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The Journal of Immunology, 1998, 161: 3292-3298.
Copyright © 1998 by The American Association of Immunologists

Superantigen-Driven, CD8+ T Cell-Mediated Down-Regulation: CD95 (Fas)-Dependent Down-Regulation of Human Ig Responses Despite CD95-Independent Killing of Activated B Cells1

William Stohl2,*, David H. Lynch{dagger}, Gary C. Starling{ddagger} and Peter A. Kiener{ddagger}

* Department of Medicine, Division of Rheumatology and Immunology, University of Southern California, Los Angeles, CA 90033; {dagger} Department of Immunobiology, Immunex Corporation, Seattle, WA 98101; and {ddagger} Department of Immunology and Inflammation, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Staphylococcal superantigens, including staphylococcal enterotoxin B (SEB), promote vigorous T cell-dependent Ig responses at low dose (0.01 ng/ml). In contrast, more mitogenic high dose SEB (100 ng/ml) profoundly inhibits the Ig responses. To assess the contribution of CD8+ T cells to this inhibition, high dose SEB-dependent killing of activated B cells and down-regulation of Ig responses were determined. Rapid killing (4 h) of activated B cells was effected by high dose SEB-activated CD8+ T cells (CD8*), but not by high-dose SEB-activated CD4+ T cells (CD4*), and required the presence of high dose SEB during the cytotoxicity assay. This killing was abrogated by chelation of extracellular calcium or by treatment with concanamycin A but was only modestly affected by treatment with brefeldin A, suggesting a perforin-based pathway of killing. Despite their widely disparate abilities to rapidly kill activated B cells, CD8* and CD4* demonstrated similar quantitative abilities to effect high dose SEB-dependent down-regulation of Ig responses. Antagonist anti-CD95 mAb substantially reversed high dose SEB-dependent down-regulation effected by CD8* but had no appreciable effects on high dose SEB-dependent killing of activated B cells. These observations strongly suggest that the small fraction of activated B cells that secrete Ig are selectively sensitive to CD95-based killing but resistant to CD95-independent killing. This finding may help explain why clinical autoimmunity associated with increased titers of autoantibodies is a predominant feature of defects in CD95 or CD95 ligand.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Naturally occurring microbial superantigens (SAg)3 trigger polyclonal activation of T cells bearing specific TCR Vß elements (1, 2). Such activation requires presentation of SAg by MHC class II+ "SAg-presenting cells" (SPC) (3, 4), a function that can be accomplished by B cells (5, 6). In addition to T cell proliferation, stimulation of T cells with SAg can lead to helper (6, 7, 8, 9, 10, 11, 12, 13, 14) and cytolytic (3, 15, 16) activities.

The balance between helper and down-regulatory activities vitally affects net SAg-driven T cell-dependent Ig-secreting cell (IgSC) responses. Staphylococcal SAg, at doses orders of magnitude lower than that needed for maximal T cell proliferation (low dose SAg), promote vigorous polyclonal IgSC generation in cultures of PBMC, unfractionated T cells + B cells, and CD4+ T cells + B cells. In contrast, more mitogenic doses of SAg (high dose SAg) in these cultures profoundly inhibit IgSC responses and result in increased B cell apoptosis with decreased numbers of viable B cells (6, 17, 18). Thus, it is likely that high dose SAg-dependent down-regulation of IgSC responses is effected, at least in part, via high dose SAg-dependent killing of target B cells by activated T cells.

In addition to CD8+ T cells effecting cytolytic activity, CD4+ T cells can also effect cytolytic activity (19, 20, 21). Although experimental conditions can be manipulated to yield CD4+ T cells that effect perforin-based killing (22, 23, 24), CD4+ T cell-mediated killing is more routinely CD95 (Fas)-based (24, 25, 26, 27, 28, 29). Indeed, using staphylococcal enterotoxin B (SEB) as a prototype SAg, we have recently demonstrated that CD4+ T cells can not only promote SEB-driven B cell differentiation but can, in the absence of other T cells or non-T/non-B cells, effect high dose SEB-dependent down-regulation of IgSC responses via a CD95-based pathway (18).

