-Dependent Kinase Inhibitor Cdkn2c Deficiency Promotes B1a Cell Expansion and Autoimmunity in a Mouse Model of Lupus

This information is current as Hari-Hara S. K. Potula, Zhiwei Xu, Leilani Zeumer, Allison of September 25, 2021. Sang, Byron P. Croker and Laurence Morel J Immunol 2012; 189:2931-2940; Prepublished online 15 August 2012; doi: 10.4049/jimmunol.1200556

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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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Cyclin-Dependent Kinase Inhibitor Cdkn2c Deficiency Promotes B1a Cell Expansion and Autoimmunity in a Mouse Model of Lupus

Hari-Hara S. K. Potula,*,1 Zhiwei Xu,*,1 Leilani Zeumer,* Allison Sang,* Byron P. Croker,*,† and Laurence Morel*

The lupus-prone NZM2410 mice present an expanded B1a cell population that we have mapped to the Sle2c1 lupus susceptibility locus. The expression of Cdkn2c, a encoding for cyclin-dependent kinase inhibitor p18Ink4c and located within Sle2c1,is significantly lower in B6.Sle2c1 B cells than in B6 B cells. To test the hypothesis that the B1a cell expansion in B6.Sle2c1 mice was due to a defective p18 expression, we analyzed the B1a cell phenotypes of p18-deficient C57BL/6 mice. We found a dose-dependent

negative correlation between the number of B1a cells and p18 expression in B cells, with p18-deficient mice showing an early Downloaded from expansion of the peritoneal B1a cell pool. p18 deficiency enhanced the homeostatic expansion of B1a cells but not of splenic conventional B cells, and the elevated number of B6.Sle2c1 B1a cells was normalized by deficiency. These data demonstrated that p18 is a key regulator of the size of the B1a cell pool. B6.p182/2 mice produced significant amounts of anti- DNA IgM and IgG, indicating that p18 deficiency contributes to humoral autoimmunity. Finally, we have shown that Sle2c1 increases lpr-associated lymphadenopathy and T cell–mediated pathology. B6.p182/2.lpr mice showed a greater lymphadenopathy than B6.Sle2c1.lpr mice, but their renal pathology was intermediate between that of B6.lpr and B6.Sle2c1.lpr mice. This indicated http://www.jimmunol.org/ that p18-deficiency synergizes, at least partially, with lpr-mediated pathology. These results show that Cdkn2c contributes to lupus susceptibility by regulating the size of the B1a cell compartment and hence their contribution to autoimmunity. The Journal of Immunology, 2012, 189: 2931–2940.

