Development of new tissue culture protocols for enrichment of CD4 T cells associated with neonatal alloimmune thrombocytopenia
ForfatterKillie, Ida Løken
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.
ForlagUniversitetet i Tromsø
University of Tromsø
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