Generation of Cytotoxic T Lymphocytes in Vitro

Abstract

Mouse cytotoxic T lymphocytes (CTL) were generated in unidirectional mixed leukocyte cultures (MLC) using normal C57BL/6 spleen cells as responding cells and irradiated DBA/2 spleen cells as stimulating cells. Cytotoxicity was assayed on 51Cr-labeled P-815 (DBA/2) target cells, and the relative frequency of CTL in individual cell populations was estimated from dose-response curves. Upon inclusion of 2-mercaptoethanol in the culture medium, it was found that significant CTL activity could be detected for as long as 3 wk in primary MLC. Reexposure of MLC cells to the original stimulating alloantigens after 14–41 days in culture resulted in significant cell proliferation and rapid regeneration of high levels of immunologically specific cytotoxicity. CTL activity in these secondary cultures increased dramatically within the first 24 h and reached higher peak levels than those found at the peak of the primary response. Furthermore, proliferation and reappearance of CTL activity could be demonstrated following each of as many as four sequential alloantigenic stimulations of the same initial cell population at 20-day intervals. Interestingly, cells recovered from MLC at the peak of the primary response on day 4 were insensitive to further allogeneic stimulation. Taken together, these results are consistent with the hypothesis that CTL differentiate in MLC to become long-lived memory cells which gradually lose their cytotoxic activity. Upon reexposure to specific alloantigen, such memory CTL rapidly regain their functional activity and proliferate to generate an expanded CTL population.

Dual function of membrane-bound heat shock protein 70 (Hsp70), Bag-4, and Hsp40: protection against radiation-induced effects and target structure for natural killer cells

“…After a 4-day stimulation period with low-dose IL-2 (100 IU/ml; Chiron) plus 2 mg/ml Hsp70 peptide TKD (aa [450][451][452][453][454][455][456][457][458][459][460][461][462][463] ; TKDNNLLGRFELSG), the cytolytic response was tested against Hsp70 high-expressing CX þ , Colo þ , and HeLa Bag-4 and low-expressing CXÀ, ColoÀ, and HeLa neo cells, either untreated or following irradiation (1 Â 10 Gy) and a recovery period of 24 h, in a standard [ 51 Cr] assay. 54 Briefly, different dilutions of effector and target cells (40 : 1 to 2 : 1) were coincubated in duplicates for 4 h at 371C, in a final volume of 200 ml. Then, supernatants (30 ml) were collected and radioactivity was measured on a g-counter (Topcount, Packard instrument).…”

Generation of Cytotoxic T Lymphocytes in Vitro

“…During the course of tumor allograft rejection in mice, the distribution in sedimentation velocity of splenic CTL changes with time, suggesting that a transition from large to small CTL may occur (7)(8)(9). Since CTL could be detected for several weeks in MLC under appropriate culture conditions (13), it was of interest to investigate the physical properties of CTL induced in vitro. For this purpose, multiple cultures of 25 x 10 ~ C57BL/6 spleen cells and 25 × 10 e irradiated DBA/2 spleen cells were established.…”

“…Since a rapid appearance of high levels of CTL activity could be demonstrated following secondary antigenic stimulation in long-term MLC (13), it was of considerable interest to investigate whether residual CTL were responsible for this activity. To this end, day 14 MLC cells were separated by velocity sedimentation at unit gravity and divided into three pooled fractions corresponding to those described in the previous section.…”

“…For purposes of comparison, the distributions of viable cells (I---O) and of LU (C)---O) for this latter separation are expressed relative to the same total number of fractionated cells as in Fig 2 a. systems, notably the MLC, has provided a basis whereby the inductive and efferent aspects of T-lymphocyte function can be studied and compared to earlier in vivo findings. By extending this in vitro model to encompass the detection and restimulation of CTL after prolonged periods of time in culture, as described here and in a previous report (13), it was hoped that an approach to the more general study of T-cell differentiation could be made.…”

Evidence for direct linkage between antigen recognition and lytic expression in effector T cells.

“…Thus, either there was no reciprocal cell death, or the a effector cells greatly exceeded the d effector cells. Although there is, as yet, no reliable indicator of the absolute number of effector cells present in a lytically active cell population, it is a common practice to consider that populations with comparable levels of cytolysis have a similar incidence of effector cells (10). As the cytolytic activity of the a anti-d and d anti-b cell populations towards homologous tumor target cells was comparable in the studies reported here, we assume that the effector cell incidence in the attacking and target populations did not markedly differ.…”