1a B cells represent a first line of defense in the serous The New Zealand Black (NZB) 3 New Zealand White (NZW) cavities by secreting germline encoded IgM, which is F1 and the NZM2410 mouse models of systemic lupus eryth- B referred to as natural Ab. Unlike conventional B2 cells, ematosus (SLE) present a greatly enlarged B1a cell pool (6), most B1a cells have a fetal origin and are maintained through self- which has been proposed to contribute to autoimmunity through renewal (1), which is achieved through a specific regulation of the several mechanisms: B1a cells produce polyreactive autoanti- by guest on September 25, 2021 (2). Cyclin D2 is required for B1a but not B2 cell de- bodies and home to CXCR5-producing inflamed tissues where velopment (3), while -deficient mice show normal B1a they can class-switch and secrete anti-dsDNA IgG (7, 8). B1a cells cell numbers and functions (4). Cyclin D3 is involved in B1a cell are also highly efficient APCs (9), and they favor the polarization proliferation, which is blocked by the temporal inactivation of of CD4+ T cells into Th17 cells (10). Recently, CD27+CD43+ cyclin D3 complexes in late (4). In addition, phorbol CD702 B cells have been identified as the human functional ester (PMA) stimulation induces B1a but not B2 cell proliferation equivalent to the murine B1a cells on the basis of spontaneous by activating cyclin D2-CDK4 complexes to phosphorylate the IgM secretion, tonic BCR signaling, and ability to activate T cells retinoblastoma gene product (pRb) (5). These results suggest that (11). B1 cells are expanded in the peripheral blood of SLE patients the regulation of the G1 phase through cyclin D2 and D3–CDK and possess a greatly enhanced T cell activation ability (12), complexes is critical for B1a cell proliferation and consequently suggesting that this function, rather than their autoantibody pro- the size of the B1a cell pool. duction, may contribute to autoimmune pathology. In the NZM2410 model, we have shown that the expansion of the B1a cell compartment is B cell intrinsic (13). The largest genetic *Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, FL 32610; and †North Florida South George Veterans Health contribution to this expansion was mapped to the NZB-derived System, Gainesville, FL 32608 Sle2c1 lupus susceptibility locus (14, 15). Sle2c1 enhances auto- 1H.-H.S.K.P. and Z.X. contributed equally to this work. immune pathology either in combination with the NZB genome Received for publication February 14, 2012. Accepted for publication July 16, 2012. (15) or with Fas deficiency (16). The Sle2c1 locus contains the This work was supported by National Institutes of Health Grants RO1 AI068965 (to Ckdn2c gene, which encodes for the cyclin-dependent kinase in- L.M.) and K01AR056725 (to Z.X.). hibitor p18INK4c (p18). p18 fine-tunes the relative amount of ac- Address correspondence and reprint requests to Dr. Laurence Morel, Department of tivated complexes formed between cyclin D2 or D3 on one hand Pathology, Immunology, and Laboratory Medicine, University of Florida, Gaines- and cyclin-dependent kinases CDK4 or CDK6 on the other hand ville, FL 32610-0275. E-mail address: morel@ufl.edu (17). p18 has been shown to be involved both in early and late The online version of this article contains supplemental material. B cell differentiation. p18 facilitates B cell differentiation from Abbreviations used in this article: ANA, anti-nuclear autoantibody; B6, C57BL/6; BM, bone marrow; DN, CD42 CD82 double-negative; ICI, interstitial chronic in- hematopoietic stem cells, and its expression can be compensated kip1 flammation; MZ, marginal zone; NZB, New Zealand Black; NZW, New Zealand partially by p27 (18). At the final stage of B cell differentia- INK4c White; p18, p18 ; PAS, periodic acid–Schiff; Pc, peritoneal cavity; SLE, sys- tion, p18 expression is responsible for the G1 cell cycle arrest that temic lupus erythematosus; Sp, splenic; Treg, Foxp3+ regulatory CD4+ T cell. characterizes plasma cells (19, 20). The expression of Cdkn2c in Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 B cells is 4-fold lower in mice expressing the Sle2c1 allele than www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200556 2932 Cdkn2c REGULATES B CELLS IN LUPUS MICE the B6 allele, and this low expression level segregated with a high gen) costained with relevant lineage markers. At least 50,000 events were number of B1a cells in Sle2c1 recombinants (15). At the molec- acquired per sample on a FACSCalibur cytometer (BD Biosciences). ular level, the Sle2c1 and B6 Cdkn2c alleles differ by a single Correlation between Cdkn2c expression and Pc B1a cell nucleotide polymorphism in the promoter (-74 C/T) that replaces percentage an Nfr2 by a YY-1 binding site adjacent to the existing YY-1 The percentage of Pc B220int CD5+ IgM+ cells was evaluated in a cohort of binding site common to both alleles (21). 2 2 2 2 2 5-mo-old B6, B6.D2 / , B6.p18 / , B6.p18+/ , B6.Sle2, and B6.Sle2c1 Based on these results, we tested the hypothesis that Cdkn2c was mice. Cdkn2c expression was measured from their splenic CD432 B cells the gene responsible for the B1a cell expansion in mice carrying by quantitative real-time PCR with a Taqman probe (ABI) normalized to the Sle2c1 locus by comparing the phenotypes of p18-deficient b-actin, as described previously (15). We have shown previously that Sp C57BL/6 (B6.p182/2) and B6.Sle2c1 mice. B6.p182/2 mice and Pc B1a cells have similar Cdkn2c expression level in (15). showed in an early expansion of the B1a cell subset corresponding Ab measurements to a preferential B1a cell homeostatic expansion. Furthermore, 2/2 Serum anti-ssDNA IgM and anti-dsDNA IgG were measured as previously B6.p18 mice produced autoantibodies, including anti-dsDNA described (23). Relative units were standardized to a 1:100 dilution of IgG and anti-nuclear autoantibody (ANA). The magnitude of these a B6.TC serum set to 100 U. Based on age-matched B6 values, sera phenotypes was greater in p18-deficient mice than in B6.Sle2c1 containing .15 U of anti-dsDNA IgG were considered to be positive for mice, demonstrating that p18 limits the size of the B1a cell this specificity. Total IgM and IgG were measured in sera diluted 1:5000 compartment in a dose-dependent manner. In addition to ex- by sandwich ELISA on plates coated with goat-anti mouse IgM or IgG (ICN/Cappel) at 1 mg/ml, and revealed with AP-conjugated goat-anti panding the B1a cell compartment, Sle2c1 greatly enhances mouse IgM or IgG (Southern Biotech). Sera from B6.Rag12/2 mice lymphadenopathy and the autoimmune pathology induced by Fas- recipients of Pc cells were diluted 1:10 for total IgM measurements. ANA Downloaded from deficiency (16). In this study, we showed that p18 deficiency stains were conducted on slides containing fixed HEp-2 cells (Inova accounts for the enhanced lymphadenopathy and IL-17 production Diagnostics) with mouse sera diluted 1:40 revealed with FITC-conjugated anti-mouse IgG (Southern Biotech) at 1:50 dilution. Staining intensity was in B6.Sle2c1.lpr mice. P18 deficiency, however, only partially calculated with image analysis software (Metamorph, Molecular Devices) contributed to the increased T cell activation characterized by the for a standardized level of green fluorescence on 10–20 randomly selected 2 2 production of CD4 CD8 double-negative (DN) T cells and ac- HEp-2 cells per sample. Samples with an average staining intensity .1 were considered positive. For ELISPOT and in vitro Ab measurements, Pc tivated memory T cells, as well as the concomitant decreased 2 + http://www.jimmunol.org/ + B1a cells were purified as Thy1 CD5 and Sp B cells were purified as production of Foxp3 regulatory T cells (Tregs). Moreover, the 2 2/2 CD43 cells with magnetic beads as described previously (15). The purity renal pathology of B6.p18 .lpr mice was intermediate between of B1a was near 90% for both strains. Total IgM and ssDNA IgM that of B6.lpr and B6.Sle2c1.lpr mice. Overall, these results ELISPOTs were performed as described previously (24). Cells (2 3 105) suggest that Cdkn2c is the Sle2c1 gene that regulates the size of were cultured in medium alone or with 5 mg/ml LPS or PMA for 5 d. Total the B1a cell compartment and therefore their contribution to au- IgM was measured by ELISA in supernatants diluted 1:10 and 1:100. toimmune pathology. In addition, our results suggest the existence Pc cell adoptive transfers of a Sle2c1 modifier gene closely linked to Cdkn2c that accen- Pc lavages were obtained from 5–7-mo-old mice and depleted from ad- tuates the effects of p18 deficiency when combined with Fas de- herent cells by a 2-h culture at 37˚C in PBS supplemented with 10% FBS. ficiency. In one cohort of mice, nonadherent cells were depleted of CD3+ cells with by guest on September 25, 2021 magnetic beads; 1–3 3 106 cells were transferred i.p. into sex-matched B6. Rag12/2 mice. Four cohorts of transfers were performed, each with donor Materials and Methods 2/2 Mice Pc cells from B6 and either B6.Sle2c1 or B6.p18 mice. Within a cohort, the same number of cells was transferred in each recipient. Flow cytometry B6.p182/2 mice (20) and B6.Ccnd22/2 (B6.D22/2) mice (22) were pro- was performed on the donor Pc cells before transfer and on the recipient Pc vided by Dr. Yue Xiong (University of North Carolina) and by Dr. T. and splenic cells 3 wk after transfer. Serum IgM was compared between Rothstein (Feinstein Institute), respectively. Screening of the genetic recipients. background of each of these two strains with 1449 single nucleotide polymorphisms (www.dartmouse.org) revealed a 9.39% and 7.52% non-B6 Renal pathology contribution, respectively. The B6.Sle2c1.Rec1 mice used in this study Kidneys of 4–5-mo-old mice were fixed and stained with H&E and peri- (referred to as B6.Sle2c1) have been described previously (15). This strain odic acid–Schiff (PAS). Renal lesions were scored as previously described carries an ∼10-Mb genomic interval that includes the Cdkn2c gene derived 2/2 (16). Glomerular lesions were classified as negative, mesangial matrix from the NZB genome on a B6 background. B6, B6.lpr, and B6.Rag1 (PAS+) or mesangial cellularity expansion (M), or proliferative glomeru- mice were originally obtained from the Jackson Laboratory. The 2/2 2/2 lonephritis (glomerular hypercellularity with capillary loop involvement, B6.Sle2c1.D2 and B6.p18 .lpr strains were generated by inter- which was global [Pg] or segmental). Extent of involvement was graded on crossing the parental strains and selecting for homozygosity at both loci. a scale of 1–4. Chronic (mononuclear cell) inflammation was classified as B6 and B6.lplr mice were used as controls for Fas-sufficient and Fas-deficient perivascular or interstitial (ICI) and graded as for glomerular inflammation. mice, respectively. Both female and male mice were used at the ages in- dicated. All experiments were conducted according to protocols approved Statistical analysis by the University of Florida Institutional Animal Care and Use Committee. Data were analyzed with GraphPad Prism 5.0 software with the statistical Flow cytometry tests indicated in the text. Nonparametric tests were used when data were not distributed normally. Means, the SEs of the mean (SEM), and the levels of Peritoneal cavity (Pc) lavages and lymph node and splenic (Sp) single-cell statistical significance for two-tailed tests are shown in the figures. suspensions were prepared by lysing RBCs with 0.83% NH4Cl. Cells were first blocked with saturating amounts of anti-CD16/CD32 (2.4G2) and then stained with FITC-, PE-, or biotin-conjugated mAbs: B220 (RA3-6B2), Results CD3e (145-2C11), CD4 (RM4–5), CD5 (53-7.3), CD8a (53-6.7), CD9 p18 expression is inversely correlated with the size of the B1a (KMC8), CD11b (M1/70), CD19 (1D3), CD21 (7G6), CD23 (B3B4), cell compartment CD24 (M1/69), CD40 (3/23), CD43 (S7), CD69 ([1H].2F3), CD80 (16- 2/2 10A), CD86 (GL1), CD93 (AA4.1), CXCR5 (2G8), and IgM (IgH6), all The Pc B1a cell compartment of B6.p18 mice was compared purchased from BD Pharmingen. Biotinylated mAbs were revealed by with that of B6.Sle2c1 and B6 mice at 2 mo (Fig. 1A) and 5–7 mo Streptavidin-PerCP-Cy5.5. Mononuclear live cells were gated on the basis (Fig. 1B) of age. As expected in both cohorts, the number and of forward and side scatter characteristics. In vivo lymphocyte proliferation percentage of B220int CD5+ B1a cells, the ratio of B1a over was analyzed either 18 h after an injection of 1.5 mg BrdU or 7 d after ad hi 2 + libitum exposure to BrdU in the drinking water (0.8 mg/ml). Proliferating B220 CD5 B2 cells (Fig. 1C), and the percentage of IgM cells were detected with a FITC-conjugated anti-BrdU Ab (BD Pharmin- CD5+ B cells (Fig. 1D) were significantly higher in B6.Sle2c1 The Journal of Immunology 2933 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 FIGURE 1. p18 deficiency increases the size of the Pc B1a cell compartment. The absolute number and percentage of B220int CD5+ IgM+ B1a cells, the ratio of B220int CD5+ IgM+ B1a to B220hi CD52 IgM+ B2 cells, and the percentage of IgMhi CD5+ B220+ cells were compared between 2 mo old (A) and 5–7-mo-old (B) B6, B6.Sle2c1 and B6.p182/2 mice. Data were compared with the Dunn multiple comparison tests. For clarity, p # 0.0001 significance levels between B6 and B6.p182/2 values are not shown when the difference between B6 and B6.Sle2c1 was significant at p # 0.001. Representative FACS plots showing the B1a (plain line) and B2 (broken line) gates (C) and the IgMhi CD5+ population in B220+ gated cells (D) in each of the three strains. (E) Correlation between Cdkn2c expression in splenic B cells and the percentage of Pc B1a cells. Each dot represents a single mouse. The p value indicates the significance of a Pearson’s correlation test. (F) Percentage of Pc B220int CD5+ IgM+ B1a cells from 5-mo-old mice. Statistical significance is shown to a two-tailed t test. *p # 0.05, **p # 0.01, ***p # 0.001, ****p # 0.0001. than in B6 mice. These values were even higher in B6.p182/2 for B6 and B6.p18+/2 Pc B1a cells (Fig. 1F). Contrary to its effect mice, which carried ∼5-fold more Pc B1a cells than B6 in both on cell numbers, p18 deficiency had a minimal effect on the age groups. The number and percentage of Pc B1a cells were also surface marker phenotypes of Pc B1a cells (Supplemental Fig. 1). significantly higher in 2-mo-old B6.p182/2 than in B6.Sle2c1 We have previously noted the presence of IgMlo B1a cells in B6. mice. Interestingly, the variance was greater in older B6.p182/2 Sle2c mice (14), and this population was also present in most B6. mice, suggesting that p18-deficiency is a primary genetic defect p182/2 mice (Supplemental Fig. 1A, IgM histogram). The ex- affecting the number of Pc B1a cells, a phenotype then subjected pression level of B220 was variable in p18-deficient Pc B1a cells, to environmental variations as the mice age. The percentage of Sp which also expressed lower amounts of CD69 and CD40 than B6. B1a cells was not affected by p18 expression at 2 mo of age, but Remarkably, p18 deficiency had no effect on the surface expres- was significantly higher in older B6.p182/2 than in B6 mice (6.07 6 sion of the same markers on Sp B cells (Supplemental Fig. 1B). 0.40% versus 3.87 6 0.42%; p = 0.003). As reported previously p18 deficiency also affected other B cell subsets besides B1a (15), no difference was observed for Sp B1a cells between B6 and cells (Table I). Overall, p182/2 bone marrow (BM) contained less B6.Sle2c1 mice in either of the two cohorts. These data suggest B220+ and IgM+ B cells. However, this reduction was not uniform that the size of the B1a cell compartment is directly correlated across B cell developmental stages, with B6.p182/2 mice pro- with p18 expression level. This hypothesis was verified with ducing more pro-B cells and fewer recirculating B cells (Fr. F) a cohort of age-matched mice from strains expressing varying than B6 cells. In the spleen, older B6.p182/2 mice showed more levels of p18 (B6, B6.D22/2, B6.p182/2, B6.p18+/2, B6.Sle2, and immature B cells; in both cohorts of B6.p182/2 mice, the per- B6.Sle2c1), and a highly significant negative correlation was ob- centage of T3 immature B cells was reduced and the percentage tained between Cdkn2c expression and the percentage of Pc B1a of marginal zone (MZ) B cells was expanded. Interestingly, T3 cells (Fig. 1E). However, p18 is haplosufficient for maintaining B cells, which have been described as anergic B cells, have been a normal frequency of Pc B1a cells with similar values obtained found in lower numbers in autoimmune mice, including the NZM 2934 Cdkn2c REGULATES B CELLS IN LUPUS MICE

