Anti-CD22 immunotherapy in diabetes
ONLINE SUPPLEMENT
MATERIALS AND METHODS
Mice
Female NOD and NOD.SCID mice of various ages were obtained from The Jackson laboratory and cared for in accordance with institutional guidelines. BDC2.5 TCR transgenic mice on the NOD genetic background were purchased from the Jackson Laboratory and bred under specific pathogen-free conditions at the Harvard Medical School Facilities for Animal Care and Housing. Protocols were approved by the Institutional Animal Care and Use Committee.
Anti-CD22/calicheamicin immunoconjugate
The immunoconjugate (referred here as anti-CD22/cal mAb, provided by Wyeth) is a conjugate of an anti–mouse CD22 mAb and N-acetyl-calicheamicin dimethyl acid, a member of the enediyne antitumor antibiotics (1). Anti–mouse CD22 is a mouse IgG1 mAb purified from Cy34.1 hybridoma (American Type Culture Collection [ATCC], Rockville, MD). Anti-CD22/cal mAb has an average loading of 17 to 30 g calicheamicin/mg antibody protein (1.2-2.6 moles calicheamicin/mol antibody). On binding to CD22-expressing mouse B cells, the conjugate is internalized and exhibits potent dose-dependent cytotoxicity due to DNA damage caused by calicheamicin (1). A mouse IgG1 anti–rat very late antigen 4 (VLA-4) mAb (which does not bind to mouse cells) conjugated to calicheamicin (GG5/cal) was used as a control.
Monitoring for Diabetes
Clinical diabetes was defined as blood glucose levels >250 mg/dL for 3 consecutive days. Blood glucose was measured using a BD Logic Blood Glucose Meter (Becton Dickinson, Franklin Lakes NJ). After all treatments, mice were monitored daily by measuring blood glucose for 3 weeks, followed by 3 times/week until the mice were sacrificed.
Flow cytometry (FACS)
Rat anti-mouse CD19 PE, CD20 PE, CD22 PE, B220 PE, B220 PerCP, CD80 (B7-1) PE, CD86 (B7-2) PE, H-2d FITC, CD4 FITC, CD23 FITC, CD25 PE, CD44 PE, CD45 FITC, CD62L APC, CD93 PE (C1qRp) and IgM PAPC, were purchased from BD Biosciences (San Jose, CA) and eBiosciences (San Diego, CA). FoxP3 APC was purchased from eBiosciences. Cells recovered from spleens and peripheral lymphoid tissues were subjected to FACS analysis and were run on a FACSCalibur™ (Becton Dickinson). Data were analyzed using FlowJo software version 6.3.2 (Treestar, Ashland, OR). The level of maturation of B cells was determined by FACS analysis by examining the expression of CD80 and 86. B cell anergic phenotype was determined by FACS using quadruple staining (for B220+CD93+CD23-IgMlo cells), as recently shown (2). FoxP3 analysis was performed following overnight permeabilization of cells extracted from spleens and peripheral lymphoid tissue using commercially available antibodies and gating on CD4+CD25+ cells.
Pancreatic digestion and lymphocyte extraction
Pancreata were collected in cold HBSS medium supplemented with 10% FBS, cut into small pieces, and pressed through a 70-µm cell strainer. After washing, mononuclear leukocytes were separated by density-gradient centrifugation using Histopaque 1077 (Sigma, St. Louis, MO). Following centrifugation for 30 min at 2500 rpm, the interphase containing the mononuclear cells was removed, and cells were washed twice with Ca2+/Mg2+-free PBS.
Islet pathology and immunohistochemistry
Immunohistochemistry was performed using 5 mm-thick formalin-fixed, paraffin-embedded tissue sections. Briefly, slides were soaked in xylene, passed through graded alcohols, and immersed in distilled water. Slides were then pre-treated with 10 mM citrate, pH 6.0 (Zymed, South San Francisco, CA) or with 1 mM EDTA (pH 8.0) in a steam pressure cooker (Decloaking Chamber, BioCare Medical, Walnut Creek, CA) as per manufacturer's instructions, followed by washing in distilled water for antigen retrieval. All further steps were performed at room temperature in a hydrated chamber. Slides were pretreated with Peroxidase Block (DAKO USA, Carpinteria, CA) for 5 minutes to quench endogenous peroxidase activity. The following primary antibodies were used (including company, clone/reference, dilution, and retrieval method, respectively): anti–CD3 (Cell Marque, CMC363, 1:1500, EDTA), anti–CD45/ B220 (BD Pharmingen, 550286, 1:200, citrate), anti–FoxP3 (eBioscience, 14-5773, 1:25, citrate), anti-insulin (DAKO, N1542, undilute, EDTA), and anti-glucagon (Abcam, Ab18461, 1:50, EDTA). All primary antibodies were applied to slides in DAKO diluent for 1 hour. Slides were then washed in 50 mM Tris-Cl, pH 7.4, and the appropriate horseradish peroxidase-coniugated secondary antibody (Envision detection kits, DAKO) was applied for 30 minutes. After further washing, immunoperoxidase staining was developed using a DAB chromogen kit (DAKO) per the manufacturer and counterstained with hematoxylin. Photomicrographs were taken on an Olympus BX41 microscope (Center Valley, PA) at indicated magnifications using an Olympus Q-color5 digital camera and analyzed with Adobe Photoshop Elements 2.0 (San Jose, CA). All photos were taken at 400x original magnification.
