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Role of CD40 in a T Cell-Mediated Negative Regulation of Ig Production¹

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

	Abstract

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To investigate the possible role of CD40 in a negative regulation of Ig production, we used the mouse Ig allotype suppression model. T splenocytes from Igh^a/a mice are able in vivo to totally and chronically inhibit the production of IgG_2a^b (IgG2a from the Igh^b haplotype). Accordingly, postnatal transfer of Igh^a/a T splenocytes into histocompatible Igh^a/b F₁ or congenic Igh^b/b mice leads to a characteristic IgG_2a^b suppression. The helper action of anti-IgG_2a^b CD4⁺ T cells is required for the recruitment of anti-IgG_2a^b CD8⁺ T suppression effectors. The latter use perforin (pore-forming protein, Pfp)- and/or Fas-dependent cytotoxic pathways to continuously eliminate B cells recently committed to IgG_2a^b production. In the present study we first showed that in vivo agonistic anti-CD40 mAb treatment of Igh^a/a mice, deprived of their CD4⁺ T cell compartment, could bypass the help of Ig allotype-specific CD4⁺ T cells and generate CD8⁺ T effector cells able to strongly inhibit IgG_2a^b production. This result demonstrates the usefulness of CD40 triggering in setting up an immune regulatory mechanism. Furthermore, with regard to the suppression-effector mechanism, we demonstrated that B cell CD40 expression was required for full suppression establishment via the Fas-dependent pathway. Indeed, Igh^a/a Pfp^°/° T cells (using exclusively the Fas pathway) induced full IgG_2a^b suppression against Igh^b/b CD40^+/+ B cells, but only partial inhibition of IgG_2a^b production against Igh^b/b CD40^°/° B cells. This finding provides the first demonstration of direct involvement of B cell CD40 expression in in vivo negative control of an Ig production.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD40, expressed by various cell types, including dendritic cells (DC),³ pre-B and B cells, is a 45- to 50-kDa glycoprotein of the TNF receptor family. Its ligand (CD40L, CD154) expressed on activated CD4⁺ T cells, is a 39-kDa glycoprotein of the TNF family (1). The CD40-CD40L pair plays important roles in CD4⁺ T cell-dependent recruitment of CTL and in initiating Ag-dependent differentiation of B cells. In cellular responses, Ag-specific CD4⁺ T cells first induce, via CD40-CD40L interaction, terminal maturation of DC. Such CD40-mediated stimulation confers onto these DC the ability to subsequently activate Ag-specific CD8⁺ T cells (2, 3, 4). This scheme explains how in the absence of CD4⁺ T cell compartment CD40 triggering can replace the helper function required for recruitment of CD8⁺ CTL. However, CD40-independent DC activation and direct CD4⁺-CD8⁺ T cell communication have also been reported as alternative mechanisms for the generation of CD8⁺ CTL (5). In humoral responses, engagement of CD40 on B cells by CD40L on CD4⁺ Th cells is essential for the formation of germinal centers, Ig class switching, and Ab production by B cells (1). Nevertheless, exclusively in vitro proof and only a few examples of CD40 involvement in negative regulation of B cell responses have been reported (6, 7, 8, 9, 10). In the present study using the well-characterized model of Ig allotype suppression (11), we first investigated the possible effect of in vivo CD40 stimulation on the recruitment of regulatory CD8⁺ T cells with the potential to negatively control an Ig production. We then sought to determine whether B cell CD40 expression was involved in the effector mechanism of this T cell-mediated negative regulation of Ig production.

T splenocytes from naive Igh^a/a mice of diverse genetic backgrounds possess an intrinsic activity exclusively directed against the expression of IgG2a from the Igh^b haplotype (12). This anti-IgG_2a^b T cell activity becomes especially obvious after sensitization of Igh^a/a mice against IgG_2a^b-producing B cells (13). Postnatal transfer of sensitized Igh^a/a splenocytes into histocompatible Igh^a/b F₁ or congenic Igh^b/b mice leads to a total, chronic, but experimentally reversible, inhibition of IgG_2a^b production, referred to as Ig allotype suppression. One can distinguish three successive phases in the IgG_2a^b suppression process: 1) amplification of the anti-IgG_2a^b T cell activity in Igh^a/a mice (sensitization), 2) induction of IgG_2a^b suppression in Igh^a/b or Igh^b/b recipients of Igh^a/a T cells, and 3) maintenance of the suppression in these hosts. Both CD4⁺ and CD8⁺ T cells are required during the amplification (14) and suppression induction (15) phases, whereas only CD8⁺ T cells are necessary to maintain suppression (16). The anti-IgG_2a^b CD4⁺ T cell help is thus indispensable to generate CD8⁺ T suppression effectors. Here we first attempted to determine whether in this experimental system an agonistic anti-CD40 mAb treatment of Igh^a/a mice during the sensitization phase could substitute for the CD4⁺ T cell help required for activation of CD8⁺ T suppression effectors.