Table I. p18 deficiency affects other B cell compartments

BM Sp

B220+ IgM+ Pro-B % B220+ Fr. F % B220+ AA4.1+ % IgM+ T3%AA4.1+ MZB % AA4.12 B6, 2 mo 23.30 6 1.09 9.60 6 0.28 41.54 6 2.59 16.62 6 2.00 3.21 6 0.35 25.88 6 1.87 12.10 6 0.75 B6.p182/2,2mo 29.20 6 1.13 12.19 6 0.49 45.63 6 1.79 11.02 6 1.25 3.34 6 0.32 14.46 6 1.07 20.39 6 0.38 B6, 5–7 mo 19.80 6 0.78 9.42 6 0.86 33.91 6 2.00 21.25 6 1.72 2.15 6 0.22 8.00 6 0.61 12.79 6 1.85 B6.p182/2, 5–7 mo 14.32 6 1.07 5.35 6 0.58 45.81 6 2.13 13.30 6 1.58 7.65 6 1.17 4.83 6 0.52 18.03 6 1.22 Mean 6 standard error; n = 7–15 mice per group. Significance of t tests between age-matched strains is shown as p , 0.05 (underlined), p , 0.01 (bold), p , 0.001 (bold and underlined). Fr. F B cells, B220+ CD24int IgMintt BM cells; MZB, IgMhi CD232 CD21hi AA4.1; Pro-B cells, B220+ CD242 IgM2 BM cells; T3, IgMint CD23+ AA4.1+. model (25, 26), whereas the expansion of MZB cell numbers has prompted us to further characterize B6.p182/2 sera for the pres- been reported in multiple autoimmune models (27). Overall, these ence of ANAs. We found robust ANA staining by B6.p182/2 and results show that p18 expression level inversely correlates the size to a lesser extent B6.Sle2c1 sera (Fig. 2B). Among the mice with of the B1a cell compartment with an early massive expansion detectable ANA staining (B6; 4/9, B6.Sle2c1: 4/9, B6.p182/2: 6/9), associated with p18 complete deficiency. p18 deficiency also the B6.p182/2 and B6.Sle2c1 sera produced a significantly more affects the differentiation into other B cell compartments, notably intense ANA staining and showed more speckled and nuclear Downloaded from the T3 and MZ B cells that have been previously associated with staining patterns (Fig. 2C), with the difference reaching sta- autoimmunity. tistical significance between B6 and B6.p182/2 (cytoplasmic versus speckled + nuclear; Fisher exact test, p = 0.035). These p18 deficiency results in the production of ANAs results showed that p18 deficiency promotes the production of B1a cells are the major producers of natural autoantibodies (28), autoantibody with specificities associated with lupus, whereas and B6.Sle2 mice produce high levels of polyclonal autoreactive the residual p18 expression protects B6.Sle2c1 mice from IgM (29). However, the individual Sle2 subloci, including Sle2c1, producing high amounts of IgG with nuclear and dsDNA http://www.jimmunol.org/ have not been associated with autoantibody production (14). We specificities. We investigated the origin of these autoantibodies revisited this topic and found that both Sle2c1 expression and p18 with ELISPOTs performed with Sp B and PC B1a cells. deficiency significantly increased total IgM and IgG levels in older Although p18 deficiency resulted in a moderate increased mice (Fig. 2A). Anti-ssDNA IgM and dsDNA IgG were found secretion of total IgM by Sp B cells, the increased secretion of only in the serum of B6.p182/2 mice, with ∼50% and 30% of the anti-ssDNA IgM was mostly limited to p182/2 Pc B1a cells (Fig. mice being positive, respectively. The presence of anti-dsDNA IgG 2D). We observed a high secretion of anti-ssDNA IgM by the Sp by guest on September 25, 2021

