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Antibody Persistence Enhanced Affinity Maturation but Impaired Humoral Immune Responses in Cr2−/− Mice: Enhanced Affinity Maturation but Impaired Antibody Persistence This information is current as Zhibin Chen, Sergei B. Koralov, Mariya Gendelman, of September 25, 2021. Michael C. Carroll and Garnett Kelsoe J Immunol 2000; 164:4522-4532; ; doi: 10.4049/jimmunol.164.9.4522 http://www.jimmunol.org/content/164/9/4522 Downloaded from References This article cites 74 articles, 38 of which you can access for free at: http://www.jimmunol.org/content/164/9/4522.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Humoral Immune Responses in Cr2؊/؊ Mice: Enhanced Affinity Maturation but Impaired Antibody Persistence1 Zhibin Chen,* Sergei B. Koralov,* Mariya Gendelman,* Michael C. Carroll,† and Garnett Kelsoe2* Deficiency in CD21/CD35 by disruption of the Cr2 loci leads to impaired humoral immune responses. In this study, we detail the role of CD21/CD35 on Ab responses to the hapten (4-hydroxy-3-nitrophenyl)acetyl conjugated to chicken gamma-globulin. Sur- ,prisingly, Cr2؊/؊ mice generate significant Ab responses and germinal center (GC) reactions to low doses of this Ag in alum although the magnitude of their responses is much reduced in comparison with those of Cr2؉/؊ and C57BL/6 controls. Increasing Ag dose partially corrected this deficit. In situ study of the somatic genetics of GC B cells demonstrated that VDJ hypermutation does not require CD21/CD35, and Cr2؊/؊ mice exhibited enhanced affinity maturation of serum Ab in the post-GC phase of the ؊ ؊ primary response. On the other hand, Cr2 / mice displayed accelerated loss of serum Ab and long-lived Ab-forming cells. These Downloaded from observations suggest that B cell activation/survival signals mediated by CD21 and/or the retention of Ag by CD21/CD35 play important roles in the generation, quality, and maintenance of serum Ab. The Journal of Immunology, 2000, 164: 4522–4532. he discovery of complement receptors on B cells by Nus- mains of human CD21 suppressed primary Ab responses to T- senzweig and colleagues (1–3) suggested that the com- dependent Ags in mice (17). Two mechanisms have been proposed T plement system might affect acquired immunity. Pepys (4, to explain enhancement of humoral immunity by CD21 (18, 19): http://www.jimmunol.org/ 5) tested this possibility by transient depletion of C3 in mice with facilitation of Ag retention by FDC in germinal centers (GCs) cobra venom factor and observed suppression of Ab responses to (20–23) and enhanced recruitment of the CD21/CD19/CD81 co- T-dependent Ags. Subsequent studies in guinea pigs (6–9), dogs receptor into the B cell Ag receptor (BCR) complex (24–27). Ag (10), humans (11), and mice (12) demonstrated that genetic defi- bound to FDCs drives the GC reaction that generates the B cell ciencies in C2, C3, or C4 resulted in similar immunological de- memory compartment, whereas the CD19/CD21 coreceptor lowers fects. Recently, Dempsey et al. (13) generated T-dependent Ags B cell activation thresholds. comprised of hen egg lysozyme and two or three tandemly aligned Several groups have used gene disruption to clarify the role(s) of copies of C3d. These novel Ags were 1,000- to 10,000-fold more CD21/35 in immunity (28–30). In mice, CD21 and CD35 are al- by guest on September 25, 2021 immunogenic in mice than the unmodified hen egg lysozyme, dra- ternatively spliced gene products encoded by the Cr2 locus (31– matically illustrating the importance of complement in enhancing 33). Disruption of the Cr2 loci abolishes expression of both CD21 Ab responses. and CD35 and results in impaired humoral immune responses (28, The effect of complement on B cell responses is thought to be 29). However, the degree of impairment is controversial. One line mediated by two distinct complement receptors, CD21 (CR2) and of Cr2Ϫ/Ϫ mice failed to generate serum Ab responses to immu- CD35 (CR1), that are expressed on B cells and follicular dendritic 7 nization with 3 ϫ 10 PFU of bacteriophage ⌽X174 in the absence cells (FDCs).3 Administration of a rat mAb specific for both CD21 of adjuvant. Nonetheless, a 10-fold increase in Ag dose elicited Ab and CD35 suppressed primary Ab responses of mice to T-depen- production but only to levels significantly below that of wild-type dent