Ligand-Mediated Fratricide Lymphocytes Susceptible to Fas

Trypanosoma cruzi Infection Selectively Renders Parasite-Specific IgG + B Lymphocytes Susceptible to Fas/Fas Ligand-Mediated Fratricide This information is current as of September 27, 2021. Elina Zuñiga, Claudia C. Motran, Carolina L. Montes, Hideo Yagita and Adriana Gruppi J Immunol 2002; 168:3965-3973; ; doi: 10.4049/jimmunol.168.8.3965

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Trypanosoma cruzi Infection Selectively Renders Parasite-Speciﬁc IgG؉ B Lymphocytes Susceptible to Fas/Fas Ligand-Mediated Fratricide1

Elina Zun˜iga,* Claudia C. Motran,* Carolina L. Montes,* Hideo Yagita,† and Adriana Gruppi2*

The control of B cell expansion has been thought to be solely regulated by T lymphocytes. We show in this study that Trypanosoma cruzi infection induces up-regulation of both Fas and Fas ligand (FasL) molecules on B cells and renders them susceptible to B cell-B cell killing (referred to as fratricide throughout this paper) mediated via Fas/FasL. Moreover, by in vivo administration of anti-FasL blocking mAb we demonstrate that Fas-mediated B cell apoptosis is an ongoing process during this parasitic infection. We also provide evidence that B cells that have switched to IgG isotype are the preferential targets of B cell fratricide. More Downloaded from strikingly, this death pathway selectively affects IgG؉ B cells reactive to parasite but not self Ags. Parasite-speciﬁc but not self-reactive B cells triggered during this response are rescued after either in vitro or in vivo FasL blockade. Fratricide among .parasite-speciﬁc IgG؉ B lymphocytes could impair the immune control of T. cruzi and possibly other chronic protozoan parasites Our results raise the possibility that the blockade of Fas/FasL interaction in the B cell compartment of T. cruzi-infected mice may provide a means for enhancing antiparasitic humoral immune response without affecting host tolerance. The Journal of Immu- nology, 2002, 168: 3965Ð3973. http://www.jimmunol.org/