The anti-IgG_2a^b CD8⁺ T effectors are MHC class I-restricted cells (17), operating concomitantly or alternatively via the Fas- or perforin (pore-forming protein, Pfp)-mediated cytotoxic pathway to chronically eliminate B cells recently committed to IgG_2a^b production (18). Under total blockage of the Pfp pathway, full suppression against IgG_2a^b-producing B cells is induced and maintained exclusively by the Fas pathway and vice versa (18). Because Fas is not expressed on the surface of mature B cells (19, 20, 21), up-regulation of this molecule would constitute a prerequisite for induction and maintenance of suppression via the Fas pathway. It has been shown that in vitro surface Ig and/or CD40 triggering up-regulate surface Fas expression on mature B cells, but CD40 triggering alone renders B cells susceptible to Fas-mediated death (22, 23). The Ig allotype suppression does not involve B cell surface Ig triggering, because Igh^a/a T cells recognize target B cells via C_2a^b-derived peptides presented by MHC molecules (24). Therefore, this model also provides the opportunity to investigate the possible involvement of CD40 expression on Igh^b/b B cells in the mechanism of suppression induction and maintenance when the Igh^a/a CD8⁺ T effectors operate exclusively via the Fas pathway.

	Materials and Methods

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Mice

BALB/c Igh^a/a and C57BL/6 Igh^b/b mice were, respectively, purchased from Iffa Credo (L’Arbresle, France) and the Center d’Elevage Janvier (Le Genest-Saint-Isle, France). Igh-congenic CB20 Igh^b/b mice have a BALB/c genetic background with the Igh^b region from the C57BL/6 strain. Conversely, Igh-congenic BC8 Igh^a/a mice possess a C57BL/6 genetic background with the Igh^a region from the BALB/c strain. BALB/c x CB20 hybrids are referred to as Igh^a/b F₁. We bred CB20 and BC8 mice in the Pasteur Institute’s animal facilities (Paris, France).

We established BC8 Igh^a/a Pfp^°/° mice by cross-breeding C57BL/6 Igh^b/b Pfp^°/° (25), purchased from The Jackson Laboratory (Bar Harbor, ME), with BC8 Igh^a/a Pfp^+/+ mice, followed by selection of Igh^a/a Pfp^°/° individuals among the offspring as detailed previously (18). CD40^°/° (26) and recombination-activating gene 2 (RAG-2^°/°) (27) mice from the sixth and the ninth backcross generations to C57BL/6 Igh^b/b strain, respectively, were obtained from the Center de Sélection d’Animaux de Laboratoire (Orléans, France). RAG-2^°/° mice were maintained in positive pressure isolators at the Pasteur Institute.

Preparation of anti-CD4 and anti-CD40 mAbs

Rat IgG2a anti-mouse CD4 (GK1.5) (28) and rat IgG2a anti-mouse CD40 (FGK45) mAbs (29) were semipurified by 18% Na₂SO₄ precipitation of ascitic fluid and extensive dialysis against saline, followed by gel filtration through a Sepharose 6B column. The anti-CD40 (FGK45) mAb is known to stimulate and not to delete CD40⁺ cells (29).

Sensitization and mAb treatments of BALB/c Igh^a/a mice and suppression induction assay in BALB/c x CB20 Igh^a/b newborns

To amplify their anti-IgG_2a^b T cell activity, adult BALB/c Igh^a/a mice received on day 0 an i.v. injection of 5 x 10⁷ living B cell-enriched splenocytes from sex-matched CB20 Igh^b/b mice (sensitization). B cell-enriched splenocytes were obtained by in vitro cytotoxic anti-Thy-1.2 (30-H-12) mAb treatment + C and contained 80% B220⁺ cells. For CD4⁺ T cell depletion, BALB/c mice received, from days -7 to 7, a daily i.v. injection of 100 µg of anti-CD4 mAb in 300 µl of saline. For in vivo anti-CD40 treatment, BALB/c mice received, from days 1 to 7, a daily i.v. injection of 100 µg of anti-CD40 mAb or an isotype-matched control Ig (provided by Dr. H. Bazin, University of Louvain, Brussels, Belgium) in 300 µl of saline. The mixture of 100 µg each of anti-CD4 and anti-CD40 (or control Ig) reagents was given in 300 µl of saline daily from days 1 to 7.

On day 8 postsensitization, T cell splenocytes from different BALB/c groups were enriched by passage through a nylon-wool column (Tsens). Living T splenocytes (1 x 10⁷; containing < 10% B220⁺ cells) in 50 µl of balanced salt solution were injected i.p. into Igh^a/b F₁ newborns. The serum IgG_2a^b levels of these recipients were then regularly monitored from 6 wk of age onward to assess the suppression induction capacity of the transferred T cells.

