Non-overlapping Fas- and BCL-2-regulated death pathways in IgG2ab-producing B cells
Laleh Majlessi and
Guy Bordenave
Unité d'Immunophysiologie Moléculaire, Institut Pasteur, 25 rue du Docteur-Roux, 75724 Paris Cedex 15, France
Correspondence to:
G. Bordenave
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Abstract
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Using perforin (Pfp)- and/or Fas-dependent cytotoxic pathways, T splenocytes from Igha/a mice are able in vivo to totally and chronically eliminate congenic Ighb/b B cells committed to IgG2ab production. This phenomenon leads to a characteristic absence of serum IgG2ab expression (IgG2ab allotype suppression) in, for instance, histocompatible Igha/b or Ighb/b mice, having neonatally received such T cells. Because the study of the protective role of BCL-2 oncoprotein against Fas-mediated cell death has generated contradictory findings, we examined the possible impact of constitutive overexpression of transgenic human BCL-2 protein in Ighb/b B cells when the latter were exposed in vivo exclusively with the Fas-dependent, anti-IgG2ab T cell activity of Igha/a Pfp0/0 mice. We observed that, despite high intracellular expression of functional transgenic BCL-2 and no up-regulation of the principal BCL-2 inhibitors in whole Ighb/b B cells, total, chronic and specific IgG2ab suppression was exerted by Igha/a Pfp0/0 cytotoxic T cells. These data show that, in this model of negative regulation of Ig production, Fas- and BCL-2-regulated mechanisms belong to non-overlapping death pathways at the level of IgG2ab-producing B cells, targets of Igha/a T cell-mediated cytotoxicity. Thus, in these mature B cells, the Fas signalingdirectly operating via caspase 8does not involve a mitochondria-dependent pathway regulated by BCL-2.
Keywords: CD95, cytotoxicity, death pathway, Ig allotype, perforin
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Introduction
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The BCL-2 oncoprotein acts as a powerful intracellular anti-apoptotic regulator in diverse cell types (14). Evidence has been provided that cell transfection with BCL-2 markedly protects lymphoid and myeloid murine cell lines from cytolysis induced by anti-Fas antibody (5). More recently, it was shown that intracellular overexpression of BCL-2 also rescued human Burkitt's lymphoma B cells from Fas-mediated cytolysis (6) and that Fas-induced death of IgG-producing B cells was associated with down-regulation of BCL-2 (7). However, these results are contradicted by independent reports indicating that BCL-2 totally failed to inhibit cytolysis, induced by Fas signaling, in lymphoma B cells and in mitogen-activated T cells (8), and thereby suggesting that Fas and BCL-2 regulate different pathways of cell death involved in the maintenance of T cell tolerance and homeostasis (9). Consequently, the question of whether Fas and BCL-2 regulate separate or common pathways of programmed cell death in hemopoietic cells remains controversial. In the present study, we took advantage of the well-characterized in vivo model of mouse Ig allotype suppression (10) to examine whether Fas-mediated death of mature B cells, involved in a T cell-induced negative regulation of Ig production, is hampered by BCL-2 or is, alternatively, independent of the mechanism regulated by this proto-oncogene product.
Normal or a fortiori specifically triggered T splenocytes from Igha/a mice are capable of total, specific and chronic, but experimentally reversible, suppression of the production of IgG2a of the Ighb haplotype (IgG2ab) (11,12). This T cell-induced IgG2ab suppression can be studied by post-natal transfer of Igha/a T splenocytes into histocompatible Igha/b or congenic Ighb/b recipients. Alternatively, the relevant congenic Ighb/b B and Igha/a T cells can be co-transferred into adult, histocompatible and immunodeficient recombination activating gene (RAG)-20/0 (13) recipients. Subsequently, the serum IgG2ab production or suppression status is determined in Igha/b or Ighb/b recipients at adulthood or in B + T cell reconstituted RAG-20/0 mice. CD4+ and CD8+ T cells, both specific to IgG2ab, are required for suppression induction (14,15), whereas the suppression effectors are anti-IgG2ab, MHC class I-restricted CD8+ T cells (16,17). We recently demonstrated that the mechanism of this Ig allotype suppression consisted of cytolysis of IgG2ab-producing B cells by Igha/a T cells which use, alternatively or concomitantly, perforin [pore-forming protein (Pfp)]- and Fas-dependent killing pathways. Indeed, Pfp0/0 (18) Igha/a T cells are able to induce complete IgG2ab suppression against wild-type Ighb/b B cells and Pfp+/+ Igha/a T cells are totally able to induce this suppression at the level of Faslpr/lpr (lymphoproliferation) Ighb/bB cells. However, suppression establishment is fully inhibited when Pfp0/0 Igha/a T cells are used to induce this suppression on Faslpr/lpr Ighb/b B cells (19).
