Tolerance Checkpoints That Restrict Pathways of -Driven Differentiation Jacqueline William, Chad Euler, Nicole Primarolo and Mark J. Shlomchik This information is current as of September 23, 2021. J Immunol 2006; 176:2142-2151; ; doi: 10.4049/jimmunol.176.4.2142 http://www.jimmunol.org/content/176/4/2142 Downloaded from

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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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

B Cell Tolerance Checkpoints That Restrict Pathways of Antigen-Driven Differentiation1

Jacqueline William,* Chad Euler,† Nicole Primarolo,† and Mark J. Shlomchik2*,†

Autoreactive B cells can be regulated by deletion, receptor editing, or anergy. Rheumatoid factor (RF)-expressing B in normal mice are not controlled by these mechanisms, but they do not secrete and were presumed to ignore self-Ag. Surprisingly, we now find that these B cells are not quiescent, but instead are constitutively and specifically activated by self-Ag. In BALB/c mice, RF B cells form germinal centers (GCs) but few Ab-forming cells (AFCs). In contrast, autoimmune mice that express the autoantigen readily generate RF AFCs. Most interestingly, autoantigen-specific RF GCs in BALB/c mice appear defective. B cells in such GCs neither expand nor are selected as efficiently as equivalent cells in autoimmune mice. Thus, our data establish two novel checkpoints of autoreactive B cell regulation that are engaged only after initial autoreactive B cell activation:

one that allows GCs but prevents AFC formation and one that impairs selection in the GC. Both of these checkpoints fail in Downloaded from . The Journal of Immunology, 2006, 176: 2142–2151.

igh autoantibody titers are a hallmark of systemic auto- syndrome, mixed cryoglobulinemia, and SLE (2, 20, 21), and by immune diseases such as rheumatoid arthritis (RA)3 and Fas-deficient lpr mice (22). H systemic lupus erythematosus (SLE) (1, 2). This must To study RF B cells, we have used AM14 H chain Tg mice. represent a breakdown of tolerance mechanisms that normally reg-

When this H chain is paired with a Tg-encoded or endogenous http://www.jimmunol.org/ ulate autoreactive B cells. prevents the develop- V␬8 L chain, the result is an Ab that binds the Fc portion of ment of high-affinity self-reactive B cells. Mechanisms of central IgG2aa, but not IgG2ab (23). The AM14 Tg mouse is an ideal tolerance, such as deletion, anergy, and receptor editing, were model because it combines specificity for a disease-related autoan- demonstrated using mice with Ig transgenes (Tg) specific for ar- tigen and the ability to study the regulation of the B cells in the tificial autoantigens, like hen egg lysozyme or MHC class I pro- presence or absence of autoantigen (in IgHa or IgHb congenic teins (3–6). There are also mechanisms for B mice). This Tg system uniquely allows for measurement and con- cells (7), but these are much less well defined. To initiate systemic trol of the presence or absence of a disease-related autoantigen. autoimmunity, either central tolerance mechanisms must be over- This ability is useful for distinguishing specific from nonspecific B come or mechanisms that regulate peripheral B cells must by guest on September 23, 2021 cell activation. We have found that B cells in autoantigen-positive break down. (IgHa) BALB/c mice Tg for both the AM14 H and L chains were Our laboratory and others have used Ig-Tg models to study how neither anergized nor deleted during development (23, 24). How- B cells specific for disease-related self-Ags are regulated (8–15). ever, in Fas-deficient H and HL Tg mice, RF B cells differentiated In SLE, only certain Ags efficiently elicit autoreactive B cells: into numerous Ab-forming cells (AFCs). The formation of AFCs chromatin, ribonucleoproteins, and IgG (2, 16). These autoanti- was autoantigen-driven, because IgHb congenic mice that lacked gens must have special properties that render them preferred tar- gets when immune regulation is abnormal. These properties, which the autoantigen failed to produce RF AFCs (25). We have recently include the ability to provide an endogenous ligand for certain further investigated the fates of RF B cells in autoimmune-prone Toll-like receptors (17, 18), are currently being elucidated. It is mice, revealing a dominant short-lived plasmablast response (26, thus important to study B cells specific for relevant autoantigens. 27). However, the differences between RF B cell fates as revealed We have focused on the regulation of rheumatoid factor (RF)- by our recent studies of MRL/lpr mice and our earlier work on expressing B cells that bind to the constant region of Ig (19). RFs BALB/c mice (23, 24) indicated the presence of additional mech- are prevalent in RA patients but are also produced by patients with anisms that regulate naive but fully developed RF B cells. These a number of other systemic autoimmune diseases such as Sjogren’s mechanisms are presumably intact in BALB/c mice but are breached in MRL/lpr mice. In this study, we have used the AM14 model to identify these *Section of Immunobiology and †Department of Laboratory Medicine, Yale Univer- sity School of Medicine, New Haven, CT 06520 mechanisms by comparing the regulation of RF B cells in MRL/lpr Received for publication July 7, 2005. Accepted for publication November 28, 2005. to the BALB/c and MRL ϩ/ϩ strains. We found that normal The costs of publication of this article were defrayed in part by the payment of page BALB/c mice do not have RF AFCs; at this level, the mice remain charges. This article must therefore be hereby marked advertisement in accordance apparently self-tolerant. However, we were surprised to find that with 18 U.S.C. Section 1734 solely to indicate this fact. despite the lack of RF AFCs, RF B cells are not quiescent in 1 This work was supported by National Institute of Health Grants P01 AI36529 and BALB/c mice. Rather, they actively participate in germinal centers R01 AR44077. 2 (GCs). Importantly, this activation of RF B cells is autoantigen- Address correspondence and reprint requests to Dr. Mark J. Shlomchik, Department b of Laboratory Medicine, Yale University School of Medicine, 333 Cedar Street, Box specific, because it is absent in the autoantigen-negative IgH 208035, New Haven, CT 06520-8035. E-mail address: [email protected] strains. Furthermore, RF B cells undergo in 3 Abbreviations used in this paper: RA, rheumatoid arthritis; SLE, systemic lupus GCs of BALB/c mice, which could potentially lead to the gener- erythematosus; Tg, transgene; RF, rheumatoid factor; AFC, Ab-forming cell; GC, germinal center; PNA, peanut agglutinin; TNP, trinitrophenol; KLH, keyhole limpet ation of high-affinity . Nonetheless, analysis of so- hemocyanin; MLN, mesenteric lymph node; IC, ; LN, lymph node. matic hypermutation patterns of V regions from RF B cells in