Cotransfer of relevant Igh^b/b B and Igh^a/a T cells into histocompatible RAG-2^°/° recipients for the suppression induction assay

Full IgG_2a^b suppression induction against homozygous Igh^b/b B cell populations generally requires two sensitizations of Igh^a/a mice against IgG_2a^b-producing B cells. Therefore, BC8 Igh^a/a Pfp^+/+ or Pfp^°/° mice received, on days 0 and 15, an i.v. injection of 5 x 10⁷ living B cell-enriched splenocytes. On day 21, 5 x 10⁷ T cell-enriched splenocytes (containing 75% Thy-1.2⁺ cells) from these sensitized BC8 Igh^a/a Pfp^+/+ or Pfp^°/° mice and 5 x 10⁷ B cell-enriched splenocytes (containing 90% B220⁺ cells) from C57BL/6 Igh^b/b CD40^+/+ or CD40^°/° mice in 500 µl of balanced salt solution were injected i.v. into sex-matched adult C57BL/6 RAG-2^°/° mice. Control RAG-2^°/° mice received 5 x 10⁷ B cell-enriched splenocytes from Igh^b/b CD40^+/+ or CD40^°/° mice without Igh^a/a T cells. The lymphocyte compartment reconstitution of the RAG-2^°/° hosts was evaluated by FACS analysis of their PBL.

ELISA and enzyme-linked immunospot (ELISPOT) assay

The serum IgG_2a^b concentrations of BALB/c x CB20 Igh^a/b F₁ or of reconstituted RAG-2^°/° mice were quantified by an ELISA with a detection limit of 5 ng/ml. The plates were coated with 1 µg/ml of anti-IgG_2a^b mAb (BG1) (30). The presence of IgG_2a^b in serial dilutions of serum was detected by biotin-labeled anti-IgG_2a^b mAb (5.7.2) (30). A standard curve was constructed using myeloma protein IgG_2a^b (CBPC101). Serum IgM^b, IgG₁^b, and IgG_2b^b allotypes were quantified by ELISA as previously described (31). The frequency of IgG_2a^b-producing B cells in Igh^b/b mice was evaluated using an IgG_2a^b-specific ELISPOT assay as detailed previously (32).

FACS analysis

To unambiguously evaluate CD4⁺ and CD40⁺ cell percentages in anti-CD4 (GK1.5)- and/or anti-CD40 (FGK45)-treated mice, we used, in FACS analyses, anti-CD4 (CT-CD4) and anti-CD40 (3/23) mAbs recognizing, respectively, distinct epitopes of CD4 and CD40. PE-conjugated anti-CD40 (3/23), anti-CD4 (CT-CD4), anti-CD8a (CT-CD8), and FITC-conjugated anti-Thy-1.2 (5a-8) mAb were obtained from Caltag (South San Francisco, CA). PE-conjugated anti-B220 was purchased from Coulter (Coultronics, Margency, France). The anti-IgD^b (H6.31) used was biotinylated. FITC-conjugated streptavidin was obtained from Amersham (Aylesbury, U.K.). Labeled cells were analyzed, after setting gates on forward vs side light scatter and living propidium iodide-negative cells, in a FACScan using CellQuest software (Becton Dickinson, Mountain View, CA).

	Results

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In vivo CD40 triggering substantially replaces the CD4⁺ T cell help in the recruitment of CD8⁺ T cell suppression effectors

We investigated the potential of agonistic anti-CD40 treatment to replace the helper function of anti-IgG_2a^b CD4⁺ T cells during the amplification of anti-Ig allotype T cell activity (sensitization). Experimental groups of BALB/c Igh^a/a mice, sensitized on day 0 against IgG_2a^b-producing B cells, received 1) nothing (n = 12), or injections of 2) anti-CD4 (GK1.5) plus anti-CD40 (FGK45) mAb (n = 12), 3) anti-CD4 plus a control Ig (isotype-matched with anti-CD40 mAb; n = 6), or 4) anti-CD40 mAb alone (n = 12). The anti-CD4 mAb was given daily from days -7 to 7, as we previously observed that this depletion treatment effectively abolished the suppression induction capacity of Igh^a/a T splenocytes (14). The anti-CD40 mAb (or control Ig) was given daily from days 1 to 7, because we reasoned that anti-CD40 stimulation would be useful only once the Igh^a/a mice had received the sensitizing Igh^b/b B cells.

Marked splenomegaly (2- to 3-fold increase in splenocyte numbers) was observed in all anti-CD40-treated mice. FACS analyses showed that the CD4⁺ T cell depletion had been effective and specific (Table I). Indeed, we detected 0.1 or 0.0% CD4⁺ T cells in pooled splenocytes from mice treated, respectively, with anti-CD4 plus anti-CD40 or anti-CD4 plus control Ig compared with 28.9% CD4⁺ T cells in their untreated counterparts. CD8⁺ T cells were not deleted in anti-CD4-treated mice. The apparent CD4⁺ and CD8⁺ percent decreases in mice treated with anti-CD40 mAb alone could be a consequence of the observed splenomegaly, probably due to proliferation of non-T populations. It is noteworthy that, as expected, this in vivo anti-CD40 mAb treatment did not induce either CD40⁺ cell depletion (no decrease, for instance, in the percentage of B220⁺ CD40⁺ B splenocytes; Table I) or internalization of surface CD40 (as assessed, at least for B220⁺ B splenocytes, by the fluorescence intensity obtained with the anti-CD40 mAb 3/23; data not shown).

