Generation of Cytotoxic T Lymphocytes in Vitro

Abstract

The cell-mediated immune response to primary allogeneic stimulation is characterized by the appearance of cytotoxic T lymphocytes (CTL) 1 (see reference 1 for review). Recently, the existence of anamnestic CTL responses to allografts has been suggested by studies in which the ability of normal and alloimmune spleen cells to respond to relevant alloantigens both in vivo (2, 3) and in vitro (4) was quantitatively compared. In other studies, physical characterization of CTL in the spleens of mice undergoing tumor allograft rejection revealed significant differences in size and density between effector cells detected early or late after immunization (5-9), the former being predominantly large and of low density, whereas the latter were mainly small and dense. In view of these observations, two important questions regarding the differentiation and ultimate fate of CTL can be raised: (a) Do the changes in physical properties of CTL with time reflect the differentiation of a single cell lineage, or alternatively the parallel development of several cell lineages? (b) Are the cells capable of mounting an anamnestic response to alloantigens derived from CTL, or are CTL truly end cells capable of no further differentiation?Until individual CTL can be identified and isolated, no definitive answer to these questions is possible. However, with the concomitant development of in vitro systems for the generation of CTL (10-12) and quantitative assays for the estimation of their relative frequency (1, 12), indirect approaches to the study of the differentiation and fate of CTL are now feasible. In a previous paper of this series (13), we presented evidence that an anamnestic response to alloantigens could be demonstrated in long-term mixed leukocyte cultures (MLC). In this

Light scatter analysis and sorting of cells activated in mixed leukocyte culture

“…That this correlation is also true in the case of MLC cells is further substantiated by comparing the FLS distributions of lymphoblasts and lymphocytes observed in this report with the distributions obtained when MLC cells are separated by velocity sedimentation at unit gravity, a technique which has been clearly shown to depend primarily on rz (8). In the latter case, we have previously shown that the modal sedimentation velocities of small T lymphocytes and T lymphoblasts are about 4 mm/hr and 6 mm/hr, respectively (5,6). This relative difference of 1.5-fold in 2 independently derived parameters related to r' would thus imply that the modal radius of T lymphoblasts exceeds that of small T lymphocytes by approximately 22%.…”

“…From previous studies in which day 5 MLC cells were separated by velocity sedimentation at unit gravity (5,6), it was established Figure 2. Modal FLS channel numbers for the two peaks were determined from the gated histograms.…”

Response of tumour-related and normal lymphocytes to antigens on fibroblasts from embryos of varying age

“…4) that the precursor cells for cytotoxic cells underwent size transitions after embryonic antigen challenge/removal in a manner reminiscent of that seen with lymphocytes from animals primed with cells differing at the MHC (see MacDonald et al, 1974;Hayry and Anderson, 1975;Hollander et al, 1974). Furthermore, both tumour bearer/pregnant animals and animals early after removal of in vivo antigen challenge were found to contain a population of cells with peak sedimentation velocity (4 5-7 0 mm/h) which could preferentially inhibit a response to embryoassociated determinants (relative to the response to allo-antigen determinants)-see Table II.…”

“…In an auxiliary test of the similarity of the findings in tumour-related and pregnant mice, we have examined the specificity of cytotoxicity to fibroblast targets of lymphocytes from tumour-resected animals re-stimulated in culture with either Comparison of these data with those of Figs (Hayry and Anderson, 1975;Hollander, Ginsburg and Feldman, 1974;MacDonald, Cerottini and Brunner, 1974). The data collected from such studies have led to the concept of alternate cycles of blastogenesis (and development of cytotoxic potential) followed by quiescence (and a return to inactive small cells).…”

Significance of cytotoxic lymphocytes after various immunizing procedures in a virus-induced non-producer syngeneic system: correlation between in vitro and in vivo lytic activity

“…For this reason, the Macdonald and/or Fitch hypotheses should be kept in mind when considering the case in hand. Of these two hypotheses we tend to prefer the first, since pre-killers, mature effectors and memory cells were shown (Macdonald et at., 1974b) to exist in different stages of cellular differentiation, where precursors and memory cells are high-density small lymphocytes and effectors are characterized as large, lower-density lymphocytes.…”