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 2143

BALB/c mice compared with those from autoimmune mice re- mune-prone/Fas-deficient (MRL/lpr) mice, spleens and mesenteric vealed a failure to efficiently select and expand RF B cell clones. lymph nodes (MLNs) of IgHa and IgHb Tg mice of ages ranging Overall, our data show that Ag-specific autoimmunity can proceed from 10 to 44 wk were examined by histology and FACS. Unex- much further than originally thought even in normal animals, but pectedly, we found that RFϩ (i.e., 4-44ϩ) GCs were common in that diversion from AFC production and regulation in the GC can the follicles of most normal Tg BALB/c spleens (Fig. 1G, M). GCs prevent the pathologic consequences. were also found in MRL ϩ/ϩ spleens (Fig. 1, H, J, N, and P). In contrast, CB.17 (IgHb) and IgHb MRL ϩ/ϩ congenic strains that Materials and Methods lacked the autoantigen had very few 4-44ϩ cells and no detectable Mice 4-44ϩ GCs (Fig. 1, E, F, K, L, Q, and R). The absence of RFϩ GCs b ϩ AM14 H Tg MRL ϩ/ϩ mice were made by backcrossing Tg BALB/c mice in the IgH mice demonstrates that the RF GCs in BALB/c and (23) at least 10 generations to MRL ϩ/ϩ mice (The Jackson Laboratory). MRL ϩ/ϩ mice were spontaneously induced by self-Ag. These Congenic Tg BALB/c IgHb mice (CB.17) were made by crossing AM14Tg data are quantitated in Fig. 2A and corroborated by FACS data BALB/c to CB.17 mice (Taconic). Tg MRL ϩ/ϩ IgHb mice were made by presented below. similar crosses between the AM14 Tg MRL ϩ/ϩ mice and MRL ϩ/ϩ b ϩ ϩ b Although both normal BALB/c and autoimmune-prone MRL IgH mice. MRL / IgH mice were made by a series of crosses of ϩ ϩ ϩ ϩ MRL/lpr IgHb (a gift from Robert Eisenberg, University of Pennsylvania, / strains had RF GCs, they had few dark staining 4-44 cells Philadelphia, PA (28)) with MRL ϩ/ϩ mice and screening for homozy- outside of follicles (Fig. 1, A–C, G–I, and S–U). In contrast, MRL/ gous Faswt mice. Mice were housed under specific pathogen-free lpr mice had very few RFϩ GCs but rather had large numbers of conditions. RF AFCs at the T zone-red pulp border (Figs. 1, C, I, and O, and Histology 2A), as reported previously (25). These darkly staining extrafol- Downloaded from licular cells very likely represent AFCs, as documented in detail in Cells were stained as described previously (29). The following Abs were used: 4-44 FITC (anti-Id), anti-CD21/35-biotin (CR1; BD Pharmingen), Ref. 27. In agreement with the histology, the median numbers of peanut agglutinin (PNA)-biotin (Vector Laboratories), 30H12-biotin (anti- AFCs/million splenocytes in the Tg BALB/c, MRL ϩ/ϩ were 25- CD90.2), and 30H12-FITC. Unless purchased as indicated, Abs were made and 16-fold lower than in MRL/lpr mice, respectively (Fig. 2B). and conjugated as described previously (23). For immunohistochemistry, Because MRL ϩ/ϩ mice develop autoimmunity much later in anti-FITC-alkaline phosphatase (Molecular Probes) and streptavidin-HRP life than MRL/lpr mice (30), we also examined spleens from Tg http://www.jimmunol.org/ (Southern Biotechnology Associates) were developed with Fast Blue BB or ϩ ϩ a 3-amino-9-ethyl-carbazole (Sigma-Aldrich), as described previously (24). MRL / IgH mice 40 wk of age and older. These spleens had RFϩ GCs (Fig. 1, J and P), like those of younger mice. However, ELISPOTs in contrast to the younger mice, the spleens of older mice had ϩ ELISPOTs were performed as described previously (24). clusters of darkly staining 4-44 cells at the T zone-red pulp bor- der, similarly to younger Tg MRL/lpr mice (Fig. 1D). Thus, with Sequencing sufficient age, RF B cells were driven by autoantigen to AFC pro- 4-44ϩ clusters were microdissected from stained sections, V␬8/J4 rear- duction, even in Fas-intact but autoimmune-prone mice. Failure to ranged DNA amplified with PFU Turbo (Stratagene), cloned, and se- regulate RF B cell AFC differentiation was specific to the MRL quenced as described previously (25). Sequences were aligned using La- by guest on September 23, 2021 sergene DNA analysis software (DNASTAR), and all mutation data was strains, because even older BALB/c mice never developed appre- compiled into a custom database program written in Filemaker Pro for ciable numbers of such cells (data not shown). further statistical analysis.

