How do cultured CD8+ murine T cell clones survive repeated ligation of the TCR?

Satoshi Sugawa,1, Deborah Palliser, Herman N. Eisen and Jianzhu Chen

Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

Correspondence to: J. Chen


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Many murine T cell clones grow continuously in culture despite weekly ligation of their TCR by antigen. To learn how the cultured cells avoid or minimize antigen-induced cell death (AICD), we compared Fas and tumor necrosis factor (TNF) receptors (TNFR) on several long-term cultured CD8+ T cell clones with those on naive and activated naive cells expressing the same TCR (2C). In contrast to the naive cells, Fas was absent on the cultured clones and the TNFR-II receptor, present initially at high levels on the cultured cells, was rapidly down-modulated in response to TCR ligation and had virtually disappeared by 2 h, when only ~10% of the cloned cells had been induced to express TNF-{alpha}. The extent of AICD of the cultured clones in response to cognate peptide–MHC on the presenting cells used for routine stimulation of the cultures was also considerably less than the massive cell death of the clones following exposure to anti-CD3 antibody plate-bound at high density.

Keywords: activation-induced cell death, cytolytic activity Fas, TCR, tumor necrosis factor receptor


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In response to ligation of the antigen-specific receptor (TCR) on naive T cells, the cells become activated: they increase in size, express altered surface molecules, secrete cytokines and proliferate. When, however, the TCR on recently activated T cells is ligated, most of the cells die [`activation-induced cell death' (AICD)] (1,2). The AICD response is mediated primarily through receptors for `death cytokines', Fas ligand (FasL) and tumor necrosis factor (TNF)-{alpha} (2). It can be elicited with diverse ligands for the TCR, including cognate peptide–MHC complexes on antigen-presenting cells (APC), anti-CD3 antibodies and several bacterial endotoxins, and it has been seen with diverse T cells (e.g. 3–8). By preventing excessive antigen-driven accumulation in vivo of T cells that are responsive to a few antigens, AICD apparently serves as one of the homeostatic mechanisms that regulates the total number and diversity of the body's T cell population.

Are murine T cell clones that are grown continuously in culture, some of them for years, an exception to the apparent universality of the AICD response by activated T cells? The question arises because in order to maintain the clones in culture they have to be stimulated at frequent intervals, typically once a week, with growth factors (such as IL-2) and APC that display cognate peptide–MHC complexes. When stimulated in this fashion and appropriately diluted, many cultured clones can increase in cell number 5- to 10-fold per week, week after week, while maintaining normal functional responses to TCR ligation. Although the cells in long-term culture seem to avoid AICD, or at least massive AICD, they are clearly capable of dying through some forms of apoptosis. They can, for example, serve as target cells and undergo lysis when specifically recognized by appropriate cytotoxic T lymphocytes (CTL) (9), a process that depends upon activation of the lysed cells' caspases (10).

Many possible explanations can be envisioned to account for the apparent ability of long-term cultured T cells to avoid or minimize AICD. One is that the cultured cells fail to produce sufficient death cytokines (FasL and TNF-{alpha}). Another is that they fail to express receptors for these cytokines. To investigate the basis for the ability of these clones to grow so successfully as cell populations we have focussed primarily on their Fas and TNF receptors (TNFR), comparing those on the cultured clones with those on CD8+ T cells taken freshly from mice, including naive cells that were either unstimulated or recently activated by exposing them to cognate peptide–MHC complexes (called `freshly activated' cells). The naive and freshly activated cells, and three of the four long-term cultured clones expressed the same TCR (2C).

