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Suppression of Immune Responses by CD8 Cells. I. Superantigen-Activated CD8 Cells Induce Unidirectional Fas-Mediated Apoptosis of Antigen-Activated CD4 Cells¹

Department of Cancer Immunology and AIDS, Dana Farber Cancer Institute, and Department of Pathology, Harvard Medical School, Boston, MA 02115

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Stimulation of mature CD4 cells through the TCR induces cellular activation and expansion that are often followed by clonal elimination by a form of apoptosis³ termed activation-induced cell death. This process of CD4 cell apoptosis is generally thought to reflect clonal suicide and to be independent of other cell types. Here we show that during the response to the superantigen Staphylococcal enterotoxin A, activated CD8 cells, but not activated CD4 cells, suppress the CD4 proliferative response. Suppression by CD8 cells reflects their ability to induce CD4 cell apoptosis via ligation of Fas. Moreover, although activated CD8 cells that express Fas ligand and Fas eliminate CD4 cells through a Fas-dependent mechanism, they are themselves resistant to Fas-dependent apoptosis. These findings indicate a fundamental difference between the two major T cell subsets with regard to sensitivity to Fas-dependent apoptosis, expression of Fas ligand, and mediation of suppressive activity following immunization with superantigen.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The issue of how an immune response, once initiated, is turned off is a central one in immunology. Not all self-reactive T cells are deleted by negative selection in the thymus, and inhibitory mechanisms leading to peripheral tolerance are required to prevent autoimmunity as well as excessive T cell responses to foreign Ag. One important mechanism of T cell down-regulation is apoptosis, which represents an important component of most immune responses and leads to elimination of reactive cells without excessive release of potentially toxic subcellular debris. T cells can undergo apoptosis by two distinct mechanisms. Activation-induced cell death (AICD)³ results after TCR ligation by Ag in the presence of IL-2 (1, 2, 3, 4), while passive cell death may be caused by a lack of stimulation or cytokines (5).

Ligation of two members of the TNF receptor family, Fas and TNF receptor, has been associated with AICD (6). Mutations of Fas or FasL result in lymphoadenopathy and autoimmunity in mice and humans (7, 8) and can exacerbate the murine AIDS syndrome (9). Studies using TCR transgenic mice carrying the lpr mutation of the Fas gene indicate that Fas:FasL interactions play an important role in peripheral T cell deletion (10, 11) and may also inhibit T cell reactions to self-Ags (12). Additional studies of the sensitivity of CD4 cells to AICD suggest that Th1 cells that express FasL can undergo AICD, but Th2 cells that do not express high levels of FasL fail to undergo AICD (13, 14).

The cellular mechanism(s) of AICD is not well understood. In vitro studies using purified CD4 T cells have suggested that AICD reflects mainly autonomous suicide of individual T cells that express both Fas and FasL (15). However, the potential role of Fas-dependent interactions between CD4 and CD8 T cell subsets that may regulate immune responses in vivo has not been investigated. This issue is important to resolve because it goes to the heart of distinguishing between models of T cell clonal expansion and deletion that postulate autonomous regulation of CD4 cell expansion through clonal suicide (16) vs models that propose additional regulation of Ag-activated CD4 cells by activated CD8 cells (17). The latter hypothesis is based on the premise that CD8 cells are especially equipped to inhibit (suppress) CD4 responses. Here we investigate mechanisms of AICD during the response to SEA in normal and Vß3⁺ TCR transgenic mice, which are hyper-responsive to SEA. We show that the in vivo CD4 response to SEA is normally down-regulated by SEA-activated CD8 cells that suppress Ag-activated CD4 cells by ligation of Fas.

	Materials and Methods

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Mice and immunizations

B6.ß₂m^-/- (MHC class I knockout) mice, normal C57BL/6 (ß₂m^+/+), MRL^+/+, and MRL-lpr/lpr mice, 6 to 12 wk old, were purchased from The Jackson Laboratory (Bar Harbor, ME). Experimental groups were age and sex matched. Vß3 tg⁺ TCR transgenic mice were a gift from Dr. A. K. Abbas (Brigham and Women’s Hospital, Boston. MA) and were on a H-2^b/b (I-E^- B6/SJL) background (18). Mice were immunized with 25 µg of SEA in 200 µl of PBS i.p. Transgenic mice expressing a Vß8.1 transgene (19) (Vß8 tg⁺ mice; provided by Dr. I. Rimm, Dana Farber Cancer Institute, Boston, MA) were injected with 50 µg of SEB i.p.

