The abrogation of TCR-independent interactions with human serum ensures a selective capture of therapeutic virus-specific CD8+ T-cells by Multimer Technology in Adoptive Immunotherapy

•MHC-multimers bind to monocytes and B lymphocytes by TCR-independent interactions•TCR-independent interactions of MHC-multimers are due to joining with the FcγR•Sample preincubation with human AB serum avoids unspecific binding of MHC-multimers•This blockage ensures a pure therapeutic product for Adoptive Immunotherapy

Multimers are complexes of recombinant MHC-class I molecules conjugated with antigenic immunodominant peptides and labeled with fluorescent molecules or magnetic microbeads that allow the quantification and selection of virus-specific cytotoxic T-cell subpopulations. Specific T-cell receptors recognize the immunodominant peptides and bind to the multimers. Although these complexes are only recognized by CD8 + T cells with specific T-cell receptors for the particular antigen, it has been observed that multimers can also bind non-specifically to CD8- cells, such as B-cells and monocytes.Using PBMCs from CMV-seropositive healthy donors, we analyze the tendency of Pentamer and Streptamer multimers towards non-specific interactions and describe a method to avoid this unwanted event. We find that a notable proportion of multimer-positive cells are likely to represent cross-contamination by cells lacking a TCR specific for pp65. In addition, we demonstrate that this unspecific interaction can be overcome by the pre-incubation of multimer-stained PBMCs with human AB serum, without altering their capacity to bind specifically to the CD8 + T cell population of interest. In conclusion, in this study we characterize a novel method to abrogate TCR-independent interactions of multimers to ensure a pure and safe therapeutic product for Adoptive Immunotherapy.