CD4+ T cell-mediated cytolytic activity can assume great clinical importance under singular physiologic circumstances. In ß2-microglobulin-deficient mice (which are profoundly deficient in CD8+ T cells), CD4+ T cells can subserve the usual protective role of cytotoxic CD8+ T cells in clearing influenza virus infection (30) and the usual pathogenic role of cytotoxic CD8+ T cells in triggering lethal meningitis following intracranial infection with lymphocytic choriomeningitis virus (31). Moreover, in irradiated hosts reconstituted with Ag-specific (transgenic) CD4+ T cells and B cells, the CD4+ T cells could effect in vivo Ag-specific elimination of the B cells (32).

Nevertheless, in hosts bearing CD8+ T cells, it is likely that the bulk of cytolytic activity is effected by CD8+ T cells, not CD4+ T cells. Since CD8+ T cells effect not just CD95-based killing but perforin- and TNF-based killing as well (25, 33, 34, 35, 36, 37, 38), we reasoned that CD8+ T cells would be especially effective high dose SEB-dependent down-regulators of Ig responses.

In this report, we demonstrate that the potency of CD8+ T cells to effect high dose SEB-dependent down-regulation of Ig responses is not much different from that of CD4+ T cells. In contrast to CD4+ T cells, which cannot effect rapid SEB-dependent killing of activated B cells, CD8+ T cells are capable of doing so via a CD95-independent, perforin-based pathway. Nevertheless, a CD95-dependent pathway predominates in CD8+ T cell-mediated high dose SAg-dependent down-regulation of Ig responses. The prominence of CD95-based down-regulation by both CD4+ T cells and CD8+ T cells in modulating SEB-driven Ig responses implies a vital role for CD95/CD95 ligand (CD95L) interactions in safeguarding against uncontrolled in vivo polyclonal Ig production following infection with SAg-producing microbial organisms.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell populations

PBMC were isolated from venous blood of healthy donors by Ficoll-Hypaque density gradient centrifugation (39). CD8- T cells (~90% CD4+), CD4- T cells (~80% CD8+), and B cells (50–90% CD20+ with undetectable CD3+ cells) were isolated by immunomagnetic bead negative selection (6, 17). In some experiments, CD4+ T cells (>99% CD4+) and CD8+ T cells (>99% CD8+) were further purified by positive-selection cell sorting with FITC-conjugated anti-CD4 mAb and phycoerythrin-conjugated anti-CD8 mAb (Dako, Carpinteria, CA).

SAg

SEB was purchased from Sigma (St. Louis, MO).

Down-regulation of Ig responses

To assess T cell-mediated down-regulation of Ig responses, two conditions had to be met. First, B cells had to be activated in a fashion that resulted in sufficient Ig production whose inhibition would be easily detectable. Second, only the experimental down-regulatory T cells could surface contact the target-activated B cells in order to eliminate potential confounding effects of other cells. To accomplish this, two separate protocols were utilized. In the first, two-chamber cultures (18) were established in RPMI 1640 medium supplemented with 10% FCS and glutamine and antibiotics in 24-well plates. Transwell inserts (Costar, Cambridge, MA) were used to separate inner chambers from outer chambers. The outer chambers contained 5 x 105 CD4+ T cells that had been activated overnight in the presence of 2 x 105 irradiated (3000 rad) B cells (serving as SPC) with high dose (100 ng/ml) SEB, and the inner chambers contained 1 x 105 B cells that had been activated overnight with high dose SEB + formalin-fixed heat-killed Staphylococcus aureus (SAC) (Life Technologies, Gaithersburg, MD; 1:105 final dilution) + rIL-2 (100 U/ml). This two-chamber design permitted the activated B cells in the inner chambers to avoid T cell surface contact while being bathed by helper factors secreted by the activated CD4+ T cells in the outer chambers. After 4 days, CD4+ or CD8+ T cells (4 x 105 unless otherwise indicated) that had been separately activated in parallel (in the presence of irradiated SPC as above) with high dose SEB (respectively denoted as CD4* and CD8*) were washed and added to the inner chambers (containing activated B cells) with or without additional high dose or low dose (0.01 ng/ml) SEB. In all experiments, the day of reconstitution of target-activated B cells with effector T cells is considered as day 0. Plaque-forming cells (PFC) in the inner chambers were determined on day 2 by the reverse hemolytic plaque assay (39, 40). Each PFC was taken as an IgSC. Alternatively, IgG and IgM levels in the culture supernatants were determined at the indicated times by ELISA (41).