Media and reagents

SEA and SEB were purchased from Toxin Technology, Inc. (Sarasota FL). Anti-CD4, anti-CD8, anti-Vß3, anti-Vß8.1/8.2, and anti-IL-4 (11B11) fluorescent mAbs; biotinylated anti-IFN- (XMG1.1), anti-FasL (Kay10), and anti-Fas (Jo2) mAbs; purified anti-CD3 (145–2C11) mAb; streptavidin-CyChrome; streptavidin-FITC; and isotype control mAbs were purchased from PharMingen (San Diego, CA). Cellect T cell purification columns were obtained from Biotex (Edmonton, Canada). Anti-rat IgG M450 Dynabeads and streptavidin M280 Dynabeads were obtained from Dynal (Lake Success, NY). Purified rat IL-2 was purchased from Collaborative Biomedical Products (Bedford, MA), and recombinant murine IL-4 and IL-12 were obtained from R&D Systems (Minneapolis, MN). [³H]thymidine and ⁵¹Cr-labeled sodium chromate were obtained from DuPont-New England Nuclear (Boston, MA). A mouse Fas/human Ig fusion protein was a gift from Dr. S. Ju (Harvard Medical School, Boston, MA). Other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). All cell preparations and fractionations were performed in DMEM/2% FBS/HEPES (1 mM). Cell culture medium was DMEM/10% FBS/L-glutamine (2 mM), sodium pyruvate (1 mM), HEPES (1 mM), nonessential amino acids (1 mM each), gentamicin (50 µg/ml), and 2-ME (50 µM). P815, 53-6.72, and 145-2C11 cell lines were obtained from American Type Culture Collection (Rockville, MD).

Cell purification

CD4 cells were purified from lymph node cells by labeling cells with 53-6.72 hybridoma (anti-CD8; American Type Culture Collection) supernatant. Labeled CD8 cells and B cells were then removed by addition of anti-rat/mouse IgG Dynabeads according to the manufacturer’s instructions. CD8 cells were purified from spleen cells using Cellect CD8 T cell purification columns according to the manufacturer’s instructions. In some experiments, CD8 cells were isolated using positive selection with anti-CD8 Dynabeads as described by the manufacturer. CD4 cells were >90% CD4⁺ and <0.2% CD8⁺; CD8 cells were >75% CD8⁺ and <1% CD4⁺. CD4 Vß3⁺ cells were enriched from spleen and lymph node cells by labeling with biotinylated anti-Vß3 mAb followed by addition of streptavidin Dynabeads.

Suppression of proliferation assay

Target and effector cells (CD4 and CD8) used for this assay were recovered from sAg-immunized mice. Ninety-six-well microtiter plates were coated with anti-CD3 (5 µg/ml in PBS) for 2 h at 37°C. Purified CD4 or CD8 target cells (5 x 10⁴) were added to each well. CD4 or CD8 suppressor cells were irradiated (7500 rad) to prevent their proliferation and added to the targets at a 1:1 ratio. PMA (10 ng/ml) and IL-2 (10 U/ml) were added, and cultures were incubated at 37°C in 5% CO₂ for 24 h before addition of [³H]thymidine (1 µCi/well). Cells were harvested after 16 h, and thymidine incorporation was counted using a Wallac 1450 Microbeta scintillation counter (Wallac, Gaithersburg, MD). All assays were performed in triplicate, and median values were used in analyses. Percent suppression values were calculated by comparing the proliferation of cultures containing irradiated, activated suppressor cells against those containing equal numbers of irradiated, unactivated CD8 cells from nonimmunized Vß3 tg⁺ mice. Suppression mediated by anti-Fas mAb was assessed as described above, but suppressor cells were replaced by streptavidin Dynabeads that had been coated with biotinylated anti-Fas mAb (5 µg/ml, 20 min) or with a negative control hamster mAb.