he immune system has a remarkable capacity to maintain of the B cell repertoire in the memory compartment (11). Fas- a state of equilibrium despite continual exposure to self deficient lpr and FasL-deficient gld mice present generalized lym- T Ags and to a large number of microbes. Although T lym- phoproliferation and produce autoantibodies resembling human phocytes have traditionally been thought to control homeostasis of systemic lupus erythematosus (12). Recent reports have demon- the immune system (1), recent reports have suggested B cells can strated FasL expression on the B cell compartment (3, 4, 13–17), also control T lymphocyte survival (2–4). One of the best-defined but the functional role of this expression is a matter of controversy. regulatory pathways of lymphocyte survival is the one mediated by In vivo experiments with mixed gld chimeras suggested that FasL 3 Fas/Fas ligand (FasL) interactions. FasL is a member of a mem- controls the expansion of lymphocytes only when expressed on T by guest on September 27, 2021 brane-bound and -shed protein belonging to the TNF family mem- cells (18). In contrast, using a model of Schistosoma mansoni in- bers, and a natural counterreceptor for the death-promoting Fas fection, FasL-expressing B cell could kill targets cells expressing molecule expressed by a variety of lymphoid and nonlymphoid Fas (4). Nevertheless, the occurrence of FasL-mediated B cell-B tissues (5). Lymphocyte apoptosis mediated by Fas/FasL pathway cell killing remains uncertain. regulates immune response (6, 7), and FasL-mediated apoptosis of Chagas disease (American trypanosomiasis), caused by leukocytes prevents inflammatory reactions at immune-privileged Trypanosoma cruzi, is a chronic and transmissible disease that sites (8). In the B cell lineage, following activation, cells rapidly affects nearly 20 million people in South and Central America up-regulate Fas expression. Engagement of Fas by FasL has been (19). During acute infection, depressed humoral and cellular im- proposed to represent an important mechanism for negative selec- mune responses coexist with a massive T and B cell polyclonal tion of autoreactive B cells (9, 10) as well as for the establishment activation (20). The chronic phase of the infection is characterized by progressive damage to heart and skeletal muscle (SM) tissues, which has been associated with either autoimmune attack or par- *Department of Clinical Biochemistry, Faculty of Chemical Science, National Uni- versity of Cordoba, Cordoba, Argentina; and †Department of Immunology, Juntendo asite persistence in host tissues (21). Besides its serious public University School of Medicine, Tokyo, Japan health and socioeconomic implications, T. cruzi infection is also an Received for publication June 19, 2001. Accepted for publication January 18, 2002. attractive model linking immunoregulatory mechanisms to those The costs of publication of this article were defrayed in part by the payment of page aimed at eliminating the pathogen. During acute phase of T. cruzi charges. This article must therefore be hereby marked advertisement in accordance infection CD4ϩ T cells undergo apoptosis in vivo and activation- with 18 U.S.C. Section 1734 solely to indicate this fact. induced cell death (AICD) in vitro (22), which is mediated by 1 This work was supported by grants from Consejo de Investigaciones Cientı´ficas y Fas/FasL interaction (23). Recently, we demonstrated that B cells Tećnicas, Fundacioń Antorchas, Agencia Co´rdoba Ciencia, and Agencia Nacional de Promocioń Cientı´ficayTećnica (to A.G.). A.G. is a member of the Scientific Career from acutely T. cruzi-infected mice display increased levels of of Consejo de Investigaciones Cientı´ficas y Tećnicas. E.Z., C.C.M., and C.L.M. are apoptosis in vitro (24). It is well documented that cell-mediated the recipients of Consejo de Investigaciones Cientı´ficas y Tećnicas fellowships. protective mechanisms are required for resistance during T. cruzi 2 Address correspondence and reprint requests to Dr. Adriana Gruppi, Departamento de Bioquı´mica Clıńica, Facultad de Ciencias Quı´micas, Universidad Nacional de infection (25, 26). However, several studies (27, 28) indicate the Co´rdoba, Pabelloń Argentina, Ala 1 Subsuelo, Ciudad Universitaria, 5000 Co´rdoba, importance of Abs for host survival and parasite clearance. Argentina. E-mail address: [email protected] In this study we investigated the mechanisms involved in B 3 Abbreviations used in this paper: FasL, Fas ligand; SMC, spleen mononuclear cell; lymphocyte apoptosis during T. cruzi infection and its biological CM, culture medium; FCM, flow cytometry; TcAg, Trypanosoma cruzi Ag; SM, skeletal muscle; AFC, Ab-forming cell; SAv, streptavidin; AICD, activation-induced implications for the humoral immune response profile. We dem- cell death; PI, propidium iodide. onstrate that B lymphocyte apoptosis is mediated by Fas/FasL