Cell isolation and FACS analysis Autoantigen drives the accumulation of RF B cells in BALB/c FACS analysis was performed as described previously (24). The following and MRL ϩ/ϩ mice Abs were used: 4-44-biotin (anti-Id), 4-44-Alexa 488, anti-phagocytic gly- In addition to differentiating into either GC cells or AFCs, as just coprotein-1-FITC (anti-CD44), anti-CD80-PE (BD Pharmingen), anti- ϩ ϩ a CD22.2-FITC (BD Pharmingen), and anti-CD22.2-PE (BD Pharmingen). described, RF (4-44 ) cells accumulated in the spleens of IgH Streptavidin-allophycocyanin (Serologicals) was used to detect biotinyl- mice. Mice were examined at 18–46 wk of age (Fas-sufficient ated reagents. Propidium iodide was used to detect dead cells. strains) or 11–22 wk of age (Fas-deficient strains). Among total ϩ Immunizations live lymphocytes, 4-44 cells comprised 4.5% in Tg BALB/c spleens vs 1.6% in CB.17 ( p Ͻ 0.0024) and 4.4% in MRL ϩ/ϩ vs H Tg mice were immunized i.p. with 50 ␮g of the 2,4,6-trinitrophenol 1.9% in MRL ϩ/ϩ IgHb ( p Ͻ 0.0001). However, presence of the (TNP)-specific IgG2a Ab, Hy1.2, complexed with 12.5 ␮g of TNP-keyhole ϩ autoantigen did not cause a significant increase in 4-44 cells in limpet hemocyanin (KLH) in alum. Alum-precipitated Hy1.2-TNP-KLH b complexes (24) were made by incubating purified Hy1.2 with TNP-KLH at the MRL/lpr vs MRL/lpr.IgH (1.5 vs 1.2%; p ϭ 0.68; Fig. 3, A–F the appropriate ratio in PBS for 1 h at 37°C. Spleens were harvested on and M), despite the increased numbers of AFCs (Fig. 2B). Because days 5, 8, and 12 of immunization. 4-44ϩ AFCs in Tg MRL/lpr IgHa mice express normal levels of surface Ig (27), they would have been included in the FACS anal- Results ϩ ysis. Their failure to accumulate is most likely because the AFCs Autoantigen drives the formation of RF GCs, but very few are plasmablasts, and their rapid death rate prevents substantial ϩ ϩ AFCs, in the spleens of BALB/c and MRL / mice numerical accumulation (27). In AM14 H Tg mice, a few percentage of B cells that naturally CD22 is down-regulated on AFCs, but expressed at normal lev- express certain V␬8 L chains are specific for IgG2aa but not els on mature and activated B cells (27). Consistent with the large IgG2ab (23, 25–27). This population is detected by the mAb 4-44, numbers of RF AFCs, a significant proportion of the 4-44ϩ cells in which is specific for the AM14 H chain when combined with one IgHa MRL/lpr mice was CD22low (36%). However, only a very of two homologous V␬8 genes, each of which confers RF speci- small percentage of the 4-44ϩ cells in BALB/c and MRL ϩ/ϩ ficity. H Tg mice are ideal to study how an autoreactive B cell is mice was CD22low (2.4 and 3.7%, respectively; Fig. 3N), again in regulated because of the ease of detecting clonal expansion and concert with the paucity of AFCs in these strains. The frequencies activation of RF B cells. of CD22low cells in BALB/c and MRL ϩ/ϩ mice were not sig- To determine how autoantigen determines the fate of RF B cells nificantly different from their IgHb congenics. Thus, the increase in in normal (BALB/c), autoimmune-prone (MRL ϩ/ϩ), or autoim- 4-44ϩ cells in BALB/c and MRL ϩ/ϩ mice is due to relative 2144 REGULATING AUTOREACTIVE B CELLS AFTER ACTIVATION Downloaded from http://www.jimmunol.org/

