NIV Congres

The presence of donor specific HLA antibodies (DSA) detectable in sensitive luminex assays is associated with graft rejection, but only in a proportion of the patients. Recently we have developed several in vitro assays to measure the number and/or reactivity of memory B cells, which may be of additional value for the risk estimation in patients with DSA. However, the current assays to measure immunoglobulin production require prolonged stimulation of B cells of typically 8 to 14 days, which makes them less useful for monitoring. Furthermore, such long B cell activation periods invariably lead to a high rate of cell death, and may therefore influence clonal distribution.

To improve on the currently available B cell stimulation protocols, we have analysed the kinetics of total B cells and FACS sorted naive (CD19⁺IgD⁺CD27^-) and memory (CD19⁺IgD^-CD27⁺) B cells upon polyclonal activation. We analysed the dead/alive cell ratios (by 7AAD and eosin staining), proliferation capacity (by thymidine incorporation) and immunoglobulin production (by IgM/IgG ELISPOT) at several time points. For antigen specific responses, tetanus toxoid (TT) antigen-specific ELISPOT assays were performed.

Upon B cell stimulation, we found low dead/alive ratios for the first 6 days of culture in all B cell fractions. Cell death gradually increased starting from day 6 up to day 10. The highest proliferation rate of memory B cells was at day 3 whereas for unsorted and naïve B cells the peak proliferation was at day 6. When we assessed immunoglobulin production by ELISPOT, we found that IgG spots were produced exclusively by memory cells and were detectable as early as day 2 of the culture period. To confirm early detection of antigen specific memory responses we performed TT antigen-specific ELISPOT assays at several time points after stimulation. In these preliminary experiments, we could readily detect TT antigen-specific memory IgG responses after 3 days of stimulation.

Our data showing low cell death up to 6 days of cell culture and the ability of detecting (antigen-specific) IgG spots produced by memory cells as early as day 2 of the culture suggest that short culture periods of 2-4 days might be sufficient to detect the memory B cell responses. We aim to extend our results to determine whether shorter culture periods may be used to detect antigen specific memory B cell responses.