In the second protocol, B cells were continuously stimulated with SAC/rIL-2 for 4 days. At this time, the activated B cells were washed and transferred to 96-well flat-bottomed plates (1 x 105 cells/0.1 ml/well). CD4* or CD8* (4 x 105 cells/0.1 ml/well) were added to the activated B cells with or without high dose or low dose SEB in the presence of rIL-2 (25 U/ml final concentration). Culture supernatants were collected 3 days later, and IgG and IgM concentrations were determined by ELISA.

CD95-based protection from SEB-dependent down-regulation of Ig responses

Antagonist anti-CD95 mAb M3 (10 µg/ml) or nonagonist/nonantagonist anti-CD95 mAb M33 (10 µg/ml) (42) were added to the experimental cultures at the time that CD4* or CD8* were added. Protection from down-regulation was calculated by the formula: [(Ig in the presence of added CD8*/SEB with test Ab) - (Ig in the presence of added CD8*/SEB without test Ab)] ÷ [(Ig in the absence of added CD8*/SEB) - (Ig in the presence of added CD8*/SEB without test Ab)].

B cell recovery

Total viable cell numbers in the inner chambers of two-chamber cultures were determined by direct cell counting in the presence of trypan blue. B cell numbers were calculated by staining the harvested cells with FITC-conjugated anti-CD20 mAb, analyzing by flow cytometry, and multiplying the total viable cell count by the percentage of CD20+ cells (18).

T cell-mediated B cell cytolysis

CD4* and CD8* were assayed on day 4 for cytolytic activity in a 4-h release assay against 51Cr-labeled activated B cells (17, 43). The target B cells either had been SAC/rIL-2-activated for 4 days or had been isolated by negative selection (6, 17) from CD4+ T cell + B cell cultures that had been stimulated with low dose SEB for 4 days. Cytotoxicity assays were performed in the presence of graded doses of SEB. Specific 51Cr release was calculated from the formula: (experimental cpm - spontaneous cpm) ÷ (maximum cpm - spontaneous cpm), where spontaneous cpm was determined from wells containing only target cells without effector cells, and maximum cpm was determined from wells containing target cells lysed by 1% Triton X-100 detergent.

Calcium dependence of cytolytic activity was assessed by adding 4 mM EGTA + 3 mM MgCl2 to the cytotoxicity assays. Repletion of extracellular calcium was accomplished by addition of 4 mM CaCl2. Sensitivity of cytotoxicity to concanamycin A (CMA, 100 nM; Sigma) or to brefeldin A (BFA, 10 µM; Sigma) was assessed by adding them to the effector cells 2 h before addition of the labeled target cells. CMA and BFA were present throughout the cytotoxicity assay. Prior studies have demonstrated that their inhibitory effects are directed only against the effector cells and not against the targets (44).

T cell-mediated B cell apoptosis

B cells were activated with SAC/rIL-2 for 4 days and labeled with the fluorescent cationic membrane tracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) (Molecular Probes, Eugene, OR) (18). The labeled B cells were cultured for two additional days with unlabeled CD8* (ratio 2:5) ± high dose SEB ± anti-CD95 mAb, stained with FITC-conjugated recombinant human annexin-V in calcium-containing binding buffer (Caltag Laboratories, Burlingame, CA), and analyzed by two-color flow cytometry. B cells undergoing apoptosis were taken as DiI-labeled (red) cells that stained positively for annexin-V (green).

Release of soluble CD95L (sCD95L)

Cultures (1 ml) of CD8+ T cells (2.5 x 106) + irradiated B cells (1 x 106) were stimulated with low dose SEB for 5 days, washed, and restimulated with graded doses of SEB. Culture supernatants were harvested at the indicated times following restimulation for sCD95L determination (18, 45).

Statistical analysis

All analyses were performed using SigmaStat software (Jandel Scientific, San Rafael, CA). The raw data were log transformed, and the paired t test and one-way repeated measures ANOVA test were used when comparing two groups and three or more groups, respectively. When the log-transformed data did not follow a normal distribution, the nonparametric Wilcoxon signed rank test and Friedman repeated measures ANOVA on ranks test were used, respectively. A p value of <0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD8* effect down-regulation of Ig responses to a degree similar to that effected by CD4*

To determine the ability of CD8* to effect high dose SEB-dependent down-regulation of IgSC responses, two-chamber cultures were established, with the CD4+ T cells in the outer chambers supplying helper factors to the activated B cells in the inner chambers. (Preliminary studies showed that Ig responses in two-chamber cultures containing activated CD4+ T cells in the outer chambers were ~10-fold greater than those in single-chamber B cell cultures activated in the absence of T cells (data not shown).) In the presence of high dose SEB, CD8* added to the inner chambers inhibited PFC responses in a dose-dependent fashion (Fig. 1GoA), similar to our previous findings with CD4* (18). Also, as previously shown for CD4* (18), PFC responses were inhibited only when the effector CD8* and the target B cells were in the same chamber (p = 0.003, Fig. 1GoB). When CD8* were added to the outer chambers rather than to the inner chambers, cell-cell interactions between the CD8* and B cells were precluded, and no significant effects on PFC responses were observed.