FIGURE 1. Histologic sites of autoantigen-driven RF B cell activation and differentiation. A–F, 4-44 spleen sections from IgHa and IgHb Tg BALB/c, MRL ϩ/ϩ, and MRL/lpr mice were stained with 4-44 (blue) and anti-Thy 1.2 (red). 4-44ϩ cells are distributed in a follicular and marginal zone pattern a ϩ a in IgH BALB/c and MRL ϩ/ϩ spleens (A and B). 4-44 cells in IgH MRL/lpr spleens have very dark (blue) cytoplasmic staining and accumulate outside by guest on September 23, 2021 the B cell follicles in the T zone-red pulp borders (C). Adjacent sections (G–L) were stained with 4-44 (blue) and PNA (red), to identify GCs (arrows). M–R, Magnifications of the boxed regions shown in G–L. Note 4-44ϩ cells in the GCs of BALB/c and MRL ϩ/ϩ Tg mice (G, H, M, and N). However, Tg MRL/lpr spleens rarely have 4-44ϩ cells in their GCs, as shown by the 4-44-negative GC in I and O. Spleens from aged (Ͼ40-wk-old) IgHa Tg MRL ϩ/ϩ mice (J and P) partially resemble Tg MRL/lpr spleens (D), but are notable for the additional presence of 4-44ϩ GCs (arrows). No accumulation of 4-44ϩ cells was noted in IgHb mice, which lack the autoantigen (E, F, K, L, Q, and R). S–X, Adjacent spleen sections of IgHa Tg BALB/c (S and V), MRL ϩ/ϩ (T and W), and MRL/lpr (U and X) mice were stained with anti-Thy1.2 (red)/4-44 (blue) (S–U) and CR1 (red)/Thy1.2 (blue) (V–X). These panels demonstrate the follicular (BALB/c and MRL ϩ/ϩ) and extrafollicular (MRL/lpr) locations of 4-44ϩ cells. For Thy1.2/PNA/4-44 stains to document GCs, data are representative of 18 BALB/c, 17 MRL ϩ/ϩ, and 72 MRL/lpr mice. CR1 staining was performed on at least four mice per group. expansion of the CD22high subpopulation in the IgHa vs IgHb analyzed expression of B cell activation markers by FACS. The strains. percentage of CD22high RF B cells that express CD44 and CD80 To determine the location of the expanded population of RF B was substantially higher in the presence of autoantigen (IgHa cells in the Tg BALB/c and MRL ϩ/ϩmice, we stained sections strains; Fig. 3, G, H, J–K, P, and Q). Interestingly, although 4-44ϩ/ with anti-CD21, which darkly stains follicular dendritic cells (Fig. CD22high compartment of cells did not expand as a percentage of 1, S–X). In Tg IgHa BALB/c and MRL ϩ/ϩ mice, 4-44ϩ cells are total lymphocytes in MRL/lpr mice compared with MRL/lpr.IgHb in follicles, colocalizing with CD21, whereas 4-44ϩ B cells in the congenics, they were nonetheless activated, as indicated by the Tg MRL/lpr mice generally do not colocalize with CD21 and are increased fraction of them that expressed CD44 and CD80 (Fig. 3, extrafollicular. Thus, the presence of self-Ag not only stimulates a I, L, P, and Q). GC response in BALB/c and MRL ϩ/ϩ mice, it also leads to Because GC cells are CD22high and express activation markers accumulation of CD22high, RF B cells that reside in follicles and like CD80, it was not surprising that we found increased numbers are neither in GCs nor are AFCs. It is important to note in com- of 4-44ϩ/CD22high cells that also had higher levels of CD44 and/or paring FACS and histologic data that the latter has a much lower CD80 in IgHa Fas-sufficient mice compared with their IgHb coun- dynamic range and that rare scattered cells are difficult to appre- terparts (Fig. 3, P and Q; see legend for p values). However, we ϩ ciate if weakly stained. In contrast, clusters of activated 4-44 were surprised to find that in these same strains, quiescent 4-44ϩ/ cells, most likely with increased intracellular and surface Ig, are CD22high cells were also increased in frequency in IgHa vs IgHb more easily appreciated on histologic analysis. mice. This was indicated by the percentages of 4-44ϩ/ high low ϩ high CD22 CD80 cells (Fig. 3O), which are substantially higher In the presence of autoantigen, 4-44 CD22 cells have in IgHa than IgHb strains (BALB/c; p ϭ 0.06; MRL ϩ; p ϭ increased expression of costimulatory molecules 0.0067). Analysis of 4-44ϩ/CD22highCD44low cells led to similar To determine whether this expansion of CD22high RF B cells in the conclusions (data not shown). Thus, Fas-sufficient mice, including Fas-sufficient strains is in part due to activation of RF B cells, we the normal strain BALB/c, not only activate RF B cells in the The Journal of Immunology 2145 Downloaded from FIGURE 2. Numbers of RFϩ AFCs and GCs in Tg BALB/c, MRL ϩ/ϩ, and MRL/lpr mice. A, The number of 4-44ϩ GCs per plane of section (bars are 1 SEM). The number of 4-44ϩ GCs in MRL/lpr spleens compared with BALB/c and MRL ϩ/ϩ spleens is significantly lower (p Ͻ 0.0001). B, 4-44ϩ AFCs were detected by ELISPOT. Each diamond is an individual mouse and bars represent means. Tg IgHa MRL/lpr spleens have much greater numbers of 4-44ϩ AFCs compared with Tg IgHa BALB/c and MRL ϩ/ϩ spleens (p Ͻ 0.0001). http://www.jimmunol.org/ presence of autoantigen, but they also accumulate RF B cells with that other accessory factors in the mice (e.g., costimulatory sig- a resting phenotype. Such accumulation could represent resting RF nals) differ in the induced vs spontaneous response. Finally, be- memory B cells that had been activated previously and/or naive B cause GCs but not AFCs were induced in both IgHa and IgHb cells that are positively selected in the presence of autoantigen but congenics, we conclude that the presence of the endogenous au- without frank activation (31–33). toantigen does not suppress the RF response.