Here, we investigated the possible impact of constitutive overexpression of BCL-2 by Ighb/b B cells on T cell-induced IgG2ab suppression induction. We used our B + T cell reconstituted RAG-20/0 mouse model, in configurations in which Ighb/b B cells from wild-type or BCL-2-22 transgenic strains (20)specifically overexpressing the human BCL-2 transgene in the B cell lineagewere exposed to Pfp+/+ or Pfp0/0 Igha/a T cells. Consequently, we evaluated the susceptibility of wild-type or BCL-2-overexpressing Ighb/b B cells to be subjected to IgG2ab suppression through Pfp/Fas-dependent or exclusively Fas-dependent cytotoxicity of anti-IgG2ab Igha/a T cells.
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Methods
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Mice
Ighb/b C57BL/6 mice were purchased from the Centre d'Elevage Janvier (Le Genest-St-Isle, France). Their congenic Igha/a BC8 mice possess, on the C57BL/6 genetic background, the Igha region from the BALB/c strain. We established Pfp0/0 Igha/a BC8 mice by crossbreeding commercially available Pfp0/0 Ighb/b C57BL/6initially obtained by homologous recombination (18)with wild-type Igha/a BC8 and by selecting Pfp0/0 Igha/a double homozygotes from the offspring, as recently described (19). We bred wild-type Pfp+/+ and Pfp0/0 mice in the Pasteur Institute's animal facilities. Faslpr/lpr C57BL/6 mice (21) were bought from the Jackson Laboratory (Bar Harbor, ME). Because RAG-20/0 mice are unable to initiate V(D)J recombination for Ig and TCR expression, they are totally devoid of mature B and T cells (13). BCL-2-22 transgenic mice (20), harboring human BCL-2 cDNA, under the control of an Ig heavy chain enhancer, constitutively express BCL-2 in all B cells. RAG-20/0 and BCL-2-22 mice, from the ninth backcross generation to C57BL/6, were obtained from the Centre de Sélection d'Animaux de Laboratoire (CNRS, Orléans, France). RAG-20/0 status and the presence of the BCL-2 transgene were verified respectively by FACS analysis and by PCR specific to human BCL-2 (see below). RAG-20/0 mice were maintained under specific pathogen-free conditions at the Pasteur Institute.
Co-transfer of different B and T cell populations into histocompatible RAG-20/0 recipients for the suppression induction assay
Adult wild-type or Pfp0/0 Igha/a BC8 mice were injected (i.v.), at a 15 day interval, with 5x107 Ighb/b C57BL/6 B splenocytes. Seven days after the second injection, T cell-enriched splenocytes (containing ~80% CD3+ and ~10% B220+ cells) were prepared by passage through a nylon-wool column. B cell-enriched splenocytes (containing ~1.0% TCR
ß+ and ~80% B220+ cells) from wild-type or BCL-2-22 transgenic Ighb/b C57BL/6 mice were obtained by in vitro cytotoxic anti-Thy-1.2 (30-H-12) mAb treatment in the presence of guinea pig serum as the source of C. Red blood cells were then removed from B cell suspensions using hemolytic Gey's solution. B cells (5x107) or sex-matched mixtures of B (5x107) + T (5x107) cells, contained in 500 µl of BSS, were injected (i.v.) into adult RAG-20/0 C57BL/6 mice. The lymphocyte compartment reconstitution of the latter was evaluated regularly, from day 8 post-transfer and thereafter by FACS analysis of their peripheral blood lymphocytes (PBL). Serum IgG2ab expression in RAG-20/0 recipients was regularly monitored to study Ig allotype-suppression induction. IgG1b, IgG2ab and IgG2bb allotype expressions were quantified by ELISA, as fully described elsewhere (19).
