Animals
At eight weeks of age, female MRL/lpr mice were acquired from SPF Biotechnology Co. Ltd, while C57BL/6J mice were obtained from Jiangsu GemPharmatech Co. LTD. Prior to use, all necessary sterilization measures were taken, including autoclaving of cages, bedding, nestlets, food, and water. The experimental protocols adhered to the animal ethical and care guidelines of the Institute of Dermatology, Chinese Academy of Medical Sciences (no. 2022-DW-004) and were approved by local government authorities to ensure compliance.
Experimental design
MRL/lpr mice
At 11 weeks of age, 30–33 female mice were randomly assigned to one of three groups (n = 10 ~ 11 per group) and received intraperitoneal injections until they reached 21 weeks of age. The treatment groups included a vehicle control (IgG isotype control, 100 µg/mouse) obtained from Bio X cell (BE0093), Cyclophosphamide (CTX) (Sigma, C0768) at a weekly dose of 30 mg/kg, or Anti-OX40L (Bio X cell, BE0033-1) at a dose of 100 µg/mouse, twice a week (Fig. 1. a). Body weight was monitored weekly throughout the study period. At 21 weeks of age, all mice were euthanized, and serum, spleen, and kidney were isolated for further analysis. Lymph nodes and spleen were also collected and weighed, with the spleen or lymph node index being calculated as the weight of the organ divided by body weight.
KLH immunization
We aimed to evaluate the impact of Anti-OX40L on immunosuppression in female C57BL/6J mice aged 8 weeks. 17 female mice were randomly assigned to one of three groups (Blank group: n = 5, no-KLH immunized mice; negative control: n = 7, KLH + IgG treated group; anti-OX40L group: n = 6, KLH + anti-OX40L treated group). The mice were subcutaneously injected with KLH (0.5 mg/ml, Sigma) emulsified in CFA on the tail, once a week for two weeks. To explore the role of Anti-OX40L, we administered anti-OX40L (100 µg) or control IgG systemically twice a week, starting at 8 weeks of age. After 2 weeks, we collected serum and spleen samples from the mice for further analysis (Fig. 4. a).
Evaluation of systemic lupus and glomerulonephritis
Serum samples were collected from MRL/lpr mice via orbital vein blood after anesthesia every 3 ~ 4 weeks. We used an ELISA kit (CUSABIO, CSB-E11194m) to detect the levels of anti-dsDNA IgG in the serum. Blood urea nitrogen levels were measured using a colorimetric method (Sangon Biotech, D799850-0100) at the end of the experiment. To quantitatively assess proteinuria, we used a Bradford assay kit (Nanjing Jiancheng Bioengineering Institute, C035-2-1).
Renal histopathology in mice was assessed by embedding the left kidneys in paraffin and cutting them into 5-µm sections. We used a standard technique to stain the sections with hematoxylin and eosin (HE) to visualize tissue morphology. The pathological scores of glomerulonephritis were determined by the average of the results from 50 glomeruli. The scoring system for glomerulonephritis was as follows: Normal (0), cell proliferation or infiltration (1), membrane proliferation, hyaline deposition, or lobulation (2), global hyalinosis or crescent formation (3). To detect IgG depositions, direct immunofluorescence (IF) was performed on paraffin-embedded kidney section via Goat Anti-Mouse IgG2a heavy chain (FITC) (Abcam, ab97244, 1:500). C3 deposits were detected using C3/C3b/C3c Polyclonal antibody (Proteintech, 21337-1-AP, 1:500) and Goat Anti-Rabbit IgG (FITC) (Proteintech, SA00003-2, 1:200) by IIF. The HE slides and immune-complex (IC) depositions in the mouse kidneys were analyzed and evaluated using a digital fluorescence microscope (Olympus, BX53F2). The fluorescent staining was recorded and analyzed using OLYMPUS cellSens Standard Imaging Software for presentation.
RNA isolation and qPCR
Total RNA of kidney was extracted using Total RNA Extraction Reagent (Vazyme, R401-01-AA). RNA quality and concentration were assessed using a NanoDrop spectrophotometer (Thermo, ND-2000). mRNA was reverse-transcribed using the HiScript III RT SuperMix for qPCR (Vazyme, R323-01) following the manufacturer’s protocol. qRT-PCR was conducted with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) using a thermocycler (Roche, LightCycler 480 II). Normalized to the control group, the gene mRNA level was calculated as 2^−ΔΔCt (ΔΔCt = ΔCt of experiment group – ΔCt of control group). mRNA primers for cytokines, chemokines, immune cell markers, and β-actin were obtained from Tsingke Biotechnology Co. (Supplemental Table 1).