Immunization with IgG2a-containing immune complexes (ICs) Activation of RF B cells in the lymph node (LN) induces a strong GC response but a very weak AFC response in Having defined autoantigen-specific RF B cell activation in the all strains spleen, we next investigated LNs. Like the spleen, MLNs of Tg by guest on September 23, 2021 Data presented above show that Fas-sufficient mice activate B cells BALB/c and MRL ϩ/ϩ mice frequently had 4-44ϩ GCs (Fig. 5). and make GCs, but generate few AFCs; whereas, Fas-deficient As for the spleen, such GCs were absent in IgHb congenics (data mice generate few GCs in spleen but large numbers of AFCs. This not shown), indicating that those in IgHa mice are autoantigen could be due to inherent differences between B cells in these driven. Despite the absence of RFϩ GCs in their spleens, a fraction strains. Alternatively, it is possible that the nature of the autoan- of MRL/lpr mice did have RFϩ GCs in MLNs (Fig. 5, C and G) tigen stimulus is different in each strain and that this determines the and Peyer’s patches (data not shown). BALB/c and MRL/lpr mice quality of the response. had similar average numbers of 4-44ϩ GCs in their MLNs, To investigate these possibilities, we immunized Tg BALB/c whereas MRL ϩ/ϩ mice had three times as many (Fig. 5D). In and MRL/lpr mice (and their IgHb congenics) with addition to GCs, most MRL/lpr and some MRL ϩ/ϩ MLNs also IgG2a-containing ICs comprised of IgG2aa anti-TNP and TNP- had numerous darkly staining 4-44ϩ cells occupying large portions KLH in alum. We used 5- to 6-wk-old mice; Tg MRL/lpr mice at of the medullary region (Fig. 5, B and C); the dark staining and this age do not have spontaneous 4-44ϩ AFCs, and their splenic characteristic location indicate that these cells represent AFCs. A architecture is normal (26). All strains formed RFϩ GCs, histo- qualitative assessment of these indicated that MRL/lpr MLNs had logically similar to those that formed spontaneously in Tg BALB/c approximately three times as many of these cells as the MRL ϩ/ϩ mice (Fig. 4, E–H). However, very few 4-44ϩ AFCs were formed, MLNs (Fig. 5H). These dark-staining cells in the medulla were even in the Fas-deficient or IgHb mice, as indicated by a lack of absent in BALB/c MLNs (Fig. 5, A and H). Thus, at the level of darkly staining 4-44ϩ cells outside of follicles (compare with Fig. GC formation, regulation of autoreactive RF B cells is lost in all 1, I and J) and as confirmed by ELISPOT analysis (data not three strains in LN, but only MRL/lpr and MRL ϩ mice had shown). Responses in all strains were also similar on days 5 (data AFCs. Consequently, in the LN, like in the spleen, a tolerance not shown) and 12 (Fig. 4, I and J, and data not shown). These mechanism that prevents AFC formation operates in BALB/c mice results indicate that ICs of IgG2a and foreign protein are sufficient but is only partially effective in MRL ϩ mice and is ineffective in to stimulate RF B cells, but they elicit mainly GCs rather than MRL/lpr mice. AFCs, regardless of strain background. Because Tg MRL/lpr mice form GCs in response to immunization, we conclude that the ab- Different patterns of somatic mutations in BALB/c and MRL/lpr sence of spontaneous RFϩ GCs in MRL/lpr spleens (Fig. 1) was GCs due neither to an inherent inability of the RF B cells to form GCs Considering that BALB/c mice are not autoimmune-prone, we did nor to an environment that could not support GCs. Rather, these not expect to find autoantigen-driven RFϩ GCs in both spleen and results make it more likely that either the endogenous form of the LNs. It would seem risky to allow selection of higher affinity mu- IgG2a autoantigen is different from injected ICs, leading to a dif- tant (autoreactive) B cells, as could occur in the GC. Therefore, it ferent quality of B cell stimulus and thus differentiation path, or was important to determine whether such selection was regulated 2146 REGULATING AUTOREACTIVE B CELLS AFTER ACTIVATION Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 3. Phenotype of activated 4-44ϩ cells in Tg BALB/c, MRL ϩ/ϩ, and MRL/lpr mice. A–F, Splenocytes from Tg IgHa BALB/c (A), MRL ϩ/ϩ (B), MRL/lpr (C), and congenic IgHb Tg mice (D–F) were analyzed by FACS for CD22 (x-axis) and 4-44 (y-axis). G–L, Histograms of gated 4-44ϩ, CD22high splenocytes showing expression of CD44 (G–I) and CD80 (J–L) for IgHa (bold line) and IgHb (dotted line) mice. M–Q, Each diamond is an individual mouse, and bars represent means. M, Percentage of splenocytes that are 4-44ϩ. N, Percentage of gated 4-44ϩ splenocytes that are CD22low. O, Percentage of live splenocytes that are resting 4-44ϩ cells, as indicated by a 4-44ϩ, CD22high and CD80low phenotype. P and Q, Percentage of gated 4-44ϩ, CD22high splenocytes that are CD44high and CD80high, respectively. IgHa strains have significantly higher percentages than their IgHb counterparts (CD44: BALB/c, p Ͻ 0.01; MRL ϩ/ϩ and MRL/lpr p ϭ 0.0005; CD80: BALB/c, p ϭ 0.023; MRL ϩ/ϩ, p ϭ 0.0011; MRL/lpr, p Ͻ 0.005).

