Although modifier genes are extensively studied in various diseases, little is known about modifier genes that regulate autoimmune diseases. Autoimmune disease caused by the Faslpr mutation depends on the genetic background of mouse strains, suggesting a crucial role of modifier genes. MRL/MpJ-Faslpr (MRL/lpr) and AKR/lpr mice develop severe and mild lupus-like autoimmune disease, respectively, whereas this mutation does not cause disease on C57BL/6 (B6) or C3H background. Both MRL and AKR carry the same haplotype of the Cd72 gene encoding an inhibitory BCR coreceptor (CD72c), and CD72c contains several amino acid substitutions and a deletion in the extracellular region compared with CD72a and CD72b. To address the role of Cd72c locus in the regulation of Faslpr-induced autoimmune disease, we generated B6.CD72c/lpr and MRL.CD72b/lpr congenic mice. Introduction of the chromosomal interval containing Cd72c did not cause disease in B6 mice by itself, but caused development of lupus-like disease in the presence of Faslpr on B6 background, clearly demonstrating that this interval contains the modifier gene that regulates Faslpr-induced autoimmune disease. Conversely, MRL.CD72b/lpr congenic mice showed milder disease compared with MRL/lpr mice. We further demonstrated that Cd72c is a hypofunctional allele in BCR signal inhibition and that CD72 deficiency induces severe autoimmune disease in the presence of Faslpr. These results strongly suggest that the Cd72c is a crucial modifier gene that regulates Faslpr-induced autoimmune disease due to its reduced activity of B cell signal regulation.

Modifier genes have been extensively studied in various diseases such as cancer, arrythmia, and cystic fibrosis, because penetrance and disease manifestations of the disease caused by disease-causing genes are extensively modified by modifier genes (13). In cystic fibrosis, contribution of modifier genes to the disease variability is almost equivalent to that of environmental factors. Mutation of the Fas gene causes autoimmune disease in both mice and human (47). Penetrance, severity, and manifestations of the disease induced by Faslpr mutation, a loss-of-function mutation of Fas, depend on the genetic background of mouse strains. MRL/MpJ-Faslpr (MRL/lpr) and AKR/lpr mice develop severe and mild lupus-like autoimmune disease, whereas Faslpr does not induce autoimmune disease in C57BL/6 and C3H mice (8, 9). Moreover, Fas-deficient BALB/c mice were recently shown to develop allergic inflammation (10). Thus, the disease caused by Faslpr or Fas deficiency is strongly regulated by modifier genes.

CD72 is a 45-kDa type II membrane protein expressed in B cells. CD72 contains a C-type lectin-like domain in the extracellular region and an immunoreceptor tyrosine-based inhibition motif (ITIM) in the cytoplasmic region (1113). CD72 negatively regulates BCR signaling by recruiting SH2-containing tyrosine phosphatase-1 at the ITIM (1216). In mice, four allelic forms of CD72 (i.e., CD72a, CD72b, CD72c, and CD72d) were serologically defined (17). CD72a, CD72b, and CD72d are highly homologous (18, 19). In contrast, the extracellular region of CD72c has a marked difference from the other alleles including a 7-aa deletion in the C-type lectin-like domain, although the amino acid sequence of the transmembrane and cytoplasmic regions of CD72c is identical to that of the other alleles (18, 19). Interestingly, MRL and AKR, both of which develop autoimmune disease in the presence of Faslpr, carry CD72c, whereas most of the other strains of mice, including BALB/c and C57BL/6 (B6), carry either CD72a or CD72b (18, 19). Moreover, studies using microsatellite markers revealed association of the loci containing Cd72 to development of glomerulonephritis in MRL/lpr mice (2022). Thus, Cd72c is a candidate for a modifier gene that regulates Faslpr-induced autoimmune disease.

In this study, we addressed the role of the Cd72c locus in the development of autoimmune disease by generating B6.CD72c and MRL.CD72b/lpr congenic mice. B cells from B6.CD72c congenic mice showed augmented BCR signaling compared with B6 B cells, and B6.CD72c/lpr developed severe autoimmune disease, whereas B6.CD72c mice showed no disease. Conversely, MRL.CD72b/lpr mice showed less severe autoimmune disease compared with MRL/lpr mice. These results suggest that Cd72c is a functionally defective allele, and the Cd72c locus does not cause any disease by itself but plays a role in development of severe autoimmune disease in MRL/lpr mice probably by augmenting BCR signaling. We further demonstrate that CD72 deficiency causes severe autoimmune disease in the presence of Faslpr by generating CD72-deficient mice. Thus, Cd72c is a modifier gene that plays a crucial role in development of Faslpr-induced autoimmune disease probably through its defective regulatory function on BCR signaling.

The mouse B cell line BAL17 and its transfectants were cultured in RPMI 1640 medium supplemented with 10% FCS, 50 μM 2-ME, 1 mM l-glutamine, and 100 U penicillin/streptomycin. The retrovirus packaging cell line PLAT-E (a gift of Dr. T. Kitamura, University of Tokyo, Tokyo, Japan) (23) was maintained in DMEM supplemented with 10% FCS, 2 mM l-glutamine, and 100 U penicillin/streptomycin. Embryonic stem (ES) cell line R-CMTI-2A derived from B6 mice was purchased from Dainippon Sumitomo Pharma (Osaka, Japan) and was cultured in DMEM medium supplemented with 15% FBS, l-glutamine, nonessential amino acids, and LIF (Chemicon International).

The CD72a and CD72c cDNA was obtained from total RNA prepared from a DBA/2 and MRL/lpr mouse spleen, respectively, by RT-PCR using a set of primers (5′-CCGAATTCATGGCTGACGCTATCACG-3′ and 5′-AAGCGGCCGCTATATCCGGTTCAGTTCAG-3′). These fragments were inserted into the retroviral vector pMX (a gift of Dr. T. Kitamura) (24). For retrovirus production, the packaging cells were transfected with retroviral vectors using a calcium phosphate method. Cells were cultured for 48 h, and the culture supernatant was collected. BAL17 cells were incubated with the supernatant containing retrovirus in the presence of 5 μg/ml polybrene for 4 h.

B6 and MRL/lpr mice were purchased from Sankyo Laboratory Service (Tokyo, Japan). B6/lpr mice were purchased from Japan SLC (Hamamatsu, Japan). QM mice were as described previously (25) (a kind gift from Dr. M. Wabl, University of California, San Francisco, San Francisco, CA). To generate CD72-dificient mice, genomic DNA fragments containing Cd72 were isolated by PCR from the bacterial artificial chromosome (BAC) clone derived from a B6 mouse. The targeting vector was constructed by inserting the neomycin resistance gene flanked by the loxP sequences upstream of the first exon of Cd72 (Supplemental Fig. 2A). The linearized targeting vector was transfected by electroporation into the R-CMTI-2A ES cells. The Cd72+/− ES cell clones 4 and 150 (Supplemental Fig. 2B) were used for blastocyst injection to generate chimeric mice. Lack of CD72 expression in Cd72−/− mice was confirmed by flow cytometry and Western blotting (Supplemental Fig. 2C, 2D). All mice used in this study were bred and maintained in a specific pathogen-free animal facility of Tokyo Medical and Dental University and handled according to our institutional guidelines.

