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Cutting Edge: Expansion and Activation of A Population of Autoreactive Marginal Zone B Cells in a Model of Estrogen-Induced Lupus

Christine M. Grimaldi^*, Daniel J. Michael^* and Betty Diamond^2,*,

Departments of ^* Microbiology and Immunology and Medicine, Albert Einstein College of Medicine, Bronx, NY 10461

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have demonstrated previously that 17 -estradiol (E2) treatment of BALB/c mice transgenic for the heavy chain of a pathogenic anti-DNA Ab induces a lupus-like phenotype with expansion of anti-DNA B cells, elevation of anti-DNA Ab titers, and glomerular immunoglobulin deposition. To understand this loss of B cell tolerance, the effects of E2 on B cell development and activation were examined. A sustained increase in E2 resulted in an altered distribution of B cell subsets, with a diminished transitional population and an increase in marginal zone B cells. Depletion of CD4⁺ T cells did not abrogate these effects. Furthermore, the B cells that spontaneously secreted anti-DNA Abs displayed a marginal zone phenotype. Thus, a sustained increase in E2 alters B cell development, leading to the survival, expansion, and activation of a population of autoreactive marginal zone B cells implicating this B cell subset in autoimmunity.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The autoimmune disease systemic lupus erythematosus is characterized by the production of Abs specific for numerous self-Ags, expansion of autoreactive B cells, and glomerular deposition of immunoglobulin complexes that contribute to kidney damage (1). Aspects of the human disease are observed in naturally occurring mouse models of systemic lupus erythematosus, such as the NZB/ZNW F₁ and MRL/lpr mouse strains. These mice spontaneously develop pathogenic autoantibodies that cause glomerulonephritis and have served to dissect the molecular pathways that regulate autoreactive B cells. Much information has also been learned from the lupus-like phenotype exhibited in nonautoimmune mice that are deficient in or overexpress key regulatory models that modulate B cell tolerance, selection, and activation.

We have shown that treatment with exogenous 17 -estradiol (E2)³ abrogates B cell tolerance in anti-DNA H chain transgenic BALB/c mice (R4A-IgG2b), which normally display effective regulation of high affinity transgene (Tg)-expressing anti-DNA B cells (2, 3, 4, 5). The lupus-like phenotype induced by E2 treatment of R4A-IgG2b BALB/c mice includes an expansion of the Tg-positive B cell population, a significant rise in IgG2b anti-dsDNA Ab titers, an increase in the number of B cells spontaneously secreting IgG2b anti-dsDNA Ab, and the presence of IgG glomerular deposits (2, 3).

In this study, we demonstrate that E2 alters the maturation of splenic B cell precursors and enhances development and activation of autoreactive marginal zone B cells. Little is known about the role of marginal zone B cells in autoimmunity, but emerging evidence suggests that marginal zone B cells contribute to the pathogenic autoantibody responses generated in NZB/NZW F₁ mice (6, 7, 8). We now provide evidence that the anti-DNA Abs induced in E2-treated nonspontaneously autoimmune mice may be secreted by autoreactive marginal zone B cells.

	Materials and Methods

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E2 treatment of mice

BALB/c mice (2–3 mo) were purchased from The Jackson Laboratory (Bar Harbor, ME). Transgenic R4A-IgG2b BALB/c mice (2–3 mo) were bred in a specific pathogen-free barrier facility. Mice were ovariectomized and treated with E2 time-release or placebo (P) pellets (Innovative Research of America, Saratoga, FL) (2).

Flow cytometry

Flow cytometry was performed using a FACSCalibur (BD Biosciences, San Jose, CA) and analyzed with FlowJo software (Tree Star, San Carlos, CA). Single cell suspensions from bone marrow or spleen were stained with mixtures of Abs specific for the following markers: B220, CD1, CD19, CD21, CD23, CD138, heat-stable Ag (HSA), IgD, IgM, B7.2, MHC class II (MHC II), CD44, and IgG2b. All Abs were purchased from BD PharMingen (San Diego, CA), except CD19 (Caltag Laboratories, Burlingame, CA).