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  		FIGURE 1. High dose SEB-dependent downregulation of PFC responses by CD8* is dose-dependent and requires intimate contact between effector CD8* and target B cells. A, The two-chamber culture design described in Materials and Methods was utilized. Graded numbers of CD8* + high dose SEB from two different donors were added to the inner chambers containing the activated B cells, and PFC in the inner chambers were assayed 2 days later. B, No cells or 4 x 105 CD8* were added to the outer chambers or to the inner chambers. High dose SEB was added in all cultures. PFC in the inner chambers were assayed 2 days later. Results are shown as geometric mean ± 1 SEM for the four donors tested.

 
No high dose SEB-dependent down-regulation was appreciated when the CD8+ T cells added to the inner chambers had not been previously activated (Fig. 2GoA). In contrast, high dose SEB-dependent down-regulation was substantial in the presence of activated CD8+ T cells, even if the CD8+ T cells had been activated by low dose SEB rather than by high dose SEB. CD8* and CD4* each effected significant high dose SEB-dependent down-regulation of IgSC responses (p < 0.001), and, strikingly, CD8* were no more potent than were CD4* in doing so (p = 0.939, Fig. 2GoB). Similar down-regulation was observed even when the effector CD8* and CD4* were generated from sort-purified CD8+ T cells and CD4+ T cells (Fig. 2GoC), indicating that the down-regulation was not due to contaminating non-CD8+ or non-CD4+ T cells, respectively.



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  		FIGURE 2. High dose SEB-dependent inhibition of IgSC responses by CD8* and CD4*. A, The two-chamber culture design was utilized. No cells (– – –) or 4 x 105 CD8+ T cells that had not been activated (none), had been activated with low dose SEB (LD), or had been activated with high dose SEB (HD) were added to the inner chambers with no (solid bars), low dose (horizontal lines), or high-dose (cross hatched lines) SEB. PFC in the inner chambers were assayed 2 days later. Results are shown for one of the three experiments performed. B, Geometric mean PFC values for the six donors tested ± 1 SEM are presented with addition to the inner chambers of high dose SEB alone (SEB), high dose SEB + CD4* (+CD4*), high dose SEB + CD8* (+CD8*), and neither T cells nor high dose SEB (none). C, No T cells (solid bars) or 4 x 105 sort-purified CD4* (horizontal lines) or sort-purified CD8* (cross-hatched lines) were added to the inner chambers. High dose SEB either was or was not also added at the same time. PFC in the inner chambers were assayed 2 days later.

 
Analysis of Ig levels in culture supernatants confirmed the PFC results. By the time CD4* or CD8* were added to the activated B cells in the inner chambers, IgG levels were already substantial. In unperturbed cultures, IgG levels steadily increased for at least eight additional days (Fig. 3GoA). Addition of CD4* alone to the B cells had little effect on IgG production, and addition of CD8* alone had, at most, only modest inhibitory effects on IgG production. In contrast, addition of either CD4* or CD8* and high dose SEB completely blocked further increases in IgG levels. IgM production in these cultures was lower, but the pattern of response to CD4* or CD8*, with or without high dose SEB, was identical (data not shown).



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  		FIGURE 3. High dose SEB-dependent inhibition of IgG production by CD8* and CD4*. A, The two-chamber culture design was utilized. No additives or CD4* or CD8* (4 x 105) were added to the inner chambers with or without high dose (HD) SEB, and aliquots of culture supernatants were harvested at the indicated times and assayed for IgG. B, B cells were activated with SAC/rIL-2 for 4 days, washed, and reconstituted with CD4*, CD8*, or no T cells. The secondary cultures were stimulated with no, low dose (LD), or high dose (HD) SEB, and culture supernatants were assayed for IgG levels on day 3 following reconstitution. Results are presented as geometric mean ± 1 SEM for the four donors tested.

