Excess CD40L does not rescue anti-DNA B cells from clonal anergy

Mohammad Aslam; Yusuke Kishi; Takeshi Tsubata

doi:10.12688/f1000research.2-218.v2

Home Browse Excess CD40L does not rescue anti-DNA B cells from clonal anergy

ALL Metrics

-

Views

-

Downloads

Get PDF

Get XML

Export

▬

✚

Research Article

Negative/null result

Revised

Excess CD40L does not rescue anti-DNA B cells from clonal anergy

[version 2; peer review: 3 approved]

Mohammad Aslam¹, Yusuke Kishi¹, Takeshi Tsubata ¹

PUBLISHED 15 Jan 2014

Author details Author details

¹ Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, 113-8510, Japan

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

CD40L, a member of the tumor necrosis factor (TNF) ligand family, is overexpressed in patients with systemic lupus erythematosus and in lupus mouse models. Previously, we demonstrated that B cells producing pathogenic anti-Sm/RNP antibodies are deleted in the splenic marginal zone (MZ), and that MZ deletion of these self-reactive B cells is reversed by excess CD40L, leading to autoantibody production. To address whether excess CD40L also perturbs clonal anergy, another self-tolerance mechanism of B cells whereby B cells are functionally inactivated and excluded from follicles in the peripheral lymphoid tissue, we crossed CD40L-transgenic mice with the anti-DNA H chain transgenic mouse line 3H9, in which Ig λ1+ anti-DNA B cells are anergized. However, the percentage and localization of Ig λ1+ B cells in CD40L/3H9 double transgenic mice were no different from those in 3H9 mice. This result indicates that excess CD40L does not perturb clonal anergy, including follicular exclusion. Thus, MZ deletion is distinct from clonal anergy, and is more liable to tolerance break.

Keywords

antibodies, B cells, T cells

Corresponding author: Takeshi Tsubata

Competing interests: No competing interests were disclosed.

Grant information: This work was supported by MEXT KAKENHI Grants Number 23390063 (TT) and 24790464 (YK).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2014 Aslam M et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

How to cite: Aslam M, Kishi Y and Tsubata T. Excess CD40L does not rescue anti-DNA B cells from clonal anergy [version 2; peer review: 3 approved]. F1000Research 2014, 2:218 (https://doi.org/10.12688/f1000research.2-218.v2) First published: 17 Oct 2013, 2:218 (https://doi.org/10.12688/f1000research.2-218.v1) Latest published: 15 Jan 2014, 2:218 (https://doi.org/10.12688/f1000research.2-218.v2)

Revised Amendments from Version 1

We thank the reviewers for their helpful comments. We have revised our manuscripts according to the suggestions, which has strengthened our paper. Specifically, we provided additional information relating to the experimental animals (including information on the number of mice we used in each group), added additional graphs in Fig. 1 to show statistical analysis comparing groups, and replaced Fig. 2C with a more representative picture. We also modified the introduction to stress that our current results are not easily foreseeable by our previous publication and that this work is a significant addition to the literature.

To read any peer review reports and author responses for this article, follow the "read" links in the Open Peer Review table.

Introduction

Antibodies to nuclear antigens such as DNA and the RNA-related Sm/RNP antigen are characteristically produced in patients with systemic lupus erythematosus (SLE), a prototype of systemic autoimmune diseases, and play a role in the development of this autoimmune disease^1–3. How B cells reactive to nuclear antigens are regulated has been extensively studied using transgenic (Tg) mice expressing auto-antibodies against DNA and RNA components, especially the anti-DNA H chain-Tg mouse lines 3H9 and 56R^4–9. Studies using these auto-antibody-Tg mice demonstrated that self-reactive B cells that produce autoantibodies to nuclear antigens are deleted by apoptosis (clonal deletion)¹⁰, are functionally inactivated (clonal anergy)¹¹ or change antigen specificity by immunoglobulin (Ig) V gene replacement (receptor editing)^8,12, in the bone marrow before they migrate to the peripheral lymphoid organs. These self-tolerance mechanisms appear to be involved in the prevention of autoantibody production in normal individuals.

CD40 is a member of the tumor necrosis factor (TNF) receptor family expressed in immune cells including B cells and dendritic cells¹³. Upon interaction with its ligand, CD40L (CD154), expressed mainly by activated T cells, CD40 transmits survival and activation signals in B cells^13,14. In both patients with SLE and in SLE mouse models, CD40L is overexpressed by T cells and ectopically expressed in B cells^15–17, and this excess CD40L expression appears to play a role in development of SLE, as treatment with antagonistic anti-CD40L antibody markedly reduces the severity of the disease in both humans and mice¹⁸.

Using CD40L/56R double transgenic mice expressing both the anti-DNA H chain 56R and CD40L in B cells, we previously demonstrated that anti-Sm/RNP B cells are regulated by a novel tolerance mechanism in peripheral lymphoid tissue, i.e., deletion in splenic marginal zone (MZ), and that the MZ deletion is perturbed by excess CD40L¹⁹. In 56R mice, B cells that produce anti-Sm/RNP antibody appear in the splenic MZ, and are subsequently deleted there by apoptosis. When 56R mice are crossed with CD40L-Tg mice in which CD40 signaling is constitutively generated in B cells²⁰, MZ deletion of anti-Sm/RNP B cells is perturbed, resulting in autoantibody production¹⁹. As anti-Sm/RNP antibodies are implicated in the pathogenesis of SLE^1,2, MZ deletion appears to be important for preventing the development of SLE through deletion of pathogenic self-reactive B cells. Hence, a defect in MZ deletion by excess CD40L¹⁹ could play a role in the development of lupus by inducing the production of pathogenic anti-Sm/RNP antibody.

