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Evidence of Alternative or Concomitant Use of Perforin- and Fas-Dependent Pathways in a T Cell-Mediated Negative Regulation of Ig Production¹

Unité d’Immunophysiologie Moléculaire, Institut Pasteur, Paris, France

	Abstract

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To study the possible involvement of perforin (Pfp)- and/or Fas-dependent cytotoxicity pathways in a T cell-mediated negative regulation of Ig production, we used the T cell-induced Ig-allotype suppression model. T splenocytes from Igh^a/a mice, when neonatally transferred into histocompatible Igh^a/b F₁ or Igh^b/b congenic hosts, are intrinsically able to totally, specifically, and chronically suppress the production of IgG_2a of the Igh^b haplotype (IgG_2a^b). It has not been established whether the suppression effectors, which are anti-IgG_2a^b MHC class I-restricted CD8⁺ T cells, cytolyse IgG_2a^b+ B targets or whether they only silence Ig production. In this study, using T cells from Igh^a/a Pfp^+/+ or Pfp^o/o mice, the latter obtained by crossbreeding, and B cells from Igh^b/b Fas^+/+ or Fas^lpr/lpr (lymphoproliferation) mice in appropriate adoptive transfer models, we demonstrated that: 1) under blockage of the Pfp-mediated pathway, Igh^a/a T cells were still able to induce suppression against wild-type IgG_2a^b+ B cells, 2) IgG_2a^b+ B cells with impaired Fas expression were also subjected to suppression by WT Igh^a/a T splenocytes, and 3) the suppression establishment was totally inhibited when both Pfp- and Fas-dependent mechanisms were simultaneously blocked, i.e., when Igh^a/a Pfp^o/o T cells were used to induce suppression against Igh^b/b Fas^lpr/lpr B cells. These results provide the first demonstration of the existence of alternative or simultaneous use of the major cytotoxic mechanisms in a T cell-mediated down-regulation of an Ig production.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Pore-forming protein (Pfp)³- or Fas-dependent pathways have been described as principal mechanisms for T cell-mediated cytotoxicity. Pfp, exhibiting close structural and functional similarities with the ninth component of complement (1), appears to be essential for killing tumor cells or cells infected with intracellular organisms (2), while Fas, belonging to the TNF-receptor family (3), seems to be involved predominantly in immunoregulatory processes (4). In the present study, we investigated the possible involvement of these molecules in a T cell-mediated negative control of Ig production, i.e., the well-characterized model of IgG_2a^b suppression, induced by constitutively emerging allotype-specific T cells (reviewed in 5).

Normal T splenocytes (Tnor) from mice of the Igh^a haplotype, for instance BC8 H-2^b mice, when postnatally transferred into histocompatible Igh^a/b heterozygotes, are characteristically able to induce the full, specific, and chronic, but experimentally reversible, suppression of IgG_2a^b production (6). This intrinsic anti-IgG_2a^b T cell activity of naive Igh^a/a H-2^b mice can be strongly amplified upon their in vivo sensitization against intact IgG_2a^b or C_2a^b-103–118 hinge region-derived peptide (7, 8). T splenocytes from such sensitized Igh^a/a mice (Tsens) acquire the capacity to induce IgG_2a^b suppression in 100% of histocompatible Igh^a/b heterozygotes or congenic Igh^b/b homozygotes into which they are transferred neonatally. Importantly, the IgG_2a^b suppression is induced and maintained by T cells of donor origin, and the recipient T compartment does not take on an autoimmune relay for suppression maintenance (9). The suppression effectors are CD8⁺ T cells (10), which act in a MHC class I-restricted manner (11), on mature B cells downstream from allelic exclusion and the switch to IgG_2a production. The action of the CD8⁺ T cells can be experimentally reversed, showing that precursors of IgG_2a^b-producing B cells are not definitively deleted in IgG_2a^b-suppressed mice (10). It is noteworthy that, in ex vivo ⁵¹Cr release assays, T splenocytes from normal or sensitized Igh^a/a mice or from IgG_2a^b-suppressed Igh^a/b or Igh^b/b mice do not display cytotoxic activity against Igh^a/b or Igh^b/b B lymphocytes or IgG_2a^b+ myeloma cells. However, this fact may reflect the low frequency of anti-IgG_2a^b CD8⁺ T cells. Therefore, it has not yet been elucidated whether these class I-restricted CD8⁺ T cells are cytotoxic effectors of IgG_2a^b+ B cells or whether they specifically negatively silence this Ig-allotype production.

Mice with a targeted mutation in the third exon of the Pfp gene (2) have normally developing lymphoid populations, particularly CD8⁺ T cells that exhibit normal activation and expansion profiles upon in vivo and further in vitro stimulations. Nevertheless, these mice present drastically lowered antiviral CD8⁺ CTL responses, 90–100% reduction of allogeneic T cell cytotoxicity (2), and significant decreases of certain spontaneous or induced organ-specific autoimmune reactions (12). The cytotoxicity of CD8⁺ type 2 T cells toward resting B cells is also abrogated in Pfp^o/o mice (13).

