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Short-Lived Plasmablasts Dominate the Early Spontaneous Rheumatoid Factor Response: Differentiation Pathways, Hypermutating Cell Types, and Affinity Maturation Outside This information is current as the of September 28, 2021. Jacqueline William, Chad Euler and Mark J. Shlomchik J Immunol 2005; 174:6879-6887; ; doi: 10.4049/jimmunol.174.11.6879 http://www.jimmunol.org/content/174/11/6879 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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Short-Lived Plasmablasts Dominate the Early Spontaneous Rheumatoid Factor Response: Differentiation Pathways, Hypermutating Cell Types, and Affinity Maturation Outside the Germinal Center1

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

We used a newly validated approach to identify the initiation of an autoantibody response to identify the sites and cell differen- tiation pathways at early and late stages of the rheumatoid factor response. The autoimmune response is mainly comprised of rapidly turning over plasmablasts that, according to BrdU labeling, TUNEL, and hypermutation data, derive from an activated precursor. Surprisingly, few long-lived plasma cells were generated. The response most likely initiates at the splenic T-B Downloaded from zone border and continues in the marginal sinus bridging channels. Both activated B cells and plasmablasts harbor V gene mutations; large numbers of mutations in mice with long-standing response indicate that despite the rapid turnover of responding cells, clones can persist for many weeks. These studies provide insights into the unique nature of an ongoing autoimmune response and may be a model for understanding the response to therapies such as B cell depletion. The Journal of Immunology, 2005, 174: 6879–6887.

ystemic autoimmune diseases are characterized by activa- cus on disease-related autoantigens, which have demonstrated a http://www.jimmunol.org/ tion of autoreactive B cells that leads both to autoantibody variety of tolerance mechanisms and in selected cases escape from S (autoAb)3 production and activation (1). Whether these in the context of a disease-prone genetic background (5, 8, this activation occurs because of a failure of (2– 11, 12, 16, 17). We have been focusing on the AM14 system, an 6), anergy (7, 8), or other mechanisms of peripheral tolerance (9– Ig-Tg model with IgG-specific (rheumatoid factor (RF)) B cells 13), or is due to activation of ignorant B cells (14, 15), it represents (14, 15, 18). RF autoAbs are found at high levels in rheumatoid a functional loss of tolerance. After the initial loss of tolerance, arthritis, Sjogren’s syndrome, in some patients with systemic lupus tissue destruction ensues, which in turn promotes further inflam- erythematosus (SLE) (19), as well as in Fas-deficient lpr mice (20,

mation, more extensive loss of self-tolerance, and ultimately 21). In AM14 Ig-Tg mice, the H chain confers specificity for the by guest on September 28, 2021 symptoms of . In addition, the self-epitopes targeted Fc portion of IgG2aa when paired with an endogenous or Tg-en- early in the response may differ from those targeted later, a phe- coded V␬8 L chain (22). AM14 binds only to IgG2a of the “a” nomenon known as epitope spreading. Thus, early and late stages allele; thus, in IgH congenic mice, RF B cells can be studied in the of autoimmunity may differ in the subsets of autoreactive cells that presence or absence (i.e., in IgHb mice) of a defined and measur- are activated, sites of activation, and the types of effector cell that able autoantigen. Using this model, we initially found that, in con- result. Immune dysregulation at the end stage of disease could be trast to most other models, the RF B cells develop normally and are of a different nature from that which occurred during the initial loss immunocompetent in normal IgHa mice (23), a phenotype referred of self-tolerance. However, because the onset of spontaneous sys- to as clonal ignorance. temic autoimmunity is not predictable from individual to individ- To determine how RF B cells become activated on an autoim- ual, it has been difficult to study early events in the loss of self- mune-prone background, we crossed the AM14 Tg onto the MRL/ tolerance and determine how they evolve into more established lpr strain (24). RF B cells in spleens of older H chain Ig-Tg MRL/ disease with time. lpr mice form clusters at the T zone-red pulp borders and undergo Ig-transgenic (Ig-Tg) mouse models have been instrumental in at this site; they are only very rarely found studies of normal B cell self-tolerance and its loss during disease in germinal centers (GCs) (15). The activation of autoreactive B (2–5, 8). Particularly interesting in this regard are models that fo- cells required the presence of the nominal autoantigen and did not occur on the IgHb background. Interestingly, there was great mouse to mouse variability in the onset and extent of RF B cell *Section of Immunobiology and †Department of Laboratory Medicine, Yale Univer- sity School of Medicine, New Haven, CT 06520 Ab-forming cell (AFC) induction. This is similar to the stochastic onset of appearance of various autoantibodies in non-Tg MRL/lpr Received for publication January 27, 2005. Accepted for publication March 16, 2005. and other autoimmune-prone mice (25). Such variability in the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance onset and nature of disease has confounded efforts to identify the with 18 U.S.C. Section 1734 solely to indicate this fact. earliest phases of autoreactive B cell activation. Consequently, 1 This work was supported by National Institutes of Health Grant P01-AI36529. whereas there are a number of studies of the autoimmune response 2 Address correspondence and reprint requests to Dr. Mark J. Shlomchik, Yale Uni- in our system and others, these data mainly reflect the later stages versity School of Medicine, 333 Cedar Street, Box 208035, New Haven, CT 06520- of established autoimmunity. 8035. E-mail address: [email protected] To understand the initiating events in the loss of B cell toler- 3 Abbreviations used in this paper: autoAb, autoantibody; Tg, transgenic; RF, rheu- matoid factor; GC, germinal center; AFC, Ab-forming cell; SLE, systemic lupus ance, we would like to identify the site(s) of initial B cell activation erythematosus; DC, dendritic cell. in secondary lymphoid organs such as the spleen; for example,

