The autoimmune disease systemic lupus erythematosus (SLE) is associated with genetic variants in the X-linked gene CXORF21, which encodes the protein TASL. TASL acts as an adaptor in the IRF5 pathway and is necessary for the phosphorylation of IRF5 in response to TLR7 or TLR9 stimulation. Here, we investigate the role of TASL in the humoral immune response, and in the development of lupus in the B6.MRL^lpr murine model of SLE. We find that while TASL is dispensable for their development, it is required for the full activation of B cells via TLR9 stimulation, and consequent interferon signaling and inflammatory cytokine expression. Additionally, TASL is crucial for the emergence of age-associated B cells (ABCs), a B cell population derived from the extrafollicular response that increases with age and is expanded in autoimmune disease, and the production of IgG2c antibodies. We also find that deletion of TASL prevents the onset of autoimmunity in the genetically-determined B6.MRL^lpr model of lupus.

Tasl deletion prevents full activation of B cells via TLR9

To investigate the role of TASL in vivo, we generated Tasl ^KO mice (S1A Fig). Steady-state peripheral populations of B cell subsets, as well as T cells, macrophages, and plasmacytoid dendritic cells (pDCs) were unaffected by the absence of TASL (S1B–S1M Fig). B cell development in the bone marrow was also normal (S1N–S1O Fig).

We first stimulated B cells in vitro with either lipopolysaccharide (LPS), imiquimod (IMQ), or CpG, agonists for TLRs 4,7, and 9 respectively. Viability and proliferation were not substantially impacted by the absence of TASL following stimulation with any of the agonists (S2A Fig). However, we observed decreased levels of the surface activation marker CD69 upon IMQ or CpG stimulation, but not with LPS, in both male and female mice (Figs 1A and S2B). Interestingly however, expression of the co-stimulatory molecule CD86 was not reduced, and was in fact relatively increased in TASL-deficient B cells following CpG stimulation (Figs 1B and 2C).

A higher proportion of B cells remained IgD⁺ when stimulated by CpG (Figs 1C,1D, and 2D), reflecting persistence of a naïve state. TASL-deficient B cells expressed lower levels of the transcription factor Prdm1, which encodes Blimp1, required for plasmablast differentiation, following CpG stimulation (Fig 1E). In keeping with this finding, loss of TASL led to a lower proportion of CD138⁺IRF4⁺ plasmablasts following stimulation with CpG (Figs 1F,1G and S2E).

We next performed RNA sequencing on CpG-stimulated Tasl ^KO and wild type B cells. We identified 633 differentially expressed genes, and performed gene set enrichment analysis (GSEA) (Fig 1H, 1I). We found downregulation of key ISGs, such as Irf7 and Ifi44, and the interferon-mediated signaling pathway in Tasl ^KO B cells. Using qPCR we confirmed reduced expression of Irf7 and the ISGs Isg15 and Mx1, and cytokines Il6 and Il10 (Fig 1J–1N). In keeping with our finding of elevated CD86 with CpG stimulation following Tasl deletion, the antigen processing and presentation pathway was upregulated.

TASL is therefore required for B cell activation and plasmablast differentiation in vitro following TLR9 stimulation.

Loss of TASL impairs humoral immunity

To understand whether TASL is important in humoral immunity, we immunized Tasl ^KO and wild type mice with 4-hydroxy-3-nitrophenylacetyl-chicken gamma globulin (NP-CGG), a protein-hapten antigen that initiates a T-dependent immune response. At 14 days post-immunization we found a reduction in the proportion and number of splenic GC B cells in Tasl ^KO mice (Fig 2A, 2B). Plasmablasts were also less abundant, but notably only in female mice (Fig 2C, 2D). This was associated with a reduction in resting serum IgG2b levels in female Tasl ^KO mice, slightly lower levels of other immunoglobulin isotypes, and reduced free kappa and lambda light chains (S3A Fig). No differences in resting immunoglobulins were observed in male mice. In response to NP-CGG immunization, there was no difference in levels of anti-NP IgG1 antibodies binding to either high (NP₂₀) or low (NP₂) conjugation-ratio proteins, reflecting low and high affinity antibodies respectively (Fig 2E). Antibody affinity maturation, measured as the ratio between NP₂ and NP₂₀ binding, was also unaffected by loss of TASL (Fig 2E). However anti-NP IgG2c antibody levels were markedly lower in both male and female Tasl ^KO mice, with lower IgG2c production in the former group confirmed using ELISpot (Fig 2G).

