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L.S. Hall et al.
Scottish National Blood Transfusion Service) were incubated in 50 mM octylglucopyranoside (Abcam, Cambridge, UK). GPIIb/IIIa was extracted from the supernatant onto sepharose beads display- ing GPIIIa-specific peptide GRGDSPK (Cambridge Research Biochemicals, Billingham, UK), and eluted with peptide GRGDSP (Merck Chemicals, Nottingham, UK). The purity of the eluted GPIIb/IIIa preparation was confirmed by western blotting.
Peptides
Peptides containing GPIIIa epitopes (Table 1) were manufac- tured to >95% purity (Cambridge Research Biochemicals). To achieve solubility in aqueous media, which is a key property for efficient production and purification, as well as for efficacy in immunotherapy,28,29,34,35 two sequences, P44 (aa331-345) and P82 (aa711-725), were extended by an arginine-lysine wrapper.
Mouse immunization and peptide treatment
Animal work was approved by the University of Aberdeen Ethical Review Committee and authorized by the UK Government Home Office (project license 70/8744). Mice trans- genic for HLA-DRA1*1010 and HLA-DRB1*1501, which express HLA-DR15 but not murine MHC class II, were originally supplied by Professor Daniel Altman (Imperial College London, UK)39 and maintained as a genotyped and phenotyped colony at the University of Aberdeen.
Anti-platelet GPIIb/IIIa responses were induced by subcuta- neous injection of mice with 10 mg purified GPIIb/IIIa antigen, repeated 2 weeks later. Mice immunized with control antigen received injections of ovalbumin (Sigma, Poole, UK) instead of GPIIb/IIIa. Mice were treated with GPIIIa peptides by subcuta- neous injection, receiving 100 mg of each peptide selected, either alone or in combination.
Blood sampling and tissue preparation
Blood samples were collected into heparinized tubes. Single cell suspensions of splenocytes were prepared using the Miltenyi spleen dissociation kitTM and gentleMacs octo-dissociatorTM
(Miltenyi Biotec, Bisley, UK). Where required, Treg were depleted from splenocytes using the Miltenyi CD4+ CD25+ regulatory T-cell isolation kit.
Cell culture
As previously described,33,34 splenocytes were cultured at 1.25x106 cells/mL in aMEM (Sigma, Poole, UK), supplemented with 0.5% syngeneic serum. Cultures were stimulated with puri- fied GPIIb/IIIa antigen at a final concentration of 1 mg/mL, or with the control antigen ovalbumin at a final concentrations of 10 mg/mL, or with DynabeadsTM Mouse T-Activator CD3/CD28 (Invitrogen, Fisher Scientific, UK), or with peptides, either individ- ually or in combination, at final concentrations of 10 mg/mL each.
Flow cytometry
After 5 days of culture, splenocytes were stained with fluores- cein isothiocyanate-conjugated anti-CD4 (eBioscience, Fisher Scientific, Loughborough, UK) and Pacific blue-conjugated anti- CD25 (Biolegend, London, UK) antibodies, permeabilized and stained with phycoerythrin-conjugated anti-FoxP3 (eBioscience). Samples were analyzed on an LSR FortessaTM flow cytometer (BD Bioscience, Oxford, UK) using FACSDivaTM software. Regulatory T cells were defined as CD4+CD25+FoxP3+.40
Proliferation assays and cytokine enzyme-linked immunosorbent assays
As described elsewhere, after 5 days of culture,33-35 splenocyte proliferation was estimated from 3H-thymidine incorporation and presented as a stimulation index, representing the ratio of counts per minute (CPM) in stimulated versus unstimulated wells (with a ratio >3 taken as being significant41). Interleukin-10 (IL-10) pro- duction in cultures was measured by enzyme-linked immunosor- bent assay (ELISA) (antibody pairs from BD Bioscience).
Measurement of serum antibody specific for glycoprotein IIb/IIIa or control antigen
ELISA to measure murine IgG antibodies specific for GPIIb/IIIa,
AB
Figure 1. Treatment of HLA-DR15 transgenic mice with individual glycoprotein IIIa peptides containing Th epitopes neither elicits antibody specific for glycoprotein IIb/IIIa, nor consistently blocks the antibody response to immunization with purified glycoprotein IIb/IIIa. (A) Purified human GPIIb/IIIa or individual peptides were administered by subcutaneous injection to HLA-DR15 transgenic mice on days 0 and 14, with the levels of plasma IgG antibodies reactive to purified GPIIb/IIIa meas- ured by enzyme-linked immunosorbent assay (ELISA) on day 28. (B) Individual peptides were administered to mice on days 0 and 14, followed by purified GPIIb/IIIa on days 28 and 42, with IgG antibodies measured by ELISA on day 70. Data points represent results from individual mice (n=3 per group). ***P<0.0001, one way analysis of variance. Ab: antibody.
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haematologica | 2019; 104(5)