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Follicular dendritic cells: beyond the necessity of T-cell help
Opinion
TRENDS in Immunology Vol.22 No.7 July 2001
Follicular dendritic cells: beyond the necessity of T-cell help John G. Tew, Jiuhua Wu, Mohamed Fakher, Andras K. Szakal and Dahui Qin Follicular dendritic cells (FDCs) are potent accessory cells for B cells, but the molecular basis of their activity is not understood. Several important molecules involved in FDC–B-cell interactions are indicated by blocking the ligands and receptors on FDCs and/or B cells. The engagement of CD21 in the B-cell coreceptor complex by complement-derived CD21 ligand on FDCs delivers a crucial signal that dramatically augments the stimulation delivered by the binding of antigen to the B-cell receptor (BCR). The engagement of Fc γ receptor IIB (FcγγRIIB) by the Ig crystallizable fragment (Fc) in antigen–antibody complexes held on FDCs decreases the activation of immunoreceptor tyrosinebased inhibition motifs (ITIMs), mediated by the crosslinking of BCR and FcγγRIIB. Thus, FDCs minimize a negative B-cell signal. In short, these ligand–receptor interactions help to signal to B cells and meet a requirement for B-cell stimulation that goes beyond the necessity of T-cell help.
John G. Tew* Jiuhua Wu Mohamed Fakher Dahui Qin Dept of Microbiology and Immunology, Andras K. Szakal Dept of Anatomy, Division of Immunobiology, Medical College of Virginia, Virginia Commonwealth University, PO Box 980678, Richmond, VA 23298-0678, USA. *e-mail: tew@ hsc.vcu.edu
We propose that FOLLICULAR DENDRITIC CELL (FDC)–Bcell interactions (see Glossary) meet a requirement for B-cell stimulation that goes beyond the necessity of T-cell help. In T-dependent antibody (Ab) responses, the T helper (Th) cells provide CD40 ligand (CD40L) and lymphokines, including chemokines, that function with cytokines produced by accessory cells to stimulate B cells. These molecules are obviously important, but in this article we review data indicating that they are not sufficient for optimal B-cell responses. Direct cell–cell interactions between B cells and FDCs allow the engagement of multiple membrane-bound receptors and ligands that are also important in B-cell signaling. In recall responses, the immunogen is immediately converted into immune complexes (ICs) by Ab persisting from previous immunizations; ICs stimulate potent recall responses if the animal has wild-type FDCs (Ref. 1). In primary responses, naive T and B-cells are apparently initially stimulated in the absence of FDCs bearing specific antigen (Ag)–Ab complexes because some Ab must first be made in order for the Ag–Ab complexes to be formed. However, it should be appreciated that as soon as Ab is produced in a primary response, Ag–Ab complexes are formed and trapped by FDCs (Ref. 2) and Ag–Ab complex–FDC–B-cell interactions play an important role in the maturation and maintenance of humoral immune responses. Specifically, interactions
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between B cells and Ag–Ab complex-bearing FDCs are linked to the formation of germinal centers, somatic mutations and memory cells, and the induction of Ab-forming cells producing large amounts of specific high-affinity Ab (Refs 3–6). In vitro studies indicate that ICs activate the immunosuppressive IMMUNORECEPTOR TYROSINE-BASED INHIBITION MOTIF (ITIM) in B cells7–9. However, in the presence of FDCs, ICs are highly immunogenic. The molecules that confer adjuvant-like accessory activity on IC-bearing FDCs have been the focus of recent research. Multiple receptors and ligands on FDCs and B cells were found to be crucial for FDC accessory activity. These receptors and ligands are illustrated in Fig. 1 and the major experimental data supporting the necessity of these molecules for optimal signaling of memory B cells are listed in Box 1. This discussion will follow the sequence of Box 1, and models involving both murine and human leukocytes will be considered.
…follicular dendritic cell–B-cell ‘… interactions meet a requirement for B-cell stimulation that goes beyond the necessity of T-cell help.’ The inhibition of recall responses: blockade of BCR or MHC class II on B cells and CD40L on T cells
The requirement for these receptors and ligands in T-cell–B-cell interactions is well known and has been confirmed using IC-bearing FDCs in in vitro studies. For example, the requirement for MHC class II was demonstrated using IMMUNE COMPLEX-COATED BODIES (iccosomes) derived from FDCs as the source of immunogen and normal FDCs for costimulatory activity. ICCOSOMAL AG, as illustrated in Fig. 1, is released by FDCs and the iccosomes are endocytosed by specific B cells. These cells process and present the Ag to appropriate T cells, which then provide the B cells with the necessary help10,11. However, we acknowledge that germinal centers with highaffinity B cells can form without T cells when Ags such as (4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll (a classical T-independent Ag) are used and B-CELL RECEPTORS (BCRs) are extensively crosslinked12. The interpretation that T cells are crucial for the simple protein Ags we typically study is supported by the observation that the murine anti-ovalbumin (antiOVA) response is inhibited by ≈95% if appropriate anti-class II monoclonal Ab (mAb) is added to the cultures. For example, the anti-OVA Ab titer induced by OVA-bearing iccosomes in the presence of FDCs from normal animals was 1370 ± 198 ng ml−1 in the presence of an isotype control mAb, in comparison with only 51 ± 12 ng ml−1 when cultures were treated with an appropriate anti-MHC class II mAb (Ref. 13).
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Box 1. Multiple receptor–ligand interactions are necessary for optimal signaling of memory B cells* T cell
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Fig. 1. Model illustrating the important receptors and ligands used by T cells and follicular dendritic cells (FDCs) in signaling to B cells. The requirement for B-cell MHC class II to present antigen (Ag)-derived peptides as ligands for the T-cell receptor (TCR) is well known, as is the involvement of CD40 on the B cell interacting with T cell CD40 ligand (CD40L). The FDC–B cell interaction delivers a primary signal through FDC Ag–B-cell receptor (BCR) interaction and a cosignal through B cell CD21 interacting with FDC CD21 ligand (CD21L). The events of a recall response are summarized as follows. (1) FDCs trap Ag–antibody (Ab) complexes and provide intact Ag for interaction with BCRs on germinal center (GC) B cells; this Ag–BCR interaction provides a positive signal for B-cell activation and differentiation. (2) FDCs provide a complement-derived CD21L for B cell CD21; its interaction with the CD21–CD19–CD81 complex delivers a positive cosignal for B-cell activation and differentiation. Coligation of BCR and CD21 (as illustrated here with a single molecule of Ag) facilitates association of the two receptors, and the cytoplasmic tail of CD19 is phosphorylated by a tyrosine kinase associated with the BCR complex51. The arrow pointing from the BCR to CD19 indicates this known interaction. (3) A high density of Fc γ receptor IIB (FcγRIIB) on FDCs binds Ig Fc in the Ag–Ab complex and consequently the signal delivered by the immunoreceptor tyrosine-based inhibition motif (ITIM) in the B cells might be blocked. This inhibitory signal is initiated by Ag–Ab complexes crosslinking BCR and FcγRIIB on B cells. Note that BCR is not crosslinked with B cell FcγRIIB in the model and thus a high concentration of FcγRIIB on FDCs minimizes a negative signal to the B cell. (4) In addition, FDCs provide immune complex-coated bodies (iccosomes), which are readily taken up by B cells52. The iccosome membrane is derived from FDC membranes that have Ag, CD21L and Ig Fc attached. Iccosomes bind tightly to B cells and are rapidly endocytosed11. We reason that binding of BCR, complement receptor 2 (CR2) and possibly B cell Fc receptor (FcR) to the iccosomal Ag–CD21L–Ig Fc complex is crucial to the process of endocytosis. The B cells process this FDC-derived Ag, present it and thus obtain T-cell help.
Blocking CD21L on the FDCs inhibits FDC accessory activity
To determine whether CD21 LIGAND/COMPLEMENT RECEPTOR 2 LIGAND (CD21L/CR2L) on FDCs plays a role in enhancing Ab responses, [COMPLEMENT RECEPTOR 2 (CR2)/CD21]2-IgG1, a soluble chimeric molecule of mouse IgG1 heavy chains and CR2 (Ref. 14), was used to block CR2L on FDCs. Binding of this soluble receptor to FDC CD21L should inhibit the binding of the CD21L to its receptor on the B cell, thus blocking FDC activity. As the dose of soluble CR2 increased, the anti-OVA IgG response was reduced to <10% of the isotype control Ab-treated FDCs (Fig. 2). Similar results were seen using human serum albumin (HSA) as the model Ag and these data are consistent with in vivo data showing that this soluble receptor dramatically inhibits Ab responses14. These in vitro results, indicating the importance of CD21L on FDCs, http://immunology.trends.com
• Blockade of B-cell receptor (BCR) or MHC class II on B cells, or CD40 ligand (CD40L) on T cells with specific antibodies inhibits recall responses induced by immune complex (IC)-bearing follicular dendritic cells (FDCs)a,b,c (J. Wu et al., unpublished). • Blockade of FDC CD21 ligand (CD21L) inhibits recall responses induced by IC-bearing FDCs (Refs d,e). • FDCs lacking complement-derived CD21L lack normal accessory activitye. • Blockade of B cell CD21 to inhibit the binding of FDC CD21L blocks the induction of recall responses by IC-bearing FDCs (Ref. e). • FDCs lacking Fc γ receptor IIB (FcγRIIB) have markedly reduced accessory activity. These FDCs cannot bind Ig Fc in ICs and allow B cell FcγRIIB and BCR to be coligatedf. • None of the important receptors or ligands on FDCs is MHC-restricted; thus FDC accessory activity crosses both MHC and species barriersg. • In the presence of FDCs, ICs are potent and efficient immunogensa,e,g (Fig. 4). *The crucial receptors and ligands are illustrated in Fig. 1. Memory B cells and T cells were obtained from blood or secondary lymphoid tissues of previously immunized and boosted humans or mice with high serum titers of IgG specific for the recall antigen. These memory cells give robust recall responses, whereas cells from nonimmunized controls fail to responda.
