Alexey Y. Karulin

Background/Objectives: Despite clonal expansion during a primary immune response, or after subsequent antigen encounters, the frequency of memory B cells (B_mem) specific for an antigen remains low, making their detection difficult. However, unlike serum antibodies, which have a short half-life in vivo and thus require continuous replenishment to maintain stable titers, circulating B_mem are long-lived; they preserve immunological preparedness through their ability to rapidly engage in recall responses and differentiate into antibody-secreting cells (ASCs) upon antigen encounter. To this end, development of assays suited for the reliable detection of rare antigen-specific B_mem is critical and can provide insights into an individual’s antigen exposure history and immune status beyond that offered by traditional serum antibody measurements alone. Methods: ImmunoSpot^® has emerged as a suitable technique for the detection of individual antigen-specific B cells through visualizing their antibody-derived secretory footprints. Here, we report the theoretical and practical foundations for detecting rare antigen-specific B_mem in human peripheral blood mononuclear cells (PBMC). Leveraging the unique availability of verifiably naïve vs. antigen-experienced human samples, we used SARS-CoV-2 Spike (S-) and Nucleocapsid (NCAP) antigens to interrogate the presence of B_mem with these respective specificities. Results: While 100% diagnostic accuracy was achieved for both antigens, detection of NCAP-specific B_mem required reducing the lower detection limit of the standard assay. Specifically, this was achieved by testing a total of 2 million PBMC across multiple replicate assay wells and assessing the cumulative number of secretory footprints detected. Conclusion: The protocols described here should facilitate the reliable detection of ASCs present at varying precursor frequencies and serve as guidance for routine immune monitoring of rare B_mem with specificity for any antigen.

Despite clonal expansion during a primary immune response, or after sub-sequent antigen encounters, the frequency of memory B cells (Bmem) specific for an antigen remains low, making their detection difficult. However, unlike serum an-tibodies, which have a short half-life in vivo and thus require continuous replen-ishment to maintain stable titers, circulating Bmem are long-lived; they preserve immunological preparedness through their ability to rapidly engage in recall re-sponses and differentiate into antibody-secreting cells (ASCs) upon antigen en-counter. Consequently, developing means for the reliable detection of anti-gen-specific Bmem promises to offer a window into an individual’s exposure history and immune status beyond that gleaned from traditional serum antibody meas-urements alone. ImmunoSpot® has emerged as a suitable technique for the de-tection of individual antigen-specific B cells through visualizing their anti-body-derived secretory footprints. Here, we report the theoretical and practical foundations for detecting rare antigen-specific Bmem in human peripheral blood mononuclear cells (PBMC). Leveraging the unique availability of verifiably naïve vs. antigen-experienced human samples, we used SARS-CoV-2 Spike (S-) and Nucleocapsid (NCAP) antigens to interrogate the presence of Bmem with these re-spective specificities. While 100% diagnostic accuracy was achieved for both an-tigens, detection of NCAP-specific Bmem required extension of the lower detection limit (LOD) of the standard assay. This could be achieved using 2 million PBMC in aggregate. The protocols described here should facilitate reliable detection of ASC present at varying precursor frequencies and serve as guidance for routine im-mune monitoring of rare Bmem with specificity for any antigen.

Over the past decade, ELISPOT has become a highly implemented mainstream assay in immunological research, immune monitoring, and vaccine development. Unique single cell resolution along with high throughput potential sets ELISPOT apart from flow cytometry, ELISA, microarray- and bead-based multiplex assays. The necessity to unambiguously identify individual T and B cells that do, or do not co-express certain analytes, including polyfunctional cytokine producing T cells has stimulated the development of multi-color ELISPOT assays. The success of these assays has also been driven by limited sample/cell availability and resource constraints with reagents and labor. There are few commercially available test kits and instruments available at present for multi-color FLUOROSPOT. Beyond commercial descriptions of competing systems, little is known about their accuracy in experimental settings detecting individual cells that secrete multiple analytes vs. random overlays of spots. Here, we present a theoretical and experimental validation study for three and four color T- and B-cell FLUOROSPOT data analysis. The ImmunoSpot^® Fluoro-X™ analysis system we used includes an automatic image acquisition unit that generates individual color images free of spectral overlaps and multi-color spot counting software based on the maximal allowed distance between centers of spots of different colors or Center of Mass Distance (COMD). Using four color B-cell FLUOROSPOT for IgM, IgA, IgG1, IgG3; and three/four color T-cell FLUOROSPOT for IL-2, IFN-γ, TNF-α, and GzB, in serial dilution experiments, we demonstrate the validity and accuracy of Fluoro-X™ multi-color spot counting algorithms. Statistical predictions based on the Poisson spatial distribution, coupled with scrambled image counting, permit objective correction of true multi-color spot counts to exclude randomly overlaid spots.

