B6(Cg)-Ifnar1^tm1.1Ees /J (The Jackson Laboratories, strain #028256) and B6.C(Cg)-Cd79a^tm1(cre)Reth /EhobJ (C57BL6.Mb1-cre) (The Jackson Laboratories, #020505) were crossed for generation of B6.BΔIFNAR mice or each backcrossed onto the B6.Nba2.ABC background (B6.Nba2) for subsequent generation of B6.Nba2.IFNAR^flx/flxMB1^cre/+ (B6.Nba2.BΔIFNAR) mice. The presence of each transgene was determined by PCR as previously described (23, 24). Primers used were: IFNAR5′: 5′ TGC TTT GAG GAG CGT CTG GA 3′ IFNAR3′: 5′ CAT GCA CTA CCA CAC CAG GCT TC 3′ IFNARΔ5′: 5′ TAG CCC CAG GGT AGT TAA CTC TTG A 3′ MB1cre F: 5′ CCC TGT GGA TGC CAC CTC 3′, MB1cre R: 5′ GTC CTG GCA TCT GTC AGA G 3′. In order to determine the presence of the NZB-derived Nba2 locus PCR analysis was done for the presence of NZB-derived D1mit36, D1mit47, D1mit113, and D1mit209 markers as previously described (25). All mice were born and housed in specific pathogen-free housing at Lerner Research Institute. Only female mice were used for this study. All animal research were approved and performed in accordance with the institutional guidelines.

B6, B6.BΔIFNAR, B6.Nba2.BΔIFNAR and B6.Nba2 female mice were harvested at 4 months of age in several separate cohorts. Spleens were harvested and weighed. A middle portion of the spleen was frozen in OCT for immunohistochemistry. A small portion of the spleen was made into a single cell suspension in 1x phosphate buffered saline (PBS) pH7.4 and incubated at 37°C for 10 mins for isolation of spleen supernatant and single cells for RNA extraction. The remaining spleen was similarly processed into single cells after which red blood cells (RBCs) were lysed using ACK (Ammonium-Chloride-Potassium) lysis buffer (0.15 M NH4Cl, 0.01 M KHCO3, 0.2 mM EDTA, pH 7.3) and the cells were used for flow cytometry. Bone marrow from the left femur and left tibia was flushed out with 1x PBS and used for flow cytometry after RBC lysis as described above. One kidney was removed and dissected into two coronal sections. One half was preserved in OCT, the other half was fixed in 10% formalin.

Splenocytes were isolated from B6.Nba2.BΔIFNAR and B6.Nba2 mice and stimulated with 1000 u/mL of recombinant mouse IFN-αA (Fisher Scientific, Hampton, NH, USA) in primary cell media (RPMI w/L-glutamine, 10% fetal bovine serum, 1% Hepes, 1% non-essential amino acids, and 1% streptomyocin) and incubated overnight. Upon harvest, cells were stained for surface markers of B cells, T cells, pDCs and cDCs, fixed and permeabilized using the Foxp3 transcription factor staining buffer set (Invitrogen, Carlsbad, CA, USA), and stained intracellularly with a rabbit anti-mouse Ifit2 antibody (26, 27) (kind gift by Dr. Sen) and a donkey anti-rabbit IgG Alexa Fluor 647-conjugated secondary antibody (eBiolegend Clone Poly4064). Levels of Ifit2 expression was determined by flow cytometry.

Flow cytometry for splenocytes and bone marrow was run on a BD LSR Fortessa™ flow cytometer (BD Biosciences, San Jose California, USA) and data was collected using BD FACSDiva™ software (BD Biosciences, San Jose California, USA). Data were analyzed using FlowJo Version 10 Software (FlowJo, Ashland, Oregon, USA). The following antibodies were used for staining and sourced from eBioscience (San Diego, CA) unless otherwise noted. Antibodies with the following specificities were used for staining: B220 Clone RA3-6B2, CD3 Clone 145-2C11, CD4 Clone GK1.5, CD11b Clone M1/70, CD11c Clone N418, CD16/32 Clone 93, CD19 Clone 1D3, CD21/35 Clone 4E3, CD23 Clone B3B4, CD38 Clone 90, CD40 Clone HM40-3, CD43 Clone R2/60, CD69 Clone H1.2F3, CD93 Clone AA4.1, CD138 Clone 281-2, CXCR5 Cat:551960 (BD Pharmigen), F4/80 Clone BM8, GL7 Clone GL-7, IgD Clone 11-26c, IgM Clone 11/41, PCDA Clone 927, PD-1 Clone J43, Siglec H Clone 440c, SignR1 Clone 22D1. Streptavidin Conjugated Antibodies were used with catalog numbers: 45-4317-82, 12-4317-87 (eBiosciences), and 405208 (Biolegend). Where indicated, specific cell subsets were isolated by cell sorting on a BD FACSAriaII. All cell subsets were gated as shown in Supplemental Figure 1 .