References a Wu, J. et al. (1996) Follicular dendritic cell (FDC) derived Ag and accessory activity in initiation of memory IgG responses in vitro. J. Immunol. 157, 3404–3411 b Gronowicz, E. and Coutinho, A. (1976) Hapten-induced B-cell paralysis. II. Evidence for trivial mechanisms of tolerance. Eur. J. Immunol. 5, 413–420 c Foy, T.M. et al. (1993) In vivo CD40–gp39 interactions are essential for thymus-dependent humoral immunity. II. Prolonged suppression of the humoral immune response by an antibody to the ligand for CD40, gp39. J. Exp. Med. 178, 1567–1575 d Hebell, T. et al. (1991) Suppression of the immune response by a soluble complement receptor of B lymphocytes. Science 254, 102–105 e Qin, D. et al. (1998) Evidence for an important interaction between a complement-derived CD21 ligand on follicular dendritic cells and CD21 on B cells in the initiation of IgG responses. J. Immunol. 161, 4549–4554 f Qin, D. et al. (2000) Fcγ receptor IIB on follicular dendritic cells regulates the B-cell recall response. J. Immunol. 164, 6268–6275 g Fakher, M. et al. (2001) Follicular dendritic cell accessory activity crosses MHC and species barriers. Eur. J. Immunol. 31, 176–185
also confirm and extend in vivo studies showing that complement protein C3 and CR2 are crucial for normal Ab responses14–20. It appears that important CD21L–CD21 interactions probably occur in germinal centers where FDCs and B cells interact.
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TRENDS in Immunology Vol.22 No.7 July 2001
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Fig. 2. The inhibitory effect of soluble complement receptor 2 (CR2/CD21) on anti-ovalbumin (anti-OVA) IgG production in vitro. OVA-primed lymphocytes (LyOVA) were isolated from OVA-immune mouse lymph nodes. OVA-bearing follicular dendritic cells (FDCOVA) were isolated from the draining lymph nodes of OVA-immune mice three days after immune challenge. Lymphocytes were cultured in a 96-well culture plate (3 × 105 well–1) with FDCOVA (5 × 104 well–1). Different doses of soluble CR2 [(CR2)2-IgG1] or an isotype-matched control antibody (Ab) were added to LyOVA + FDCOVA cultures. The culture supernatant fluids were harvested on day seven and new culture medium was added. Anti-OVA IgG in the supernatant fluid was measured by enzyme-linked immunosorbent assay (ELISA) on day 14. Controls, including LyOVA cultured alone (no FDCs added) and FDCOVA cultured alone, produced <10 ng of anti-OVA IgG ml–1. (Reproduced, with permission, from Ref. 21, 1998, The American Association of Immunologists.)
FDCs lacking complement-derived CD21L lack normal accessory activity
We reasoned that FDCs isolated from C3 knockout mice16 would not bear CD21Ls generated from C3 fragments (iC3b, C3d or C3dg) and would not be able to enhance humoral immune responses as effectively as FDCs from wild-type mice. To test this prediction, FDCs were isolated from the lymph nodes of C3−/− mice and used in lymphocyte cultures. The ability of FDCs from C3−/− mice to enhance IgG responses was reduced by ≈80% (Ref. 21). These data support the concept that C3-derived CD21L is a major stimulator of CR2, but other potential CD21Ls are present on FDCs and could also be involved22,23. Blocking CD21 on the B cell inhibits FDC accessory activity
C3-derived CD21Ls on FDCs might enhance Ab responses by engaging CD21 on B cells; in this case, the FDC-derived cosignal should be blocked if CD21 on the B cell is blocked. To test this prediction, mAbs against CD21 (clones 7G6 against murine CD21 and B-ly4 against human CD21) were used to mask CD21 on B cells, and Ab responses were monitored after stimulation with appropriate ICs and FDCs. Use of http://immunology.trends.com
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anti-CD21 mAb suppressed 80–95% of the specific Ab expression induced by HSA or OVA in the murine system and tetanus toxoid (TT) in the human system21,24. The human model allowed us to set up the experiment such that the murine anti-CD21 could only bind CD21 on human B cells and not the CD21 on murine FDCs, thus eliminating concern about an effect of the mAb binding to FDC CD21 (Ref. 24). The anti-human CD21 mAb also does not crossreact with CD35, as the 7G6 mAb does, thus eliminating concern about the consequences of binding CD35. We also reasoned that FDC accessory activity would be reduced on B cells lacking CD21 because FDC CD21L could not exert its costimulatory effect. Not surprisingly, CD21−/− mice have a profound deficit in their ability to mount T-dependent Ab responses and it is not possible to study typical recall responses in these mice20. Nevertheless, a dramatic stimulatory effect of FDC CD21L can be obtained when the primary signal is given by a polyclonal B-cell activator; lipopolysaccharide (LPS) works well in the murine system and poke weed mitogen in the human system21,24. B cells from CD21−/− mice respond to LPS but the addition of FDCs has very little costimulatory effect. This contrasts with the dramatic effect of adding FDCs bearing CD21L to LPS-stimulated wild-type B cells21. These results further reinforce the importance of CD21L–CD21 in FDC–B-cell interactions. FDCs lacking FcγγRIIB have markedly reduced accessory activity
FDCs label intensely for FC γ RECEPTOR IIB (FCγRIIB) in reactive follicles containing germinal centers. In fact, when compared with FDCs, the B cells in the germinal centers and follicular mantel appear negative for FcγRIIB, indicating that the concentration of FcγRIIB on FDCs is much higher than on B cells1,22. As illustrated in Fig. 1, FDCs bearing FcγRIIB should bind Ig crystallizable fragment (Fc) in ICs and minimize the coligation of B-cell FcγRIIB and BCR by ICs, thus interfering with the delivery of a negative B-cell signal through activation of the ITIM. Indeed, ICs stimulate potent recall responses in the presence of FDCs (Ref. 21). We proposed that FDCs from FcγRIIB−/− mice, which will still bind ICs through complement receptors, would not provide normal FDC accessory activity to wildtype B cells because the negative regulation could still be delivered by free Ig Fc in the ICs. To test this, we compared the accessory activity of FDCs from wildtype and FcγRIIB−/− mice (Fig. 3). Substitution of knockout for wild-type FDCs reduced the recall response by ≈90%. This was confirmed in an in vivo study where memory T and B-cells from wild-type mice were adoptively transferred into FcγRIIB−/− mice, where they would depend on accessory activity from FDCs lacking FcγRIIB. Again, recall responses in these mice were profoundly suppressed1. However, when B cells from FcγRIIB−/− mice are used, the FcγRIIBs on FDCs do not appear to be crucial and
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were compared, as well as human and murine FDCs. Human and murine FDCs converted ICs into potent immunogens and MHC barriers did not restrict this activity. Furthermore, human FDCs worked with murine lymphocytes and murine FDCs worked with human lymphocytes24. In short, there was no lack of activity (specific Ab increased from background levels to thousands of ng ml−1) when crossing either MHC or species barriers24.
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Fig. 3. Follicular dendritic cell (FDC) Fc γ receptor IIB (FcγRIIB) is important for FDCs to convert immune complexes (ICs) into an effective immunogen. FDCs were cultured with memory T or B-cells (Ly) from ovalbumin (OVA)-immune mice in the presence of OVA–anti-OVA ICs, and their ability to convert the ICs into an effective immunogen was measured. The ICs (50 ng of OVA) were formed near equivalence. Cultures were washed on day seven to remove soluble ICs and anti-OVA IgG was measured on day 14. The FDCs were obtained from FcγRIIB−/− and wild-type mice. Controls included normal memory T or B-lymphocytes cultured alone, FDCs cultured alone (FDC-FcγRIIB+/+ and FDC-FcγRIIB−/−), and memory T or B-cells plus OVA–anti-OVA ICs (no FDCs); all produced <10 ng of anti-OVA IgG ml–1. (Reproduced, with permission, from Ref. 1, 2000, The American Association of Immunologists.)