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 6880 PLASMABLASTS DOMINATE THE EARLY RF RESPONSE does RF B cell activation occur at the T-B border or the marginal Sequencing sinus-bridging channel? In addition, we would like to know FACS-sorted cells were digested overnight at 37°C in 10 ␮l of 0.8 mg/ml whether induction of autoimmunity occurs at other secondary lym- proteinase K, 50 mM Tris, pH 8, 50 mM KCl, 0.63 mM EDTA, 0.22% phoid sites and whether it is synchronous with activation in the Igepal, and 0.22% Tween 20. Sequencing was performed as described (15). spleen. Finally, it is important to define the cell types and differ- In brief, V␬8/J4 rearranged sequences were amplified by nested PCR using Ј entiation states found during initial RF B cell activation as well as Pfu Turbo (Stratagene): external primers 5 -AGCTATGCATTTTCACT GTC-3Ј and 5Ј-AGCCCTCTCCATTTTCTC-3Ј, and internal primers 5ЈTT how the response evolves over time. Recently, we developed a GTGATGACACAGTCTCC-3Ј and 5ЈAAGTTACCCAAACAGAACC-3Ј. system to identify which mice were at the early stages of initial RF Amplified DNA was cloned using the TOPO-Zero blunt cloning kit (Invitro- B cell activation based on the appearance of activated RF B cells gen Life Technologies) and further amplified by picking transformed colonies in the peripheral blood (53). In the present study, we use this directly into a PCR amplification reaction using primers M13 forward 5Ј- GTAAAACGACGGCCAG-3Јand M13 reverse 5Ј-CAGGAAACAGCTAT system to address some of the questions defined above concerning GAC-3Ј provided in the kit. The PCR product was purified using the QIAquick the sites, tissues, and cell types involved in the early phases of a PCR purification kit (Qiagen), mixed with the sequencing primer T3 5Ј- spontaneous autoimmune response and how these evolve ATTAACCCTCACTAAAGGGA-3Ј and sequenced by the Keck Biotechnol- with time. ogy Resource Laboratory at Yale University.

Materials and Methods Hybridomas and RF affinity ELISAs Mice Splenocytes from an aged AM14 MRL/lpr mouse were fused to SP2/0 cells and resultant hybridomas screened for idϩ Ab secretion. Two hybridomas The AM14 H Tg is a conventional IgM-only Tg. AM14 H Tg mice (22) secreting the AM14 H chain/V␬8 L chain pair were recovered and tested were backcrossed at least 10 generations onto the MRL/lpr background. for affinity to the autoantigen IgG2a as described (29). Briefly, plates were Downloaded from

All mice were housed under specific pathogen-free conditions. coated with the (4-hydroxy-3-nitro-2-iodo-phenyl)acetyl (NIP)26- BSA, blocked with PBS/1% BSA, and then incubated with increasing con- Identification of converted mice and FACS analysis centrations with 23.3, a NIP-specific IgG2a. After washing, plates were Mice that had initiated an RF B cell AFC response in the spleen (“con- incubated with hybridoma or spleen cell supernatants, followed by detec- verted mice”) were identified by FACS on PBL, detecting RF B cells using tion with anti-IgM-alkaline phosphatase (Southern Biotechnology Associ- the 4-44 anti-Id Ab, as described.4 Splenocytes were prepared as described ates) and development with para-nitrophenyl phosphate substrate (Sigma- (18). FACS analysis of PBL and splenocytes was performed as described Aldrich). To measure RF affinity from polyclonal AFCs in converted mice, http://www.jimmunol.org/ (18). Propidium iodide was used to exclude dead cells. For detection of splenocytes from aged H MRL/lpr mice were cultured in 10% complete intracellular Ab, membrane-stained cells were resuspended in 1% parafor- RPMI overnight and supernatants tested as described above. maldehyde solution/PBS for 20 min on ice. Following fixation, the cells were washed in FACS buffer and resuspended in permeabilization buffer Results (PBS/0.3% saponin/1% BSA/0.05% sodium azide) at 4°C. After 2 h, the The early splenic response in converted AM14 MRL/lpr mice is cells were washed and resuspended in labeled 4-44 Ab, diluted in perme- low ϩ abilization buffer with 10% rat serum, and incubated overnight at 4°C. marked by the appearance of a CD22 population of 4-44 B Cells were then washed in permeabilization buffer followed by FACS cells buffer and analyzed by FACS. Ethidium monoazide bromide (Molecular We recently showed (53) that the appearance of Id 4-44ϩ (RFϩ)B