We found pronounced differences between death receptors on the cultured clones, and on naive and freshly activated cells. We also found that the long-term T cell clones underwent extensive cell death when stimulated by anti-CD3 antibody plate-bound at high density. When, however, the stimulus was provided by the APC used for routine maintenance of the cultures (e.g. irradiated tumor cells displaying cognate peptide–MHC complexes) the extent of cell death was, on the whole, considerably less. Finally, we observed that three 2C clones independently derived from 2C TCR transgenic mice differed in their susceptibility to TCR-mediated cell death, and that these differences roughly paralleled variations between the clones in their cytolytic activity and in the rate at which they up-regulated CD69 in response to TCR ligation.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells
CD8+ CTL clones L3.100, K and G3.1 expressing the 2C TCR were derived from mice expressing the 2C TCR transgenes on the H-2b background (11). The 2C TCR responds strongly to SIYRYYGL (referred to hereafter as SIRGL) peptide in association with MHC class I Kb molecules (syngeneic) and to allogeneic Ld+ cells, particularly in association with QL9 peptide (QLSPFPFDL) (1214). All three clones were maintained in long-term culture by stimulating them at 7-day intervals with irradiated P815 (Ld+) cells (15,000 rad). The CD8+ clone 4G3, which recognizes an ovalbumin peptide (SIINFEKL) in association with Kb (15), was stimulated at 7-day intervals with irradiated EL4 cells that expressed the ovalbumin transgene (16). All clones were grown in supplemented RPMI 1640 containing 10% heat-inactivated FCS, 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM ß-mercaptoethanol, 4% rat concanavalin A supernatant (supernatant from rat spleen cells stimulated with concanavalin A, a source of IL-2) and 4% {alpha}-methylmannoside.

Naive 2C T cells were collected from inguinal, brachial, axillary, mesenteric and periaortic lymph nodes from RAG-1-deficient (RAG-1-/-) mice expressing the 2C TCR transgenes (2C/RAG mice) (17). Over 95% of lymph node cells were 2C T cells: they were suspended in the supplemented RPMI 1640 medium described above, but without the rat concanavalin A supernatant and {alpha}-methylmannoside, and used directly as naive T cells in the AICD assays.

Some naive 2C T cells were stimulated ex vivo with irradiated C57BL/6 (B6, Kb) splenocytes (4000 rad) plus 1x10-8 M SYRGL peptide. After 3–5 days these cells, termed `activated' 2C cells, were examined in AICD assays. Memory 2C cells were from lymph nodes of RAG-1-/- recipient mice that had been adoptively transferred with naive 2C cells and immunized with SIRGL peptide in complete Freund's adjuvant 6 months previously (18). Over 70% of the lymph node cells were memory 2C T cells and were used directly for AICD assay.

Assay for AICD
To assay for AICD, 1x105 cells in 100 µl supplemented RPMI 1640 lacking T cell growth factors (rIL-2 or rat concanavalin A supernatant) were added to wells of 96-well plates (Immulon 4 HBX; Dynex, Chantilly, VA) with or without various stimuli. The principal stimuli were (i) plate-bound anti-mouse CD3{varepsilon} antibody (2C11) (19) added to wells initially at 10 µg/ml or (ii) irradiated allogeneic P815 cells (1x105 cells/well). In a few experiments cells were also stimulated with irradiated syngeneic B6 splenocytes (1x105 cells/well) in the presence or absence of 1x10-8 M of SIRGL peptide. Also tested as stimuli for AICD were plate-bound antibody to mouse Fas (PharMingen, San Diego, CA) at 10 µg/ml, or polyclonal goat IgG to mouse TNFR-I and -II (R & D Systems, Minneapolis, MN) at 10 µg/ml. For blocking experiments, TNFR–Ig fusion protein, a gift from Dr Paul Rennert of Biogen (20), was used at 25 µg/ml. After 24 h the cells were vigorously re-suspended, stained with anti-CD8 antibody, and mixed with a known number of microbeads (5.9 µm; Duke Scientific, Palo Alto, CA) and propidium iodide (PI) at 1 µg/ml. Viable CD8+ cells were counted by flow cytometry in a FACSCalibur (Becton Dickinson, San Jose, CA) by using forward light scattering parameters to include blast cells, gating on CD8+ PI- cells and collecting a fixed number of microbead events. All assays were performed in triplicate. The results (mean ± SD) are expressed as the percentage of viable cells that survived the treatment normalized to control wells, which contained T cells in the absence of TCR-ligating reagents.

Flow cytometry
Antibodies to CD8, IL-2R{alpha} (CD25), CD69, and Fas were purchased as conjugates from PharMingen. Biotinylated antibodies to TNFR-I and -II (R & D Systems) were detected by streptavidin–phycoerythrin. Clonotypic antibody for 2C TCR (1B2) (21) was labeled with FITC. To detect intracellular TNF-{alpha}, cells were stimulated with irradiated B6 splenocytes in the presence of 1x10-8 M of SIRGL peptide and 10 µg/ml of Brefeldin A, surface-stained with the 1B2 antibody, and permeabilized in PBS containing 1% paraformaldehyde and 0.01% Tween 20 for 1 h on ice. The cells were finally incubated with anti-TNF-{alpha} (rat IgG; PharMingen) and fluorescent anti-rat IgG (Kirkegaard & Perry, Gaithersburg, MD), and examined in a FACSCalibur using CellQuest software.