Some self-reactive B cells including a part of anti-DNA B cells are silenced by clonal anergy in which B cells persist in the peripheral lymphoid organs but are unresponsive to antigen stimulation. Anergized self-reactive B cells are excluded from follicles or the MZ of the spleen. Instead, they are located in the red pulp and the T cell zone of the spleen, especially in the border between the T cell zone and the follicles, and undergo apoptosis²¹. Ig λ1 L chain⁺ B cells in the anti-DNA H chain-Tg mouse line 3H9 are reactive to DNA and are anergized^9,22,23. Previously, we demonstrated that excess CD40L does not induce anti-DNA antibody production in 3H9 mice¹⁹. Nonetheless, it is possible that anti-DNA B cells in these mice are rescued from anergy by excess CD40L but are tolerized by some other mechanism as there are multiple tolerance mechanisms at different developmental stages of B cells. To address whether excess CD40L can reverse the anergy of self-reactive B cells, we crossed CD40L-Tg mice with 3H9 mice and examined the percentage and localization of Ig λ1⁺ B cells. Our results demonstrated that excess CD40L does not expand anergized anti-DNA B cells or reverse their follicular exclusion, indicating that excess CD40 does not reverse anergy of self-reactive B cells. As excess CD40L does perturb MZ deletion of anti-Sm/RNP B cells, clonal anergy appears to be distinct from MZ deletion, although both of them induce apoptosis of self-reactive B cells in peripheral lymphoid tissue.

Materials and methods

Mice

The conventional Tg mouse line expressing the H chain of the anti-DNA antibody 3H9 on the BALB/c background⁵ was a kind gift of Dr. M. Weigert (The University of Chicago). We previously generated CD40L-Tg mice on the C57BL/6 background²⁰. CD40L-Tg mice were crossed with 3H9-Tg mice to generate wild type, 3H9-Tg and CD40L/3H9 double Tg mice on (BALB/c × C57BL/6)F1 background. Mice were genotyped by PCR reaction using specific pairs of primers for the CD40L and 3H9 transgenes, respectively^14,19. All mice were housed and bred at our specific pathogen-free facility. Groups of 3 mice were kept in conventional shoebox-type polycarbonate cages, which were changed every 7 days. All animals were provided with food (CE-2, CLEA Japan, Inc.) and water ad libitum and were maintained on a 12-hour light/dark cycle. All procedures followed the guidelines of Tokyo Medical and Dental University for animal research and were approved by Institutional Animal Care and Use Committee, Tokyo Medical and Dental University. For each of 3H9, CD40L and CD40L/3H9-Tg mice, 3 or 4 female mice at 11–34 weeks of age were analyzed by flow cytometry and immunohistochemistry, respectively. Mice were euthanized by cervical dislocation after CO2-induced unconsciousness and whole spleens were removed aseptically.

Flow cytometry

Spleens were finely minced over a wire mesh, and spleen cells were collected and suspended in PBS containing 2% FCS (Cell Culture Bioscience, Japan) and 0.1% NaN₃ (Nacalai Tesque, Inc., Japan). Cells (1 × 10⁷/ml) were then stained with the following antibodies and reagents. The dilutions at which the antibodies were used are indicated in parentheses. Alexa Fluor 647-conjugated rat anti-mouse B220 (RA3-6B2, BioLegend, USA) (1:100), Pacific Blue-conjugated anti-mouse Ig λ1 (LS-136, a kind gift of Dr. G. Kelsoe at Duke University) (1:100)²⁴ and FITC-conjugated goat anti-mouse Ig λ chain antibody (Cat No. 1060-02, Southern Biotech, USA) (1:1000). Lymphoid cells were gated by FSC/SSC dot plots, and analyzed on a CyAn ADP flow cytometer (Beckman Coulter, USA).

Immunohistochemistry

Tissues were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek U.S.A., Inc., U.S.A.), snap-frozen in liquid nitrogen, and stored at −80°C. Cryostat (Cryostat 1720, Leica Microsystems GmbH, Germany) sections of 5 μm in thickness were mounted onto micro slide glass (Matsunami Glass Ind. Ltd., Japan), air dried, and fixed in acetone at room temperature for 20 min. The sections were incubated with blocking buffer (PBS containing 2.0% (wt/vol) FCS (Cell Culture Bioscience, Japan), 0.05% NaN3 (Nacalai Tesque, Inc., Japan)) for 30 min and were stained at room temperature for 60 min with biotin-conjugated rat anti-mouse MOMA-1 (Cat No. T-2021, BMA Biomedicals, Switzerland), Alexa Fluor 647-conjugated streptavidin (Cat No. S21374, Invitrogen, USA), Pacific Blue-conjugated rat anti-mouse B220 (RA3-6B2, BioLegend, USA) and FITC-conjugated goat anti-mouse λ chain antibody (Cat No. 1060-02, Southern Biotech, USA). All the antibodies and reagents were used at a 1:100 dilution.

Statistical analysis

Spleen cells were compared between animals. Statistical analysis of data by two-tailed Student t test was performed using Prism 5.0 software (GraphPad). p < 0.05 was considered statistically significant.

Results

Excess CD40L fails to expand anergized anti-DNA B cells

To address whether excess CD40L perturbs clonal anergy of anti-DNA B cells, we crossed CD40L Tg mice with 3H9 mice, because B cells expressing Ig λ1 L chain and 3H9 H chain are reactive to DNA and show characteristics for anergy including follicular exclusion, shortened life span and failure of antibody production²³. When spleen B cells of wild type, 3H9 Tg and CD40L overexpressing CD40L/3H9 double Tg, mouse lines were analyzed by flow cytometry, the percentage of λ1⁺ B cells in total B cells expressing a B cell marker B220 was markedly reduced in 3H9 mice compared to wild type mice (Figure 1A) (p < 0.01) as λ1⁺ B cells in 3H9 but not wild type mice are self-reactive. The percentage of λ1⁺ B cells in CD40L/3H9 double Tg mice was equivalent to that in 3H9 mice, indicating that excess CD40L does not expand anergized anti-DNA B cells.