Formalin-fixed kidneys were transferred to 80% ethanol and embedded in paraffin. Three µm sections were cut and stained with hematoxylin/eosin (Histology Core, Lerner Research Institute) or Periodic acid shiff (PAS) (Thermo Scientific, Waltham, MA, USA) for detection of kidney morphology, damage and cellular infiltrates. Sections were scored in a blinded fashion by a renal pathologist (JN) at the Cleveland Clinic. Kidneys were evaluated on a scale of 0–5 for mesangial and endothelium hypercellularity. Glomerular area was calculated by measuring the area of 5–15 individual glomeruli per section for each mouse. Immunofluorescence was performed on 5µm sections of kidneys preserved in OCT™ (Scigen, Paramount, CA, USA). Sections were thawed, fixed in acetone, blocked with 10% normal goat serum, and stained using Texas-Red conjugated anti-IgG or anti-IgM antibodies (SouthernBiotech, Birmingham, AL, USA) and FITC-conjugated anti- Complement factor C′3 antibodies (Immunology Consultants Laboratory Inc., Portland, OR, USA). Imaging was done on a Keyence BZ-X700 All-in-one microscope (Keyence, Osaka, Osaka, Japan) and images were quantified using the Keyence BZ-X analysis software (Keyence, Osaka, Osaka, Japan). A total of 5–15 glomeruli were quantified for each section and the average calculated per mouse.

For immunofluorescent staining of spleens, 5µm sections were cut of spleens preserved in OCT™ compound. On day one of staining, sections were thawed, fixed in acetone, blocked with 10% normal goat serum, and stained using FITC conjugated anti-B220 IgG2a (eBioscience, San Diego, CA, USA) and biotin conjugated anti-GL-7 IgM antibodies (eBioscience, San Diego, CA, USA) followed by Texas-Red conjugated streptavidin (Southern Biotech, Birmingham, AL, USA). Imaging and quantification was done using the same process as for kidney immunofluorescence.

RNA was isolated from frozen splenocytes using the RNeasy mini kit (Qiagen, Valencia, CA, USA) and quantified using nanodrop technology (Nanodrop ND-1000 Spectrophotometer, Thermo Fisher). Complementary DNA (cDNA) was made using qScript™ DNA supermix (Quanta BioSciences, Gaithersburg, MD) and quantified using nanodrop technology and diluted for qPCR. qPCR was performed with 100ng cDNA using SYBR™ green PerfeCTa^® SYBR^® Green FastMix^® ROX (Quanta BioSciences) and run on a Step One Plus real time PCR system (Applied Biosciences, Foster City, CA, USA). All transcripts were analyzed using β-actin expression as a control. Primers (Integrated DNA Technologies, Skokie, IL, USA) used are listed below:

qPCR primers; β-actin F: 5′ TGG GAA TGG GTC AGA AGG AC 3′β-actin R: 5′ GGT CTC AAA CAT GAT CTG GG 3′, Mx1 F: 5′ TTC CTG AAG AGG CGG CTT T 3′ Mx1 R: 5′ GGT TAA TCG GAG AAT TTG CCA A 3′, IL-1β F: 5′ CCC TGC AGC TGG AGA GTG TGG A 3′ IL-1β R: 5′ CTG AGC GAC CTG TCT TGG CCG 3′, Bcl6 F: 5′ CCT GAG GGA AGG CAA TAT CA 3′ Bcl6 R: 5′ CGG CTG TTC AGG AAC TCT TC 3′, Il6 F: 5′ ACA CAT GTT CTC TGG GAA ATC GT 3′ Il6 R: 5′ AAG TGC ATC ATC GTT GTT CAT ACA 3′, Il21 F: 5′ CGC CTC CTG ATT AGA CTT CG 3′ Il21 R: 5′ TGG GTC TCC TTT TCT CAT ACG 3′, Blimp F: 5′ TAG ACT TCA CCG ATG AGG GG 3′ Blimp R: 5′ GTA TGC TGC CAA CAA CAG CA 3′, Baff F: 5′ CAG CGA CAC GCC GAC TAT AC 3′ Baff R: 5′ CCT CCA AGG CAT TTC CTC TTT T 3′, Irf4 F: 5′ GCC CAA CAA GCT AGA AAG 3′ Irf4 R: 5′ TCT CTG AGG GTC TGG AAA CT 3′, Ifi202 F: 5′ GGT CAT CTA CCA ACT CAG AAT 3′ Ifi202 R: 5′ CTC TAG GAT GCC ACT GCT GTT G 3′, Bcl2 F 5′: TGA GTA CCT GAA CCG GCA TCT Bcl2 R 5′: GCA TCC CAG CCT CCG TTA T, Bim F 5′ CGG ATC GGA GAC GAG TTC A 3′ Bim R 5′ TTC CAG CCT CGC GGT AAT CA, Bclxl F 5′: TGG AGT AAA CTG GGG GTC GCA TCG Bclxl R 5′: AGC CAC CGT CAT GCC CGT CAG G.

Statistical analyses were performed in GraphPad Prism (La Jolla, CA, USA). All comparisons between two groups were done by Student’s T test with Welch’s correction. Statistical significance is defined as p < 0.05.

To study how IFNαβ stimulation of B cells specifically alters lupus-like disease and contributes to symptom presentation, we created B cell-specific IFNAR-deficient mice (BΔIFNAR) on the lupus-prone B6.Nba2 background ( Figure 1A ). To functionally verify our model we stimulated primary splenocytes with murine recombinant IFN-αA overnight, and measured cell surface markers and intracellular Ifit2, an IFNαβ-induced protein, using flow cytometry (28). B cells from B6.Nba2.BΔIFNAR mice did not show production of Ifit2 in response to IFN-αA stimulation, whereas B cells from WT B6.Nba2 lupus-prone control mice upregulated Ifit2 levels by ~25 fold ( Figure 1B ). This effect was specific to B cells, as both T cells and dendritic cells from B6.Nba2.BΔIFNAR mice upregulated Ifit2 expression after stimulation ( Figure 1B and data not shown).

To confirm that B6.Nba2.BΔIFNAR mice were not displaying B cell developmental defects, frequencies of early B progenitor cells, pro-B cells, pre-B cells and immature B cells were determined. No differences were identified between B6.Nba2 and B6.Nba2.BΔIFNAR mice ( Supplemental Figure 2 ). Interestingly, we observed significantly reduced levels of total splenic pDCs in B6.Nba2.BΔIFNAR mice, however levels of interferon-producing SiglecH+ pDCs were unchanged ( Figures 1C, D ), suggesting that IFNαβ levels would be unaffected. Studies of the expression of interferon stimulated genes (ISGs) in spleen and peripheral blood mononuclear cells (PBMCs) from B6.Nba2 and B6.Nba2.BΔIFNAR mice confirmed that IFNαβ levels were indeed unchanged in these mice ( Figures 1E–G ).

Hypergammaglobulinemia and elevated levels of serum ANA are hallmarks of B6.Nba2 lupus-like disease (22, 25). We compared serum antibodies in 4 months old B6.Nba2 and B6.Nba2.BΔIFNAR mice and found no significant differences in the levels of serum IgM ( Figure 2A ), IgG ( Figure 2B ), and IgA (data not shown). In contrast, B6.Nba2.BΔIFNAR mice displayed significantly reduced levels of serum anti-dsDNA IgG, serum anti-chromatin IgG ( Figures 2C, D ) and reduced levels of serum anti-nRNP IgG ( Figure 2E ). There were no differences in total serum IgG subclasses ( Figure 2F ), however, anti-dsDNA and anti-nRNP IgG_2c antibodies, but not other IgG subclasses, were decreased in serum of B6.Nba2.BΔIFNAR mice ( Figures 2G, H ).

