In vivo elimination of viral superantigen-activated CD4+ T cells: apoptosis occurs at a distance from the activation site
Agnès Le Bon,
Anne-Claude Waché and
Martine Papiernik
INSERM U345, Institut Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France
Correspondence to:
M. Papiernik
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Abstract
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Local injection of mouse mammary tumor virus (MMTV) induces a local immune response, with activation of viral superantigen (vSAG)-specific T cell subsets followed by their clonal deletion. We investigated the fate of vSAG-reactive T cells following footpad injection of MMTV(SW) to mice. Activated T cells accumulated in draining lymph nodes. However, we demonstrated that apoptosis did not occur at the activation site, on the contrary of what has been shown after bacterial SAG activation. Although activated T cells were already shown to have the capacity to migrate to the gut, the fate of gut homing cells remains unclear. We demonstrate that the number of vSAG-specific T cells activated in the periphery was increasing in the follicles of gut-associated lymphoid organs, together with the number of apoptotic cell clusters. These results strongly suggested that gut-associated lymphoid tissue was the specific graveyard for apoptotic vSAG-activated CD4 T cells.
Keywords: apoptosis, clonal deletion, viral superantigen
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Introduction
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The in vivo response to antigens leads to the generation of effector T cells, memory T cells and/or tolerance induction by cellular anergy or elimination. The control mechanisms leading to memory and tolerance are difficult to study with conventional antigens, as the frequency of responding cells is low. Mice transgenic for an antigen-specific TCR and injection of superantigens (SAG) recognized by entire families of T cells bearing a SAG-specific Vß chain are both useful models to address these questions. In mice transgenic for a specific TCR, interaction of transgenic T cells with the nominal antigen or peptide leads first to their activation and proliferation, then to their partial elimination (13). Injection of bacterial SAG (bSAG)- and of viral SAG (vSAG)-presenting cells (Mls-1a) into Mls-1b host mice both also lead to activation and subsequent elimination of SAG-reactive T cells (410). Elimination of specific clones after the initial expansion phase is a mechanism of tolerance induction and may be also necessary to control a normal immune response.
The way in which such activated T cells are eliminated is unclear, although cell death by apoptosis has been observed in some circumstances (3,7,1115). We analyzed the mechanisms of cell elimination induced by an infectious mouse mammary tumor virus (MMTV). All known MMTV express vSAG (1618) encoded by the open reading frame of their 3' long terminal repeat (19,20). MMTV(SW) is an infectious MMTV coding for a SAG with the same Vß specificity as Mtv-7 (Mls-1a) (21,22). In this model, the viral infection spreads gradually (23,24) and the vSAG persists. MMTV(SW)-SAG-reactive T cells of mice infected at birth are gradually deleted (25). By contrast, injection of MMTV(SW) into the footpads of adult mice induces an acute immune response in the draining popliteal lymph nodes (LN) characterized by a rapid increase in the frequency of vSAG-specific Vß6+ cells in the CD4+ T cell population and in the absolute number of Vß6+ CD4+T cells (26,27). This acute response involves preferential trapping of vSAG-reactive T cells within the draining LN, their local activation (28) and their rapid elimination (2425). To study the fate of these activated CD4+ T cells, we investigated the site of apoptosis during the course of clonal elimination. Unexpectedly, cells did not die at their site of activation, as it is the case following bSAG activation (7,1214). Increased numbers of apoptotic cell clusters and vSAG-reactive T cells in gut-associated lymphoid tissues suggested that vSAG-activated T cells died at a distance from their activation site.
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Methods
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Mice and in vivo treatments
BALB/c (H-2d, Mls-1b) mice were purchased from Charles River (Cléon, France).
MMTV(SW) preparation and injection
Mammary gland tumors from 1-year-old MMTV(SW)-infected BALB/c mice (Iffa Credo, L'Abresle, France) were cut into small pieces and homogenized in 0.01 M Tris, pH 8, 0.1 M NaCl, 1 mM EDTA and centrifuged at 600 g for 5 min. The supernatant was then centrifuged at 12,400 g for 10 min. The resulting supernatant was ultracentrifuged at 105,000 g for 1 h. The pellet was then suspended in PBS containing 0.5 M sucrose. Aliquots were kept in liquid nitrogen. The activation capacity of the vSAG encoded by MMTV(SW) was tested by injecting serial dilutions of the virus into the footpads of BALB/c mice. We used the dilution giving the highest activation of vSAG-reactive Vß6+ T cells in the draining LN. The same batch of virus was used in all experiments and increased the percentage of Vß6+ cells in CD4+ T cells from 10 to 30% in draining LN 5 days after injection.