Probes) was used to exclude dead cells in procedures involving fixation by guest on September 28, 2021 (26). Unfixed cells were incubated for 15 min in the dark with ethidium cells in PBL correlated strongly with the presence of elevated monoazide bromide, exposed to light for 10 min, then washed before numbers of RF AFC in the spleen and conversely that the absence fixation. of such cells in PBL predicted the presence of only very low num- Reagents bers of RF AFC in spleen. From these data we reasoned that the initial appearance of RFϩ B cells in PBL marked the onset of the Abs prepared in our laboratory as described (22) were: 4-44-biotin (anti- initial RF autoimmune response in the spleen, a phenomenon we Id), 4-44 FITC, 4-44-Alexa Fluor 647, 4-44-Alexa Fluor 488, 30H12-biotin (anti-CD90.2), C363–29B-FITC (anti-CD3), RA3–6B2-FITC (anti-B220), termed “conversion.” We therefore used this approach to identify Pgp-1-FITC (anti-CD44). DS1-PE (anti-mouse IgM), anti-CD21/35-FITC, early converts and then study the nature of the response. ϩ anti-CD23-FITC, anti-CD80-PE, anti-CD86-PE, anti-I-Ak-FITC, anti- In early converts, 4-44 cells accumulated and mutated primar- CD22.2-FITC and PE, anti-CD138 (conjugated to PE and unlabeled) were ily at the T zone-red pulp border (Fig. 1A) (15), similar to aged obtained from BD Pharmingen. Anti-BrdU FITC (Phoenix Flow), peanut IgHa MRL/lpr mice (15). We used FACS and immunohistology to agglutinin-biotin (Vector), and MOMA-1-FITC (anti-marginal zone met- understand the origin and identity of these unusual mutating cells. allophilic macrophages; Serotec) were used as indicated. Stromal cell-de- ϩ rived factor-1-human Fc fusion protein (for CXCR4 detection) and rabbit 4-44 cells from IgHb and nonconverted IgHa mice made up 1–2% anti-CXCR5 were both kind gifts from Dr. Jason Cyster (University of of the splenic population and were uniformly CD22high (Fig. 1B). California, San Francisco). Streptavidin-allophycocyanin (Serologicals) In recently converted mice, there was a significant expansion of a and streptavidin-PE (Molecular Probes) were used to detect biotinylated ϩ low reagents. Anti-human IgG-PE (Jackson ImmunoResearch Laboratories) novel 4-44 , CD22 population (compare upper left quadrants in and goat anti-rabbit Alexa Fluor 633 (Molecular Probes) were used as Fig. 1, B–D, summarized in Fig. 1E). This population of cells secondary reagents for stromal cell-derived factor-1-Fc and anti-CXCR5, expanded further in mice that had converted at least 2 wk ago (Fig. respectively. 1E). In contrast, the CD22high population rarely expanded at any low Histology stage of conversion (Fig. 1, B–E). The CD22 population was B220low, CR1/2low, CD23low, CD44high, CD80high, CD86high, Sections were prepared and stained as described (27). MOMA-FITC stain- class IIint/hi, CXCR5low, and CXCR4high (Fig. 1, F–N). The ex- ing was amplified using anti-FITC Alexa Fluor 488 (Molecular Probes) as a secondary. Nuclei were identified with 4Ј,6Ј-diamidino-2-phenylindole pression patterns of CXCR4 and CXCR5 are consistent with lo- (Molecular Probes). Fluorescent images were captured on an Olympus calization of the CD22low population outside of follicles and in the BX-40 microscope using a SPOT-RT Slider (Scanalytics) digital camera. marginal sinus-bridging channels (30). The CD22high population in high high Cell proliferation and apoptosis assays both converts and nonconverts was B220 , CR1/2 , CD23 variable, class IIhigh, CXCR5high, and CXCR4low (Fig. 1, F–N). Mice were given an i.p. injection of 2 mg of BrdU (Sigma-Aldrich) in PBS These markers, particularly the chemokine receptors, are consis- at 2, 12, 24, or 72 h before sacrifice. Splenocytes were first membrane- tent with a follicular localization (30). In converted mice, a frac- stained, and BrdU labeling was detected as previously described (28). Ap- high optosis was detected with the CaspGLOW fluorescein active caspase stain- tion of the CD22 population reproducibly demonstrated in- ing kit (Biovision). creased expression of the activation markers CD44, CD80 and, to The Journal of Immunology 6881 Downloaded from http://www.jimmunol.org/