mAb and FACS analysis
FITC-labeled anti-human BCL-2 (bcl-2/100) was obtained from Ancell (Coger, Paris, France). Phycoerythrin (PE)-conjugated anti-CD4 (CT-CD4), anti-CD8 (CT-CD8) and anti-B220 (RA3-6B2) mAb, FITC-conjugated anti-Thy-1.2 (5a-8) mAb, and `Perm and Fix' cytoplasmic staining solutions were from Caltag (San Francisco, CA). We prepared biotinylated anti-IgDb mAb (H63.1) (22). Gates were set on forward/side scatter and propidium iodide (PI) cells to analyze viable labeled cells in a FACScan using CellQuest software (Becton Dickinson, Mountain View, CA). Cells were sorted in an Epics Elite ESP system (Coulter, Hialeah, FL).
Cell culture
Dexamethasone was purchased from Sigma-Aldrich (St Quentin Fallavier, France) and was dissolved in pure ethanol as a 103 M stock solution. Splenocytes were cultured, for 24 h, at 8x105 cells/ml/well in complete RPMI 1640 + 5% FCS in the presence of increasing concentrations of Dexamethasone or corresponding dilutions of ethanol vehicle. B cell viabilities were determined by PE-conjugated anti-B220 mAb and PI staining followed by FACS analysis. Results are expressed, for each point, as the ratio of B220+ PI cell percent in the presence of Dexamethasone to that in the presence of the corresponding vehicle dilutionsx100.
Primers
The BCL-2 primers (5'-TGG ATC CAG GAT AAC GGA GG-3' and 5'-TGT TGA CTT CAC TTG TGG CC-3') yielded 168 bp fragments from human, but not from mouse, BCL-2 gene. The bcl-x primers (5'-TGA GAG AGG CAG GCG ATG AGT TTG-3' and 5'-CCA CAG TCA TGC CCG TCA GGA ACC-3') yielded respectively 203 and 393 bp fragments from bcl-xS and bcl-xL mouse cDNA. The bax primers (5'-ATG GAC GGG TCC GGG GAG CAG C-3' and 5'-ATC TTC TTC CAG ATG GTG AGC GA-3') generated 579 bp fragments from mouse cDNA.
PCR and semi-quantitative RT-PCR
Genomic DNA was extracted from PBL according to a standard protocol. Total RNA was prepared, using RNA Plus solution (Bioprobe System, Montreuil-sous-Bois, France), from 25x105 B220+ IgDb+ sorted B splenocytes (~96% pure) from individual reconstituted RAG-20/0 mice. cDNA was then synthesized by use of MMLV reverse transcriptase SuperScript (Gibco, Erancy, France). PCR were performed using Goldstar DNA Taq polymerase purchased from Advanced Biotechnologies (Eurogentec, Seraing, Belgium), in the presence of 12.5 pmol of appropriate primers in a PTC-100 programmable thermal controller (Prolabo, Fontenay-sous-Bois, France). In appropriate ranges of cDNA serial dilutions and after 3035 PCR amplification cycles, a semi-logarithmic relationship existed between the input cDNA level and the band intensities of RT-PCR products. Samples were first adjusted to equal cDNA concentrations based on their amounts of ß-actin RT-PCR products. Serial dilutions from equal amounts of cDNA were then amplified in the presence of bcl-x or bax primers. RT-PCR reaction samples were electrophoresed in 2% agarose gels containing 0.15 µg/ml of ethidium bromide. RT-PCR band intensities were quantified using BioCapt and Bio-1D software (Vilber Lourmat, Marne-la-Vallée, France).