Flow cytometry

Immunophenotyping of spleen or lymph node cells was performed by staining 10⁶ cells with 0.5 µg of each mAb, followed by washing in PBS and analysis on an EPICS XL flow cytometer (Coulter Corp., Miami, FL). Peripheral blood was collected from eye bleeds into sodium citrate for labeling with mAbs as described above. Erythrocytes were lysed with ammonium chloride lysis buffer, and cells were washed in 2% FBS/PBS. For analysis of apoptosis by the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay, cells were stained for surface molecules, fixed, and permeabilized, and DNA degradation was assessed using the fluorescein in situ cell death detection kit (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s instructions. Apoptotic cells were identified as FITC positive by flow cytometry. Surface expression of Fas and FasL was detected on purified CD4 and CD8 cells from the spleen of previously SEA-immunized or nonimmunized Vß3 tg⁺ mice. Cells from each purified subpopulation were cultured separately in IL-2-supplemented (10 U/ml) medium for 16 h before staining. Dead cells were excluded from the analyses using forward and side scatter gating. Cells undergoing apoptosis exhibit decreased forward scatter (20). For intracellular labeling of cytokines, cells that had been stimulated with PMA (10 ng/ml) and ionomycin (400 ng/ml) in the presence of the protein transport inhibitor monensin (3 µM) for 5 h were stained for cell surface molecules as described above. Cells were permeabilized by washing in PBS containing 0.03% saponin and 1% BSA before staining with anti-cytokine mAbs or isotype control mAbs. Cells were then washed in permeabilizing buffer and fixed with 1% paraformaldehyde in PBS.

Anti-CD3-redirected cytolysis assay

P815 mastocytoma cells were labeled with ⁵¹Cr and placed in U-shaped microwells (5 x 10⁴ cells/well). Effector CD4 or CD8 blast cells were added at various E:T target ratios, and anti-CD3 (1/10 dilution of 145-2C11 hybridoma supernatant) was added. Cells were cultured for 14 h, and ⁵¹Cr release into the supernatant was determined by gamma counting. Percent lysis values were calculated using target cells alone and Triton X-100-lysed cells to determine spontaneous and total ⁵¹Cr release, respectively.

	Results

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Enhanced CD4 T cell responses to SEA in ß₂m^-/- and lpr mice

ß₂m^-/- mice are deficient in cell surface expression of MHC class I products and, consequently, lack mature CD8 T cells. To determine the effect of the absence of CD8 T cells on the response of CD4 cells to SEA, C57BL6/ß₂m^-/- mice and control C57BL/6 mice were injected with 25 µg of SEA i.p., and the proportion of Vß3⁺ CD4 cells in peripheral blood was determined by flow cytometry (Fig. 1A). In control (ß₂m^+/+) mice, the proportion of Vß3⁺ cells in the CD4 population doubled by day 5 before declining to below preimmunization levels by day 10. In ß₂m^-/- mice, the proportion of Vß3⁺ cells tripled and, in contrast to that in control Vß8 tg mice, remained elevated through day 10. The levels of Vß8⁺ CD4 cells, which are unresponsive to SEA, were similar in the presence and the absence of ß₂m (Fig. 1C).

View larger version (29K): [in this window] [in a new window]   FIGURE 1. Responses to SEA in ß2m and MRL mice. Groups of three C57BL/6 ß2m-/- and C57BL/6 ß2M+/+ control mice or MRL-lpr/lpr and MRL+/+ control mice were injected with 25 µg of SEA i.p., and numbers of Vß3+ or Vß8.1/8.2+ cells in the CD4 population were monitored by flow cytometry of PBL from serial eye bleeds. Data shown are the mean ± SEM.

SEA-primed CD8 cells suppress proliferation of CD4 T cells after in vitro restimulation

The above data suggested a role for both CD8 T cells and Fas/FasL in regulating the expansion of CD4 cells in response to SEA. We next examined interactions between CD4 and CD8 cells during the SEA response using Vß3 tg⁺ mice. Injection of SEA into these mice induces an initial increase in CD4 T cells in the spleen by day 4 (Fig. 2A) followed by a decline to about 50% of the initial (preimmune) levels by day 10, similar to the CD4 response of nontransgenic mice. CD8 cells in Vß3 tg⁺ mice, which are normally present in reduced numbers because the transgenic TCR is specific for a class II-restricted peptide, expanded more dramatically than CD4 cells and reached maximal levels by day 7 (Fig. 2A), at which time the Vß3⁺ CD4 population was declining rapidly.