 
To document that the reduction of Ig levels in the two-chamber cultures was not due to CD4* or CD8* blocking B cell responsiveness to the helper factors secreted by the CD4+ T cells in the inner chambers, SAC/rIL-2-activated B cells were washed and transferred into single-chamber cultures (Fig. 3GoB). The modest IgG production was significantly retarded when CD4* or CD8* were added with high dose SEB (p < 0.001). In the absence of added CD4* or CD8*, high dose SEB had no inhibitory effects (p = 0.930), and neither CD4* nor CD8* were inhibitory even in the presence of low dose SEB (p = 0.339). Of note, CD8* were statistically again no more potent down-regulators than were CD4* in the presence of high dose SEB (p = 0.071).

CD8* effect high dose SEB-dependent killing of activated B cells more potently than do CD4* via a calcium-dependent, CMA-sensitive, CD95-independent pathway

Despite the similar down-regulatory potencies of CD4* and CD8*, CD8*, but not CD4*, effected considerable high dose SEB-dependent killing of activated B cells in a 4-h 51Cr release assay (Fig. 4GoA). Chelation of extracellular calcium with EGTA/MgCl2 profoundly inhibited activated B cell killing by CD8*, and such cytotoxicity was restored by replenishment of extracellular calcium with CaCl2 (Fig. 4GoB), suggesting a prominent role for perforin-based cytotoxicity (46, 47, 48, 49). Unfortunately, perforin-based cytotoxicity cannot be inhibited with available anti-perforin mAb {delta}G9 (50) (data not shown), either because the mAb does not block the biologic activity of perforin or because the anti-perforin mAb cannot sterically reach the intimate cell-cell contact points between the CTL effectors and the target cells. Nevertheless, involvement of a perforin-based pathway can be inferred by pretreatment of effector CTL with the vacuolar type H+-ATPase inhibitor, CMA, a process that inhibits perforin-based cytotoxicity but does not affect CD95-based cytotoxicity (44, 51). CMA completely blocked high dose SEB-dependent killing of activated B cell targets (Fig. 4GoC). In contrast, BFA, an inhibitor of intracellular glycoprotein transport that profoundly inhibits CD95-based cytotoxicity but has only modest effect on perforin-based cytotoxicity (44), had only a modest inhibitory effect on high dose SEB-dependent activated B cell killing (Fig. 4GoC). In addition, a CD95L fusion protein (52), which induces CD95-based apoptosis of activated B cells after overnight incubation, did not promote any specific 51Cr release in the 4-h assay (data not shown). Taken together, these experiments suggest that high dose SEB triggers a cytolytic perforin-based, CD95-independent pathway in CD8* that is either absent or markedly diminished in CD4*.



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  		FIGURE 4. CD8*, but not CD4*, effect rapid high dose SEB-dependent cytolysis of activated B cells via a calcium-dependent, CMA-sensitive pathway. A, To generate B cell targets, CD4+ T cell + B cell cultures were stimulated with low dose SEB for 4 days, at which time the B cells were isolated by negative selection and labeled with 51Cr. CD4* (open symbols) and CD8* (closed symbols) were assayed for cytolytic activity in the presence of no SEB (circles), low dose SEB (squares), or high dose SEB (triangles). Error bars indicate 1 SEM of the replicates. B, CD4* and CD8* were assayed against 51Cr-labeled 4-day SAC/rIL-2-activated B cells in the presence of no SEB (lines rising to right), low dose SEB (LD SEB, lines rising to left), high dose SEB (HD SEB, horizontal lines), high dose SEB + EGTA/MgCl2 (solid bars), or high dose SEB + EGTA/MgCl2 + CaCl2 (cross-hatched lines). Results are shown for E:T ratio of 20:1. C, CD8* were assayed against 51Cr-labeled 4-day SAC/rIL-2-activated B cells in the presence of high dose SEB (circles), high dose SEB + CMA (squares), high dose SEB + BFA (triangles), or no SEB (diamonds). CMA and BFA were added to the effector cells 2 h before addition of the 51Cr-labeled targets. Error bars indicate 1 SEM of the replicates.

