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M. Rijkers et al.
A and B.3 Binding of antibodies to HLA class I on donor platelets results in the formation of IgG-opsonized platelets which are rapidly cleared from the circulation. Several parameters may contribute to the efficacy of HLA antibody-induced platelet clearance. Firstly, HLA density on platelets may differ between individuals. A recent study showed that platelets from donors with consistently low HLA-B8, B12 or B35 displayed a strongly reduced antibody-mediated internalization by macrophages.9 Furthermore, low levels of HLA antibodies were not asso- ciated with platelet refractoriness in the TRAP (Trial to Reduce Alloimmunization to Platelets) study.10 Results from the same study revealed that high levels of anti-HLA antibodies were clearly related to refractoriness to platelet transfusion.10 Transfusions with HLA-compatible platelets have been shown to be effective in patients with pre- existing HLA antibodies.11 In an early clinical trial no ben- eficial effect of treatment with HLA-matched platelet con- centrates was observed.12 These findings indicate that platelet refractoriness is of non-immune origin in a signif- icant number of patients and, collectively, suggest that HLA antibodies, dependent on their titer and the HLA density on donor platelets, can induce platelet refractori- ness. Whether additional mechanisms contribute to the observed clinical effects of anti-HLA antibodies has not yet been clearly delineated.
Apart from the transfusion setting, a pathogenic role for platelet-specific antibodies has been described in several diseases. Patients with immune thrombocytopenia often have autoantibodies against glycoprotein (GP)Ib/IX or GPIIbIIIa, frequently coinciding with refractoriness.13–15 Anti-GPIbα has been associated with Fcγ receptor IIa (FcγRIIa)-independent platelet activation, through loss of sialic acid and subsequent clearance via the Ashwell Morell receptor localized on hepatocytes.16 Alternatively, in heparin-induced thrombocytopenia, antibodies direct- ed to platelet factor 4/heparin complex induce platelet
Human platelets
Citrated whole blood was obtained from healthy human volun- teers with known HLA type (second field) in accordance with Dutch regulations and after approval from the Sanquin Ethical Advisory Board in accordance with the Declaration of Helsinki. Written informed consent was given by all participants. Platelets were isolated and washed as described elsewhere,24 and resus- pended in platelet assay buffer (10 mM HEPES, 140 mM NaCl, 3 mM KCl, 0.5 mM MgCl2, 10 mM glucose and 0.5 mM NaHCO3, pH 7.4).
Platelet activation
Washed platelets (2.5x108 platelets/mL) were incubated with HLA monoclonal antibodies or patients’ sera (1:50) containing HLA antibodies for 1 h at room temperature. Where appropriate, platelets were pre-incubated with FcγRIIa blocking antibody IV.3, Syk inhibitor IV or intravenous immunoglobulin.
Flow cytometry
For flow cytometry measurements, platelets were fixed in 1% PFA and diluted in platelet assay buffer. Anti-CD62P, anti-PAC-1 and anti-IgG antibodies were used to stain the platelets. Analysis was performed using a FACSCanto II (Becton Dickinson) flow cytometer.
Internalization of opsonized platelets by macrophages
Internalization of platelets by macrophages was determined as previously described.9 In short, PKH26-labeled platelets were opsonized with HLA monoclonal antibodies in the presence or absence of Syk inhibitor IV and incubated with monocyte-derived macrophages. Platelet internalization was quantified by imaging
®
flow cytometry (ImageStream X Mark II Imaging Flow
Cytometer, Merck Millipore, Amsterdam, the Netherlands).
Data and statistical analysis
Flow cytometry data were analyzed using FlowJo version 10 (Ashland, OR, USA). Data are represented as either mean ± stan- dard deviation (SD) or all data points are shown. Statistical analy- ses were performed using GraphPad Prism 7 version 7.02 (La Jolla, CA, USA), with the analyses used specified in the respective figure legends. Differences were considered statistically significant when P values were <0.05.
Further details on materials and methods can be found in the Online Supplementary Data.
Results
HLA monoclonal antibodies induced platelet α-granule release
Eight human HLA-specific monoclonal antibodies were used to study the effect of HLA antibodies on platelets from healthy donors (Table 1). These antibodies all recog- nize different HLA epitopes, of which some are specific for a particular HLA antigen (e.g. GV2D5 binds only HLA- A1) and others are broadly reactive (e.g. WIM8E5, binding to HLA-A1/A10(A25/A26/A34/A43/A66)/A11/A9(A23/ A24)/A29/A30/A31/A33/A28(A68/A69)).21,25 Donors were selected in such a way that platelets expressed an HLA type matching the specificity of antibodies used in each experiment. A similar level of binding of HLA monoclonal antibodies to the matched platelets was obtained in all experiments, as verified by flow cytometry (Figure 1A). The ability of HLA monoclonal antibodies to induce α- granule release was assessed by measuring CD62P expo-
17,18 clearance and FcγRIIa-dependent platelet activation.
Previous studies have described on FcγRIIa-dependent activation of platelets by (non-physiological) crosslinking of the murine pan-HLA class I antibody W6/32.19 Complement-dependent platelet aggregation induced by HLA antibodies has also been reported.20 Based on these findings we hypothesized that a subset of human HLA antibodies may be able to activate platelets. To address this issue we tested a panel of well-characterized human monoclonal HLA antibodies and HLA antibody-contain- ing sera from platelet-transfusion refractory patients for their ability to activate platelets.
Methods
HLA monoclonal antibodies and patients’ sera
Human HLA-specific monoclonal antibodies, all of IgG1 iso- type, were produced by hybridoma technology as described pre- viously.21,22 Blood samples of patients refractory to platelet transfu- sion were sent to the Department of Immunohematology Diagnostic Services, Sanquin, Amsterdam, the Netherlands. Leftover material was used according to the Dutch established codes of conduct for responsible use of patients’ material and as approved by our institute.23 HLA antibody specificities in patients’ sera were determined by a single antigen bead assay (Luminex). Thirteen sera positive for HLA antibodies and negative for other platelet-specific antibodies were used.
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haematologica | 2018; 103(10)