View larger version (15K): [in this window] [in a new window]   FIGURE 1. Suppression induction capacity of Tsens from BALB/c Igha/a mice treated with anti-CD4 and/or anti-CD40 (or control Ig) mAbs. Serum IgG2ab concentrations in 13-wk-old BALB/c x CB20 Igha/b F1 (untreated controls or recipients of different preparations of Tsens). n, number of Igha/b F1 tested. Vertical bars indicate the mean IgG2ab concentrations.

Induction of full IgG_2a^b suppression via the Fas-dependent cytotoxic pathway necessitates Igh^b/b B cell CD40 expression

We then sought to determine whether CD40 expression on IgG_2a^b-producing cells took part in the suppression induction/maintenance, particularly when this negative regulation was mediated via the Fas pathway. We investigated the susceptibility of Igh^b/b CD40^+/+ or CD40^°/° B cells to suppression induction by Igh^a/a Pfp^+/+ T cells (using Fas- and/or Pfp-mediated pathways) or by Igh^a/a Pfp^°/° T cells (using exclusively the Fas-mediated pathway). To this end we used an alternative experimental model of Ig allotype suppression induction that consists of cotransfer of histocompatible Igh^b/b B and/or Igh^a/a T cells of wild-type or diverse knockout origins into immunodeficient RAG-2^°/° mice. This model has the advantage that the implantation of transferred wild-type or different mutant B or T cells can be directly checked by FACS analysis of PBL from RAG-2^°/° hosts, which are totally devoid of endogenous mature B and T cells. Moreover, with this system the IgG_2a^b expression or suppression status can be determined as early as 2–3 wk after B plus T cell transfer. For these experiments, we also switched to the BC8 Igh^a/a-C57BL/6 Igh^b/b congenic mouse system because of the availability of the required knockout mice with the C57BL/6 genetic background.

Groups of RAG-2^°/° mice received Igh^b/b CD40^+/+ (n = 2) or CD40^°/° (n = 3) B cells alone or a mixture of Igh^b/b CD40^+/+ or CD40^°/° B cells plus Igh^a/a Pfp^+/+ or Pfp^°/° T cells (n = 3/group). As shown in Table II, comparable repopulations of the B cell compartment were obtained with Igh^b/b CD40^+/+ or CD40^°/° B cells in RAG-2^°/° recipients of B cells alone or of B plus T cell mixtures. Successful CD4⁺ and CD8⁺ T cell repopulations were observed in RAG-2^°/° recipients of Igh^a/a Pfp^+/+ or Pfp^°/° T cells. As we described previously (18), the CD4⁺/CD8⁺ ratio in PBL of reconstituted RAG-2^°/° mice was inverted compared with that of the initial transferred cell suspensions. Some CD4⁺ and CD8⁺ T cells were found in RAG-2^°/° recipients of only Igh^b/b B cells, a consequence of the presence of 2–4% residual T cells in the transferred Igh^b/b B cell-enriched preparations. Of course, untreated RAG-2^°/° controls had no B or T cells (Table II, first line). These engraftment data showed that appropriate conditions had been obtained for IgG_2a^b suppression induction assays by Igh^a/a Pfp^+/+ or Pfp^°/° T cells against Igh^b/b CD40^+/+ or CD40^°/° B cells.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Susceptibility of CD40+/+ (A) or CD40°/° (B) B cells from C57BL/6 Ighb/b mice to suppression induction by Pfp+/+ or Pfp°/° T cells from BC8 Igha/a mice. Data obtained by ELISA represent binding of IgG2ab present in serial dilutions of sera from individual C57BL/6 RAG-2°/° mice reconstituted with diverse B cell or B plus T cell populations on day 45 post-transfer.

Enhanced serum IgG_2a^b production by Igh^b/b CD40^°/° B cells engrafted into a histocompatible RAG-2^°/° environment