FIGURE 2. p18 deficiency results in the production of autoantibodies. (A) Serum total IgM, anti-ssDNA IgM, total IgG, and anti-dsDNA IgG in 7–12- mo-old B6, B6.Sle2c1, and B6.p182/2 mice. (B) Representative ANA IgG staining of Hep-2 cells incubated with serum from each of the three strains. Original magnification 320. The B6.Sle2c1 sample shows a specked pattern, and the B6.p182/2 sample shows a nuclear pattern. (C) ANA pattern dis- tribution and staining intensity in pixels among the mice in each strain that showed a positive ANA stain. (D) ELIPSOT analyses of total IgM production by Sp B cells (left) and anti-ssDNA IgM production by Pc (P) and Sp (S) B cells (right). (E) IgM secretion by Pc B1a or Sp B cells purified from B6 and B6. p182/2 mice, cultured for 5 d in medium alone, with LPS or PMA. Each dot represents a single mouse. For (A), (C), and (D), data were compared with Mann-Whitney tests between B6 and B6.Sle2c1 or B6.p182/2 .In(E), t tests were used to compare the two strains for each condition. *p # 0.05, **p # 0.01, ***p # 0.001. C, Cytoplasmic; N, nuclear; S, speckled. The Journal of Immunology 2935

B cells of one of six B6.p182/2 mice. These results indicate that cells (data not shown). Finally, the transfer of B6.p182/2 Pc cells p18 deficiency not only increases the number of Pc B1a cells, but resulted in an increased production of serum IgM compared with also skews their repertoire toward more autoreactive specificities. B6 donors (Fig. 3C). These results show a preferential expansion The B6.p182/2 genome derives a significant contribution from of p18-deficient Pc B1a cells in response to homeostatic signals, 129/Sv, and ANA susceptibility loci have been well documented which suggests that p18 deficiency accelerates the cell cycle of in the combination of 129/Sv and B6 genomes (30, 31). We deter- B1a cells, but not of the other B cell subsets. mined that these susceptibility loci are of B6 origin in B6.p182/2, 2 2 Sle2c1-induced expansion of Pc B1a cells is corrected by except one SNP in Sle16 that is of 129/Sv origin in both B6.p18 / 2 2 cyclin D2 deficiency and B6.D2 / mice. Because this locus is ∼1 Mb telomeric to the Fcr and the Slamf gene clusters, and that B6.D22/2 mice do not p18 regulates the activity of cyclin D2 and cyclin D3 through produce ANA (data not shown), it is unlikely that this locus CDK4 and CDK6. Cyclin D2 is the first cyclin to be activated in contributes to ANA production by B6. p182/2 mice. PMA-stimulated B1a cells (5). Deficiency in cyclin D3 is com- One of the characteristics of B1a cells is their ability to spon- pensated by cyclin D2 in B1a cells (4), whereas cyclin D2 defi- taneously secrete IgM. Pc B1a cells purified from B6.p182/2 mice ciency results in a drastic reduction in the number of B1a cells (3). produced significantly more IgM in medium alone than B6 (Fig. We therefore reasoned that if the B1a cell expansion in B6.Sle2c1 2E). B6.p182/2 Pc B1a cells also produced more IgM than B6 in mice is due to an accelerated cell cycle resulting from a decreased response to either LPS or PMA (Fig. 2E). The purity of the B220lo p18 expression, the number of B1a cells in B6.Sle2c1 mice should CD5+ or IgM+ CD5+ cell populations used for these in vitro assays be normalized by cyclin D2 deficiency. The percentage and were equivalent between the two strains (data not shown), indi- number of B1a cells and the B1a/B2 ratio were significantly re- Downloaded from 2 2 cating that p18-deficiency results in a greater intrinsic IgM se- duced in B6.Sle2c1.D2 / compared with B6.Sle2c1 mice at ei- cretion by B1a cells. LPS stimulation resulted in a similar IgM ther 2 mo (Fig. 4A) or 5–7 mo (Fig. 4B) of age. These results production by B6.p182/2 and B6 Sp B cells (Fig. 2E). As ex- validate the hypothesis that the expansion of Pc B1a cells in pected, B6 Sp B cells did not secrete IgM in medium alone or after B6.Sle2c1 mice are driven by an accelerated cyclin-D2–initiated PMA stimulation, and p18 deficiency did not have any effect in cell cycle mediated by a low expression of p18.

these conditions. These results suggest that p18 deficiency spe- http://www.jimmunol.org/ P18 deficiency synergizes with the lpr mutation to induce T cell cifically increased the Ab production and autoreactivity by B1a hyperplasia and autoimmune pathology cells, but has less effect on conventional B cells. This finding is consistent with B1a cells differentiating into Ab-secreting cells We have previously shown that Sle2c1 greatly enhanced the through a mechanism that does not involve G1 phase arrest. lymphadenopathy and T cell–driven autoimmune pathology in- duced by Fas deficiency (16). To test whether this synergy was due p18 deficiency preferentially enhances B1a cell homeostatic to p18 deficiency, we compared B6.p182/2.lpr and B6.Sle2c1.lpr expansion mice aged along with B6.lpr controls. Most B6.p182/2.lpr and The preferential expansion of Pc B1a cells relative to other B cell B6.Sle2c1.lpr mice had to be euthanized for animal welfare issues, 2/2 types in B6.p18 mice suggested that p18-deficient Pc B1a cells when their cervical lymph node mass reached an external diameter by guest on September 25, 2021 have a greater capacity for homeostatic expansion than other of ∼1.5 cm. Some mice were also sacrificed when they developed B cells. Intraperitoneal transfers of nonadherent B6.Sle2c1 or severe dermatitis, which occurred at a similar frequency in B6.p182/2 Pc cells into B6.Rag12/2 mice resulted in significantly B6.p182/2.lpr and B6.Sle2c1.lpr mice (5/36 versus 13/84), and only more Pc B1a cells than transfers of B6 cells (Fig. 3A). Both a few mice were found moribund. Given these endpoint parame- T cells (data not shown) and total IgM+ B cells expanded sig- ters, survival of B6.p182/2.lpr mice was significantly reduced nificantly more from B6.p182/2 than from B6 donors (Fig. 3B). compared with B6.Sle2c1.lpr mice (Fig. 5A; median survival, 120 Among the B cells, the difference was significant only for B1a versus 146.5 d; log-rank test, p , 0.01). All the B6.lpr mice (n = cells, although the same trend was observed in the other cell types. 22) that were sacrificed as age-matched controls were healthy, and The expansion of B6.Sle2c1 B1a cells was intermediate between their cervical lymph nodes did not reach the end-point diameter that of B6 and B6.p182/2 donors (Fig. 3B). Few if any B cells (data not shown). Both B6.p182/2.lpr and B6.Sle2c1.lpr mice de- were found in the spleens of the recipient Rag12/2 mice, and no veloped significant lymphadenopathy of the spleen and even more difference were observed between donor strains. The same results of the lymph nodes (Fig. 5B), which was matched by accordingly were obtained with transfers of nonadherent CD3-depleted Pc increased cell numbers (data not shown). There was no significant

