Development of new tissue culture protocols for enrichment of CD4 T cells associated with neonatal alloimmune thrombocytopenia

Abstract

Neonatal alloimmune thrombocytopenia (NAIT) is most commonly caused by destruction of foetal platelets by maternal antibodies reactive to human platelet antigen (HPA)-1a. The activation of antigen-primed B cells to differentiate to antibody-secreting plasma cells usually requires help from CD4 T cells. The strong association between anti-HPA-1a-production and the MHC allele HLA-DRB3*0101 supports that this notion is also valid in the context of NAIT, and suggests the activation of HPA-1a-specific T cells as the most critical event of immunization.

In this study, an improved protocol for enrichment, identification and efficient isolation of HPA-1a-specific CD4 T cells is presented. By replacing foetal bovine serum with human serum, enrichment of antigen-specific CD4 T cells improved dramatically. Identification and isolation of HPA-1a-specific CD4 T cells greatly improved when combining the CFSE proliferation assay with a second stimulation with antigen and subsequent assay for surface detection of TNF production. HPA-1a-specific CD4 T cells could also be identified in the CFSE proliferation assay as proliferating T cells with down-regulated expression of CD4.

HPA-1a-specific T cells isolated from immunized women may serve as useful tools for investigating the cellular immune response to HPA-1a, and for developing strategies to prevent immunization in HPA-incompatible pregnancies, e.g. through TCR epitope mapping and examinations of the immunogenicity of the HPA-1a antigen at the amino-acid level.