Assay for cytolytic activity
51Cr-labeled T-2Kb target cells were incubated with CTL clones in the presence of various concentrations of SIRGL peptide. After 4 h, 51Cr in the supernatants was counted. All samples were assayed in triplicate. Percentages of specific lysis was calculated as [(experimental count – spontaneous count)/(maximum count–spontaneous count)]x 100. Maximum counts were determined with the labeled target cells in 0.1% NP-40. Background values (in the absence of added peptide) were <2%.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TCR-mediated changes in viable cell numbers of CD8+ 2C T cells
To determine if CTL clones undergo AICD, the survival of cells from clone L3.100 expressing the 2C TCR was examined on day 7, the same day the clone was due to be re-stimulated by allogeneic P815 (Ld) cells for routine maintenance. For comparison, various primary T cells, which expressed the same TCR, were evaluated. These included naive and memory 2C cells freshly isolated from mice or activated 2C cells generated by stimulating naive cells for 3 days with syngeneic B6 (Kb) splenocytes plus cognate SIRGL peptide in the presence of IL-2. We assayed cell survival instead of cell death for quantifying the extent of AICD because the number of live cells can be counted accurately, whereas dead cells decompose and counting them is subject to substantial error. Live cell number is an aggregate of cell survival, death and proliferation. However, under the experimental condition used for inducing AICD in this report (no IL-2 added), 2C clones did not proliferate within 48 h after anti-CD3 stimulation as revealed by labeling cells with the cell division-tracking dye CFSE (data not shown). Thus, the number of live cells 24 h after incubation with various stimuli is a valid indicator of the extent of AICD.

The results of a representative experiment with various CD8+ T cells are shown in Fig. 1Go. Approximately 50% of the naive 2C cells were alive after 24 h culture in medium in the absence of IL-2. Stimulation of these cells with plate-bound anti-CD3{varepsilon} antibody, or allogeneic P815 cells, or syngeneic B6 splenocytes plus the SYRGL peptide resulted in an increase in the relative number of viable cells although few cells proliferated during this time period (Fig. 1AGo and data not shown). In contrast, with freshly activated 2C T cells the same stimuli induced a profound drop (~75%) in the number of viable cells. A substantial loss of viable cells was also seen after 2C clone L3.100 had been incubated with the anti-CD3{varepsilon} antibody (at least 50% and as much as ~90%); but when these cells were stimulated with allogeneic P815 cells or syngeneic B6 splenocytes plus the SYRGL peptide, the resulting AICD was less (Fig. 1CGo and unpublished data). The anti-CD28 antibody had no effect, alone or together with the plate-bound anti-CD3{varepsilon} antibody, on all three types of cells, consistent with previous observations (22). In addition, a decrease in the number of viable cells was detected when memory 2C cells were stimulated with plate-bound anti-CD3 antibody (Fig. 1DGo). To avoid the confounding effect of fratricide (15) due to addition of the potent cognate peptide, SIRGL, we used anti-CD3 antibody and allogeneic P815 cells to induce AICD in the remaining studies.



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Fig. 1. Comparison of AICD among various primary 2C T cells and a 2C clone. T cells (1x105/well) were either untreated or treated with various stimuli in the absence of IL-2 for 24 h. Viable cells were counted by flow cytometry (see Methods). All assays were performed in triplicate. Viable cells that survived treatment are calculated as a percentage of cells that survived without any treatment (no treatment). Results shown are means ± SD (error bars). Some results are shown without error bars because the SD is too small to be shown by the graphic software program (<3%). {alpha}CD3, anti-CD3{varepsilon} antibody; {alpha}CD28, anti-CD28 antibody; P815, irradiated P815 cells; B6 spl, irradiated C57BL/6 splenocytes; pep, SYRGL peptide.