Figure 1. Excess CD40L does not expand anergic anti-DNA B cells in 3H9 mice.

Spleen cells from 11- to 34-wk-old wild type (WT), 3H9, and CD40L/3H9 mice were stained for B220 (A and B), Ig λ1 chain (A and B) and Ig λ chain (B), and B220⁺ cells were analyzed by flow cytometry. Percentages of λ1⁺ cells in total B220⁺ cells (A), percentages of λ⁺ cells in total B220⁺ cells (B, upper panel) and percentages of λ1⁺ cells in total λ⁺ cells (B, lower panel) are indicated (left panels). Representative data from three independent experiments. Graphs show mean ± SD, three mice per genotype (right panels). * p<0.05, ** p<0.01, ns = not significant.

When we used anti-λ antibody that reacts to multiple Ig λ chain subtypes such as λ1 and λ2, the percentage of total λ⁺ cells were only slightly higher than that of λ1⁺ cells in wild type, 3H9 and CD40L/3H9 mice (Figure 1A and B upper panels), suggesting that most of the λ⁺ cells express the λ1 subtype in all these mice and are thus reactive to DNA. This is confirmed by determining percentage of λ1⁺ cells in total λ⁺ cells. In both 3H9 and CD40L/3H9 mice, the percentage of λ1⁺ cells in total λ⁺ cells is slightly reduced compared to that in wild type mice (p < 0.05 and 0.01, respectively), but λ1⁺ cells constitute the majority of the λ⁺ cells in these mice as well as wild type mice (Figure 1B lower panels).

Excess CD40L does not reverse follicular exclusion of anergized anti-DNA B cells

To address whether excess CD40L reverses follicular exclusion of anergized anti-DNA B cells, we examined spleen sections of 3H9 and 3H9/CD40L Tg mice using an anti-λ antibody but not anti-λ1 antibody, as the anti-λ1 antibody did not yield any staining when used for immunohistochemistry. Nonetheless, most of the B cells stained with anti-λ antibody in these mice express λ1 (Figure 1B, lower panels) and are thus reactive to DNA. In wild type spleen, λ⁺ cells were located mostly in the follicle where B220⁺ B cells accumulate (Figure 2A). In contrast, λ⁺ cells were found mostly in the border between T cell zone and follicle, T cell zone and red pulp in both 3H9 and CD40L/3H9 spleens (Figure 2B and C), indicating that anti-DNA B cells are excluded from follicles in CD40L/3H9 mice as well as 3H9 mice. Thus, excess CD40L does not reverse follicular exclusion of anergized anti-DNA B cells.

Figure 2. Excess CD40L does not reverse follicular exclusion of anergic anti-DNA B cells in 3H9 mice.

Sections of spleen from 11- to 34-wk-old wild type (WT) (A), 3H9 (B), and CD40L/3H9 (C) mice were stained for MOMA-1, a marker for a subset of splenic macrophages (red), B220, a B cell marker (blue), Ig λ L chain (green), and analyzed by confocal microscopy. Ig λ⁺ B cells in 3H9 mice are DNA-reactive. B cells positive for B220 are located in the follicles, and MOMA-1⁺ macrophages are located at the outer part of the follicles. T cell zone and red pulp are located inside and outside of the follicles, respectively. Representative data from four independent experiments. Four mice in each group were analyzed. Original magnifications, 20X. Scale bar (yellow line): 100 μm.

Discussion

In this study, we crossed CD40L-Tg mice with anti-DNA H chain-Tg 3H9 mice, and demonstrated that the percentage and location of λ1⁺ anergic anti-DNA B cells are not altered in CD40L/3H9 double Tg mice compared to those in 3H9 mice, suggesting that anergy of λ1⁺ anti-DNA B cells is not reversed in CD40L/3H9 mice. This result is consistent with our previous finding that CD40L/3H9 mice do not produce anti-DNA antibodies in sera, whereas another anti-DNA H chain Tg mouse line 56R in which self-reactive B cells are deleted at MZ did produce autoantibodies in sera when crossed with the same CD40L Tg mice¹⁹. Thus, excess CD40L does not perturb anergy of anti-DNA B cells.

Although excess CD40L fails to perturb clonal anergy of anti-DNA B cells in 3H9 mice, anergic B cells are not insensitive to CD40L. A previous study by Lesley et al. demonstrated that anergic self-reactive B cells expand by receiving stimulation from a low level expression of CD40L in unstimulated T cells²⁵. Thus, anergic B cells sensitively respond to a low level CD40L expression, but do not further respond to excess CD40L expression. The mechanisms behind this phenomenon however, are not yet clear.

Using the same CD40L-Tg mice that we used in the present study, we previously demonstrated that excess CD40L inhibits apoptosis of anti-Sm/RNP B cells in MZ, and that excess CD40L induces autoantibody production¹⁹. This suggests that MZ deletion is distinct from clonal anergy, although both anergic B cells and B cells that are deleted in MZ appear in peripheral lymphoid tissue and are eliminated by apoptosis.

