Response under pressure: deploying emerging technologies to understand B-cell-mediated immunity in COVID-19

Abstract

Critical technological advances have enabled the rapid investigations into the immune responses elicited by SARS-CoV-2, the pathogen responsible for the COVID-19 pandemic. In this Comment, we discuss the cutting-edge methods used to deconvolute the B cell responses against this virus, and the significant impact they have had in the ongoing public health crisis.

Figure 1.. Efficient utilization of patient blood in B cell-focused investigations of COVID-19

Routine EDTA-tube blood collection from human patients with COVID-19 can be readily processed into plasma and PBMC fractions for downstream investigation. Application of high-dimensional flow cytometry panels to collected PBMCs can reveal alterations in B cell activation pathways, antigen specificity tracking, memory emergence and persistence, and anticipate B cell effector functions. Cell sorting of these analysis platforms allows for the single-cell functional testing of antibody secreting cells to identify clonotype specificity, neutralizing potential, and real-time screening for therapeutic potential. Cells either directly sorted based on fluorescent markers or recovered from single cell functional assays can then be shunted into single cell sequencing applications to investigate the transcriptomic, epigenetic, and repertoire features of interest from selected B cell-derived populations. Using this multi-omics approach, single cells of interest can be identified for mAb production to screen and identify potential binding partners. Resulting mAbs, patient plasma, or ex vivo ASC (MENSA) cultures can then be applied to multiplex antigen screening tools to identify relevant viral and autoantigen reactivities across a multitude of platforms.

Figure 2.. Function-based pre-selection of Ag-specific ASC.

(Left) Development of plasma cell survival system (PCSS). Primary mesenchymal stromal cells (MSC) derived from human bone marrow (BM) are expanded and irradiated. The resulting culture medium is then collected and supplemented with APRIL and hypoxic conditions to promote ASC survival. (Middle) Single cell detection of antigen (Ag)-specific ASC by an in-channel binding assay. A single-assay mixture of Ag-coated beads and fluorescently labeled detection antibodies (Abs) is imported into the channel above the NanoPen chambers. PCSS is perfused through the chip to maintain survival of penned ASC, whose secreted Abs diffuse into the channel and bind the beads. Accumulation of fluorescence on the beads leads to the development of fluorescent halos (“blooms”) in the channel adjacent to the pens that contain Ag-specific ASC. (Right) Single cell functional screening for Ag-specific ASC by an in-pen receptor blocking assay. After positioning Ag-coated beads into pens and saturating the Ag, an assay mixture of fluorescent receptor (ACE2) and fluorescently labeled detection Abs targeting Ag-specific (RBD) binding Abs is imported into the channel and allowed to diffuse into pens. PCSS is perfused through the chip to maintain survival of penned ASC. (Top panel) RBD-binding, ACE2 non-blocking Abs result in accumulation and detection of both fluorophores; (bottom panel) ACE2 blocking Abs exhibit only signals for RBD-binding Abs. ASC secreting blocking Abs are then exported from specific pens for downstream interrogation.