in any way within BALB/c GCs. Because GCs were present in the 4-44ϩ GCs from MLNs of Tg BALB/c, MRL ϩ/ϩ, and MRL/ MLN of all three strains, there was an opportunity to compare lpr mice were microdissected, and the endogenous V␬8 L chains across strains the degree of somatic hypermutation and the patterns were sequenced (25). PCR products using V␬8-specific primers of these mutations that reflect selection. (and containing V␬8–19 or V␬8–28 genes; Ref. 25) were readily The Journal of Immunology 2147

FIGURE 4. Immunization of Tg BALB/c and MRL/lpr mice with IgG2a-containing ICs results in the formation of RFϩ GCs but few AFCs. Tg IgHa and Tg IgHb BALB/c and MRL/lpr mice, as indicated on the individual panels, were immunized with either IgG2a-containing complexes (I.C.) in alum (E–J) or with alum alone (A–D). All spleen sections were stained with 4-44 (blue) and PNA (red). Images are from spleens harvested at day 8 (A–H)or day 12 (I and J). Alum-only immunized mice form no purple 4-44ϩ GCs, although there are GCs of unknown specificity (red). Note the lack of histologically identifiable 4-44ϩ AFCs, which would stain more darkly than the GCs and would be at the border of the red pulp or in the red pulp. Each panel is representative of at least three mice per group, all with similar appearance. Downloaded from

recovered from nearly all microdissections, confirming the histo- shown). The smaller number of mutations and greater number of logic identification of 4-44ϩ GCs. Sequence analysis revealed sig- clones in BALB/c GCs both indicate less clonal expansion com- nificant differences among the phenotypically similar GCs in the pared with the MRL strains. three strains. There were 2.6, 4.2, and 5.1 mutations per sequence The differences in mutation frequency and clonal composition http://www.jimmunol.org/ in the BALB/c, MRL ϩ/ϩ, and MRL/lpr GCs, respectively ( p Ͻ also suggest that selection of RF B cells was different in these GCs. 0.001 for BALB/c vs MRL/lpr; p ϭ 0.03 for BALB vs MRL ϩϩ; Differences in selection are also reflected in the shapes of the ge- and p ϭ not significant for MRL ϩ/ϩ vs MRL/lpr). The BALB/c nealogic trees that are derived from the patterns of shared and GCs were also more polyclonal: for every 100 cells within the unique mutations (34). From each microdissection, we used max- BALB/c, MRL ϩ/ϩ, and MRL/lpr GCs, there were 12, 9, and 5 imum parsimony to reconstruct genealogic trees that reflect clonal different clones, respectively (Fig. 6, A–C, and analysis not evolution. To quantitate the extent of branching in these trees, each by guest on September 23, 2021

FIGURE 5. MLNs in all three strains contain 4-44ϩ GCs, but AFCs are found only in MRL strains. A–C and E–G, MLNs of IgHa Tg BALB/c, MRL ϩ/ϩ, and MRL/lpr mice stained with PNA (red) and 4-44 (blue). A–C, Magnification, ϫ100. E–G, Enlargement of boxed areas in A–C, demonstrating the presence of numerous 4-44ϩ cells in GCs in all three strains. Note the large number of darkly staining 4-44ϩ cells in the medullary region of MLNs of Tg IgHa MRL ϩ/ϩ and MRL/lpr (B and C) but not BALB/c (A) mice. D, The number of 4-44ϩ GCs per LN section was tallied in each mouse, and averages were plotted (bars are 1 SEM). Data are compiled from representative single sections from 11 BALB/c, 14 MRL ϩ/ϩ, and 44 MRL/lpr mice with p values for MRL ϩ/ϩ vs the other two strains Ͻ0.002, and p ϭ not significant between BALB/c and MRL/lpr. H, The number of AFCs in each LN section was estimated using an AFC score that represents the approximate percentage of each MLN spanned by cells with dark cytoplasmic staining (see Materials and Methods). Histograms represent averages of 11 BALB/c, 14 MRL ϩ/ϩ, and 54 MRL/lpr LN examined. BALB/c vs MRL ϩ/ϩ and vs MRL/lpr are both highly significant (p Ͻ 0.0001), whereas MRL ϩ/ϩ and MRL/lpr are not significantly different (p ϭ 0.07). 2148 REGULATING AUTOREACTIVE B CELLS AFTER ACTIVATION Downloaded from http://www.jimmunol.org/

