2, could be inhibited by anti-CD3 and by anti-HLA-A2 antibodies or by modulation of the CD3 complex. Cold-target competition studies showed that K562, but not VSKB-LCL, could compete with Me665/2 for lysis by CTL 49. However, unlike K562, Me665/2 could be lysed by CTL 49 in a Ca2(+)-independent fashion in 4 h and 18 h assays. CTL 49 expressed mRNA specific for tumor necrosis factor (TNF alpha) and, to a lesser extent, for lymphotoxin (TNF beta). Exposure of the clone to anti-CD3 antibodies induced the expression of interferon(IFN)-gamma-specific mRNA. Antibodies to TNF alpha, TNF beta and IFN reduced the lysis of Me665/2, but not of K562, by CTL 49 in 18-h cytotoxic assays. Antibodies to TNF alpha and to IFN gamma almost completely inhibited the lysis seen on Me665/2 (but not on K562), in 96-h assays, by supernatants isolated from VSKB-LCL- or anti-CD3-stimulated CTL 49 cells. Taken together, these data indicate that major-histocompatibility-complex-independent lysis of autologous tumor cells and of natural killer reference targets by the same alloreactive T cell clone are activities related at the level of target recognition but distinct at the level of the lytic hit. Thus, efficient lysis of autologous tumor cells results from a complex mechanism based upon direct effector-target interaction as well as on cytokine-mediated cytolytic effects.

Cell retargeting by bispecific monoclonal antibodies. Evidence of bypass of intratumor susceptibility to cell lysis in human melanoma.

“…Unlike the effector cells used in other studies (22)(23)(24)(25)(26)(27), the CTL 49 clone used in the present study is per se capable of recognizing and lysing the autologous melanoma cells in a TCR-independent fashion. As previously reported (13), the killing activity of CTL 49 cells takes place through multiple lytic mechanisms mediated by both direct cell-cell contacts and by the release of soluble cytotoxic factors.…”

“…These observations clearly indicate that HMW-MAA, which is recognized by Ep2 mAb, represents an ideal target, being expressed by the majority of melanoma cells with an epitope density able to mediate antibody binding and cell killing. The CTL 49 cells (CD3+, WT3 1+, CD8+) were selected as the effector clone because of their ability to lyse the autologous melanoma cell lines in a TCR-independent fashion ( 13). CTL 49 cells were tested for recognition by all the parental and the bsmAbs used in the study.…”

“…Effector cells CTL 49 is an alloreactive T cell clone directed to HLA-A2 related determinants not expressed on the autologous melanoma cells ( 13). The clone was obtained as follows from PBL of a melanoma patient (patient 665), from whom tumor cells lines and clones were originated (see Tumor cells and clones): 5 x 106 PBL ofthe patient were stimulated with 5 x 106 irradiated (8,000 rad) VSKB-LCL cells (HLA-A2,3; B44,35; CW4; DR1,4; DP4,DP6) in 10 ml of 20% heat-inactivated human serum (HS) in RPMI 1640 medium.…”

“…Simple genetic complementation produces vectorcontaining particles, which then are released in the culture medium and infect the hybridoma cells. Such procedures allow the recovery of 30-50% resistant cells within [10][11][12][13][14] ( 10). The results ofthe present study clearly show that bsmAbs can significantly increase the lysis oftumor cell lines and clones exerted by autologous lymphoid cells via a T cell receptor (TCR)-independent pathway, thus overcoming intratumor heterogeneity of susceptibility to CTL lysis.…”

Generation and characterization of two human alpha/beta T cell clones. Recognizing autologous breast tumor cells through an HLA- and TCR/CD3-independent pathway.

“…Available experimental evidence indicates, in fact, that the CD3-TCR complex is unlikely to play a functional role in this type of effector-target recognition (27). Although the ability of our clones to retain their tumor specificity over a long period of time strongly suggests that they may belong to a specific effector lineage, additional experimental work is needed to confirm this hypothesis.…”