Aged B6.Nba2 mice present with a moderate kidney phenotype including IgG-immune complex (IgG-IC) deposition and complement C′3 fixation in the glomeruli, immune cell infiltration, and mesangial cell proliferation (29). We found no difference in mesangial hypercellularity and glomerular area between B6.Nba2.BΔIFNAR mice and B6.Nba2 controls at 4 months of age ( Supplemental Figure 3 ). Similarly, we found no difference in the glomerular deposition of IgG- or IgM-IC and no difference in complement C′3 fixation ( Supplemental Figure 3 and data not shown). Surprisingly, we did not also see differences in total IgG2c-IC between B6.Nba2 and B6.Nba2.BΔIFNAR mice ( Supplemental Figure 3 ).

Splenomegaly, a pronounced presentation of B6.Nba2 lupus-like disease, was quantified and found to be significantly reduced in B6.Nba2.BΔIFNAR mice as compared with B6.Nba2 age-matched mice ( Figure 3A ). Further analyses of the spleen composition, revealed no significant differences in the frequency of total B cells, total T cells, macrophages, conventional DCs (cDC) and neutrophils ( Figures 3B–D and data not shown). As previously mentioned, total pDCs were significantly reduced in BΔIFNAR mice while the subset of IFNαβ producing pDCs was unchanged (see above, Figures 1C, D ). Levels of transitional B cells ( Figure 3E ) were similarly unchanged, as were transcriptional levels of Baff and splenocyte secreted BAFF ( Figures 3F, G ). Populations of follicular mature B cells and marginal zone B cells also remained unchanged ( Supplemental Figures 4A, B ). However, frequencies of CD69+ and CD40+ B cells were significantly reduced in B6.Nba2.BΔIFNAR mice ( Figures 3H, I ). This was not due to a general reduction in the level of cellular activation, as the CD69+ activated T cell population was unaffected ( Figure 3J ). Thus, expression of IFNAR by B cells in B6.Nba2 mice contributes to splenomegaly and activation of B cells specifically, but alters neither the total B cell nor T cell, macrophage and dendritic cell populations.

Corresponding with the reduced levels of ANAs, B6.Nba2.BΔIFNAR mice displayed significantly reduced levels of plasma blasts/plasma cells (PB/PC) and memory B cell ( Figures 4A, B ). This reduction was specific to the B6.Nba2 model as levels of PB/PC and memory B cells were unchanged between B6 and B6.BΔIFNAR mice ( Figures 4A, B ). It should be noted that the decrease in splenic PB/PC in B6.Nba2.BΔIFNAR mice was not due to an increase in homing to the bone marrow, as bone marrow PB/PC levels were unchanged ( Supplemental Figure 1E ). Interestingly, we found no significant differences in splenic transcript levels of Blimp ( Figure 4C ) and Irf4 ( Figure 4D ) both of which are required for plasma cell differentiation (30–32), suggesting that the differentiation process to PB/PC and memory B cells was unaffected by IFNAR-deficiency.

We quantified the GC B cells to determine if the significant decrease in PB/PC and memory B cells was due to reduced GC B cell population. Flow cytometric analyses identified a significant decrease in the GC B cell population of B6.Nba2.BΔIFNAR mice ( Figure 4E ), although immunohistochemistry analyses did not support differences in GC area ( Figures 4F, G ). The reduction in GC B cell numbers, was not due to changes in the TFH cell population ( Figure 5A ) or in levels of IL-6, IL-1β, and IL-21, known to be involved in the GC reaction ( Figures 5B–D ). Similarly, expression levels of Bcl6, a crucial transcription factor for TFH and GC B cells (33–35), was unchanged ( Figure 5E ).

IFNα signaling has been found to drive Bcl2 expression in lymphocytes, suggesting that a lack of IFNα signaling may affect B cell survival (36). We evaluated expression of the anti-apoptotic factors Bcl2 and Bclxl in total splenocytes and in sorted GC B cells and PB/PC. While there was no change in expression in total splenocytes ( Figure 6A ), GC B cells from B6.Nba2.BΔIFNAR mice showed decreased expression of Bcl2 and Bclxl (p < 0.05 and p = 0.1428, respectively), and a trend towards elevated expression of Bim (p = 0.1202) ( Figure 6B ). In contrast, there were no differences in the expression level of either factor in PB/PC cells ( Figure 6C ). In support of a role for IFNα in regulating Bcl2, splenic B cells from the B6.Nba2 mice showed increased intracellular Bcl2 protein ex vivo in response to recombinant IFN-αA stimulation, while B cells from B6.Nba2.BΔIFNAR mice were unable to upregulate intracellular Bcl2 in response to the same conditions (data not shown).