FIGURE 3. p18 deficiency results in the homeostatic expansion of Pc B1a cells. (A) Absolute numbers of Pc CD5+ B220lo IgM+ B1a cells before (donors [D]) and after (recipients [R]) transfer into B6.Rag12/2 mice according to the donor cells’ strain of origin. Data were compared using paired t tests. (B) Fold expansion of the Pc B cells 3 wk after transfer according to the donor cells’ strain of origin. Expansion was calculated for total IgM+ cells, B1a: CD5+ B220int IgM+ cells, B1b: CD52 B220int IgM+ cells, and B2: CD52 B220hi IgM+ cells. (C) Serum IgM in the recipient B6.Rag12/2 mice according to the donor cells’ strain of origin. For (B) and (C), data were compared with t tests between B6 and B6.Sle2c1 or B6.p182/2.*p # 0.05, **p # 0.01, ***p # 0.001. 2936 Cdkn2c REGULATES B CELLS IN LUPUS MICE Downloaded from

FIGURE 4. Cyclin D2 deficiency abrogates Sle2c1-induced Pc B1a cell expansion. The absolute number and percentage of B220int CD5+ IgM+ B1a cells, the ratio of B220int CD5+ IgM+ B1a to B220hi CD52 IgM+ B2 cells, and the percentage of IgMhi CD5+ B220+ cells were compared between 2-mo-old 2 2 2 2 (A) and 5–7-mo-old (B) B6, B6.Sle2c1, B6.Sle2c1.D2 / , and B6.D2 / mice. Data were compared with the Dunn multiple comparison tests. *p # 0.05, http://www.jimmunol.org/ **p # 0.01, ***p # 0.001, ****p # 0.0001. difference between the two strains, but the B6.p182/2.lpr mice old mice and observed similar differences (data not shown). were significantly younger, indicating that p18 deficiency accel- Overall, these data suggest that p18 deficiency contributes to, but erated lpr-induced lymphadenopathy as compared with Sle2c1 is not entirely responsible for, the expansion of DN T cells and expression. As previously shown for B6.Sle2c1.lpr (16), lymph- skewing of Fas-deficient CD4+ T cells toward an activated adenopathy in B6.p182/2.lpr mice was mostly due to T cells (Fig. memory phenotype. 5C). In vivo BrdU incorporation showed high proliferation rates We have reported an expansion of the Th17 subset, a known by guest on September 25, 2021 in CD4+ and DN T cells, which were significantly higher in contributor to lupus renal pathology (33), in B6.Sle2c1.lpr lym- B6.p182/2.lpr than in B6.lpr mice (Fig. 6A, 6B). Proliferation phoid organs and kidneys (16). B6.p182/2.lpr mice presented was lower in B cells, and a difference between strains appeared significantly more IL-17+ CD4+ (Fig. 7A, 7B) and IL-17+ DN only after long term BrdU exposure (Fig. 6B). These results T cells (Fig. 7C) in their spleens than did B6.lpr mice. Impor- suggest that p18 and Fas deficiency synergize to enhance prolif- tantly, a greater percentage of B6.p182/2.lpr DN T cells produced eration, and that this synergy affects T cells more than B cells, IL-17 in the kidneys (Fig. 7D), although the number of renal DN which corresponds to the relative increased of T cell numbers in T cells was similar between B6.p182/2.lpr and B6.lpr mice (data these mice. not shown). As the percentage of CD3+ and DN T cells were The percentage of CD3+ T cells was lower in B6.p182/2.lpr decreased in B6.p182/2.lpr compared with B6.Sle2c1.lpr (Fig. than in B6.Sle2c1.lpr lymph nodes. In the spleen, the percent- 6C, 6D), there was a corresponding decreased in the percentage of age of CD3+ T cells was not significantly different between IL-17+ CD3+ splenocytes; however, the percentage of T cells that B6.p182/2.lpr and B6.lpr, and it was lower than in B6.Sle2c1.lpr were IL-17+ was equivalent between the two strains (data not mice (39.91 6 1.79 versus 46.35 6 2.14%; p = 0.03). Both Sle2c1 shown). As another biomarker of autoimmune pathology, we expression and p18 deficiency significantly expanded the number compared serum anti-dsDNA IgG, which was found in similar of B220+ T cells (Fig. 5D) and DN T cells (Fig. 5E), two subsets amounts in the three Fas-deficient strains (Fig. 7E). However, that are characteristic of Fas-deficient mice (32). As for the per- whereas all B6.Sle2c1.lpr mice were producing anti-dsDNA IgG, centage of total CD3+ T cells, the B6.p182/2.lpr values for B220+ it was the case for only 60% of the B6.lpr and 75% of the and DN T cells were intermediate between that of B6.lpr and B6.p182/2.lpr mice (Fig. 7F). This finding corroborated the small B6.Sle2c1.lpr mice. This was more pronounced in the spleen, effect of Sle2c1 expression on ANA production that we have re- where the percentage of DN T cells was not significantly different ported in older Fas-deficient mice (16). It also indicates that p18 between B6.lpr (45.41 6 1.23%) and B6.p182/2.lpr (41.54 6 deficiency–induced ANA (Fig. 3) does not synergize with lpr-in- 1.96%). Sle2c1 expression tilts the balance in the favor of CD44+ duced ANA. Finally, renal pathology in B6.p182/2.lpr mice was memory T cells to the expense of Foxp3+ Tregs (16). The same also intermediate between B6.lpr and B6.Sle2c1.lpr mice. Glo- trend was observed in B6.p182/2.lpr mice, although the difference merulonephritis was largely limited to mesangial expansion in with B6.lpr did not reach significance for Tregs (Fig. 5F). The B6.p182/2.lpr mice compared with proliferative glomerulonephri- Foxp3+/CD44+ ratio was significantly lower in B6.p182/2.lpr tis, which was the predominant presentation of the B6.Sle2c1.lpr mice than in B6.lpr (0.22 6 0.2 versus 0.30 6 0.02; p , 0.01). renal pathology (Fig. 7G). Moreover, interstitial and perivascular The overall weaker T cell phenotypes in B6.p182/2.lpr compared chronic infiltrates (i.e., ICI) were rarely present in B6.p182/2.lpr with B6.Sle2c1.lpr mice were not due to B6.p182/2.lpr mice kidneys, but abundant in B6.Sle2c1.lpr kidneys (Fig. 7H). Taken being younger on average. Indeed, we compared a cohort of 3-mo- together, cellular analysis of lymphoid tissues, anti-dsDNA IgG The Journal of Immunology 2937 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. p18 deficiency synergizes with the lpr mutation to induce T cell hyperplasia and activation. (A) Survival of B6.p182/2.lpr (n = 36) and B6.Sle2c1.lpr (n = 84) mice, which were sacrificed when their cervical lymph node mass reached 1.5 cM, displayed open sore dermatitis or were found sick. (B) Spleen and total lymph node weights at sacrifice. (C) Percentage of CD3+ lymphocytes in the lymph nodes. Percentage of CD3+ T cells expressing B220 (D) and DN T cells (E). (F) Expression of Foxp3 and CD44 on CD3+CD4+ gated cells, with a graph showing the percentage of Foxp3+ CD4+ T cells. For (D)–(F), a representative FACS plot is shown for each strain with the gates on interest, and values for the whole cohorts are shown on the right. Mice were between 4 and 6 mo old. Data in (B)–(F) were compared with the Dunn multiple comparison tests. *p # 0.05, **p # 0.01, ****p # 0.0001. production, and renal pathology indicate that B6.p182/2.lpr mice mune pathology (23). We have shown that the age-dependent have an intermediate phenotype between B6.lpr and B6.Sle2c1.lpr accumulation of Pc B1a cells in B6.Sle2 mice was B cell intrin- mice. sic and that it was sustained by several mechanisms, including a greater differential proliferation compared with conventional Discussion B cells, and a greater output of B1a cells from adult lymphoid The lupus-prone NZM2410 mice present an expanded B1a cell organs (13). Congenic recombinant screening mapped the greatest population (9) that we have mapped to the Sle2 locus (29). Ex- contribution to Pc B1a cell expansion to a 10-Mb NZB-derived pression of Sle2 by itself on a nonautoimmune background is not interval that we called Sle2c1 (14, 15). analysis pathogenic, but its addition to the Sle1/Sle3 lupus susceptibility of Sle2c1 Pc B1a cells identified a differential expression of cell loci greatly enhances the penetrance and the severity of autoim- cycle , and among them, Cdkn2c, encoding for cyclin- 2938 Cdkn2c REGULATES B CELLS IN LUPUS MICE Downloaded from http://www.jimmunol.org/