Like CD40L, B cell activating factor (BAFF), another member of the TNF ligand family, induces B cell survival and is overexpressed in patients with SLE^26–28 and its mouse models^29,30. Anergic B cells show increased dependency on BAFF for survival, and this increased dependency appears to be involved in rapid elimination of anergic B cells by competition with non-self-reactive B cells^31,32. In the presence of non-self-reactive B cells, anergic B cells may not be able to interact with a sufficient level of BAFF required for their survival, due to competition for a limited amount of BAFF³². Nonetheless, excess BAFF fails to fully reverse anergy of self-reactive B cells. Lesley et al.³¹ demonstrated that excess BAFF expands anergic B cells but fails to reverse follicular exclusion. Thien et al.³² demonstrated that excess BAFF expands and reverses follicular exclusion in only anergic B cells with intermediate affinity but not those with high affinity. In contrast, cognate T cell help perturbs follicular exclusion and induces autoantibody production in anergic self-reactive B cells³³. Thus, reversing clonal anergy requires strong T cell help, and excess BAFF or CD40L alone may be insufficient. In contrast, we previously demonstrated by crossing the CD40L-Tg mice with another anti-DNA H chain Tg mice 56R that excess CD40L perturbs MZ deletion of self-reactive B cells and induces autoantibody production, suggesting that MZ deletion is more sensitive to a tolerance break than clonal anergy¹⁹. As excess CD40L is found in patients with SLE and various SLE model mice, MZ deletion is likely to be defective in lupus, and its defect may be involved in development of lupus.

Author contributions

TT conceived the study. MA, YK and TT designed the experiments, and carried out the research. MA and TT wrote the manuscript.

Competing interests

No competing interests were disclosed.

Grant information

This work was supported by MEXT KAKENHI Grants Number 23390063 (TT) and 24790464 (YK) and a grant from SENSHIN Medical Research Foundation.(TT)

Acknowledgements

We thank Dr. Martin Weigert for the 3H9 mice, and Dr. Garnett Kelsoe for the anti-mouse Ig λ1 antibody producing hybridoma cells (LS-136).

Faculty Opinions recommended

References

1. Berland R, Fernandez L, Kari E, et al.: Toll-like receptor 7-dependent loss of B cell tolerance in pathogenic autoantibody knockin mice. Immunity. 2006; 25(3): 429–440. PubMed Abstract | Publisher Full Text
2. Christensen SR, Shupe J, Nickerson K, et al.: Toll-like receptor 7 and TLR9 dictate autoantibody specificity and have opposing inflammatory and regulatory roles in a murine model of lupus. Immunity. 2006; 25(3): 417–428. PubMed Abstract | Publisher Full Text
3. Tan EM: Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv Immunol. 1989; 44: 93–151. PubMed Abstract | Publisher Full Text
4. Chen C, Radic MZ, Erikson J, et al.: Deletion and editing of B cells that express antibodies to DNA. J Immunol. 1994; 152(4): 1970–1982. PubMed Abstract
5. Erikson J, Radic MZ, Camper SA, et al.: Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice. Nature. 1991; 349(6307): 331–334. PubMed Abstract | Publisher Full Text
6. Chen C, Nagy Z, Radic MZ, et al.: The site and stage of anti-DNA B-cell deletion. Nature. 1995; 373(6511): 252–255. PubMed Abstract | Publisher Full Text
7. Fukuyama H, Nimmerjahn F, Ravetch JV: The inhibitory Fcgamma receptor modulates autoimmunity by limiting the accumulation of immunoglobulin G+ anti-DNA plasma cells. Nat Immunol. 2005; 6(1): 99–106. PubMed Abstract | Publisher Full Text
8. Gay D, Saunders T, Camper S, et al.: Receptor editing: an approach by autoreactive B cells to escape tolerance. J Exp Med. 1993; 177(4): 999–1008. PubMed Abstract | Publisher Full Text | Free Full Text
9. Noorchashm H, Bui A, Li HL, et al.: Characterization of anergic anti-DNA B cells: B cell anergy is a T cell-independent and potentially reversible process. Int Immunol. 1999; 11(5): 765–776. PubMed Abstract | Publisher Full Text
10. Nemazee DA, Burki K: Clonal deletion of B lymphocytes in a transgenic mouse bearing anti-MHC class I antibody genes. Nature. 1989; 337(6207): 562–566. PubMed Abstract | Publisher Full Text
11. Goodnow CC, Crosbie J, Adelstein S, et al.: Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature. 1988; 334(6184): 676–682. PubMed Abstract | Publisher Full Text
12. Radic MZ, Erikson J, Litwin S, et al.: B lymphocytes may escape tolerance by revising their antigen receptors. J Exp Med. 1993; 177(4): 1165–1173. PubMed Abstract | Publisher Full Text | Free Full Text