ϩ interaction and preferentially affects IgG highly activated B (Fc block) for 30 min at 4°C. Cells were then incubated with each PE-, cells specific for parasite Ags. FITC-, or biotin-conjugated Ab for 30 min at 4°C and washed with FCM buffer. For apoptotic cell detection, PI staining was performed as described (30). Briefly, cells were washed twice with HBSS and fixed in 1 ml 70% Materials and Methods ethanol at 4°C. Cell pellets were gently resuspended in 1 ml hypotonic Infection with T. cruzi and anti-FasL treatment fluorochrome solution (50 ␮g/ml PI diluted in 4 mM sodium citrate plus 0.3% Nonidet P-40) and kept at 4°C for 18 h in the dark. Data were BALB/c mice (6–8 wk old, obtained from Comisioń Nacional de Energıá acquired on a Cytoron Absolute cytometer (Ortho Diagnostics, Raritan, Ato´mica, Buenos Aires, Argentina) were infected i.p. with 500 trypomas- NJ) and analyzed using WinMDI 2.7 software (J. Trotter, Scripps Research tigotes from T. cruzi (Tulahueń strain) as described (24). Age-matched Institute, La Jolla, CA). uninfected normal littermates were used as controls. Spleen cells were harvested 15 days after infection. For in vivo treatment with anti-FasL, neutralizing anti-FasL mAb MFL-4 (0.5 mg per mouse) or normal hamster Transmission electron microscopy IgG (0.5 mg per mouse) was injected i.p. into infected mice twice a week Purified splenic B cells, from control or infected mice, were fixed in 1% starting on day 4 postinfection. glutaraldehyde diluted in 0.1 M cacodylate buffer (pH 7.3). Samples were postfixed in 1% OsO , dehydrated, and embedded in araldite (Ciba-Geigy, Abs and reagents 4 Summit, NJ). Thin sections were cut in a Porter-Blum MT2 ultramicrotome PE-labeled anti-mouse MHC class II (2G9), PE-labeled and biotinylated (Munich, Germany) and examined in a Zeiss 109 electron microscope anti-mouse CD19 (1D3), FITC-labeled anti-mouse CD3 (145-2C11), (Zeiss, Overkochen, Germany). Photographs were taken on Kodak electron FITC-labeled anti-mouse Mac-1 (M1/70), PE-labeled anti-mouse FasL imaging film (Eastman Kodak, Rochester, NY). (MFL-3), FITC-labeled anti-mouse Fas (Jo2), FITC-labeled anti-mouse IgM mAbs, anti-mouse FasL mAb (NA/LE; MFL-3), PE-labeled anti- B cell culture and spontaneous proliferation in vitro Downloaded from mouse Syndecan-1 (281-2), as well as streptavidin (SAv)-CyChrome and SAv-FITC, were purchased from BD PharMingen (San Diego, CA). FITC- B cells (1 ϫ 106cells/well) were cultured (1 ml) in flat-bottom 48-well labeled anti-mouse IgG, peroxidase-conjugated or nonconjugated anti- tissue culture plates (Techno Plastic Product, Trasadingen, Switzerland) in mouse IgG and IgM, biotinylated anti-goat IgG, SAv-alkaline phosphatase, culture medium (CM), which consisted of RPMI 1640 medium supple- 2-amino-2 methyl-1-propanol, and 4-bromo-4-chloro-indolyl phosphate mented with 10% FBS and 40 ␮g/ml gentamicin, for the indicated periods were from Sigma-Aldrich (St. Louis, MO). Anti-mouse FasL mAb in the presence of either anti-mouse FasL mAb (10 ␮g/ml) or a control (MFL-4) for in vivo treatment was prepared as described previously (29). hamster IgG mAb (10 ␮g/ml). To assess spontaneous proliferation, B cells 5 RPMI 1640, Percoll, and propidium iodide (PI) were also purchased from (2 ϫ 10 cells/well) were cultured in CM for6hintheabsence of any http://www.jimmunol.org/ Sigma-Aldrich. exogenous stimuli in the presence of [3H]TdR (1 ␮Ci/well; New England Nuclear, Boston, MA) following the protocol of Minoprio et al. (31). Cells Cell preparations were harvested and [3H]TdR incorporation into DNA was measured by liquid scintillation spectroscopy. Results are mean and SD of triplicate Splenocytes from infected or normal mice were obtained by homogeniza- cultures. tion in a tissue grinder. To obtain spleen mononuclear cells (SMC), eryth- rocytes were lysed in RBC lysis buffer (Sigma-Aldrich). For B cell puri- fication, monocytes were removed by adherence to plastic (1.5 ϫ 107 cells Ag preparation per 10-cm petri dish; a 1-h incubation at 37°C), and T cells were depleted T. cruzi Ags (TcAg) were prepared from epimastigote (Tulahueń strain) by magnetic cell sorting using anti-Thy 1.2-coated magnetic beads (Dynal, harvested from cultures in monophasic medium (32). The epimastigote Compie´gne, France) following the manufacturer’s instructions. This pro- by guest on September 27, 2021 ϩ ϩ homogenate was centrifuged at 105,000 ϫ g, and the supernatant was used cedure yielded Ͼ95% of CD19 B cells (Fig. 1A), with Ͻ2% CD3 cells ϩ for ELISA and ELISPOT assays. Mouse SM extract was from muscle (Fig. 1B) and Ͻ3% Mac-1 cells (Fig. 1C), as determined by flow cytom- tissue homogenized in 10 vol of ice-cold KCl buffer (0.3 M KCl, 15 mM etry (FCM) analysis. K HPO (pH 6.5), 5 mM MgCl , 20 mM EDTA, 1 mM PMSF). After B cells were further fractionated in low- and high-density cells. Briefly, 2 4 2 centrifugation at 10,000 ϫ g for 20 min at 4°C, the supernatant was col- purified B cells were layered on a Percoll gradient and centrifuged for 15 lected and stored at Ϫ70°C (33). min at 4°C at 1200 ϫ g. Low-density (large) B cells were collected above the 50–60% interface, while high-density (small) B cells were collected bellow the 60–66% interface. ELISPOT assay FCM analysis and apoptosis assay The presence of anti-TcAg or anti-mouse SM Ab-forming cells (AFCs) in large and small B cells was determined by ELISPOT as described (34). In B cell suspensions were washed twice in ice-cold FCM buffer (HBSS, 1% brief, serial dilutions of B cells or SMC (starting from 200,000 cells/well) ␮ BSA, 0.1% NaN3) and preincubated with anti-mouse CD32/CD16 mAb were cultured in CM in wells coated with 10 g/ml TcAg or mouse SM.