CD4⁺ T cell depletion

Mice received i.p. injections (500 µg) of purified anti-CD4 GK1.5 mAb for 2 consecutive days. On day 3, E2 or P pellets were implanted in the mice. This protocol was repeated every 3 wk and was sufficient to deplete >98% of CD4⁺ T cells during the 5- to 7-wk E2 treatment period.

Immunohistochemistry

Frozen splenic sections (5 µm) were fixed in acetone. After blocking with 3% BSA/PBS, the slides were incubated with a 1/200 dilution of 7-amino-4-methylcoumarin-3-acetic acid-labeled anti-IgM (Jackson ImmunoResearch Laboratories, West Grove, PA) and Texas Red-labeled anti-IgG2b (BD PharMingen).

ELISPOT assay

Splenocytes from three E2-treated or three P-treated R4A-IgG2b BALB/c mice were pooled. B cells were isolated by depletion with biotin-labeled anti-CD43 and streptavidin-labeled Dynabeads (Dynal, Lake Success, NY) and stained with FITC-labeled anti-CD21 and PE-labeled anti-CD23. Follicular B cells (CD21^int/CD23⁺) and marginal zone B cells (CD21^high/CD23^neg-low) were isolated using a FACSVantage cell sorter (BD Biosciences) with >90% purity. Two-fold serial dilutions of cells starting from 1 x 10⁵ follicular B cells or 4 x 10⁴ marginal zone B cells were added in duplicate to microtiter wells coated with dsDNA (100 µg/ml) and incubated for 16 h at 37°C. IgG2b anti-DNA B cells were revealed by the addition of biotin-labeled anti-IgG2b and alkaline phosphatase-labeled streptavidin as previously described (2).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
E2 treatment activates a small population of B cells

To understand how a sustained increase in E2 abrogates B cell tolerance in R4A-IgG2b BALB/c mice, we asked whether E2-treated mice contain a large population of activated Tg⁺ B cells consistent with an E2-induced polyclonal activation. Flow cytometry was performed on splenocytes with Abs to B7.2, CD44, and MHC II and IgG2b to distinguish Tg⁺ B cells from the endogenous B cell population. The Tg⁺ B cell population of E2-treated mice displayed an increase in both the level of B7.2 and the percentage of B7.2⁺ B cells (Table I). However, there was only a modest increase in the level of CD44 expression and no change in the level of MHC II expression. Furthermore, there was no apparent increase in B cell size based on forward scatter flow cytometric profiles (data not shown). E2-treated mice displayed a 2- to 3-fold increase in Tg⁺ cells that were positive for CD138, a marker for plasma cells (Table I). Although the increase in B7.2 and CD138⁺ cells suggests that some of the Tg⁺ B cells were activated, the majority of splenic Tg⁺ B cells were not, arguing against the hypothesis that a sustained increase in E2 results in polyclonal B cell activation (9).

We examined the impact of a sustained increase of E2 on the distribution of splenic B cell subsets in E2-treated nontransgenic BALB/c mice, because it has been shown that transgenic expression of rearranged Ig genes may alter the normal distribution of B cell subsets (10). Furthermore, E2-treated BALB/c mice also exhibit a modest increase in anti-DNA Ab titers (Ref. 11 , and data not shown), suggesting that similar cellular activation occurs in R4A-IgG2b and nontransgenic mice. In agreement with previous reports of decreased lymphopoiesis with E2 treatment (12, 13, 14), flow cytometry revealed a reduction in bone marrow B220⁺ B cells in E2-treated BALB/c mice (Table II). The percentage of splenic B cells was unchanged; however, there was a shift in the immature and mature B cell compartments with reduced numbers of B220⁺/HSA^high transitional B cells and increased numbers of B220⁺/HSA^low-int mature B cells (Fig. 1A). The reduction of B220⁺/HSA^high B cells was due to a decrease in the percentage of CD21^neg/HSA^high transitional type 1 (T1) B cells, which represent the most immature splenic B cell and give rise to transitional type 2 (T2) B cells (15, 16). The CD21^high/HSA^high transitional T2 population, which differentiates into the mature splenic B cell populations, was slightly reduced in E2-treated mice. In fact, in E2-treated mice, there are similar numbers of T1 and T2 B cells, whereas in untreated mice, there are twice as many T1 as T2 B cells (Fig. 1B and Table II). The increased number of B220⁺/HSA^low-int mature splenic B cells in E2-treated mice was largely due to the 3- to 5-fold increase in CD21^high/HSA^int marginal zone B cells (Fig. 1B and Table II).