13. Quezada SA, Jarvinen LZ, Lind EF, et al.: CD40/CD154 interactions at the interface of tolerance and immunity. Annu Rev Immunol. 2004; 22: 307–328. PubMed Abstract | Publisher Full Text
14. Tsubata T, Wu J, Honjo T: B-cell apoptosis induced by antigen receptor crosslinking is blocked by a T-cell signal through CD40. Nature. 1993; 364(6438): 645–648. PubMed Abstract | Publisher Full Text
15. Blossom S, Chu EB, Weigle WO, et al.: CD40 ligand expressed on B cells in the BXSB mouse model of systemic lupus erythematosus. J Immunol. 1997; 159(9): 4580–4586. PubMed Abstract
16. Desai-Mehta A, Lu L, Ramsey-Goldman R, et al.: Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production. J Clin Invest. 1996; 97(9): 2063–2073. PubMed Abstract | Publisher Full Text | Free Full Text
17. Koshy M, Berger D, Crow MK: Increased expression of CD40 ligand on systemic lupus erythematosus lymphocytes. J Clin Invest. 1996; 98(3): 826–837. PubMed Abstract | Publisher Full Text | Free Full Text
18. Peters AL, Stunz LL, Bishop GA: CD40 and autoimmunity: the dark side of a great activator. Semin Immunol. 2009; 21(5): 293–300. PubMed Abstract | Publisher Full Text | Free Full Text
19. Kishi Y, Higuchi T, Phoon S, et al.: Apoptotic marginal zone deletion of anti-Sm/ribonucleoprotein B cells. Proc Natl Acad Sci U S A. 2012; 109(20): 7811–7816. PubMed Abstract | Publisher Full Text | Free Full Text
20. Higuchi T, Aiba Y, Nomura T, et al.: Cutting Edge: Ectopic expression of CD40 ligand on B cells induces lupus-like autoimmune disease. J Immunol. 2002; 168(1): 9–12. PubMed Abstract
21. Cyster JG, Hartley SB, Goodnow CC: Competition for follicular niches excludes self-reactive cells from the recirculating B-cell repertoire. Nature. 1994; 371(6496): 389–395. PubMed Abstract | Publisher Full Text
22. Mandik-Nayak L, Bui A, Noorchashm H, et al.: Regulation of anti-double-stranded DNA B cells in nonautoimmune mice: localization to the T-B interface of the splenic follicle. J Exp Med. 1997; 186(8): 1257–1267. PubMed Abstract | Publisher Full Text | Free Full Text
23. Radic MZ, Mascelli MA, Erikson J, et al.: Ig H and L chain contributions to autoimmune specificities. J Immunol. 1991; 146(1): 176–182. PubMed Abstract
24. Reth M, Imanishi-Kari T, Rajewsky K: Analysis of the repertoire of anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies in C 57 BL/6 mice by cell fusion. II. Characterization of idiotopes by monoclonal anti-idiotope antibodies. Eur J Immunol. 1979; 9(12): 1004–1013. PubMed Abstract | Publisher Full Text
25. Lesley R, Kelly LM, Xu Y, et al.: Naive CD4 T cells constitutively express CD40L and augment autoreactive B cell survival. Proc Natl Acad Sci U S A. 2006; 103(28): 10717–10722. PubMed Abstract | Publisher Full Text | Free Full Text
26. Groom J, Kalled SL, Cutler AH, et al.: Association of BAFF/BLyS overexpression and altered B cell differentiation with Sjögren's syndrome. J Clin Invest. 2002; 109(1): 59–68. PubMed Abstract | Publisher Full Text | Free Full Text
27. Mariette X, Roux S, Zhang J, et al.: The level of BLyS (BAFF) correlates with the titre of autoantibodies in human Sjögren's syndrome. Ann Rheum Dis. 2003; 62(2): 168–171. PubMed Abstract | Publisher Full Text | Free Full Text
28. Mackay IR, Groom J, Mackay CR: Levels of BAFF in serum in primary biliary cirrhosis and autoimmune diabetes. Autoimmunity. 2002; 35(8): 551–553. PubMed Abstract | Publisher Full Text
29. Mackay F, Woodcock SA, Lawton P, et al.: Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations. J Exp Med. 1999; 190(11): 1697–1710. PubMed Abstract | Publisher Full Text | Free Full Text
30. Khare SD, Sarosi I, Xia XZ, et al.: Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice. Proc Natl Acad Sci U S A. 2000; 97(7): 3370–3375. PubMed Abstract | Publisher Full Text | Free Full Text
31. Lesley R, Xu Y, Kalled SL, et al.: Reduced competitiveness of autoantigen-engaged B cells due to increased dependence on BAFF. Immunity. 2004; 20(4): 441–453. PubMed Abstract | Publisher Full Text
32. Thien M, Phan TG, Gardam S, et al.: Excess BAFF rescues self-reactive B cells from peripheral deletion and allows them to enter forbidden follicular and marginal zone niches. Immunity. 2004; 20(6): 785–798. PubMed Abstract | Publisher Full Text
33. Seo SJ, Fields ML, Buckler JL, et al.: The impact of T helper and T regulatory cells on the regulation of anti-double-stranded DNA B cells. Immunity. 2002; 16(4): 535–546. PubMed Abstract | Publisher Full Text