B cells can up-regulate their Fas expression (reviewed in 14) upon activation (15, 16, 17) and constitute potential targets for Fas-dependent apoptosis (18 , 19 , and reviewed in 20). In vitro, Fas-Fas ligand (FasL) interaction has been shown to be implicated in T cell cytotoxicity against mitogen-activated B cells (18) and adequate APC primed with self-derived peptides (21, 22), and to be involved in the negative regulation of proliferative B cell responses (23). In vivo, Fas plays a role in the homeostasis of T and B lymphocyte expansions by regulating activation-induced cell death (14). The lymphoproliferation (lpr) mutation consists of the insertion of an early transposon into the second intron of the Fas gene, thereby resulting in aberrant Fas-mRNA transcription. In spite of the leaky character of this mutation and the weak expression of WT Fas mRNA in Fas^lpr/lpr mice (24, 25, 26, 27, 28), the drastic decrease of Fas expression (29) has been shown to prevent programmed cell death mechanisms and to be responsible for certain lymphoproliferative disorders (30).

In the present study, we transferred by crossbreeding, the Pfp^o mutation (2) from Igh^b/b Pfp^o/o C57BL/6 mice into Igh^a/a BC8 congenic mice. The capacity of Igh^a/a Tsens to induce the suppression against WT IgG_2a^b+ B cells was then investigated under total inhibition of Pfp production by T effectors. In addition, the availability of Igh^b/b C57BL/6 mice in which the lpr mutation had been transferred by backcrossing (31) allowed us to attempt, by means of WT Igh^a/aTsens, suppression induction against IgG_2a^b+ B cell targets with impaired Fas expression. Finally, we examined the suppression-induction ability when both Pfp- and Fas-dependent cytotoxicity pathways were simultaneously blocked, i.e., by suppression-induction assays with Igh^a/a Pfp^o/o Tsens against Igh^b/b Fas^lpr/lpr B targets. These attempts were made either with appropriate Igh^a/b newborns, recipients of Igh^a/a Tsens, or in mice deficient for recombination-activating gene 2 (RAG2^o/o) (32) coreconstituted with different mixtures of Igh^a/a T cells + Igh^b/b B cells.

	Materials and Methods

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Mice

Igh^b/b C57BL/6 mice were obtained from the Centre d’Elevage Janvier (Le Genest-Saint-Isle, France). BC8 mice have a C57BL/6 genetic background, with the Igh^a region from the BALB/c strain, and are therefore Igh congenic to the C57BL/6 strain. We bred BC8 mice in the Pasteur Institute’s animal facilities (Paris, France). RAG2^o/o mice, obtained by homologous recombination (32), have a severe combined immunodeficiency because of their total inability to initiate V(D)J rearrangements for TCR and Ig expression. The RAG2^o/o mice used in this study were from the ninth backcross generation to C57BL/6 and were purchased from the Centre de Sélection d’Animaux de Laboratoire, Centre National de la Recherche Scientifique (CNRS, Orléans, France). RAG2^o/o mice were maintained under specific pathogen-free conditions at the Pasteur Institute. Igh^b/b Fas^lpr/lpr (31) or Pfp^o/o (2) C57BL/6 mice, both from the eighth backcross generation to C57BL/6, were bought from The Jackson Laboratory (Bar Harbor, ME). The RAG2^o/o or Fas^lpr/lpr status was always verified by cytofluorometric analysis, respectively, on PBL or thymocytes.

Generation of Igh^a/a Pfp^o/o mice

Igh^b/b Pfp^o/o C57BL/6 mice were crossed against congenic Igh^a/a Pfp^+/+ BC8 mice to generate Igh^a/b Pfp^+/o double heterozygotes. The latter were then inbred, and Igh^a/a Pfp^o/o double homozygotes were selected from the offspring. The Igh genotype was first determined at the C locus by FACS analysis of PBL using a combination of anti-B220 and anti-IgD^b mAbs. C^a/a individuals were then tested for the allotypy of their C_2a locus by C_2a^a- or C_2a^b-specific PCR on genomic PBL DNA. C^a/a individuals were always of the C_2a^a/a genotype, concording with the fact that the Igh genes are inherited as a very tightly linked loci (33). The selected Igh^a/a mice were then screened for the Pfp locus by PCR specific to WT Pfp⁺ or mutated Pfp^o alleles. For more details, see below.

Sensitization of Igh^a/a mice and suppression induction in Igh^a/b or Igh^b/b newborns

Adult Igh^a/a WT or Pfp^o/o mice received, at a 15-day interval, an i.v. injection of 5 x 10⁷ viable B splenocytes from sex-matched Igh^b/b C57BL/6 mice. Seven days after the last injection, a T cell-enriched fraction of splenocytes (containing 80% CD3⁺ and 10% B220⁺ cells) was prepared by passage through a nylon wool column (34). Living Tsens, respectively, 5 x 10⁷ or 1 x 10⁷/50 µl, were postnatally injected (i.p.) into Igh^b/b congenic mice or histocompatible Igh^a/b F₁ born to Igh^a/a BC8 females and Igh^b/b C57BL/6 males. Serum IgG_2a^b expression was then monitored regularly as of 6 wk of age to assess suppression induction.