Genomic DNA was extracted from mouse tail and genotyping was done by PCR. Microsatellite primers D4Mit268, D4Mit193, D4Mit196, D4Mit91, D4Mit241, D4Mit17, D4Mit9, D4Mit308, and D4Mit203, located at 8.73, 13.99, 20.16, 23.04, 30.48, 33.96, 43.34, 57.66, and 63.26 cM distal from the centromere on chromosome 4, respectively, were synthesized according to Mouse Genome Informatics (The Jackson Laboratory). The Cd72b and Cd72c allele were specifically amplified using the following primers sets: Cd72b forward, 5′-ACATATTACCAGAAGTGGGA-3′ and reverse, 5′-GGTTAAGGATGTAGGTCACAAGGTCTT-3′; and Cd72c forward, 5′-ATATATAACAAGAAGTGGGC-3′ and reverse, 5′-GGTTAAGGATGTAGGTCACAAGGTCTT-3′. FasWT and Faslpr were specifically amplified using the following primers: forward primer, 5′-TTTACTCATTGACTTATCAAGT-3′; reverse primer specific for FasWT, 5′-AGCCTCCAGGGCCTTCACCTTCTCA-3′; and a reverse primer specific for Faslpr, 5′-CAAATTTTATTGTTGCGAC-3′.

Single-cell suspensions were prepared and stained with the following Abs: FITC-conjugated anti-CD3ε mAb (145-2C11), PE-conjugated anti-CD5 mAb (53-7.3), FITC-conjugated anti-CD21 mAb (7E9) (BioLegend); PE-conjugated anti-CD23 mAb (B3B4), PE-conjugated anti-CD72b+c mAb (JY/93; BD Pharmingen); FITC-conjugated anti-CD72a+b mAb (K10.6; a kind gift from Dr. N. Tada, Tokai University, Tokyo, Japan) (26), Alexa Fluor 647–labeled anti-B220 Ab (RA3-6B2), PE-conjugated anti-CD138 mAb (c363.16A; eBioscience), and FITC-conjugated goat anti-mouse IgM (Southern Biotechnology Associates). Data was collected by a FACSCalibur (BD Biosciences) or a CyAn ADP (DakoCytomation) and analyzed using the FlowJo software (Tree Star) or Summit software (DakoCytomation), respectively.

Serum levels of total IgG were measured by standard sandwich ELISA. Titers of IgG Ab to dsDNA, ssDNA, and chromatin were measured by ELISAs as described previously (27). Briefly, ELISA plates were coated with 10 μg/ml dsDNA, 10 μg/ml ssDNA, or 4 μg/ml chromatin. After blocking with 0.5% BSA in PBS, 50 μl diluted serum samples were added and incubated for 60 min at room temperature. Plates were then washed and incubated with alkaline phosphatase–conjugated goat anti-mouse IgG Ab (Southern Biotechnology Associates). After washing, plates were reacted by phosphatase substrate (Sigma-Aldrich), and the absorbance at 405 nm was measured on a Vmax kinetic microplate reader (Molecular Devices). Autoantibody titers were determined using the sera pooled from (NZB × NZW) F1 mice >8 mo old as a standard.

B cells were purified from mouse spleen as described previously (28) and were stimulated with 0.2 μg/ml 4-hydroxy-3-nitrophenyl acetyl (NP)-BSA or 10 μg/ml F(ab′)2 fragments of goat anti-mouse IgM Ab (Jackson ImmunoResearch Laboratories). Alternatively, BAL17 cells were stimulated with 10 μg/ml F(ab′)2 fragments of goat anti-mouse IgM Ab at 37°C. Cells were lysed in Triton X-100 lysis buffer (1% Triton X-100, 10% glycerol, 150 mM sodium chloride, 20 mM Tris-HCl, 2 mM EDTA, 0.02% sodium azide, 10 μg/ml PMSF, and 1 mM sodium orthovanadate) and immunoprecipitated with rat anti-CD72 mAb JY/93 (BD Pharmingen) using protein G-Sepharose (Amersham Biosciences). Total cell lysates or immunoprecipitates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were incubated with goat anti-mouse IgM Ab (Southern Biotechnology Associates), rabbit anti-CD72 Ab (Santa Cruz Biotechology), or rabbit anti-p42/44 ERK Ab (Cell Signaling Technology), followed by reaction with HRP-conjugated donkey anti-goat IgG Ab (Santa Cruz Biotechnology) or HRP-conjugated goat anti-rabbit IgG Ab (Southern Biotechology Associates). Alternatively membranes were incubated with mouse anti–β-tubulin Ab TUB2.1 (Seikagaku Kogyo), followed by reaction with HRP-conjugated goat anti-mouse IgG Ab (Southern Biotechology Associates). Proteins were then visualized using ECL system (Amersham Biosciences). The intensity of protein bands was quantified using the Image J software (National Institutes of Health).

Spleen B cells were purified as described previously (29). BAL17 cells and its transfectants or purified spleen B cells (1 × 106) were incubated in culture medium containing 5 μg Fluo-4/AM (Molecular Probes) at 37°C for 30 min. Cells were stimulated with 10 μg/ml F(ab′)2 fragments of anti-IgM Ab or 0.2 μg/ml NP-BSA, and fluorescence was continuously measured by an FACSCalibur (BD Bioscience) for a total of 300 s.

Spleen B cells were purified as described previously (28) and labeled with 5 μM CFSE (Molecular Probes). The purity of purified cells was determined by flow cytometry using anti-B220 Ab staining (purity >93%). Cells (2 × 105/ well) were then seeded into 96-well plate and cultured in RPMI 1640 medium supplemented with 10% FCS, 50 μM 2-ME, 1 mM l-glutamine, and 100 U penicillin/streptomycin with or without 10 μg/ml anti-CD40 Ab (FGK45) (30), 10 μg/ml F(ab′)2 fragment of goat anti-mouse IgM Ab (Jackson ImmunoResearch Laboratories), or 10 ng/ml CpG oligomer (ODN 1668) (31) at 37°C for 72 h. The fluorescence of CFSE was measured by a CyAn ADP (DakoCytomation).

Mice were sacrificed, and tissues were fixed in neutral buffered formalin and embedded in paraffin according to standard practices. Tissue sections (5 μm) were stained with either H&E or periodic acid–Schiff and hematoxylin (PASH). Glomerular damages were scored as described previously (32). For immunohistochemical analysis, portions of kidney were embedded in Tissue-Tek OCT compound (Sakura) and snap frozen in liquid nitrogen. Cryostat sections (7-μm thickness) were mounted onto slide glass. The sections were incubated with blocking buffer (PBS containing 0.5% BSA and 0.05% sodium azide) for 30 min and stained with FITC-conjugated anti-mouse IgG Ab (Cappel) or FITC-conjugated anti-mouse C3 Ab (Cappel) at room temperature for 1 h. Sections were analyzed using a laser-scanning microscope Leica DMI6000B (Leica Microsystems).

The protein level of mouse urine was semiquantitatively analyzed as described previously (33).

The data are presented as the means ± SEM, and all statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad). The p values were calculated with the two-tailed Student t test (*p < 0.05, **p < 0.01, ***p < 0.001).