Cell surface staining and flow cytometry
Cells were suspended with a Potter homogenizer, washed and counted. For immunofluorescence labeling, cells were washed in ice-cold PBS supplemented with 5% FCS and 0.2% sodium azide.
The following antibodies were used: anti-CD4 (clone GK 1-5) (29), anti-Vß6 (clone 44.22.1) (30), anti-Vß8.2 (clone F23.2) (31) and anti-B220 (clone 6B2) (32). Antibodies were directly coupled to FITC or phycoerythrin (PE), or biotinylated. Cells were first incubated with biotinylated antibody, then with PE-labeled antibody and FITC-conjugated antibody for triple surface staining. The biotinylated antibodies were revealed with streptavidinTriColor (Caltag, San Francisco, CA).
Cell acquisition and analysis were performed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) using Lysys II software.
Injection of FITC-labeled purified CD4+ T cells
CD4+ T cells were purified from 6- to 8-week-old BALB/c mouse LN cells. CD8+ T cells were eliminated by anti-CD8 Ig binding followed by depletion with magnetic beads coated with sheep anti-rat Ig (Dynabeads; Dynal, Oslo, Norway) and B cells were eliminated after incubation with magnetic beads coated with sheep anti-mouse Ig (Dynabeads). Purified CD4+ T cells were labeled with 500 mg/ml FITC (Sigma, St Louis, MO) in PBS containing 2% FCS for 15 min at 37°C. Cells were pelleted for 5 min at 600 g through a cushion of FCS to eliminate free FITC and then washed 3 times in PBS. FITC labeling was checked by flow cytometry. Purified CD4+ FITC+ cells (2.5x106) were then injected i.v into each mouse.
Detection of apoptotic cells by TUNEL assay
Flow cytometry
Cells from various lymphoid organs were isolated and cells were incubated for 3 h in complete medium (2x106 cells/ml) at 37°C. After surface labeling, PE and FITC double-stained cells were fixed in 200 ml of 1% paraformaldehyde containing 0.01% Tween 20 (PFAT; Sigma) overnight at 4°C. Cells were then washed in PBS and in TdT buffer (0.1 M sodium cacodylate/0.1 M DTT/100 mg/ml BSA), and incubated at 37°C for 30 min in TdT buffer with 5 mM Biotin-16-dUTP (Boehringer, Mannheim, Germany) and 5 U of terminal transferase (Boehringer). After a wash with PBS, lymphocytes were incubated with streptavidinTriColor (Caltag) in PBS/0.5% Tween 20, washed and resuspended in PBS. Immunolabeled cells were analyzed in a FACScan flow cytometer (Becton Dickinson) using Lysys II software.
Sections
Fresh lymphoid tissues immersed in OCT compound (Bayer Diagnostic, Tarrytown, NY) were frozen in isopentane cooled in liquid nitrogen and stored at 80°C. Sections of 5 mm were cut with a cryostat (Cryocut 1800), air-dried overnight and fixed for 10 min in acetone. Sections were stored at 20°C until use. The protocol used for TUNEL labeling has been described elsewhere (33). Briefly, sections were rehydrated in PBS and incubated in PBS 0.02% H2O2 to inhibit endogenous peroxidase. TUNEL labeling was performed as for flow cytometry. Sections were washed in PBS, incubated for 20 min at room temperature with streptavidin conjugated with biotinylated horseradish peroxidase (Amersham, Amersham, UK) and revealed with the peroxidase substrate 3-amino-9-ethylcarbazole (0.2 mg/ml in 1 volumes of N,N-dimethylformamide, 10 volumes of 0.2 M sodium acetate, pH 5, 10 volumes of H2O). The enzyme reaction was stopped in H2O. Sections were lightly counterstained in hematoxylin. Positive controls were thymus sections from mice injected i.v. with 650 µg of dexamethazone-21-phosphate (Sigma) 16 h previously.