Multi-color FLUOROSPOT assays for simultaneous detection of several T-cell cytokines and/or classes/sub-classes of immunoglobulins secreted by B cells have recently become a major new avenue of development of ELISPOT technology. Advances in assay techniques and the availability of commercial test kits stimulated development of multi-color FLUOROSPOT data analysis platforms. The ImmunoSpot^® Fluoro-X™ Software Suite was developed by CTL as an integrated data acquisition, analysis, and management solution for automated high-throughput processing of multi-color T- and B-cell FLUOROSPOT assay plates. The Fluoro-X™ software counting module is based on SmartSpot™/AutoGate™ technologies and utilizes CTL’s Center of Mass Distance algorithm for the detection of multi-color spots. The Fluoro-X™ software provides an objective, user error-free means for analyzing multi-color FLUOROSPOT data. An integrated quality control module, with optional GLP and CFR Part 11 compliant package and role-based security, enables data validation, review, and approval with complete audit trails. The extensive multi-format data output and presentation capabilities of the Fluoro-X™ software allow further analysis of FLUOROSPOT data using any commercial flow cytometry software and facilitate the generation of professional reports and presentation. In this article, we present a detailed step-by-step workflow for the analysis of a human four-color IFN-γ, IL-2, TNF-α, and GzB antigen-specific T-cell assay using the Fluoro-X Software Suite.

In individuals who have once developed humoral immunity to an infectious/foreign antigen, the antibodies present in their body can mediate instant protection when the antigen re-enters. Such antigen-specific antibodies can be readily detected in the serum. Long term humoral immunity is, however, also critically dependent on the ability of memory B cells to engage in a secondary antibody response upon re-exposure to the antigen. Antibody molecules in the body are short lived, having a half-life of weeks, while memory B cells have a life span of decades. Therefore, the presence of serum antibodies is not always a reliable indicator of B cell memory and comprehensive monitoring of humoral immunity requires that both serum antibodies and memory B cells be assessed. The prevailing view is that resting memory B cells and B cell blasts in peripheral blood mononuclear cells (PBMC) cannot be cryopreserved without losing their antibody secreting function, and regulated high throughput immune monitoring of B cell immunity is therefore confined to—and largely limited by—the need to test freshly isolated PBMC. Using optimized protocols for freezing and thawing of PBMC, and four color ImmunoSpot^® analysis for the simultaneous detection of all immunoglobulin classes/subclasses we show here that both resting memory B cells and B cell blasts retain their ability to secrete antibody after thawing, and thus demonstrate the feasibility of B cell immune monitoring using cryopreserved PBMC.

One of the primary effector functions of immune cells is the killing of virus-infected or malignant cells in the body. Natural killer (NK) and CD8 effector T cells are specialized for this function. The gold standard for measuring such cell-mediated cytolysis has been the chromium release assay, in which the leakage of the radioactive isotope from damaged target cells is being detected. Flow cytometry-based single cell analysis of target cells has recently been established as a non-radioactive alternative. Here we introduce a target cell visualization assay (TVA) that applies similar target cell staining approaches as used in flow cytometry but based on single cell computer image analysis. Two versions of TVA are described here. In one, the decrease in numbers of calcein-stained, i.e., viable, target cells is assessed. In the other, the CFSE/PI TVA, the increase in numbers of dead target cells is established in addition. TVA assays are shown to operate with the same sensitivity as standard chromium release assays, and, leaving data audit trails in form of scanned (raw), analyzed, and quality-controlled images, thus meeting requirements for measuring cell-mediated cytolysis in a regulated environment.