B6/lpr and C3H /lpr mice are reported to show no or only a mild autoimmune disease (8, 9), whereas AKR/lpr and MRL/lpr mice develop moderate and severe lupus-like disease, respectively, suggesting that some genes carried by AKR and MRL mice are required for Faslpr-induced autoimmune disease. To address whether such a gene is located in the Cd72c locus carried by both AKR and MRL, we generated B6.CD72c congenic mice carrying the MRL-derived Cd72c haplotype by selective backcrossing of the F1 hybrid between B6 carrying Cd72b and MRL mice to B6 mice for 11 generations. Microsatellite marker analysis revealed that B6.CD72c mice carry an MRL/lpr-derived interval on chromosome 4 containing the Cd72c locus (Supplemental Fig. 1A). We then generated B6.CD72c/lpr mice by crossing B6.CD72c mice with B6/lpr mice. B6.CD72c/lpr mice at 12 mo of age showed marked splenomegaly (Fig. 1A) and moderate lymphadenopathy (data not shown) compared with B6/lpr mice, whereas the spleen weight in B6.CD72c mice was similar to that in B6 mice. Flow cytometry analysis revealed that percentages of T cells and B cells in both spleen and lymph nodes (LNs) of B6.CD72c mice were similar to those of B6 mice (Table I). In contrast, B6.CD72c/lpr mice showed marked reduction in the percentage of B cells and increase in the percentage of T cells compared with B6/lpr mice. The percentage of B220+CD3+ lpr T cells in B6.CD72c/lpr mice was not increased compared with B6/lpr mice. Thus, introduction of the interval of chromosome 4 containing Cd72c locus induced marked splenomegaly and altered T cell to B cell ratio in B6/lpr but not B6 mice, suggesting that the chromosomal interval containing Cd72c locus does not modulate immune homeostasis by itself, but does so in the presence of Faslpr mutation.

FIGURE 1.

Lupus-like disease in B6.CD72c /lpr congenic mice. (AE) One-year-old female B6, B6.CD72c, B6/lpr, and B6.CD72c/lpr mice (n = 6–11) were analyzed. (A) Spleen weight. (B) Concentrations of total IgG and titers of indicated autoantibodies in sera. Horizontal bars represent mean values. For determining autoantibody titers, pooled sera from >8-mo-old (NZB × NZW) F1 mice are used as a standard (1000 U/ml). (C) PASH staining of glomeruli. Severity of glomerular damage was scored as described previously (32). Grade 0, no involvement; grades 1, 2, and 3, changes in 0–25%, 25–50%, and 50–75% of total glomeruli, respectively; grade 4, sclerosis or crescent formation in >90% of glomeruli. Scale bars, 50 μM. (D) Immunohistochemical analyses for IgG and C3 in glomeruli. Scale bars, 50 μM. (E) H&E staining of liver and lung. Representative data of more than five mice in each genotype are shown (original magnification ×100). Severity of the disease was scored according to the degree of lymphocyte infiltration. Grade 0, no lymphocyte infiltration; grade 1, moderate lymphocyte infiltration; and grade 2, severe lymphocyte infiltration. (F) Serum titer of anti-ssDNA IgG. Six-month-old female B6, B6.CD72c, B6/lpr, B6.CD72c/lpr, and MRL/lpr mice were analyzed (n = 3–10). *p < 0.05, **p < 0.005, ***p < 0.001.

FIGURE 1.

Lupus-like disease in B6.CD72c /lpr congenic mice. (AE) One-year-old female B6, B6.CD72c, B6/lpr, and B6.CD72c/lpr mice (n = 6–11) were analyzed. (A) Spleen weight. (B) Concentrations of total IgG and titers of indicated autoantibodies in sera. Horizontal bars represent mean values. For determining autoantibody titers, pooled sera from >8-mo-old (NZB × NZW) F1 mice are used as a standard (1000 U/ml). (C) PASH staining of glomeruli. Severity of glomerular damage was scored as described previously (32). Grade 0, no involvement; grades 1, 2, and 3, changes in 0–25%, 25–50%, and 50–75% of total glomeruli, respectively; grade 4, sclerosis or crescent formation in >90% of glomeruli. Scale bars, 50 μM. (D) Immunohistochemical analyses for IgG and C3 in glomeruli. Scale bars, 50 μM. (E) H&E staining of liver and lung. Representative data of more than five mice in each genotype are shown (original magnification ×100). Severity of the disease was scored according to the degree of lymphocyte infiltration. Grade 0, no lymphocyte infiltration; grade 1, moderate lymphocyte infiltration; and grade 2, severe lymphocyte infiltration. (F) Serum titer of anti-ssDNA IgG. Six-month-old female B6, B6.CD72c, B6/lpr, B6.CD72c/lpr, and MRL/lpr mice were analyzed (n = 3–10). *p < 0.05, **p < 0.005, ***p < 0.001.

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Table I.
Flow cytometry analysis of spleen and LN cells from B6, B6.CD72c, B6/lpr, and B6.CD72c/lpr mice
TissueCell PopulationB6B6.CD72cB6/lprB6.CD72c/lpr
Spleen Total cell number (×106145.0 ± 11.0 127.0 ± 9.2 320.0 ± 100.0 764.3 ± 38.9* 
 Phenotype (%)a     
  B220+CD3 (B cells) 55.9 ± 3.1 56.0 ± 6.9 46.4 ± 3.9 18.1 ± 5.8** 
  (× 106)b (80.1 ± 4.8) (69.2 ± 11.9) (133.4 ± 38.3) (137.6 ± 47.6) 
  CD3+B220 (T cells) 25.5 ± 3.2 20.3 ± 6.3 24.3 ± 3.4 40.5 ± 4.5* 
  (× 106(37.9 ± 6.4) (24.3 ± 9.9) (89.2 ± 35.4) (315.8 ± 43.4)** 
  B220CD3 16.8 ± 1.9 21.4 ± 8.1 22.5 ± 4.4 34.5 ± 10.1* 
  (× 106(25.3 ± 4.6) (27.6 ± 9.6) (84.0 ± 30.9) (255.4 ± 75.8)** 
  B220+CD3+ (lpr T cells) 1.6 ± 0.1 1.2 ± 0.4 6.7 ± 2.3 7.3 ± 2.7 
  (× 106(3.4 ± 1.3) (1.6 ± 0.6) (13.4 ± 3.7) (55.5 ± 21.2)* 
LN Phenotype (%)b     
  B220+CD3 (B cells) 52.9 ± 6.3 44.2 ± 6.9 43.3 ± 3.0 17.9 ± 7.5* 
  CD3+B220 (T cells) 40.2 ± 6.3 45.1 ± 9.9 24.7 ± 4.1 52.0 ± 8.2* 
  B220CD3 5.8 ± 1.1 9.4 ± 3.7 4.1 ± 2.6 1.9 ± 0.5 
  B220+CD3+ (lpr T cells) 0.9 ± 0.2 1.1 ± 0.3 27.1 ± 1.3 27.3 ± 3.0 
TissueCell PopulationB6B6.CD72cB6/lprB6.CD72c/lpr
Spleen Total cell number (×106145.0 ± 11.0 127.0 ± 9.2 320.0 ± 100.0 764.3 ± 38.9* 
 Phenotype (%)a     
  B220+CD3 (B cells) 55.9 ± 3.1 56.0 ± 6.9 46.4 ± 3.9 18.1 ± 5.8** 
  (× 106)b (80.1 ± 4.8) (69.2 ± 11.9) (133.4 ± 38.3) (137.6 ± 47.6) 
  CD3+B220 (T cells) 25.5 ± 3.2 20.3 ± 6.3 24.3 ± 3.4 40.5 ± 4.5* 
  (× 106(37.9 ± 6.4) (24.3 ± 9.9) (89.2 ± 35.4) (315.8 ± 43.4)** 
  B220CD3 16.8 ± 1.9 21.4 ± 8.1 22.5 ± 4.4 34.5 ± 10.1* 
  (× 106(25.3 ± 4.6) (27.6 ± 9.6) (84.0 ± 30.9) (255.4 ± 75.8)** 
  B220+CD3+ (lpr T cells) 1.6 ± 0.1 1.2 ± 0.4 6.7 ± 2.3 7.3 ± 2.7 
  (× 106(3.4 ± 1.3) (1.6 ± 0.6) (13.4 ± 3.7) (55.5 ± 21.2)* 
LN Phenotype (%)b     
  B220+CD3 (B cells) 52.9 ± 6.3 44.2 ± 6.9 43.3 ± 3.0 17.9 ± 7.5* 
  CD3+B220 (T cells) 40.2 ± 6.3 45.1 ± 9.9 24.7 ± 4.1 52.0 ± 8.2* 
  B220CD3 5.8 ± 1.1 9.4 ± 3.7 4.1 ± 2.6 1.9 ± 0.5 
  B220+CD3+ (lpr T cells) 0.9 ± 0.2 1.1 ± 0.3 27.1 ± 1.3 27.3 ± 3.0 

Data were obtained from 12–14-mo-old mice and are expressed as mean ± SEM (n = 6 to 7).

a

Percentages of cells expressing the indicated surface markers in total lymphocyte-gated cells from spleen and LN.

b

Absolute cell numbers are indicated in parentheses. Statistical significance was calculated between B6 and B6.CD72c mice and between B6/lpr and B6/lpr.CD72c mice.

