The Clonal Selection Hypothesis Evaluated by Grafted Cells Reacting Against their Hosts

335

Using limiting dilutions of responding cells in mouse mixed leukocyte cultures, we obtained direct estimates of the minimum frequency of precursors of cytotoxic T lymphocytes (CTL.P) for a variety of antigens. Depending on the strain combination, there were as many as 4-15 CTL.P reactive to DBA/2 among 10(4) lymph node cells. Taking into account that only 5-10% of peripheral T lymphocytes have the potential to develop into cytotoxic T lymphocytes (CTLs) (6), this implies that at least 1-2% of all CTL.P are responsive to any given H-2 haplotype difference. Precursors of cytotoxic cells thus have the same high frequency of cells reactive to alloantigens of the major histocompatibility complex as found among proliferating cells in graft-vs.-host reactions and mixed lymphocyte interactions. The frequencies of CTL.P reactive to xenoantigens (rat) or trinitrophenyl-modified self were less than half the frequency of alloreactive CTL.P. A minority of the CTL.P specific for one H-2 haplotype were also reactive to a third party H-2 haplotype, presumably on the basis of recognition of shared determinants. By dilution of sensitized cells from single microcultures, it was shown that a single CTL.P undergoes a minimum of three to four cell divisions and generates at least 8-16 CTLs after antigenic activation.

Section: Assessments Of Proliferation and Cytotoxicmentioning

confidence: 56%

Histocompatibility antigen-activated cytotoxic T lymphocytes. II. Estimates of the frequency and specificity of precursors.

Lindahl

Wilson

1977

335

“…Making use of the strong histocompatibility H-2 system of mice, Simonsen has shown that deliberate sensitization of cell donors against prospective host antigens does not invariably increase GVH immunologic reactivity of subsequently transferred cells. This finding has been interpreted to indicate that the size of the clone of reactive cells in a normal donor against a particular histocompatibility antigen system is already so large that deliberate immunization does not significantly increase its size (5).…”

Section: Discussionmentioning

confidence: 97%

Quantitative Studies on the Mixed Lymphocyte Interaction in Rats

Wilson

Blyth

Nowell̀

1968

249

The proliferative interaction of cultured rat lymphocytes of immunogenetically disparate origin—the mixed lymphocyte interaction—was employed as an experimental model to examine the initial stages of the immune response mechanism. Using mixed cultures of cells derived from parental strain and F1 hybrid rats, in which only the parental lymphocytes respond, the following observations were made on the magnitude and kinetics of the reaction. After initiation of the cultures, there was a latent period of approximately 40 hours during which time no mitotic activity was detected. This inactive phase was followed by a period of proliferation in which previously nondividing cells entered the mitotic cycle for the first time. Activity in the cultures, as detected by incorporation of radioactive thymidine and measured by radioautography or scintillation spectrometry, increased exponentially with a doubling time (T2) of 9–10 hr. In this exponential proliferative phase, lasting approximately 100 hr, the dividing cells underwent a series of rapid sequential divisions with a generation time (Tc) of 8 hr, and few, if any, dropped out of the mitotic cycle. In addition to the cells which first entered mitosis at the beginning of the proliferative phase and then proceeded through multiple divisions, significant numbers of new, previously nondividing cells continued to enter the mitotic cycle during the entire exponential growth phase. The total number of these newly responsive, first division cells throughout the total culture period amounted to 1–3% of the original parental cell inoculum. This is a surprisingly large proportion of peripheral blood lymphocytes with demonstrable reactivity to a particular antigen system, if it is assumed that these first division cells in vitro are functionally related to the hypothetical antigen-sensitive cells which proliferate and differentiate into immunological effector cells. At present there is no entirely satisfactory explanation for this large number of reactive cells in the mixed lymphocyte interaction.

“…Studies comparing the reactivity of normal and alloimmune lymphoid cells, as assessed by a graft-vs, host assay in vivo (13) or by a proliferative assay in vitro (14) were unable to detect significant differences between the two cell populations. On the other hand, skin grafts appear to be rejected more rapidly by specifically alloimmune hosts (15), and a more rapid and quantitatively greater generation of specific CTL has been observed in the spleens of mice twice immunized with allogeneic tumor cells (4).…”

Section: Discussionmentioning

confidence: 99%

“…More recently, using improved culture conditions identical to those described in this report, we have found that the generation of CTL in MLC may be increased approximately fivefold when the responding spleen cells are taken from alloimmune mice (5). However, since the primary response to alloantigens appears to involve a relatively large number of cells (13,14), it is difficult in each of these model systems to compare the secondary response in the absence of a strong concomitant primary response.…”

Section: Discussionmentioning

confidence: 99%

Generation of Cytotoxic T Lymphocytes in Vitro

MacDonald

Engers

Cerottini

et al. 1974

166

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.