potent recall responses are observed25. This is reasonable, as these B cells lack the potential for coligation of BCR and FcγRIIB and there is therefore no need for FDCs to minimize this effect. According to our model, FDCs bind Ig Fc and minimize the crosslinking of BCR and FcγRIIB, and the activation of ITIM in B cells. Activation of ITIM in B cells results in phosphorylation of Src homology 2 domain-containing inositol polyphosphate 5′-phosphatase (SHIP)26. Thus, we reasoned that the presence of FDCs should reduce SHIP phosphorylation in B cells. Data supportive of this hypothesis was obtained using anti-Ig Ab, a wellknown inducer of SHIP phosphorylation, as an analog for ICs (Ref. 27). As predicted, the addition of wildtype FDCs plus anti-Ig Ab to B-cell cultures reduced the level of anti-Ig Ab-induced SHIP phosphorylation compared with control B cells in the absence of FDCs. In marked contrast, FDCs from FcγRIIB−/− mice were unable to reduce the level of anti-Ig Ab-induced SHIP phosphorylation in B cells (J. Wu et al., unpublished). FDC accessory activity crosses MHC and species barriers
The only ligand–receptor interaction shown in Fig. 1 that is MHC-restricted is the interaction between the T cell and the B cell involving T-cell receptor (TCR) and class II MHC. Thus, FDC accessory activity should cross MHC and even species barriers. Murine FDCs expressing three different MHC haplotypes http://immunology.trends.com
Clearly, B cells specifically trap free Ag using BCRs, and Ag-presentation experiments indicate that B cells process free Ag very efficiently28. As discussed previously, free ICs inhibit B-cell function and are poorly immunogenic in the absence of FDCs. The amount of IC trapped by FDCs is in the pg–low ng range in vivo and if these ICs are to have significant immunogenicity they must be used efficiently. The ICs on the FDCs are converted into iccosomes, which are rapidly endocytosed and processed by B cells. The uptake and processing of iccosomal Ag by B cells prompted us to postulate that ICs would be a potent form of immunogen13, perhaps as potent as free Ag in stimulating B cells. A comparison between free Ag and ICs was conducted using human peripheral-blood lymphocytes (PBLs) responding to TT. TT–anti-TT ICs elicit no anti-TT Ab response in PBLs from immune subjects in the absence of FDCs; however, in the presence of FDCs the TT–anti-TT ICs elicited >103 ng of anti-TT Ab. By contrast, free TT elicited only a few ng of anti-TT Ab with PBLs; the addition of FDCs did increase the response but to a lesser extent than with ICs. This prompted dose–response studies comparing free TT with TT–anti-TT ICs in the presence of FDCs. A representative experiment is shown in Fig. 4. ICs were approximately tenfold more stimulatory than free Ag, with 0.5 ng of TT–anti-TT ICs giving a detectable immune response. Clearly, ICs are potent, efficient immunogens in the presence of FDCs. This might relate to the presentation of Ag and CD21L in a form that allows for crosslinking of BCR and CD21 (Fig. 1). B-cell CD21 delivers a powerful costimulatory signal when coligated with BCR (Refs 29,30) and this might explain the potency and efficiency of ICs on FDCs (Ref. 13). Concluding remarks Ag in germinal centers and Ab production in the bone marrow
Immunogens are converted into ICs as soon as natural Ab or Ab induced as a consequence of infection is encountered. Ag-transporting cells (ATCs) trap ICs and traffic to the draining lymph nodes within minutes, and the ICs are present on FDCs in the follicles within five hours2. Recall responses develop in germinal centers in response to ICs trapped on FDCs (Refs 6,31–33). As reviewed here, crucial
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inhibitor38,43. In short, we suggest that severe bleeding reduces the feedback inhibitor and allows Ag persisting on FDCs to elicit a recall response, resulting in an Ab rebound and a burst of new memory B cells. Thus, although persisting Ag might not be required for the long-term maintenance of memory B cells or serum Ab levels it could still help regulate and modulate these responses. FDC CD21L and the efficiency and potency of the specific immunogen
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Fig. 4. Immune complexes (ICs) are as efficient or more efficient than free antigen (Ag) in stimulating recall responses in the presence of follicular dendritic cells (FDCs). Peripheral blood lymphocytes (PBLs) were isolated from a tetanus toxoid (TT)-immune volunteer in the laboratory. FDCs were isolated from the lymph nodes of normal BALB/c mice. Addition of TT or TT–anti-TT ICs to PBLs (1.0 × 106 ml−1) plus murine FDCs (0.2 × 106 ml−1) stimulated a recall response. Controls included PBLs cultured alone, FDCs cultured alone, PBLs plus TT (no FDCs) and PBLs plus TT–anti-TT ICs (no FDCs); all produced <15 ng of anti-TT IgG ml−1 (M. Fakher et al., unpublished).
ligand–receptor reactions involved in the interaction between FDCs and B cells include Ag–BCR, CD21L–CD21 and Fc–FcR. Within three days of a secondary challenge, plasmablasts emerge from these germinal centers and home to the bone marrow, where they become mature plasma cells and produce the vast majority of the Ig in recall responses33,34. FDCs and the regulation and maintenance of recall responses
Plasma cells persist in the bone marrow and are capable of maintaining serum Ab levels in the absence of persisting Ag on FDCs (Ref. 35). Similarly, memory B cells can persist without restimulation by Ag on FDCs (Ref. 36). This raises the issue of the role of the Ag that does persist on FDCs for months or even years37. We suspect that this Ag does modulate recall responses. For example, removing draining lymph nodes that contain the persisting Ag, does reduce the level of persisting Ab by ≈50%, whereas removing nondraining lymph nodes has no effect38. However, consistent with recent data, removal of the FDCs and associated persisting Ag clearly does not ablate the long-term Ab response. Furthermore, Uhr and colleagues demonstrated that severe bleeding of immune animals can elicit a dramatic rebound in specific serum Ab titer, which exceeds the prebleeding level, and that this Ab response is subject to Ab feedback inhibition39–41. FDCs bearing persisting Ag can elicit just such a rebound phenomenon in vitro42 when cultured in the absence of high concentrations of specific Ab. This ‘spontaneous response’ can be ablated by the addition of specific Ab as a feedback http://immunology.trends.com
It is well known that recall responses can be studied in vitro by simply adding Ag to cultures containing T cells and memory B cells, but these responses are seldom impressive. For example, recall responses to TT averaged only 25 ng ml−1 over a 12 day culture period in a large study with PBLs (Ref. 44). This anti-TT Ab response has been manipulated but responses remain low45–47. Data often depend on sensitive systems for detecting the small amounts of Ab produced by single cells, reported as Ab-forming cells (AFCs). Recall responses in vivo are initiated in secondary lymphoid tissues where FDCs reside. We reason that the addition of FDCs and ICs to memory lymphocyte cultures helps recreate the in vivo microenvironment and results in IgG responses in the 103 ng ml−1 range24.
…complement proteins C3 and ‘… CR2 are crucial for normal Ab …’ responses… The B-cell coreceptor complex consisting of CD19, CD21 and CD81 (Ref. 48) is crucial to the function of FDCs. It has been known for >25 years that C3 plays a crucial role in the induction of T-dependent antibody responses. However, the mechanisms involved in this complement-mediated enhancement have begun to be understood only recently. Initial experiments indicated that in vivo treatment with cobra venom factor, or other agents that destroy or block expression of C3, resulted in dramatically diminished Ab responses17–19. Subsequent studies showed that injecting soluble CR2 (the chimeric IgG1 containing CD21 used in Fig. 2) also caused a profound immunosuppression, indicating that engagement of CD21 by a C3 fragment was crucial14. Consistent with this observation are results indicating that animals lacking CR2 on B cells are profoundly immunosuppressed20. Furthermore, the level of BCR stimulation required could be reduced by two orders of magnitude by simultaneously stimulating CD19–CD21–CD81, and recent studies using fusion proteins of C3d and hen egg lysozyme demonstrate a 1000–10 000-fold increase in efficiency when the immunogen includes a high density of C3d (Refs 29,30). Many microbial Ags stimulate the alternative complement pathway and it is reasoned that decorating microbial Ags with CD21L would dramatically increase the efficiency of the primary response29,49.
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Glossary B-cell receptor (BCR) This receptor, also known as cell-surface Ig, is responsible for the recognition of specific antigen (Ag) by the B cell. Complement receptor 2 (CR2) A receptor on follicular dendritic cells (FDCs) and B cells that binds the complement component 3 (C3) fragments iC3b, C3d or C3dg, which are covalently bound to Ag–antibody (Ab) complexes. CD21 ligand/complement receptor 2 ligand (CD21L/CR2L) Ligands include iC3b, C3d and C3dg, which decorate the surface of the FDC and the immune complexes (ICs) on FDCs. Follicular dendritic cells (FDCs) A novel cell type with dendritic morphology that traps and retains Ag–Ab complexes and is restricted to the light zone of germinal centers. Fc γ receptor IIB (FCγγRIIB) The Fc portion of IgG molecules in Ag–Ab complexes binds to this receptor on FDCs or B cells. Crosslinking of BCR and B cell FcγRIIB activates immunoreceptor
Acknowledgements This work was supported by National Institutes of Health grants AI-17142 and DE-10703.
The efficiency and potency of the FDC-associated Ag is also remarkable. FDCs retain only pg levels of Ag but a few pgs of FDC–Ag are adequate to elicit potent recall responses13, and we have been able to induce Ab production using <1 pg of FDC-associated Ag. As illustrated in Fig. 1, the presentation of ICs by FDCs minimizes the potential for coligation of BCR and FcγRIIB on B cells and promotes the simultaneous stimulation of BCR and the CD19–CD21–CD81 complex needed for efficient signaling. Interestingly, FDCs are heavily decorated with C3 fragments and
References 1 Qin, D. et al. (2000) Fcγ receptor IIB on follicular dendritic cells regulates the B-cell recall response. J. Immunol. 164, 6268–6275 2 Szakal, A.K. et al. (1983) Transport of immune complexes from the subcapsular sinus to lymph node follicles on the surface of nonphagocytic cells, including cells with dendritic morphology. J. Immunol. 131, 1714–1727 3 MacLennan, I.C.M. and Gray, D. (1986) Antigendriven selection of virgin and memory B cells. Immunol. Rev. 91, 61–85 4 Berek, C. et al. (1991) Maturation of the immune response in germinal centers. Cell 67, 1121–1129 5 Berek, C. and Ziegner, M. (1993) The maturation of the immune response. Immunol. Today 14, 400–404 6 Tew, J.G. et al. (1997) Follicular dendritic cells and presentation of antigen and costimulatory signals to B cells. Immunol. Rev. 156, 39–52 7 Phillips, N.E. and Parker, D.C. (1983) Fc-dependent inhibition of mouse B-cell activation by whole antimu antibodies. J. Immunol. 130, 602–606 8 D’Ambrosio, D. et al. (1995) Recruitment and activation of PTP1C in negative regulation of antigen receptor signaling by FcγRIIB1. Science 268, 293–297 9 Bolland, S. et al. (1998) SHIP modulates immune receptor responses by regulating membrane association of Btk. Immunity 8, 509–516 10 Kosco, M.H. et al. (1988) In vivo-obtained antigen presented by germinal center B cells to T cells in vitro. J. Immunol. 140, 354–360 11 Szakal, A.K. et al. (1988) A novel in vivo follicular dendritic cell-dependent iccosome-mediated mechanism for delivery of antigen to antigenprocessing cells. J. Immunol. 140, 341–353 12 de Vinuesa, C.G. et al. (2000) Germinal centers without T cells. J. Exp. Med. 191, 485–494
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tyrosine-based inhibition motifs (ITIMs) in the B cells and is immunosuppressive. Binding of Fc by this receptor on FDCs minimizes the inhibitory signal to the B cells. Immune complex-coated bodies (iccosomes) These Ag-coated liposome-like particles (0.25–0.38 µm in diameter) are released from FDC dendrites and are rapidly endocytosed by germinal center B cells. This mechanism allows B cells in germinal centers to obtain Ag required for processing and presentation to T cells. Iccosomal Ag Indicates that FDC Ag is not in peptide form displayed on MHC molecules but is an intact protein complexed with specific Ab and attached by Ig Fc and C3 fragments. Immunoreceptor tyrosine-based inhibition motif (ITIM) The tails of some receptors, including FcγRIIB, contain motifs that, when activated, bind phosphatases; these phosphatases inhibit tyrosine activation motifs that promote cell activation (e.g. BCR).
the high concentration of these molecules is probably important to function50. We reason that large amounts of FDC CD21L could be engaged by numerous CD21 molecules on the B cell simultaneously with FDC–Ag–BCR. Given the necessity for FDC Ag to be used efficiently it would follow that blocking the CD21L–receptor system on the FDC, by eliminating C3 or by the use of soluble CD21, would have the profound effects noted. In our opinion, this FDC-based ligand–receptor system is essential to basic immune function and is just beginning to be appreciated.