FIGURE 1. RF B cell activation and differentiation upon conversion. A, Thy1.2 (blue), MOMA-1 (green), and 4-44ϩ (red) staining of a spleen section of an IgHa Tg MRL/lpr mouse demonstrating that 4-44ϩ cells expand primarily at the T zone red pulp border (arrow) following conversion. B–D, ϩ low b a 4-44 /CD22 cells appear following conversion. FACS analysis of live-gated splenocytes from an IgH Tg MRL/lpr mouse (B), a nonconverted IgH by guest on September 28, 2021 Tg MRL/lpr mouse (C), and a recently converted IgHa Tg MRL/lpr mouse (D), stained with 4-44 and anti-CD22. Numbers in quadrants are percentages of live cells. E, CD22low cells continue to expand following conversion. Mean percentages of CD22low/4-44ϩ (hatched) and CD22high/4-44ϩ (open) splenocytes from IgHa Tg MRL/lpr mice at stages pre- and postconversion. F–N, FACS overlays for 4-44ϩ/CD22low (thick red) and 4-44ϩ/CD22high (thin blue) splenocytes from converted Tg IgHa MRL/lpr mice. CD22high 4-44ϩ splenocytes from nonconverted mice (dashed green) are controls, and they resemble plots of CD22high 4-44ϩ cells from IgHb mice (not shown).

a slight degree, CD86. This can be seen in Fig. 1, I–K, comparing Surprisingly, although the CD22low cells were AFCs, they were the blue, thin solid line to the dashed green one. Note that the not classical plasma cells. They had normal levels of expression of CD22high cells from nonconverted mice (Fig. 1) lack the popula- surface Ig and class II, unlike terminally differentiated plasma tion of cells with higher expression of these markers; this popu- cells, which lack surface Ig and class II (31, 32). Rather, these lation is also lacking in IgHb mice (not shown). AFCs had features more consistent with plasmablasts, given their persistent expression of activation markers and surface Ig. In fact, low CD22 cells are AFCs that reside at the T zone red pulp these plasmablast-like cells were the dominant population, as very border and maintain normal levels of surface Ig few sIglo/neg classical plasma cells could be identified in converted Consistent with the FACS results, immunohistochemical analysis mice. Rather, although most of the cells with high intracellular Ig of frozen sections from converted mice showed that most of the staining (a marker of both plasma cells and plasmablasts, but not 4-44ϩ cells in the bridging channels were CD22low/syndecanhigh resting or activated B cells) were CD22low (Fig. 2M), they had (Fig. 2, A–G). At this site, there were also smaller numbers of normal levels of surface Ig (Fig. 2, K and L; compare yellow R6 CD22high (Fig. 2C) as well as syndecan-negative cells (Fig. 2G). gate with green and red R4 and R5 gates in Fig. 2L). ϩ high Conversely, most of the 4-44 cells in the follicles were CD22 low (Fig. 2, B and D). FACS analysis confirmed that most CD22low CD22 cells with a similar, but not identical, FACS profile are cells were expressing high levels of syndecan (Fig. 2H, red line) in found in PBL contrast to CD22high cells, which were syndecan negative (Fig. 2H, We wondered whether either of the two populations of RF B cells blue and green lines). The FACS-sorted CD22low population was in the spleens of converted mice corresponded to the 4-44ϩ cells responsible for virtually all of the Ab secretion (17% of 4-44ϩ that signify conversion in blood. FACS analysis showed that RFϩ CD22low cells formed ELISpots vs only 0.33% of 4-44ϩ CD22high B cells in blood of converted mice (Fig. 3, solid line) are indeed cells), demonstrating that CD22low cells were indeed AFCs, con- similar to the CD22low subset in that most have down-regulated sistent with the syndecan staining and their appearance on cytospin CD22, as well as CR1/2 (Fig. 3, B and C). Also like the cells in (Fig. 2, L and M). spleen, 4-44ϩ cells among PBL had increased levels of CD86 and 6882 PLASMABLASTS DOMINATE THE EARLY RF RESPONSE Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. Location and identity of CD22low and CD22high RF B cells. A–D, Splenic follicle stained for Thy1.2 (red) and CD22 (blue) (A) and CD22 (blue) and 4-44 (red) (B). Most of the CD22high cells reside in the follicles and most of the CD22low 4-44ϩ cells are at the edge of the T zone and red pulp. Boxed areas are enlarged in C and D. There are numerous scattered 4-44ϩ/CD22ϩ cells at the T zone red pulp border (C); there are 4-44ϩ/CD22high follicular cells that stain more weakly than extrafollicular CD22low cells (D) since they lack the cytoplasmic Ig (see I and J). E–G, Dark-staining 4-44ϩ cells costain with syndecan. E, 4-44 (red) and CD3 (blue); F, syndecan (red) and CD3 (blue); G, 4-44 (red) and syndecan (blue). In G, note the doubly stained, almost black 4-44ϩ/syndecanϩ cells as well as scattered cells staining with only one or the other marker. H, 4-44ϩ/CD22low cells (thick red) from recent converts are syndecanϩ and all of the 4-44ϩ/CD22high cells (thin blue) are syndecanϪ, regardless of conversion status. As a control, 4-44ϩ/CD22high cells from a young nonconverted mouse (green dashed) are shown. I–J, Cytospins of sorted CD22low/4-44ϩ (I) and CD22high/4-44ϩ splenocytes (J) demonstrating strong cytoplasmic staining in all of the CD22low cells. Green ϭ 4-44 and blue ϭ 4Ј,6Ј-diamidino-2-phenylindole. K–M, Most of the 4-44ϩ/CD22low AFCs in converted mice have normal surface Ig levels, and very few resemble true plasma cells. Splenocytes were stained for membrane and intracellular 4-44 using two different fluorochromes. Plots are gated on cells that were alive before fixation. Very few cells in the nonconverted mouse (K) have intracellular 4-44ϩ staining. Plasma cells are expected to have bright intracellular staining but weak surface staining (R6, orange); few were seen in the converted mouse (L), although membrane 4-44ϩ cells with intermediate (R4) and high (R5) intracellular Ig were present. M, CD22 expression in the 4-44ϩ populations gated in the plot of the converted mouse in L. Nearly all of the cells with elevated intracellular staining (R4–6) are CD22low, and all of the cells with very low intracellular staining (R3) are CD22high.