Adoptive cotransfer of different B and T cell populations into histocompatible RAG2^o/o hosts

T cell-enriched splenocytes were prepared as described above. Igh^b/b B cell-enriched splenocytes were obtained by ex vivo negative selection using a mixture of rat anti-mouse Thy-1.2 (30-H-12), CD4 (GK1.5), and CD8 (H35.17.2) mAbs + guinea pig serum as the source of C. RBC were removed from cell suspensions using hemolytic Gey’s solution according to the standard protocol. B cell suspensions contained 1.5% CD3⁺ and at least 73–83% Ig⁺ cells. Viable T cells (5 x 10⁷), B cells (5 x 10⁷), or sex-matched mixtures of them (5 x 10⁷ B cells + 5 x 10⁷ T cells), in a volume of 500 µl, were injected into the tail vein of adult, histocompatible, and sex-matched RAG2^o/o mice. The reconstitution of their lymphocyte compartments was evaluated regularly, from day 7 or day 16 to day 92 posttransfer, by FACS analysis of their PBL isolated from blood collected on heparin (Laboratoire Choay, Gentilly, France), and centrifuged on Lympholyte M (Cedarlane Laboratories, Hornby, Ontario, Canada). Spleen reconstitution was studied at the end of the experiment, i.e., on day 92 posttransfer.

mAbs used in FACS analysis

Phycoerythrin-conjugated anti-CD4 (CT-CD4), anti-CD8 (CT-CD8), anti-B220 (RA3-6B2) mAbs, and FITC-conjugated anti-Thy-1.2 (5a-8) mAb were from Caltag (South San Francisco, CA). FITC-conjugated anti-CD3 (145-2C11) mAb was a gift of Dr. Truffa-Bachi (Pasteur Institute). We prepared FITC-conjugated anti-TCRß (H57.597) (35) and biotinylated anti-IgD^b (H63.1) (36) mAbs. FITC-labeled anti-mouse Fas (Jo2) was obtained from PharMingen (San Diego, CA). After setting a combination of gates on forward/side scatter and propidium iodide^- cells, viable labeled cells (1 x 10⁶) were analyzed in a FACScan system (Becton Dickinson, Mountain View, CA) using Cell Quest software.

Serum Ig-allotype monitoring

IgG₁^b- and IgG_2b^b-allotype serum expressions were quantified by ELISA. Plates were coated with 50 µl of 5 µg/ml of, respectively, goat anti-mouse IgG₁ or IgG_2b anti-isotype Abs (Southern Biotechnology, Birmingham, AL). The presence of IgG₁^b or IgG_2b^b in serial dilutions of serum was then, respectively, detected by biotin-labeled IgG_2b anti-IgG₁^b (412-79) or IgG₁ anti-IgG_2b^b (412-72) anti-allotype mAb (PharMingen). Standard curves were constructed with BALMOPC245 (IgG₁^b) or BPC4 (IgG_2b^b). The serum IgG_2a^a expression was quantified by ELISA. Plates were coated with 50 µl of 5 µg/ml of anti-IgG_2a^a (20.8.3) (37) anti-allotype mAb. The presence of IgG_2a^a in sera was detected by biotin-labeled anti-IgG_2a^a (20.6.B8) (37) anti-allotype mAb. A standard curve was constructed with HOPC.1 (IgG_2a^a) myeloma Ig. All myeloma Ig were purified from ascitic fluid by 18% Na₂SO₄ precipitation, followed by gel filtration through a Sepharose-6B column. BALMOPC245, BPC4, HOPC.1, and CBPC101 myeloma cells were kindly provided by Dr. M. Potter (National Cancer Institutes Contract N-01-CB-71 085). Alkaline phosphatase-labeled streptavidin (Southern Biotechnology) was used to detect binding. The serum IgG_2a^b expression was first visualized by immunoprecipitation in 1% agar-gel medium and was then quantified by ELISA (with a detection limit of 0.3 µg/ml), as fully described elsewhere (11). Only a mouse with undetectable serum IgG_2a^b was considered to be IgG_2a^b suppressed.

PCR and RT-PCR

Genomic DNA was prepared from PBL using a standard protocol. Total RNA was extracted from 1 x 10⁷ viable splenocytes using RNA-Plus (Bioprobe System, Montreuil-sous-Bois, France) and was reverse transcribed into cDNA using SuperScript (Life Technologies, Erancy, France).