13 Wu, J. et al. (1996) Follicular dendritic cell (FDC) derived Ag and accessory activity in initiation of memory IgG responses in vitro. J. Immunol. 157, 3404–3411 14 Hebell, T. et al. (1991) Suppression of the immune response by a soluble complement receptor of B lymphocytes. Science 254, 102–105 15 Ahearn, J.M. et al. (1996) Disruption of the Cr2 locus results in a reduction in B-1a cells and in an impaired B-cell response to T-dependent antigen. Immunity 4, 251–262 16 Fischer, M.B. et al. (1996) Regulation of the B-cell response to T-dependent antigens by classical pathway complement. J. Immunol. 157, 549–556 17 Bottger, E.C. et al. (1986) Impaired humoral immune response in complement C3-deficient guinea pigs: absence of secondary antibody response. Eur. J. Immunol. 16, 1231–1235 18 Pepys, M.B. (1974) Role of complement in induction of antibody production in vivo. Effect of cobra factor and other C3-reactive agents on thymus-dependent and thymusindependent antibody responses. J. Exp. Med. 140, 126–145 19 Pepys, M.B. (1972) Role of complement in induction of the allergic response. Nat. New Biol. 237, 157–159 20 Croix, D.A. et al. (1996) Antibody response to a T-dependent antigen requires B-cell expression of complement receptors. J. Exp. Med. 183, 1857–1864 21 Qin, D. et al. (1998) Evidence for an important interaction between a complement-derived CD21 ligand on follicular dendritic cells and CD21 on B cells in the initiation of IgG responses. J. Immunol. 161, 4549–4554 22 Maeda, K. et al. (1992) Murine follicular dendritic cells (FDC) and low affinity Fc-receptors for IgE (FcεRII). J. Immunol. 148, 2340–2347
23 Aubry, J.P. et al. (1992) CD21 is a ligand for CD23 and regulates IgE production. Nature 358, 505–507 24 Fakher, M. et al. (2001) Follicular dendritic cell accessory activity crosses MHC and species barriers. Eur. J. Immunol. 31, 176–185 25 Takai, T. et al. (1996) Augmented humoral and anaphylactic responses in FcγRII-deficient mice. Nature 379, 346–349 26 D’Ambrosio, D. et al. (1996) The SHIP phosphatase becomes associated with FcγRIIB1 and is tyrosine phosphorylated during ‘negative’ signaling. Immunol. Lett. 54, 77–82 27 Chacko, G.W. et al. (1996) Negative signaling in B lymphocytes induces tyrosine phosphorylation of the 145 kDa inositol polyphosphate 5-phosphatase, SHIP. J. Immunol. 157, 2234–2238 28 Lanzavecchia, A. (1990) Receptor-mediated antigen uptake and its effect on antigen presentation to class II-restricted T lymphocytes. Annu. Rev. Immunol. 8, 773–793 29 Dempsey, P.W. et al. (1996) C3d of complement as a molecular adjuvant: bridging innate and acquired immunity. Science 271, 348–350 30 Carter, R.H. and Fearon, D.T. (1992) CD19: lowering the threshold for antigen receptor stimulation of B lymphocytes. Science 256, 105–107 31 Szakal, A.K. et al. (1989) Microanatomy of lymphoid tissue during the induction and maintenance of humoral immune responses: structure–function relationships. Annu. Rev. Immunol. 7, 91–109 32 Tew, J.G. et al. (1990) Follicular dendritic cells as accessory cells. Immunol. Rev. 117, 185–211 33 Tew, J.G. et al. (1992) Germinal centers and antibody production in bone marrow. Immunol. Rev. 126, 1–14
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34 Benner, R. et al. (1981) The bone marrow: the major source of serum immunoglobulins, but still a neglected site of antibody formation. Clin. Exp. Immunol. 46, 1–8 35 Slifka, M.K. et al. (1998) Humoral immunity due to long-lived plasma cells. Immunity 8, 363–372 36 Maruyama, M. et al. (2000) Memory B-cell persistence is independent of persisting immunizing antigen. Nature 407, 636–642 37 Mandel, T.E. et al. (1980) The follicular dendritic cell: long term antigen retention during immunity. Immunol. Rev. 53, 29–59 38 Tew, J.G. et al. (1980) The maintenance and regulation of the humoral immune response: persisting antigen and the role of follicular antigen-binding dendritic cells as accessory cells. Immunol. Rev. 53, 175–201 39 Bystryn, J.C. et al. (1971) A model for the regulation of antibody synthesis by serum antibody. In Progress in Immunology (Amos, B., ed.), pp. 628–636, Academic Press 40 Graf, M.W. and Uhr, J.W. (1969) Regulation of antibody formation by serum antibody. I. Removal
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of specific antibody by means of immunoadsorption. J. Exp. Med. 130, 1175–1186 Bystryn, J.C. et al. (1970) Regulation of antibody formation by serum antibody. II. Removal of specific antibody by means of exchange transfusion. J. Exp. Med. 132, 1279–1287 Qin, D. et al. (1999) Follicular dendritic cells mediated maintenance of primary lymphocyte cultures for long-term analysis of a functional in vitro immune system. J. Immunol. Methods 226, 19–27 Tew, J.G. et al. (1973) The spontaneous induction of anamnestic antibody synthesis in lymph node cell cultures many months after primary immunization. J. Immunol. 111, 416–423 Lum, L.G. and Culbertson, N.J. (1985) The induction and suppression of in vitro IgG antitetanus toxoid antibody synthesis by human lymphocytes stimulated with tetanus toxoid in the absence of in vivo booster immunizations. J. Immunol. 135, 185–191 Tew, J.G. et al. (1987) Fusobacterium nucleatum mediated immunomodulation of the in vitro secondary antibody response to tetanus toxoid and Actinobacillus actinomycetemcomitans. J. Periodontal Res. 22, 506–512
Mac-1+ myelopoiesis induced by CFA: a clue to the paradoxical effects of IFN-γγ in autoimmune disease models Patrick Matthys, Kurt Vermeire and Alfons Billiau The mechanisms accounting for the protective role of endogenous interferon γ (IFN-γγ) in certain murine autoimmune disease models, versus a diseasepromoting role in others, have remained elusive. The protective effect of IFN-γγ might be unique to models that rely on the use of complete Freund’s adjuvant (CFA) and whose pathogenesis is predominantly driven by delayed-type hypersensitivity. In these models, IFN-γγ counteracts disease development by inhibiting CFA-induced proliferation of a pathogenically important Mac-1+ cell population(s). This calls into question our usual conceptualization of the balance between innate and specific immunity in these models, as well as their clinical relevance, particularly when the role of IFN-γγ or related cytokines is considered.
The effect of endogenous interferon γ (IFN-γ) in autoimmune disease constitutes an enigma that compromises the T helper 1 (Th1)/Th2 paradigm as a
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46 Peters, M. and Fauci, A.S. (1983) Selective activation of antigen-specific human B cells in recently immunized individuals by nonspecific factors in the absence of antigen. J. Immunol. 130, 678–680 47 Gerrard, T.L. and Fauci, A.S. (1982) Activation and immunoregulation of antigen-specific human B lymphocyte responses: multifaceted role of the monocyte. J. Immunol. 128, 2367–2372 48 Fearon, D.T. and Carter, R.H. (1995) The CD19–CR2–TAPA-1 complex of B lymphocytes: linking natural to acquired immunity. Annu. Rev. Immunol. 13, 127–149 49 Fearon, D.T. (1998) The complement system and adaptive immunity. Semin. Immunol. 10, 355–361 50 Yamakawa, M. and Imai, Y. (1992) Complement activation in the follicular light zone of human lymphoid tissues. Immunology 76, 378–384 51 Carter, R.H. et al. (1997) Membrane IgM-induced tyrosine phosphorylation of CD19 requires a CD19 domain that mediates association with components of the B cell antigen receptor complex. J. Immunol. 158, 3062–3069 52 Tew, J.G. et al. (1989) The alternative antigen pathway. Immunol. Today 10, 229–232
basis for explaining cytokine-mediated regulation of autoimmune disease. Although it is an unequivocal Th1-type cytokine (for review, see Ref. 1), endogenous IFN-γ acts as a protective factor in experimental autoimmune encephalomyelitis (EAE)2–6 and in the related disease experimental autoimmune uveitis (EAU)7,8. Both are prototype Th1-driven diseases with a predominant delayed-type hypersensitivity (DTH)-mediated tissue involvement9. The protective effect of both endogenous and exogenous IFN-γ in these models counters the well-known Th1-directed immunopotentiating effects of IFN-γ. It also contrasts with the disease-promoting effects of IFN-γ in other autoimmune disease models, such as spontaneously developing lupus in MRL/lpr mice10–13 or insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice14–16. In a third functional category, IFN-γ can either promote or inhibit disease depending on the experimental circumstances, for example, in collagen-induced arthritis (CIA)17–19. Careful study of the pathogenesis of this model has enabled the definition of a distinct pathway by which IFN-γ can counteract Th1-driven autoimmune pathogenesis17. The clue to the enigma appeared to be the use of complete Freund’s adjuvant (CFA) in the induction procedure. CFA and the protective effect of IFN-γγ in CIA
CIA, a model for rheumatoid arthritis (RA) in humans, is classically induced in DBA/1 mice by immunization with collagen type II (CII) in CFA. Knockout mice deficient in the IFN-γ receptor (IFN-γR-KO mice) develop CIA more readily than do their wild-type counterparts, clearly indicating that in this model, as in EAE or EAU, endogenous IFN-γ plays a protective role. However, when mycobacteria
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Follicular dendritic cells: beyond the necessity of T-cell help John G. Tew, Jiuhua Wu, Mohamed Fakher, Andras K. Szakal and Dahui Qin Follicular dendritic cells (FDCs) are potent accessory cells for B cells, but the molecular basis of their activity is not understood. Several important molecules involved in FDC–B-cell interactions are indicated by blocking the ligands and receptors on FDCs and/or B cells. The engagement of CD21 in the B-cell coreceptor complex by complement-derived CD21 ligand on FDCs delivers a crucial signal that dramatically augments the stimulation delivered by the binding of antigen to the B-cell receptor (BCR). The engagement of Fc γ receptor IIB (FcγγRIIB) by the Ig crystallizable fragment (Fc) in antigen–antibody complexes held on FDCs decreases the activation of immunoreceptor tyrosinebased inhibition motifs (ITIMs), mediated by the crosslinking of BCR and FcγγRIIB. Thus, FDCs minimize a negative B-cell signal. In short, these ligand–receptor interactions help to signal to B cells and meet a requirement for B-cell stimulation that goes beyond the necessity of T-cell help.