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Results
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Co-reconstitution of RAG-20/0 mice with wild-type or BCL-2-22 Ighb/b B cells and Pfp+/+ or Pfp0/0 Igha/a T cells
To investigate the IgG2ab suppression-induction potential of Pfp+/+ or Pfp0/0 Igha/a T cells at the level of wild-type or BCL-2-22 Ighb/b B cells, we opted for our experimental model of RAG-20/0 mice, co-reconstituted with Ighb/b B + Igha/a T cell mixtures. With this model, the IgG2ab expression or suppression status can be determined as early as 23 weeks after B + T cell transfer. Furthermore, this reconstituted RAG-20/0 mouse model has the advantage that the successful implantation of wild-type or different transferred mutant B or T cell partners can be preliminarily verified by FACS analysis of PBL from RAG-20/0 recipients, entirely devoid of endogenous mature B and T cells.
Experimental groups of RAG-20/0 mice received a mixture of wild-type Ighb/b B + Pfp+/+ Igha/a T cells (n = 3), wild-type Ighb/b B + Pfp0/0 Igha/a T cells (n = 3), BCL-2-22 Ighb/b B + Pfp+/+ Igha/a T cells (n = 3), BCL-2-22 Ighb/b B + Pfp0/0 Igha/aT cells (n = 3) or BCL-2-22 Ighb/b B cells alone (n = 3). The repopulation of the mature B and T cell compartments of the RAG-20/0 recipients was regularly monitored by FACS analysis of their PBL. As shown in Fig. 1
, as early as day 15 post-transfer, readily detectable B cell implantations were found with wild-type or BCL-2-22 Ighb/b B cells in recipients of B + T mixtures or B cells alone. Considerable CD4+ and CD8+ T subset reconstitutions were obtained in recipients of Pfp+/+ or Pfp0/0 Igha/a T cells, regardless of the origin of co-transferred Ighb/b B cells. In accordance with our previous observation (19), the CD4+/CD8+ ratio in the peripheral T cell compartment of reconstituted RAG-20/0 mice was often inverted compared to that of the initial transferred T cell suspensions. Note that ~3% CD4+, but no detectable CD8+, T cells were found in RAG-20/0 recipients of BCL-2-22 Ighb/b B cells alone (Fig. 1
), resulting from the systematic presence of 12% residual CD4+ T cells contaminating our B cell-enriched suspensions. These engraftment data showed that suitable experimental conditions had been obtained to investigate IgG2ab suppression-induction on wild-type or BCL-2-overexpressing Ighb/b B cells by Pfp+/+ (Pfp/Fas-dependent) or Pfp0/0 (only Fas-dependent) cytotoxic Igha/a T cells.

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Fig. 1. Implantation of B and T cells in RAG-20/0 recipients of wild-type or BCL-2-22 Ighb/b B + Pfp+/+ or Pfp0/0 Igha/a T cell mixtures or the same preparation of BCL-2-22 Ighb/b B cells alone. Similar engraftment profiles were obtained with wild-type or BCL-2-22 Ighb/b B cells, and with CD4+ and CD8+ T cells of Pfp+/+ or Pfp0/0 Igha/a origin. FACS analyses of PBL from individual representative mice were conducted on day 15 post-transfer. Percentages are those of double-labeled cells. No B220+ IgDb+, Thy-1.2+CD4+ or Thy-1.2+CD8+ cells were detected in RAG-20/0 mice before cell transfers.
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Effect of constitutive overexpression of BCL-2 by Ighb/b B cells on IgG2ab suppression induction
From day 23 post-transfer until the end of the experiment, i.e. day 87, we observed that RAG-20/0 recipients of wild-type Ighb/b B + Pfp+/+ Igha/a T cell or wild-type Ighb/b B + Pfp0/0 Igha/a T cell mixtures were subjected to total IgG2ab suppression. In addition, we reproduced here our previous observation that all recipients of a Faslpr/lpr Ighb/b B + Pfp0/0 Igha/a T cell mixture escaped suppression induction and expressed high serum levels of IgG2ab (410 ± 140 µg/ml on day 23 post-transfer) (Fig. 2
). We previously observed that RAG-20/0 recipients of a Faslpr/lpr Ighb/b B + Pfp+/+ Igha/a T cell mixture were submitted to total IgG2ab suppression (19). Thus, upon total blockade of the Pfp-dependent pathway, Igha/a T cells induced total IgG2ab suppression, exclusively via the Fas-dependent cytotoxic pathway.