View larger version (14K): [in this window] [in a new window]   FIGURE 2. Left panel, Responses of CD4 and CD8 T cells to SEA in Vß3 tg+ mice. Mice were injected with 25 µg of SEA, and splenocytes were stained for CD4 and CD8 at various time intervals. Data shown are the mean ± SEM from groups of two to five mice. Right panel, Time course of CD8 suppressor activity after injection of SEA into Vß3 tg+ mice. CD8 cells were purified from mice primed for various time intervals with SEA, irradiated, and added to SEA-primed (day 3) CD4 cells in the presence of immobilized anti-CD3, PMA, and IL-2. [3H]thymidine incorporation was determined after 24 h. Suppression of CD4 cell proliferation was calculated relative to the activity induced by unprimed control CD8 cells. The graph shows the mean ± SEM from triplicate cultures.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. A, Suppression of proliferation is mediated by CD8 cells on CD4 cells only. CD4 or CD8 T cells from Vß3 tg+ mice primed 3 days previously with SEA were restimulated in the presence of irradiated effectors cells as described in Materials and Methods. The percent suppression of proliferation by either T cell subset was determined. B, CD8 suppressor activity is not specific for Vß expression or dependent on MHC class I. Activated CD8 cells from SEA-primed Vß3 tg+ mice were added to CD4 cells from SEA-primed Vß3 or SEB-primed Vß8+ tg+ donors (upper panel) or to purified Vß3+ cells from either class I-deficient (ß2m-/- Vß3+) or control (ß2m+/+ Vß3+) mice injected 3 days previously with SEA (lower panel). Vß8.1,8.2 transgenic mice (Vß8 tg+) were immunized with SEB. Suppression of proliferation was determined. Data shown are the mean ± SEM from three independent experiments.

Since CD8 can inhibit the CD4 response to sAg via recognition of TCR Vß associated with the ß₂m-associated Qa-1 surface protein (26), we determined whether CD8 suppression required recognition of Vß3⁺ CD4 cells and/or a ß₂m-associated protein. CD8 cells from Vß3 tg⁺ mice injected 3 days previously with SEA suppressed the proliferative response of both Vß3⁺ CD4 SEA-primed blasts and SEB-stimulated Vß8⁺ CD4 blasts (Fig. 3B), indicating that suppression was not specific for Vß expression. Moreover, SEA-induced proliferation of Vß3⁺ CD4 cells from ß₂m^-/- mice or C57BL/6 (ß₂m^+/+) control mice was suppressed equally well by Vß3 tg⁺ CD8 blasts, indicating that inhibitory activity did not depend on expression of a ß₂m-associated protein, such as Qa-1 (Fig. 3B).

Since CD4 T cells were not effectively deleted by SEA in lpr mice (Fig. 1), we asked whether CD8 suppression depended on Fas/FasL-mediated apoptosis. A human Fas-Ig fusion protein that prevents anti-CD3-induced death of a T cell hybridoma (not shown) also effectively and specifically inhibited CD8-dependent suppression (Fig. 4).

View larger version (18K): [in this window] [in a new window]   FIGURE 4. CD8 suppressor activity is dependent on Fas ligation. Irradiated CD8 cells were added to CD4 cells from SEA-primed Vß3 tg+ mice, and levels of suppression were determined in the presence of various concentrations of mouse Fas-Ig fusion protein or human IgG (hu IgG). Data shown are the mean ± SEM from three independent experiments.

To confirm that CD8-dependent suppression was due to apoptosis, we used TUNEL analysis to detect early signs of DNA degradation in restimulated CD4 cells in the presence or the absence of CD8 cells. CD4 cells from Vß3 tg⁺ mice injected 3 days previously with SEA were incubated with immobilized anti-CD3 and CD8 cells for 6 h before detection of CD4 apoptosis by flow cytometry (Fig. 5). Cultures of CD4 cells restimulated with anti-CD3 contained very few apoptotic cells, and this was only slightly increased by the addition of unprimed CD8 cells. In contrast, addition of activated CD8 cells from SEA-primed mice increased the percentage of apoptotic CD4 cells from 5 to 43%, confirming that CD8 cells promote AICD.

View larger version (16K): [in this window] [in a new window]   FIGURE 5. CD8 cells promote AICD in restimulated CD4 blasts. SEA-primed CD4 cells (day 3) were restimulated in vitro with immobilized anti-CD3, PMA, and IL-2, with or without the addition of unprimed or SEA-stimulated (day 3) CD8 cells. After 6 h, cells were stained with anti-CD4-CyChrome mAb, and apoptotic cells were detected by TUNEL. Histograms show levels of FITC staining, indicating incorporation of dUTP-FITC into DNA strand breaks of apoptotic cells (x-axes), vs numbers of gated CD4+ cells (y-axes). A, CD4 cells alone; B, CD4 cells and unprimed CD8 cells; C, CD4 cells and activated CD8 cells. Numbers indicate the percentages of apoptotic CD4 cells. Results are representative of four independent experiments.