John G. Tew* Jiuhua Wu Mohamed Fakher Dahui Qin Dept of Microbiology and Immunology, Andras K. Szakal Dept of Anatomy, Division of Immunobiology, Medical College of Virginia, Virginia Commonwealth University, PO Box 980678, Richmond, VA 23298-0678, USA. *e-mail: tew@ hsc.vcu.edu
We propose that FOLLICULAR DENDRITIC CELL (FDC)–Bcell interactions (see Glossary) meet a requirement for B-cell stimulation that goes beyond the necessity of T-cell help. In T-dependent antibody (Ab) responses, the T helper (Th) cells provide CD40 ligand (CD40L) and lymphokines, including chemokines, that function with cytokines produced by accessory cells to stimulate B cells. These molecules are obviously important, but in this article we review data indicating that they are not sufficient for optimal B-cell responses. Direct cell–cell interactions between B cells and FDCs allow the engagement of multiple membrane-bound receptors and ligands that are also important in B-cell signaling. In recall responses, the immunogen is immediately converted into immune complexes (ICs) by Ab persisting from previous immunizations; ICs stimulate potent recall responses if the animal has wild-type FDCs (Ref. 1). In primary responses, naive T and B-cells are apparently initially stimulated in the absence of FDCs bearing specific antigen (Ag)–Ab complexes because some Ab must first be made in order for the Ag–Ab complexes to be formed. However, it should be appreciated that as soon as Ab is produced in a primary response, Ag–Ab complexes are formed and trapped by FDCs (Ref. 2) and Ag–Ab complex–FDC–B-cell interactions play an important role in the maturation and maintenance of humoral immune responses. Specifically, interactions
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between B cells and Ag–Ab complex-bearing FDCs are linked to the formation of germinal centers, somatic mutations and memory cells, and the induction of Ab-forming cells producing large amounts of specific high-affinity Ab (Refs 3–6). In vitro studies indicate that ICs activate the immunosuppressive IMMUNORECEPTOR TYROSINE-BASED INHIBITION MOTIF (ITIM) in B cells7–9. However, in the presence of FDCs, ICs are highly immunogenic. The molecules that confer adjuvant-like accessory activity on IC-bearing FDCs have been the focus of recent research. Multiple receptors and ligands on FDCs and B cells were found to be crucial for FDC accessory activity. These receptors and ligands are illustrated in Fig. 1 and the major experimental data supporting the necessity of these molecules for optimal signaling of memory B cells are listed in Box 1. This discussion will follow the sequence of Box 1, and models involving both murine and human leukocytes will be considered.
…follicular dendritic cell–B-cell ‘… interactions meet a requirement for B-cell stimulation that goes beyond the necessity of T-cell help.’ The inhibition of recall responses: blockade of BCR or MHC class II on B cells and CD40L on T cells
The requirement for these receptors and ligands in T-cell–B-cell interactions is well known and has been confirmed using IC-bearing FDCs in in vitro studies. For example, the requirement for MHC class II was demonstrated using IMMUNE COMPLEX-COATED BODIES (iccosomes) derived from FDCs as the source of immunogen and normal FDCs for costimulatory activity. ICCOSOMAL AG, as illustrated in Fig. 1, is released by FDCs and the iccosomes are endocytosed by specific B cells. These cells process and present the Ag to appropriate T cells, which then provide the B cells with the necessary help10,11. However, we acknowledge that germinal centers with highaffinity B cells can form without T cells when Ags such as (4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll (a classical T-independent Ag) are used and B-CELL RECEPTORS (BCRs) are extensively crosslinked12. The interpretation that T cells are crucial for the simple protein Ags we typically study is supported by the observation that the murine anti-ovalbumin (antiOVA) response is inhibited by ≈95% if appropriate anti-class II monoclonal Ab (mAb) is added to the cultures. For example, the anti-OVA Ab titer induced by OVA-bearing iccosomes in the presence of FDCs from normal animals was 1370 ± 198 ng ml−1 in the presence of an isotype control mAb, in comparison with only 51 ± 12 ng ml−1 when cultures were treated with an appropriate anti-MHC class II mAb (Ref. 13).
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Box 1. Multiple receptor–ligand interactions are necessary for optimal signaling of memory B cells* T cell
Ag
TCR
FDC BCR
CD40L
MHC II
CD19
CD40
B cell
FcγRIIB CD21L
CD21
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Iccosome TRENDS in Immunology
Fig. 1. Model illustrating the important receptors and ligands used by T cells and follicular dendritic cells (FDCs) in signaling to B cells. The requirement for B-cell MHC class II to present antigen (Ag)-derived peptides as ligands for the T-cell receptor (TCR) is well known, as is the involvement of CD40 on the B cell interacting with T cell CD40 ligand (CD40L). The FDC–B cell interaction delivers a primary signal through FDC Ag–B-cell receptor (BCR) interaction and a cosignal through B cell CD21 interacting with FDC CD21 ligand (CD21L). The events of a recall response are summarized as follows. (1) FDCs trap Ag–antibody (Ab) complexes and provide intact Ag for interaction with BCRs on germinal center (GC) B cells; this Ag–BCR interaction provides a positive signal for B-cell activation and differentiation. (2) FDCs provide a complement-derived CD21L for B cell CD21; its interaction with the CD21–CD19–CD81 complex delivers a positive cosignal for B-cell activation and differentiation. Coligation of BCR and CD21 (as illustrated here with a single molecule of Ag) facilitates association of the two receptors, and the cytoplasmic tail of CD19 is phosphorylated by a tyrosine kinase associated with the BCR complex51. The arrow pointing from the BCR to CD19 indicates this known interaction. (3) A high density of Fc γ receptor IIB (FcγRIIB) on FDCs binds Ig Fc in the Ag–Ab complex and consequently the signal delivered by the immunoreceptor tyrosine-based inhibition motif (ITIM) in the B cells might be blocked. This inhibitory signal is initiated by Ag–Ab complexes crosslinking BCR and FcγRIIB on B cells. Note that BCR is not crosslinked with B cell FcγRIIB in the model and thus a high concentration of FcγRIIB on FDCs minimizes a negative signal to the B cell. (4) In addition, FDCs provide immune complex-coated bodies (iccosomes), which are readily taken up by B cells52. The iccosome membrane is derived from FDC membranes that have Ag, CD21L and Ig Fc attached. Iccosomes bind tightly to B cells and are rapidly endocytosed11. We reason that binding of BCR, complement receptor 2 (CR2) and possibly B cell Fc receptor (FcR) to the iccosomal Ag–CD21L–Ig Fc complex is crucial to the process of endocytosis. The B cells process this FDC-derived Ag, present it and thus obtain T-cell help.
Blocking CD21L on the FDCs inhibits FDC accessory activity
To determine whether CD21 LIGAND/COMPLEMENT RECEPTOR 2 LIGAND (CD21L/CR2L) on FDCs plays a role in enhancing Ab responses, [COMPLEMENT RECEPTOR 2 (CR2)/CD21]2-IgG1, a soluble chimeric molecule of mouse IgG1 heavy chains and CR2 (Ref. 14), was used to block CR2L on FDCs. Binding of this soluble receptor to FDC CD21L should inhibit the binding of the CD21L to its receptor on the B cell, thus blocking FDC activity. As the dose of soluble CR2 increased, the anti-OVA IgG response was reduced to <10% of the isotype control Ab-treated FDCs (Fig. 2). Similar results were seen using human serum albumin (HSA) as the model Ag and these data are consistent with in vivo data showing that this soluble receptor dramatically inhibits Ab responses14. These in vitro results, indicating the importance of CD21L on FDCs, http://immunology.trends.com
• Blockade of B-cell receptor (BCR) or MHC class II on B cells, or CD40 ligand (CD40L) on T cells with specific antibodies inhibits recall responses induced by immune complex (IC)-bearing follicular dendritic cells (FDCs)a,b,c (J. Wu et al., unpublished). • Blockade of FDC CD21 ligand (CD21L) inhibits recall responses induced by IC-bearing FDCs (Refs d,e). • FDCs lacking complement-derived CD21L lack normal accessory activitye. • Blockade of B cell CD21 to inhibit the binding of FDC CD21L blocks the induction of recall responses by IC-bearing FDCs (Ref. e). • FDCs lacking Fc γ receptor IIB (FcγRIIB) have markedly reduced accessory activity. These FDCs cannot bind Ig Fc in ICs and allow B cell FcγRIIB and BCR to be coligatedf. • None of the important receptors or ligands on FDCs is MHC-restricted; thus FDC accessory activity crosses both MHC and species barriersg. • In the presence of FDCs, ICs are potent and efficient immunogensa,e,g (Fig. 4). *The crucial receptors and ligands are illustrated in Fig. 1. Memory B cells and T cells were obtained from blood or secondary lymphoid tissues of previously immunized and boosted humans or mice with high serum titers of IgG specific for the recall antigen. These memory cells give robust recall responses, whereas cells from nonimmunized controls fail to responda.
