Optimizing PBMC Cryopreservation and Utilization for ImmunoSpot Analysis of Antigen-Specific Memory B Cells

Background: Measuring frequencies of antigen-specific memory B cells (Bmem), their immunoglobulin (Ig) class and subclass usage, cross-reactivity and affinity can provide insights into the efficacy of future antibody responses in case of antigen re-encounter. B cell ImmunoSpot assays can provide such information; however, like most cell-based tests, they too require considerable amounts of blood to be drawn from the donor and this has hindered their inclusion into clinical trials and routine immune diagnostics. Methods: We introduce strategies for reducing the cell numbers required to 2-3 million peripheral blood mononuclear cells (PBMC) per antigen, obtainable from 2-3 mL of blood from healthy adult donors. Results: Except when Bmem frequencies were very low, we found that testing PBMC in singlet wells, but in serial dilution, enables as reliable Bmem frequency assessments as testing replicate wells at a single fixed cell number. Additionally, B cell ImmunoSpot assays can be multiplexed for detecting four Ig classes, or IgG subclasses, simultaneously and without loss of sensitivity. The requirement for low cell numbers and the retention of B cell functionality of cryopreserved PBMC equivalent to freshly isolated material implies that fewer than the standard 10 million PBMC per vial must be conserved. This would reduce the number of individuals who could not be tested for Bmem due to insufficient availability of PBMC, a common problem with such assays. Conclusions: The predictable need for, and recovery of cryopreserved PBMC facilitates planning of, and optimal cell utilization in B cell ImmunoSpot assays and increases the practical feasibility of extensive Bmem characterization in larger cohorts.