The C_2a^a primers (5'-AGA ACC ATC TCA AAA CCC AA-3' and 5'-GGA GTA GCT ATT TCT TTC CAC-3') yielded a 385-bp fragment from Igh^a, but not from Igh^b genomic DNA. The C_2a^b primers (5'-AAA ACC ATC TCA AAA CCC AG-3' and 5'-GA GCA GGC GAA AAG ACT TCC-3') yielded, from the C_2a^b, but not the C_2a^a sequence, respectively, a 391- or 279-bp fragment from genomic DNA or cDNA. The specificities of these C_2a PCR were verified by cloning and product sequencing using, respectively, pMOSBlue T-vector (Amersham, Buckinghamshire, U.K.) and TaqTrack Sequencing Systems kits (Promega, Madison, WI). The Pfp primers were 5'-CCC CTG CAC ACA TTA CTG GAA G-3', 5'-CGC GTC CTG AAC TCC TGG CCA-3', and 5'-CTC GTG CTT TAC GGT ATC GC-3'. The combination of the first and the second yielded a 320-bp fragment for the WT Pfp+ allele. The combination of the second and the third (the latter specific to the neo insert) gave a 156-bp fragment for the mutated Pfp^o allele. All primers were synthesized by Eurobio (Les Ulis, France). Goldstar DNA Taq polymerase was purchased from Advanced Biotechnologies (Eurogentec, Seraing, Belgium). The PCR were performed in a PTC-100 programmable thermal controller (Prolabo, Fontenay-sous-Bois, France).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
IgG_2a^b-suppression induction in WT Igh^a/b recipients by T splenocytes from Igh^a/a Pfp^o/o donors

We investigated the possible role of Pfp-dependent cytotoxicity in the T cell-induced suppression against IgG_2a^b+ B cells. We first obtained Igh^a/a Pfp^o/o double homozygous mice on a C57BL/6 genetic background. In a preliminary study of the T cell compartment constitution of these generated Igh^a/a Pfp^o/o mice, we compared their TCR-Vß repertoire with that of their Igh^a/a Pfp^+/+ counterparts by using a broad panel of anti-TCR-Vß mAbs (specific to Vß2, Vß3, Vß4, Vß5.1–2, Vß6, Vß7, Vß8.1–2-3, Vß9, Vß10, Vß13, and Vß14) in cytofluorometric analysis. We observed similar TCR-Vß repertoire profiles in the CD4⁺ and CD8⁺ T subset splenocytes of these two strains (data not shown). Subsequently, Igh^a/a Pfp^+/+ or Pfp^o/o mice were sensitized, and the resulting Tsens were postnatally transferred into Igh^a/b BC8 x C57BL/6 F₁ for the IgG_2a^b suppression-induction assays. Using specific PCR, we determined that, at 6 wk of age, the WT Igh^a/b recipients of Igh^a/a Pfp^o/oTsens had PBL with the neo insert at the level of the third exon of the Pfp gene that characterizes the targeted Pfp° mutation (data not shown). Thus, the donor Igh^a/a Pfp^o/o Tsens were successfully engrafted into their WT Igh^a/b recipients.

The serum IgG_2a^b expression of the Igh^a/b F₁, untreated controls and recipients of Igh^a/a Pfp^+/+ or Pfp^o/oTsens was regularly followed between 6 and 31 wk of age. All untreated controls (n = 20) continuously produced serum IgG_2a^b, while all Igh^a/b recipients of Igh^a/a Pfp^+/+Tsens (n = 14) or of Igh^a/a Pfp^o/o Tsens (n = 18) were chronically subjected to total IgG_2a^b suppression. As determined in 19-wk-old Igh^a/b individuals, the serum IgG_2a^b concentration was 480 ± 130 µg/ml in untreated controls and under the detectable limit in recipients of Igh^a/a Pfp^+/+ or Pfp^o/oTsens (Fig. 1). Thus, when the Pfp-dependent pathway was completely blocked, T splenocytes from Igh^a/a mice remained able to induce Ig-allotype suppression against WT IgG_2a^b+ B cells.

View larger version (12K): [in this window] [in a new window]   FIGURE 1. IgG2ab-suppression induction in WT Igha/b recipients of Igha/a Pfp+/+ or Pfpo/o Tsens. Serum IgG2ab concentrations in groups of 19-wk-old Igha/b BC8 x C57BL/6 F1: untreated controls, recipients of transferred Tsens from Igha/a Pfp+/+ or Pfpo/o donors. The horizontal bar is the mean concentration.

We then studied the possible involvement of Fas expression by IgG_2a^b+ B cells in the suppression induction by transfer of Tsens from WT Igh^a/a BC8 donors into Igh^b/b Fas^+/+ or Fas^lpr/lpr C57BL/6 newborns. Igh^a/a donor lymphocytes failed to implant and thereby to induce suppression in Fas^lpr/lpr Igh^b/b recipients (n = 28), in contrast to 100% successful cell implantation and suppression induction in Igh^b/b Fas^+/+ recipients (n = 27). For instance, among the splenocytes of Fas^lpr/lpr recipients in vitro stimulated with Con A, Fas⁺ CD4⁺ or Fas⁺ CD8⁺ T cells of donor origin were undetectable. At the same time, the presence of 5–10% IgM^a+ residual B cells in the transferred Tsens population enabled us to evaluate the implantation of donor Igh^a/a B cells in Igh^b/b Fas^+/+ recipients, but not in their Fas^lpr/lpr Igh^b/b counterparts (data not shown). These observations agree with the existence, in Fas^lpr/lpr mice, of abnormal Thy-1.2⁺ FasL⁺⁺ CD4^- CD8^- double-negative (DN) T lymphocytes, exhibiting high cytotoxic activity against potentially Fas⁺ non-lpr cells (see Discussion). Thus, in the context of Igh^a/a T cell transfer into Igh^b/b Fas^lpr/lpr recipients, it was not possible to study the potential role of Fas in this T cell-induced negative regulation of Ig production.