 
To determine whether the observed AICD of clone L3.100 was representative of diverse 2C CTL clones, we compared three clones, L3.100, K and G3.1, which expressed roughly the same levels of TCR and somewhat varied levels of CD8 (legend, Fig. 2Go) but differed in their cytolytic activity and activation kinetics. As shown in Fig. 2AGo, the three clones showed the expected dependency of cytolytic activity on the agonist (SIRGL) peptide, but differed greatly in the extent to which they elicited target cell lysis after 4 h. Clone L3.100 was most effective, clone G3.1 was least effective and clone K showed intermediate activity. CD69 up-regulation after stimulation with anti-CD3 antibody correlated with cytolytic activity: it was most rapid on clone L3.100 and slowest on clone G3.1 with clone K in between (Fig. 2BGo). Exposure to anti-CD3 antibody led to a substantial loss of cell viability of all three clones within 24 h (Fig. 2C–EGo). The extent of cell death roughly paralleled cytolytic activity and CD69 up-regulation, i.e. clone L3.100 showed the greatest TCR-mediated cell death, whereas clones K was intermediate and G3.1 was least susceptible. These results show that CTL clones undergo AICD upon TCR ligation and that the extent of AICD is proportional to the intensity of other responses by these clones to TCR ligation.



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Fig. 2. Comparison of three different 2C clones for their antigen-stimulated responses and susceptibility to AICD. (A) Comparison of cytolytic activity. 51Cr-labeled T-2Kb target cells (T) were incubated with CTL clones (E) at an E:T cell ratio of 10:1 in the presence of different concentrations of SIRGL peptide. Percentages of specific lysis were determined after 4 h. (B) Time-course of CD69 up-regulation. Cloned T cells were stimulated with anti-CD3{varepsilon} antibody and CD69 expression was assayed by flow cytometry at the times indicated. Fluorescence intensity (geometric mean) is shown as a function of time. (C–E) Comparison of AICD of the three 2C clones. Cloned T cells cultured either in medium alone (no treatment) or in the presence of various antibodies (no added IL-2) were assayed and results are shown as in Fig. 1Go. Error bars are omitted when the SD is too small to be shown (<3%). Geometric mean fluorescence (GMF) values for the 2C TCR on the three clones were (mean ± SD of five determinations): 123 ± 7 for L3.100, 173 ± 9 for K and 199 ± 7 for G3.1. For CD8 the corresponding average GMF values were 1352 ± 226 for L3.100, 4126 ± 85 for K and 2308 ± 231 for G3.1.

 
The 2C CTL clones were maintained in culture for many months by stimulating them at 7-day intervals with irradiated allogeneic P815 cells plus rat concanavalin A supernatant (a source of IL-2). To investigate the survival of L3.100 during the weekly stimulation cycle, the total cell number and percentages of apoptotic cells were monitored everyday for an entire week. As shown in Fig. 3Go, within the first 2 days of stimulation, the total numbers of viable cells were slightly lower than those initially plated. Correspondingly, a significant but small percentage of cells were TUNEL+, consistent with their undergoing some AICD in response to the P815 cells. However, cell numbers started to increase after 2 days, continued to increase until day 4 and were then maintained at a relatively steady level. Using CFSE-labeled cells, we showed that by day 3 all surviving cells underwent proliferation although at various rates (data not shown). These results indicate that the extent to which cloned 2C cells undergo AICD during the weekly maintenance cycle is small (~25%): most of the cells survive and are able to proliferate to expand the culture.



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Fig. 3. Changes in viable cell numbers during one cycle (7 days) of stimulation. L3.100 cells (4x105) were cultured with an equal number of irradiated allogeneic P815 cells in the presence of IL-2. At the indicated time, viable cells were counted by flow cytometry as in Fig. 1Go and apoptotic cells were assayed by TUNEL flow cytometry. (A) Total viable cells at each day over the 7-day period. Error bars are omitted when the SD is too small to be shown (<3%). (B) Percentages of TUNEL+ cells over the 7-day period.

 
Death receptors expressed by cultured CTL clones
AICD of T cells is mainly mediated via the death cytokine receptors Fas and TNFR (I and II) (2,3). To investigate how CTL clones have adapted to survive and expand after repeated stimulation through TCR, the cell-surface abundance of death receptors, including Fas, TNFR-I and -II, and changes in their abundance after TCR ligation, were assayed on various cells at different times after stimulation. The level of Fas was low on naive 2C T cells but increased over time after stimulation (Fig. 4BGo). The level of this receptor was higher on the freshly activated 2C cells and increased still further after re-stimulation (Fig. 4A and BGo), consistent with previous observations (23). However, Fas was not detectable on any of the three 2C CTL clones before or after TCR-stimulation, consistent with the recent findings of Walker et al. (29). TNFR-II, in contrast, was low on naive 2C cells and was at a higher level on the activated 2C cells (Fig. 4A and CGo), also consistent with previous observations (5). On the 2C CTL clones this receptor was present at much higher levels, especially on the most reactive clone L3.100. Upon TCR ligation, TNFR-II was rapidly down-modulated and its expression was not restored to the original level until 24 h later (see also 25). TNFR-I was essentially at or very near the background level on naive, freshly activated, and cultured 2C T cells before and after stimulation (Fig. 4AGo and data not shown).



