Generation of cytotoxic T lymphocytes in vitro. IV. Functional activation of memory cells in the absence of DNA synthesis.

Abstract

The generation of mouse cytolytic T lymphocytes (CTL)' in mixed leukocyte cultures (MLC) is a well characterized model system in which both the afferent and efferent phases ofT-cell-mediated immunity can be studied (1-3). Recently, the potential usefulness of this model has been extended by the demonstration that anamnestic CTL responses can occur under appropriate culture conditions . In particular, re-exposure of long-term MLC cells to the original stimulating alloantigens was found to result in significant proliferation and in rapid reappearance of high levels of CTL activity (4, 5) .Kinetic studies of the regeneration of CTL activity under these conditions revealed that cytotoxic activity increased significantly within the first 24 h with no detectable increase in viable cell number, but that activity and cell number increased in parallel thereafter (4). These results hence suggested the possibility that CTL activation may be dissociated from proliferation during the initial 24 h of alloantigenic re-exposure.In this report, we-have taken a more direct approach to the investigation of the relationship between differentiation and proliferation of restimulated CTL. By re-exposing long-term MLC cells to alloantigens during 24 h in the presence of cytosine arabinoside (ARA-C), a potent inhibitor of DNA synthesis, we have found that functional activation of CTL occurs even when DNA synthesis is completely blocked. Furthermore, morphological, immunological, and cell separation experiments indicate that this functional activation is accompanied by an enlargement of the responding small T lymphocytes.

Primary generation of cytolytic T lymphocytes in the absence of DNA synthesis.

“…Furthermore, CTL generated in the presence of ARA-C wcrc characterized as large cells by vclocitysedimentation cell separation (H. R. MacDonald, and R. K. Lees, unpublished data). Because the functional activation of memory CTL in a secondary response is known to bc independent of DNA synthesis (4,8,16), it could bc argued that the cells in normal splccn which arc stirnulatcd by Con A arc memory CTL resulting from immunization with cross-rcacting environmental antigens. This would seem unlikely, however, because no PHA-dcpendcnt cytolytic activity was observed either in fresh spleen cells or after 24-h culture in the presence of Con A.…”

Cytotoxic T-cell activation by polyribonucleotides: DNA synthesis is not required.

“…Since this induction is polyclonal, a large proportion of T cells should be activated, abrogating the need for clonal expansion required in an antigendependent clonally restricted response. A similar situation exists in the secondary CML response where cytotoxic cells are rapidly induced in the absence of DNA synthesis (27).…”

“…It has been reported that DNA synthesis is required for the generation of cytotoxic cells by alloantigens or mitogens (8-10). In contrast, there is at least one report of cytotoxic cell activation independent of DNA synthesis (26,27). To determine whether DNA synthesis accompanied poly A:U activation of cytotoxic cells, normal A/sn or CBA spleen cells were activated by poly A:U (100/~g/ml) during a 2 h incubation at 37°C.…”

Tumor metastases and cell‐mediated immunity in a model system in DBA/2 mice.: Iv. Antigenic differences between a metastasizing variant and the parental tumor line revealed by cytotoxic T lymphocytes

“…CMC (1) by its quicker CD ur ~i m as susuumlul u x s \R = L A iv , * iiviii IV down to 6 x lo4). The effector cells generated in these cultures were then tested for cytotoxic activity at an E:T ratio of 50:l against either Eb or ESb tumor target development (Engers and MacDonald, 1976), (2) by the fact that it does not require proliferation (MacDonald et al, 1975), and (3) by the less stringent requirements of antigen presentation for lymphocyte activation (Wagner et ul., 1976). In the present study we have made use of the high cytotoxic activity of a sec.…”