Comments on this article Comments (0)

Version 2

VERSION 2 PUBLISHED 17 Oct 2013

Author details Author details

¹ Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, 113-8510, Japan

Competing interests

No competing interests were disclosed.

Grant information

This work was supported by MEXT KAKENHI Grants Number 23390063 (TT) and 24790464 (YK).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (2)

version 2

Revised

Published: 15 Jan 2014, 2:218

https://doi.org/10.12688/f1000research.2-218.v2

version 1

Published: 17 Oct 2013, 2:218

https://doi.org/10.12688/f1000research.2-218.v1

Copyright

© 2014 Aslam M et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Download

Export To

metrics

	Views	Downloads
F1000Research	-	-
PubMed Central Data from PMC are received and updated monthly.	-	-

Citations

0

SEE MORE DETAILS

CITE

how to cite this article

Aslam M, Kishi Y and Tsubata T. Excess CD40L does not rescue anti-DNA B cells from clonal anergy [version 2; peer review: 3 approved]. F1000Research 2014, 2:218 (https://doi.org/10.12688/f1000research.2-218.v2)

NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.

track

receive updates on this article

Track an article to receive email alerts on any updates to this article.

Open Peer Review

Current Reviewer Status: ?

Key to Reviewer Statuses VIEW HIDE

ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions

Version 2

VERSION 2

PUBLISHED 15 Jan 2014

Revised

Views

14

Reviewer Report 05 Mar 2014

Jennifer Marshall, University of Birmingham, Birmingham, UK

Approved

https://doi.org/10.5256/f1000research.3592.r3145

With the addition of the animal numbers used, the graphs of data in figure 1 and the new images submitted I think that the data, and therefore the paper, is much improved. I would very much like to thank the authors ... Continue reading

CITE

Report a concern

Respond or Comment

Views

35

Reviewer Report 28 Jan 2014

David Nemazee, Department of Immunology and Microbial Science, Scripps Research Institute, La Jolla, CA, USA

Approved

https://doi.org/10.5256/f1000research.3592.r3359

The paper has been much improved, as it now provides more data on the statistical significance of the ... Continue reading

CITE

Report a concern

Respond or Comment

Version 1

VERSION 1

PUBLISHED 17 Oct 2013

Views

68

Reviewer Report 28 Nov 2013

David Nemazee, Department of Immunology and Microbial Science, Scripps Research Institute, La Jolla, CA, USA

Not Approved

https://doi.org/10.5256/f1000research.2308.r2590

The paper by Aslam, Kishi and Tsubata addresses an interesting question about the effects of bystander stimulation through CD40L on immunological self tolerance of B cells. Previous studies from this group showed that transgene-enforced CD40L expression on B cells promoted ... Continue reading

The paper by Aslam, Kishi and Tsubata addresses an interesting question about the effects of bystander stimulation through CD40L on immunological self tolerance of B cells. Previous studies from this group showed that transgene-enforced CD40L expression on B cells promoted a lupus-like autoimmunity in mice with a polyclonal B cell population. A later study showed that autoreactive B cells in a transgenic mouse with a restricted antibody repertoire biased to DNA reactivity (mice carrying 3H9-56R DNA-reactive H-chain) were promoted to central tolerance in the presence of excess CD40L, whereas 3H9-H mice lacking that mutation, while still expressing a repertoire biased to autoreactivity, failed to show a clear break in tolerance.

The present study is a rather minor addition to this body of work, providing additional data on the fate of B cells expressing a λ1 light chain partner, which have high anti-DNA affinity when expressed with 3H9H. The data show that 3H9H/λ1 B cells are not rescued in their development or altered in their anatomical localization (follicular exclusion) when developing in an environment with excess B cell-expressed CD40L. The data are consistent with the earlier, more extensive study, but do provide some new information. However, the data shown have no indication of reproducibility or provide more than a single mouse per group. There is no molecular characterization of the cells to verify that the receptor expression expected is indeed confirmed. Overall, this is an incremental contribution to the literature.

Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

CITE

Report a concern

Respond or Comment

Views

34

Reviewer Report 25 Nov 2013

Jennifer Marshall, University of Birmingham, Birmingham, UK

Approved with Reservations

https://doi.org/10.5256/f1000research.2308.r2424

This report from Aslam et al., provides data on the lack of an effect of over-expression of CD40L on the number and location of anergic 3H9 B cells that respond to DNA. This follows on from their 2012 PNAS study ... Continue reading

This report from Aslam et al., provides data on the lack of an effect of over-expression of CD40L on the number and location of anergic 3H9 B cells that respond to DNA. This follows on from their 2012 PNAS study which used similar methods investigating the effects of CD40L on anergic B cells from 56R mice.

The paper is well constructed and all sections are clear. However, I believe that the data could be presented in a manner more helpful to the reader and provide a firmer foundation for their conclusions. I think that these changes (especially those concerning figure 2) are necessary to substantiate their conclusions and abstract.

Both figure legends should contain information on how many mice were used in each group. This information is essential for all publications involving animal work and is necessary to allow proper judgment of data. Whilst the methods do state that the mice are caged in groups of 3 this is within the details of animal husbandry and, as such, it is not clear if this related to the experimental group size.

In addition to the representative flow cytometry plots provided Figure 1A and B would benefit from a graph that shows the percentage of splenic Ig λ1 L chain B cells of each mouse in the experiment. This would provide an opportunity to be made aware of the variation between the mice within each group as well as compare between groups. If there are sufficient mouse numbers a statistical analysis comparing groups would also be useful.

I am puzzled by the choice of images for Figure 2. Firstly, due to the shape of the follicle it is difficult to compare the location of Ig λ L chain cells in 2C with those in 2A and B. As the follicular exclusion of λ L chain B cells within the CD40L/3H9 mouse is a key conclusion of this paper it would be helpful for the reader if 2C was replaced with a representative white pulp area that provides a clearer demarcation between follicle and the T zone. Secondly, I would suggest that Figure 2B makes it appear that the 3H9 mice have substantially fewer Ig λ L chain B cells than the CD40L/3H9 mice shown in Figure 2C. If it is representative of all 3H9 spleens I do not think that this corroborates the conclusions drawn from Figure 1. As they stand the images make my suggestions regarding an extra graph for Figure 1, and the mouse numbers used, more compelling.

Without these changes I cannot truly say that the title and conclusions are appropriate. However, these are quick changes and I trust that it is just a bad image choice.

Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

CITE

Report a concern

Respond or Comment

Views

34

Reviewer Report 31 Oct 2013

Marko Radic, Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN, USA

Approved

https://doi.org/10.5256/f1000research.2308.r2273

The brief report by Aslam et al. develops a theme that was the initial focus of the Tsubata lab in the early 90’s. The authors follow up on the observation that CD40L is overexpressed on T cells in SLE patients ... Continue reading

The brief report by Aslam et al. develops a theme that was the initial focus of the Tsubata lab in the early 90’s. The authors follow up on the observation that CD40L is overexpressed on T cells in SLE patients and, ectopically, on B cells. As CD40L provides an important second signal to B cells, the question was whether it can overcome negative regulation of certain B cell states. The current work continues upon Kishi et al. (PNAS 2012) who used anti-DNA transgenic mice to examine the effect of CD40L overexpression on the regulation of autoreactive B cells. Kishi et al. established that an avid anti-DNA transgene, 56R, led to B cell sequestration in the splenic marginal zone (MZ) and death by apoptosis. CD40L overexpression was able to inhibit apoptosis in the MZ and allow expression of certain H/L pairs, most notably those with anti-Sm specificity. Here, the authors use a closely related anti-DNA H chain, 3H9, which is of lower affinity for DNA compared to 56R.