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Fig. 4. Expression of Fas and TNFR-I and -II on various 2C T cells before and after anti-CD3 stimulation. T cells were stimulated with plate-bound anti-CD3{varepsilon} antibody, harvested, and assayed for cell surface Fas, TNFR-I and -II by flow cytometry at the indicated times. (A) Representative histograms showing the levels of Fas, TNFR-I and -II expression (logarithmic scale). (B and C) Fluorescence intensity (arbitrary units) of Fas and TNFR-II is shown as a function of time after stimulation.

 
Mechanisms involved in AICD of cultured CTL clones
To determine which receptors could initiate cell death, we compared the effects of antibodies to Fas and to TNFR-I and TNFR-II on naive, activated, and cultured 2C T cells, and on a CD8+ CTL clone specific for the ovalbumin peptide SIINFEKL in association with Kb. As shown in Fig. 5Go, the four clones in long-term culture behaved alike, and differed markedly from the naive and activated 2C cells. In the clones, anti-Fas had no effect, in accord with the apparent absence of Fas on these cells, but anti-TNFR-II caused considerable cell death. In contrast, with the freshly activated 2C cells anti-Fas was highly effective and anti-TNFR-II was less effective. Neither anti-Fas nor anti-TNFR-II had a discernible effect on the naive cells.



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Fig. 5. Comparison of cell death among different T cells induced by anti-CD3{varepsilon}, anti-Fas and anti-TNFR-II antibodies. T cells were stimulated with various antibodies for 24 h in the absence of IL-2 and viable cells were counted by flow cytometry. All assays were performed in triplicate. Viable cells that survived treatment are calculated as a percentage of cells that survived without any treatment (no treatment). Results shown are mean ± SD (error bars). Error bars are omitted when the SD is too small to be shown (<3%). L3.100, K and G3.1 are 2C CTL clones; 4G3 is an anti-ovalbumin CTL clone; naive and freshly activated 2C T cells are as in Fig. 1Go.

 
Binding of TNF-{alpha} and lymphotoxin (LT)-{alpha} to TNFR normally induces apoptosis of activated T cells (5,6). If TNFR-II were responsible for initiating AICD of the clones, these cells would be expected to express TNF-{alpha} and/or LT-{alpha}. As shown in Fig. 6Go, only a few L3.100 cells expressed TNF-{alpha} and few more were induced to express TNF-{alpha} by anti-CD3 stimulation in a time-dependent manner. By 2 h after stimulation 13% of the cells were clearly positive for TNF-{alpha}. In the other 2C clones, which were less reactive, the induction of TNF-{alpha} was detected but to a lesser extent (data not shown). In contrast, TNF-{alpha} was not significantly induced in naive 2C cells within 2 h of anti-CD3 stimulation.



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Fig. 6. Time-course of TNF-{alpha} induction in naive and cloned 2C T cells after TCR ligation. T cells were stimulated with irradiated C57BL/6 splenocytes plus SYRGL peptide in the presence of Brefeldin A. TNF-{alpha} expression was assayed at different time points by intracellular staining followed by flow cytometry. Percentages of TNF-{alpha}+ cells are shown.

 
To further investigate whether the induced expression of TNF-{alpha} is directly involved in AICD of T cell clones, we assayed the ability of the TNFR–Ig fusion protein to block AICD of the T cell clones. TNFR–Ig was used because it can bind both TNF-{alpha} and LT-{alpha}, and thus is more broadly effective than neutralizing antibodies specific to TNF-{alpha} or LT-{alpha} alone (20). The presence of TNFR–Ig fusion protein significantly reduced the percentage of T cells that were TUNEL+ following stimulation with either anti-CD3{varepsilon} or irradiated P815 cells (Fig. 7Go). However, TNFR–Ig fusion protein did not abolish AICD completely, either because it failed to reduce TNF-{alpha} levels sufficiently or because while ligation of TNFR contributes to the death of T cell clones following stimulation through TCR, additional AICD pathways may also be involved (2). The latter possibility is consistent with the observation that anti-CD3{varepsilon} treatment was more effective than anti-TNFR antibody in inducing death of T cell clones (Fig. 5Go).