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Fig. 2. Assay of IgG2ab suppression induction, by Pfp+/+ or Pfp0/0 Igha/a T cells, at the level of wild-type, Faslpr/lpr or BCL-2-22 Ighb/b B cells. Serum IgG2ab concentrations in BCL-2-22 B or diverse B + T cell reconstituted RAG-20/0 mice on day 23 post-transfer. The vertical bars represent the mean concentrations.
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Furthermore, we observed that the RAG-20/0 recipients of BCL-2-22 Ighb/b B + Pfp+/+ Igha/a T cell or BCL-2-22 Ighb/b B + Pfp0/0 Igha/a T cell mixtures were all chronically subjected to full IgG2ab suppression, while RAG-20/0 recipients of the same preparation of BCL-2-22 Ighb/b B cells alone continuously expressed high serum concentrations of IgG2ab (800 ± 180 µg/ml on day 23 post-transfer) (Fig. 2
). Importantly, all reconstituted RAG-20/0 mice had circulating IgG1b and IgG2bb allotypes, showing the strict specificity of the T cell-induced suppression of the IgG2ab allotype (data not shown). Consequently, like wild-type B cells, BCL-2-overexpressing Ighb/b B cells can receive cytotoxic signals released by Pfp/Fas-dependent or exclusively Fas-dependent Igha/a T cells. In particular, the full IgG2ab-suppression induction by Pfp0/0 Igha/a T cells in contact with BCL-2-22 Ighb/b B cells demonstrated that the Fas-mediated cytotoxicity against mature IgG2ab-producing B cells, in this in vivo negative regulation model, is totally distinct from the death mechanism impeded by BCL-2.
Inability of BCL-2-22 Ighb/b B cells to escape suppression induction was not due to their failure to express functional transgenic BCL-2 or to up-regulation of the principal BCL-2 inhibitors
We wondered whether the suppression induction against BCL-2-22 Ighb/b B cells was due to a possible failure of transgenic BCL-2 expression or function in whole transplanted B cells. Consequently, RAG-20/0 recipients of wild-type Ighb/b B cells + Pfp0/0 Igha/a T cells, BCL-2-22 Ighb/b B cells + Pfp+/+ or Pfp0/0 Igha/a T cells or BCL-2-22 Ighb/b B cells alone were evaluated, on day 52 post-transfer, for intracellular human BCL-2 expression. FACS analysis showed comparable transgenic BCL-2+ B cell percentages with similar fluorescence intensities among PBL from IgG2ab-suppressed or IgG2ab-unsuppressed RAG-20/0 mice, recipients of BCL-2-22 Ighb/b B cells alone or a B + T mixture (Fig. 3
).

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Fig. 3. Intracellular expression of transgenic human BCL-2 in B lymphocytes from individual RAG-20/0 mice reconstituted with wild-type or BCL-2-22 Ighb/b B + Pfp+/+ or Pfp0/0 Igha/a T cell mixtures or BCL-2-22 Ighb/b B cells alone. FACS analysis of PBL was carried out on day 52 post-transfer. Percentages are those of double-labeled cells.
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BCL-2 has been described to confer resistance to glucocorticoid-induced cell death (23). To examine if transgenic BCL-2 protein was not only present but also functional in BCL-2-22 B cells transferred into the RAG-20/0 environment, we compared in vitro the viability of the transferred BCL-2-22 B cells in the presence of Dexamethasone to that of their wild-type counterparts. In the absence of any particular treatment, 36% of endogenous B splenocytes from wild-type or BCL-2-22 mice survive after 24 h of culture. Such population, from BCL-2-22 origin, displayed significant resistance to Dexamethasone-induced death (Fig. 4A
). In contrast to B splenocytes from RAG-20/0 mice reconstituted with wild-type Ighb/b B cells, B splenocytes from RAG-20/0 recipients of BCL-2-22 Ighb/b B cells showed marked resistance to Dexamethasone-induced death (Fig. 4B
) indicating that the transgenic BCL-2 protein in BCL-2-22 cells reconstituting RAG-20/0 mice was functional.