FIGURE 1. Puriﬁcation grade of B cells obtained from BALB/c mice. B cells were puriﬁed from SMC by per- forming plastic adherence and magnetic negative selection to eliminate macrophages and T cells, respectively (for details see Materials and Meth- ods). The cell population obtained was stained with biotin anti-mouse CD19 followed by SAv-CyChrome (A), with FITC-labeled anti-mouse CD3 (B) or with FITC-labeled anti- mouse Mac-1 (C). M1 indicates the percentage of CD19, CD3, and Mac-1ϩ cells, respectively. Staining with control isotype is shown as open histograms. The Journal of Immunology 3967

Following overnight incubation at 37°C, the wells were washed and incu- Table I. Activation grade of B cells obtained from normal and T. bated overnight at 4°C with anti-IgG or anti-IgM Abs, and then with al- cruzi-infected mice kaline phosphatase-conjugated anti-goat IgG, and washed after each incubation. AFCs were visualized by adding a 1/4 mix of distilled water Spontaneous containing 1.25 mg/ml 4-bromo-4-chloro-indolyl phosphate in 2-amino-2 Purified MHC Class IIhigh Proliferation methyl-1-propanol buffer (1 M 2-amino-2 methyl-1-propanol, MgCl2, Tri- B Cells Blasts (%) B Cells (%) (cpm Ϯ SD) ton X-450, NaN3 (pH 10.2)). Normal ELISA Small 8.8 22 0.30 Ϯ 0.60 Large 12.3 27 0.57 Ϯ 0.17 After a 96-h cell culture either in the absence or in the presence of anti- Infected FasL blocking mAb (MFL-3), supernatants from B cells (from either nor- Small 26.4 58 1.95 Ϯ 0.25 mal or infected mice) were collected for TcAg or mouse SM IgM and IgG Large 45.8 71 6.30 Ϯ 1.80 determination. Levels of specific Abs were detected by ELISA following the procedure described previously (35). Each sample was assayed in triplicate and the values were expressed as mean of OD read at 490 nm in an ELISA reader (Bio-Rad, Hercules, CA). mice were further separated on Percoll gradient into large and small B cells. As shown in Table I, large B cells from infected Statistical analysis mice exhibited the highest degree of activation, as demonstrated by Statistical comparisons were performed by using unpaired Student’s t test the marked increase in percentages of blast cells and MHC class or ANOVA test as indicated in the figures. All data were considered sta- IIhigh cells, and their spontaneous proliferation. B cells from non- tistically significant if p values were Ͻ0.05.