Insulitis score
Insulitis scoring was performed on hematoxylin- and eosin-stained pancreatic sections. A score from 0 to 4 was assigned based on islet infiltration as previously described (3). At least 30 islets per group were analyzed, pooled from different mice. Insulitis score was graded as follows: grade 0, normal islets; grade 1, mild mononuclear infiltration (<25%) at the periphery; grade 2, 25-50% of the islets infiltrated; grade 3, more than 50% of the islets infiltrated; grade 4, islets completely infiltrated with no residual parenchyma remaining.
In vitro generation of bone marrow (BM)-derived dendritic cells (DC)
DC were isolated by culturing murine BM cells from NOD or BALB/c mice. Briefly, the femurs and tibiae of mice were flushed, and the cells were seeded in Petri dishes at a density of 2 x 106/ml of RPMI-1640 medium supplemented with 20 ng/ml of recombinant murine granulocyte-monocyte colony stimulating factor (rmGM-CSF, R&D Systems, Minneapolis, MN). At days 3, 6, and 8, additional medium and rmGM-CSF were added. On day 9, non-adherent cells were harvested for use.
CD4+ and CD19+ cells extraction
CD4+ and CD19+ cells were extracted from B cell-depleted and control splenocytes from NOD or BDC2.5 mice with magnetic microbeads (Miltenyi Biotec, Auburn, CA). Purity of more than 95% was assessed by FACS analysis (data not shown). A purity of 99% was obtained for gene expression profile using multiple columns and isolations with the magnetic device (data not shown).
ELISpot
To measure cytokine production by CD4+ cells extracted from B cell-depleted and control NOD mice, our ELISpot assay was used as previously described (4). Briefly, Millipore immunospot plates (Millipore Corporation, Bedford, MA) are coated with capture antibodies (BD Biosciences). Plates are blocked with 1% bovine serum albumin (BSA) to prevent non-specific binding. 1x105 NOD BM-derived DC are used as stimulators, while 2x105 NOD CD4+ cells extracted from B cell-depleted and control mice are used as responders in the presence of 150 mg/ml BDC2.5 peptide, (Ac-RTRPLWVRME amide, purity>90%, QCB, Hopkinton MA). After 4 days of culture in 10% FCS 1% penicillin-streptomycin RPMI at 37° C and 5% CO2, plates were washed, and biotinylated antibodies specific for each cytokine were added to wells and incubated at 4° C for 12 hours. Plates were then washed and incubated at room temperature with streptavidin-horseradish peroxidase (HRP) for 2 hours and developed using aminoethyl carbazole (AEC, Sigma Aldrich) diluted in N,N-dimethylformamide. Spots were counted on an immunospot analyzer (Cellular Technology Ltd., Cleveland, OH). In another ELISpot experiment, 1x105 NOD CD19+ cells from treated or untreated NOD mice were used as stimulators of 1x105 CD4 cells from BDC2.5 mice in the presence of 15 ng/ml BDC2.5 peptide for 24h. The supernatant of each culture was used for Luminex analysis as well.
Adoptive cell transfer
5x106 diabetogenic CD4+ cells extracted from hyperglycemic NOD mice were adoptively transferred alone or in combination with 5x106 of re-emerging or naïve CD19+ cells (B cells) into NOD.SCID hosts. Naïve B cells were extracted from both normoglycemic and hyperglycemic NOD mice, while re-emerging B cells were extracted from B cell-depleted NOD mice after the recovery of the B cell pool. Diabetes onset was monitored in the following weeks. We also analyzed the percentage of CD4+CD25+FoxP3+ cells in the spleen of NOD.SCID recipients of diabetogenic CD4+ T cells and re-emerging B cells or controls (B cells from hyperglycemic animals or no cells) at day 30 post-adoptive transfer.
In vivo tracking of autoreactive T cells
For in vivo tracking of autoreactive T cells, we used CD4+ cells extracted from BDC2.5 mice, so that autoreactive CD4+ cells can be easily tracked using the anti-ideotypic antibody against the Vb4 chain of the TCR receptor. CD4+ cells were adoptively transferred into a NOD.SCID host previously reconstituted with splenocytes extracted from NOD mice and depleted of B cells or left untreated. After 72 hours cells were recovered, autoreactive T cells were counted and proliferation of CD4+Vb4+ autoreactive T cells was evaluated.