View larger version (65K): [in this window] [in a new window]   FIGURE 1. Altered distribution of B cell subsets in E2-treated mice. Splenocytes from nontransgenic BALB/c mice treated with E2 or P pellets (n = 3–7) were stained for B220, CD21, CD23, and HSA (A and B) or B220, CD1, CD23, and IgD (C and D). The immature HSAhigh population was reduced and the mature HSAlow-int population was increased in E2-treated mice. B, Analysis of CD21 and HSA revealed that the marginal zone (MZ) B cells were expanded, the transitional T1 and transitional T2 B cells were reduced, and the follicular cells (FO) were slightly increased in E2-treated mice. C, Staining with CD23 and CD1 revealed a similar distribution of B subsets in E2-treated mice as CD21 and HSA. D, Gating on CD23+/CD1high (dashed line) and CD23neg-low/CD1high cells (heavy line) to assess IgD expression confirmed that these populations correspond to transitional T2 (IgD+) and marginal zone (IgDneg-low) B cells, respectively.

The Tg⁺ transitional T1 B cell population was significantly increased following E2 treatment from 25 to 50% (p < 0.02; data not shown), suggesting that the expansion of Tg⁺ B cells occurs before the development of immature B cells into transitional T1 B cells and may reflect impaired negative selection in the bone marrow. In addition, a significant increase in the number of Tg-expressing CD1^high/CD21^high transitional T2 and marginal zone B cells, and CD1^int/CD21^int follicular B cells, was observed in E2-treated mice (Fig. 2). The expansion of Tg⁺ B cell subsets was not dependent on cognate CD4⁺ T cell interactions or CD4⁺ T cell-derived cytokines because depletion of CD4⁺ T cells did not block this expansion (Fig. 2). Thus, the rescued population of Tg⁺ B cells in E2-treated R4A-IgG2b mice differentiated into distinct B cell compartments before a stage at which cognate T cell help or T cell-derived cytokines are required.

View larger version (47K): [in this window] [in a new window]   FIGURE 2. Expansion of Tg+ B cells in the absence of CD4+ T cells. Splenocytes from P (n = 7), E2 (n = 7) and E2-CD4+ T cell-depleted (n = 5) transgenic mice were stained for CD19, CD1, CD21, and IgG2b. Expansion of the Tg+ B cells in both CD19+/CD1high/CD21high (T2/MZ populations) and CD1int/CD21int (FO) gated populations was observed in E2-treated mice. Depletion of CD4+ T cells before E2 treatment did not block expansion of mature Tg+ subsets. Representative profiles are shown. Percentage of Tg+ TZ/MZ B cells in P-treated mice = 25.6 ± 9.3%, E2-treated mice = 50.0 ± 8.5%, and E2-treated/CD4+-depleted = 55.9 ± 11.2%; the percentage of Tg+ FO B cells in P-treated mice = 12.9 ± 5.0%, E2-treated mice = 25.2 ± 6.2%, and E2-treated/CD4+-depleted = 27.4 ± 8.1%. A two-tailed Student’s t test revealed that the differences between the values of P and E2 or E2-CD4+ T cell-depleted-treated mice are significant (p < 0.005).