Compared with Igh^b/b CD40^+/+ mice, the Igh^b/b CD40^°/° mice used here had equivalent serum IgG₁^b and IgG_2b^b concentrations, but 140 times less serum IgG_2a^b (Table III). Unexpectedly, when Igh^b/b CD40^°/° B cell-enriched splenocytes alone were transplanted into histocompatible RAG-2^°/° mice, strikingly increased serum IgG_2a^b levels, comparable to those in RAG-2^°/° recipients of Igh^b/b CD40^+/+ B cells alone or in Igh^b/b CD40^+/+ mice, were detected (Table III). Using the IgG_2a^b-specific ELISPOT assay, we determined that, compared with Igh^b/b CD40^+/+ mice, Igh^b/b CD40^°/° mice had dramatically lower frequencies and absolute numbers of IgG_2a^b-producing splenocytes. Importantly, RAG-2^°/° recipients of Igh^b/b CD40^+/+ or CD40^°/° B cells alone had markedly higher frequencies and absolute numbers of these cells, respectively, than Igh^b/b CD40^+/+ or CD40^°/° mice (Table III).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Using the experimental system of Ig allotype suppression, which provides a good example of CD4⁺-dependent generation of CD8⁺ CTL (14, 15), we first showed that CD4⁺ T cell-depleted Igh^a/a mice, subjected to agonistic anti-CD40 treatment and sensitized against congenic Igh^b/b B cells, were able to recruit CD8⁺ T cells with strong potential to inhibit IgG_2a^b production in Igh^a/b F₁ recipients. Thus, the generation of these regulatory CD8⁺ T cells is in agreement with the model of CD8⁺ T cell activation by CD40-triggered DC. The engagement of CD40 on DC by CD40L on activated CD4⁺ T cells up-regulates the expression of cytokines, adhesion and costimulatory molecules in DC, thereby endowing them with an enhanced ability to stimulate Ag-specific CD8⁺ CTL (2, 3, 4). According to this model, the in vivo Ag presentation to the anti-IgG_2a^b Igh^a/a T cells would essentially be due to the uptake of IgG_2a^b from sensitizing Igh^b/b B cells and presentation of C_2a^b-derived peptides by DC. Importantly, despite their significant ability to negatively regulate IgG_2a^b production, the anti-IgG_2a^b CD8⁺ T cells recruited by CD40 triggering in CD4⁺ T cell-depleted Igh^a/a mice, failed to induce full suppression in all their Igh^a/b F₁ recipients and were thus less effective than the anti-IgG_2a^b CD8⁺ T cells normally stimulated in the presence of CD4⁺ T cells. This incomplete replacement of CD4⁺ T cell help by in vivo CD40 triggering could reflect, as recently suggested (5), the need for a parallel CD40-independent DC stimulation or direct CD4⁺-CD8⁺ T cell communication via cytokines for the priming of CD8⁺ T cells. The efficacy of in vivo agonistic anti-CD40 treatment for the generation of Ag-specific and anti-tumor CTL has been well documented (2, 3, 4, 33, 34, 35). Our data provide further evidence that such treatment also helps to recruit cytotoxic CD8⁺ T cells involved in immune regulatory functions.

CD8⁺ T cell suppression effectors involved in this model use Fas- and Pfp-mediated pathways alternatively or concomitantly to chronically cytolyse the IgG_2a^b-producing B targets (18). Because Fas is not expressed on mature B cells (19, 20, 21), its up-regulation on IgG_2a^b-producing B cells is plausibly essential for Fas-mediated suppression induction. CD40 triggering in the absence of Ig stimulation up-regulates B cell Fas expression, rendering these cells vulnerable to Fas-mediated cytolysis (22, 23). In this study we also demonstrated that the expression of CD40 on IgG_2a^b-producing B targets was required for full suppression induction via the Fas pathway. Indeed, implantation of appropriate Igh^a/a T and Igh^b/b B cells into histocompatible RAG-2^°/° recipients and measurement of their serum Ig allotype production showed that Igh^a/a Pfp^°/° T cells (operating exclusively through the Fas pathway) failed to establish full suppression against Igh^b/b CD40^°/° B cells, while they were able to totally induce it against Igh^b/b CD40^+/+ B cells. Notably, with Igh^a/a Pfp^°/° T cells, Igh^b/b CD40^°/° B cells were only subjected to a partial negative regulation of IgG_2a^b production. It can be hypothesized that the significantly lower, but permanent and easily detectable, IgG_2a^b production in RAG-2^°/° recipients of Igh^b/b CD40^°/° B plus Igh^a/a Pfp^°/° T cells would be due to the persistence of low frequencies (< 1/1 x 10⁷ lymphocytes; below the detection limit of our ELISPOT assay) of potential suppression targets unable to be cytolysed via the Fas pathway in the absence of CD40 expression. In accordance with this hypothesis, one can imagine the existence of two kinds of IgG_2a^b-producing B cells: 1) a highly represented population, readily susceptible to Fas-induced suppression, which has to up-regulate Fas through a CD40-independent mechanism; and 2) a weakly represented population, escaping Fas-induced suppression in the absence of CD40-mediated Fas up-regulation. Alternatively, when CD40-dependent Fas up-regulation is impossible, Fas-mediated cytolysis of B cells recently committed to IgG_2a^b production would be somewhat delayed before they would become susceptible to CD40-independent Fas-mediated death. During this latent period, even if such B cells would not produce enough IgG_2a^b to be visualized in ELISPOT assay, minute amounts of this Ig allotype could be secreted per cell and would lead to the presence of weak, but detectable, serum IgG_2a^b.

We have to emphasize that under simultaneous blockage of Pfp and Fas pathways no evident decrease in IgG_2a^b production was observed (18). This finding indicates that Pfp- and Fas-dependent pathways most probably constitute exclusive suppression mechanisms and thereby eliminate the possibility that CD40 would be involved in an additional suppression mechanism, independent of the former.

