Streptamer technology allows accurate and specific detection of CMV-specific HLA-A*02 CD8+ T cells by flow cytometry

Abstract

Background: Multimer technology is widely used to screen antigen-specific immune recovery after allogeneic hematopoietic stem cell transplantation (allo-HSCT) as it enables identification, enumeration, phenotypic characterization and isolation of virus-specific T-cells. Novel approaches of multimerization might improve on classical tetramer staining; however, their use as standard monitoring technique to quantify antigen-specific cells has not been validated yet. We have compared two of these available multimeric complexes: pentamer and streptamer to select the best strategy for the incorporation into clinical monitoring practice.

Methods: CMVpp65^495-503 -specific HLA-A*02:01 CD8⁺ T lymphocytes (CTL^A *^02:01 -CMVpp65^495-503 ) were examined with pentamer and streptamer in peripheral blood cells of 77 healthy volunteers. Quantitative and qualitative analyses were performed to compare the precision and repeatability, sensitivity and accuracy and specificity of both technologies by flow cytometry.

Results: Standard deviation for both techniques was less than 0.05 showing that they are repetitive and precise. Both techniques significantly correlated at high frequencies (r_Spearman = 0.9422; P < 0.0001) but it was lost at lower levels (<1%) of CTL^A *^02:01 -CMVpp65^495-503 (r_Spearman = 0.3351; P = 0.1376). Streptamer is more accurate for the detection of CTL^A *^02:01 -CMVpp65^495-503 providing significantly closer values to the theoretical ones (P < 0.0001) as pentamer binds unspecifically to a notable proportion of non-CMV-specific CD8⁺ T-cells.

Conclusion: Our results suggest that streptamer multimer provides precise, accurate and specific results to detect CTL^A *^02:01 -CMVpp65^495-503 by flow cytometry. Streptamer multimer can be used not only for the monitoring of early CTL^A *^02:01 -CMVpp65^495-503 reconstitution in immunosuppressed patients following allo-HSCT but also, in conjunction with its reversibility role, for the isolation of CTL^A *^02:01 -CMVpp65^495-503 for its future use in adoptive immunotherapy. © 2016 International Clinical Cytometry Society.

Keywords: CMV-specific CTL; immune monitoring; pentamer; polychromatic flow cytometry; streptamer.