Mixed lymphocyte reaction (MLR)
Cultured BM-derived DC from BALB/c mice were used after irradiation with 3000 rads to stimulate CD4+ NOD cells isolated from splenocytes obtained from B cell-depleted and control NOD mice by magnetic bead separation (Miltenyi Biotec) at a ratio of 1:1 DC-splenocytes. Proliferation was measured at day 3 of incubation at 37° C and 5% CO2 following pulsing with [3H]TdR (Perkin Elmer, Wellesley, MA) using a liquid scintillation counter. Splenocytes were also stimulated with con A as a positive control.
Luminex serum cytokine determination
B cell-depleted and control age-matched NOD mice were bled via tail perforation at selected times (0, 2, 4, 6, 8, and 10 weeks) post-injection in our prevention studies (every other week up to 10 weeks after injection) or at 0, 10, and 100 days post-injection in the reversal studies, and serum samples were collected. Supernatant from in vitro studies (ELISpot assay) was also collected to analyze cytokine profile. The Beadlyte Mouse Multi-Cytokine Beadmaster Kit (Millipore, Billerica, MA) was used according to the manufacturer’s protocol to determine cytokine levels of IL-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p70, IL-15, IL-17, IFN-g, and TNF-a as previously described (5). Briefly, supernatant samples are incubated overnight at room temperature with beads conjugated to the aforementioned cytokines; matched biotinylated reporters are added and incubated for 1.5 hours, and streptavidin-phycoerythrin solution is incubated with samples for 30 minutes. Following addition of stop solution, sample cytokine levels are calculated from a standard curve using a Luminex100 reader from Luminex Corporation (Austin, TX).
Trascriptome analysis
RNA amplification and hybridization on Illumina beadchip mouse6v1.1
The integrity of RNA was evaluated using an Experion™ (Bio-RAD).100 ng total RNA was used to generate cRNA by the use Illumina TotalPrep RNA Amplification Kit (Ambion, Austin TX). Reverse transcription was performed with the T7 oligo (dT) primer to obtain first-strand cDNA, which then underwent second-strand synthesis and RNA degradation by DNA Polymerase and RNase H followed by clean up. In vitro transcription (IVT) technology along with biotin UTP was employed to generate multiple copies of biotinylated cRNA. The labeled cRNA was purified via Filter Cartridge and quantified by NanoDrop and RiboGreen® (Molecular Probes Inc. Ugene, OR).
The labeled cRNA target (1.5 mg each) was used for hybridization to each array according to Illumina Sentrix beadchip array mouse-6v1.1 protocol. A maximum of 10 ml cRNA was mixed with 20 mL GEX-HYB hybridization solution. The preheated 30 ml assay sample was dispensed onto the large sample port of each array and was incubated for 18 hours at 58 ºC at a rocker speed of 5. Following hybridization, the samples were washed according to the protocol and scanned with a BeadArray Reader (Illumina, San Diego, CA).
Beadarray data analysis
The bead array data was analyzed by Bead Studio Version 2 (Illumina). The differential expression of genes between the treated group and control group and between the treated group and hyper group was obtained after rank invariant normalization. The criteria for this analysis is differing score less than -13 or more than 13, which is corresponding to P value less than 0.05. The heat map of differential expression genes was generated by Multiple Array Viewer software after data standardization.
Autoantibody evaluation (IAA assay)
We evaluated serum antibodies in NOD mice during treatment in collaboration with the Barbara Davis Center in Colorado. Briefly, IAA (IA2) were measured with a standard IAA radioassay (6, 7) utilizing competition with unlabeled insulin with 600 µl of sera per determination (150 µl duplicates with and without unlabeled insulin). After a 7-day incubation at 4°C, antibody-antigen complexes were precipitated with polyethylene glycol 8000, and the results were calculated as the difference between samples without cold insulin and samples with cold insulin and were expressed as nU/ml.
Statistical analyses
Data are expressed as mean±standard error. Kaplan-Meier analysis was used for survival analysis. ANOVA (for parametric data) and Kruskal-Wallis test (for non-parametric data) were used. Chi-square test for categorical variables was used when necessary. When groups were compared cross-sectionally, two-sided unpaired Student t-test (for parametric data) or Mann-Whitney tests (for non-parametric data) were used according to distribution. A P value of less than 0.05 (by two-tailed testing) was considered an indicator of statistical significance. Analysis of data was done using an SPSS statistical package for Windows (SPSS Inc., Chicago, Illinois). Prism software was used for drawing graphs (GraphPad Software, Inc., San Diego, CA).
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