The biochemistry of intracytoplasmic events leading to up-regulation of Fas by CD40 engagement has not been entirely elucidated. The cytoplasmic tail of CD40 lacks enzymatic activity, but CD40-CD40L interaction induces trimerization of CD40 and its assembly to certain adaptor proteins of the TNF receptor-associated factor (TRAF) family, i.e., TRAF2, -3, -5, and -6, able to recruit other downstream mediators (36). To examine the possible involvement of such TRAF molecules in CD40-dependent Fas up-regulation in mature B cells, it will be informative to compare the susceptibility to Fas-mediated suppression of B cells from CD40^°/° mice to that of B cells from mouse strains deficient for these adaptor proteins (37, 38, 39, 40). In this respect, despite the short life expectancy of most of the TRAF-deficient mice (2 wk), it remains possible to reconstitute lymphocyte compartments of immune-deficient hosts, for instance RAG-2^°/° recipients, with fetal liver cells from TRAF-deficient donors to investigate the behavior of their B cells in this suppression induction process.

The negative influence of CD40 involvement on B cell functions has previously been observed in a few in vitro models (6, 7, 8, 9, 10). For instance, CD40 ligation on human peripheral B cells by high amounts of soluble or membrane-bound CD40L reduced Ig production, especially by activated B cells that had undergone switch recombination (10). It is likely that after initial activation by CD40 engagement, B cells became more susceptible to inhibition by further CD40 ligation (8, 10). In those previous studies the CD40 ligation on activated B cells induced inhibition of Ig production without blockage of B cell proliferation. Comparatively, in the Ig allotype suppression model, CD40 plays a role in the negative control of Ig production by B cells that have switched to IgG_2a^b production, but this regulation leads to Fas-mediated death of these Ig-producing cells.

Injection of CD40^°/° embryonic stem cells into RAG-2^°/° blastocysts generates chimeric mice with CD40^°/° mature lymphocytes developing in an environment principally made of CD40^+/+ RAG-2^°/° cells (41). Like CD40^°/° mice (26), these chimeras have low serum IgG2a levels due to defective isotype switching. In the present study we observed that transfer of peripheral mature B cells into RAG-2^°/° hosts strongly favored switch recombination to IgG2a and that this effect was particularly striking for CD40^°/° B cells. This finding contrasts with that observed in CD40^°/°-RAG-2^°/° chimeras, seemingly because in our model mature CD40^°/° B cells are directly transferred into RAG-2^°/° hosts, whereas CD40^°/° B cell progenitors have to develop and differentiate into potent B cells in CD40^°/°-RAG-2^°/° chimeras. Our observation strongly suggests that in the RAG-2^°/° environment, mature B cells find conditions enhancing a CD40-independent mechanism leading to switch recombination, at least to IgG2a. The elucidation of this phenomenon would be useful in the perspective of correcting Ig class switching in diseases such as human X-linked hyper-IgM syndrome due to genetic alterations of the CD40L gene (42).

	Acknowledgments

 
We thank Christèle Sellier for excellent technical assistance, Pascal Dardenne for expert assistance in animal care and performing all the bleedings, Andrée Goyat for serum preparations, 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 Center National de la Recherche Scientifique (1961), and the Association pour la Recherche sur le Cancer (9955).

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.

³ Abbreviations used in this paper: DC, dendritic cells; CD40L, CD40 ligand; Pfp, pore-forming protein; RAG-2, recombination-activating gene 2; TRAF, TNF receptor-associated factor; Tsens, nylon wool nonadherent Igh^a/a T splenocytes sensitized against congenic Igh^b/b B splenocytes; ELISPOT, enzyme-linked immunospot.