References a Wu, J. et al. (1996) Follicular dendritic cell (FDC) derived Ag and accessory activity in initiation of memory IgG responses in vitro. J. Immunol. 157, 3404–3411 b Gronowicz, E. and Coutinho, A. (1976) Hapten-induced B-cell paralysis. II. Evidence for trivial mechanisms of tolerance. Eur. J. Immunol. 5, 413–420 c Foy, T.M. et al. (1993) In vivo CD40–gp39 interactions are essential for thymus-dependent humoral immunity. II. Prolonged suppression of the humoral immune response by an antibody to the ligand for CD40, gp39. J. Exp. Med. 178, 1567–1575 d Hebell, T. et al. (1991) Suppression of the immune response by a soluble complement receptor of B lymphocytes. Science 254, 102–105 e Qin, D. et al. (1998) Evidence for an important interaction between a complement-derived CD21 ligand on follicular dendritic cells and CD21 on B cells in the initiation of IgG responses. J. Immunol. 161, 4549–4554 f Qin, D. et al. (2000) Fcγ receptor IIB on follicular dendritic cells regulates the B-cell recall response. J. Immunol. 164, 6268–6275 g Fakher, M. et al. (2001) Follicular dendritic cell accessory activity crosses MHC and species barriers. Eur. J. Immunol. 31, 176–185
also confirm and extend in vivo studies showing that complement protein C3 and CR2 are crucial for normal Ab responses14–20. It appears that important CD21L–CD21 interactions probably occur in germinal centers where FDCs and B cells interact.
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LyOVA + FDCOVA + isotype-matched control Ab LyOVA + FDCOVA + (CR2)2-IgG1 14 000
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Fig. 2. The inhibitory effect of soluble complement receptor 2 (CR2/CD21) on anti-ovalbumin (anti-OVA) IgG production in vitro. OVA-primed lymphocytes (LyOVA) were isolated from OVA-immune mouse lymph nodes. OVA-bearing follicular dendritic cells (FDCOVA) were isolated from the draining lymph nodes of OVA-immune mice three days after immune challenge. Lymphocytes were cultured in a 96-well culture plate (3 × 105 well–1) with FDCOVA (5 × 104 well–1). Different doses of soluble CR2 [(CR2)2-IgG1] or an isotype-matched control antibody (Ab) were added to LyOVA + FDCOVA cultures. The culture supernatant fluids were harvested on day seven and new culture medium was added. Anti-OVA IgG in the supernatant fluid was measured by enzyme-linked immunosorbent assay (ELISA) on day 14. Controls, including LyOVA cultured alone (no FDCs added) and FDCOVA cultured alone, produced <10 ng of anti-OVA IgG ml–1. (Reproduced, with permission, from Ref. 21, 1998, The American Association of Immunologists.)
FDCs lacking complement-derived CD21L lack normal accessory activity
We reasoned that FDCs isolated from C3 knockout mice16 would not bear CD21Ls generated from C3 fragments (iC3b, C3d or C3dg) and would not be able to enhance humoral immune responses as effectively as FDCs from wild-type mice. To test this prediction, FDCs were isolated from the lymph nodes of C3−/− mice and used in lymphocyte cultures. The ability of FDCs from C3−/− mice to enhance IgG responses was reduced by ≈80% (Ref. 21). These data support the concept that C3-derived CD21L is a major stimulator of CR2, but other potential CD21Ls are present on FDCs and could also be involved22,23. Blocking CD21 on the B cell inhibits FDC accessory activity
C3-derived CD21Ls on FDCs might enhance Ab responses by engaging CD21 on B cells; in this case, the FDC-derived cosignal should be blocked if CD21 on the B cell is blocked. To test this prediction, mAbs against CD21 (clones 7G6 against murine CD21 and B-ly4 against human CD21) were used to mask CD21 on B cells, and Ab responses were monitored after stimulation with appropriate ICs and FDCs. Use of http://immunology.trends.com
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anti-CD21 mAb suppressed 80–95% of the specific Ab expression induced by HSA or OVA in the murine system and tetanus toxoid (TT) in the human system21,24. The human model allowed us to set up the experiment such that the murine anti-CD21 could only bind CD21 on human B cells and not the CD21 on murine FDCs, thus eliminating concern about an effect of the mAb binding to FDC CD21 (Ref. 24). The anti-human CD21 mAb also does not crossreact with CD35, as the 7G6 mAb does, thus eliminating concern about the consequences of binding CD35. We also reasoned that FDC accessory activity would be reduced on B cells lacking CD21 because FDC CD21L could not exert its costimulatory effect. Not surprisingly, CD21−/− mice have a profound deficit in their ability to mount T-dependent Ab responses and it is not possible to study typical recall responses in these mice20. Nevertheless, a dramatic stimulatory effect of FDC CD21L can be obtained when the primary signal is given by a polyclonal B-cell activator; lipopolysaccharide (LPS) works well in the murine system and poke weed mitogen in the human system21,24. B cells from CD21−/− mice respond to LPS but the addition of FDCs has very little costimulatory effect. This contrasts with the dramatic effect of adding FDCs bearing CD21L to LPS-stimulated wild-type B cells21. These results further reinforce the importance of CD21L–CD21 in FDC–B-cell interactions. FDCs lacking FcγγRIIB have markedly reduced accessory activity
FDCs label intensely for FC γ RECEPTOR IIB (FCγRIIB) in reactive follicles containing germinal centers. In fact, when compared with FDCs, the B cells in the germinal centers and follicular mantel appear negative for FcγRIIB, indicating that the concentration of FcγRIIB on FDCs is much higher than on B cells1,22. As illustrated in Fig. 1, FDCs bearing FcγRIIB should bind Ig crystallizable fragment (Fc) in ICs and minimize the coligation of B-cell FcγRIIB and BCR by ICs, thus interfering with the delivery of a negative B-cell signal through activation of the ITIM. Indeed, ICs stimulate potent recall responses in the presence of FDCs (Ref. 21). We proposed that FDCs from FcγRIIB−/− mice, which will still bind ICs through complement receptors, would not provide normal FDC accessory activity to wildtype B cells because the negative regulation could still be delivered by free Ig Fc in the ICs. To test this, we compared the accessory activity of FDCs from wildtype and FcγRIIB−/− mice (Fig. 3). Substitution of knockout for wild-type FDCs reduced the recall response by ≈90%. This was confirmed in an in vivo study where memory T and B-cells from wild-type mice were adoptively transferred into FcγRIIB−/− mice, where they would depend on accessory activity from FDCs lacking FcγRIIB. Again, recall responses in these mice were profoundly suppressed1. However, when B cells from FcγRIIB−/− mice are used, the FcγRIIBs on FDCs do not appear to be crucial and
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were compared, as well as human and murine FDCs. Human and murine FDCs converted ICs into potent immunogens and MHC barriers did not restrict this activity. Furthermore, human FDCs worked with murine lymphocytes and murine FDCs worked with human lymphocytes24. In short, there was no lack of activity (specific Ab increased from background levels to thousands of ng ml−1) when crossing either MHC or species barriers24.
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Fig. 3. Follicular dendritic cell (FDC) Fc γ receptor IIB (FcγRIIB) is important for FDCs to convert immune complexes (ICs) into an effective immunogen. FDCs were cultured with memory T or B-cells (Ly) from ovalbumin (OVA)-immune mice in the presence of OVA–anti-OVA ICs, and their ability to convert the ICs into an effective immunogen was measured. The ICs (50 ng of OVA) were formed near equivalence. Cultures were washed on day seven to remove soluble ICs and anti-OVA IgG was measured on day 14. The FDCs were obtained from FcγRIIB−/− and wild-type mice. Controls included normal memory T or B-lymphocytes cultured alone, FDCs cultured alone (FDC-FcγRIIB+/+ and FDC-FcγRIIB−/−), and memory T or B-cells plus OVA–anti-OVA ICs (no FDCs); all produced <10 ng of anti-OVA IgG ml–1. (Reproduced, with permission, from Ref. 1, 2000, The American Association of Immunologists.)