Immunohistochemistry
Peyer's patches were removed from 6- or 7-day MMTV(SW)-infected and uninfected mice. Sections of Peyer's patches were immunolabeled with biotinylated anti-Vß6 antibody (rat IgG2a anti-mouse, clone 44.22.1) or a biotinylated isotype control antibody (rat IgG2a, clone R35.95; PharMingen, San Diego, CA). Biotinylated antibodies were revealed using the biotinyl tyramide detection system (TSA Indirect Tyramide Signal Amplification; DuPont-NEN, Boston, MA), using Texas Redstreptavidin (Amersham) as fluorochrome. At least 20 sections of Peyer's patch follicles from each mouse were examined. The number of Vß6+ cells per follicle section was counted blindly under a fluorescence microscope (Leica, Rueil-Malmaison, France).
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Results
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Local activation and selective migration induced by MMTV(SW) in draining LN is a long-lasting phenomenon and is associated with specific cell decay in non-draining LN
BALB/c mice (68 weeks old) were inoculated with MMTV(SW) into each hind footpad. Draining (popliteal) and non-draining LN (axillary) were removed at various times after infection and cell phenotypes were determined by flow cytometry. The frequencies of vSAG-reactive Vß6+ and non-specific Vß8.2+ among CD4+ T cells, and the absolute number of Vß6+ CD4+ and Vß8.2+ CD4+ cells, were determined from day 4 to day 30 after MMTV(SW) injection (Fig. 1
). In the draining LN, the percentage of Vß6+ among CD4+ T cells peaked on day 5 (33.3 ± 0.4 compared to 11.4 ± 0.3% in control mice); absolute numbers of Vß6+ CD4+ T cells increased 33-fold compared to control (Fig. 1A and C
) and then decreased. The absolute number of Vß6+ CD4+ T cells was still 2.5 times higher than control 30 days after infection (Fig. 1C
). In non-draining LN, the Vß6+ cell percentage within the CD4+ subset started to fall as early as day 5 (7.9 ± 0.4 versus 11.4 ± 0.2% in control) and was clearly below the control value on day 30 after injection (4.2 ± 0.3 versus 11.9 ± 0.4%). A similar fall in absolute numbers was also found (4-fold fewer Vß6+ CD4+ T cells on day 30 than in control) (Fig. 1B and D
). In the draining LN, accumulation of non-reactive Vß8.2+ CD4+ T cells was also observed (13-fold more than in control 5 days after injection), suggesting that activated draining LN recruited non-specific cells through inflammatory processes. These results suggested that the decay of vSAG-specific T cells started in non-draining LN. To explain these results we postulated that draining LN recruited and trapped specific cells from the circulation during a long period. Indeed, we had previously found that the initial accumulation of MMTV(SW)-SAG reactive T cells was due to selective influx rather than local proliferation (28). To determine if this process was restricted to the early response, we studied cell migration in vivo. At various times after MMTV(SW) injection, 6- to 8-week-old BALB/c mice were injected i.v. with 2.5x106 syngeneic purified naive CD4+ cells labeled ex vivo with FITC. Two days after cell injection, mice were killed and absolute numbers of Vß6+ and Vß8.2 CD4+ FITC+ migrating cells were determined in draining and in non-draining LN (Fig. 2
). Preferential migration of Vß6+ CD4+ T cells to draining LN was detected, for as long as 1 month after MMTV(SW) injection (Fig. 2A
). Their migration was under the control level in non-draining LN (Fig. 2B
). Migration of vSAG-non-reactive subpopulation (Vß8.2+ CD4+ T cells) into draining LN was also enhanced, although less than vSAG-specific cell migration.

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Fig. 1. Immune response to MMTV(SW) injection. BALB/c mice (68 weeks old) were inoculated with MMTV(SW) into each footpad. Popliteal and axillary LN were removed at various times after MMTV(SW) injection and cells were labeled for immunofluorescence analysis. Results are expressed as the percentage of Vß6+ or Vß8.2+ cells gated on CD4+ T cells (A and B) and as absolute numbers of Vß6+ or Vß8.2+ CD4+ T cells (C and D). Mean values ± SE for three to nine mice.