Received for publication August 18, 2000. Accepted for publication October 17, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Van Kooten, C., J. Banchereau. 2000. CD40-CD40 ligand. J. Leukocyte Biol. 67:2.[Abstract]
2. Ridge, J. P., R. F. Di, P. Matzinger. 1998. A conditioned dendritic cell can be a temporal bridge between a CD4⁺ T-helper and a T-killer cell. Nature 393:474.[Medline]
3. Bennett, S. R., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. Miller, W. R. Heath. 1998. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393:478.[Medline]
4. Schoenberger, S. P., R. E. M. Toes, E. I. H. van der Voort, R. Offringa, C. J. M. Melief. 1998. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393:480.[Medline]
5. Lu, Z., L. Yuan, X. Zhou, E. Sotomayor, H. I. Levitsky, D. M. Pardoll. 2000. CD40-independent pathways of T cell help for priming of CD8⁺ cytotoxic T lymphocytes. J. Exp. Med. 191:541.[Abstract/Free Full Text]
6. Heath, A. W., R. Chang, N. Harada, L. Santos-Argumedo, J. Gordon, C. Hannum, D. Campbell, A. B. Shanafelt, E. A. Clark, R. Torres, et al 1993. Antibodies to murine CD40 stimulate normal B lymphocytes but inhibit proliferation of lymphoma cells. Cell. Immunol. 152:468.[Medline]
7. Callard, R. E., J. Herbert, S. H. Smith, R. J. Armitage, K. E. Costelloe. 1995. CD40 cross-linking inhibits specific antibody production by human B cells. Int. Immunol. 7:1809.[Abstract/Free Full Text]
8. Silvy, A., C. Lagresle, C. Bella, T. Defrance. 1996. The differentiation of human memory B cells into specific antibody-secreting cells is CD40 independent. Eur. J. Immunol. 26:517.[Medline]
9. Bergman, M. C., J. F. Attrep, A. C. Grammer, P. E. Lipsky. 1996. Ligation of CD40 influences the function of human Ig-secreting B cell hybridomas both positively and negatively. J. Immunol. 156:3118.[Abstract]
10. Miyashita, T., M. J. McIlraith, A. C. Grammer, Y. Miura, J. F. Attrep, Y. Shimaoka, P. E. Lipsky. 1997. Bidirectional regulation of human B cell responses by CD40-CD40 ligand interactions. J. Immunol. 158:4620.[Abstract]
11. Majlessi, L., N. Rujithamkul, G. Bordenave. 1995. Mechanisms of T-cell-induced allotypic suppression of mouse IgG_2a^b and of tolerance acquisition to this allotype. Res. Immunol. 146:213.[Medline]
12. Benaroch, P., G. Bordenave. 1987. Normal T splenocytes are able to induce immunoglobulin allotypic suppression in F₁ hybrid mice. Eur. J. Immunol. 17:167.[Medline]
13. Benaroch, P., G. Bordenave. 1988. Enhancement in Igh^a mouse strain of the "natural" suppressive activity of normal T splenocytes against the expression of Igh-1b allotype. I. Molecular aspects of the chronic suppression obtained. Eur. J. Immunol. 18:51.[Medline]
14. Majlessi, L., P. Benaroch, C. Denoyelle, G. Bordenave. 1993. Role of CD4⁺ and CD8⁺ cell subsets during amplification of natural T cell activity against IgG_2a^b in Igh^a mice and during induction of IgG_2a^b allotype suppression in Igh^a/b mice. J. Immunol. 151:1859.[Abstract]
15. Benaroch, P., G. Bordenave. 1989. T cell-induced Ig allotypic suppression in mice. II. Both CD4⁺CD8^- and CD4^-CD8⁺ T cell subsets from sensitized Igh^b mice are required to induce suppression of Igh-1b allotype expression. J. Immunol. 142:1.[Abstract]
16. Benaroch, P., E. Georgatsou, G. Bordenave. 1988. Cellular induction of chronic allotype suppression of IgG_2a in Igh^b/b homozygous mice and its abrogation by in vivo treatment with anti-CD8 monoclonal antibody. J. Exp. Med. 168:891.[Abstract/Free Full Text]
17. Majlessi, L., G. Bordenave. 1997. The T/B cell interaction involved in induction of the mouse IgG_2a^b suppression is restricted by major histocompatibility complex class I, but not class II molecules. Eur. J. Immunol. 27:1346.[Medline]
18. Majlessi, L., G. Bordenave. 1999. Evidence of alternative or concomitant use of perforin- and Fas-dependent pathways in a T cell-mediated negative regulation of Ig production. J. Immunol. 162:4391.[Abstract/Free Full Text]
19. Drappa, J., N. Brot, K. B. Elkon. 1993. The Fas protein is expressed at high levels on CD4⁺CD8⁺ thymocytes and activated mature lymphocytes in normal mice but not in the lupus-prone strain, MRL lpr/lpr. Proc. Natl. Acad. Sci. USA 90:10340.[Abstract/Free Full Text]
20. Watanabe, D., T. Suda, S. Nagata. 1995. Expression of Fas in B cells of the mouse germinal center and Fas-dependent killing of activated B cells. Int. Immunol. 7:1949.[Abstract/Free Full Text]
21. Wang, J., I. Taniuchi, Y. Maekawa, M. Howard, M. D. Cooper, T. Watanabe. 1996. Expression and function of Fas antigen on activated murine B cells. Eur. J. Immunol. 26:92.[Medline]
22. Rothstein, T. L., J. K. Wang, D. J. Panka, L. C. Foote, Z. Wang, B. Stanger, H. Cui, S. T. Ju, R. A. Marshak. 1995. Protection against Fas-dependent Th1-mediated apoptosis by antigen receptor engagement in B cells. Nature 374:163.[Medline]