*

p < 0.05, **p < 0.01.

Next we examined development of autoimmune disease in B6.CD72c/lpr mice. Sera from 12-mo-old B6.CD72c/lpr mice contained a much larger amount of IgG autoantibodies such as anti-dsDNA, anti-ssDNA, and anti-chromatin Abs compared with B6/lpr mice (Fig. 1B). Histopathological and immunohistological analysis of kidney revealed that B6.CD72c/lpr mice developed more severe glomerular lesions with more prominent immune complex deposition compared with B6/lpr mice (Fig. 1C, 1D). Moreover, B6.CD72c/lpr mice showed severe cell infiltration in lung and liver, whereas cell infiltration in these organs was mild in B6/lpr mice (Fig. 1E). In contrast, B6.CD72c mice did not show any pathological findings including kidney, lung, and liver. Thus, the chromosomal interval containing Cd72c carries a modifier gene that regulates Faslpr-induced autoimmune disease.

To compare the autoimmune disease in B6.CD72c/lpr mice to that in MRL/lpr mice, we examined autoantibody production in 6-mo-old B6.CD72c/lpr mice and MRL/lpr mice because MRL/lpr mice do not survive until 12 mo old. The titer of serum anti-DNA IgG in B6.CD72c/lpr mice was significantly lower than that in MRL/lpr mice (Fig. 1F). Thus, MRL loci other than the Cd72c locus are also involved in the development of severe autoimmune disease in MLR/lpr mice.

To further address whether Cd72c locus regulates autoimmune disease in MRL/lpr mice, we generated MRL.CD72b/lpr congenic mice by selective backcrossing of the F1 hybrid between MRL/lpr and B6 (Cd72b) mice to MRL/lpr mice for 12 generations. Analysis with microsatellite markers demonstrated that a B6-derived interval on chromosome 4 containing the Cd72 locus was introduced into MRL.CD72b/lpr mice (Supplemental Fig. 1B). We analyzed 6-mo-old female MRL.CD72b/lpr mice and age-matched female MRL/lpr mice. Compared to MRL/lpr mice, spleen weight (Fig. 2A), the percentage of lpr T cells in spleen (Fig. 2B), and the serum titer of anti-dsDNA and anti-ssDNA IgG (Fig. 2C) were markedly reduced in MRL.CD72b/lpr mice. Although histological score on renal disease based on the percentage of the affected glomeruli was not much improved in MRL.CD72b/lpr mice (Fig. 2D), these mice showed smaller glomerular size (Fig. 2E), reduced immune complex deposition (Fig. 2F), and reduced urine protein level (Fig. 2G), suggesting improvement of renal disease in MRL/lpr mice by replacing the chromosomal interval including Cd72 locus by the B6-derived interval. Taken together, the chromosomal interval including Cd72c plays a role in development of autoimmune disease in MRL/lpr mice, especially in expansion of lpr T cells and autoantibody production.

FIGURE 2.

Reduced severity of autoimmune disease in MRL.CD72b/lpr mice. Female MRL/lpr and MRL.CD72b/lpr mice at 6 mo old were analyzed. (A) Spleen weights (n = 10–16). (B) Percentages of lpr T cells (B220+CD3+), B cells (B220+CD3), and T cells (B220CD3+) in total lymphocyte-gated splenocytes were measured by flow cytometry (n = 3). (C) Concentrations of total IgG and titers of anti-dsDNA and anti-ssDNA IgG in sera were measured by ELISA (n = 5–9). For determining autoantibody titers, pooled sera from >8-mo-old (NZB × NZW) F1 mice are used as a standard (1000 U/ml). (D) PASH staining of glomeruli (n = 3–5). Severity of glomerular damage was scored as in the legend to Fig. 1C. Glomeruli are shown at the same magnification. Scale bars, 50 μM. (E) Size of glomeruli (n = 3–5). The diameter of glomeruli from vascular pole of five randomly selected glomeruli was measured on PASH-stained sections of kidneys (original magnification ×400). Each dot represents the mean value of the glomerular diameter for each mouse. (F) Immunohistochemical analysis of glomeruli for IgG and C3. Representative data of more than three mice in each genotype are shown. Scale bars, 50 μM. (G) Urine protein level. Urine was spotted on filter paper, and the protein level was semiquantitatively measured (n = 7–12). The grade of proteinuria was defined as follows: grade 6, equivalent to 30 mg/ml BSA; grade 5, 10 mg/ml BSA; grade 4, 3.3 mg/ml BSA; grade 3, 1.1 mg/ml BSA; grade 2, 0.74 mg/ml BSA; and grade 1, 0.37 mg/ml BSA. *p < 0.05, **p < 0.005.

FIGURE 2.

Reduced severity of autoimmune disease in MRL.CD72b/lpr mice. Female MRL/lpr and MRL.CD72b/lpr mice at 6 mo old were analyzed. (A) Spleen weights (n = 10–16). (B) Percentages of lpr T cells (B220+CD3+), B cells (B220+CD3), and T cells (B220CD3+) in total lymphocyte-gated splenocytes were measured by flow cytometry (n = 3). (C) Concentrations of total IgG and titers of anti-dsDNA and anti-ssDNA IgG in sera were measured by ELISA (n = 5–9). For determining autoantibody titers, pooled sera from >8-mo-old (NZB × NZW) F1 mice are used as a standard (1000 U/ml). (D) PASH staining of glomeruli (n = 3–5). Severity of glomerular damage was scored as in the legend to Fig. 1C. Glomeruli are shown at the same magnification. Scale bars, 50 μM. (E) Size of glomeruli (n = 3–5). The diameter of glomeruli from vascular pole of five randomly selected glomeruli was measured on PASH-stained sections of kidneys (original magnification ×400). Each dot represents the mean value of the glomerular diameter for each mouse. (F) Immunohistochemical analysis of glomeruli for IgG and C3. Representative data of more than three mice in each genotype are shown. Scale bars, 50 μM. (G) Urine protein level. Urine was spotted on filter paper, and the protein level was semiquantitatively measured (n = 7–12). The grade of proteinuria was defined as follows: grade 6, equivalent to 30 mg/ml BSA; grade 5, 10 mg/ml BSA; grade 4, 3.3 mg/ml BSA; grade 3, 1.1 mg/ml BSA; grade 2, 0.74 mg/ml BSA; and grade 1, 0.37 mg/ml BSA. *p < 0.05, **p < 0.005.

