HSP-encoding constructs
Full-length cDNAs of human HSP60, HSP90 genes were cloned individually into the pcDNA3 vector (Invitrogen) under the control of the human CMV promoter (Supplementary Fig. 1A). Briefly, cDNA encoding HSP60 or HSP90 in pGEM was amplified using specific oligonucleotides containing restriction sites for BamHI or HindIII. The amplicon and pcDNA3 vectors were purified and digested with BamHI/HindIII. The digested PCR product coding for the respective HSP (HSP60 or HSP90) and linearized pcDNA3 vector were ligated using T4 DNA ligase according to standard protocols. The ligated plasmids were used to transform E. coli and later sequenced to confirm the correct insertion of the cDNAs. For vaccination, the individual DNA constructs—named pHSP60 and pHSP90 when encoding HSP60 and HSP90, respectively—were prepared in large scale using the EndoFree Plasmid Mega Kit (Qiagen, Santa Clarita, CA). After DNA precipitation with ethanol and resuspension in sterile PBS, endotoxin levels were checked by Limulus amebocyte lysate and always found to be under acceptable levels for in vivo use (< 0.02 endotoxin U/μg DNA). Construct quality and purity were reassessed before use by enzymatic digestion, to reconfirm vector map sites.
HSP90 expression
Translation of HSP90 protein in vitro from pHSP90 was assessed by SDS-PAGE and autoradiography following incubation with 35S-methionine (Perkin Elmer, Akron, OH). A 90-kDa protein was detected in the pHSP90 samples, while no 35S-labeled product was detected from the control pcDNA3 vector (Supplementary Fig. 1B). The few minor bands detected in the HSP90 preparations were likely degradation products recognized by anti-HSP90 antibodies.
Mice
(NZB x NZW)F1 (NZB/W) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained at the University of California Los Angeles under pathogen-free conditions. Only female mice were used and treated according to the National Institutes of Health guidelines for the use of experimental animals under protocols approved by the institutional Animal Research Committee. Mice were divided into pHSP90 and pHSP60 groups and two control groups (pcDNA3 and PBS), each containing 10 mice. At 8 weeks of age and 5 days after injection with 50 μl cardiotoxin (10 μM) in the right tibialis anterior muscle, mice were injected in the same area with pHSP60 or pHSP90 constructs or pCDNA3 empty vector (each with 100 μl at 1 μg/μl) or vehicle (100 μl PBS). The mice received a second injection of the same amount of plasmid or vehicle in the left tibialis anterior muscle the following week, having been injected 5 days earlier with cardiotoxin.
Measurement of antibodies
Blood samples were collected at the beginning of the study and 12 days after the end of the regime of DNA vaccination. Serum was stored at − 20 °C until use. HSP90- and HSP60-specific antibodies were measured by ELISA in flat-bottom microtiter plates precoated overnight with 0.1 μg/well recombinant HSP90 or HSP60 (Boston Biochem, Cambridge, MA) or glutathione S-transferase as control (Sigma Aldrich, St. Louis, MO) in carbonate buffer at 4 °C. Non-specific binding was blocked by incubation with 1% skim milk for 2 h at 37 °C. Serum samples were added diluted 1:100 and incubated for 3 h at 37 °C. Bound IgG were detected using alkaline phosphatase-conjugated goat anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) using substrate for alkaline phosphatase (Sigma Aldrich).
Proteinuria
Albustix reagent strips for urinalysis (Bayer, Pittsburgh, PA) were used to monitor urine protein content. Proteinuria was defined as > 100 mg/dl at two different measurements 1 day apart.
Elisa
ELISA measured total anti-dsDNA antibodies (Alpha Diagnostic Intl., San Antonio, TX). Subtyping of the anti-dsDNA antibodies by ELISA was done using as secondary antibodies HRP-conjugated anti-IgG1, IgG2a, IgG2b, or IgG3 (eBioscience, San Diego, CA).
Flow cytometry
Popliteal lymph node cells (draining from the muscle injected with plasmid constructs) were costained for CD11 and MHC class II, CD40, CD80, CD86 using fluorochrome-labeled monoclonal antibodies (mAb). T regulatory cells (Tregs) were costained with fluorochrome-labeled mAb to CD3, CD4, CD25, and Foxp3 (the latter after using the Cytofix/Cytoperm kit, eBioscience). All mAb were from eBioscience. Cells were acquired on a FACSCalibur™ flow cytometer with CellQuest™ software (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (Tree Star, Ashland, OR).
In vitro assays
Dendritic cells (DCs) from popliteal lymph nodes were isolated on an AutoMACS® separator with the Pan Dendritic Cell Isolation kit (both from Miltenyi Biotec) and cultured in the presence of 10 ng/ml GM-CSF. For extracellular HSP expression, 3 × 105 DCs/well were cultured in 6-well plates for 3 days at 37 °C/5% CO2 in the presence of 1.25 μg recombinant HSP90 or vehicle in HL-1 medium (Lonza, Benicia, CA). Cocultures included CD4+CD25− T cells isolated as negative fraction with the Mouse CD4+CD25+ Regulatory T Cell Isolation kit (Miltenyi Biotec). For intracellular HSP90 expression in DCs, Chariot™ transfection reagent (Active Motif, Carlsbad, CA) was used according to the manufacturer’s instructions. Briefly, 1.25 μg recombinant HSP90 was complexed to Chariot™ in 100 μl PBS volume prior to transfection of sorted DCs plated at a concentration of 3 × 105 cells/well in 6-well plates. HSP-transfected DCs were then cultured for 3 days at 37 °C/5% CO2 in HL-1 medium with CD4+CD25− T cells that had been isolated as negative fraction with the Miltenyi Biotec Mouse CD4+CD25+ Regulatory T Cell Isolation kit. Intracellular expression of HSP90 in DCs after transfection with Chariot™ reagent was confirmed by western blot 6 h post-transfection of HSP90 in the comparison with non-transfected DCs cultured for 6 h in the presence of the same amount of HSP90 in medium, using anti-HSP90 mAb (BD Biosciences, Franklin Lakes, NJ) followed by detection with ECL Plus reagents (GE Healthcare Chicago, IL).
Histology
Kidneys from mice of 30–32 weeks of age were embedded in OCT compound, snap frozen, and stored at − 80 °C. For staining, sections of 4 μm were put in cold acetone for 5 min, washed, and blocked with 2% BSA for 1 h before indirect immunofluorescence using FITC-conjugated rabbit anti-mouse IgG (Thermo Fisher Scientific, Waltham, MA). Sections were counterstained with H&E for assessments of glomerular activity score (G.A.S.) and tubulointerstitial activity score (T.I.A.S.) in a blinded manner, using a scale of 0–3, where 0 is no lesions, 1 is lesions in < 30% of glomeruli, 2 is lesions in 30–60% of glomeruli, and 3 is lesions in > 60% of glomeruli. The G.A.S. includes glomerular proliferation, karyorrhexis, fibrinoid necrosis, inflammatory cells, cellular crescents, and hyaline deposits. The T.I.A.S. includes interstitial inflammation, tubular cell necrosis and/or flattening, and epithelial cells or macrophages in the tubular lumen. The raw scores were averaged to obtain a mean score for each feature, and mean scores were summed to obtain an average score from which a composite kidney biopsy score was derived [14].
Statistical analyses
Differences between individual groups were evaluated using the Student’s t test with GraphPad Prism 4 software (San Diego, CA). P < 0.05 was considered significant.