 
CD8* effect high-dose SEB-dependent down-regulation of IgSC responses via a CD95-based pathway

We have previously demonstrated that CD4+ T cell-mediated high-dose SEB-dependent down-regulation of IgSC responses is CD95-based. This is associated with increased release of sCD95L by CD4+ T cells following stimulation with high dose, but not low dose, SEB (18). Given the considerable CD95-independent killing of activated B cells by CD8* in the presence of high dose SEB, we anticipated that high-dose SEB-dependent down-regulation of IgSC responses mediated by CD8* would also be largely CD95 independent. Surprisingly, antagonist anti-CD95 mAb M3, but not nonagonist/nonantagonist anti-CD95 mAb M33, had a considerable protective effect (Fig. 5Go). In the four experiments performed, addition of CD8* to cultures containing high dose SEB resulted in a 94% drop in geometric mean PFC response. Addition of mAb M3 to such cultures restored the geometric mean PFC response to 58% of the level in cultures not containing CD8* (p = 0.033), resulting in a 65% geometric mean protective effect by mAb M3. In contrast, addition of CMA had no protective effect. However, since addition of CMA in the absence of CD8* or high dose SEB inhibited PFC responses by ~80% (data not shown), the lack of protection by CMA should be interpreted with great caution. Consistent with CD95-based down-regulation is that high dose, but not low dose, SEB markedly increased soluble CD95L (sCD95L) levels in CD8+ T cell + irradiated B cell cultures (Fig. 6Go), similar to previous observations with CD4* (18).



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  		FIGURE 5. Protection by antagonist anti-CD95 mAb M3 against high dose SEB-dependent down-regulation of PFC responses mediated by CD8*. The two-chamber culture design was utilized. No additives (none), high dose SEB only (SEB), high dose SEB + CD8* without anti-CD95 mAb (SEB/CD8*), high dose SEB + CD8* + mAb M3 (SEB/CD8*/M3), or high dose SEB + CD8* + mAb M33 (SEB/CD8*/M33) were added to the inner chambers, and PFC were assayed 2 days later. One representative experiment of the four performed is shown.

 


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  		FIGURE 6. Increased sCD95L levels following stimulation with high dose SEB. CD8+ T cell + irradiated B cell cultures were stimulated for 5 days with low dose SEB, washed, and restimulated with no, low dose (LD), or high dose (HD) SEB. Culture supernatants were harvested on day 1 (circles) and day 2 (diamonds) and assayed for sCD95L levels by ELISA. Results are presented as geometric mean ± 1 SEM for the four donors tested.

 
Antagonist anti-CD95 mAb M3 does not protect against high dose SEB-dependent activated B cell killing mediated by CD8*

CD4* are capable of effecting CD95-based, high dose SEB-dependent killing of activated B cells (18). Although such killing is not detectable in a 4-h 51Cr release assay (Fig. 4Go, A and B), it is readily detectable in a 2-day apoptosis assay (18). Thus, it was possible that CD95-based, high dose SEB-dependent killing of activated B cells by CD8* would be appreciated in the latter assay. However, in contrast to the protective effect of mAb M3 against high dose SEB-dependent B cell apoptosis mediated by CD4* (18 and data not shown), apoptosis of activated B cells incubated for 2 days with CD8* + high dose SEB was unaffected by antagonist anti-CD95 mAb M3 (Fig. 7Go).



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  		FIGURE 7. Lack of protection by antagonist anti-CD95 mAb M3 against high dose SEB-dependent activated B cell apoptosis mediated by CD8*. B cells were activated with SAC/rIL-2 for 4 days and labeled with DiI. These DiI-labeled activated B cells were cultured for 2 days with CD8* (2:5 ratio) + high-dose SEB in the presence of no anti-CD95 mAb, mAb M33, or mAb M3. The cells were stained with FITC-conjugated annexin-V and analyzed by flow cytometry. B cells undergoing apoptosis displayed both red (DiI) and green (FITC-annexin-V) fluorescence. The numbers adjacent to the upper right quadrants indicate the percentages of labeled B cells (DiI+) positively staining with annexin-V.