The lack of difference in levels of anti-NP IgG1 antibodies, with no impairment of affinity maturation, suggested that TASL may be dispensable for the GC reaction. To test this, we generated competitive mixed bone marrow chimeras, using CD45.1 wild type and CD45.2 wild type or Tasl ^KO bone marrow cells. Following reconstitution, mice were immunized with sheep red blood cells (SRBC), a T-dependent antigen, and the GC response examined at day nine. The relative proportion of wild type and Tasl ^KO B cells in the GC reaction and in the plasmablast population was not different (Fig 2H, 2I). We also generated in vitro GC B cells (iGCBCs) using the iGB system, in which B cells are cultured on a fibroblast layer which expresses BAFF and CD40L, in the presence of IL-4, for 5 days [18]. We did not detect a difference in the capacity of wild type and Tasl ^KO B cells to differentiate into iGCBCs (S4A–S4B Fig).

We then immunized mice with NP-Ficoll, a T-independent antigen which elicits a predominantly extrafollicular response [19], characterized by IgM antibodies. We found that Tasl ^KO mice of both sexes had a markedly reduced level of anti-NP IgM antibodies at day seven following immunization (Fig 2J), and decreased plasmablast formation was seen in female mice (Fig 2K). Using a competitive bone marrow chimera, as before with CD45.1 wild type, and either wild type or Tasl ^KO CD45.2 bone marrow cells, following NP-Ficoll immunization there was a reduction in the proportion of Tasl ^KO plasmablasts compared to CD45.1 wild type competitors at day seven (Fig 2L).

Together these data demonstrate that TASL is primarily required for the extrafollicular response, with a limited role in the GC reaction.

TASL is required for the formation of age-associated B cells

Age-associated B cells (ABCs) are a B cell subset which accumulates with age and in autoimmune disease, and are proportionately much more frequent in female mice and humans [17,20,21]. ABCs are of predominantly extrafollicular origin, and we therefore examined whether TASL was required for their accumulation. We found that as expected, ABCs were more abundant in aged female wild type mice, but were markedly reduced in Tasl ^KO mice of both sexes at eight months of age (Fig 3A–3C).

To determine whether this effect was cell intrinsic, we cultured isolated B cells from wild type and Tasl ^KO mice with the TLR7 agonist IMQ, agonistic anti-CD40, anti-IgM F(ab’)₂, and IL-21 to induce ABC differentiation (Fig 3D) [22–24]. ABC formation was substantially reduced in B cells from Tasl ^KO mice. ABCs are potent producers of IgG2c antibodies, and its level in the culture supernatant was also much decreased (Fig 3E–3F).

TASL is therefore required for the emergence of ABCs in aged mice, and following culture in vitro with ABC-inducing stimuli.

Absence of TASL prevents the development of lupus in B6.MRL^lpr mice

We next examined whether TASL was necessary for the development of lupus. We crossed Tasl ^KO mice with the B6.MRL-Fas^lpr/J congenic lupus mouse model, generating B6.MRL^Lpr × Tasl ^KO mice (hereafter referred to as MRL^Lpr × Tasl ^KO). These mice were aged to 20 weeks to allow disease to develop, and compared with B6.MRL^lpr mice, or wild type C57BL/6 controls.

We found that the absence of TASL decreased splenomegaly and lymphadenopathy to wild type levels (Fig 4A–4C). The spontaneous GCs seen in MRL^Lpr mice were absent in MRL^Lpr × Tasl ^KO mice (Fig 4D–4F), and the frequency and count of plasmablasts was also decreased, to a larger extent in females than in males (Fig 4G–4H). There was also a marked reduction in effector (CD62L^loCD44^high) T cells and increased numbers of naive T cells (CD62L^hiCD44^low) (Fig 4I, 4J), but this was only seen in female mice, although a trend in the same direction was observed in males. We also observed no difference in the frequencies or numbers of pDCs in MRL^Lpr × Tasl ^KO compared to MRL^Lpr mice, but they were less activated, with a smaller proportion CD86⁺ (S5A–S5C Fig).

ABCs are a key element of the pathogenesis of lupus [14,15,25]. We therefore investigated whether the absence of TASL impacted the development of this population in MRL^Lpr × Tasl ^KO mice.