Ags, whereas an Ab specific for CD35 alone had a more Ϫ Ϫ controls (28). Reconstitution of lethally irradiated Cr2 / mice modest immunosuppressive effect (14–16). This suggested that ϩ ϩ with bone marrow from MHC-matched Cr2 / donors repaired CD21 may play a more prominent role in mediating complement’s this defect, indicating that impaired Ab production was a defect of immune-enhancing effects than does CD35. Supporting this argu- B cells (28). Supporting this notion, mice lacking CD21/35 only on ment, a fusion protein containing the complement-binding do- B cells failed to generate Ab responses to 10 ␮g (4-hydroxy-3- nitrophenyl)acetyl (NP)-keyhole limpet hemocyanin in alum adju- *Department of Immunology, Duke University Medical Center, Durham, NC 27710; vant (30). An independently generated line of Cr2Ϫ/Ϫ mice also † and Center for Blood Research, Harvard Medical School, Boston, MA 02115 displayed markedly reduced primary humoral responses but re- Received for publication September 14, 1999. Accepted for publication February sponded to both high and low doses of SRBCs (29). In addition, 18, 2000. defective Ab responses were observed even when this line of The costs of publication of this article were defrayed in part by the payment of page Ϫ/Ϫ ϩ/ϩ charges. This article must therefore be hereby marked advertisement in accordance Cr2 mice was reconstituted with Cr2 bone marrow, sug- with 18 U.S.C. Section 1734 solely to indicate this fact. gesting a significant role for CD21/CD35 on FDCs (34). 1 This work was supported in part by U.S. Public Health Service Grants AI-24335 and CD21 has also been shown to promote the survival of GC B AG-13789 (to G.K.), AI-39246, AI-36389 (to M.C.C.), and AG-10207 (to G.K. and M.C.C.). cells. Stimulation of human tonsillar GC B cells with anti-CD21 mAbs in vitro induced the expression of Bcl-2 and reduced levels 2 Address correspondence and reprint requests to Dr. Garnett Kelsoe, Department of Ϫ/Ϫ Immunology, Box 3010, Duke University Medical Center, Durham, NC 27710. E- of apoptosis (35, 36). Adoptively transferred, Ag-specific Cr2 mail address: [email protected] B cells did not persist in the GCs of wild-type mice, despite ex- 3 Abbreviations used in this paper: FDC, follicular dendritic cell; GC, germinal cen- pression of BCRs with high affinity for Ag (37). Nonetheless, in ter; BCR, B cell Ag receptor; NP, (4-hydroxy-3-nitrophenyl)acetyl; AFC, Ab-forming the absence of competition from CD21/CD35ϩ cells, B lympho- cell; CG, chicken gamma-globulin; ELISPOT, enzyme-linked immunospot; PNA, Ϫ Ϫ peanut agglutinin. cytes in Cr2 / mice can form GCs (28, 29, 34), although the Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 4523 magnitude of the reaction is significantly reduced (28). The quality Mice were immunized with a single i.p. injection of NP12-CG precipitated of the GC reaction in the absence of CD21/CD35, e.g., hypermu- in alum. tation, clonal selection and affinity maturation, and maintenance of B cell memory compartments, remains unknown. Quantification of serum NP-specific Abs To investigate further the function of CD21/CD35 in B cell responses, we bred the Cr2Ϫ/Ϫ mutation onto the Ighb genetic Serum IgM, IgG1, or ␭1 Ab specific for NP was quantified by ELISA on NP6-BSA or NP20-BSA (48). In brief, 96-well plates (Falcon 3912; Becton background of C57BL/6 mice to study immune responses to NP. In ␮ b ␭ Dickinson, Oxnard, CA) were coated with 20 g/ml NP-BSA in 0.1 M all inbred mouse strains carrying Igh and Ig 1, primary Ab re- carbonate buffer (pH 8.8) at 4°C overnight and then were blocked and sponses to immunogenic conjugates of NP are clonally restricted washed with PBS (pH7.4) containing 0.1% Tween 20 (Sigma, St. Louis, (38). Virtually all NP-specific Abs in Ighb mice bear the ␭1 light MO) and 1% BSA. Serially diluted sera were added to duplicate wells and chain (39, 40) and use V 186.2 and DFL16.1 gene segments to incubated for1hatroom temperature. Each plate included standard con- H ␭ ␥ ␭ encode the Ig heavy chain (41–43). This response is compartmen- trols of serially diluted monoclonal IgG1/ 1 Abs, H33L 1/ 1, and pE- ␥ Х ϫ 7 VHC 1 (50). These Abs bind NP with different affinities (Ka 2.0 10 talized into pauciclonal foci of Ab-forming cells (AFCs) along the Ϫ1 ϫ 6 Ϫ1 ␭ M and 1 10 M , respectively).
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