Enhanced detection of antigen-specific T cells by a multiplexed AIM assay
Audrée Lemieux, Gérémy Sannier, Alexandre Nicolas, Manon Nayrac, Gloria-Gabrielle Delgado, Rose Cloutier, Nathalie Brassard, Mélanie Laporte, Mélina Duchesne, Alina Maria Sreng Flores, Andrés Finzi, Olivier Tastet, Mathieu Dubé, Daniel E. Kaufmann
Abstract
Broadly applicable methods to identify and characterize antigen-specific CD4+ and CD8+ T cells are key to immunology research, including studies of vaccine responses and immunity to infectious diseases. We developed a multiplexed activation-induced marker (AIM) assay that presents several advantages compared to single pairs of AIMs. The simultaneous measurement of four AIMs (CD69, 4-1BB, OX40, and CD40L) creates six AIM pairs that define CD4+ T cell populations with partial and variable overlap. When combined in an AND/OR Boolean gating strategy for analysis, this approach enhances CD4+ T cell detection compared to any single AIM pair, while CD8+ T cells are dominated by CD69/4-1BB co-expression. Supervised and unsupervised clustering analyses show differential expression of the AIMs in defined T helper lineages and that multiplexing mitigates phenotypic biases. Paired and unpaired comparisons of responses to infections (HIV and cytomegalovirus [CMV]) and vaccination (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) validate the robustness and versatility of the method.