We found that CD11c⁺T-bet⁺ ABCs were almost completely absent in the spleens of both male and female MRL^Lpr × Tasl ^KO mice (Fig 4K–4M). In addition, MRL^Lpr × Tasl ^KO mice had drastically reduced anti-nuclear antibodies (S5D Fig), and much lower levels of IgG2c anti-dsDNA antibodies (S5E Fig). Anti-Sm/RNP antibodies were also decreased, but only significantly in female mice (S5F Fig).

Deletion of Tasl therefore abrogates the development of clinical and immunologic features of lupus and is required for the formation of ABCs in the B6.MRL-Fas^lpr/J mouse model.

Ethics statement

All procedures and experiments were performed in accordance with the UK Scientific Procedures Act (1986) under a project license authorized by the UK Home Office (PPL no. PP1971784).

Mice

B6.MRL-Fas^lpr/J (strain 000482) were purchased from the Jackson Laboratory. C57BL/6J-Tasl^em3H/H mice were generated by the Mary Lyon Centre at MRC Harwell, via CRISPR/Cas9-induced mutation deleting 10 nucleotides in exon ENSMUSE00000697605 of the Tasl gene. B6.SJL.CD45.1 mice were obtained from the central breeding facility of the University of Oxford.

Males and females between the ages of 8–16 weeks were used, except where stated separately for individual experiments. Mice were bred and maintained under specific pathogen-free conditions at the Kennedy Institute of Rheumatology, University of Oxford.

Immunization

50μg NP_(30–39)-CGG (Biosearch Tech) or NP-Ficoll was mixed with alum (Thermo Fisher Scientific) at a 1:1 ratio and rotated at room temperature for 30 min, before being injected intraperitoneally. For NP-CGG and NP-Ficoll immunisations, day 14 and day 7 were used as timepoints respectively, unless stated otherwise.

For SRBC immunization, 1 ml of sterile SRBCs in Alsever’s solution (Thermo Fisher Scientific) was washed twice with 10 ml ice-cold PBS, then reconstituted in 2–3 ml of PBS. 200 μl was then injected intraperitoneally.

Bone marrow chimera generation

Recipient female B6.SJL.CD45.1 mice received two doses of 5.5Gy irradiation 4 hours apart, and were then injected intravenously with 4 × 10⁶ bone marrow (BM cells) in 200μl PBS, made up of 50:50 CD45.2⁺ Tasl ^KO and CD45.1⁺ WT, or CD45.2⁺ WT and CD45.1⁺ WT BM cells from sex- and age-matched donor mice. Recipient mice were given antibiotics (Baytril, Bayer corporation) in their drinking water for two weeks. Eleven weeks after BM reconstitution, recipient mice were immunized with either SRBC or NP-Ficoll. Spleens were analyzed 9 days post-immunization with SRBCs and 7 days post-immunization with NP-Ficoll.

Flow cytometry

Spleens were mashed through a 70 μm strainer (VWR). Red blood cells were lysed with ACK Lysis buffer (Gibco). Single-cell suspensions were incubated with Fixable Viability Dye eFluor 780 (eBioscience) or Zombie UV Fixable Viability Dye (BioLegend) in PBS for 30 min on ice, then Fc block (BD Biosciences) for 5 mins, followed by surface antibodies in a 50:50 solution of FACS buffer and Brilliant Stain buffer (BD Biosciences) for 30 mins on ice. Cells were fixed in freshly prepared 4% PFA for 10 mins at room temperature.

For intracellular staining, cells were fixed using FoxP3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent (eBioscience) for 30 mins at room temperature, then permeabilised in Permeabilization buffer (eBioscience). Cells were then incubated with intracellular antibodies diluted in permeabilization buffer for 45 mins at room temp.

The following antibodies were used (Table 1):

Cells were resuspended in FACS buffer and acquired on either a BD Fortessa X20 or Cytek Aurora flow cytometer.

Cell isolation and culture

Splenic B cells were isolated by using the Pan B cell Isolation II Kit (Milteny Biotec), with resulting purity of over 90%. For proliferation analysis, cells were incubated with Cell Trace Violet (Life Technologies) in PBS for 20 mins at room temperature, before adding complete media and washing. Isolated B cells were then cultured in RPMI-1640 medium (Gibco) with 10% FCS (Gibco), 50 μM 2-mercaptoethanol (Gibco), 1 mM glutamine (Gibco), 1 mM sodium pyruvate (Gibco), 10 mM HEPES buffer (Gibco), and 100 IU/ml penicillin and streptomycin (Life Technologies Ltd).

