Platelets from Donors with Consistent Low HLA-B8, B12 or B35 Expression Do Not Undergo Antibody-Mediated Internalization

Introduction Patients refractory to platelet (PLT) transfusions due to HLA specific alloantibodies require HLA-matched PLTs. However, even with large HLA-typed donor pools patients with rare HLA haplotypes may not have suitable donors. In order to improve the efficiency of HLA-matching, PLTs with low HLA class I expression may be an alternative for fully HLA matched transfusions. This study established the proportion of donors with consistent low expression of HLA-B8, B12, and B35 on PLTs and studied the effect of this low HLA expression on antibody-mediated internalization of PLTs by macrophages.

Methods HLA-B8, B12, or B35 expression on PLTs of HLA typed blood donors was determined using AlexaFluor488 labeled human monoclonal antibodies (mAbs) specifically targeted to either one of these antigens. Donors were tested several times for their specific HLA class I expression. HLA typed PLTs with low (<2SDs above background) or high (>median expression) expression were opsonized with saturating concentrations of human IgG1 mAbs targeting either HLA-B8, B12 or B35. In addition, PLTs were opsonized with increasing concentrations of anti-HPA1a humanized IgG1 or anti-pan HLA class I mouse IgG2a. Subsequently, these opsonized PLTs were incubated with macrophages and antibody-mediated internalization was determined using imaging flow cytometry.

Results The expression of HLA-B8 (n=113), B12 (n=98) or B35 (n=66) on PLTs was extremely variable between individuals (coefficients of variation: 47.6, 73.6% and 41.4, respectively). For HLA-B8, but not for B12 or B35, this variation was in part explained by hetero- and homozygosity. The observed variations were most pronounced in, but not exclusive to PLTs. In contrast, expression within one donor was consistent over time. Remarkably, 32% of HLA-B8, 34% of HLA-B12 and 9% of HLA-B35 donors showed PLT antigen expression that was not or only minimally (<2SDs) above background. Opsonization of PLTs with low HLA expression did not affect internalization, whereas PLTs with high HLA expression did show significant antibody-mediated internalization. Furthermore, this internalization of PLTs by macrophages correlated with the level of antibody opsonization and PLT antigen expression.

Discussion Our findings show high inter-individual variation for HLA-B8, B12 and B35 expression on PLTs, with 9 to 34% of donors showing reproducibly undetectable or low expression. Because of this consistency, donors can be marked as 'low expressors' in the HLA-typed donor file which might greatly facilitate HLA-matching efficiency. In line with this hypothesis, antibody-mediated internalization showed a strong correlation with mAb opsonization and PLT antigen expression and was not observed in PLTs with low antigen expression. As in vitro antibody-mediated internalization has been correlated to in vivo clearance, this suggest that PLTs from 'low expressors' may be used to treat refractory patients despite HLA mismatches.