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Fig. 4. Transgenic BCL-2 protein is functional in BCL-2-22 B cells transferred into RAG-20/0 recipients. B220+ cell viability of wild-type or BCL-2-22 splenocytes (A) or of splenocytes of RAG-20/0 mice reconstituted with wild-type or BCL-2-22 B cells (B) in the presence of increasing concentrations of Dexamethasone. Results are mean viabilities ± SD obtained in three independent experiences.
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It is widely accepted that cell death/survival can be controlled by competition between pro- and anti-apoptotic BCL-2 family proteins (24,25). Therefore, it was important to evaluate the expression levels of the major BCL-2 inhibitors, i.e. BCL-XS (short isoform of BCL-X protein) and BAX (BCL-2-associated X protein) when wild-type and BCL-2-22 B cells were transplanted into RAG-20/0 recipients. B220+ IgDb+ sorted B splenocytes from different reconstituted RAG-20/0 groups were analyzed, by semi-quantitative RT-PCR, for their expression of bcl-xS and bax mRNA. The bcl-xS transcripts were undetectable and therefore were not up-regulated in sorted B cell cDNA from all these experimental groups (data not shown), a finding which is in concordance with previous observations (26). Comparable levels of bax mRNA were detected in IgG2ab-suppressed RAG-20/0 recipients of wild-type Ighb/b B cells + Pfp0/0 Igha/a T cells, or BCL-2-22 Ighb/b B cells + Pfp+/+ or Pfp0/0 Igha/a T cells, and in IgG2ab-expressing RAG-20/0 recipients of BCL-2-22 Ighb/b B cells alone (Fig. 5
).

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Fig. 5. Comparable levels of bax mRNA expression in sorted B splenocytes from RAG-20/0 recipients of wild-type or BCL-2-22 Ighb/b B + Pfp+/+ or Pfp0/0 Igha/a T cells, or BCL-2-22 Ighb/b B cells alone.
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Thus, the absence of suppression inhibition with BCL-2-22 Ighb/b B cells was not due to the failure of intracellular expression of functional transgenic BCL-2, or the up-regulation or overexpression of major BCL-2 antagonists in whole Ighb/b B cells transplanted into RAG-20/0 recipients.
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Discussion
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Contradictory data have been obtained concerning the potential of the BCL-2 oncoprotein to prevent Fas-mediated cell death. The T cell-induced Ig allotype suppression model provides an opportunity to address this issue in the context of an in vivo negative regulation of the B cell compartment. Using Fas- and Pfp-dependent cytotoxicity pathways, in an alternative or concomitant manner, T splenocytes from Igha/amice are constitutively able to induce full and chronic suppression of IgG2ab production by congenic Ighb/b B cells. The programmed death of IgG2ab-producing B cells, provoked by Pfp0/0 Igha/a T cells, thus operates exclusively via the Fas-mediated pathway (19). In the present investigation, considering the latter configuration and the availability of the BCL-2-22 Ighb/b mouse strain (20), we examined whether the involved Fas-dependent B cell death shares a common death pathway with the BCL-2-regulated one.