For TLR stimulation, either 100 nM CpG-B (ODN1826) (Miltenyi Biotec), 5 μg/ml IMQ (Invitrogen), or 10 μg/ml LPS (Merck) was used, for times indicated in figures.

For ABC in vitro culture, a differentiation cocktail of 0.5 μg/ml IMQ, 1 μg/ml anti-CD40 (clone FGK45.4, Miltenyi Biotec), 1 μg/ml anti-IgM F(ab’)₂ (Jackson), and 50ng/ml IL-21 (PeproTech) was used.

iGB culture system

The iGB culture system was used as previously described [18,35]. The 40 LB fibroblast cell line was cultured in high-glucose DMEM with Glutamax (Thermofisher) medium containing 10% FCS (Gibco) and 50 IU/ml penicillin and streptomycin (Life Technologies Ltd) in T75 tissue culture flasks (Sarstedt). When cells were confluent, they were detached using trypsin/EDTA (Gibco), washed, then resuspended in 5 ml medium in 15 ml tubes and irradiated with a dose of 80 Gy. Irradiated cells were then washed, and seeded at 0.5 × 10⁶ in 6-well culture plates (Falcon), then incubated overnight at 37°C and 5% CO₂.

Naïve B cells were isolated using anti-CD43 microbeads (Miltenyi) and added to wells with irradiated 40 LBs cells for 5 days in complete RPMI medium, as described above, with 1ng/ml IL-4 (Peprotech). Cells were then collected and analyzed by flow cytometry. iGCBCs were defined as CD19⁺IgD^loCD38^loGL-7⁺.

ELISA

96-well plates were coated with either NP₂ or NP_>20 conjugated to BSA, at 5 μg/ml in bicarbonate/carbonate coating buffer at 4 °C overnight. Plates were washed in PBS, then blocked with 5% skimmed milk in PBS for 2 hour at 37 °C. After washing with PBS, serum from immunized mice was diluted in 1% skimmed milk and then added to plates and incubated for 1 hour at 37 °C. Plates were then washed multiple times with PBS-Tween20 (0.05%) (PBS-T), before alkaline phosphatase-conjugated goat anti-mouse IgG, IgG1, IgG2c, or IgM detection antibodies (SouthernBiotech) were added and incubated at 37 °C for 1 hour. After washing, alkaline phosphatase substrate (Sigma) was added to plates and left to develop for 5 min at room temperature, after which readings were taken every 5 mins on a FLUOstar Omega plate reader.

For Ig isotyping, Mouse Ig Isotyping Uncoated ELISA kit (Thermo Fisher Scientific) was used according to manufacturer’s protocol.

For detection of auto-antibodies, 96-well plates were coated with UltraPure Salmon Sperm DNA Solution (Invitrogen) at 100 μg/ml in PBS at 4 °C overnight for anti-dsDNA antibodies, or 1 μg/ml Sm/RNP (Arotec Diagnostics) in PBS at 4 °C overnight for anti-Sm/RNP antibodies. Plates were washed in PBS, then blocked with 2% BSA for 1 hour at 37 °C. Plates were then washed in PBS, and mouse serum diluted in PBS containing 1% BSA was added and incubated for 2 hour at 37 °C. After multiple washes in PBS-T, alkaline phosphatase-conjugated goat anti-mouse IgG, IgG2c, or IgM detection antibodies (SouthernBiotech) were then added and incubated at 37 °C for 1 hour. Plates were then washed with PBS-T, then alkaline phosphatase substrate (Sigma) was added to plates and left to incubate for 5 mins at room temperature. Readings were then taken every 3 mins on a FLUOstar Omega plate reader at 405 nm.

ELISpot assay

Low fluorescent PVDF Fluorospot plates (Mabtech) were coated with anti-IgG2c (Southern Biotech) overnight at 4 °C. Plates were washed with PBS and then blocked with PBS containing 1% gelatin. After washing with PBS, single-cell splenocyte suspensions from mice 14 days after immunization with NP-CGG were added to plates in complete RPMI and incubated overnight at 37 °C. Plates were then washed with distilled water-0.1% Tween and PBS-0.05% Tween. Alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Biotech) was then added and incubated for 1 hour at 37 °C. Plates were then washed with PBS-0.05% Tween and AMP/BCIP substrate solution overnight at 4 °C. Plates were then washed with distilled water and left to dry, before being analyzed on an AID ELISpot reader.