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Fig. 2. Selective accumulation of FITC-labeled Vß6+ CD4+ T cells in draining LN. BALB/c mice were inoculated with MMTV(SW) into the hind footpad. At various times after MMTV(SW) injection, 2.5x106 purified FITC-labeled CD4+ T cells were injected i.v. Popliteal and axillary LN cells were harvested 2 days after cell inoculation and surface stained with anti-CD4 and anti-Vß6 or anti-Vß8.2. Results are expressed as absolute numbers of Vß6+ or Vß8.2+ CD4+ FITC+ T cells. Control mice were injected with FITC-labeled cells only and were tested on day 2. One representative experiment out of three: mean value for three mice.
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These migration experiments clearly showed that vSAG-reactive T cell disappearance from non-draining LN was associated with long-lasting migration and trapping in draining LN.
No apoptosis of lymphocytes isolated from draining LN
Vß6+ CD4+ T cell absolute numbers fell rapidly between days 5 and 10, although specific cells were still recruited to draining LN. To determine if programmed cell death occurred at this time, popliteal and axillary LN cells were tested for apoptosis using the TUNEL technique between these two time points (Fig. 3
). The percentage of apoptotic Vß6+ CD4+ T cells was the same in axillary LN cells from infected and control mice. Surprisingly, we observed no increase in the apoptotic cell percentage among Vß6+ CD4+ T cells in draining LN; on the contrary, values were significantly lower than controls.

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Fig. 3. Apoptosis is not detected in draining popliteal LN. BALB/c mice were injected with MMTV(SW) into the hind footpad. At various times after infection, popliteal LN, axillary LN, mesenteric LN and Peyer's patch cells were incubated for 3 h at 37°C and surface-stained with anti-CD4 and anti-Vß6. B cells were removed from Peyer's patches with magnetic beads coated with sheep anti-mouse antibodies. Apoptotic cells were revealed by the TUNEL technique. Results are expressed as percentages of TUNEL-positive cells among Vß6+ CD4 T cells. The control values (mean for two mice tested) are given for each group of experimental mice tested. Axillary and popliteal LN: mean value ± SE for three experimental mice tested the same day (one experiment out of three performed). Mesenteric LN and Peyer's patches: mice infected for 5, 7, 8, 9 and 10 days were killed and tested the same day. Cells of three individual mice per group were pooled. One experiment out of three performed.
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These results showed that the drastic fall in CD4+ Vß6+ T cell absolute numbers was not due to apoptosis in situ. As it remained possible that vSAG-specific cells migrated to other sites before being eliminated, we investigated apoptosis in other lymphoid organs. The percentage of apoptotic cells among Vß6+ CD4+ T cells in mesenteric LN and Peyer's patches remained close to the control level. No significant increase in apoptosis was found in the spleen or liver (data not shown).
Thus, no significant increase in apoptosis of vSAG-reactive T cells was detected in cell suspensions from any site, including the activation site.
Increased numbers of apoptotic cell clusters in mucosa-associated lymphoid organs
To determine if vSAG-reactive T cells were rapidly cleared by local phagocytosis, we tested for the presence of engulfed apoptotic cells in sections of popliteal LN, axillary LN, mesenteric LN, spleen, liver and Peyer's patches from 410-day MMTV(SW)-injected-mice by the TUNEL technique. No increase in apoptotic cells was observed in popliteal or axillary LN, or in the spleen and liver. In contrast, sections of mesenteric LN and Peyer's patches contained a large number of apoptotic cell clusters. These clusters were concentrated within the deeper part of the follicle (Fig. 4
) where the `tingible body macrophages' (TBM) have been localized within the germinal centers (34,35); they were also detected in gut-associated lymphoid organs of control mice. To determine if their number was increased in infected mice, sections of popliteal and mesenteric LN and Peyer's patches (four Peyer's patches per mouse) were analyzed. At least 10 sections of each structure, taken at different levels, were TUNEL labeled and apoptotic cell clusters were blindly counted. As shown in Table 1
, few if any clusters were detected in draining popliteal LN. The number of apoptotic clusters was increased from days 4 to 7 in the mesenteric LN, reached a maximum on day 7 (6 times higher than in control mesenteric LN; P < 0.001) and decreased thereafter. In the Peyer's patches, the number of apoptotic cell clusters was high in control mice, possibly owing to permanent activation by high local concentrations of antigens derived from food or microorganisms. However, the number of apoptotic cell clusters was significantly higher 7 days after MMTV(SW) infection (P < 0.004 compared to control). The apparent decrease of the apoptotic cell clusters 4 days after infection was not statistically significant.