FIGURE 6. p18 deficiency preferentially enhances the in vivo proliferation of lpr T cells. Five-month-old B6.lpr and B6.p182/2.lpr mice were exposed to BrdU for either 18 h (A)or7d(B, C). BrdU incorporation was measured in splenic (A, B) and lymph node (C) DN, CD4+ T cells, or CD32 B220+ B cells. Data were compared with t tests. BrdU incorporation in the lymph nodes after 18 h exposure was low and not different between the two strains (data not by guest on September 25, 2021 shown). *p # 0.05, **p # 0.01. dependent kinase inhibitor p18INK4c, is located within Sle2c1.A arrest is a major checkpoint for the size of the Pc B1a cell com- 4-fold lower expression of Cdkn2c in B cells, which corresponds partment. CDK4 and CDK6 are the targets of p18 inhibition, and to a higher cell cycle activity, segregated with the expansion of Pc CDK62/2 and as well as CDK4 and CDK6 double-deficient mice B1a cells in Sle2c1 recombinants (15). A C-to-T transition in the present reduced numbers of lymphocytes in which there is a Cdkn2c promoter results in a differential binding for the tran- delayed G1 progression (34). It is not known whether these muta- scription factor YY-1 to two binding sites in the Sle2c1 allele as tions have any specific effect on the B1a cell subset, which might compared with the single binding site in the B6 allele, and this be difficult to evaluate because CDK2 binding to the D-cyclin can accounts for the decreased transcription efficiency of the NZB compensate for the absence of CDK4 and CDK6 (35). allele (21). Taken together, these results identified Cdkn2c as the Besides Pc B1a cells, p18 deficiency also affected BM B cell prime candidate to control the size of the B1a cell compartment in differentiation, with a greater production of pro-B cells and a B6.Sle2c1 mice. Although Cdkn2c expression has not been pre- lower amount of mature recirculating B cells. A small interfering viously associated with B1a cell differentiation or function, it is RNA knock-down approach has shown a requirement for p18 well documented that cell cycle is regulated differently between expression in B cell lymphopoiesis that can be compensated by p27 B1a and conventional B cells (2). This corresponds to the fact that, expression (18). We have not observed differences in B cell contrary to conventional B cells, B1a cells do not proliferate in lymphopoiesis between B6 and B6.Sle2c1 mice, indicating that response to BCR crosslinking, but maintain a stem cell–like the residual p18 expression in B6.Sle2c1 mice, possibly in com- ability for self-renewal and proliferation. bination with p27, was sufficient to ensure normal B cell differ- To formally prove that a reduced p18 expression was responsible entiation. p18-deficient mice also showed an expanded MZB cell for the Pc B1a cell expansion in B6.Sle2c1 mice, we compared the population. MZB and B1a cells present many functional overlaps, B6.Sle2c1 and B6.p182/2 B1a cell populations and found a dose- and these data suggest that they may also share some mechanisms dependent negative correlation between the size of the Pc B1a cell of cell cycle regulation. compartment and Cdkn2c expression in B cells. In addition, p18- Consistent with a larger number of B1a cells, B6.p182/2 and deficiency resulted in the selectively enhanced homeostatic ex- B6.Sle2c1 mice present elevated levels of serum IgM. p18-deficient pansion of Pc B1a cells, and the number of B6.Sle2c1 Pc B1a cells Pc B1a cells produced more IgM in vitro, either spontaneously or was normalized by cyclin D2 deficiency. These data demonstrated in response to PMA and LPS stimulation. This finding indicated that p18-deficiency was sufficient to greatly expand the number of that p18 deficiency has opposite effects on differentiation into Ab- Pc B1a cells and established that p18 enforcement of cell cycle producing cells, which is enhanced in B1a cells and impaired in The Journal of Immunology 2939 Downloaded from

FIGURE 7. B6.p182/2.lpr mice develop a level of renal pathology intermediate between B6.lpr and B6.Sle2c1.lpr. (A) Representative histograms showing intracellular IL-17 in CD3+CD4+ T cells from B6.p182/2.lpr (black) and B6.lpr (filled gray) spleens. The horizontal bar shows the gate used in (B)–(D). Percentage of splenic CD3+CD4+ IL-17+ (B), splenic CD3+ DN IL-17+ (C), and renal CD3+ DN IL-17+ (D) lymphocytes. (E) Anti-dsDNA IgG present in the sera of the three strains of Fas-deficient mice. (F) Percentages of dsDNA IgG+ mice in these cohorts. (G) Distribution of glomerulonephritis http://www.jimmunol.org/ (GN) scores between the three strains (n = 13–15 mice per strain), with either mesangial expansion (M) or proliferative global (Pg) scores on a scale of 1–4. The B6.p182/2.lpr and B6.Sle2c1.lpr distributions were compared with a x2 test. (H) Percentage of mice showing ICI. All mice were 4–5 mo old. In (F) and (H), data were compared with two-tailed Fisher exact tests. *p # 0.05, **p # 0.01, ***p # 0.001, ****p # 0.0001. conventional B cells. In addition, B6.p182/2 mice produced tivation, however, was not as pronounced in B6.p182/2.lpr mice a significant amount of IgM and IgG autoantibodies, including compared with B6.Sle2c1.lpr mice, considering the expansion of ANAs, which were found at much lower levels in B6.Sle2c1 mice. DN T cells and memory CD4+ T cells, the reduced Foxp3+ Treg While the production of ssDNA IgM is likely to be a direct compartment, or the expression of activation markers on CD4+ consequence of the expansion of the number of B1a cells, the T cells. B6.p182/2.lpr T cells produced a significant amount of by guest on September 25, 2021 production of class-switched anti-dsDNA Abs was less expected. IL-17, consistent with this phenotype being the consequence of an B1a cells have the ability to produce anti-dsDNA IgG in diseased expanded B1a cell compartment. p18 has been reported to inhibit NZB/W F1 mice (7). However, the inflammatory conditions en- Ag-induced T cell proliferation (39). Therefore, the T cell acti- countered in this lupus model are absent in B6.p182/2 mice. It is vation phenotypes in B6.p182/2.lpr and B6.Sle2c1.lpr mice most possible that all B1a cells have a small intrinsic probability to likely result from a combination of T cell–intrinsic and T cell– produce class-switched autoantibodies, and the mere increase in extrinsic B1a cell–dependent factors. The fact that these pheno- number of these cells results in the presence of enough anti- types are more severe in B6.Sle2c1.lpr than in B6.p182/2.lpr mice dsDNA IgG-producing B1a cells to result in substantial anti- is due either to the existence of a closely linked modifier gene in dsDNA IgG production. Alternatively, an expanded B1a cell the Sle2c1 interval or the residual p18 expression, most likely in compartment may contribute indirectly to anti-dsDNA IgG pro- T cells. We are currently screening Fas-deficient Sle2c1 congenic duction through Th17 induction. B1a cells favor Th17 polarization recombinants to test the former hypothesis. The latter will require (10), and Th17 T cells promote the production of autoreactive a detailed analysis of the cell cycle in p18-deficient and Sle2c1 B cells (36). The fact that T cell–activating CD11b+ B1 cells, but T cells with and without Fas expression. not spontaneously IgM-secreting CD11b2 B1 cells, are expanded These results show that Cdkn2c is a lupus susceptibility gene in in SLE patients (12) favors the latter hypothesis. p18 deficiency, the NZM2410 model by regulating the size of and the repertoire however, impairs Ab secretion from conventional B cells (19, 20), of the B1a cell compartment and hence their contribution to au- which we confirmed in B6.Sle2c1 mice (15). Considering these toimmunity. This conclusion is based on results obtained with potentially conflicting contributions, the origin of anti-dsDNA IgG Cdk2nc-deficient mice. A complete demonstration would require in B6.p182/2 mice will have to be addressed directly in future a rescue of the phenotypes by overexpression of the gene. How- studies. ever, to our knowledge this is not feasible because Cdkn2c over- Sle2c1 expression synergizes with Fas-deficiency to enhance expression induces cell cycle arrest (19, data not shown). In not only lymphoproliferation, but also T cell–mediated immune addition, we cannot exclude at this point that the change in B1a pathology (16). B6.p182/2.lpr mice showed an even more en- cell repertoire in the B6.p182/2 mice is due to the expression of hanced lymphoproliferation, which indicates that, in combination 129/Sv loci rather that p18 deficiency, although a careful exami- with lpr, p18-deficiency affects the proliferation of cells other than nation of the 129/Sv genome segregation in that strain makes B1a cells. Nonapoptotic functions have been reported for FADD- it unlikely. Other cyclin-dependent kinase inhibitors have been binding death receptor, including proliferation (37). It is possible previously associated with lupus. Polymorphisms in human that the accelerated cell cycle owing to p18 deficiency synergizes CDKN1A leading to decreased p21CIP1/WAF1 levels have been with the residual Fas expression found in lpr mice (38) in a novel associated with SLE (40), and -deficient mice develop a lupus- manner that will have to be elucidated. The extent of T cell ac- like disease through the accumulation of activated and memory 2940 Cdkn2c REGULATES B CELLS IN LUPUS MICE