infected mice showed background levels of activation. Each of Downloaded from these cell populations was analyzed for apoptosis by PI staining in Results either freshly explanted or cultured B cells. B cells from T. cruzi-infected mice undergo apoptosis in vivo As expected, large and small B cells from normal mice show and in vitro background levels of apoptosis (Fig. 3, A and B), while a signif- B cells from T. cruzi-infected mice show increased spontaneous icant proportion of large and small B cells from infected mice apoptosis in vitro (24). To investigate whether B cell apoptosis undergo increased levels of apoptosis both in vivo (Fig. 3, C and http://www.jimmunol.org/ occurs in vivo, puriﬁed B cell populations were obtained from D, left panels) and in vitro (Fig. 3, C and D, right panels). More- normal or T. cruzi-infected mice and immediately processed for over, freshly explanted large B cells from T. cruzi-infected mice analysis of DNA content and transmission electronic microscopy. showed an increase in the percentage of apoptotic cells compared Around 18% freshly of explanted B cells from T. cruzi-infected with small B cells (Fig. 3, C vs D, left panels). Consistently, after mice undergo apoptosis, as evidenced by the presence of cells 18 h of culture in medium alone strongly activated large B cells bearing hypodiploid nuclei (Fig. 2A, right panel), which is 3-fold from infected mice show higher levels of spontaneous apoptosis the background levels seen in uninfected mice (Fig. 2A, left panel). (40%) compared with small B cells, which exhibit 28% of hypo- A representative electron micrograph of B cells from T. cruzi- diploid nuclei (Fig. 3, C vs D, right panels). In summary, B cells infected mice is shown in Fig. 2B, right panel, in comparison with strongly activated by T. cruzi infection present the highest levels of by guest on September 27, 2021 B cells from normal mice (Fig. 2B, left panel). B cells from in- apoptosis both in vivo and in vitro, and the death rate is associated fected mice showed the typical ultrastructural features of apopto- with the B cell activation stage. sis, including reduction of the cytoplasmic volume, loss of surface microvilli, chromatin condensation, and margination along the in- Inﬂuence of Fas/FasL pathway in regulation of T. cruzi-induced ner surface of the nuclear envelope. B cell apoptosis To investigate whether B cell apoptosis correlated with the cell In the next series of experiments we attempted to examine the role activation stage, B cells from either normal or T. cruzi-infected of Fas/FasL interactions in regulating B cell survival during T.

FIGURE 2. B cells from T. cruzi-infected mice undergo apoptosis in vivo. B cells from normal (left panels)orT. cruzi-infected (rights panels) mice were processed for analysis of apoptosis. A, B cell nuclei were stained with PI and the cells were subjected to hypodiploid DNA content analysis by FCM. M1 indicates the percentage of cells with hypodiploid DNA content. B, Representative electron micrographs at ϫ11,000. Data are representative of two to four independent experiments. 3968 Fas/FasL-MEDIATED B CELL FRATRICIDE DURING T. cruzi INFECTION Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. Large B cells from T. cruzi-infected mice exhibit the highest level of apoptosis in vivo and in vitro. Small (A and C) and large (B and D) B cell subpopulations from normal (A and B) or from T. cruzi-infected (C and D) mice were separated by a discontinuous Percoll gradient. Hypodiploid DNA content analysis for each isolated subpopulation was performed in freshly explanted (in vivo, left panels) cells or after an 18-h culture with medium alone (in vitro, right panels). M1 indicates the percentage of cells with hypodiploid DNA content. Data are representative of two independent experiments.

cruzi infection. First, we analyzed the expression of Fas and FasL sion of these molecules (data not shown). Accordingly, Percoll- on B cells from control or T. cruzi-infected mice. By FCM analysis separated large B cells from infected mice exhibit the highest ex- we detected that T. cruzi infection induced marked up-regulation pression of both Fas (Fig. 4B) and FasL (Fig. 4C). In contrast, of both Fas (data not shown) and FasL (Fig. 4A) on CD19ϩ B cells. small B cells from infected mice had only slightly up-regulated Fas Moreover, CD19ϩ B cells with FSChigh show the highest expres- and FasL expression (Fig. 4, B and C) compared with controls. The Journal of Immunology 3969 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. Fas and FasL expression on B cells from normal or T. cruzi-infected mice. A, Total SMC from normal or T. cruzi-infected mice were stained with both PE-labeled anti-mouse CD19 and anti-mouse FasL followed by biotinylated anti-hamster IgG and SAv-FITC. Staining with isotype control is shown in left panel. B and C, Small (left panels) and large (right panels) B cells from normal or T. cruzi-infected mice were incubated with FITC-labeled anti-mouse Fas (B) or PE-labeled anti-mouse FasL (C). M1 indicates the percentage of Fasϩ (B) or FasLϩ (C) B cells. Staining with control isotype is shown as open histograms.