Development of an alternative in vivo model for adequate coengraftment of WT Igh^a/a T cells together with Fas^lpr/lpr Igh^b/b B cells in a shared histocompatible environment

To circumvent the problem of nonimplantation of WT Igh^a/a BC8 T cells in Igh^b/b Fas^lpr/lpr C57BL/6 recipients and to make lymphocyte populations of these origins engraft in a shared histocompatible environment, different mixtures of B and T lymphocytes were adoptively cotransferred into immunodeficient RAG2^o/o C57BL/6 mice. It has been demonstrated that the majority of Fas^lpr/lpr DN T cells was derived from the CD8⁺ T lineage (38, 39), but that a minor subset also seemed to be of CD4⁺ origin (40). Thus, to eliminate the risk of DN T cell emergence in B populations, we negatively selected B splenocytes from Fas^lpr/lpr (or control Fas^+/+) Igh^b/b C57BL/6 mice, by ex vivo depletion of Thy-1.2⁺, CD4⁺, and CD8⁺ T cells. WT Igh^a/a Tnor or Tsens were prepared according to our usual protocol. Each of these cell populations was injected alone or as a sex-matched B + T mixture into adult RAG2^o/o mice. Thus, groups of RAG2^o/o mice received nothing (n = 3) or a single preparation of Fas^+/+ (n = 2) or Fas^lpr/lpr (n = 2) Igh^b/b B cells, a single preparation of Tnor (n = 2) or Tsens (n = 2), or a mixture of Igh^b/b Fas^+/+ B cells + Tnor (n = 3), Igh^b/b Fas^lpr/lpr B cells + Tnor (n = 3), Igh^b/b Fas^+/+ B cells + Tsens (n = 3), or Igh^b/b Fas^lpr/lpr B cells + Tsens (n = 3).

The reconstitution of the B and T cell compartments of the RAG2^o/o hosts was regularly monitored by FACS analysis. Table I reports implantation data obtained with PBL from all different RAG2^o/o hosts on day 16. Despite the weak implantation of transferred B220⁺ IgD^b+ cells detected among PBL (3.3% of total lymphocytes), these B cells were easily identifiable because of their strong fluorescent intensity. Fig. 2 shows representative reconstitutions of the splenocyte populations of RAG2^o/o recipients of Igh^b/b Fas^+/+ or Fas^lpr/lpr B cells + Tsens compared with untreated controls on day 92. The B splenocyte compartment was markedly reconstituted by both Fas^+/+ or Fas^lpr/lpr Igh^b/b B cells (Fig. 2). Moreover, substantial CD4⁺ and CD8⁺ T subsets were repopulated in the presence of Fas^+/+ or Fas^lpr/lpr B cells (Table I and Fig. 2). Interestingly, CD4⁺ and CD8⁺ T cell implantations were higher when Tnor or Tsens had been injected alone than in a B + T mixture. This observation could reflect the space available for cell expansion. Sometimes, the mean CD8⁺ implantation was consistently higher than that of CD4⁺ cells, which gave rise to the inverse of the situation obtained with the T cell preparations initially injected. CD4⁺ reconstitution with Tsens was lower in the presence of Fas^lpr/lpr B cells than in the presence of Fas^+/+ B cells (Table I). However, as we will see below, these quantitative differences did not influence the T cell function investigated in this study, i.e., T cell-induced IgG_2a^b suppression. We could not detect the accumulation of any potentially aggressive Thy-1.2⁺ B220⁺ DN population among the PBL or splenocytes of RAG2^o/o recipients of Fas^lpr/lpr B cells (data not shown). Using our panel of anti-TCR-Vß mAbs (see above), we observed that the TCR-Vß repertoire of CD4⁺ and CD8⁺ splenocytes from diverse reconstituted RAG2^o/o mice was quite comparable with that of Igh^a/a donors (data not shown). Taken together, these findings showed that the appropriate conditions had been met for IgG_2a^b-suppression induction monitoring in the presence or absence of Fas expression at the level of target Igh^b/b B cells.

View larger version (33K): [in this window] [in a new window]   FIGURE 2. Successful reconstitution of RAG2o/o lymphocyte compartments by transferred Ighb/b Fas+/+ or Faslpr/lpr B cells + Igha/a Tsens: comparable percentages of engraftment of non-lpr Igha/a Tsens in the presence of Ighb/b Fas+/+ or Faslpr/lpr B cells. Representative data obtained by FACS analysis of splenocytes (on day 92) from individual RAG2o/o mice; untreated controls, recipients of Ighb/b Fas+/+ or Faslpr/lpr B cells + Igha/a Tsens. Percentages of double-labeled cells, as a function of total lymphocytes, are given in the upper right-hand corner of each plot.