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Fig. 4. Localization of apoptotic cells in the Peyer's patches of MMTV(SW)-infected mice. Using the TUNEL technique, sections of popliteal LN, axillary LN, mesenteric LN, spleen, liver and Peyer's patches were labeled in control and day-6-MMTV(SW)-infected mice. We show here the localization of apoptotic cells (arrows) in the deeper part of the germinal center of Peyer's patch follicles (A) (x100). Clusters of apoptotic cells (B) co-localized with TBM (x250). High magnification showing apoptotic nuclei (C) (x630). (See Table 1 for the number of apoptotic clusters in infected and control mice.)
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These results showed that vSAG-reactive T cells were not cleared by local macrophages in the draining LN. Surprisingly, the number of apoptotic cell clusters which co-localized with the site of TBM in the germinal centers (36) was clearly increased in gut-associated lymphoid organs, with a peak 7 days after infection.
Increased Vß6+ T cell numbers in the follicles of Peyer's patches from infected mice
TBM clear dying cells from germinal centers (37,38). These dying cells are thought to be mainly B cells (3941). Although T cells are present in the germinal centers, they have not been observed to die locally. We therefore investigated whether the increased number of apoptotic cell clusters in the zone of TBM localization reflected the clearance of vSAG-reactive T cells by TBM. Vß6+ T cell distribution was analyzed on Peyer's patch sections by immunohistochemistry. Scattered Vß6+ T cells were detected in the T cell area and in the villi (not shown). Vß6+ T cells were clearly detected in the follicles of day 6-infected mice; some were located in the apical zone, but most were found in the deeper part of the follicle (Fig. 5A and B
). Very few were detected in the follicles of control mice (Fig. 5C
). To quantify T cells, at least 20 sections of Peyer's patches were labeled with anti-Vß6 or anti-Vß8.2 antibody at each time point. The numbers of Vß6+ or Vß8.2+ T cells were blindly counted within the follicles and the results were expressed as the number of labeled cells per section of follicle. The number of Vß6+ T cells per section was strongly increased 6 and 7 days after MMTV(SW) injection whereas the number of Vß8.2 T cells remained unchanged (Fig. 6
). Interestingly, the number of Vß6+ T cells was not increased at day 4 in the Peyer's patch follicles, when the reaction was maximum in the draining popliteal LN (Fig. 6
).

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Fig. 5. Localization of vSAG-reactive, Vß6+ T cells in Peyer's patch follicles. Vß6+ T cells were localized on sections of Peyer's patches by immunohistochemistry. Vß6+ T cells were mainly located in the dark zone of germinal center macrophages. (A) Vß6 T cells in the Peyer's patches of a mouse infected by MMTV(SW) 7 days previously. (B) Same section showing autofluorescent macrophages. (arrows: localization in the macrophage zone of 3 Vß6+ T cells shown in the upper part of (A). (C) Detection of Vß6+ T cells on a section of control Peyer's patches. (D) A Vß6+ T cell (arrow) close to autofluorescent macrophages (E). (AC: x250; D and E: x630.)
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Fig. 6. Increased numbers of Vß6+ T cells in Peyer's patch follicles after MMTV(SW) infection. Sections of Peyer's patches from control and 4-, 6- and 7-day-infected mice were immunolabeled with anti-Vß6 or anti-Vß8.2 antibodies. The number of Vß+ T cells was blindly counted on at least 20 sections. Mean numbers of Vß+ T cells per section of Peyer's patch follicle ± SE.
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No specific labeling was detected on tingible bodies engulfed in the TBM. The membranes of engulfed lymphocytes were either destroyed or non-accessible within the TBM. Interestingly, however, Vß6+ T cells were often located in the vicinity of TBM (Fig. 5A, B, D and E
).