potent recall responses are observed25. This is reasonable, as these B cells lack the potential for coligation of BCR and FcγRIIB and there is therefore no need for FDCs to minimize this effect. According to our model, FDCs bind Ig Fc and minimize the crosslinking of BCR and FcγRIIB, and the activation of ITIM in B cells. Activation of ITIM in B cells results in phosphorylation of Src homology 2 domain-containing inositol polyphosphate 5′-phosphatase (SHIP)26. Thus, we reasoned that the presence of FDCs should reduce SHIP phosphorylation in B cells. Data supportive of this hypothesis was obtained using anti-Ig Ab, a wellknown inducer of SHIP phosphorylation, as an analog for ICs (Ref. 27). As predicted, the addition of wildtype FDCs plus anti-Ig Ab to B-cell cultures reduced the level of anti-Ig Ab-induced SHIP phosphorylation compared with control B cells in the absence of FDCs. In marked contrast, FDCs from FcγRIIB−/− mice were unable to reduce the level of anti-Ig Ab-induced SHIP phosphorylation in B cells (J. Wu et al., unpublished). FDC accessory activity crosses MHC and species barriers
The only ligand–receptor interaction shown in Fig. 1 that is MHC-restricted is the interaction between the T cell and the B cell involving T-cell receptor (TCR) and class II MHC. Thus, FDC accessory activity should cross MHC and even species barriers. Murine FDCs expressing three different MHC haplotypes http://immunology.trends.com
Clearly, B cells specifically trap free Ag using BCRs, and Ag-presentation experiments indicate that B cells process free Ag very efficiently28. As discussed previously, free ICs inhibit B-cell function and are poorly immunogenic in the absence of FDCs. The amount of IC trapped by FDCs is in the pg–low ng range in vivo and if these ICs are to have significant immunogenicity they must be used efficiently. The ICs on the FDCs are converted into iccosomes, which are rapidly endocytosed and processed by B cells. The uptake and processing of iccosomal Ag by B cells prompted us to postulate that ICs would be a potent form of immunogen13, perhaps as potent as free Ag in stimulating B cells. A comparison between free Ag and ICs was conducted using human peripheral-blood lymphocytes (PBLs) responding to TT. TT–anti-TT ICs elicit no anti-TT Ab response in PBLs from immune subjects in the absence of FDCs; however, in the presence of FDCs the TT–anti-TT ICs elicited >103 ng of anti-TT Ab. By contrast, free TT elicited only a few ng of anti-TT Ab with PBLs; the addition of FDCs did increase the response but to a lesser extent than with ICs. This prompted dose–response studies comparing free TT with TT–anti-TT ICs in the presence of FDCs. A representative experiment is shown in Fig. 4. ICs were approximately tenfold more stimulatory than free Ag, with 0.5 ng of TT–anti-TT ICs giving a detectable immune response. Clearly, ICs are potent, efficient immunogens in the presence of FDCs. This might relate to the presentation of Ag and CD21L in a form that allows for crosslinking of BCR and CD21 (Fig. 1). B-cell CD21 delivers a powerful costimulatory signal when coligated with BCR (Refs 29,30) and this might explain the potency and efficiency of ICs on FDCs (Ref. 13). Concluding remarks Ag in germinal centers and Ab production in the bone marrow
Immunogens are converted into ICs as soon as natural Ab or Ab induced as a consequence of infection is encountered. Ag-transporting cells (ATCs) trap ICs and traffic to the draining lymph nodes within minutes, and the ICs are present on FDCs in the follicles within five hours2. Recall responses develop in germinal centers in response to ICs trapped on FDCs (Refs 6,31–33). As reviewed here, crucial
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inhibitor38,43. In short, we suggest that severe bleeding reduces the feedback inhibitor and allows Ag persisting on FDCs to elicit a recall response, resulting in an Ab rebound and a burst of new memory B cells. Thus, although persisting Ag might not be required for the long-term maintenance of memory B cells or serum Ab levels it could still help regulate and modulate these responses. FDC CD21L and the efficiency and potency of the specific immunogen
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Fig. 4. Immune complexes (ICs) are as efficient or more efficient than free antigen (Ag) in stimulating recall responses in the presence of follicular dendritic cells (FDCs). Peripheral blood lymphocytes (PBLs) were isolated from a tetanus toxoid (TT)-immune volunteer in the laboratory. FDCs were isolated from the lymph nodes of normal BALB/c mice. Addition of TT or TT–anti-TT ICs to PBLs (1.0 × 106 ml−1) plus murine FDCs (0.2 × 106 ml−1) stimulated a recall response. Controls included PBLs cultured alone, FDCs cultured alone, PBLs plus TT (no FDCs) and PBLs plus TT–anti-TT ICs (no FDCs); all produced <15 ng of anti-TT IgG ml−1 (M. Fakher et al., unpublished).
ligand–receptor reactions involved in the interaction between FDCs and B cells include Ag–BCR, CD21L–CD21 and Fc–FcR. Within three days of a secondary challenge, plasmablasts emerge from these germinal centers and home to the bone marrow, where they become mature plasma cells and produce the vast majority of the Ig in recall responses33,34. FDCs and the regulation and maintenance of recall responses
Plasma cells persist in the bone marrow and are capable of maintaining serum Ab levels in the absence of persisting Ag on FDCs (Ref. 35). Similarly, memory B cells can persist without restimulation by Ag on FDCs (Ref. 36). This raises the issue of the role of the Ag that does persist on FDCs for months or even years37. We suspect that this Ag does modulate recall responses. For example, removing draining lymph nodes that contain the persisting Ag, does reduce the level of persisting Ab by ≈50%, whereas removing nondraining lymph nodes has no effect38. However, consistent with recent data, removal of the FDCs and associated persisting Ag clearly does not ablate the long-term Ab response. Furthermore, Uhr and colleagues demonstrated that severe bleeding of immune animals can elicit a dramatic rebound in specific serum Ab titer, which exceeds the prebleeding level, and that this Ab response is subject to Ab feedback inhibition39–41. FDCs bearing persisting Ag can elicit just such a rebound phenomenon in vitro42 when cultured in the absence of high concentrations of specific Ab. This ‘spontaneous response’ can be ablated by the addition of specific Ab as a feedback http://immunology.trends.com
It is well known that recall responses can be studied in vitro by simply adding Ag to cultures containing T cells and memory B cells, but these responses are seldom impressive. For example, recall responses to TT averaged only 25 ng ml−1 over a 12 day culture period in a large study with PBLs (Ref. 44). This anti-TT Ab response has been manipulated but responses remain low45–47. Data often depend on sensitive systems for detecting the small amounts of Ab produced by single cells, reported as Ab-forming cells (AFCs). Recall responses in vivo are initiated in secondary lymphoid tissues where FDCs reside. We reason that the addition of FDCs and ICs to memory lymphocyte cultures helps recreate the in vivo microenvironment and results in IgG responses in the 103 ng ml−1 range24.
…complement proteins C3 and ‘… CR2 are crucial for normal Ab …’ responses… The B-cell coreceptor complex consisting of CD19, CD21 and CD81 (Ref. 48) is crucial to the function of FDCs. It has been known for >25 years that C3 plays a crucial role in the induction of T-dependent antibody responses. However, the mechanisms involved in this complement-mediated enhancement have begun to be understood only recently. Initial experiments indicated that in vivo treatment with cobra venom factor, or other agents that destroy or block expression of C3, resulted in dramatically diminished Ab responses17–19. Subsequent studies showed that injecting soluble CR2 (the chimeric IgG1 containing CD21 used in Fig. 2) also caused a profound immunosuppression, indicating that engagement of CD21 by a C3 fragment was crucial14. Consistent with this observation are results indicating that animals lacking CR2 on B cells are profoundly immunosuppressed20. Furthermore, the level of BCR stimulation required could be reduced by two orders of magnitude by simultaneously stimulating CD19–CD21–CD81, and recent studies using fusion proteins of C3d and hen egg lysozyme demonstrate a 1000–10 000-fold increase in efficiency when the immunogen includes a high density of C3d (Refs 29,30). Many microbial Ags stimulate the alternative complement pathway and it is reasoned that decorating microbial Ags with CD21L would dramatically increase the efficiency of the primary response29,49.
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Glossary B-cell receptor (BCR) This receptor, also known as cell-surface Ig, is responsible for the recognition of specific antigen (Ag) by the B cell. Complement receptor 2 (CR2) A receptor on follicular dendritic cells (FDCs) and B cells that binds the complement component 3 (C3) fragments iC3b, C3d or C3dg, which are covalently bound to Ag–antibody (Ab) complexes. CD21 ligand/complement receptor 2 ligand (CD21L/CR2L) Ligands include iC3b, C3d and C3dg, which decorate the surface of the FDC and the immune complexes (ICs) on FDCs. Follicular dendritic cells (FDCs) A novel cell type with dendritic morphology that traps and retains Ag–Ab complexes and is restricted to the light zone of germinal centers. Fc γ receptor IIB (FCγγRIIB) The Fc portion of IgG molecules in Ag–Ab complexes binds to this receptor on FDCs or B cells. Crosslinking of BCR and B cell FcγRIIB activates immunoreceptor
Acknowledgements This work was supported by National Institutes of Health grants AI-17142 and DE-10703.
The efficiency and potency of the FDC-associated Ag is also remarkable. FDCs retain only pg levels of Ag but a few pgs of FDC–Ag are adequate to elicit potent recall responses13, and we have been able to induce Ab production using <1 pg of FDC-associated Ag. As illustrated in Fig. 1, the presentation of ICs by FDCs minimizes the potential for coligation of BCR and FcγRIIB on B cells and promotes the simultaneous stimulation of BCR and the CD19–CD21–CD81 complex needed for efficient signaling. Interestingly, FDCs are heavily decorated with C3 fragments and
References 1 Qin, D. et al. (2000) Fcγ receptor IIB on follicular dendritic cells regulates the B-cell recall response. J. Immunol. 164, 6268–6275 2 Szakal, A.K. et al. (1983) Transport of immune complexes from the subcapsular sinus to lymph node follicles on the surface of nonphagocytic cells, including cells with dendritic morphology. J. Immunol. 131, 1714–1727 3 MacLennan, I.C.M. and Gray, D. (1986) Antigendriven selection of virgin and memory B cells. Immunol. Rev. 91, 61–85 4 Berek, C. et al. (1991) Maturation of the immune response in germinal centers. Cell 67, 1121–1129 5 Berek, C. and Ziegner, M. (1993) The maturation of the immune response. Immunol. Today 14, 400–404 6 Tew, J.G. et al. (1997) Follicular dendritic cells and presentation of antigen and costimulatory signals to B cells. Immunol. Rev. 156, 39–52 7 Phillips, N.E. and Parker, D.C. (1983) Fc-dependent inhibition of mouse B-cell activation by whole antimu antibodies. J. Immunol. 130, 602–606 8 D’Ambrosio, D. et al. (1995) Recruitment and activation of PTP1C in negative regulation of antigen receptor signaling by FcγRIIB1. Science 268, 293–297 9 Bolland, S. et al. (1998) SHIP modulates immune receptor responses by regulating membrane association of Btk. Immunity 8, 509–516 10 Kosco, M.H. et al. (1988) In vivo-obtained antigen presented by germinal center B cells to T cells in vitro. J. Immunol. 140, 354–360 11 Szakal, A.K. et al. (1988) A novel in vivo follicular dendritic cell-dependent iccosome-mediated mechanism for delivery of antigen to antigenprocessing cells. J. Immunol. 140, 341–353 12 de Vinuesa, C.G. et al. (2000) Germinal centers without T cells. J. Exp. Med. 191, 485–494
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tyrosine-based inhibition motifs (ITIMs) in the B cells and is immunosuppressive. Binding of Fc by this receptor on FDCs minimizes the inhibitory signal to the B cells. Immune complex-coated bodies (iccosomes) These Ag-coated liposome-like particles (0.25–0.38 µm in diameter) are released from FDC dendrites and are rapidly endocytosed by germinal center B cells. This mechanism allows B cells in germinal centers to obtain Ag required for processing and presentation to T cells. Iccosomal Ag Indicates that FDC Ag is not in peptide form displayed on MHC molecules but is an intact protein complexed with specific Ab and attached by Ig Fc and C3 fragments. Immunoreceptor tyrosine-based inhibition motif (ITIM) The tails of some receptors, including FcγRIIB, contain motifs that, when activated, bind phosphatases; these phosphatases inhibit tyrosine activation motifs that promote cell activation (e.g. BCR).
the high concentration of these molecules is probably important to function50. We reason that large amounts of FDC CD21L could be engaged by numerous CD21 molecules on the B cell simultaneously with FDC–Ag–BCR. Given the necessity for FDC Ag to be used efficiently it would follow that blocking the CD21L–receptor system on the FDC, by eliminating C3 or by the use of soluble CD21, would have the profound effects noted. In our opinion, this FDC-based ligand–receptor system is essential to basic immune function and is just beginning to be appreciated.