Appropriate mixtures of wild-type, BCL-2-22 or Faslpr/lpr Ighb/b B cells + Pfp+/+ or Pfp0/0 Igha/a T cells were transferred into histocompatible RAG-20/0 recipients. Analysis of serum Ig allotype expression in these mice, subsequent to repopulation of their B, CD4+ T and CD8+ T compartments, revealed that Pfp+/+ (Pfp/Fas-dependent) cytotoxic Igha/a T cells induced equally well the IgG2ab suppression on wild-type or BCL-2-22 Ighb/b B cells. Thus, despite their characteristic prolonged cell survival (20), BCL-2-overexpressing Ighb/b B cells were not able to escape the cytotoxicity exerted by Pfp+/+ Igha/a T cells. Most importantly, Pfp0/0 (exclusively Fas-dependent) cytotoxic Igha/a T cells also induced this Ig allotype suppression on wild-type or BCL-2-22, but not on Faslpr/lpr, Ighb/b B cells. The behavior of BCL-2-overexpressing Ighb/b B cells was therefore opposite to that of their Fas-deficient counterparts when subjected to Fas-mediated cytotoxicity. This full suppression induction on BCL-2-22 B cells by Pfp0/0 Igha/a T cells demonstrated that constitutive overexpression of BCL-2 did not rescue IgG2ab-producing B cells from Fas-mediated death. In this context, the suppression observed, i.e. no BCL-2 effect, was not due to a failure to express functional transgenic human BCL-2 or to up-regulation or overexpression of the two principal antagonists of BCL-2 (BCL-XS and BAX oncoproteins) in whole Ighb/b B cells transplanted into RAG-20/0 recipients. Importantly, no species barrier seems to be evident since transgenic human BCL-2 protein has been shown to be functional in numerous mouse experimental models (1,5,8,9,27).
The overexpression of BCL-2 is known to disturb B cell homeostasis by generating excess mature B cells (25). We examined whether the suppression induction on BCL-2-22 B cells, compared to their wild-type counterparts, would necessitate greater expansion of suppression inducer/effector T cells, possibly oligoclonally expressing certain TCR Vß chain(s). By use of anti-TCR Vß mAb (specific to Vß2, Vß3, Vß4, Vß5.1-2, Vß6, Vß7, Vß8.1-2-3, Vß9, Vß10 and Vß14) in FACS analyses, investigation of the TCR Vß repertoires of T splenocytes from IgG2ab-suppressed RAG-20/0 recipients of wild-type or BCL-2-22 Ighb/b B + Pfp+/+ or Pfp0/0 Igha/a T cell mixtures showed similar patterns, suggesting that Igha/a T cell suppression induction on BCL-2-22 B cells would not require oligoclonal expansion of anti-IgG2ab T population(s).
It is noteworthy that, as the lpr mutation alone already allowed Ighb/b B cells to escape completely from the cytotoxicity exerted by Pfp0/0 Igha/a T cells, it would not have been informative to explore the fate of BCL-2-22 Faslpr/lpr double-mutant Ighb/b B cells exposed to Pfp0/0 Igha/a T cells. Moreover, we did not study the effect of Pfp+/+ Igha/a T cells on BCL-2-22 Faslpr/lpr Ighb/b B cells to assess the possible protective role of BCL-2 against exclusively Pfp-mediated B cell death. Actually, only weak BCL-2-mediated protection has been reported against Pfp-mediated cytotoxicity (27). More recently, it was shown that BCL-2 only inhibited cell death induced by a combination of purified Pfp and granzyme B but failed to prevent cell death provoked by intact cytotoxic T lymphocytes (CTL) which involves the mobilization of complex cytotoxic granules, and their release of Pfp, granzymes and numerous other proteolytic enzymes (28).
sIg and/or CD40 triggering induce surface Fas expression on mature B cells (2931). Nevertheless, only CD40 triggering, in the absence of sIg stimulation, rendered B cells susceptible to Fas-mediated cell death (32,33). sIg triggering can promote up-regulation (3437) or inhibit down-regulation (7) of BCL-2/BCL-XL proteins and perhaps, in this way, can protect CD40-activated B cells from Fas-induced cytolysis. It has been suggested that the absence of BCL-2/BCL-XL-mediated protection of CD40-activated B cells against Fas-mediated death would constitute a potential negative regulatory mechanism controlling B cell responses (6,32). The T cell-induced Ig-allotype suppression may represent a relevant example of such a mechanism. Indeed, this model does not involve B cell sIg triggering, i.e. activation by antigen, but necessitates Igha/a T cell recognition of IgG2ab-producing B cells via C
2ab-derived peptides, presented by MHC molecules (38). It is conceivable that cellcell contact between IgG2ab-producing B cells and CD4+ T suppression inducers would up-regulate B cell Fas expression, e.g. via CD40CD40 ligand interaction. According to the above scenario, in the absence of sIg signaling, the BCL-2-regulated pathway would remain disconnected from the Fas-induced death pathway and even BCL-2 overexpression would not be able to protect such CD40-triggered Ighb/b B cells from Fas-mediated cytotoxicity exerted by CD8+ T cell suppression effectors.