T cells without a major effect on B cells (41). In addition, B cell 15. Xu, Z., H. H. Potula, A. Vallurupalli, D. Perry, H. Baker, B. P. Croker, I. Dozmorov, and L. Morel. 2011. Cyclin-dependent kinase inhibitor Cdkn2c homeostasis is regulated by the RAPL-mediated translocation of regulates B cell homeostasis and function in the NZM2410-derived murine lupus Kip1 Cdkn1b/p27 to the nucleus, and the forced sequestration of susceptibility locus Sle2c1. J. Immunol. 186: 6673–6682. p27 in the cytoplasm leads to a lupus-like phenotype (42). We 16. Xu, Z., C. M. Cuda, B. P. Croker, and L. Morel. 2011. The NZM2410-derived lupus susceptibility locus Sle2c1 increases Th17 polarization and induces ne- have not found any association between differences in expression phritis in fas-deficient mice. Arthritis Rheum. 63: 764–774. of either p21, p27, or p19Ink4d and more B1a cells in mice 17. Sherr, C. J., and J. M. Roberts. 1999. CDK inhibitors: positive and negative expressing in the NZB allele of p18 (data not shown), indicating regulators of G1-phase progression. Genes Dev. 13: 1501–1512. 18. Wang, Y. Y., Z. Li, D. Jiao, Z. Zhang, X. Shao, J. Yuan, and P. Yu. 2010. RNA a unique role for p18 in this cell type. These data suggest that the interference reveals a requirement for both p18INK4c and p27Kip1 in B lym- size of lymphocyte subsets is limited by specific cyclin-dependent phopoiesis. J Mol Cell Biol 2: 209–216. 19. Morse, L., D. Chen, D. Franklin, Y. Xiong, and S. Chen-Kiang. 1997. Induction kinase inhibitors, and that impaired expression of each of them of cell cycle arrest and B cell terminal differentiation by CDK inhibitor p18 may result in systemic autoimmunity. It should be noted that the (INK4c) and IL-6. Immunity 6: 47–56. analysis of gene-targeted mice has identified mechanisms other 20. Tourigny, M. R., J. Ursini-Siegel, H. Lee, K. M. Toellner, A. F. Cunningham, D. S. Franklin, S. Ely, M. H. Chen, X. F. Qin, Y. Xiong, et al. 2002. CDK in- than cell cycle that regulate the size of the B1a cell pool, including hibitor p18(INK4c) is required for the generation of functional plasma cells. the strength of the BCR signaling (1). To our knowledge, our Immunity 17: 179–189. results are the first to link a natural polymorphism in a cyclin- 21. Potula, H. H., and L. Morel. 2012. Genetic variation at a Yin-Yang 1 response site regulates the transcription of cyclin-dependent kinase inhibitor p18(INK4C) dependent kinase inhibitor with a dysregulated cell cycle that transcript in lupus-prone mice. J. Immunol. 188: 4992–5002. expands B1a cells and contributes to autoimmunity. The recent 22. Sicinski, P., J. L. Donaher, Y. Geng, S. B. Parker, H. Gardner, M. Y. Park, R. L. Robker, J. S. Richards, L. K. McGinnis, J. D. Biggers, et al. 1996. Cyclin finding of the expansion of B1 cells in lupus patients (12) raises D2 is an FSH-responsive gene involved in gonadal cell proliferation and onco- the possibility that an accelerated cell cycle may be also involved genesis. Nature 384: 470–474. Downloaded from in expanding human B1 cells. 23. Morel, L., B. P. Croker, K. R. Blenman, C. Mohan, G. Huang, G. Gilkeson, and E. K. Wakeland. 2000. Genetic reconstitution of systemic lupus erythematosus immunopathology with polycongenic murine strains. Proc. Natl. Acad. Sci. USA Acknowledgments 97: 6670–6675. We thank Dr. Yue Xiong and Dr. T. Rothstein for the p18-deficient and 24. Niu, H., E. S. Sobel, and L. Morel. 2008. Defective B-cell response to T- dependent immunization in lupus-prone mice. Eur. J. Immunol. 38: 3028–3040. cyclin D2-deficient mice, respectively, and Nathalie Kanda for excellent 25. Merrell, K. T., R. J. Benschop, S. B. Gauld, K. Aviszus, D. Decote-Ricardo, animal care. L. J. Wysocki, and J. C. Cambier. 2006. Identification of anergic B cells within http://www.jimmunol.org/ a wild-type repertoire. Immunity 25: 953–962. 26. Xu, Z., B. Duan, B. P. Croker, and L. Morel. 2006. STAT4 deficiency reduces Disclosures autoantibody production and glomerulonephritis in a mouse model of lupus. The authors have no financial conflicts of interest. Clin. Immunol. 120: 189–198. 27. Nashi, E., Y. Wang, and B. Diamond. 2010. The role of B cells in lupus path- ogenesis. Int. J. Biochem. Cell Biol. 42: 543–550. 28. Haas, K. M., J. C. Poe, D. A. Steeber, and T. F. Tedder. 2005. B-1a and B-1b References cells exhibit distinct developmental requirements and have unique functional 1. Berland, R., and H. H. Wortis. 2002. Origins and functions of B-1 cells with roles in innate and adaptive immunity to S. pneumoniae. Immunity 23: 7–18. notes on the role of CD5. Annu. Rev. Immunol. 20: 253–300. 29. Mohan, C., L. Morel, P. Yang, and E. K. Wakeland. 1997. Genetic dissection of 2. Piatelli, M. J., D. Tanguay, T. L. Rothstein, and T. C. Chiles. 2003. Cell cycle systemic lupus erythematosus pathogenesis: Sle2 on murine 4 leads