View larger version (65K): [in this window] [in a new window]   FIGURE 3. Localization of Tg+ anti-DNA B cells. A, Frozen splenic sections were stained for IgM (blue) to identify B cell regions and for IgG2b (yellow) to localize Tg+ B cells. The Tg+ B cells of P-treated mice were localized predominantly to the T/B cell interface (T). The spleens of E2-treated mice exhibited an increased number of Tg+ B cells that were found in B cell follicles (B), around the marginal sinus (M), and in the red pulp (R). Magnification, x10. B, ELISPOT assay was performed using cell-sorted marginal zone and follicular B cells. Cells were incubated in duplicate in dsDNA-coated microtiter wells. Both the marginal zone and follicular B cell populations isolated from E2-treated mice displayed an increase in the number of anti-DNA-secreting cells as compared with cells from P-treated mice; however, the increase in the number of anti-DNA marginal zone B cells was most prominent.

To determine which mature subsets were activated by E2 in vivo to secrete anti-DNA Ab, an ELISPOT assay was performed with isolated follicular and marginal zone B cells. Increases in both Tg⁺ follicular and marginal zone anti-DNA B cells from E2-treated R4A-IgG2b mice were observed (Fig. 3). However, the frequency of anti-DNA-secreting Tg⁺ marginal zone B cells was 10-fold higher than anti-DNA Tg⁺ follicular B cells. Thus, it appears that the increase in anti-dsDNA Ab titers in E2-treated R4A-IgG2b mice is largely due to the activation of autoreactive Tg⁺ marginal zone B cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
A sustained increase in E2 has been shown to accelerate the onset of disease and decrease the life span of mouse models of lupus (17, 18, 19) and to induce a lupus-like phenotype in some nonautoimmune-prone mouse strains (9, 11). We have been studying the impact of E2 administration on the regulation of autoreactive B cells. R4A-IgG2b BALB/c transgenic mice treated with E2 demonstrate survival and activation of B cells that have high affinity for DNA and unmutated light chain genes (2, 3), showing that E2 alters the selection of the naive repertoire of B cells exiting the bone marrow, and leads to the activation of these cells.

We found an altered maturation of transitional B cells with either rapid transit from the T1 to the T2 stage and/or a lack of deletion at the T2 stage. During the transitional stage, negative selection of autoreactive B cells that have not encountered self-Ag in the bone marrow is believed to occur (20, 21). Transitional T1 B cells are thought to arise from bone marrow immature B cells and to be direct precursors of transitional T2 B cells (15). Cross-linking of the B cell receptor mediates signal transduction events that direct the differentiation of transitional B cells into the mature marginal zone or follicular B cell subset (10, 15). The expansion of marginal zone B cells occurs in both R4A-IgG2b and nontransgenic BALB/c mice treated with E2. Therefore, it is possible that E2 alters signaling pathways to skew B cell development toward a marginal zone phenotype. Interestingly, mice transgenic for B cell activation factor from the TNF family display a lupus phenotype and exhibit a decrease in transitional T1 B cells and an expansion of transitional T2 and marginal zone B cells (16). This appears to be the result of impaired negative selection of autoreactive B cells and leads to a disproportionate number of marginal zone B cells in these mice (16).