Hence, during T. cruzi infection Fas and FasL expression is of CD19ϩ B cells was evaluated by FCM on day 15. As clearly strongly associated with B cell activation status. shown in Fig. 5A, T. cruzi-infected mice treated with anti-FasL To investigate whether Fas/FasL interactions limit B cell expan- mAb presented a signiﬁcant increase in the total number of B cells sion during infection in vivo, a neutralizing anti-FasL mAb was compared with mice treated with control hamster IgG or PBS. administered into infected mice. Anti-FasL mAb was injected According to this, we observed that in vivo anti-FasL treatment twice a week starting on day 4 postinfection and the total number markedly reduces the amount of B cell apoptosis compared with 3970 Fas/FasL-MEDIATED B CELL FRATRICIDE DURING T. cruzi INFECTION Downloaded from http://www.jimmunol.org/

FIGURE 5. Anti-FasL blocking mAb abrogates IgGϩ large B cell apoptosis from T. cruzi-infected mice. A, SMC from T. cruzi-infected mice treated by guest on September 27, 2021 with anti-FasL mAb (0.5 mg per mouse), control hamster IgG (0.5 mg per mouse), or PBS were stained with biotin anti-mouse CD19 followed by SAv-CyChrome and analyzed by FCM. The number of CD19ϩ B cells present was determining by referring the percentage of CD19ϩ cells to the total number of SMC. B cells from normal mice were processed in parallel. Each symbol represents the results for individual mice, and means are depicted by horizontal lines. The results were statistically evaluated by ordinary ANOVA and Tukey-Kramer multiple comparisons test. B, Puriﬁed B cells from normal or T. cruzi-infected mice were cultured during 18 h in presence of 10 ␮g/ml anti-FasL mAb or isotype-matched control mAb and processed for apoptotic detection. Results are shown as the mean of B cell apoptosis Ϯ SD. The results were statistically evaluated by Student’s t test. C, Small and large B cells from normal or T. cruzi-infected mice were cultured during 18 h in presence of 10 ␮g/ml anti-FasL mAb or isotype-matched control mAb and processed for apoptotic detection by staining with PI. Results are shown as the mean of B cell apoptosis Ϯ SD. The results were statistically evaluated by Student’s t test. D, B cells from T. cruzi-infected mice were cultured during 18 h in presence of 10 ␮g/ml anti-FasL (ﬁlled histogram) or isotype-matched control (open histogram) mAb. The cells were then analyzed for IgG expression by FCM using FITC-labeled anti-mouse IgG.

mice treated with control hamster IgG (data not shown). These Fas/FasL-mediated fratricide preferentially targets IgGϩ highly results support the idea that Fas/FasL interaction triggers B cell activated B lymphocytes death during in vivo acute T. cruzi infection. In an attempt to evaluate the relative importance of FasL-mediated Considering that during T. cruzi infection B cells undergo spon- fratricide in T. cruzi infection, we determined by FACS the per- taneous apoptosis in the complete absence of T cells (24), we next centage of IgGϩ and IgMϩ B cells after culture in the presence of investigated whether FasL-mediated B cell-B cell fratricide ac- anti-FasL. FasL blockade barely decreased the percentage of IgMϩ counted for the increased rate of B cell apoptosis. Purified B cells, B cells from infected mice compared with control isotype (data not completely depleted from T cells (Fig. 1B), taken from infected or shown) but rescued 10% of IgGϩ B cells (Fig. 5D). These data control mice were cultured for 18 h in the presence of anti-FasL correspond to the overall antiapoptotic effect of FasL blockade blocking mAb or an isotype control. Anti-FasL mAb partially cur- seen in our cultures and indicate that B cells strongly activated by tails apoptosis of B cells from infected mice but not normal mice T. cruzi infection develop a killer capacity that preferentially tar- (Fig. 5B). Moreover, anti-FasL mAb efficiently inhibited apoptosis gets cells that have switched to IgG. of large (but not small) B cells from infected mice, resulting in a reduction from 34 to 18% of subdiploid DNA content (Fig. 5C). Influence of Fas/FasL B cell fratricide on the humoral immune These data add a new dimension to humoral immune response response profile during T. cruzi infection regulation, because they demonstrate for the first time that exten- Apoptosis of B cell population may be beneficial or detrimental for sively activated large B cells from T. cruzi-infected mice could the host, depending on the specificity of the clones affected. We control their survival via Fas/FasL interactions. evaluated the effect of FasL blockade on the frequency of AFCs The Journal of Immunology 3971