13 Wu, J. et al. (1996) Follicular dendritic cell (FDC) derived Ag and accessory activity in initiation of memory IgG responses in vitro. J. Immunol. 157, 3404–3411 14 Hebell, T. et al. (1991) Suppression of the immune response by a soluble complement receptor of B lymphocytes. Science 254, 102–105 15 Ahearn, J.M. et al. (1996) Disruption of the Cr2 locus results in a reduction in B-1a cells and in an impaired B-cell response to T-dependent antigen. Immunity 4, 251–262 16 Fischer, M.B. et al. (1996) Regulation of the B-cell response to T-dependent antigens by classical pathway complement. J. Immunol. 157, 549–556 17 Bottger, E.C. et al. (1986) Impaired humoral immune response in complement C3-deficient guinea pigs: absence of secondary antibody response. Eur. J. Immunol. 16, 1231–1235 18 Pepys, M.B. (1974) Role of complement in induction of antibody production in vivo. Effect of cobra factor and other C3-reactive agents on thymus-dependent and thymusindependent antibody responses. J. Exp. Med. 140, 126–145 19 Pepys, M.B. (1972) Role of complement in induction of the allergic response. Nat. New Biol. 237, 157–159 20 Croix, D.A. et al. (1996) Antibody response to a T-dependent antigen requires B-cell expression of complement receptors. J. Exp. Med. 183, 1857–1864 21 Qin, D. et al. (1998) Evidence for an important interaction between a complement-derived CD21 ligand on follicular dendritic cells and CD21 on B cells in the initiation of IgG responses. J. Immunol. 161, 4549–4554 22 Maeda, K. et al. (1992) Murine follicular dendritic cells (FDC) and low affinity Fc-receptors for IgE (FcεRII). J. Immunol. 148, 2340–2347
23 Aubry, J.P. et al. (1992) CD21 is a ligand for CD23 and regulates IgE production. Nature 358, 505–507 24 Fakher, M. et al. (2001) Follicular dendritic cell accessory activity crosses MHC and species barriers. Eur. J. Immunol. 31, 176–185 25 Takai, T. et al. (1996) Augmented humoral and anaphylactic responses in FcγRII-deficient mice. Nature 379, 346–349 26 D’Ambrosio, D. et al. (1996) The SHIP phosphatase becomes associated with FcγRIIB1 and is tyrosine phosphorylated during ‘negative’ signaling. Immunol. Lett. 54, 77–82 27 Chacko, G.W. et al. (1996) Negative signaling in B lymphocytes induces tyrosine phosphorylation of the 145 kDa inositol polyphosphate 5-phosphatase, SHIP. J. Immunol. 157, 2234–2238 28 Lanzavecchia, A. (1990) Receptor-mediated antigen uptake and its effect on antigen presentation to class II-restricted T lymphocytes. Annu. Rev. Immunol. 8, 773–793 29 Dempsey, P.W. et al. (1996) C3d of complement as a molecular adjuvant: bridging innate and acquired immunity. Science 271, 348–350 30 Carter, R.H. and Fearon, D.T. (1992) CD19: lowering the threshold for antigen receptor stimulation of B lymphocytes. Science 256, 105–107 31 Szakal, A.K. et al. (1989) Microanatomy of lymphoid tissue during the induction and maintenance of humoral immune responses: structure–function relationships. Annu. Rev. Immunol. 7, 91–109 32 Tew, J.G. et al. (1990) Follicular dendritic cells as accessory cells. Immunol. Rev. 117, 185–211 33 Tew, J.G. et al. (1992) Germinal centers and antibody production in bone marrow. Immunol. Rev. 126, 1–14
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34 Benner, R. et al. (1981) The bone marrow: the major source of serum immunoglobulins, but still a neglected site of antibody formation. Clin. Exp. Immunol. 46, 1–8 35 Slifka, M.K. et al. (1998) Humoral immunity due to long-lived plasma cells. Immunity 8, 363–372 36 Maruyama, M. et al. (2000) Memory B-cell persistence is independent of persisting immunizing antigen. Nature 407, 636–642 37 Mandel, T.E. et al. (1980) The follicular dendritic cell: long term antigen retention during immunity. Immunol. Rev. 53, 29–59 38 Tew, J.G. et al. (1980) The maintenance and regulation of the humoral immune response: persisting antigen and the role of follicular antigen-binding dendritic cells as accessory cells. Immunol. Rev. 53, 175–201 39 Bystryn, J.C. et al. (1971) A model for the regulation of antibody synthesis by serum antibody. In Progress in Immunology (Amos, B., ed.), pp. 628–636, Academic Press 40 Graf, M.W. and Uhr, J.W. (1969) Regulation of antibody formation by serum antibody. I. Removal
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of specific antibody by means of immunoadsorption. J. Exp. Med. 130, 1175–1186 Bystryn, J.C. et al. (1970) Regulation of antibody formation by serum antibody. II. Removal of specific antibody by means of exchange transfusion. J. Exp. Med. 132, 1279–1287 Qin, D. et al. (1999) Follicular dendritic cells mediated maintenance of primary lymphocyte cultures for long-term analysis of a functional in vitro immune system. J. Immunol. Methods 226, 19–27 Tew, J.G. et al. (1973) The spontaneous induction of anamnestic antibody synthesis in lymph node cell cultures many months after primary immunization. J. Immunol. 111, 416–423 Lum, L.G. and Culbertson, N.J. (1985) The induction and suppression of in vitro IgG antitetanus toxoid antibody synthesis by human lymphocytes stimulated with tetanus toxoid in the absence of in vivo booster immunizations. J. Immunol. 135, 185–191 Tew, J.G. et al. (1987) Fusobacterium nucleatum mediated immunomodulation of the in vitro secondary antibody response to tetanus toxoid and Actinobacillus actinomycetemcomitans. J. Periodontal Res. 22, 506–512
Mac-1+ myelopoiesis induced by CFA: a clue to the paradoxical effects of IFN-γγ in autoimmune disease models Patrick Matthys, Kurt Vermeire and Alfons Billiau The mechanisms accounting for the protective role of endogenous interferon γ (IFN-γγ) in certain murine autoimmune disease models, versus a diseasepromoting role in others, have remained elusive. The protective effect of IFN-γγ might be unique to models that rely on the use of complete Freund’s adjuvant (CFA) and whose pathogenesis is predominantly driven by delayed-type hypersensitivity. In these models, IFN-γγ counteracts disease development by inhibiting CFA-induced proliferation of a pathogenically important Mac-1+ cell population(s). This calls into question our usual conceptualization of the balance between innate and specific immunity in these models, as well as their clinical relevance, particularly when the role of IFN-γγ or related cytokines is considered.
The effect of endogenous interferon γ (IFN-γ) in autoimmune disease constitutes an enigma that compromises the T helper 1 (Th1)/Th2 paradigm as a
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46 Peters, M. and Fauci, A.S. (1983) Selective activation of antigen-specific human B cells in recently immunized individuals by nonspecific factors in the absence of antigen. J. Immunol. 130, 678–680 47 Gerrard, T.L. and Fauci, A.S. (1982) Activation and immunoregulation of antigen-specific human B lymphocyte responses: multifaceted role of the monocyte. J. Immunol. 128, 2367–2372 48 Fearon, D.T. and Carter, R.H. (1995) The CD19–CR2–TAPA-1 complex of B lymphocytes: linking natural to acquired immunity. Annu. Rev. Immunol. 13, 127–149 49 Fearon, D.T. (1998) The complement system and adaptive immunity. Semin. Immunol. 10, 355–361 50 Yamakawa, M. and Imai, Y. (1992) Complement activation in the follicular light zone of human lymphoid tissues. Immunology 76, 378–384 51 Carter, R.H. et al. (1997) Membrane IgM-induced tyrosine phosphorylation of CD19 requires a CD19 domain that mediates association with components of the B cell antigen receptor complex. J. Immunol. 158, 3062–3069 52 Tew, J.G. et al. (1989) The alternative antigen pathway. Immunol. Today 10, 229–232
basis for explaining cytokine-mediated regulation of autoimmune disease. Although it is an unequivocal Th1-type cytokine (for review, see Ref. 1), endogenous IFN-γ acts as a protective factor in experimental autoimmune encephalomyelitis (EAE)2–6 and in the related disease experimental autoimmune uveitis (EAU)7,8. Both are prototype Th1-driven diseases with a predominant delayed-type hypersensitivity (DTH)-mediated tissue involvement9. The protective effect of both endogenous and exogenous IFN-γ in these models counters the well-known Th1-directed immunopotentiating effects of IFN-γ. It also contrasts with the disease-promoting effects of IFN-γ in other autoimmune disease models, such as spontaneously developing lupus in MRL/lpr mice10–13 or insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice14–16. In a third functional category, IFN-γ can either promote or inhibit disease depending on the experimental circumstances, for example, in collagen-induced arthritis (CIA)17–19. Careful study of the pathogenesis of this model has enabled the definition of a distinct pathway by which IFN-γ can counteract Th1-driven autoimmune pathogenesis17. The clue to the enigma appeared to be the use of complete Freund’s adjuvant (CFA) in the induction procedure. CFA and the protective effect of IFN-γγ in CIA
CIA, a model for rheumatoid arthritis (RA) in humans, is classically induced in DBA/1 mice by immunization with collagen type II (CII) in CFA. Knockout mice deficient in the IFN-γ receptor (IFN-γR-KO mice) develop CIA more readily than do their wild-type counterparts, clearly indicating that in this model, as in EAE or EAU, endogenous IFN-γ plays a protective role. However, when mycobacteria
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