were very bright for CD44 (Fig. 3, E and F) and had down-regu- enously positive for B220 (Fig. 3A), which was variably down- lated MHC class II molecules (Fig. 3D), perhaps to an even greater regulated on CD22low cells in spleen (Fig. 1F). Interestingly, the extent than splenic CD22low cells (Fig. 1L). However, unlike their cells were syndecan-negative (not shown), in contrast to the CD22low counterparts in spleen, the cells in PBL were homog- splenic counterparts (Fig. 2, F–H). Thus, RFϩ cells in PBL were The Journal of Immunology 6883 Downloaded from http://www.jimmunol.org/

FIGURE 3. Phenotype of IgMaϩ/4-44ϩ cells in the peripheral blood. (A-F) Histograms of gated IgMaϩ,4-44ϩ PBLs in converted (thick line) and nonconverted (thin line) mice, showing expression of B220 (A), CD22 (B), CD21/35 (C), class II (D), CD86 (E), and CD44 (F). IgMϩ/4-44Ϫ cells from the converted mice (dashed line) have similar profiles to the IgMϩ/4-44ϩ cells from non-converted mice. clearly activated and had some features of plasmablasts. The lack CD22high cells of converts than of nonconverted or IgHb mice. of syndecan, which is not uniformly expressed on plasmacytes (33, However, unlike the CD22low cells, only a small percentage of ϩ 34), may reflect the migratory nature of the PBL cells, as syndecan CD22high cells remained BrdU after 24 h (Fig. 4A). Since the by guest on September 28, 2021 is an adhesion molecule and when expressed may prevent migra- initial proliferation rate in both populations is similar, the tion into blood. The exact significance and origin of the PBL cells CD22high cells either must die faster than the CD22low cells or remains to be worked out; it will be of interest as these cells do differentiate quickly into CD22low cells once activated. To distin- resemble cells found in PBL of SLE patients with active disease guish these possibilities, we measured apoptosis using TUNEL. If (34). To begin to address this, we have performed limited V␬ the more rapid loss of BrdU label from the CD22high population sequence analysis of cells sorted from PBL. We could readily re- were due to apoptosis, we would have expected a greater fre- cover V␬8 sequences even from small numbers of such cells, con- quency of TUNEL-positive cells in this population than in the firming their identity. More importantly, these cells did contain CD22low one. However, on average, 38.4% of the CD22low cells mutations (average of 2 mutations per sequence), establishing that were undergoing apoptosis directly ex vivo, whereas only 22.2% these B cells circulate in response to autoantigen-driven stimula- of the CD22high cells were undergoing apoptosis (Fig. 4B). This tion and that they derive from a compartment of B cells undergo- degree of apoptosis was associated with activation, because only ing mutation or that had mutated in the past. Such precursors are 7.8% and 4.1% of CD22high cells from IgHb mice and IgHa non- presumably in the spleen, given that their appearance only corre- converts, respectively, were apoptotic. It thus seems likely that the lated with the activation of RF B cells in spleen and not LN (53). more rapid loss of label from the CD22high population must be attributable to differentiation to CD22low cells. Therefore, CD22low low high Both CD22 and CD22 cells in spleen are actively cells derive from CD22high cells as well as from self-renewal. The low proliferating, but CD22 cells have higher rates of apoptosis apparent slower decay of BrdUϩ CD22low cells at later points fol- The AFC/CD22low populations in PBL and spleen resembled plas- lowing the pulse label (Fig. 4A) is also consistent with this inter- mablasts more than classical plasma cells. Plasmablasts are divid- pretation since labeled CD22high cells would be differentiating into ing cells, and in our view, cell division definitively separates non- labeled CD22low cells, thus replenishing the latter compartment. dividing plasma cells from plasmablasts (35). To determine Attempts to further investigate this in cell transfer experiments whether the CD22low population was dividing, mice were given were unsuccessful due to limited cell recoveries and a propensity BrdU and sacrificed at various intervals. After2hoflabeling, both of CD22low cells to die ex vivo during purification. low high CD22 and CD22 cells in converted mice had similar pro- low high portions (10–20%) of BrdU-labeled, 4-44ϩ cells (Fig. 4A). Ap- Somatic mutation in CD22 and CD22 cells pearance of labeled cells after only2hofBrdU exposure indicates Since there were two populations of proliferating RF B cells at the active proliferation and further establishes the CD22low cells as T zone-red pulp border, neither of which is classically thought to plasmablasts. BrdU-labeling also demonstrated that the CD22high undergo somatic hypermutation, we wanted to determine which of population in converted mice was activated and proliferating, as these cell types was actively mutating. To determine mutational the proportion of BrdUϩ cells was substantially higher among content in the two populations, we FACS-sorted both, sequenced 6884 PLASMABLASTS DOMINATE THE EARLY RF RESPONSE Downloaded from http://www.jimmunol.org/