FIGURE 6. In vitro FasL blockade selectively rescues T. cruzi-spe- ciﬁc IgG-secreting cells. The presence of AFCs secreting IgM (upper panels) or IgG (lower panels) reactive to parasite Ags (TcAg, left panels) or self Ags (SM, right panels) were determined by ELISPOT. Cells were processed freshly explanted (open bars) or after culturing during

36 h in the presence of anti-FasL Downloaded from mAb (p) or isotype-matched control mAb (o). The results were statistically evaluated by repeated measures ANOVA and Tukey-Kramer multiple comparisons test. http://www.jimmunol.org/ by guest on September 27, 2021 reactive with either TcAg or mouse SM self Ag. We selected as These results indicate that FasL-mediated fratricide selectively self Ag mouse SM antigenic fraction because several studies in targets parasite-specific IgG-producing cells. humans and animals have detected humoral and cellular autoreac- tivity against this tissue during T. cruzi infection and demonstrated that SM is one of the target structures involved in the pathogenesis Discussion of this disease (36). The AFC frequency before and after FasL In this study, we demonstrate that B cell apoptosis is an ongoing blockade was compared with an isotype control and evaluated by process in vivo during T. cruzi infection that is B cell sufficient and ELISPOT analysis (Fig. 6). Both large and small B cells freshly is mediated by the Fas/FasL pathway. Moreover, our data indicate explanted (Fig. 6, open bars) from T. cruzi-infected mice exhibited that FasL-mediated B cell fratricide selectively eliminates IgGϩ a high frequency of anti-parasite and self Ag-specific IgM and IgG activated B cell reactive against parasite but not self Ags. AFCs. Highly activated large B cells showed the highest frequency We have previously reported that B cells from T. cruzi-infected of IgG reactive with TcAg. After 36 h of culture (Fig. 6, hatched mice undergo an increased rate of apoptosis in culture with me- bars), we detected a drastic reduction in the frequency of both dium alone (24). Herein, we extend these findings by showing that anti-parasite and SM-specific IgM and IgG AFCs. Surprisingly, B cells also undergo apoptosis in vivo during infection. These blockade of Fas/FasL pathway rescued only T. cruzi-specific IgG results are consistent with the notion that B lymphocyte death is a secreting cells, as judged by the increase in the frequency of these hallmark event during the acute phase of many infectious pro- treated cells compared with isotype control (Fig. 6, lower left pan- cesses (17, 38, 39). We found that susceptibility to apoptosis is el). Parasite-specific IgG production after 96 h of culture was also influenced by the activation state of the B cell. Our data agree with significantly augmented in the supernatant when blocking FasL the proposal that activated B lymphocytes are more prone to die molecule (data not shown). than resting ones (40). Cell death of proliferating lymphocytes is To evaluate the influence of in vivo Fas/FasL blockade on the necessary to preserve a healthy and balanced immune system (6). humoral immune response triggered during T. cruzi infection, we Nevertheless, during an infectious process pathogen-specific lym- analyzed its impact on AFCs. We observed that the percentage of phocyte apoptosis, at a stage when the pathogen has not been CD19ϩ B cells expressing Syndecan-1, a marker of AFC (37), is cleared, would be deleterious, as it would not only restrict the significantly increased in mice treated with anti-FasL mAb in com- magnitude of the effector response but it would also facilitate es- parison with mice treated with control hamster IgG or PBS (Fig. tablishment of the microorganism and thus chronicity of the in- 7A). According to in vitro results, the in vivo anti-FasL mAb treat- fection (41). In this regard, the delaying of pathogen-specific lym- ment selectively increases the number of TcAg (but not SM)-spe- phocyte apoptosis would be an attractive strategy to enhance cific IgG-secreting cells. protective immunity and eradicate the infectious agent. Therefore, 3972 Fas/FasL-MEDIATED B CELL FRATRICIDE DURING T. cruzi INFECTION Downloaded from