It appears that contradictory data on BCL-2 inhibition of Fas-mediated death have been more frequently observed in in vitro models making use of different BCL-2-transfected target cells, on the one hand, and of different anti-Fas mAb or FasL+ T cell clones or primary CTL, on the other (5,6,8,23,3942). Therefore, it seems that various combinations of targetantibody or targetCTL, as well as differences in expression/distribution profiles of BCL-2and its antagonistscondition in vitro BCL-2-mediated protection against Fas-induced cell death. In contrast, in vivo model systems, mainly using BCL-2 transgenic mouse strains, led to more harmonious observations, at least, with hemopoietic cells. Indeed, in most in vivo situations, BCL-2- and Fas-regulated death pathways have been shown to be totally or partially distinct. For instance, Fas-deficiency and BCL-2-overexpression acted in an additive or synergistic manner respectively to generate lymphadenopathy or to prolong survival of superantigen-activated T cells (8). In addition, Fas deficiency and BCL-2 overexpression in a TCR-transgenic model system did not result in overlapping characteristics in terms of T cell homeostasis and clonal deletion by tolerogen administration (9). Moreover, Fas deficiency and BCL-2 overexpression have been reported to act synergistically in the malignant transformation occurring in acute myeloid leukemia (43). Our investigation of the in vivo roles of these two pathways in a T cell-mediated negative regulation of an Ig production supports these latter findings by demonstrating that the Fas-mediated total elimination of IgG2ab-producing B cells by Igha/a CD8+ T suppression effectors was fully functional when Ighb/b B cells constitutively overexpressing active BCL-2 protein were involved.
Recently, regarding the Fas-signaling death pathway, two types of lymphoid cell lines have been described (44). (i) Type I cells, in which Fas signaling bypasses the mitochondria-dependent death pathway, and induces rapid and direct recruitment of high levels of caspase 8 which activates the other downstream caspases. (ii) In type II cells, Fas signaling essentially leads to a mitochondria-dependent activation of caspase 8. The fact that BCL-2 and BCL-XL are only able to block the mitochondrial pathway explains why in type I cell lines, Fas-induced death is not inhibitable by overexpression of BCL-2. Based on this cell type classification, thymic and peripheral T cells belong to the type I phenotype while hepatocytes present type II characteristics (44). Our present data allow us to propose that mature IgG2ab-producing B cells, subjected to Fas-induced T cell-mediated cytotoxicity despite BCL-2 overexpression, constitute cells from the type I.
As we mentioned elsewhere (45), reasons for the existence of anti-IgG2ab T cell activity in Igha/a mice of different genetic backgrounds remain unknown. This anti-Ig allotype activity could represent a vestige of an anti-Ig isotype regulatory mechanism. The fact that the actual C
2aa and C
2ab alleles constituted, in the past, two tandemly organized isotype genes reinforces this hypothesis (46). Alternatively, this T cell activity could result from an accidental cross-reactivity with certain anti-infectious or regulatory T cell function. Based on sequence comparison of C
2ab peptides recognized by anti-IgG2ab TCR to sequence data banks, experiments are in progress to attempt to test the latter hypothesis.
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Acknowledgments
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This work was supported by grants from the Institut Pasteur (3540), the Centre National de la Recherche Scientifique (URA 1961), the Association Franciaise contre les Myopathies and the Association pour la Recherche sur le Cancer. We are indebted to Christèle Sellier for excellent technical assistance, Pascal Dardenne for animal care and performing all the bleedings, and Janet Jacobson for correcting the English version of this paper.
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Abbreviations
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CTL cytotoxic T lymphocyte |
lpr lymphoproliferation |
PBL peripheral blood lymphocyte |
PE phycoerythrin |
Pfp pore-forming protein (perforin) |
PI propidium iodide |
RAG recombination activating gene |
WT wild type |
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Notes
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Transmitting editor: L. Du Pasquier
Received 4 January 2000,
accepted 3 March 2000.
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