Dozens of labs have used the 3H9 and the 56R H-chain transgenic mouse lines which have become emblematic for the type of H chain that is immunodominant and – with different L chains – has the potential to encode a range of self-specificities, including anti-DNA, anti-phospholipid, anti-nucleosome and also anti-apoptotic cell antibodies. Earlier work by the Erikson lab had established that 3H9VH in combination with λ1 gives a B cell receptor that induces anergy and leads to a characteristic extrafollicular localization. Aslam et al. observe that the 3H9/λ1 combination produces the same number of splenic B cells whether or not it is crossed to CD40L and that the splenic location of the single and double transgenic B cells is identical.

The significance of this work is that it draws an important distinction between tolerance mechanisms that result in anergy vs. MZ deletion. Only the latter is sensitive to rescue by CD40L signals. This serves to clarify important characteristics of splenic B cell regulation. It will be informative to follow these studies with additional approaches to test whether B cell signalling pathways are perturbed by the presence of CD40L, whether increased CD40L expression on B or T cells is involved in suppressing apoptosis in the MZ, and whether CD40L overexpression could affect other types of 56R “incomplete editing” such as in the case of intracellular Golgi retention that is observed with 56R and Vk38c and λX L chains.

The title is appropriate for the content of the article and the abstract represents a suitable summary of the work.
The design, methods and analysis of the results from the study have been explained and they are appropriate for the topic being studied. The conclusions are sensible, balanced and justified on the basis of the results of the study and enough information has been provided to be able to replicate the experiment.

Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

CITE

Report a concern

Respond or Comment

Comments on this article Comments (0)

Version 2

VERSION 2 PUBLISHED 17 Oct 2013

Open Peer Review

Reviewer Status

Reviewer Reports

	Invited Reviewers
	1	2	3
Version 2 (revision) 15 Jan 14		read	read
Version 1 17 Oct 13	read	read	read

Marko Radic, University of Tennessee Health Science Center, Memphis, TN, USA
Jennifer Marshall, University of Birmingham, Birmingham, UK
David Nemazee, Scripps Research Institute, La Jolla, CA, USA

Competing Interests

No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Respond to this report

Responses (0)

[1] 1. Berland R, Fernandez L, Kari E, et al.: Toll-like receptor 7-dependent loss of B cell tolerance in pathogenic autoantibody knockin mice. Immunity. 2006; 25(3): 429–440. PubMed Abstract | Publisher Full Text

[2] 2. Christensen SR, Shupe J, Nickerson K, et al.: Toll-like receptor 7 and TLR9 dictate autoantibody specificity and have opposing inflammatory and regulatory roles in a murine model of lupus. Immunity. 2006; 25(3): 417–428. PubMed Abstract | Publisher Full Text

[3] 3. Tan EM: Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv Immunol. 1989; 44: 93–151. PubMed Abstract | Publisher Full Text

[4] 4. Chen C, Radic MZ, Erikson J, et al.: Deletion and editing of B cells that express antibodies to DNA. J Immunol. 1994; 152(4): 1970–1982. PubMed Abstract

[5] 5. Erikson J, Radic MZ, Camper SA, et al.: Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice. Nature. 1991; 349(6307): 331–334. PubMed Abstract | Publisher Full Text

[6] 6. Chen C, Nagy Z, Radic MZ, et al.: The site and stage of anti-DNA B-cell deletion. Nature. 1995; 373(6511): 252–255. PubMed Abstract | Publisher Full Text

[7] 7. Fukuyama H, Nimmerjahn F, Ravetch JV: The inhibitory Fcgamma receptor modulates autoimmunity by limiting the accumulation of immunoglobulin G+ anti-DNA plasma cells. Nat Immunol. 2005; 6(1): 99–106. PubMed Abstract | Publisher Full Text

[8] 8. Gay D, Saunders T, Camper S, et al.: Receptor editing: an approach by autoreactive B cells to escape tolerance. J Exp Med. 1993; 177(4): 999–1008. PubMed Abstract | Publisher Full Text | Free Full Text

[9] 9. Noorchashm H, Bui A, Li HL, et al.: Characterization of anergic anti-DNA B cells: B cell anergy is a T cell-independent and potentially reversible process. Int Immunol. 1999; 11(5): 765–776. PubMed Abstract | Publisher Full Text

[10] 10. Nemazee DA, Burki K: Clonal deletion of B lymphocytes in a transgenic mouse bearing anti-MHC class I antibody genes. Nature. 1989; 337(6207): 562–566. PubMed Abstract | Publisher Full Text

[11] 11. Goodnow CC, Crosbie J, Adelstein S, et al.: Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature. 1988; 334(6184): 676–682. PubMed Abstract | Publisher Full Text

[12] 12. Radic MZ, Erikson J, Litwin S, et al.: B lymphocytes may escape tolerance by revising their antigen receptors. J Exp Med. 1993; 177(4): 1165–1173. PubMed Abstract | Publisher Full Text | Free Full Text

[13] 13. Quezada SA, Jarvinen LZ, Lind EF, et al.: CD40/CD154 interactions at the interface of tolerance and immunity. Annu Rev Immunol. 2004; 22: 307–328. PubMed Abstract | Publisher Full Text

[14] 14. Tsubata T, Wu J, Honjo T: B-cell apoptosis induced by antigen receptor crosslinking is blocked by a T-cell signal through CD40. Nature. 1993; 364(6438): 645–648. PubMed Abstract | Publisher Full Text

[15] 15. Blossom S, Chu EB, Weigle WO, et al.: CD40 ligand expressed on B cells in the BXSB mouse model of systemic lupus erythematosus. J Immunol. 1997; 159(9): 4580–4586. PubMed Abstract