The serum IgG_2a^b concentrations (mean ± SD) in RAG2^o/o mice reconstituted with a single preparation of Igh^b/b Fas^+/+ or Fas^lpr/lpr B cells were, respectively, 500 ± 30 µg/ml (n = 2) or 570 ± 60 µg/ml (n = 2), as determined on day 92 after transfer. However, the RAG2^o/o recipients of Igh^b/b Fas^+/+ B cells + Tsens (n = 3) or Igh^b/b Fas^lpr/lpr B cells + Tsens (n = 3) mixture both exhibited full IgG_2a^b suppression (Fig. 3), which was chronically observed from day 16 to day 92 (the last time tested). The unamplified anti-IgG_2a^b T cell activity of transferred Tnor was also sufficient, in this cotransfer model, for total suppression induction against Igh^b/b Fas^+/+ B cells (n = 3). Tnor mixed with Igh^b/b Fas^lpr/lpr B cells also induced total suppression (in 1/3 mice) or highly significant inhibition of IgG_2a^b production (in 2/3 mice) (Student’s t test, p < 0.05).

View larger version (15K): [in this window] [in a new window]   FIGURE 3. IgG2ab suppression against Ighb/b Faslpr/lpr B cells is induced by WT Igha/a Tsens. Serum IgG2ab concentrations in RAG2o/o mice (on day 92) reconstituted with a single preparation of Fas+/+ or Faslpr/lpr Ighb/b B cells, or with mixture of Ighb/b Fas+/+ or Faslpr/lpr B cells + WT Igha/a Tnor or Tsens.

Thus, adaptation of the T cell-induced IgG_2a^b-suppression model to B + T-reconstituted RAG2^o/o mice demonstrated that IgG_2a^b+ B cells with impaired Fas expression are still fully able to receive suppression signal(s) from WT Igh^a/a T cells.

Total inhibition of IgG_2a^b-suppression induction under simultaneous blockage of Pfp- and Fas-dependent cytotoxicity pathways

We wondered whether this negative regulation of Ig production was induced under conditions of concomitant blockage of Pfp- and Fas-dependent mechanisms. To address this question, we injected into RAG2^o/o mice a mixture of Igh^b/b Fas^+/+ B cells + Igh^a/a Pfp^o/oTsens (n = 3) or Igh^b/b Fas^lpr/lpr B cells + Igh^a/a Pfp^o/o Tsens (n = 3). The reconstitution of the B and T cell compartments of RAG2^o/o recipients was again regularly followed by FACS analysis. Representative data are shown for the PBL on day 13 postinjection (Fig. 4). The engraftment of Igh^b/b Fas^+/+ or Fas^lpr/lpr B cells and Igh^a/a Pfp^o/o T cells (Fig. 4) was comparable with that obtained in the previous experiment with RAG2^o/o mice reconstituted with Igh^b/b Fas^+/+ or Fas^lpr/lpr B cells and Igh^a/a WT Tsens (Table I).

View larger version (28K): [in this window] [in a new window]   FIGURE 4. Successful reconstitution of RAG2o/o lymphocyte compartments by transferred Ighb/b Fas+/+ or Faslpr/lpr B cells + Igha/a Pfpo/o Tsens: similar implantation patterns of Igha/a Pfpo/o Tsens in the presence of Ighb/b Fas+/+ or Faslpr/lpr B cells. Representative FACS data obtained with PBL (day 13) from individual RAG2o/o mice: untreated controls, recipients of Ighb/b Fas+/+, or Faslpr/lpr B cells + Igha/a Pfpo/o Tsens.

View larger version (16K): [in this window] [in a new window]   FIGURE 5. Total inhibition of IgG2ab-suppression induction under simultaneous blockage of Pfp- and Fas-dependent cytotoxicity pathways. Serum IgG2ab concentrations in RAG2o/o mice (on day 92) reconstituted with Igha/a Pfpo/o Tsens + Ighb/b Fas+/+ or Faslpr/lpr B cells. The horizontal bar is the mean concentration.

Consequently, these experiments demonstrated that, under the conditions of simultaneous blockage of Pfp- and Fas-dependent cytotoxicity pathways, the induction of the negative control of IgG_2a^b production by Igh^a/a T cells is totally and specifically inhibited.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fas- or Pfp-mediated cell death pathways are major mechanisms for T cell-mediated cytotoxicity. Many observations indicate the involvement of the Fas system in homeostatic regulation of the B cell compartment. Importantly, the lpr B cell defect had been shown to be involved in vivo in autoantibody production (42). Moreover, Ag-specific T cell clones exerted in vitro Fas-dependent cytotoxicity against Ag-primed B cells (18). In the opposite, few data support the direct immune control of the nonmalignant B cell compartment via Pfp-dependent T cell-mediated cytotoxicity. To the best of our knowledge, only an example of CD8⁺ type 2 T cells has been described that involves a Pfp-dependent cytotoxicity toward resting B cells (13).