FIGURE 4. 4-44ϩ/CD22low and CD22high cells from converted mice have V gene mutations and are proliferating and undergoing apoptosis. A, Kinetics of BrdU labeling. Converted mice were injected with BrdU and sacrificed 2, 12, 24, or 72 h later. Labeling was detected by FACS. Like the CD22high/4-44Ϫ population (square), very few CD22high 4-44ϩ cells from IgHb Tg MRL/lpr were in cycle, as assessed by the 2-h pulse (triangle). Each symbol represents the mean of at least three mice; error bars are SEM. B, Increased apoptosis among CD22low/4-44ϩ and CD22high/4-44ϩ cells from converted IgHa Tg MRL/lpr mice. Percentages of CaspGLOWϩ apoptotic cells in the indicated gates are shown. Similar data were obtained with TUNEL staining (not shown). C, Mutational content of V␬8 L chain sequences from sorted CD22low/4-44ϩ and CD22high/4-44ϩ splenocytes. Mutational content increases with con- version. Between 11 and 42 sequences were obtained for each sorted population. None of the V␬8 sequences from nonconverted mice had mutations. D and E, Histogram of the distribution of number of mutations per sequence in recent (D) and older (E) converted mice. by guest on September 28, 2021 the endogenous V␬8 L chain, and calculated the average number Affinity maturation of the RF response low of mutations/sequence (Fig. 4C). In recent converts, CD22 cells Somatic mutations can lead to higher affinity Abs and these can be had on average twice as many mutations as their CD22high coun- selected for during Ag-driven immune responses (36, 37). There is terparts ( p ϭ 0.004). However, this difference is accounted for by evidence that similar selection can lead to higher affinity autoan- a higher fraction of unmutated sequences in the CD22high cells, as tibodies to DNA, as determined in hybridoma studies (38, 39). shown in the histogram of the distribution of the number of mu- However, there is little evidence for affinity maturation of RF B tations/sequence (Fig. 4D); considering only the mutated se- cells (40), although elevated R/S ratios in CDRs appeared to reflect quences, the frequency of mutations is similar in the two popula- selection in one study (41). To determine whether the response tions (CD22low ϭ 2.5 Ϯ 0.3 mutations, CD22high ϭ 2.0 Ϯ 0.2 occurring in the spleen of converted AM14 H Tg MRL/lpr mice mutations, p ϭ 0.22). led to the selection of higher affinity RF B cells, we used an ELISA Interestingly, the average number of mutations increases signif- icantly in old converts (Fig. 4C, p ϭ 0.03). Among these more- assay as previously described (29) that can determine relative af- mutated sequences, a clear bimodal distribution emerges in those finity of RF B cells. When the target substrate on the ELISA plate from the CD22high population (Fig. 4E). The bimodal distribution is diluted in concentration, the binding of lower affinity RF is re- could be explained if the CD22high population includes both acti- duced before that of higher affinity RF. A similar assay has been vated (mutating) and resting (unmutated cells), consistent with the described to detect relative affinity of nitrophenyl-specific Ab phenotypic heterogeneity of the CD22high population described based on the reduced density of hapten using differentially hapte- H J nated substrate (42). We tested two different sources of RF. First, above (see Fig. 1, – ). Since both populations contain mutations, ϩ then either both are mutating or only one is mutating and it then we recovered three 4-44 /RF hybridomas from a fusion of spon- differentiates into the other. Assuming that the CD22high cells dif- taneously activated B cells from the spleen of a converted mouse. ferentiate into the CD22low cells, as suggested by the BrdU-label- These RFs expressed V␬8 light chains (data not shown). Notably, ing kinetics and TUNELϩ frequencies, then the CD22high popu- these hybridomas had apparently higher affinities than the germline lation must be mutating. However, this explanation does not AM14 IgM Ab transfectoma 400t␮23 (14) (Fig. 5A and data not exclude that the CD22low cells are also mutating. Some ongoing shown). The higher affinities are indicated by the stronger relative mutation in the CD22low population is quite plausible, since it is binding at low densities of IgG2a target Ag. We also cultured clearly undergoing active cell division; in fact, a proportion of the whole splenocytes from converted mice overnight to allow the CD22low sequences has more mutations than any of the CD22high resident plasmablasts to secrete RF into the medium. These super- sequences (Fig. 4, D and E), suggesting that some additional mu- natants were then tested in the differential substrate assay (Fig. tation can take place among CD22low cells. 5B). Again, supernatants demonstrated higher affinity than the The Journal of Immunology 6885

