(253) Investigating the initiation of humoral immunity using Ce3D microscopy

Introduction/Rationale: Effective immune responses require rapid threat identification and correctly targeted immunity, as can be reflected by T cell polarization and the isotype of secreted immunoglobulins. Recent work indicates that T cells instruct critical B cell fate decisions, such as class-switch recombination (CSR), before the germinal center reaction begins. However, due to the rarity of antigen-specific lymphocytes at such early timepoints (e.g. day 1 to 4), it has been challenging to understand the first moments of critical signal interchange that shape the trajectory of the unfolding response. Therefore, superior detection strategies must be applied.

Methods: Clearing-enhanced 3 dimensional (Ce3D) confocal microscopy involves the capture of high-resolution images of cleared tissue sections up to 300µm thick, providing a large search space for rare cells. A high-sensitivity flow cytometry strategy was also employed.

Results: We found that Ce3D microscopy facilitated the detection of physiologic numbers of adoptively-transferred antigen-specific B and T cell lymphocytes in the early day 1 to 4 window, capturing rare events including lymphocytes mid-division or undergoing multi-partner interactions. Notably, fluorescent reporters of signaling or transcription, as well as staining of phosphorylated epitopes after methanol fixation, were both compatible with this technique, revealing functional spatial patterning. We also optimized a flow-cytometry-based approach with sufficient sensitivity to detect early antigen-specific lymphocytes at physiologic frequencies, enabling kinetic analyses of surface marker expression over time and cell divisions.

Conclusion: Our Ce3D- and flow cytometry-based strategies facilitate the study of early, rare lymphocytes to interrogate how rapid and correctly targeted humoral immunity is first coordinated. Ongoing and future work will characterize transcriptional profiles of interacting lymphocytes to determine how signal exchange coordinates CSR and other important fate decisions.