Flow cytometry
Spleen tissues were mechanically disrupted and filtered to obtain cell suspensions. About 1.5×106 cells were incubated with mouse Fc-R block (BD Pharmingen, 553141) for 15 minutes (min) at 20–25°C, following with the surface staining using antibodies against specific markers for 45 min at 4°C, away from dark. Intracellular staining was performed via fixing and permeabilizing the cells with a Transcription Factor Buffer Set (BD Pharmingen, 562574) and staining with fluorescent antibodies for 1.5 h at 4°C. To assess cytokine production by lymphocytes in spleens, 2×106 cells/well were stimulated with the Leukocyte Activation Cocktail (BD GolgiPlug™ antibody, BD 550583) in 6-well plates for 6–8 hours at 37°C with 5% CO2. Flow cytometry results were downloaded analyzed using FlowJo software 10.8.1. The following antibodies were used: FITC anti-mouse CD3 Antibody (BioLegend, 100204), Alexa Fluor® 700 anti-mouse CD4 Antibody (BioLegend, 100430), CD25 Monoclonal Antibody (PC61.5), Super Bright™780 (eBioscience, 78-0251-82), Zombie Aqua™ Fixable Viability Kit (BioLegend, 423101), PE-CF594 Rat Anti-Mouse IFN-γ (BD Pharmingen, 562303), IL-17A Monoclonal Antibody (eBio17B7), eFluor™ 450 (eBioscience, 48-7177-82), PE/Cyanine7 anti-mouse IL-4 Antibody (BioLegend, 504118), FOXP3 Monoclonal Antibody (FJK-16s), PE (eBioscience, 12-5773-82), PerCP/Cyanine5.5 anti-mouse/human CD45R/B220 Antibody (BioLegend, 103236), PE anti-mouse CD19 Antibody (BioLegend, 152408), IgD Monoclonal Antibody (11-26c (11–26)), Super Bright™ 600 (eBioscience, 12-5773-82), Brilliant Violet 421™ anti-mouse CD138 (Syndecan-1) Antibody (BioLegend, 142523), Alexa Fluor® 647 anti-mouse/human GL7 Antigen (T and B cell Activation Marker) Antibody (BioLegend, 144606), PE-CF594 Hamster Anti-Mouse CD95 (BD Pharmingen, 562499), CD8a Monoclonal Antibody (53 − 6.7), APC-eFluor™ 780 (eBioscience, 47-0081-82), PE/Cyanine7 anti-mouse CD62L Antibody (BioLegend, 104418), Brilliant Violet 785™ anti-mouse/human CD44 Antibody (BioLegend, 103059), Biotin Rat Anti-Mouse CD185 (CXCR5) (BD Pharmingen, 551960), APC Streptavidin (BioLegend, 405207), BB700 Hamster Anti-Mouse CD279 (PD-1) (BD Pharmingen, 566514). Flow cytometry was performed using a Cytek NL-CLC V16B14R8 instrument.
Cytometric bead array
Serum samples from KLH-immunized mice and supernatants from cultured splenic B cells were collected and analyzed using a LEGENDplex™ Mouse Immunoglobulin Isotyping Panel (6-plex) (BioLegend, 740492), following the manufacturer's protocols. The concentrations of mouse Igs (IgA, IgM, IgG1, IgG2a, IgG2b, and IgG3) were caculated through LEGENDplex software after flow cytometry analysis on a BD FACSVerse™ Flow Cytometer instrument.
Isolation and stimulation of CD4+ T cells
Spleen cells of 8-week-old C57BL/6J mice were isolated and purified using the MojoSort™ Mouse CD4 T Cell Isolation Kit (BioLegend, 480006) and added at an intensity of 5×105 cells/well in a 24-well plate. The cells were activated using plate-bound anti-CD3 (Invitrogen, 16-0031-86, 3 µg/ml) and anti-CD28 (Invitrogen, 16-0281-85, 2 µg/ml) antibodies for 3 days with the addition of isotype IgG or anti-OX40L (100 µg/ml), furthermore, blank group without CD3/CD28 stimulation was also established. Subsequently, Th1, Th2, Th17, Tfh, and Treg cells were detected by flow cytometry.
Culture of splenic B cell
To obtain Mouse Pan B cells, splenocytes were isolated and purified using the MojoSort™ Mouse Pan B Cell Isolation Kit II (BioLegend, 480088). The purified B cells were then cultured in RPMI 1640 (Gibco, 21870076) supplemented with IL-4 (PeproTech, 214-14-20, 30 ng/ml) and R848 (Sigma, SML0196-10MG, 100 ng/ml) to promote activation as described previously [22]. Cultures were set up in 24-well plates with 5×105 cells/well in 500 µl of medium at 5% CO2 and 37°C. To block OX40/OX40L signaling, anti-OX40L (100 µg/ml) or IgG (100 µg/ml) was added to the culture. Blank control was set up without the addition of IL-4 and R848 in the medium. After 72 h, all cells were harvested for the evaluation of B cell differentiation and the supernatant was collected to detect antibody production using flow cytometry. Next, we investigated the effect on transcription factors related to B-cell proliferation and differentiation using western blot assay, after 48 h culture.
Western blotting
Mouse splenic B cells were isolated after 2 days of culture, and lysed using RIPA buffer (Beyotime, P0013K), and the protein was quantified via a BCA Protein Assay Kit (Beyotime, P0011). A 10% SDS-page was loaded with the protein and then transferred to polyvinylidene difluoride membranes. Subsequently, western blotting was performed using antibodies that were specific to various proteins such as Blimp-1 (CST, 9115S, 1:1000), BCL6 (CST, 5650S, 1:1000), XBP1 (Affinity, AF5110, 1:1000), IRF8 (Proteintech, 18977-1-AP, 1:500), PAX5 (Affinity, AF5110, 1:500), SPI-B (Santa Cruz, sc-517204, 1:1000) and GAPDH (CST, 5174S, 1:2000). The blots were then visualized using a Tanon 5200 imaging system.
Statistical analysis
The statistical analysis was performed using GraphPad Prism 9.5.0. The results are displayed as mean ± SEM, and the data were analyzed for normal distribution and equal variance between groups. Two-tailed unpaired Student's t-tests were used to compare two groups. For multigroup comparisons, we used and one-way analysis of variance (ANOVA) with appropriate post hoc tests. The 2-tailed Mann-Whitney U test was performed when the data had unequal variances or were not normally distributed between two groups. The sample sizes were not predetermined using a statistical method, and mice were randomly assigned to different groups.