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To elucidate whether CD72c is functionally distinct from CD72b, we crossed B6.CD72c mice with the QM mice on a B6 background expressing CD72b to generate QM. CD72c mice. As almost all B cells from QM mice express BCR reactive to hapten NP due to their expression of knocked-in VH17.2.25 and λ L chain (25), we ligated BCR in spleen B cells from QM mice and QM.CD72c mice using an Ag NP-conjugated BSA and examined BCR signaling by analyzing calcium mobilization and phosphorylation of ERK. Although the Ca2+ response in QM.CD72c B cells was similar to that in QM B cells (Fig. 3A), QM.CD72c B cells showed augmented ERK phosphorylation compared with QM B cells (Fig. 3B).

FIGURE 3.

CD72c is a poor negative regulator of BCR signaling and B cell activation. Spleen B cells were purified from 8–12-wk-old QM and QM.CD72c mice. Fluo-4/AM-loaded (A) or untreated (B) cells were stimulated with 0.2 μg/ml NP-BSA. Intracellular free calcium ion level was measured by flow cytometry (A). The arrowhead indicates the time point when NP-BSA was added. Alternatively, total cell lysates were analyzed for phosphorylation of ERK by Western blotting (B). The same membrane was reprobed with anti–β-tubulin Ab to ensure equal loading. Representative data of three experiments are shown. (C) Purified spleen B cells from B6 or B6.CD72c mice were labeled with CFSE and cultured with indicated reagents for 72 h. CFSE fluorescence was analyzed by flow cytometry. The percentages of proliferated cells are indicated (left panel). Mean ± SD of triplicate is shown (right panel). Data are representative of three independent experiments. (D) CD72 expression in indicated BAL17 transfectants were analyzed by flow cytometry using anti-CD72 Abs K10.6 reactive to CD72a and CD72b and JY/93 reactive to CD72b and CD72c. Mean fluorescence intensity (MFI) is indicated. Unstained cells were used as negative controls (shaded histograms). Fluo-4/AM–loaded (E) or untreated (F) BAL17 transfectants were stimulated with 10 μg/ml anti-IgM Ab. Calcium ion concentration was analyzed by flow cytometry (E). The arrowhead indicates the time point when anti-IgM Ab was added. Representative data of five experiments are shown. Total cell lysates were analyzed for phosphorylation of ERK by Western blotting (F). The same membrane was reprobed with anti–β-tubulin Ab to ensure equal loading. Representative data of three experiments are shown. (G) Total cell lysates of purified spleen B cells from B6 and B6.CD72c mice were immunoprecipitated (IP) with anti-CD72 or control Ab and analyzed by Western blotting using anti-IgM and anti-CD72 Abs. Representative data of three experiments are shown. ***p < 0.001.

FIGURE 3.

CD72c is a poor negative regulator of BCR signaling and B cell activation. Spleen B cells were purified from 8–12-wk-old QM and QM.CD72c mice. Fluo-4/AM-loaded (A) or untreated (B) cells were stimulated with 0.2 μg/ml NP-BSA. Intracellular free calcium ion level was measured by flow cytometry (A). The arrowhead indicates the time point when NP-BSA was added. Alternatively, total cell lysates were analyzed for phosphorylation of ERK by Western blotting (B). The same membrane was reprobed with anti–β-tubulin Ab to ensure equal loading. Representative data of three experiments are shown. (C) Purified spleen B cells from B6 or B6.CD72c mice were labeled with CFSE and cultured with indicated reagents for 72 h. CFSE fluorescence was analyzed by flow cytometry. The percentages of proliferated cells are indicated (left panel). Mean ± SD of triplicate is shown (right panel). Data are representative of three independent experiments. (D) CD72 expression in indicated BAL17 transfectants were analyzed by flow cytometry using anti-CD72 Abs K10.6 reactive to CD72a and CD72b and JY/93 reactive to CD72b and CD72c. Mean fluorescence intensity (MFI) is indicated. Unstained cells were used as negative controls (shaded histograms). Fluo-4/AM–loaded (E) or untreated (F) BAL17 transfectants were stimulated with 10 μg/ml anti-IgM Ab. Calcium ion concentration was analyzed by flow cytometry (E). The arrowhead indicates the time point when anti-IgM Ab was added. Representative data of five experiments are shown. Total cell lysates were analyzed for phosphorylation of ERK by Western blotting (F). The same membrane was reprobed with anti–β-tubulin Ab to ensure equal loading. Representative data of three experiments are shown. (G) Total cell lysates of purified spleen B cells from B6 and B6.CD72c mice were immunoprecipitated (IP) with anti-CD72 or control Ab and analyzed by Western blotting using anti-IgM and anti-CD72 Abs. Representative data of three experiments are shown. ***p < 0.001.

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Next, we addressed proliferative response of Cd72c-carrying B cells to various stimuli by CFSE dilution assay. When purified spleen B cells from B6 and B6.CD72c mice were stimulated with CpG oligomers or anti-CD40 Ab, percentage of proliferated cells were significantly higher in B6.CD72c cells than in B6 B cells (Fig. 3C). Percentage of divided cells after anti-IgM stimulation was not increased in B6.CD72c B cells compared with B6 B cells probably because B6 B cells fully proliferated to this stimulation. Thus, Ag-induced ERK activation and proliferative response to CpG and anti-CD40 Ab were augmented in B6.CD72c B cells, suggesting that CD72c negatively regulates B cell activation less efficiently than CD72b does, although the possibility that the other genes in the MRL-derived interval in B6.CD72c mice regulate B cell activation is not excluded.

To directly demonstrate that CD72c poorly regulates BCR signaling, we transduced CD72c and CD72a, the latter of which is highly homologous to CD72b, to the mouse B cell line BAL17 and examined their capacity to regulate BCR signaling. As BAL17 cells express endogenous CD72b (34), we examined the expression of the transduced CD72a and CD72c by flow cytometry using anti-CD72 mAbs reactive to CD72a and CD72b and that reactive to CD72b and CD72c, respectively. The expression level of CD72 in BAL17-CD72a transfectant and BAL17-CD72c transfectant are 1.74 and 3.58 times higher than parent BAL17 cells, respectively (Fig. 3D). We are not able to exclude the possibility that these anti-CD72 Abs react to different CD72 allelic forms with different efficiency or the possibility that transduced CD72 affects expression of endogenous CD72 or forms a heterodimer with endogenous CD72. Nonetheless, our result on flow cytometry suggests that CD72c expression in BAL17-CD72c cells is higher than CD72a expression in BAL17-CD72a cells. We then ligated BCR on BAL17 transfectants using anti-IgM Ab and analyzed calcium mobilization and ERK phosphorylation. Both calcium mobilization and ERK phosphorylation induced by BCR ligation were reduced in BAL17-CD72a transfectant compared with control transfectant (Fig. 3E, 3F), indicating that CD72a negatively regulates BCR signaling in agreement with previous findings (14). In contrast, CD72c expression reduced both calcium mobilization and ERK phosphorylation only marginally if any (Fig. 3E, 3F), although the CD72 expression level in CD72c transfectant is higher than that in CD72a transfectant. Thus, CD72c regulates BCR signaling less efficiently than CD72a in both primary B cells and BAL17 cells.

CD72c differs from CD72a or CD72b at the extracellular part but not the cytoplasmic region including ITIM. To address how the extracellular part of CD72c reduces its negative-regulatory activity on B cell activation, we examined association of CD72 to BCR. When we immunoprecipitated CD72b and CD72c from lysates of B6 and B6.CD72c B cells, respectively, CD72b coprecipitated more IgM than CD72c did (Fig. 3G). This result suggests that CD72c associates with BCR less strongly than CD72b does, although we are not able to exclude the possibility that anti-CD72 Abs differently react to the different CD72 allelic forms, resulting in different immunoprecipitation and detection efficiency depending on the allelic forms. Taken together, CD72c regulates B cell signaling and B cell activation inefficiently probably due to its weak association to BCR.