In the in vivo model of T cell-induced IgG_2a^b suppression, IgG_2a^b-specific CD4⁺ and CD8⁺ T cells from Igh^a/a donors cooperate during the induction of this negative regulation of Ig-allotype production (43, 44). Recently, the use of Igh^b/b C57BL/6 mice, genetically deficient for MHC class I or class II molecules, enabled us to demonstrate that only expression of the former at the surface of IgG_2a^b-producing B cell targets was necessary to establish suppression. Therefore, despite the essential role of CD4⁺ T cells during the induction phase, the suppression mechanism does not require an MHC class II-restricted T-B interaction (11). The suppression effectors are MHC class I-restricted CD8⁺ T cells (10, 11) acting on IgG_2a^b+ B cells. The suppression can be intentionally reversed with in vivo anti-CD8 mAb treatment (10). It was reasonable to speculate, but had not yet been experimentally demonstrated, that IgG_2a^b+ B cells were cytolysed by CD8⁺ T cell suppression effectors. Alternatively, these CD8⁺ T cells could negatively regulate the IgG_2a^b production without B cell destruction.

In the present investigation, we first showed that Tsens from Igh^a/a Pfp^o/o donors were as effective as Tsens from their Igh^a/a Pfp^+/+ counterparts in IgG_2a^b-suppression induction in WT Igh^a/b recipients. Therefore, under the sole inhibition of Pfp-mediated cytotoxicity, Igh^a/a anti-IgG_2a^b CD8⁺ T cells were still able to achieve total suppression. We then looked for the possible role of IgG_2a^b+ B cell Fas expression in receiving the negative regulatory signal(s) from the WT Tsens. We observed the failure of WT Igh^a/a Tsens to engraft in Igh^b/b Fas^lpr/lpr congenic recipients. This finding fits well with the phenomenon of accumulation, in Fas^lpr/lpr mice, of unusual DN T lymphocytes with abundant and constitutive FasL expression, which confers upon them a spontaneous, TCR- and MHC-independent cytotoxicity against potentially Fas⁺ non-lpr cells (45, 46, 47, 48). Consequently, great care should be taken in experiments implying adoptive transfer models using cell populations of Fas^lpr/lpr and Fas^+/+ origins.

Our strategy to study possible IgG_2a^b-suppression induction against Igh^b/b Fas^lpr/lpr B cells, while minimizing non-lpr T cell rejection, was to cotransfer into a fully immunodeficient but histocompatible RAG2^o/o environment, negatively selected B splenocytes (depleted of Thy-1.2⁺, CD4⁺, and CD8⁺ T cells) from Igh^b/b Fas^lpr/lpr (or Fas^+/+ control) mice with Igh^a/a T cells. Under these conditions, presumably due to the elimination of potential sources of abnormal DN T cells, no obstacle was encountered in the coengraftment of the transferred non-lpr T and Fas^lpr/lpr B cells. IgG_2a^b+ B lymphocytes from both Igh^b/b Fas^+/+ or Fas^lpr/lpr mice were subjected to the specific and full IgG_2a^b suppression in the presence of WT Igh^a/a Tsens. Thus, under the conditions of blockage of only the Fas-dependent cytotoxicity, it was also quite possible to induce full suppression against IgG_2a^b+ B cells. It is important to note that the IgG_2a^b T cell-induced suppression in the system of adoptive cotransfer of B and T cells into histocompatible RAG2^o/o mice presents the same characteristics, in terms of full and chronic suppression, allotype restriction, and low C_2a^b-mRNA levels, as the usual Ig-allotype-suppression model that we developed using neonatal transfer of Igh^a/a T splenocytes into histocompatible Igh^a/b F₁ or Igh^b/b congenic recipients.

Using the B + T cell-reconstituted RAG2^o/o mouse model, we investigated the potential of Igh^a/a Pfp^o/oTsens to induce IgG_2a^b suppression against Igh^b/b Fas^+/+ or Fas^lpr/lpr B cells. This experiment first confirmed that, in the absence of the Pfp pathway, Tsens were able to induce the suppression against WT B cells. Most importantly, we observed that, under simultaneous blockage of Pfp- and Fas-dependent pathways, respectively, at the levels of Igh^a/a T effectors and Igh^b/b B targets, the IgG_2a^b-suppression induction was totally inhibited. Taken together, our results show the existence in vivo of alternative or concomitant use of Pfp- and Fas-mediated cytotoxic mechanisms in this T cell-induced suppression. Two hypotheses can be advanced to explain our observations: 1) a population of Igh^a/a anti-IgG_2a^b T cells acts, on the same IgG_2a^b+ B cells, alternatively or simultaneously, via Pfp- or Fas-dependent cytotoxic mechanisms, or 2) two distinct populations of Igh^a/a suppression-effector T cells exert the suppression on the same IgG_2a^b+ B targets, one via the Pfp- and the other via the Fas-dependent pathway, and each being sufficient alone to induce total suppression. Moreover, it is likely that, at any stage during their development, the IgG_2a^b+ B cells are susceptible to both Pfp- and Fas-dependent T cell-induced negative regulation because upon blockage of only one or the other pathway, the IgG_2a^b suppression is fully achieved and no IgG_2a^b production is detected.