 
To confirm the lack of discernible protection by antagonist anti-CD95 mAb against B cell death, B (CD20+) cell numbers in the inner chambers of two-chamber cultures were assessed on day 2 (the time when PFC responses were assessed). When CD8* were added to the inner chambers without high dose SEB, geometric mean B cell recovery (x 10-3) in the four experiments performed was 86.9. When high dose SEB was also added, geometric mean B cell recovery fell to 58.5. Addition of neither nonagonist/nonantagonist anti-CD95 mAb M33 nor antagonist anti-CD95 mAb M3 had any restorative effects on B cell recovery (respective geometric mean B cell recoveries 47.2 and 57.9). That is, with CD8* as effector cells, there was a striking dichotomy between the considerable ability of antagonist anti-CD95 mAb M3 to protect against high dose SEB-dependent down-regulation of Ig responses and the lack of mAb M3 to protect against high dose SEB-dependent killing of activated B cells. Thus, although CD8*, in the presence of high dose SEB, are potent killers of activated B cells via a perforin-based pathway, it is via a CD95-based pathway that CD8* effect the majority of their down-regulation of Ig responses.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Microbial SAg elaborated by ubiquitous disease-promoting infectious organisms can lead to T cell-dependent polyclonal B cell differentiation in vitro (6, 7, 8) and in vivo (53, 54, 55) with increased titers of autoantibodies. Indeed, a compelling argument promoting a role for microbial SAg in the generation of pathogenic autoantibodies in vivo has been made (56). By inference, the normal ability to down-regulate SAg-driven polyclonal Ig responses may play an important role in maintaining an autoimmune disease-free state.

Regulation of polyclonal Ig responses to SAg, at least in vitro, is critically dependent upon SAg concentration. As long as surface contact between CD4+ T cells and B cells can occur, low dose SAg promotes vigorous Ig responses in CD4+ T cell + B cell cultures, whereas high dose SAg profoundly inhibits Ig responses (17, 18). This CD4+ T cell-mediated high dose SEB-dependent down-regulation is effected via a cytolytic CD95-based pathway (18).

Since the bulk of cytolytic activity under physiologic conditions is likely effected by CD8+ T cells, we focused our attention on CD8* as effectors of SEB-dependent down-regulation of Ig responses. As expected, CD8* were much more proficient than were CD4* in effecting high dose SEB-dependent killing of activated B cells (Fig. 4GoA). Importantly, the mechanism underlying the ability of CD8* to rapidly (4 h) kill activated B cell targets appears to be perforin-based and not CD95-based. First, cytotoxicity was highly calcium-dependent (Fig. 4GoB), and perforin-based pore formation and cytotoxicity is exquisitely dependent upon extracellular calcium (46, 47, 48, 49). Second, cytotoxicity was highly sensitive to CMA but only modestly sensitive to BFA (Fig. 4GoC), consistent with perforin-based cytotoxicity but not with CD95-based cytotoxicity (44). Third, a biologically potent CD95L fusion protein (52) was incapable of inducing detectable 51Cr release in a 4-h time period (data not shown).

Despite this greater perforin-based killing of activated B cells by CD8* than by CD4*, high dose SEB-dependent down-regulation effected by CD8* was no greater than that effected by CD4* (Figs. 2Go and 3Go). Restimulation of CD8* with high dose, but not low dose, SEB resulted in a dramatic increase in release of sCD95L (Fig. 6Go), similar to previous observations with CD4* (18). This suggested that a CD95-based pathway may substantially contribute to high dose SEB-dependent down-regulation effected by CD8*. Indeed, even though antagonist anti-CD95 mAb M3 had no appreciable effect on high-dose SEB-dependent killing of activated B cells mediated by CD8* (Fig. 7Go), antagonist anti-CD95 mAb M3 was highly protective of IgSC responses (Fig. 5Go). Thus, not only is a CD95-based pathway critical to Ig down-regulation effected by CD4* (18), but it is vital to Ig down-regulation effected by CD8* as well. This high dose SEB-dependent down-regulatory effect of CD8* on B cell function is a direct one requiring no other cells as intermediaries and requiring surface contact between effector CD8* and target activated B cells (Fig. 1Go). This effect on B cells is distinct from a recently described CD95-based down-regulatory effect of murine SEA-activated CD8+ T cells on SEA-activated CD4+ T cells (57). It may be that CD95-based, SAg-dependent, CD8+ T cell-mediated down-regulation is realized in vivo via effects on both B cells and on CD4+ T cells. In any case, the redundancy of CD95-based down-regulation effected by both CD4+ T cells and CD8+ T cells may be an important physiologic protective mechanism against development of autoimmunity even under conditions of selective T cell subset depletion (e.g., AIDS).

