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Anti-HLA antibodies with complementary and synergistic interaction
Online Supplementary Figure S3). Although some associa- tion between IgG binding and complement deposition and FcγRIIa-dependent platelet activation was observed, some sera displaying only limited IgG binding were still capable of inducing complement activation or Syk-depen- dent activation. This suggests that only a subset of anti- HLA antibodies present in patient sera can induce C3b deposition on platelets.
Discussion
Development of HLA alloantibodies remains a major cause of refractoriness to platelet transfusions. As yet, the pathogenic properties of HLA alloantibodies have not been sufficiently characterized. Here, we have shown that HLA antibodies are capable of inducing complement acti- vation on platelets, leading to C4b and C3b deposition on platelet surfaces and the formation of a functional MAC.
It was previously shown that platelet activation can lead to the binding of C3b,9 inducing complement activation via the alternative pathway. We have previously shown that HLA antibodies can induce FcγRIIa-dependent platelet activation, leading to CD62P surface exposure.8 Therefore, we tested whether the complement activation by HLA antibodies occurs via the classical pathway, or whether CD62P surface exposure directly leads to C3b binding followed by activation of the alternative comple- ment pathway. Since a blocking antibody directed to C1q completely blocked C3b and C4b deposition, complement activation by HLA antibodies appears to be fully depend- ent on the classical pathway. In line with this observation, blocking FcγRIIa-dependent activation had no effect on C3b deposition on platelets. Similar results were obtained in patient sera containing HLA antibodies. Also, the ability of HLA antibody-containing sera to induce complement activation was not directly correlated to FcγRIIa-depen- dent platelet activation. Some sera were capable of induc- ing both complement- and FcγRIIa-dependent activation of platelets.
We observed enhanced CD62P exposure in the samples in which complement was activated. In the presence of a complement source, CD62P exposure was only partly blocked upon inhibition of FcγRIIa-signaling. Simultaneous blocking of complement activation and FcγRIIa-signaling resulted in complete inhibition of platelet activation as measured by CD62P exposure. This suggests that HLA antibodies can induce platelet activa- tion by promoting complement deposition as well as FcγRIIa-dependent signaling. We speculate that both mechanisms may contribute to the rapid clearance of platelets in refractory patients.
In our study, we demonstrated that not all HLA mAbs and patient sera induce complement activation to a similar extent. Specific combinations of mAbs showed signifi- cantly enhanced C3b deposition; strong, synergistic com- plement deposition induced by incubation of platelets with low concentrations of two distinct HLA mAbs (SN607D8 and SN230G6) was observed. This suggests that structural organization of platelet bound IgG is crucial for initiation of the complement cascade. It has been shown that C1q binds most efficiently to IgG hexamers, for which Fc:Fc interactions are needed.23,30,31 In line with these observations, we could completely block comple- ment deposition induced by HLA mAbs by adding an IgG-
Fc blocking peptide24 described to prevent these Fc:Fc interactions. The same blocking peptide inhibited C3b deposition on platelets induced by HLA antibody-contain- ing patient sera. These results suggest that the propensity of polyclonal (patient)-derived HLA antibodies to form IgG hexamers on the platelet surface provides a binding platform for C1q which finally promotes the formation of C5b-9 complexes on platelet membranes. Additional properties of HLA antibodies such as specific subclass or composition of the glycan in the Fc part of IgGs are expected to potentially modulate complement activation of HLA antibodies on platelets.
In this respect, it is interesting to note that glycosylation of IgG1 has been described to affect the binding of IgG1 to C1q which also might affect the ability of individual antibodies to induce complement activation.32 Altogether, this might explain why only a subset of HLA mAbs and patient sera containing HLA antibodies induce comple- ment activation. Interestingly, sera containing HLA anti- bodies matching the HLA type of the platelet donor did not always induce complement activation. This may part- ly be explained by differences in concentration and bind- ing specificity of the different antibodies in these poly- clonal sera, and varying levels of HLA expression on the donor platelets used in this study.7 Also, the presence of specific IgG subclasses might play a role, as different sub- types bind with different affinity to C1q.33 In this study, we show that low concentrations of specific HLA anti- bodies can induce complement activation in a synergistic manner. This observation suggests that depending on the HLA profile and HLA density on donor platelets, specific combinations of patient-derived HLA antibodies can potently induce complement activation. Our results indi- cate that the propensity of HLA antibodies to induce com- plement activation is not per se related to the titer and binding specificity of HLA antibodies in serum. Rather, our findings suggest that the ability of HLA antibodies to form hexameric IgG complexes on platelets determines their ability to induce complement activation.
Ideally, the ability of HLA antibodies of individual patients to induce platelet activation8 and /or complement deposition on donor platelets should be assessed before transfusion. Previous studies have shown that platelet cross match does increase platelet recovery in patients, but data on occurrence of hemorrhage and mortality are lack- ing.34 Implementation of functional tests focusing on HLA antibody-induced platelet activation and complement deposition may have the potential to functionally stratify platelet concentrates for specific HLA alloimmunized patients. This would further increase the pool of suitable donor platelets for treatment of HLA alloimmunized patients who are currently dependent on treatment with HLA-matched platelet concentrates. This would increase transfusion safety, lower the costs and avoid delays in pro- vision of donor platelets, which is important for transfu- sion support in chronically transfused patients.
There are several treatment options for refractory thrombocytopenia, which from an immunological per- spective could target both FcγR- and complement- dependent clearance of donor platelets. Immunoglobulins (IVIg) could inhibit C3b deposition on platelets induced by HLA mAbs. Inhibition of complement deposition by IVIg has previously been shown in a number of autoimmune disorders.35,36 A randomized trial in HLA alloimmunized patients treated with IVIg revealed increased platelet-cor-
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