The rescued autoreactive B cell population in R4A-IgG2b mice consists predominantly of marginal zone B cells. The role of marginal zone B cells in autoimmunity is not well understood. Anti-DNA Abs associated with lupus in both humans and mouse models display somatic mutations and have undergone isotype class switching, hallmarks of a T cell-dependent immune response (22, 23). Furthermore, the T cell-dependent follicular B cell subset is clearly a source of pathogenic autoantibodies, because CD4⁺ T cell depletion (24) or blockade of T cell costimulation (25) reduces autoantibody titers and prolongs the life of NZB/NZW F₁ mice. However, autoantibody production may not be limited to the follicular B cell subset, and the T cell-independent marginal zone B cell subset may also contribute to an autoimmune response. Marginal zone B cells are increased in young NZB/NZW F₁ mice (8). This increase is linked to the Nba2 locus that associates with nephritis (7). Furthermore, CD1^high B cells produce large amounts of anti-dsDNA IgM Ab in NZB/NZW F₁ mice (6), although it still remains to be established that marginal zone B cells secrete Abs that are pathogenic. It has also been suggested that T cell-independent B cells assist in the development of T cell-dependent B cell immune responses by providing a source of Ag-Ab complexes that are deposited on follicular dendritic cells (26). Marginal zone B cells may act as APCs (27), potentially triggering a T cell-dependent follicular B cell response. It has also been shown that CD40-CD40 ligand interactions activate marginal zone B cells (28). It is interesting to note that in the absence of CD4⁺ T cells, the E2-induced expansion of the Tg⁺ marginal zone B cell population still occurred, but did not result in elevated serum IgG2b anti-DNA Ab titers (data not shown).

An expansion of Tg⁺ follicular B cells and a modest increase in the number of anti-dsDNA follicular B cells occur in E2-treated R4A-IgG2b mice. These cells may also contribute to the serum anti-dsDNA response. We propose that E2 rescues autoreactive transitional B cells from deletion; most differentiate to marginal zone B cells, and some to follicular B cells. Activation of Tg⁺ follicular B cells in E2-treated mice would require the presence of activated autoreactive T cells because it has been shown that anti-dsDNA B cells are routinely generated in BALB/c mice (29, 30), but are not typically activated due to an absence of Ag-specific T cells (31, 32). We hypothesize that there is a small number of anti-DNA follicular B cells that are rescued but not activated by E2. Perhaps these cells differ in fine specificity from anti-DNA marginal zone B cells, as there is evidence that the antigenic specificity of B cells contributes to the determination of their subset phenotype. Whether the antigenic specificities of the Tg⁺ marginal zone actually differ from those of follicular B cells needs further study.

In conclusion, E2 treatment of BALB/c mice alters B cell development and expands the population of marginal zone B cells. The increased maturation and activation of autoreactive marginal zone B cells in E2-treated mice, as well as in B cell activation factor for the TNF family mice and NZB/NZW F₁ mice, may be mediated by a common defect in the tolerance induction pathway. Continued analysis of autoreactive marginal zone B cells should provide insight into the role of T cell-independent B cell responses in the development of B cell-mediated autoimmune diseases.

	Acknowledgments

 
We thank Matthew Scharff and Elena Peeva for critical review of this manuscript.

	Footnotes

 
¹ This work was supported by grants from the National Institutes of Health (to B.D.) and the New York Chapter of the Arthritis Foundation (to C.M.G.). C.M.G. is a fellow of the Irvington Institute for Immunological Research, and D.J.M. is funded by National Institutes of Health Medical Scientist Training Grant T32-GM07288.

² Address correspondence and reprint requests to Dr. Betty Diamond, Albert Einstein College of Medicine, Department of Microbiology and Immunology, 1300 Morris Park Avenue, Forchheimer Building 405, Bronx, NY 10461. E-mail address: diamond{at}aecom.yu.edu

³ Abbreviations used in this paper: E2, 17 -estradiol; P, placebo; Tg, transgene; MFI, mean fluorescence intensity; HSA, heat-stable Ag; MHC II, MHC class II; T1, type 1; T2, type 2.

Received for publication April 23, 2001. Accepted for publication June 20, 2001.