FIGURE 6. Decreased clonal expansion and diversification in Tg IgHa BALB/c MLN GCs. Sample genealogical trees generated from sequences derived a ϩ ϩ

from microdissections of individual MLN GCs of Tg IgH BALB/c (A), MRL / (B), and MRL/lpr (C) mice. Individual clones were assigned to a by guest on September 23, 2021 position in a genealogical tree on the basis of shared and unique mutations. All members of a tree are from a single GC, although some GCs yielded more than one tree (e.g., A). Bases of trees show the six nucleotides at the VJ junction of the parental germline rearrangement. By convention, sequences that shared junctions but no other mutations are depicted in separate trees, because there are many clones with the same canonical VJ junction. The serial numbers of sequenced clones from each pick are written inside the circles, with the pick name followed by the clone number. Numbers in parenthesis indicate the number of identical clones retrieved from each pick. For example, in the BALB/c tree (A), the sequence 20b4-47 was identical with four other sequences obtained from the 20b4 pick. Mutations and their codon position are written on the sides of branches, with affected regions (either CDR or framework region (FR)) in parenthesis. Amino acid changes are written below the codons. Silent mutations are in italics. Sequences written in red are nonviable sequences that have resulted from an insertion, deletion, or a stop codon mutation. Dark circles represent inferred precursors and intermediates in clonal evolution, which were not recovered. D–F, Pie charts representing the distribution of mutations among the branches of genealogical trees inferred from V region sequences from GCs of each strain. Mutations were classified according to their occurrence in the genealogical trees as follows: T(Un) and T(Br) are both mutations shared by all sequences of a tree. Unbranched (Un) mutations are those in trunks without branches above them, and branched (Br) mutations are those in trunks that do have branches extending from them. Branch mutations were labeled B(1-n), depending on the level of branching on the tree. Examples of each of these categories are written in blue on the trees (A–C). Data are derived from microdissections of 10 BALB/c, 9 MRL ϩ/ϩ, and six MRL/lpr GCs from which were obtained 62, 49, and 36 unique sequences.

mutation was given a “branch” or “trunk” designation, depending did not survive, either because of negative selection against auto- on its position in the genealogical tree (see Fig. 6, A–C, and leg- reactive cells or more likely failure to positively select. Lack of end); then the distributions of all mutations from each strain were selection that would lead to oligoclonal expansion is consistent plotted as pie charts (Fig. 6, D–F). with the larger number of clones and few mutations/sequence Genealogic trees without any selection should have many found in BALB/c GCs. In contrast, trees from Tg MRL ϩ/ϩ and branches, and nearly all mutations should be branch mutations, a MRL/lpr GCs were bushier, with a much higher proportion of result of the mutation rate of ϳ1 mutation/cell/division (35). Se- branch mutations, as well as trunks with branches above them (Fig. lection of mutant cells (either positive or negative) will distort the 6, B, C, E, and F). This indicates that, at the least, there is positive “natural” shape of the trees, and the type of selection will affect the selection for certain mutants and may also indicate less stringent neg- resultant shape (36). It is clear that trees from BALB/c LN are ative selection. This view is also consistent with the low replacement: shaped differently from the MRL ϩ/ϩ and MRL/lpr-derived trees. silent ratios in CDRs of BALB/c GCs (data not shown). These dif- BALB/c trees had shapes resembling blades of grass. Most of the ferences in mutation number, clonal composition, and tree shape all mutations were “trunk unbranched” mutations, with very few indicate that BALB/c GCs, in contrast to MRL ϩ/ϩ or MRL/lpr GCs, branch mutations. This indicates that most of the BALB/c mutants do not efficiently generate expanded clones of mutant RF B cells. The Journal of Immunology 2149

Discussion must play a role in promoting AFC formation in the Fas-sufficient B cells that recognize self can escape the dominant mechanisms of MRL mice. Although chimera experiments suggest a role for in- central tolerance (23, 37–39). It has been widely assumed that trinsic B cell defects in MRL ϩ mice (53, 59), whether any of the these escaped cells are quiescent in the peripheral lymphoid tissues already-identified MRL genes are expressed in B cells has not been of normal animals (40). It is felt that peripheral autoreactive B cells reported. Nonetheless, in New Zealand Black/New Zealand White- are regulated by or are clonally ignorant and do not based models of systemic autoimmunity, there are clear examples sense the presence of self-Ag; or if activated, they are quickly of genes with polymorphisms associated with autoimmunity that eliminated (41). In this study, we demonstrate that this is not al- are expressed in B cells (60–62). ways the case: we show that RF B cells are activated and partic- B cell-extrinsic factors could also determine whether autoreac- ipate in GC reactions in the periphery of BALB/c mice. Moreover, tive B cells differentiate into AFCs and are selected in GCs. These this activation specifically requires the presence of self-Ag. include the form and concentration of the autoantigen as well as This demonstration that disease-related autoreactive B cells can stimulatory and regulatory roles of T cells. Certain forms of the RF be activated by autoantigen to the point of GC development in autoantigen may be required to provide the signals needed to in- normal individuals is important because it expands and alters our duce AFCs. IgG2a complexed with chromatin or CpG-containing view of how autoimmunity is normally kept in check. All of the DNA (17, 63) is a potent mitogen for RF B cells in vitro. This previously known regulatory checkpoints for self-reactive B cells ligand provides a combination of BCR and TLR-mediated signals. operate to prevent initial activation or to eliminate cells quickly These signals also promote the initiation of AFC differentiation after they are activated (41–46). In this study, we have found two (Ann Marshak-Rothstein, personal communication). In contrast, Downloaded from new ways by which autoreactive B cells are prevented from caus- IgG2a complexed with proteins is a poor inducer of RF B cell ing harm to the host even after they have been activated. proliferation in vitro (17, 63). It is possible that these in vitro First, the generation of RF AFCs was suppressed in BALB/c findings are paralleled in vivo in AM14 Tg mice. Supporting this mice, whereas MRL/lpr mice and older MRL ϩ/ϩ mice generated notion, immunization with IgG2a anti-protein ICs did not induce large numbers of AFCs (Figs. 1–3). We propose that the suppres- AFCs in any of the strains but instead elicited a GC response (Fig. sion of AFC formation serves to prevent the harmful effects of