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Fig. 7. Inhibition of AICD by TNFR–Ig fusion protein. Clone L3.100 was cultured for 24 h with anti-CD3{varepsilon} antibody or with irradiated P815 cells in the absence or presence of TNFR–Ig fusion protein or an equal amount of control IgG. Cell death was determined by TUNEL assay stained with dUTP–FITC. Percentages of TUNEL+ cells are shown.

 
Figure 7Go also shows that the frequency of TUNEL+ cells was lower in the cells that were stimulated by the irradiated APC (P815) used to maintain the clones in culture than by the plate-bound anti-CD3{varepsilon} antibody.

Survival of cultured CTL clones
Given that the CTL clones express high levels of TNFR-II and can be induced to express TNF-{alpha}, the question arises as to how the CTL clones survive the repeated TCR stimulation. One possibility is that the TNFR is rapidly lost from the cell surface (Fig. 3Go), before TNF-{alpha} is produced. (25,26). As shown in Fig. 8Go, in response to anti-CD3 stimulation, TNFR-II disappeared very rapidly from both clone L3.100 and freshly activated 2C T cells, and by 2 h was virtually gone from the cultured clone. At this time only ~10% of the cells were expressing TNF-{alpha} (Fig. 6Go). On naive 2C T cells, TNFR-II was not expressed at an appreciable level and the level was not altered significantly within 2 h of TCR ligation. It is of incidental interest that in response to ligating the TCR, this receptor's down-regulation was considerably slower on activated and cultured cells than the rate at which the TNFR-II receptor was lost (Fig. 8Go). Ligation of TCR on the naive cells also caused no significant down-regulation of the TCR within a 2-h time period, consistent with our previous observation (18).



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Fig. 8. Comparison of TNFR-II and TCR down-modulation on naive, activated and cloned 2C T cells. T cells were stimulated with plate-bound anti-CD3{varepsilon} antibody, harvested, and assayed for cell surface TNFR-II and TCR by flow cytometry at the indicated times. Relative TNFR-II (A) and TCR (B) levels are shown as a percentage of TNFR-II and TCR levels on T cells that were incubated in the absence of anti-CD3 antibody.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Although most T cells die when subjected to repeated TCR-mediated stimulation (1,2) many murine CD8+ CTL cell clones can be propagated indefinitely in culture, some continuously for years, while receiving, and indeed requiring, repeated (weekly) TCR-mediated stimulation with cognate peptide–MHC complexes on APC. To understand how the cultured cells avoid or minimize AICD, we examined their expression of Fas, and TNFR-II, the principal receptors for the FasL and TNF-{alpha} `death cytokines' (2). We also compared the receptors on these cells with those on naive cells that were unstimulated or had been recently stimulated (`freshly activated') T cells. Our results show that unlike the naive and freshly activated T cells, the cultured clones did not express Fas and that they rapidly and extensively down-modulated TNFR-II in response to TCR ligation. After this study was completed, Walker et al. (29) reported absence of Fas on other murine CD8+ clones (specific for the antigen called Cw3). They observed, moreover, a progressive decrease in the level of Fas when peritoneal exudate CTL were subjected ex vivo to weekly stimulation in culture and found that Fas had virtually disappeared by the seventh week in culture. (They did not, however, describe the TNFR on these cells.)

The rapid TCR-mediated down-modulation of TNFR-II described here is also consistent with earlier work of Ware et al., who studied human T cell lines and clones (25). Unlike murine T cell clones, human T cell clones rarely survive in culture for long periods. (It is, for example, exceedingly rare to see the same human clone used repeatedly in diverse studies.) Nevertheless, Ware et al. described virtually complete disappearance of TNFR-II from their human cell lines and clones, evidently due to proteolytic cleavage of the receptor's extracellular domain (26). It appeared, moreover, that loss of this receptor was essentially complete within an hour after TCR ligation, before the T cells secreted TNF-{alpha}. Our results, described in Figs 4, 6 and 7GoGoGo, are consistent with theirs. Together, the absence of Fas, and the rapid and extensive loss of TNFR-II, before much TNF-{alpha} is produced, can account for the reduced susceptibility to AICD of murine T cells clones that are adapted to long-term growth in culture.

