The impact of antibody subclasses on anti-HPA-1a mediated platelet phagocytosis

Background

Accelarated clerance of maternal IgG antibody (anti-HPA-1a)-opsonized fetal platelets by phagocytes represents the major mechanism in cases of fetal and neonatal alloimmune thrombocytopenia (FNAIT). Until today, limited studies examining the impact of IgG antibody subclasses have been conducted. Such study is mainly hampered by the absence of good standards for anti-HPA-1a antibody subclasses and reliable platelet phagocytosis assay. In this study, both limitiations are carefully addressed.

Methods

Different IgG subclasses (IgG1, -IgG2, -IgG3 and -IgG4) of recombinant monoclonal antibody anti-HPA-1a (moAb 26.4) and effector-silent moAb 26.4 (IgG1-LALAP) were produced. Binding of antibody subclasses onto platelets was tested by flow cyometry using fluorescence labelled subclass-specific and Fcγ-specific secondary antibodies. The clearance of anti-HPA-1a sensitized platelets was analyzed by Whole Blood Platelet Phagocytosis Assay (WHOPPA). Platelets were labelled with pH sensitive dye (pHrodo-SE), opsonized with different moAb 26.4 subclasses and subjected to WHOPPA. The rate of pHrodo entgulfed platelets by monocytes was measured by flow cytometry.

Results

All 26.4 IgG subclasses did not interact with HPA-1bb, but bound equally to HPA-1aa typed platelets when tested with Fcγ-specific secondary antibodies by flow cytometry. In contrast, IgG subclass bound moAb 26.4 could only be detected by subclass specific secondary antibodies; bound moAb 26.4 IgG1 only reacted with anti-IgG1, but not anti-IgG2, -3 or -4. This specific reaction was also found with IgG2, -3 and -4. Analysis by WHOPPA showed that except effector-silent 25.4-IgG1 LALAP, all 26.4 IgG sublasses induced significant platelet phagocytosis by monocytes, but not by neutrophils. Highest phagocytosis rate was observed with 26.4-IgG3 (˜78.7%), followed by IgG1 (˜51.1%), IgG4 (˜35.4%), and IgG2 (˜19.1%).

Conclusion

Here, we enable reliable analysis of platelet phagocytosis caused by anti-HPA-1a IgG subclass using different subclasses of moAb 26.4 as standard. Our observation that moAb 26.4 IgG3 subclass induced platelet phagocytosis most strongly is interesting and is in line with the high capability of IgG3 to induce effector functions, most probably via high affinity FcγRI receptor on monocytes. The question wheather anti-HPA-1a IgG3 abs are dominated in maternal sera with severe FNAIT is intriguing.

Offenlegung Interessenkonflikt:

Alle Autoren haben kein Interessenkonflikt