Immunohistochemistry

Spleens were fixed overnight at 4 °C in Antigenfix (DiaPath), then washed in PBS and left in 30% sucrose in PBS overnight at 4 °C. Spleens were then snap frozen in methanol on dry ice and stored at −70 °C. Spleens were sectioned at 8−12 μm thickness and rehydrated in PBS at room temperature, before being permeabilised and blocked in PBS containing 10% goat serum and 0.1% Tween-20. Spleens were then stained overnight at 4 °C with anti-GL-7 AF488 (Biolegend) and anti-CD21/35 AF647 (Biolegend). Slides were then washed and mounted before being imaged with an Airyscan 2 and analyzed in ImageJ.

Anti-nuclear antibody imaging

Mouse serum was added to Kallestad Hep-2 slides (BioRad) and incubated for 30 mins at room temperature in the dark. Slides were washed in PBS, then AF488-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) was added to slides and incubated for 30 mins at room temperature in the dark. Slides were then washed and mounted, before being imaged with a ZEISS LSM 980 and analyzed in ImageJ.

Western blotting

Cells were lysed in RIPA buffer (Merck) supplemented with phosphatase and protease inhibitors (cOmplete ULTRA Tablets and PhosSTOP, Roche) for 40 mins with regular vortexing, before being centrifuged at 15,000 g for 15 mins at 4 °C. The supernatant was then collected. Protein was quantified with a Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Samples were denatured in Laemmli buffer (BioRad) containing 10% 2-mercapthoethanol for 5 mins at 95 °C, then cooled immediately on ice. Samples were then run on a 10% gel (Mini-PROTEAN TGX Precast Gels, BioRad).

Protein was then transferred to a PVDF membrane (BioRad). Membranes were blocked in 2.5% skimmed milk in TBS for 1h, washed with TBS-Tween20 (0.05%) (TBS-T), then incubated overnight at 4 °C with primary antibodies anti-TASL (1:1000) (Atlas) and anti-vinculin (1:1000) (Cell Signaling Technology) diluted in TBS-T containing 2.5% BSA. Membranes were then washed with TBS-T before adding HRP-conjugated goat anti-rabbit secondary antibody (Cell Signaling Technology), and incubating for 1 hour at room temperature. SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher) was then added to membranes, and chemiluminescence was used to detect signal.

Quantitative PCR

Cells were lysed in RLT Plus (Qiagen) containing 1% 2-mercaptoethanol. RNA was isolated using an RNeasy Plus Mini kit (Qiagen), according to manufacturer’s instructions. Isolated RNA was reverse transcribed to cDNA using the High Capacity RNA-to-cDNA kit (Thermo Fisher).

qPCR reaction was performed using TaqMan Fast Advanced Master Mix (Applied Biosystems) and TaqMan primers for Isg15 (Mm01705338_s1), Il6 (Mm00446190_m1), Il10 (Mm01288386_m1), Irf7 (Mm00516793_g1), Mx1 (Mm00487796_m1), Prdm1 (Mm00476128_m1), and Ubc (Mm00476128_m1). Data was acquired on an Applied Biosystems Viia7 System, and expression was normalized to Ubc expression using the ΔΔCT method.

Bulk RNA sequencing analysis

Isolated splenic B cells were stimulated for 24 hour with 100 nM CpG. Cells were then lysed in RLT plus containing 1% 2-mercaptoethanol, and RNA was isolated using RNeasy Plus Mini kit (Qiagen). Next generation paired-end sequencing was performed by Azenta using the Illumina NovaSeq 6000 platform, with 20 M reads taken per sample. Transcripts were counted using Salmon.

After standard processing, data was filtered to genes that had expression higher than 1CPM in at least 50% of samples. Normalization and differential expression analysis were then performed with edgeR.

For gene set enrichment analysis (GSEA), the ClusterProfiler package was used with gene ontology terms related to biological processes (GO BP). The Benjamini-Hochberg method was used for P-value adjustment to control for false discovery rate.

S1 Fig.