Another factor that affects the long-term culture of many murine CD8+ T cell clones is the choice of ligand for their TCR. For the clones studied here irradiated tumor cells, displaying indigenous peptide–MHC I complexes, were introduced weekly to stimulate the cells. For the 2C clones, P815 cells displaying Ld and peptides from {alpha}-ketoglutarate dehydrogenase were used, and for the 4G3 clone, EL4 cells displaying Kb with the SIINFEKL peptide from transfected ovalbumin were used (14,16,27,28). The extent of AICD induced by irradiated APC (e.g. P815 tumor cells or Kb+ splenocytes presenting the SIRGL peptide) was, on the whole, considerably less than that induced by anti-CD3 antibody (Figs 1C and 3GoGo, and especially unpublished results, D. Palliser and C. McKinley). The greater effect of anti-CD3 could be due to the persistent stimulation it provides, in contrast to the peptide–MHC-presenting tumor cells, which are expected to be largely lysed by the CTL in several hours. Even without this time difference, it is clear that the amount of plate-bound anti-CD3{varepsilon} antibody used routinely in the present work (10 µg/ml to coat the plates) provoked more intense TCR-mediated responses than cognate peptide–MHC complexes on APC. Thus, diminished cell death was seen when the amount of plate-bound antibody was titrated down (S. Sugawa, unpublished observation).

Differences among the three 2C CTL clones examined here likewise suggests that the extent of antigen-stimulated apoptosis reflects the intensity of other TCR-mediated responses (22). Thus, the clone that lysed target cells most effectively and up-regulated CD69 most rapidly also exhibited the most extensive AICD (L3.100; Fig. 2Go). In contrast, the clone (G3.1) that lysed target cells least and up-regulated CD69 at the slowest rate, had the lowest level of AICD. The third clone (K) was intermediate in cytolytic activity, CD69 up-regulation and AICD. The three clones expressed the 2C TCR at about the same level (see legend, Fig. 5Go). They also were all CD8+ and while they differed somewhat in CD8 levels, the differences were unrelated to differences in activity (see legend, Fig. 5Go). In cytolytic assays, moreover, the clones required about the same peptide concentration for half-maximal cytolytic activity (amounting to a requirement for the same epitope density, since the same target cells were used) (Fig. 2AGo). Taken together, all of these findings suggest that the clonal variation in susceptibility to AICD, like the variation in cytolytic activity, arises from differences among the clones in downstream signaling rather than from differences in the initial, activating TCR–peptide–MHC interaction. The variation in susceptibility to AICD raises the possibility that adaptation of CD8 T cells for growth in long-term culture might select against the most susceptible clones. If the most susceptible clones are also the most responsive to TCR ligation and capable of the highest level of cytolytic activity (such as L3.100), then the clones that are finally propagated successfully in culture, even the most highly cytolytic ones, may under-represent the efficacy of many active clones in vivo.


    Acknowledgments
 
We thank Drs D. Loh, D. M. Kranz and S.Tonegawa for 2C transgenic mice and RAG-1-deficient mice; Dr P. Rennert for TNFR–Ig fusion protein; Carol McKinley for maintaining CTL clones and help with cell counting; G. Paradis for assistance in flow cytometry; Drs Q. Ge and R. Varada for providing memory 2C mice; Drs B. Cho, E. Guillen and members of the Chen laboratory for helpful discussions; and Dr L. Van Parijs for critical reading of the manuscript. We are grateful to Mimi Rasmussen for having derived 2C clones L3.100, K and G3.1. This work was supported in part by NIH grants AI44478 (to J. C.), AI44477 and CA60686 (to H. N. E.), and Cancer Center core grant CA14051 (to R. O. Hynes). S. S. was supported in part by Mitsubishi Chemical Corp. and D. P. is a StressGen postdoctoral fellow.


    Abbreviations
 
AICD activation-induced cell death
APC antigen-presenting cell
CTL cytotoxic T lymphocyte
FasL Fas ligand
LT lymphotoxin
TNF tumor necrosis factor
TNFR tumor necrosis factor receptor

    Notes
 
1 Present address: Yokohama Research Center, Mitsubishi Chemical Corp., Yokohama, 227–8502, Japan Back

Transmitting editor: M. J. Bevan

Received 12 February 2001, accepted 24 September 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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