[16] 16. Desai-Mehta A, Lu L, Ramsey-Goldman R, et al.: Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production. J Clin Invest. 1996; 97(9): 2063–2073. PubMed Abstract | Publisher Full Text | Free Full Text

[17] 17. Koshy M, Berger D, Crow MK: Increased expression of CD40 ligand on systemic lupus erythematosus lymphocytes. J Clin Invest. 1996; 98(3): 826–837. PubMed Abstract | Publisher Full Text | Free Full Text

[18] 18. Peters AL, Stunz LL, Bishop GA: CD40 and autoimmunity: the dark side of a great activator. Semin Immunol. 2009; 21(5): 293–300. PubMed Abstract | Publisher Full Text | Free Full Text

[19] 19. Kishi Y, Higuchi T, Phoon S, et al.: Apoptotic marginal zone deletion of anti-Sm/ribonucleoprotein B cells. Proc Natl Acad Sci U S A. 2012; 109(20): 7811–7816. PubMed Abstract | Publisher Full Text | Free Full Text

[20] 20. Higuchi T, Aiba Y, Nomura T, et al.: Cutting Edge: Ectopic expression of CD40 ligand on B cells induces lupus-like autoimmune disease. J Immunol. 2002; 168(1): 9–12. PubMed Abstract

[21] 21. Cyster JG, Hartley SB, Goodnow CC: Competition for follicular niches excludes self-reactive cells from the recirculating B-cell repertoire. Nature. 1994; 371(6496): 389–395. PubMed Abstract | Publisher Full Text

[22] 22. Mandik-Nayak L, Bui A, Noorchashm H, et al.: Regulation of anti-double-stranded DNA B cells in nonautoimmune mice: localization to the T-B interface of the splenic follicle. J Exp Med. 1997; 186(8): 1257–1267. PubMed Abstract | Publisher Full Text | Free Full Text

[23] 23. Radic MZ, Mascelli MA, Erikson J, et al.: Ig H and L chain contributions to autoimmune specificities. J Immunol. 1991; 146(1): 176–182. PubMed Abstract

[24] 24. Reth M, Imanishi-Kari T, Rajewsky K: Analysis of the repertoire of anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies in C 57 BL/6 mice by cell fusion. II. Characterization of idiotopes by monoclonal anti-idiotope antibodies. Eur J Immunol. 1979; 9(12): 1004–1013. PubMed Abstract | Publisher Full Text

[25] 25. Lesley R, Kelly LM, Xu Y, et al.: Naive CD4 T cells constitutively express CD40L and augment autoreactive B cell survival. Proc Natl Acad Sci U S A. 2006; 103(28): 10717–10722. PubMed Abstract | Publisher Full Text | Free Full Text

[26] 26. Groom J, Kalled SL, Cutler AH, et al.: Association of BAFF/BLyS overexpression and altered B cell differentiation with Sjögren's syndrome. J Clin Invest. 2002; 109(1): 59–68. PubMed Abstract | Publisher Full Text | Free Full Text

[27] 27. Mariette X, Roux S, Zhang J, et al.: The level of BLyS (BAFF) correlates with the titre of autoantibodies in human Sjögren's syndrome. Ann Rheum Dis. 2003; 62(2): 168–171. PubMed Abstract | Publisher Full Text | Free Full Text

[28] 28. Mackay IR, Groom J, Mackay CR: Levels of BAFF in serum in primary biliary cirrhosis and autoimmune diabetes. Autoimmunity. 2002; 35(8): 551–553. PubMed Abstract | Publisher Full Text

[29] 29. Mackay F, Woodcock SA, Lawton P, et al.: Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations. J Exp Med. 1999; 190(11): 1697–1710. PubMed Abstract | Publisher Full Text | Free Full Text

[30] 30. Khare SD, Sarosi I, Xia XZ, et al.: Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice. Proc Natl Acad Sci U S A. 2000; 97(7): 3370–3375. PubMed Abstract | Publisher Full Text | Free Full Text

[31] 31. Lesley R, Xu Y, Kalled SL, et al.: Reduced competitiveness of autoantigen-engaged B cells due to increased dependence on BAFF. Immunity. 2004; 20(4): 441–453. PubMed Abstract | Publisher Full Text

[32] 32. Thien M, Phan TG, Gardam S, et al.: Excess BAFF rescues self-reactive B cells from peripheral deletion and allows them to enter forbidden follicular and marginal zone niches. Immunity. 2004; 20(6): 785–798. PubMed Abstract | Publisher Full Text

[33] 33. Seo SJ, Fields ML, Buckler JL, et al.: The impact of T helper and T regulatory cells on the regulation of anti-double-stranded DNA B cells. Immunity. 2002; 16(4): 535–546. PubMed Abstract | Publisher Full Text

Excess CD40L does not rescue anti-DNA B cells from clonal anergy

Abstract

Keywords

Revised Amendments from Version 1

Introduction

Materials and methods

Mice

Flow cytometry

Immunohistochemistry

Statistical analysis

Results

Excess CD40L fails to expand anergized anti-DNA B cells

Figure 1. Excess CD40L does not expand anergic anti-DNA B cells in 3H9 mice.

Excess CD40L does not reverse follicular exclusion of anergized anti-DNA B cells

Figure 2. Excess CD40L does not reverse follicular exclusion of anergic anti-DNA B cells in 3H9 mice.

Discussion

Author contributions

Competing interests

Grant information

Acknowledgements

References

Comments on this article Comments (0)

Open Peer Review

Comments on this article Comments (0)

Open Peer Review

Reviewer Status

Reviewer Reports

Comments on this article

Browse by related subjects

Competing Interests Policy

Stay Updated