23. Rathmell, J. C., S. E. Townsend, J. C. Xu, R. A. Flavell, C. C. Goodnow. 1996. Expansion or elimination of B cells in vivo: dual roles for CD40- and Fas (CD95)-ligands modulated by the B cell antigen receptor. Cell 87:319.[Medline]
24. Majlessi, L., C. Roth, P. Benaroch, J.-M. Claverie, P. Kourilsky, G. Bordenave. 1993. Identification of T-cell peptides from IgG_2a^b involved in IgG_2a^b T-cell-induced allotypic suppression: relationship with the MHC haplotype of the suppressor T-cell-donor Igh^a mice. J. Immunol. 151:1263.[Abstract]
25. Kagi, D., B. Ledermann, K. Burki, P. Seiler, B. Odermatt, K. J. Olsen, E. R. Podack, R. M. Zinkernagel, H. Hengartner. 1994. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369:31.[Medline]
26. Kawabe, T., T. Naka, K. Yoshida, T. Tanaka, H. Fujiwara, S. Suematsu, N. Yoshida, T. Kishimoto, H. Kikutani. 1994. The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation. Immunity 1:167.[Medline]
27. Shinkai, Y., G. Rathbun, K. P. Lam, E. M. Oltz, V. Stewart, M. Mendelsohn, J. Charron, M. Datta, F. Young, A. M. Stall, et al 1992. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68:855.[Medline]
28. Dialynas, D. P., Z. S. Quan, K. A. Wall, A. Pierres, J. Quintans, M. R. Loker, M. Pierres, F. W. Fitch. 1984. Characterization of the murine T cell surface molecule, designated L3T4, identified by monoclonal antibody GK1.5: similarity of L3T4 to human Leu3/4 molecule. J. Immunol. 131:2445.[Abstract]
29. Rolink, A., F. Melchers, J. Andersson. 1996. The SCID but not the RAG-2 gene product is required for Sµ-S heavy chain class switching. Immunity 5:319.[Medline]
30. Huang, C. M., M. Parsons, V. T. Oi, H. J. S. Huang, L. A. Herzenberg. 1983. Genetic characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies. Immunogenetics. 18:311.[Medline]
31. Majlessi, L., M.-A. R. Marcos, P. Benaroch, C. Denoyelle, G. Bordenave. 1994. T cell-induced Ig allotypic suppression in mice: basis for emergence or tolerization, during the perinatal period, of natural T cells specific to the IgG_2a^b allotype. J. Immunol. 152:3342.[Abstract]
32. Benaroch, P., E. Georgatsou, G. Bordenave. 1993. T cell-induced suppression of IgG_2a^b expression in vivo leads to a large reduction of C_2a^b mRNA levels. J. Immunol. 150:858.[Abstract]
33. French, R. R., H. T. C. Chan, A. L. Tutt, M. J. Glennie. 1999. CD40 antibody evokes a cytotoxic T-cell response that eradicates lymphoma and bypasses T-cell help. Nat. Med. 5:548.[Medline]
34. Diehl, L., A. T. den Boer, S. P. Schoenberger, E. I. H. van der Voort, T. N. M. Schumacher, C. J. M. Melief, R. Offringa, R. E. M. Toes. 1999. CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance and augments anti-tumor vaccine efficacy. Nat. Med. 5:774.[Medline]
35. Sotomayor, E. M., I. Borrello, E. Tubb, F.-M. Rattis, H. Bien, Z. Lu, S. Fein, S. Schoenberger, H. I. Levitsky. 1999. Conversion of tumor-specific CD4⁺ T-cell tolerance to T-cell priming through in vivo ligation of CD40. Nat. Med. 5:780.[Medline]
36. Inoue, J.-I., T. Ishida, N. Tsukamoto, N. Kobayashi, A. Naito, S. Azuma, T. Yamamoto. 2000. Tumor necrosis factor receptor-associated factor (TRAF) family: adaptor proteins that mediate cytokine signaling. Exp. Cell. Res. 254:14.[Medline]
37. Yeh, W. C., A. Shahinian, D. Speiser, J. Kraunus, F. Billia, A. Wakeham, J. de la Pompa, D. Ferrick, B. Hum, N. Iscove, et al 1997. Early lethality, functional NF-B activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice. Immunity 7:715.[Medline]
38. Xu, Y., G. Cheng, D. Baltimore. 1996. Targeted disruption of TRAF3 leads to postnatal lethality and defective T-dependent immune responses. Immunity 5:407.[Medline]
39. Nakano, H., S. Sakon, H. Koseki, T. Takemori, K. Tada, M. Matsumoto, E. Munechika, T. Sakai, T. Shirasawa, H. Akiba, et al 1999. Targeted disruption of Traf5 gene causes defects in CD40- and CD27-mediated lymphocyte activation. Proc. Natl. Acad. Sci. USA 96:9803.[Abstract/Free Full Text]
40. Lomaga, M. A., W. C. Yeh, I. Sarosi, G. S. Duncan, C. Furlonger, A. Ho, S. Morony, C. Capparelli, G. Van, S. Kaufman, et al 1999. TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev. 13:1015.[Abstract/Free Full Text]
41. Castigli, E., F. W. Alt, L. Davidson, A. Bottaro, E. Mizoguchi, A. K. Bhan, R. Geha. 1994. CD40-deficient mice generated by recombination-activating gene-2-deficient blastocyst complementation. Proc. Natl. Acad. Sci. USA 91:12135.[Abstract/Free Full Text]
42. Ramesh, N., M. Seki, L. D. Notarangelo, R. Geha. 1998. The hyper-IgM (HIM) syndrome. Springer Semin. Immunopathol. 19:383.[Medline]

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