Comparative analysis of the germinal center response by flow cytometry and immunohistology

Germinal centers (GCs) are structures formed within B cell follicles critical for the generation of high affinity antibodies. The evaluation of GCs in secondary lymphoid tissues has emerged as a valuable means for understanding the immunological activity in vaccine responses, autoimmunity and cancer. The analysis has been facilitated by advances in sampling techniques, including non-invasive lymph node collection and fine needle aspiration. In this study, we performed a systematic comparison between immunohistology and flow cytometry for analysis of GCs with the major aim to identify strategies for data analysis that would allow to relate data acquired by the two methods. Lymph nodes from rhesus macaques were divided in half and analysed as either cryosections or cell suspensions. Using human markers such as PD-1 and Ki67 to identify T follicular helper (T_FH) cells and GC B cells, we developed a method for GC analysis by immunohistology using CellProfiler™ software and a flow cytometry panel with relatively limited numbers of antibodies to be scalable and feasible for most laboratories to perform. While some discrepancies between the two methods were identified, T_FH cells and GC B cells normalized by total CD3⁺ T cell or CD20⁺ B cell numbers, respectively, in immunohistology correlated well with matched data from flow cytometry. GC area normalized by section area in immunohistology also correlated well with T_FH cells per total live cells from flow cytometry. Performing this type of data analysis would therefore facilitate comparison of results generated between the two methods.