In Ig allotype-suppressed mice, although no IgG_2a^b is detectable in total cytosolic extracts of B lymphocytes, the presence of weak levels of C_2a^b mRNA strongly suggests the existence of small amounts of these Ig allotype-derived peptides in B cells committed to IgG_2a^b production (41). Seemingly, the presentation of such peptides in a MHC class I-restricted manner makes these B cells potent targets for suppression-effector CTL that operate via Pfp- and/or Fas-mediated mechanisms. For the time being, it is technically not possible to directly visualize probable DNA fragmentation in IgG_2a^b+ B targets. Indeed, using IgG_2a^b-specific ELISA spot assay, the frequency of IgG_2a^b-producing B cells in T cell-depleted splenocytes of normal Igh^b/b mice has been estimated to 80/10⁶, while corresponding cell preparations from their IgG_2a^b-suppressed counterparts were totally exempt of such B cells (41). Nevertheless, 1) this absence of IgG_2a^b-producing B cells, 2) the C_2a^b mRNA levels, barely detectable with a sensitive RT-PCR, and 3) involvement of both Pfp and Fas cell death factors in this model strongly suggest that apoptosis constitutes the mechanism of this T cell-mediated Ig-allotype suppression.

The Fas molecule is not detectable cytofluorometrically on unstimulated bone marrow or spleen B cells (15, 17). Indeed, in situ hybridization on histologic spleen sections revealed the presence of Fas mRNA only in B220⁺ B cells of the germinal centers, and especially in B cells Ag activated after in vivo immunization (16). Nevertheless, Fas expression can be up-regulated and associated with the intracellular death pathway of naive B cells, particularly upon CD40 triggering (17, 49, 50). Given these data and the CD4⁺-CD8⁺ T cell cooperation required for IgG_2a^b-suppression establishment, it can be imagined that, in the case of Fas-mediated Ig-allotype suppression, the IgG_2a^b+ B cell targets would first up-regulate their Fas expression during their contact with CD4⁺ T cell suppression inducers, for instance via CD40-CD40 ligand interaction. Sequentially, IgG_2a^b+ B cell targets, rendered susceptible to Fas-mediated cell death, would be cytolysed by CD8⁺FasL⁺ T cell-suppression effectors. Our present results show that the T cell-induced IgG_2a^b suppression does not imply silencing Ig production. In contrast, this phenomenon involves B cell destruction by CD8⁺ T cells using the Fas- and/or Pfp-dependent cytotoxicity.

As we have detailed elsewhere (5, 8), the reason for the selection and maintenance, during mouse evolution, of this anti-IgG_2a^b T cell activity still remains unknown. At least three hypotheses can be put forward to explain this reason. 1) Nucleotide-sequence comparison between C_2a^a and C_2a^b alleles strongly suggests that these genes could have distinct isotypic origins. Moreover, these two genes are still tandemly organized in wild mice (51, 52, 53). Therefore, one could speculate that the present anti-IgG_2a^b anti-allotype activity would represent a sequelae of a past negative regulation of an Ig-isotype production. 2) TCR specific to certain pathogenic or structural peptides, involved in a much more essential function in mouse species survival, would recognize by fortuitous cross-reactivity IgG_2a^b-derived peptides. 3) In normal Igh^a/b or Igh^b/b mice, we cannot exclude that anti-IgG_2a^b T cells could play a negative regulatory role in this Ig-allotype production without achieving total suppression. The high IgG_2a^a/IgG_2a^b concentration ratio in sera of Igh^a/b mice (8) strengthens this hypothesis. Moreover, we have demonstrated that anti-IgG_2a^b T cell activity spontaneously emerges in Igh^a/b mice, perinatally deprived of their B cell compartment. At adulthood, these individuals are subjected to an autoimmune, chronic, and T cell-mediated IgG_2a^b suppression (54). One can speculate that the alternative or concomitant use of both Pfp- and Fas-dependent cytotoxicity pathways, each being sufficient to ensure this phenomenon, would represent a kind of safety to prevent probable disorders provoked in the absence of this T cell function.

	Acknowledgments

 
We thank Christèle Sellier for excellent technical assistance, Pascal Dardenne for animal care and for performing all of the bleedings, Andrée Goyat for serum preparations, Gérard Dumas for his advice on the characterization of RT-PCR products, and Janet Jacobson for correcting the English version of this paper.

	Footnotes

 
¹ This work was supported by grants from the Institut Pasteur (3540), the Centre National de la Recherche Scientifique (URA 1961), and the Association Française Contre les Myopathies.

² Address correspondence and reprint requests to Dr. Guy Bordenave, Unité d’Immunophysiologie Moléculaire, Institut Pasteur, 25, rue du Docteur-Roux, 75724 Paris Cedex 15, France. E-mail address:

³ Abbreviations used in this paper: Pfp, pore-forming protein; DN, double-negative; FasL, Fas ligand; lpr, lymphoproliferation; RAG2, recombination-activating gene 2; Tnor, nylon wool nonadherent T splenocytes from normal Igh^a/a mice; Tsens, nylon wool nonadherent T splenocytes from Igh^a/a mice sensitized twice against B splenocytes from their Igh^b/b congenic counterparts; WT, wild type.

Received for publication December 9, 1998. Accepted for publication January 13, 1999.

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