The ability of SEA-activated CD8 cells to induce apoptosis of activated CD4 cells might reflect increased expression of FasL on CD8 cells after immunization with SEA. Although resting or activated CD4 and CD8 cells expressed similar levels of Fas (not shown), activated CD8 cells expressed enhanced levels of FasL compared with unstimulated CD8 cells. In contrast, activated CD4 cells did not express increased levels of FasL (Fig. 6A). To assess whether the increase in FasL expression on CD8 cells was associated with enhanced levels of Fas/FasL-dependent cytotoxic activity, activated CD4 or CD8 cells were added to ⁵¹Cr-labeled P815 target cells in the presence of anti-CD3. The cytotoxic activity of CD8 cells against P815 targets was much greater than that of activated CD4 cells (Fig. 6B).

View larger version (23K): [in this window] [in a new window]   FIGURE 6. Left panel, Expression of FasL by CD4 and CD8 cells from Vß3 tg+ mice after SEA stimulation. Splenocytes were prepared from mice immunized 3 days previously with SEA (activated) or from nonimmunized controls (resting). Purified CD4 and CD8 cells were cultured separately, and the relative percentages of FasL+ CD4 and CD8 cells were determined by flow cytometry. The data shown are representative of three similar experiments. Right panel, Cytotoxic activity of SEA-primed CD4 and CD8 T cells. Cells from 3-day immunized or nonimmunized mice were added to 51Cr-labeled P815 target cells at a 3:1 ratio and cultured for 14 h in the presence of anti-CD3 (1/10 dilution of 145-2C11 supernatant). The release of 51Cr into the supernatants was used to calculate the percent lysis compared with that in target cells alone. Assays were performed in triplicate, and data shown are the mean ± SEM from three independent experiments.

The finding that activated CD8 cells express particularly high levels of FasL compared with activated CD4 cells is consistent with the ability of CD8 cells, but not CD4 cells, to induce apoptosis of CD4 target cells. However, the resistance of CD8 targets (compared with CD4 targets) to Fas-dependent apoptosis and suppression by CD8 cells is not explained by these data. We asked whether direct Ab-dependent ligation of Fas on these T cell subsets might reveal a difference in their response (Fig. 7) to activation by anti-CD3. Although Fas ligation suppressed CD4 cell proliferation to anti-CD3, CD8 cell proliferation to anti-CD3 was not suppressed by any dose of anti-Fas-coated beads and was actually increased by low levels of Fas ligation. Thus, despite similar levels of expression of Fas on CD4 and CD8 cells, these data demonstrate that the Fas-coupled signaling pathway in these two subsets leads to dissimilar functional outcomes and may be biochemically distinct.

View larger version (25K): [in this window] [in a new window]   FIGURE 7. Fas ligation inhibits proliferation of CD4 blasts, but stimulates CD8 cell growth after restimulation. CD4 and CD8 T cells were purified from Vß3 tg+ mice 3 days after SEA injection and restimulated with immobilized anti-CD3, PMA, and IL-2 (5 x 104/well). Streptavidin Dynabeads were coated with biotinylated anti-Fas mAb or with a negative control hamster mAb and added to the restimulated cells at the indicated ratios. [3H]thymidine incorporation was determined after 24 h.

Since differential expression of FasL on Th1 and Th2 clones has been reported (13, 14), we tested whether skewing of CD4 cells toward the Th1 or the Th2 phenotype might render them differentially susceptible to Fas-mediated lysis by activated CD8 cells. CD8-depleted spleen and lymph node cells from untreated Vß3 tg⁺ mice were stimulated in vitro with SEA alone or in the presence of IL-4 or IL-12 before detection of intracellular cytokines by flow cytometry (27). CD4 cells primed with SEA in vitro for 3 days did not produce IL-10 (a Th2 cytokine) unless cultured in the presence of IL-4 (Fig. 8A). Addition of IL-12 had no effect on IL-10 synthesis, but did increase priming for IFN- (Fig. 8A).