FIGURE 5. RF AFCs generated in the spontaneous response have higher affinity than the germline AM14 HL combination. Low affinity RF loses binding when the Ag coating is low density (29). This is seen by comparing 20.8.3 (solid line, round symbols in A and B), a very high affinity RF clone, and 400t␮23 (light line, squares in A and B), the original AM14 low affinity unmutated RF clone. y-axis shows binding relative to maximal binding for each sample to normalize for different starting concentrations of samples. The x-axis represents the concentration of IgG2a target bound to the plate. Note that with decreasing target Ag the binding of high affinity 20.8.3 is relatively unaffected whereas most of the binding by low affinity 400t␮23 is lost. A, relative binding to varying concentrations of coated IgG2a target for two Idϩ RF hybridomas (dashed lines) made by fusion of spleens from a converted Downloaded from H Tg MRL/lpr mouse as described in Materials and Methods. At concentrations of IgG2aa, Ͻ0.5 ␮g/ml, these hybridoma RFs show increased IgG2aa binding relative to 400t␮23. The unusual prozone effect for 15C was a reproducible observation. B, Splenocytes from three aged H MRL/lpr mice that had high numbers of idϩ ELISpots (dashed lines, diamonds) were cultured overnight and supernatants assessed by ELISA as shown in A. The solid thin line (triangles) represents supernatant from spleens of double HL Tg BALB/c mice in which the Tg L chain is unable to mutate. Like 400t␮23, binding by this supernatant falls off at the lower concentrations of coated target. In contrast, note that the titration curves for splenocyte supernatants from the H MRL/lpr mice approach that of the high affinity 20.8.3 clone. http://www.jimmunol.org/ germline. Together, these two experiments indicate that the pro- advantage of the ability to identify the onset of the RF response in cess of RF B cell activation by autoantigen selects for higher af- individual animals, a process we termed conversion. We found that finity RF B cells. there were both CD22low plasmablasts and CD22high-activated B cells in converted mice. These populations were only found in Discussion mice with ongoing autoimmunity. We were surprised to clearly Systemic autoimmunity is a complex process involving initial loss identify a plasmablast—as opposed to a —as the dom- of B and T cell tolerance followed by engagement of multiple inant producer of RF autoAb. The normal plasmablasts that dom- by guest on September 28, 2021 effector functions that lead to tissue destruction. The initial inflam- inate early immune responses to foreign Ags are not completely mation undoubtedly leads to subsequent activation of further characterized; however, the population we observed seemed to re- waves of autoreactive lymphocytes and repetition of the cycle until semble the normal population, as evidenced by high expression of a state of chronic inflammation ensues. Most studies of humans CD44 and syndecan and low expression of CD22 (43, 44). Despite and mice have only been able to identify and characterize disease this, it remains possible that there are subtle differences between at this late stage. In part, this is because early stages of disease may plasmablasts secreting RF autoantibody in autoimmune-prone be asymptomatic and because stochastic or environmental events mice and those responding to foreign Ag in normal mice. An even play a major role in determining the onset and nature of autoim- more complete characterization of both populations is required. munity—even identical twins are not always concordant, and in- The identification of the AFC as a plasmablast has important bred mice in the same cage can have markedly different kinetics implications. These plasmablasts have characteristics that would and features of autoimmunity. allow presentation of Ag to T cells and thus may be active in this It is critical to obtain more information about the nature of early role. Further, plasmablasts would be expected to respond quite events in the process. This has been very difficult precisely because differently to certain therapeutic interventions; for example, their the environmental and stochastic factors that incite autoimmunity are not under experimental control, and thus it is difficult to know surface phenotype including the lack of typical B cell markers like at what stage of disease any individual is at a given age or time CD20 and CD22 is important if one is designing Ab-mediated point. We previously devised and validated a method to identify therapy to eliminate AFCs. Plasmablasts can interact with den- the onset of the RF B cell response in a cohort of autoimmune- dritic cells (DCs) and may require factors from DCs for their sur- vival (45, 46). Indeed, we previously showed that RF AFCs were prone mice carrying a Tg for the H chain of an RF autoantibody. ϩ These mice are destined to make an RF autoantibody response, but in contact with CD11c DCs in the spleens of converted mice the time of onset varies over a period of several months (53). We (15). If these interactions are important for plasmablast survival also showed in this system that older mice that had made an RF then disrupting them could represent a therapeutic strategy. For response demonstrated a predominant extrafollicular Ab-forming example, plasmablasts in vitro respond to BAFF (46), a myeloid cell response without an RF GC response. These extrafollicular B product (47), and this could explain the efficacy of inhibition of cells were quite unusual in that they were undergoing somatic BAFF/APRIL in vivo in MRL/lpr mice (48). The abnormal regu- hypermutation but were not further characterized (15). lation of AFCs in NZM2410 mice (49) further highlights the im- portance of understanding the genesis of AFCs in autoimmunity. B cell subpopulations that develop during the RF response The present study makes an important contribution by identifying The major goal of the present work was to define the populations the key role of the plasmablast and defining its phenotype and that were participating in the early splenic RF response, taking relationship to an activated B cell precursor. 6886 PLASMABLASTS DOMINATE THE EARLY RF RESPONSE