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				J. Venkatesh, E. Peeva, X. Xu, and B. Diamond Cutting Edge: Hormonal Milieu, Not Antigenic Specificity, Determines the Mature Phenotype of Autoreactive B Cells J. Immunol., March 15, 2006; 176(6): 3311 - 3314. [Abstract] [Full Text] [PDF]

				L. S. Ackerman Sex hormones and the genesis of autoimmunity. Arch Dermatol, March 1, 2006; 142(3): 371 - 376. [Abstract] [Full Text] [PDF]

				C. M. Grimaldi, V. Jeganathan, and B. Diamond Hormonal Regulation of B Cell Development: 17beta-Estradiol Impairs Negative Selection of High-Affinity DNA-Reactive B Cells at More Than One Developmental Checkpoint. J. Immunol., March 1, 2006; 176(5): 2703 - 2710. [Abstract] [Full Text] [PDF]

				C. S. Goodyear, F. Sugiyama, and G. J. Silverman Temporal and Dose-Dependent Relationships between In Vivo B Cell Receptor-Targeted Proliferation and Deletion-Induced by a Microbial B Cell Toxin J. Immunol., February 15, 2006; 176(4): 2262 - 2271. [Abstract] [Full Text] [PDF]

				L. Delpy, V. Douin-Echinard, L. Garidou, C. Bruand, A. Saoudi, and J.-C. Guery Estrogen Enhances Susceptibility to Experimental Autoimmune Myasthenia Gravis by Promoting Type 1-Polarized Immune Responses J. Immunol., October 15, 2005; 175(8): 5050 - 5057. [Abstract] [Full Text] [PDF]

				J. E. Wither, C. Loh, G. Lajoie, S. Heinrichs, Y.-C. Cai, G. Bonventi, and R. MacLeod Colocalization of Expansion of the Splenic Marginal Zone Population with Abnormal B Cell Activation and Autoantibody Production in B6 Mice with an Introgressed New Zealand Black Chromosome 13 Interval J. Immunol., October 1, 2005; 175(7): 4309 - 4319. [Abstract] [Full Text] [PDF]

				K. D. Salazar, P. de la Rosa, J. B. Barnett, and R. Schafer The Polysaccharide Antibody Response after Streptococcus pneumoniae Vaccination Is Differentially Enhanced or Suppressed by 3,4-Dichloropropionanilide and 2,4-Dichlorophenoxyacetic Acid Toxicol. Sci., September 1, 2005; 87(1): 123 - 133. [Abstract] [Full Text] [PDF]

				A. M. Jacobi and B. Diamond Balancing diversity and tolerance: lessons from patients with systemic lupus erythematosus J. Exp. Med., August 1, 2005; 202(3): 341 - 344. [Abstract] [Full Text] [PDF]

				E. Peeva, J. Venkatesh, and B. Diamond Tamoxifen Blocks Estrogen-Induced B Cell Maturation but Not Survival J. Immunol., August 1, 2005; 175(3): 1415 - 1423. [Abstract] [Full Text] [PDF]

				Z. Xu, B. Duan, B. P. Croker, E. K. Wakeland, and L. Morel Genetic Dissection of the Murine Lupus Susceptibility Locus Sle2: Contributions to Increased Peritoneal B-1a Cells and Lupus Nephritis Map to Different Loci J. Immunol., July 15, 2005; 175(2): 936 - 943. [Abstract] [Full Text] [PDF]

				Y. Chen, D. Perry, S. A. Boackle, E. S. Sobel, H. Molina, B. P. Croker, and L. Morel Several Genes Contribute to the Production of Autoreactive B and T Cells in the Murine Lupus Susceptibility Locus Sle1c J. Immunol., July 15, 2005; 175(2): 1080 - 1089. [Abstract] [Full Text] [PDF]

				J. Rolf, V. Motta, N. Duarte, M. Lundholm, E. Berntman, M.-L. Bergman, L. Sorokin, S. L. Cardell, and D. Holmberg The Enlarged Population of Marginal Zone/CD1dhigh B Lymphocytes in Nonobese Diabetic Mice Maps to Diabetes Susceptibility Region Idd11 J. Immunol., April 15, 2005; 174(8): 4821 - 4827. [Abstract] [Full Text] [PDF]