As CD72c regulates BCR signaling less efficiently than CD72a, we next addressed whether CD72 deficiency induces severe autoimmune disease in the presence of the Faslpr gene by generating Cd72−/− mice on a B6 background (Supplemental Fig. 2). When we examined signaling properties of Cd72−/− B6 B cells, BCR ligation–induced ERK phosphorylation was augmented compared with that in wild-type B6 B cells expressing CD72b (Fig. 4A), whereas calcium signaling in Cd72−/− B cells was similar to that in wild-type B6 B cells (Fig. 4B). Thus, CD72 appears to efficiently regulate BCR ligation–induced ERK phosphorylation but not calcium signaling in agreement with our results in QM.CD72c B cells and BAL17 transfectants (Fig. 3). Proliferative response to CpG and anti-CD40 Ab was augmented in Cd72−/− B cells compared with wild-type B6 B cells (Fig. 4C), as is the case for B6.CD72c B cells. Thus, signaling and proliferative properties of Cd72−/− B cells are similar to those of Cd72c-carrying B cells.

FIGURE 4.

Severe autoimmune disease in Cd72−/− B6/lpr mice. (A and B) Spleen B cells were purified from 8–12-wk-old B6 and Cd72−/− B6 mice. Total cell lysates were analyzed for phosphorylation of ERK by Western blotting (A). The same membrane was reprobed with anti–β-tubulin Ab to ensure equal loading. The intensity of the protein bands was quantified, and the relative amounts of phosphorylated ERK and β-tubulin are indicated. Representative data of three experiments are shown. Alternatively, Fluo-4/AM–loaded cells were stimulated with 10 μg/ml anti-IgM Ab (B). Intracellular free calcium ion level was measured by flow cytometry. The arrowhead indicates the time point when anti-IgM Ab was added. (C) Purified B cells from B6 and Cd72−/− B6 mice were labeled with CFSE and cultured with indicated reagents for 72 h. CFSE fluorescence was analyzed by flow cytometry. The percentages of proliferated cells are indicated (left panel). Mean ± SD of triplicate is shown (right panel). Data are representative of three independent experiments. ***p < 0.0001. (DH) Female wild-type B6, Cd72−/− B6, B6/lpr, and Cd72−/− B6/lpr mice at 6 mo old were analyzed. (D) Spleen weight (n = 6–12). (E) Concentrations of total IgG and titers of anti-dsDNA, anti-ssDNA, and anti-chromatin IgG in sera were determined by ELISA. For determining autoantibody titers, pooled sera from >8-mo-old (NZB × NZW) F1 mice are used as a standard (1000 U/ml). Horizontal bars represent mean values (n = 10–14). (F) PASH staining of glomeruli. Severity of glomerular damage was scored as in the legend to Fig. 1C. Scale bars, 50 μM. (G) Immunohistochemical analyses for IgG and C3 in glomeruli. Scale bars, 50 μM. (H) H&E staining of liver and lung (original magnification ×100). Severity of the disease was scored as in the legend to Fig. 1E. Representative data of more than six mice in each genotype are shown. *p < 0.05, **p < 0.005, ***p < 0.001.

FIGURE 4.

Severe autoimmune disease in Cd72−/− B6/lpr mice. (A and B) Spleen B cells were purified from 8–12-wk-old B6 and Cd72−/− B6 mice. Total cell lysates were analyzed for phosphorylation of ERK by Western blotting (A). The same membrane was reprobed with anti–β-tubulin Ab to ensure equal loading. The intensity of the protein bands was quantified, and the relative amounts of phosphorylated ERK and β-tubulin are indicated. Representative data of three experiments are shown. Alternatively, Fluo-4/AM–loaded cells were stimulated with 10 μg/ml anti-IgM Ab (B). Intracellular free calcium ion level was measured by flow cytometry. The arrowhead indicates the time point when anti-IgM Ab was added. (C) Purified B cells from B6 and Cd72−/− B6 mice were labeled with CFSE and cultured with indicated reagents for 72 h. CFSE fluorescence was analyzed by flow cytometry. The percentages of proliferated cells are indicated (left panel). Mean ± SD of triplicate is shown (right panel). Data are representative of three independent experiments. ***p < 0.0001. (DH) Female wild-type B6, Cd72−/− B6, B6/lpr, and Cd72−/− B6/lpr mice at 6 mo old were analyzed. (D) Spleen weight (n = 6–12). (E) Concentrations of total IgG and titers of anti-dsDNA, anti-ssDNA, and anti-chromatin IgG in sera were determined by ELISA. For determining autoantibody titers, pooled sera from >8-mo-old (NZB × NZW) F1 mice are used as a standard (1000 U/ml). Horizontal bars represent mean values (n = 10–14). (F) PASH staining of glomeruli. Severity of glomerular damage was scored as in the legend to Fig. 1C. Scale bars, 50 μM. (G) Immunohistochemical analyses for IgG and C3 in glomeruli. Scale bars, 50 μM. (H) H&E staining of liver and lung (original magnification ×100). Severity of the disease was scored as in the legend to Fig. 1E. Representative data of more than six mice in each genotype are shown. *p < 0.05, **p < 0.005, ***p < 0.001.

Close modal

We then bred Cd72−/− mice with B6/lpr mice and analyzed 6-mo-old female Cd72−/− B6/lpr mice. These mice showed severe splenomegaly (Fig. 4D) and lymphadenopathy (data not shown) and marked expansion of lpr T cells in spleen, LNs, and peritoneal cavity (peritoneal exudate cells [PEC]) (Tables II, III), whereas expansion of lpr T cells is mild in B6/lpr mice. As is the case for B6.CD72c/lpr mice, percentages of T cells and B cells were increased and decreased, respectively, in Cd72−/− B6/lpr mice (Table II). In contrast, Cd72−/− mice showed mild splenomegaly but no distorted proportions of T and B cells. Thus, CD72 deficiency induces marked splenomegaly synergistically with the Faslpr gene and accelerates expansion of lpr T cells.