A recent report presented a characterization of AFCs in the has a higher affinity than the germline AM14 Ab. This is consistent spleen of autoimmune New Zealand Black/White mice (54). Al- with affinity maturation due to somatic hypermutation. Supporting though this study did not determine the stage of disease we did this interpretation is the finding of certain recurrent replacement here or focus on RF B cells, they did find a substantial proportion mutations in CDRs in our collection of V region sequences (Ref. of short-lived plasmablasts. The authors highlighted the finding of 15 and unpublished data). Thus, we have now shown that both long-lived plasma cells seen in their model, which were not ob- mutation and selection occur outside the GC in the spontaneous RF served in ours. Whether this represents a difference in strain, spec- response in MRL/lpr mice. ificity, stage of disease, or methodology is unclear. Nonetheless, the presence of short-lived plasmablasts is a common feature. Conclusion Cell death and division in the autoimmune B cell response We have used the RF Tg system to develop a detailed picture of both the onset and evolution of a model autoantibody response in Analysis of cell division and death revealed an ongoing, highly the spleen. Like the established response (15), the early response dynamic process of autoimmunity. Both the CD22high and low does not include GCs. Instead, it begins as scattered foci of ex- CD22 populations were undergoing very rapid cell division and trafollicular sites of proliferation and differentiation of RF B cells apoptosis. The revelation that the autoimmune reaction in the (this study).4 CD22high activated B cells are very likely the pre- spleen is so dynamic, if it parallels the human situation, could cursors of CD22low plasmablasts. This event is accompanied by explain the efficacy of cytotoxic and anti-proliferative agents in the appearance of a plasmablast-like cell in the PBL. The entire SLE as well as the rapid time course of such responses. Emerging process is highly dynamic, particularly in the plasmablast com-

pos Downloaded from data from humans treated with anti-CD20 to deplete CD20 B high low neg partment. CD22 and possibly CD22 cells are undergoing cells (but presumably not CD20 plasmablasts or plasma cells somatic hypermutation as well as selection for higher affinity. The directly) demonstrate that some but not all autoantibody titers fall response is progressive and includes at least some clones that live relatively quickly (50, 51). This could be explained if the plasma- ϩ for a long period. blast population were being renewed by a proliferating CD20 - This picture of a dynamic ongoing response has implications for activated B cell population such as the CD22high cells, which are low our understanding of spontaneous systemic autoimmune disease evidently precursors of the CD22 plasmablasts in our system. In and strategies for its treatment. In future work, we would like to http://www.jimmunol.org/ this context, the experiment of depleting CD20 B cells in humans determine whether this scenario applies to other autoAbs and other with autoimmunity suggests that, as in our model, a substantial murine models of autoimmunity as well as human disease. Given proportion of autoantibody is generated from a dynamic process of the common autoAb profiles of MRL/lpr mice and human disease, plasmablast differentiation. we expect that our findings will be generalizable; however, even Mutation and selection in the spleen potential differences will be interesting because they will probably reflect the range of clinical disease and autoAb profiles that vary The identification of two proliferating populations raised the ques- greatly among patients with SLE. tion of which was undergoing somatic mutation (15). However, since we found that both populations contained mutations, we still by guest on September 28, 2021 cannot distinguish whether one or both populations is mutating. Acknowledgments Nonetheless, the BrdU and apoptosis data, along with the fact that We thank Ann Haberman, Shannon Anderson, and Martin Weigert for plasmablasts normally derive from activated B cells, suggest that critical reading of the manuscript. at least the CD22high population is mutating. This does not exclude the possibility that the plasmablasts could also be mutating. The Disclosures presence of a subset of CD22low cells with substantially more mu- The authors have no financial conflict of interest. tations suggests that there may be some ongoing mutation in this population as well. However, resolving this question definitively References has proved difficult. 1. Lipsky, P. E. 2001. 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