				C. M. Grimaldi, R. Hicks, and B. Diamond B Cell Selection and Susceptibility to Autoimmunity J. Immunol., February 15, 2005; 174(4): 1775 - 1781. [Abstract] [Full Text] [PDF]

				R. Brummel and P. Lenert Activation of Marginal Zone B Cells from Lupus Mice with Type A(D) CpG-Oligodeoxynucleotides J. Immunol., February 15, 2005; 174(4): 2429 - 2434. [Abstract] [Full Text] [PDF]

				D. Panagopoulos, P. Victoratos, M. Alexiou, G. Kollias, and G. Mosialos Comparative Analysis of Signal Transduction by CD40 and the Epstein-Barr Virus Oncoprotein LMP1 In Vivo J. Virol., December 1, 2004; 78(23): 13253 - 13261. [Abstract] [Full Text] [PDF]

				J. M. Ward, N. P. Nikolov, J. R. Tschetter, J. B. Kopp, F. J. Gonzalez, S. Kimura, and R. M. Siegel Progressive Glomerulonephritis and Histiocytic Sarcoma Associated with Macrophage Functional Defects in CYP1B1-Deficient Mice Toxicol Pathol, October 1, 2004; 32(6): 710 - 718. [Abstract] [PDF]

				S. Atencio, H. Amano, S. Izui, and B. L. Kotzin Separation of the New Zealand Black Genetic Contribution to Lupus from New Zealand Black Determined Expansions of Marginal Zone B and B1a Cells J. Immunol., April 1, 2004; 172(7): 4159 - 4166. [Abstract] [Full Text] [PDF]

				N.-H. Chang, R. MacLeod, and J. E. Wither Autoreactive B Cells in Lupus-Prone New Zealand Black Mice Exhibit Aberrant Survival and Proliferation in the Presence of Self-Antigen In Vivo J. Immunol., February 1, 2004; 172(3): 1553 - 1560. [Abstract] [Full Text] [PDF]

				M. Batten, C. Fletcher, L. G. Ng, J. Groom, J. Wheway, Y. Laabi, X. Xin, P. Schneider, J. Tschopp, C. R. Mackay, et al. TNF Deficiency Fails to Protect BAFF Transgenic Mice against Autoimmunity and Reveals a Predisposition to B Cell Lymphoma J. Immunol., January 15, 2004; 172(2): 812 - 822. [Abstract] [Full Text] [PDF]

				Y. Qian, H. Wang, and S. H. Clarke Impaired Clearance of Apoptotic Cells Induces the Activation of Autoreactive Anti-Sm Marginal Zone and B-1 B Cells J. Immunol., January 1, 2004; 172(1): 625 - 635. [Abstract] [Full Text] [PDF]

				J.-Q. Yang, A. K. Singh, M. T. Wilson, M. Satoh, A. K. Stanic, J.-J. Park, S. Hong, S. D. Gadola, A. Mizutani, S. R. Kakumanu, et al. Immunoregulatory Role of CD1d in the Hydrocarbon Oil-Induced Model of Lupus Nephritis J. Immunol., August 15, 2003; 171(4): 2142 - 2153. [Abstract] [Full Text] [PDF]

				T. G. Phan, M. Amesbury, S. Gardam, J. Crosbie, J. Hasbold, P. D. Hodgkin, A. Basten, and R. Brink B Cell Receptor-independent Stimuli Trigger Immunoglobulin (Ig) Class Switch Recombination and Production of IgG Autoantibodies by Anergic Self-Reactive B Cells J. Exp. Med., April 7, 2003; 197(7): 845 - 860. [Abstract] [Full Text] [PDF]

H. Amano, E. Amano, T. Moll, D. Marinkovic, N. Ibnou-Zekri, E. Martinez-Soria, I. Semac, T. Wirth, L. Nitschke, and S. Izui The Yaa Mutation Promoting Murine Lupus Causes Defective Development of Marginal Zone B Cells J. Immunol., March 1, 2003; 170(5): 2293 - 2301. [Abstract] [Full Text] [PDF]