control mechanisms in B-1 and B-2 lymphoid subsets. Immunol. Res. 27: 31–52. to B cell hyperactivity. J. Immunol. 159: 454–465. by guest on September 25, 2021 3. Solvason, N., W. W. Wu, D. Parry, D. Mahony, E. W. Lam, J. Glassford, 30. Heidari, Y., A. E. Bygrave, R. J. Rigby, K. L. Rose, M. J. Walport, H. T. Cook, G. G. Klaus, P. Sicinski, R. Weinberg, Y. J. Liu, et al. 2000. Cyclin D2 is es- T. J. Vyse, and M. Botto. 2006. Identification of chromosome intervals from 129 sential for BCR-mediated proliferation and CD5 B cell development. Int. and C57BL/6 mouse strains linked to the development of systemic lupus Immunol. 12: 631–638. erythematosus. Genes Immun. 7: 592–599. 4. Mataraza, J. M., J. R. Tumang, M. R. Gumina, S. M. Gurdak, T. L. Rothstein, 31. Carlucci, F., L. Fossati-Jimack, I. E. Dumitriu, Y. Heidari, M. J. Walport, and T. C. Chiles. 2006. Disruption of cyclin D3 blocks proliferation of normal B- M. Szajna, P. Baruah, O. A. Garden, H. T. Cook, and M. Botto. 2010. Identifi- 1a cells, but loss of cyclin D3 is compensated by cyclin D2 in cyclin D3-deficient cation and characterization of a lupus suppressor 129 locus on chromosome 3. J. mice. J. Immunol. 177: 787–795. Immunol. 184: 6256–6265. 5. Tanguay, D. A., T. P. Colarusso, S. Pavlovic, M. Irigoyen, R. G. Howard, 32. Cohen, P. L., and R. A. Eisenberg. 1992. The lpr and gld genes in systemic J. Bartek, T. C. Chiles, and T. L. Rothstein. 1999. Early induction of cyclin D2 autoimmunity: life and death in the Fas lane. Immunol. Today 13: 427–428. expression in phorbol ester-responsive B-1 lymphocytes. J. Exp. Med. 189: 33. Crispı´n, J. C., and G. C. Tsokos. 2010. Interleukin-17-producing T cells in lupus. 1685–1690. Curr. Opin. Rheumatol. 22: 499–503. 6. Duan, B., and L. Morel. 2006. Role of B-1a cells in autoimmunity. Autoimmun. 34. Malumbres, M., R. Sotillo, D. Santamarı´a, J. Gala´n, A. Cerezo, S. Ortega, Rev. 5: 403–408. P. Dubus, and M. Barbacid. 2004. Mammalian cells cycle without the D-type 7. Enghard, P., J. Y. Humrich, V. T. Chu, E. Grussie, F. Hiepe, G. R. Burmester, cyclin-dependent kinases Cdk4 and Cdk6. Cell 118: 493–504. A. Radbruch, C. Berek, and G. Riemekasten. 2010. Class switching and con- 35. Berthet, C., and P. Kaldis. 2006. Cdk2 and Cdk4 cooperatively control the ex- secutive loss of dsDNA-reactive B1a B cells from the peritoneal cavity during pression of Cdc2. Cell Div. 1: 10. murine lupus development. Eur. J. Immunol. 40: 1809–1818. 36. Doreau, A., A. Belot, J. Bastid, B. Riche, M. C. Trescol-Biemont, B. Ranchin, 8. Moreth, K., R. Brodbeck, A. Babelova, N. Gretz, T. Spieker, J. Zeng-Brouwers, N. Fabien, P. Cochat, C. Pouteil-Noble, P. Trolliet, et al. 2009. Interleukin 17 acts J. Pfeilschifter, M. F. Young, R. M. Schaefer, and L. Schaefer. 2010. The pro- in synergy with B cell-activating factor to influence B cell biology and the teoglycan biglycan regulates expression of the B cell chemoattractant CXCL13 pathophysiology of systemic lupus erythematosus. Nat. Immunol. 10: 778–785. and aggravates murine lupus nephritis. J. Clin. Invest. 120: 4251–4272. 37. Strasser, A., P. J. Jost, and S. Nagata. 2009. The many roles of FAS receptor 9. Mohan, C., L. Morel, P. Yang, and E. K. Wakeland. 1998. Accumulation of signaling in the immune system. Immunity 30: 180–192. splenic B1a cells with potent antigen-presenting capability in NZM2410 lupus- 38. Kobayashi, S., T. Hirano, M. Kakinuma, and T. Uede. 1993. Transcriptional prone mice. Arthritis Rheum. 41: 1652–1662. repression and differential splicing of Fas mRNA by early transposon (ETn) 10. Zhong, X., W. Gao, N. Degauque, C. Bai, Y. Lu, J. Kenny, M. Oukka, insertion in autoimmune lpr mice. Biochem. Biophys. Res. Commun. 191: 617– T. B. Strom, and T. L. Rothstein. 2007. Reciprocal generation of Th1/Th17 and 624. T(reg) cells by B1 and B2 B cells. Eur. J. Immunol. 37: 2400–2404. 39. Kovalev, G. I., D. S. Franklin, V. M. Coffield, Y. Xiong, and L. Su. 2001. An 11. Griffin, D. O., N. E. Holodick, and T. L. Rothstein. 2011. Human B1 cells in important role of CDK inhibitor p18(INK4c) in modulating antigen receptor- umbilical cord and adult peripheral blood express the novel phenotype CD20+ mediated T cell proliferation. J. Immunol. 167: 3285–3292. CD27+ CD43+ CD70-. J. Exp. Med. 208: 67–80. 40. Kim, K., Y. K. Sung, C. P. Kang, C. B. Choi, C. Kang, and S. C. Bae. 2009. A 12. Griffin, D. O., and T. L. Rothstein. 2011. A small CD11b(+) human B1 cell regulatory SNP at position -899 in CDKN1A is associated with systemic lupus subpopulation stimulates T cells and is expanded in lupus. J. Exp. Med. 208: erythematosus and lupus nephritis. Genes Immun. 10: 482–486. 2591–2598. 41. Arias, C. F., A. Ballesteros-Tato, M. I. Garcı´a, J. Martı´n-Caballero, J. M. Flores, 13. Xu, Z., E. J. Butfiloski, E. S. Sobel, and L. Morel. 2004. Mechanisms of peri- C. Martı´nez-A, and D. Balomenos. 2007. p21CIP1/WAF1 controls proliferation toneal B-1a cells accumulation induced by murine lupus susceptibility locus of activated/memory T cells and affects homeostasis and memory T cell Sle2. J. Immunol. 173: 6050–6058. responses. J. Immunol. 178: 2296–2306. 14. Xu, Z., B. Duan, B. P. Croker, E. K. Wakeland, and L. Morel. 2005. Genetic 42. Katagiri, K., Y. Ueda, T. Tomiyama, K. Yasuda, Y. Toda, S. Ikehara, dissection of the murine lupus susceptibility locus Sle2: contributions to in- K. I. Nakayama, and T. Kinashi. 2011. Deficiency of Rap1-binding creased peritoneal B-1a cells and lupus nephritis map to different loci. J. RAPL causes lymphoproliferative disorders through mislocalization of p27kip1. Immunol. 175: 936–943. Immunity 34: 24–38.