View larger version (32K): [in this window] [in a new window]   FIGURE 8. CD4 cells polarized toward the Th2 phenotype are less susceptible to CD8 cell-mediated suppression. A, CD8-depleted spleen and lymph node cells (1:1 ratio) from untreated Vß3 tg+ mice were stimulated in vitro with SEA (0.1 µg/ml) for 3 days in the presence or the absence of IL-4 (20 ng/ml) or IL-12 (20 ng/ml) as indicated. Cells were washed; restimulated with PMA, ionomycin, and monensin for 5 h; and stained for intracellular accumulation of cytokines. Representative two-parameter histograms showing IL-10 staining (y-axes) vs IFN- staining (x-axes) in gated, CD4+ cells are shown. The percentages of cytokine-positive cells are indicated in the relevant quadrants. B, CD8-depleted cells were cultured in vitro with SEA and cytokines for 3 days and restimulated with immobilized anti-CD3, anti-PMA, and anti-IL-2 mAb in the presence of irradiated CD8 suppressor cells from SEA-primed Vß3 tg+ mice. The percent suppression of CD4 proliferation was determined as described in Materials and Methods. Data shown are the mean ± SEM from three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study supports an important role for CD8 T cells in the mediation of CD4 cell AICD. This mechanism is at least partially dependent on Fas, is not Ag specific, and requires activated CD4 and CD8 T cells. Deletion of CD4 cells by this mechanism results in the selective expansion of Vß3⁺ CD4 cells after SEA immunization of ß₂m^-/-, but not ß₂m^+/+, mice. One mode of CD8-dependent inhibition of SEA-activated CD4 cells may depend on recognition of Vß-specific peptide/Qa-1 complexes displayed by activated Th cells, which allows killing by CD8 (26). We show here that CD8 suppression may also be independent of MHC class I expression and specific TCR Vß expression. Nonetheless, CD8 suppression is focussed on responding CD4 cells and not bystanders, since Ag-activated CD4 cells, but not resting cells, are susceptible to Fas-dependent killing by CD8 cells. These findings and the observation that CD8-suppressive activity is detectable only 2 to 4 days after SEA stimulation suggest that Fas-dependent, non-Ag-specific suppression represents a rapid and early response that limits early CD4 cell expansion in response to strong stimuli by accelerating AICD of CD4 cells. In contrast, the mechanism of CD8 suppression described by Jiang et al. involving specific CTL responses to TCR-specific peptides may be necessary for long term deletional effects (26). It is not yet known whether long term Ag-specific suppression may also depend on a Fas-dependent CD8 suppression, although the observation that Fas-deficient MRL-lpr/lpr mice fail to delete CD4 cells responding to sAg late after immunization suggests that this is the case.

AICD has been interpreted as a mechanism of suicide in which enhanced expression of Fas and FasL on individual cells induces apoptosis, while contact-dependent mechanisms may operate at higher cell densities in vitro (15, 28). However, these studies used purified CD4 or CD8 cells and did not account for the effect of CD4:CD8 T cell interactions during AICD. Our observation that activated CD4 or CD8 cells fail to suppress their own proliferative responses following Vß3 TCR ligation supports a suicidal pathway. However, activated CD8 cells also suppress the proliferative response of CD4 cells in a Fas-dependent manner. Moreover, our experiments suggest that CD8 T cell suppression of CD4 cell responses may be as important as suicidal AICD in regulating in vivo immune responses of CD4 cells, thus reflecting the increased efficacy of interactions between T cell subsets in vivo compared with in vitro culture systems. One characteristic of AICD is that a proportion of the cells survive restimulation and can proliferate to new Ag (24, 25). Our results using TUNEL analysis and a suppression assay indicate that CD8 cells enhance the rate of CD4 cell apoptosis and decrease the proportion of CD4 cells that survive this process and can proliferate in response to additional stimulation. This T:T interaction, therefore, may play an important role in down-regulating the immune response in the face of repeated restimulation of T cells, for instance by autoantigen or viral Ags.

Injection of SEA into Vß3 tg⁺ mice resulted in a greater increase in cell surface FasL expression and cytolytic activity in CD8 cells than in CD4 cells. Preferential expression of FasL by activated CD8 T cells may play a key role in the ability of CD8 cells to suppress immune responses by induction of CD4 cell apoptosis. Differential expression of FasL on T cell subsets would allow more sophisticated regulation of T cell expansion and death compared with a simple suicide model and may be a central effector mechanism in a number of models in which CD8 T cell suppression has been described (reviewed in 29 . Differential susceptibility of subsets of Th cells to AICD induced by CD8 cells provides a further level of regulation, since CD4 cells that express a Th2 cytokine profile are less prone to CD8 cell-mediated AICD. The latter property of CD8-mediated suppression may allow preferential removal of Th1-type CD4 cells and enhanced development of Th2 responses during an immune response.

While it is clear that activated CD4 T cells are sensitive to Fas:FasL-mediated AICD, our results also indicate that CD8 cells are resistant to death induced by Fas ligation and that ligation of Fas may actually enhance CD8 cell proliferation. The inherent resistance of CD8 cells to Fas-mediated cell death may provide a survival mechanism that prevents the suicide of individual CD8 cells and allows the development of effective Fas/FasL-dependent CD8 regulatory responses. CD8 T cells may instead be susceptible to AICD through a TNF-dependent pathway (30). TNF receptor knockout lpr mice show increased lymphoproliferation (31), which is thought to be CD8 cell dependent (32).