Table II.
Flow cytometry analysis of spleen cells from B6, Cd72−/− B6, B6/lpr, and Cd72−/− B6/lpr mice
Cell PopulationB6Cd72−/− B6B6/lprCd72−/− B6/lpr
Total cell number (×10698.8 ± 9.3 160.2 ± 15.4 121.6 ± 12.4 308.8 ± 92.8 
Phenotype (%)a     
 B220+CD3 (B cells) 45.6 ± 2.2 40.0 ± 2.4 55.1 ± 1.3 21.6 ± 3.6** 
  (× 106)b (44.5 ± 3.9) (63.2 ± 6.2) (66.9 ± 6.5) (54.4 ± 6.6) 
 CD3+B220 (T cells) 41.4 ± 2.7 41.7 ± 1.2 19.4 ± 1.2 31.0 ± 4.2* 
  (× 106(41.3 ± 5.3) (66.3 ± 5.6) (23.8 ± 3.6) (108.7 ± 49.6)* 
 B220CD3 12.1 ± 1.4 16.3 ± 2.4 8.0 ± 1.0 16.0 ± 2.5* 
 (× 106(12.0 ± 1.6) (27.3 ± 5.5) (9.6 ± 1.2) (41.9 ± 4.9)** 
 B220+CD3+ (lpr T cells) 0.9 ± 0.1 2.0 ± 0.5 17.5 ± 1.2 31.3 ± 3.7** 
  (× 106(0.9 ± 0.2) (3.4 ± 1.0) (21.3 ± 2.6) (103.8 ± 40.5)* 
Phenotype of B220+ B cells (%)b     
 CD21±CD23hi (FO B cells) 76.1 ± 2.1 52.6 ± 3.1* 46.3 ± 1.4 15.8 ± 5.0*** 
 CD21hiCD23± (MZ B cells) 11.0 ± 0.9 7.2 ± 0.7* 10.8 ± 0.9 0.6 ± 0.3*** 
Cell PopulationB6Cd72−/− B6B6/lprCd72−/− B6/lpr
Total cell number (×10698.8 ± 9.3 160.2 ± 15.4 121.6 ± 12.4 308.8 ± 92.8 
Phenotype (%)a     
 B220+CD3 (B cells) 45.6 ± 2.2 40.0 ± 2.4 55.1 ± 1.3 21.6 ± 3.6** 
  (× 106)b (44.5 ± 3.9) (63.2 ± 6.2) (66.9 ± 6.5) (54.4 ± 6.6) 
 CD3+B220 (T cells) 41.4 ± 2.7 41.7 ± 1.2 19.4 ± 1.2 31.0 ± 4.2* 
  (× 106(41.3 ± 5.3) (66.3 ± 5.6) (23.8 ± 3.6) (108.7 ± 49.6)* 
 B220CD3 12.1 ± 1.4 16.3 ± 2.4 8.0 ± 1.0 16.0 ± 2.5* 
 (× 106(12.0 ± 1.6) (27.3 ± 5.5) (9.6 ± 1.2) (41.9 ± 4.9)** 
 B220+CD3+ (lpr T cells) 0.9 ± 0.1 2.0 ± 0.5 17.5 ± 1.2 31.3 ± 3.7** 
  (× 106(0.9 ± 0.2) (3.4 ± 1.0) (21.3 ± 2.6) (103.8 ± 40.5)* 
Phenotype of B220+ B cells (%)b     
 CD21±CD23hi (FO B cells) 76.1 ± 2.1 52.6 ± 3.1* 46.3 ± 1.4 15.8 ± 5.0*** 
 CD21hiCD23± (MZ B cells) 11.0 ± 0.9 7.2 ± 0.7* 10.8 ± 0.9 0.6 ± 0.3*** 

Data were obtained from 6-mo-old mice and are expressed as mean ± SEM (n = 5 to 6). Statistical significance was calculated between B6 and Cd72−/− B6 mice and between B6/lpr and Cd72−/− B6/lpr mice.

a

Percentages of cells expressing the indicated surface markers in lymphocyte-gated cells.

b

Absolute cell numbers are indicated in parentheses.

*

p < 0.05, **p < 0.01, ***p < 0.001.

FO, Follicular; MZ, marginal zone.

Table III.
Flow cytometric analysis of LN, bone marrow, and PEC from B6, Cd72−/− B6, B6/lpr, and Cd72−/− B6/lpr mice
TissueCell PopulationB6Cd72−/− B6B6/lprCd72−/− B6/lpr
LN B220+CD3 (B cells) 28.0 ± 2.1 29.2 ± 2.2 42.2 ± 3.6 13.6 ± 4.2** 
 CD3+B220 (T cells) 69.3 ± 2.2 64.8 ± 2.0 10.9 ± 1.0 26.1 ± 2.8** 
 B220+CD3+ (lpr T cells) 0.4 ± 0.1 0.6 ± 0.1 42.0 ± 4.3 57.7 ± 2.2* 
BM B220+IgM (pre-B cells) 10.8 ± 1.1 19.5 ± 2.2* 15.4 ± 2.5 8.3 ± 1.6* 
 B220lowIgMlow (immature B cells) 6.5 ± 0.8 6.6 ± 0.3 5.0 ± 0.7 2.2 ± 0.4* 
 B220+IgMhigh (transitional B cells) 2.7 ± 0.5 1.5 ± 0.3 1.8 ± 0.3 0.7 ± 0.4* 
 B220highIgMlow (mature B cells) 6.5 ± 0.8 4.9 ± 0.6 3.4 ± 1.2 2.6 ± 0.6 
PEC IgMhighCD5+ (B1-a cells) 12.2 ± 2.9 11.6 ± 3.8 3.1 ± 0.2 4.5 ± 0.1 
 IgMB220+CD5+ (lpr T cells) ND ND 32.9 ± 6.6 56.0 ± 0.7* 
TissueCell PopulationB6Cd72−/− B6B6/lprCd72−/− B6/lpr
LN B220+CD3 (B cells) 28.0 ± 2.1 29.2 ± 2.2 42.2 ± 3.6 13.6 ± 4.2** 
 CD3+B220 (T cells) 69.3 ± 2.2 64.8 ± 2.0 10.9 ± 1.0 26.1 ± 2.8** 
 B220+CD3+ (lpr T cells) 0.4 ± 0.1 0.6 ± 0.1 42.0 ± 4.3 57.7 ± 2.2* 
BM B220+IgM (pre-B cells) 10.8 ± 1.1 19.5 ± 2.2* 15.4 ± 2.5 8.3 ± 1.6* 
 B220lowIgMlow (immature B cells) 6.5 ± 0.8 6.6 ± 0.3 5.0 ± 0.7 2.2 ± 0.4* 
 B220+IgMhigh (transitional B cells) 2.7 ± 0.5 1.5 ± 0.3 1.8 ± 0.3 0.7 ± 0.4* 
 B220highIgMlow (mature B cells) 6.5 ± 0.8 4.9 ± 0.6 3.4 ± 1.2 2.6 ± 0.6 
PEC IgMhighCD5+ (B1-a cells) 12.2 ± 2.9 11.6 ± 3.8 3.1 ± 0.2 4.5 ± 0.1 
 IgMB220+CD5+ (lpr T cells) ND ND 32.9 ± 6.6 56.0 ± 0.7* 

Data were obtained from 6-mo-old mice and are expressed as mean ± SEM (n = 5 to 6). Values represent the percentages of cells expressing the indicated surface makers in total lymphocyte-gated cells from bone marrow (BM), PEC, and LN. Statistical significance was calculated between B6 and Cd72/ B6 mice and between B6/lpr and Cd72/ B6/lpr mice.

*

p < 0.05, **p < 0.001.

ND, Not detected.

Although the serum IgG level in Cd72−/− B6 and Cd72−/− B6/lpr mice at 6 mo of age were comparable to that in age-matched Cd72+/+ B6 mice, the high titer of anti-chromatin IgG was produced in both Cd72−/− and Cd72−/− B6/lpr mice (Fig. 4E). Although the titers of anti-dsDNA and anti-ssDNA IgG were significantly increased in Cd72−/− B6 mice compared with Cd72+/+ B6 mice, the titers of these autoantibodies were markedly higher in Cd72−/− B6/lpr mice than in Cd72−/− B6 mice (Fig. 4E), suggesting that CD72 deficiency induces production of a large amount of anti-DNA Abs in the presence of the Faslpr gene. Histopathological analysis revealed development of glomerulonephritis with immune complex deposition (Fig. 4F, 4G) and cell infiltration in lung (Fig. 4H) in Cd72−/− B6 mice, which are consistent with the previous report (35). Cd72−/− B6/lpr mice developed more severe glomerulonephritis and cell infiltration in lung and liver than Cd72−/− B6 or B6/lpr mice, suggesting that CD72 deficiency induces development of autoimmune glomerulonephritis and cell infiltration in lung and liver synergistically with the Faslpr gene.