In summary, a significant increase in TBM and Vß6+ T cell numbers was observed in Peyer's patch follicles during the rapid phase of vSAG-specific T cell disappearance from draining LN. These results can be interpreted as evidence for vSAG-specific T cell destruction within the follicles of gut-associated lymphoid organs.
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Discussion
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Local MMTV(SW) injection induces rapid activation of MMTV(SW)-vSAG-reactive T cells. Their number increases in draining LN and they acquire activation markers (28). Subsequently, their number declines in all lymphoid areas (2125). The site(s) at which these cells died remained to be identified. In this study, we found that although draining LN trapped these cells for as long as 1 month, they were not the site of clonal elimination.
Draining LN trapped vSAG-reactive T cells after MMTV(SW) injection (28). Therefore, despite efficient viral spread (24), draining LN remained the only site of vSAG-induced T cell accumulation after local MMTV(SW) injection. This may be explained by a high concentration of cells able to present the SAG in the LN draining the site of infection. We have already described the clonal deletion kinetics of MMTV(SW)-SAG-reactive T cells: `clonal deletion' was detectable 10 days after infection in all non-draining lymphoid areas, i.e. axillary LN, mesenteric LN and the spleen, and slightly later in Peyer's patches (24). Data on the kinetics of activation and clonal elimination and those on cell migration clearly suggest a correlation between the decline in vSAG-reactive T cell numbers in non-draining LN and their trapping by draining LN (2125). However, even though draining LN were still able to trap specific T cells, a large number of specific T cells disappeared from the draining LN between days 5 and 10. The most likely mechanism of clonal elimination is apoptosis. Indeed, disappearance of activated T cells in vivo is related to apoptosis in various models. Several groups have shown that staphylococcal enterotoxin B induces apoptosis of SAG-specific cells in situ (7,1214). Treatment with anti-CD3 antibodies also drives apoptosis in vivo (42). The use of mice transgenic for a TCR and injection of the specific peptide enables apoptosis to be visualized in peripheral lymphoid organs (3,15). This cell elimination by in situ apoptosis has been termed activation-induced cell death (AICD) and has been linked to a prior extensive proliferation phase (43). AICD is also preceded by DNA synthesis (12,44).
In the case of MMTV(SW) infection, strong activation restricted to draining LN is induced by vSAG (28). However, we detected no increase in apoptotic cell numbers (`free' or engulfed) in draining LN, showing that vSAG-activated T cells were not susceptible to AICD at the activation site. We have previously demonstrated, using BrdUrd labeling in vivo, that vSAG-reactive T cells proliferate little between day 1 and day 8 after MMTV(SW) injection (28). MMTV infection in vivo thus induced neither strong proliferation nor in situ AICD. The amount of antigen could be responsible for this difference with other models, because in all cases in which AICD has been demonstrated the amount of antigen was large (12,15), whereas chronic exposure to low doses of SEA leads to clonal elimination without prior proliferation (45). Reactive T cell numbers could also determine the site and mechanism of their elimination. Indeed, Kearney et al. developed chimera in which transgenic cells represented only 0.2% of total T cells. After in vivo challenge of antigen-specific T cells, cell cycle analysis suggests that their secondary decrease following activation is not linked to local apoptosis (46).