Regardless of the mechanism of AICD resistance by CD8 cells, these findings indicate that differential susceptibility to Fas-mediated apoptosis may represent a fundamental biologic difference between the two major T cell subsets. Additional studies of the signaling events coupled to Fas ligation in CD8 and CD4 cells are necessary to define the differential outcome of Fas ligation in biochemical terms.

	Acknowledgments

 
The authors thank Drs. A. K. Abbas and I. Rimm for transgenic mice, Dr. S. Ju for provision of Fas-Ig fusion protein, and Alison Angel for assistance with the preparation of this manuscript.

	Footnotes

 
¹ This work was supported in part by Research Grants AI37833 and AI13600.

² Address correspondence and reprint requests to Dr. Harvey Cantor, 44 Binney St., Boston, MA 02115.

³ Abbreviations used in this paper: AICD, activation-induced cell death; FasL, Fas ligand; SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin B; sAg, superantigen; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.

Received for publication May 9, 1997. Accepted for publication September 3, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Russell, J. H., C. L. White, D. Y. Loh, P. Meleedy-Rey. 1991. Receptor-stimulated death pathway is opened by antigen in mature T-cells. Proc. Natl. Acad. Sci. USA 88:2151.[Abstract/Free Full Text]
2. Lenardo, M. J.. 1991. Interleukin-2 programs mouse alpha beta T lymphocytes for apoptosis. Nature 353:858.[Medline]
3. Kneitz, B., T. Herrmann, S. Yonehara, A. Schimpl. 1995. Normal clonal expansion but impaired Fas-mediated cell death and anergy induction in interleukin-2-deficient mice. Eur. J. Immunol. 25:2572.[Medline]
4. Radvanyi, L. G., G. B. Mills, R. G. Miller. 1993. Religation of the T cell receptor after primary activation of mature T cells inhibits proliferation and induces apoptotic cell death. J. Immunol. 150:5704.[Abstract]
5. van Parijs, L., A. Ibraghimov, A. K. Abbas. 1996. The roles of costimulation and Fas in T-cell apoptosis and peripheral tolerance. Immunity 4:321.[Medline]
6. Su, X., T. Zhou, Z. Wang, P. Yang, R. S. Jope, J. D. Mountz. 1995. Defective expression of hematopoietic cell protein tyrosine phosphatase (HCP) in lymphoid cells blocks Fas-mediated apoptosis. Immunity 2:353.[Medline]
7. Nagata, S., P. Golstein. 1995. The Fas death factor. Science 237:1449.
8. Sneller, M. C., S. E. Straus, E. S. Jaffe, J. S. Jaffe, T. A. Fleisher, M. Stetler-Stevenson, W. Strober. 1992. A novel lymphoproliferative/autoimmune syndrome resembling murine lpr/gld disease. J. Clin. Invest. 90:334.
9. Kanagawa, O., B. A. Vaupel, S. J. Korsmeyer, J. H. Russell. 1995. Apoptotic death of lymphocytes in murine acquired immunodeficiency syndrome: involvement of Fas-Fas ligand interaction. Eur. J. Immunol. 25:2421.[Medline]
10. Singer, G. G., A. K. Abbas. 1994. The Fas antigen is involved in peripheral but not thymic deletion of T lymphocytes in T cell receptor transgenic mice. Immunity 1:365.[Medline]
11. Sytwu, H. -K., R. S. Liblau, H. O. McDevitt. 1996. The roles of Fas/APO-1 (CD95) and TNF in antigen-induced programmed cell death in T cell receptor transgenic mice. Immunity 5:17.[Medline]
12. Griffith, T. S., X. Yu, J. M. Herndon, D. R. Green, T. A. Ferguson. 1996. CD95-induced apoptosis of lymphocytes in an immune privileged site induces immunological tolerance. Immunity 5:7.[Medline]
13. Ramsdell, F., M. S. Seaman, R. E. Miller, K. S. Picha, M. K. Kennedy, D. H. Lynch. 1994. Differential ability of Th1 and Th2 T cells to express Fas ligand and to undergo activation-induced cell death. Int. Immunol. 6:1545.[Abstract/Free Full Text]