The preeminence of CD95-dependent down-regulation by CD8* despite their ability to rapidly kill activated B cells via a CD95-independent pathway suggests that the activated B cell targets of CD95-independent killing are preferentially not IgSC or their precursors. Only a small fraction of activated B cells differentiate into IgSC, and the degree of activated B cell death under our experimental conditions was always considerably less than 100% (Figs. 4Go and 7Go and data not shown). Thus, IgSC or their precursors may be selectively sensitive to CD95-based killing but relatively resistant to perforin-based killing. CD95-independent (perforin-based) killing of activated B cells could be quantitatively abundant while having a disproportionately low effect on ultimate IgSC responses. Nevertheless, our results suggest some role for CD95-independent down-regulation by CD8*. Although we previously showed that antagonist anti-CD95 mAb M3 completely (geometric mean 106%) protected against high dose SEB-dependent down-regulation mediated by CD4* (18), mAb M3 incompletely (geometric mean 65%) protected against identical down-regulation mediated by CD8* (Fig. 5Go).

The modest CD95-independent contribution to high dose SEB-dependent down-regulation of Ig responses notwithstanding, our results underscore the vital role for CD95/CD95L interactions in Ig homeostasis. Systemic lupus erythematosus-like illness (including increased autoantibody production) has been described in association with genetic defects in CD95 or CD95L in man (58, 59, 60, 61) and mouse (34, 62, 63, 64, 65, 66, 67, 68). In contrast, although mice deficient in perforin have impaired abilities to clear in vivo viral infections (35, 36), the importance of perforin to the regulation of B cell function is uncertain. In fact, studies in a MHC-matched allogeneic bone marrow transplantation model have raised doubt regarding the role of perforin-based effector function in B cell elimination in vivo. In these studies, transfer of a sublethal dose of donor T cells from wild-type mice resulted in marked lymphoid hypoplasia in the recipients with almost complete eradication of CD19+ B cells. When donor T cells were from perforin-deficient mice, an identical pattern was observed in the recipients. However, when donor T cells were from CD95L-deficient mice, resulting B cell numbers in the recipients were normal (69), suggesting that in vivo elimination of B cells may be far more dependent upon CD95-based mechanisms than on perforin-based mechanisms.

Adoptive transfer studies have also supported a vital role for CD95/CD95L interactions in elimination of B cells in vivo. Anergic B cells transgenic for hen egg lysozyme (HEL) and HEL-specific T cells (both CD4+ and CD8+) were adoptively transferred into nontransgenic (perforin-intact) recipients. When the adoptively transferred B cells were derived from CD95-intact mice, the number of HEL-binding B cells detected in vivo was very low. When the adoptively transferred B cells were derived from CD95-deficient mice, the number of HEL-binding B cells detected in vivo was very great (32). Nevertheless, more recent studies using mice transgenic for CD86 have suggested that in vivo B cell elimination is also significantly affected by non-CD95-based mechanisms (70), and perforin deficiency exacerbated autoimmune features in mice whose background genes were 50% derived from autoimmune-prone MRL mice (71). Additional studies will be necessary to assess the in vivo effects of SAg on B cell function and B cell elimination (effected via CD95- and non-CD95-based pathways) and their ramifications for clinical autoimmunity.


   Acknowledgments
 
We thank Drs. Stephen Stohlman, David Horwitz, and Günther Dennert for helpful suggestions and critical review of this manuscript; Julie Elliott, Anna Gilmore, and Dong Xu for technical assistance; and Iris Williams for assistance with the flow cytometry.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grant AR41006, by a grant from the Arthritis Foundation Southern California Chapter, and by a grant from the Robert E. and May R. Wright Foundation. Back

2 Address correspondence and reprint requests to Dr. William Stohl, Division of Rheumatology, University of Southern California, 2011 Zonal Ave., HMR 711, Los Angeles, CA 90033. E-mail address: Back

3 Abbreviations used in this paper: SAg, superantigen; BFA, brefeldin A; CD4*, high dose SEB-activated CD4+ T cells; CD8*, high dose SEB-activated CD8+ T cells; CD95L, CD95 ligand; CMA, concanamycin A; DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; HEL, hen egg lysozyme; IgSC, Ig-secreting cell; PFC, plaque-forming cell; SAC, formalin-fixed heat-killed Staphylococcus aureus; sCD95L, soluble CD95L; SE, staphylococcal enterotoxin; SPC, SAg-presenting cell(s). Back

Received for publication February 19, 1998. Accepted for publication May 28, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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