A. AImmunoblot of TASL in WT and Tasl ^KO splenocytes. Vinculin is used as a loading control. Representative of 2 independent experiments. B. Flow cytometric quantification of frequency of live CD19⁺ B cells in spleens of WT (n = 10) and Tasl ^KO (n = 8) mice. Data representative of and pooled from 3 independent experiments. C. Count of live CD19⁺ B cells in spleens of WT (n = 5) and Tasl ^KO (n = 5) mice. Data representative of and pooled from 2 independent experiments. D. Representative gating and quantification of frequency of CD19⁺CD23⁺CD21^int follicular B cells and CD19⁺CD23⁻CD21⁺ marginal zone (MZ) B cells in spleens of WT (n = 12) and Tasl ^KO (n = 8) B cells. Data representative of and pooled from 3 independent experiments. E. Count of follicular B cells and MZ B cells, gated as in D, in spleens of WT (n = 7) and Tasl ^KO (n = 5) mice. Data representative and pooled from 2 independent experiments. F. Representative gating and quantification of frequency of CD19⁺CD93⁺ T1 (CD23^-IgM⁺), T2 (CD23⁺IgM⁺), and T3 (CD23⁺IgM⁻) B cells in spleens of WT (n = 12) and Tasl ^KO (n = 8) mice. Data representative of and pooled from 3 independent experiments. G. Count of transitional B cell populations, gated as in F, in spleens of WT (n = 7) and Tasl ^KO (n = 5) mice. Data representative of and pooled from 2 independent experiments. H. Quantification of total TCRβ⁺ T cell frequency in spleens of WT (n = 10) and Tasl ^KO (n = 7) mice. Data representative of and pooled from 3 independent experiments. I. Quantification of total TCRβ⁺ T cell count in spleens of WT (n = 7) and Tasl ^KO (n = 5) mice. Data representative of and pooled from 2 independent experiments. J. Representative gating and quantification of frequency and count of splenic CD11b⁺F4/80⁺ macrophages in spleens of WT (n = 12) and Tasl ^KO (n = 8) mice. Data representative of and pooled from 3 independent experiments. K. Count of macrophages, as gated as in F, in spleens of WT (n = 7) and Tasl ^KO (n = 5) mice. Data representative of and pooled from 2 independent experiments. L. Representative gating and quantification of frequency of plasmacytoid dendritic cells (pDCs) in spleens of WT (n = 12) and Tasl ^KO (n = 8) mice. Data representative and pooled from 3 independent experiments. M. Count of pDCs, gated as in F, in spleens of WT (n = 7) and Tasl ^KO (n = 5) mice. Data representative of and pooled from 2 independent experiments. N. Representative gating and quantification of frequency and count of for IgM⁻IgD⁻B220⁺ pre-pro-, pro- and pre- B cells in the bone marrow of WT (n = 4) and Tasl^KO (n = 4) mice. Equivalent Hardy fractions are shown in parentheses. Data representative of 2 independent experiments. O. Representative gating and quantification of frequency and count of B220⁺IgD⁻IgM⁺ immature B cells and B220⁺IgD⁺ mature B cells in the bone marrow of WT (n = 4) and Tasl ^KO (n = 4) mice. Equivalent Hardy fractions are shown in parentheses. Data representative of 2 independent experiments. Statistical significance was calculated using two-tailed unpaired t test (B–E, H–M) or two-way ANOVA with Šidák’s multiple testing correction (F, G, N, and O). Data are presented as mean ± S.E.M. Each point represents a single mouse. The data underlying this figure can be found at: DOI https://doi.org/10.5281/zenodo.18649676

https://doi.org/10.1371/journal.pbio.3003342.s001

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S2 Fig.