Where then do activated T cells die in the absence of in situ AICD? In some circumstances, apoptosis of CD8+ T cells has been shown to occur in the liver rather than in peripheral lymphoid organs (4243) but we failed to find more apoptotic cells in the liver after MMTV(SW) injection (data not shown). However, these studies focused on CD8+ T cell elimination and CD4+ T cells may die elsewhere. Work by Sprent showed in vivo that alloantigen- or SAG-stimulated T cells could migrate to the gut and gut-associated lymphoid organs, especially to Peyer's patches. However, the fate of gut homing cells remains unclear (49). We therefore looked for apoptosis in mesenteric LN and Peyer's patches. Although few if any free apoptotic cells were found, an enhanced number of apoptotic cell clusters was detected in the follicles of Peyer's patches and mesenteric LN. These clusters co-localized with TBM previously described in the dark zone of germinal centers (35). These `tingible bodies' within macrophages are nuclei of engulfed apoptotic cells. TBM are the site of B cell destruction within the germinal center (40,41) and the only site where TUNEL-positive cells can be demonstrated during the course of the affinity maturation process (50). However, T cell destruction has not been described there. To identify a link between the increase in apoptotic cell clusters and clonal elimination of vSAG-reactive T cells, we assessed Vß6 expression on sections of Peyer's patches. Although we were unable to detect apoptotic vSAG-reactive T cells directly, we found that Vß6+ T cell numbers increased in the follicles concomitantly with the number of apoptotic cell clusters. The increase of the T cells in the follicle is restricted to the MMTV(SW)-SAG-reactive T cells. Moreover, Vß6+ T cells within the follicles were often located in the vicinity of TBM. This influx of vSAG-reactive T cells into the follicles of Peyer's patches, and their local destruction, may explain why the fall in the percentage of Vß6+ cells among CD4+ T cells within Peyer's patches during the clonal deletion process was slightly delayed compared to all other non-draining lymphoid organs (24). Our results then demonstrate that the vSAG-activated CD4+ T cells are not dying at their activation site and suggest that they are dying into the follicles of the gut-associated lymphoid organs. Using the Mls-1a system, Webb and Sprent have also studied the fate of in vivo Mtv-7-activated T cells. They have showed that these activated T cells die by apoptosis in 24 h in vitro and mentioned that they were unable to find any peripheral apoptotic cells ex vivo. Furthermore, very similar to our results, they have discussed the fact that there were Mls-1a-reactive T cells in non-lymphoid organs and especially in the lamina propria of the gut (51). However, they never found any apoptotic cells in the lamina propria (S. Webb, pers. commun.). It could be that one part of the vSAG-reactive T cells migrate into the lamina propria and disappear by an unknown mechanism, and one part dies in the follicle of the Peyer's Patches by apoptosis.
Why activated T cell elimination in the course of a vSAG-induced immune response takes place after migration to gut-associated lymphoid organs rather than at the activation site is intriguing. Similar results have been obtained in two other viral infection models. During acute infection with an enteropathic strain of SIV (52), and with LPBM5, which induces murine acquired immunodeficiency syndrome (53), apoptosis occurs preferentially in Peyer's patches. Thus, elimination of activated T cells may occur at different sites, including gut-associated lymphoid organs. As regards the signals driving cells to migrate to gut-associated lymphoid organs, changes in their membrane antigens at the activation site (mainly specific integrins) may be one explanation. However, integrin
4ß7, which is thought to be involved in specific migration to mucosal structures, was not detected on vSAG-activated T cells (data not shown). Rather than an acquired capacity of these cells to migrate specifically to mucosal lymphoid areas, it could be that activated T cells need germinal center structures to die. As germinal centers are abundant in gut-associated lymphoid organs, vSAG-activated T cells may have a higher probability of migrating and dying there.
The mechanisms by which apoptosis occurs in this model are unknown. We have previously shown that the FasFas ligand interaction is not a prerequisite for apoptosis, as clonal deletion induced by MMTV(SW) took place normally in MRL lpr/lpr mice (54). The mechanism by which Vß6+ CD4+-activated T cells disappear in vivo is thus Fas independent. Tumor necrosis factor (TNF)TNF receptor interaction may be an alternative pathway (55). Mice lacking TNF receptor I (p55) have defective Peyer's patch organogenesis (56). Thus, in addition to impaired cell death signaling, these mice may have abnormal migration to gut-associated lymphoid organs. The TNFTNF receptor pathway may not only be involved as a death signal but might also be required for activated T cells to die in gut-associated lymphoid organs. The role of the TNFTNF receptor pathway will be the subject of future studies.
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Acknowledgments
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We thank Claude Pénit, Bruno Lucas and Delphine Guy-Grand for critical review of the manuscript, and Sonia Hamon for help in manuscript presentation. This work was partly supported by the `Association pour la Recherche contre le Cancer' (ARC).
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Abbreviations
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AICD | activation-induced cell death |
bSAG | bacterial superantigen |
LN | lymph nodes |
MMTV | mouse mammary tumor virus |
SAG | superantigen |
TBM | tingible body macrophage |
TNF | tumor necrosis factor |
vSAG | viral superantigen |
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Notes
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Transmitting editor: J.-F. Bach
Received 2 July 1998,
accepted 10 November 1998.
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