Autoimmune disease caused by Faslpr depends on the genetic background of mouse strains. In this study, we demonstrate that introduction of the MRL-derived chromosomal interval containing Cd72c into B6 mice did not cause any disease but markedly enhanced severity of autoimmune disease in B6/lpr mice. This result clearly demonstrates that this locus contains a modifier gene that regulates Faslpr-induced autoimmune disease. Conversely, introduction of the B6-derived interval containing Cd72b reduced the severity of the disease in MRL/lpr mice further support the crucial role of this locus in regulation of Faslpr-induced autoimmune disease. We also demonstrated that CD72c is hypofunctional in regulating BCR signaling and B cell activation and that CD72 deficiency induces severe autoimmune disease in the presence of Faslpr. Thus, in B6.CD72c/lpr mice, the hypofunctional Cd72c allele but not other genes in the MRL-derived chromosomal interval appears to be responsible for induction of severe autoimmune disease, and Cd72c is a modifier gene that regulates Faslpr-induced autoimmune disease.

Our finding on the role of Cd72c in development of autoimmune disease is also supported by the finding by Oishi et al. (36). They generated MRL/lpr mice carrying a BAC transgene encoding Cd72b and a mutant BAC in which exon 8 encoding a part of the extracellular region of Cd72 was replaced by the exon 8 derived from Cd72c. The Cd72b-containing BAC but not the mutant BAC markedly reduced BCR signaling and severity of the autoimmune disease in MRL/lpr mice, clearly demonstrating distinct functional activity of CD72c and its role in development of autoimmune disease in MRL/lpr mice, in agreement with our finding. Previously, Li et al. (35) demonstrated that Cd72−/− mice spontaneously develop autoimmune manifestations including glomerulonephritis and inflammatory infiltration of the lung and salivary glands at 1 y of age, and we confirmed this finding in the independently established Cd72−/− mouse line (Fig. 4). Thus, CD72 deficiency but not Cd72c causes mild lupus-like disease by itself. Cd72c may not cause autoimmune disease by itself as it probably retains its regulatory activity to some extent. Thus, a hypofunctional allele of a gene that is crucial for preventing autoimmune disease can play a role as a modifier gene.

An old study demonstrated that the Faslpr locus induces autoimmune disease in mice with AKR but not C3H or B6 backgrounds (8). Because AKR as well as MRL carries Cd72c, Cd72c may be a modifier gene involved in the development of autoimmune disease in AKR/lpr as well as MRL/lpr mice. In human, mutations of Fas cause autoimmune lymphoproliferative syndrome (ALPS), in which penetrance is variable among families (6, 7, 37). As there is a functional difference between human CD72 haplotypes (38, 39), CD72 polymorphism may play a crucial role in the regulation of penetrance and disease manifestations in ALPS. Modifier genes are extensively studied in various diseases including cystic fibrosis, arrhythmia, and cancer because modifier genes extensively regulate penetrance, severity, and manifestations of these diseases (13). Also, modifier genes can be a good target of therapy and prevention if it is difficult to correct the defect caused by disease-causing mutations. However, little is known about modifier genes that regulate autoimmune diseases. The Yaa gene may be another modifier gene that regulates autoimmune diseases because it is required for development of autoimmune disease in BXSB mice but does not induce autoimmune disease by itself in the B6 background (40), although Yaa is naturally found only in BXSB mice. As most cases of autoimmune diseases appear to involve multiple genes, all of which contribute to a minor component (41), it may not be straightforward to distinguish modifier genes from disease-causing genes in autoimmune diseases, except for the cases in which a single gene plays a dominant role, such as patients with ALPS and MRL/lpr mice.

In this study, the autoimmune disease in B6.CD72c/lpr mice is less severe than that in MRL/lpr mice, indicating involvement of other MRL-derived genes in development of the severe disease. This is consistent with the previous findings on the association of other genetic loci such as the Opn (20) and FcγRIIB loci (42, 43) with development of autoimmune disease in MRL/lpr mice. Thus, multiple genes including Faslpr and Cd72c are involved in development of severe autoimmune disease in MRL/lpr mice. Lack of these genes other than Faslpr and Cd72c may explain why AKR/lpr develops milder autoimmune disease than MRL/lpr does. Identification of the modifier genes in the MRL background that are involved in the autoimmune disease enables us to study how these genes interact with each other and ultimately induce the autoimmune disease.

It is already well established that CD72 is a negative regulator of BCR signaling (1216). Expression of CD72a negatively regulates both calcium signaling and ERK phosphorylation induced by BCR ligation in BAL17 cells (Fig. 3). In contrast, Cd72−/− B cells show augmented ERK phosphorylation but no alteration in calcium signaling induced by treatment with anti-IgM Ab (Fig. 4A, 4B). However, a previous study demonstrated that BCR ligation-induced calcium response is enhanced in Cd72−/− B cells (15, 16). Thus, CD72-mediated regulation of BCR signaling depends on experimental conditions. In the current study, we demonstrated that CD72c is hypofunctional. This property of CD72c may cause enhanced B cell activation, which may be involved in development of autoimmune disease through augmented autoantibody production. The hypofunctional property of CD72c may not be due to its expression efficiency on the cell surface as surface expression level of CD72c in B6.CD72c B cells appears to be equivalent to that of CD72b in B6 B cells, although it is not proven if the anti-CD72 Ab used for measuring CD72 expression level detects CD72b and CD72c equally (Supplemental Fig. 3). In contrast, CD72c coprecipitates BCR less efficiently than CD72a (Fig. 3G), suggesting that interaction of CD72c with BCR is weaker than that of CD72a. As interaction and colocalization of inhibitory coreceptors including FcγRIIB with BCR is crucial for their inhibitory activity (44, 45), less efficient interaction of CD72c with BCR may reduce its regulatory activity on BCR signaling. CD72c contains several amino acid substitutions and a 7-aa deletion in the extracellular region compared with CD72a or CD72b, whereas the cytoplasmic region of CD72c is identical to that of CD72a or CD72b (18, 19). Alterations in the extracellular region of CD72 may change its association with BCR or binding to its ligands, leading to reduction in its colocalization with BCR either directly or indirectly. Although CD100 was reported to be a ligand of CD72 (46), there might be other ligands. Thus, full elucidation of the interaction of CD72 with BCR and ligands may be crucial to understand the defect in signaling function of CD72c and its role in development of autoimmune disease.

We thank Dr. T. Kitamura (University of Tokyo, Tokyo, Japan) for cell lines, Dr. M. Wabl (University of California, San Francisco) for QM mice, Dr. N. Tada (Tokai University, Tokyo, Japan) for reagent, Dr. M. Nose (Ehime University, Ehime Prefecture, Japan) for discussion, T. Usami (Tokyo Medical and Dental University) for technical assistance, and S.S. Devi for initial work on the generation of B6.CD72c mice.

This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Japan Society for the Promotion of Science.

The online version of this article contains supplemental material.

Abbreviations used in this article:

ALPS

autoimmune lymphoproliferative syndrome

B6

C57BL/6

BAC

bacterial artificial chromosome

BM

bone marrow

ES

embryonic stem

ITIM

immunoreceptor tyrosine-based inhibition motif

LN

lymph node

MRL/lpr

MRL/MpJ-Faslpr

NP

4-hydroxy-3-nitrophenyl acetyl

PASH

periodic acid–Schiff and hematoxylin

PEC

peritoneal exudate cells.

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The authors have no financial conflicts of interest.