4). If form of autoantigen controls the nature of the response, we http://www.jimmunol.org/ pathogenic autoantibodies. Blocking AFC generation, however, would expect BALB/c mice to have relatively small amounts of would not fully prevent the adverse effects of activating autoreac- DNA-containing ICs and possibly more of classical antiprotein tive B cells. Such B cells could also promote autoimmunity by ICs. In this view, MRL/lpr mice should have high levels of DNA- acting as APCs for pathogenic self-reactive T cells (47, 48). Thus, containing ICs, as has been shown to be the case (64). A number the generation in GCs of high-affinity RF B cells is likely to be of genes that predispose to systemic autoimmunity might control detrimental. The second means by which RF responses were re- the levels of DNA-containing ICs and thus the form of the RF stricted in BALB/c mice addresses this potential problem: there autoantigen. For example, natural and artificial mutations that af- was less clonal expansion and weaker selection of mutants in GCs fect clearance of apoptotic cells and ICs, such as deficiency in of normal BALB/c mice than in MRL/lpr or MRL ϩ/ϩ GCs (Figs. by guest on September 23, 2021 early complement components or the tyrosine kinase Mer, are as- 5 and 6). Such regulation of clonal expansion and selection of sociated with systemic autoimmunity (65, 66). We think that this mutants would naturally limit the generation of high-affinity RF B explanation could well account for the differences between normal cells. We propose that controlling selection and clonal expansion and autoimmune-prone mice and could be a way that genetic pre- of autoreactive cells in the normal GC is an additional form of disposition could be linked to regulation of B cell activation. self-tolerance. The influence of T cells could also be important in controlling How are AFC formation and selection in GCs regulated in the fates of activated RF B cells. This is easiest to understand for BALB/c mice compared with MRL mice? Such regulation could GCs, the complete development of which is known to be - be B cell intrinsic, or relate to exogenous factors like T cell help dependent. T cells have been shown to be hyperreactive in MRL or regulation or even the form of autoantigen present in the dif- ferent strains. Whether the regulation is B cell intrinsic or simply mice and in patients with SLE (67, 68); hyperreactive T cells in reflects the requirement for certain forms of Ag that are more prev- MRL mice could provide signals that override normal controls that alent in autoimmune-prone mice, such mechanisms would consti- operate in BALB/c mice. Conversely, there is some evidence that ϩ ϩ tute tolerance checkpoints in that they result in the restriction of CD4 CD25 T cells can regulate the extent of the GC reaction in autoreactive B cell immune responses in terms of differentiation normal mice (69). Such cells can also regulate organ-specific au- and affinity maturation. toimmunity in a variety of model systems (70). This has been less With regard to B cell intrinsic mechanisms, MRL ϩ or MRL/lpr well-investigated in systemic autoimmunity, with evidence for a B cells themselves may be more likely to undergo AFC differen- role in some but not other model systems (71). Lupus patients are ϩ ϩ tiation and/or to survive in the GC. The differences between Fas- reportedly deficient in CD4 CD25 cell numbers (72). However, deficient and sufficient strains demonstrate that Fas plays a role. we are not aware of a direct test of the function of these cells in This could be intrinsic to the B cell, because GC B cells express regulating the activation of autoreactive B cells in MRL mice, Fas (49–51). There is genetic evidence in MRL/lpr of a B cell- although in a transfer system they can prevent the activation of intrinsic defect that leads to autoimmunity (52–54). Fas, which anti-DNA B cells by T cells that recognize Ag constitutively pre- transmits a death signal (55), could therefore serve to regulate GC sented by these B cells (73). ϩ responses as has been suggested for GC responses to model Although BALB/c spleens and LNs lack RF AFCs, the pres- ϩ Ags (56). Fas is also expressed on AFCs (49, 50), but there is little ence of RF GCs in BALB/c secondary lymphoid tissue poses a data on whether Fas normally influences AFC development. constant risk for functional autoimmunity. Left unchecked, prolif- Because older MRL ϩ/ϩ, but not BALB/c, mice do make RF erating and mutating low-affinity self-reactive GC B cell could AFCs, Fas cannot be the only explanation for RF AFC develop- differentiate into high-affinity memory clones or long-lived AFCs ment in the autoimmune-prone strains. Some of the polymorphic (74–77). Thus, the normal control of GC dynamics in BALB/c genes associated with the MRL autoimmune phenotype (57, 58) LNs could be an important fail-safe mechanism of tolerance. If GC 2150 REGULATING AUTOREACTIVE B CELLS AFTER ACTIVATION tolerance mechanisms were to be breached, the type of autoimmu- 9. Chen, C., Z. Nagy, M. Z. Radic, R. R. Hardy, D. Huszar, S. A. Camper, and nity associated with SLE would result: the generation of high- M. Weigert. 1995. The site and stage of anti-DNA B-cell deletion. Nature 373: 252–255. affinity, -switched autoantibodies. 10. Li, Y., H. Li, and M. Weigert. 2002. 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