FIGURE 7. In vivo FasL blockade increases the percentage of plasma cells and the number of T. cruzi-speciﬁc IgG-secreting cells. A, B cells from normal or T. cruzi-infected mice injected with PBS, control hamster IgG (0.5 mg per mouse), or anti-FasL mAb (0.5 mg per mouse) were stained with PE-labeled anti-mouse Syndecan-1 and analyzed by FCM. M1 indicates the percentages of Syndecan-1ϩ cells. Staining with control isotype is shown as open histograms. B, Freshly explanted SMC were obtained from normal or T. cruzi-infected mice injected with PBS, control hamster IgG (0.5 mg per mouse), or anti-FasL mAb (0.5 mg per mouse). The presence of AFCs secreting IgM or IgG reactive to parasite (TcAg) or self (SM) Ags were determined

by ELISPOT. The results were statistically evaluated by ordinary ANOVA and Tukey-Kramer multiple comparisons test. http://www.jimmunol.org/

identification of lymphocyte apoptotic mechanisms triggered dur- be representative of infectious processes where pathogens replicate ing chronic infections emerges as an important issue from a clin- intensively in host cells and display a complex antigenicity (in- ical point of view. cluding molecular mimetism and mitogens). In keeping with this, Fas-induced apoptosis contributes to the maintenance of ho- it has been proposed that the type of homeostatic process termi- meostasis in both B and T lymphocyte-mediated immunity (42). nating an immune response may vary according to the nature of the by guest on September 27, 2021 Fas-mediated apoptosis of T lymphocytes has been reported in Ag (40). Our data indicate that only IgGϩ AFCs specific for par- several infections (39, 43, 44), including T. cruzi infection, where asite Ags are rescued by FasL blockade, indicating that during T. CD4 T cells up-regulate both Fas and FasL and undergo AICD cruzi infection this apoptotic mechanism preferentially targets B upon stimulation (23). B cells increase Fas expression and become cells reactive against parasite but not self Ags. The question as to susceptible to Fas-mediated apoptosis in response to activation sig- why Ag specificity determines B cell susceptibility to FasL-medi- nals (45, 46). Accordingly, we found increased Fas expression on ated fratricide remains unanswered. Considering that B cell apo- B lymphocytes activated in vivo by T. cruzi infection. Our study ptosis is an immune-regulatory mechanism triggered by the host to provides the first experimental evidence of the influence of Fas/ control the excessive expansion of these cells, it is likely that T. FasL pathway on B cell killing in the context of an infectious cruzi-induced massive B lymphocyte activation would be an eva- process. However, the partial reduction of B cell death after FasL sive mechanism developed by this parasite to trigger host homeo- blockade suggests that other apoptotic mechanisms also contribute static control and interfere with protective Ab response. Further- to B cell apoptosis. more, a comparable mechanism might also reduce the efficiency of B cell killing through Fas was initially thought to be executed the immune control of other protozoan parasites. Because parasite- only by T cells expressing FasL (1, 9). However, this idea is now specific Ab production is required for full resistance during T. controversial, because several recent reports indicate that activated cruzi infection, the increment of T. cruzi-specific IgG production B cells can express FasL (3, 13–17). Furthermore, FasL expressed on B cells activated by S. mansoni soluble egg Ags is functional, raised by FasL blockade would favor the control of this infection. because it is able to kill a Fas-bearing target cell line (4). We show Our results suggest that the blockade of Fas/FasL pathway specif- that FasL is expressed on highly activated large B cells during ically in the B cell compartment of T. cruzi-infected mice could acute T. cruzi infection and provide evidence that T. cruzi infection provide a means for enhancing antiparasitic humoral responses programs B cells to induce Fas/FasL-mediated B cell fratricide. without affecting host tolerance. However, our findings should be Furthermore, we have found that this death pathway preferentially considered with caution. First, this treatment should be conducted targets highly activated B cells that have switched to IgG, the main during a limited period of acute infection to enhance the protective isotype involved in defense and pathogenesis of this parasitic dis- immunity without promoting immunopathological damage. Sec- ease (36). Further work is required to elucidate why isotype ond, we do not know whether B cells reactive to tissue-restricted switching influences susceptibility to FasL-induced death. and/or widely disseminated self Ags other than SM could be The main targets of Fas-mediated B cell apoptosis have so far affected by FasL blockade. Fas-mediated elimination of parasite- been shown to be autoreactive lymphocytes (9, 11). Fas-mediated specific B cells during acute infection adds to other reports AICD has been shown to be important for eliminating T cells showing that death of effector T lymphocytes limits the ability reactive against self Ags but not foreign Ags (47). Nevertheless, of the host to achieve complete elimination of the pathogen these experiments have used simple foreign Ag, and this may not (43, 48–51). The Journal of Immunology 3973

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