14. Hahn, S., T. Stalder, M. Wernli, D. Burgin, J. Tschopp, S. Nagata, P. Erb. 1995. Down-modulation of CD4⁺ T helper type 2 and type 0 cells by T helper type 1 cells via Fas/Fas-ligand interaction. Eur. J. Immunol. 25:2679.[Medline]
15. Russell, J. H., B. Ruch, C. Weaver, R. Wang. 1993. Mature T-cells of autoimmune lpr/lpr mice have a defect in antigen-stimulated suicide. Proc. Natl. Acad. Sci. USA 90:4409.[Abstract/Free Full Text]
16. McCormack, J. E., J. E. Callahan, J. Kappler, P. C. Marrack. 1993. Profound deletion of mature T cells in vivo by chronic exposure to exogenous superantigen. J. Immunol. 150:3785.[Abstract]
17. Murphy, D. B.. 1993. T cell mediated immunosuppression. Curr. Opin. Immunol. 5:411.[Medline]
18. Berg, L. J., B. F. de St. Groth, A. M. Pullen, M. M. Davis. 1989. Phenotypic differences between alpha beta versus beta T-cell receptor transgenic mice undergoing negative selection. Nature 340:559.[Medline]
19. Blackman, M. A., H. Gerhard-Burgert, D. L. Woodland, E. Palmer, J. W. Kappler, P. Marrack. 1990. A role for clonal inactivation in T cell tolerance to Mls-1a. Nature 345:540.[Medline]
20. Darzynkiewicz, Z., S. Bruno, G. Del Bino, W. Gorczyca, M. A. Hotz, P. Lassota, F. Traganos. 1992. Features of apoptotic cells measured by flow cytometry. Cytometry 13:795.[Medline]
21. Kawabe, Y., A. Ochi. 1991. Programmed cell death and extrathymic reduction of Vß8⁺ CD4⁺ T cells in mice tolerant to Staphylococcus aureus enterotoxin B. Nature 349:245.[Medline]
22. Scott, D. E., W. J. Kisch, A. D. Steinberg. 1993. Studies of T cell deletion and T cell anergy following in vivo administration of SEB to normal and lupus-prone mice. J. Immunol. 150:664.[Abstract]
23. Mogil, R. J., L. Radvanyi, R. Gonzalez-Quintial, R. Miller, G. Mills, A. N. Theofilopoulos, D. R. Green. 1995. Fas (CD95) participates in peripheral T cell deletion and associated apoptosis in vivo. Int. Immunol. 7:1451.[Abstract/Free Full Text]
24. Ettinger, R., D. J. Panka, J. K. M. Wang, B. Z. Stanger, S.-T. Ju, A. Marshak-Rothstein. 1995. Fas ligand-mediated cytotoxicity is directly responsible for apoptosis of normal CD4⁺ T cells responding to a bacterial superantigen. J. Immunol. 154:4302.[Abstract]
25. Russell, J. H.. 1995. Activation-induced death of mature T cells in the regulation of immune responses. Curr. Opin. Immunol. 7:382.[Medline]
26. Jiang, H., R. Ware, A. Stall, L. Flaherty, L. Chess, B. Pernis. 1995. Murine CD8⁺ T cells that specifically delete autologous CD4⁺ T cells expressing Vß8 TCR: role of the Qa-1 molecule. Immunity 2:185.[Medline]
27. Openshaw, P., E. E. Murphy, N. A. Kosken, V. Maino, K. Davis, K. Murphy, A. O’Garra. 1995. Heterogeneity of intracellular cytokine synthesis at the single-cell level in polarized T helper 1 and T helper 2 populations. J. Exp. Med. 182:1357.[Abstract/Free Full Text]
28. Vignaux, F., P. Goldstein. 1994. Fas-based lymphocyte-mediated cytotoxicity against syngeneic activated lymphocytes: a regulatory pathway?. Eur. J. Immunol. 24:923.[Medline]
29. Bloom, B. R., R. L. Modlin, P. Salgame. 1992. Stigma variations: observations on suppressor T cells and leprosy. Annu. Rev. Immunol. 10:453.[Medline]
30. Zheng, L., G. Fisher, R. E. Miller, J. Peschon, D. H. Lynch, M. J. Lenardo. 1995. Induction of apoptosis in mature T cells by tumor necrosis factor. Nature 377:348.[Medline]
31. Zhou, T., C. K. Edwards III, P. Yang, Z. Wang, H. Bluethmann, J. D. Mountz. 1996. Greatly accelerated lymphadenopathy and autoimmune disease in lpr mice lacking tumor necrosis factor receptor I. J. Immunol. 156:2661.[Abstract]
32. Maldonado, M. A., R. A. Eisenberg, E. Roper, P. L. Cohen, B. L. Kotzin. 1995. Greatly reduced lymphoproliferation in lpr mice lacking major histocompatibility complex class I. J. Exp. Med. 181:641.[Abstract/Free Full Text]

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