A. Representative histogram and proliferation index of isolated splenic B cells from WT or Tasl ^KO mice (n = 4) stimulated for 72 hour with either lipopolysaccharide (LPS), imiquimod (IMQ), or CpG. Data representative of 2 independent experiments. B. Flow cytometric measurement of CD69 gMFI in isolated splenic B cells from either WT (n = 4) or Tasl ^KO (n = 4) male mice after stimulation with either LPS, IMQ, or CpG for 24 hour. Representative of two independent experiments. C. Flow cytometric measurement of CD86 gMFI in isolated splenic B cells from either WT (n = 4) or Tasl ^KO (n = 4) male mice after stimulation with either LPS, IMQ, or CpG for 24 hour. Representative of two independent experiments. D. Quantification of IgD⁺ B cells as percentage of total B cells, after stimulation of isolated splenic B cells from male WT (n = 4) or Tasl ^KO (n = 4) mice with either LPS, IMQ, or CpG for 72 hour. Representative of 2 independent experiments. E. Quantification of plasmablasts as CD19⁺IgD⁻CD138⁺IRF4⁺ B cells after culture of male B cells with either LPS, IMQ, or CpG for 72 hour (n = 4). Representative of 2 independent experiments. Statistical significance was calculated using two-tailed unpaired t test (A, G) or two-way ANOVA with Šidák’s multiple testing correction (B–E). Data are presented as mean ± S.E.M. Each point represents a single mouse. The data underlying this figure can be found at: DOI https://doi.org/10.5281/zenodo.18649676

https://doi.org/10.1371/journal.pbio.3003342.s002

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S3 Fig.

A. Immunoglobulin isotyping of unimmunized female WT (n = 5) or Tasl ^KO (n = 3) or male WT (n = 4) or Tasl ^KO (n = 4) mice. Data are representative of 2 independent experiments. Statistical significance calculated by two-way ANOVA with Šidák’s multiple testing correction. Data are presented as mean ± S.E.M. Each point represents a single mouse. The data underlying this figure can be found at: DOI https://doi.org/10.5281/zenodo.18649676

https://doi.org/10.1371/journal.pbio.3003342.s003

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S4 Fig.

A. Schematic of iGB culture system. Isolated naïve splenic B cells from WT (n = 7) or Tasl ^KO (n = 7) mice were seeded on a layer of irradiated fibroblasts expressing BAFF and CD40L, with IL-4 and incubated for 5 days. B. Quantification of CD38^-GL-7⁺ iGCB cells from (F). Statistical significance was calculated using two-tailed unpaired t test. Data are presented as mean ± S.E.M. Each point represents a single mouse. The data underlying this figure can be found at: DOI https://doi.org/10.5281/zenodo.18649676S

https://doi.org/10.1371/journal.pbio.3003342.s004

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S5 Fig.

A. Representative flow cytometric gating of CD19⁻TCRb⁻CD11c⁺pDCA1⁺ pDCs in MRL^Lpr and MRL^Lpr ×Tasl ^KO mice. B. Quantification of pDC proportion and count in 20-week old female WT (n = 4), MRL^Lpr (n = 6), and MRL^Lpr ×Tasl ^KO (n = 5) mice, and male WT (n = 3), MRL^Lpr (n = 4), and MRL^Lpr ×Tasl ^KO (n = 5) mice. Data representative of and pooled from 2 independent experiments. C. Quantification of CD86^hi pDCs in 20-week old female WT (n = 4), MRL^Lpr (n = 6), and MRL^Lpr ×Tasl ^KO (n = 5) mice, and male WT (n = 3), MRL^Lpr (n = 4), and MRL^Lpr ×Tasl ^KO (n = 5) mice. Data representative of and pooled from 2 independent experiments. D. Representative images of anti-nuclear antibodies (ANAs) in serum of 20-week old WT, MRL^Lpr and MRL^Lpr ×Tasl ^KO mice, detected by immunofluorescence. Scale bar represents 100μm. E. ELISA quantification of anti-dsDNA IgG2c in sera of 20-week old female WT (n = 4), MRL^Lpr (n = 3), and MRL^Lpr ×Tasl ^KO (n = 7) mice, and male WT (n = 3), MRL^Lpr (n = 10), and MRL^Lpr ×Tasl ^KO (n = 7) mice. Data representative of and pooled from 3 independent experiments. F. ELISA quantification of anti-Sm/RNP IgG2c in sera of 20-week old female WT (n = 4), MRL^Lpr (n = 3), and MRL^Lpr ×Tasl ^KO (n = 9) mice, and male WT (n = 3), MRL^Lpr (n = 10), and MRL^Lpr ×Tasl ^KO (n = 7) mice. Data representative of and pooled from 3 independent experiments. Statistical significance was calculated using two-way ANOVA with Šidák’s multiple testing correction. Data are presented as mean ± S.E.M. Each point represents a single mouse. The data underlying this figure can be found at: DOI https://doi.org/10.5281/zenodo.18649676

https://doi.org/10.1371/journal.pbio.3003342.s005

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