Anti-HLA-I IgG Subclass Influences Antibody-Mediated Platelet Activation in the Context of Platelet Transfusion Refractoriness

Background:

Patients requiring extensive platelet transfusion support are prone to platelet transfusion refractoriness (PTR), a therapeutic failure of platelet transfusion due to the rapid clearance of transfused platelets from the circulation. PTR can be attributed to the presence of allogeneic anti-HLA class I (HLA-I) antibodies (Abs) within the recipient's circulation. This leads to the rapid elimination of transfused platelets through mechanism that remains poorly understood. Strikingly, not all patients with anti-HLA-I Abs develop PTR upon platelet transfusions raising the question whether intrinsic properties of Abs determine their pathogenic ability to trigger PTR. Among the characteristics of IgG molecules, the IgG subclass could play an essential role in the pathogenicity of Abs. IgG subclasses have several functional differences notably in their ability to activate complement or engage Fc receptors, two mechanisms reported to affect platelet activation and subsequent platelet elimination.

Aim:

To examine the anti-HLA-I IgG subclass as a pathogenicity criterion in platelet transfusion-refractory state.

Methods: Sera from patients with or without PTR were collected in the frame of a prospective clinical trial and anti-HLA-I IgG were analyzed by Luminex®. Chimeric Abs (hIgG1-, hIgG2- and hIgG3-W6/32) were generated by fusion of the VH/VL part of the murine pan anti-HLA-I antibody (clone W6/32) and the Fc part of human IgG1, IgG2 or IgG3. Hirudinated platelet-rich plasma (PRP) or washed platelets from healthy voluntary donors were incubated with these chimeric antibodies before assessing platelet activation status by aggregation tests and flow cytometry experiments. In some conditions, platelets were incubated either with Eculizumab (100 µg/mL), IV.3 (10µg/mL) or IdeS (Imlifidase, 20 µg/mL) before incubation with the different chimeric antibodies in order to inhibit certain activation pathways.

Results: We performed a prospective clinical study describing the occurrence and the factors associated with PTR in a cohort of patients with onco-hematological disorders. Not all alloimmunized patients developed invariably a PTR, even when exhibiting a comparable level of Abs. This observation underlines the importance of intrinsic features of Abs associated with PTR. In vitro, chimeric hIgG1-, hIgG2- and hIgG3-W6/32 Abs led to platelet aggregation (73± 2, 84±3 and 72±2% max aggregation (% A_max) for hIgG1, hIgG2 and hIgG3-W6/32 respectively). Flow cytometry analysis confirmed Abs-induced platelet activation through P-selectin (P-sel) exposure and Annexin V binding. Interestingly, the activation mechanisms differ depending on the IgG subclass. The inhibition of the CD32A receptor by incubation with mAb IV.3 resulted in the complete abrogation of hIgG2-induced activation (6±3% A_max; n=3), while hIgG1-induced activation remained unaffected (71±1% A_max; n=3). Conversely, complement inhibition with Eculizumab, an anti-C5 antibody, seems to affect more the activation mediated by hIgG3 (3±1% A_max; n=3) as compared to that induced by hIgG2 (58±28% A_max; n=3). Accordingly, hIgG2 but neither hIgG1 nor hIgG3 induced P-sel exposure and a complete aggregation of washed platelets, confirming the involvement of a plasma component in the platelet activation mediated by hIgG1- and hIgG3-W6/32 subclasses. Finally, Abs-induced platelet activation is dependent on the Fc part since F(ab’)₂ of W6/32 mAb could not induce platelet activation (1.8±1.5% Psel⁺, n=4). Consistently, a pre-incubation with the cysteine protease IdeS totally inhibited the hIgG2-induced activation effects confirming the important of the Fc part in this activation (38.5 ± 16 % Psel⁺ for IgG2 vs 3.6 ± 1.8 Psel⁺ for IgG2+IdeS, n=4, p=0.04).

Conclusions:

Our findings indicate there is no definitive correlation between the presence of anti-HLA-I Abs and PTR. This suggests that some intrinsic features of anti-HLA-I IgG should be considered in this context. Our research reveals that platelet activation mechanisms in response to anti-HLA-I antibodies depend on the IgG subclass. Cleavage of anti-HLA-I IgG by IdeS abolishes platelet activation mediated by antibodies, which opens the door for developing strategies to safely transfuse platelets despite the limitations imposed by alloantibodies.