Cryopreserved peripheral blood mononuclear cells (PBMC) constitute an important component of immune monitoring studies as they allow for efficient batch- testing of samples as well as for the validation and extension of original studies in the future. In this study, we systematically test the permutations of PBMC thawing practices commonly employed in the field and identify conditions that are high and low risk for the viability of PBMC and their functionality in downstream ELISPOT assays. The study identifies the addition of ice-chilled washing media to thawed cells at the same temperature as being a high risk practice, as it yields significantly lower viability and functionality of recovered PBMC when compared to warming the cryovials to 37 °C and adding a warm washing medium. We found thawed PBMC in cryovials could be kept up to 30 minutes at 37 °C in the presence of DMSO before commencement of washing, which surprisingly identifies exposure to DMSO as a low risk step during the thawing process. This latter finding is of considerable practical relevance since it permits batch-thawing of PBMC in high-throughput immune monitoring environments.

Detection of antigen-specific memory B cells for immune monitoring requires their activation, and is commonly accomplished through stimulation with the TLR7/8 agonist R848 and IL-2. To this end, we evaluated whether addition of IL-21 would further enhance this TLR-driven stimulation approach; which it did not. More importantly, as most antigen-specific B cell responses are T cell-driven, we sought to devise a polyclonal B cell stimulation protocol that closely mimics T cell help. Herein, we report that the combination of agonistic anti-CD40, IL-4 and IL-21 affords polyclonal B cell stimulation that was comparable to R848 and IL-2 for detection of influenza-specific memory B cells. An additional advantage of anti-CD40, IL-4 and IL-21 stimulation is the selective activation of IgM⁺ memory B cells, as well as the elicitation of IgE⁺ ASC, which the former fails to do. Thereby, we introduce a protocol that mimics physiological B cell activation through helper T cells, including induction of all Ig classes, for immune monitoring of antigen-specific B cell memory.

Experimental autoimmune encephalomyelits (EAE) has been widely studied as a model for multiple sclerosis (MS). EAE also holds a special place in basic autoimmune research. It is induced by immunizing healthy, naïve mice with neuroantigen. Unlike in spontaneous autoimmune models, one can therefore clearly define the initiation time point, the inducing antigen, the circumstances of the immunization that elicit a pathogenic—or nonpathogenic—T cell response, and many other parameters that are required for the induction and perpetuation of autoimmune central nervous system pathology. In the following, we will provide an overview of our current understanding of the discrete steps that lead to the pathogenesis of EAE, and we will highlight several junctions at which the perpetuation or abortive course of the disease is defined. It has become abundantly clear that the induction of a pathogenic CD4⁺ T cell response is a necessary requirement for the induction of EAE. However, many downstream mechanisms need to be considered if we want to understand the pathomechanisms that define the variable outcomes of EAE, and by inference, of MS.

T cell immunity is traditionally assessed through functional recall assays, which detect the consequences of the T cells' antigen encounter, or via fluorescently labeled multimers that selectively bind peptide-specific T cell receptors. Using either approach, if the wrong antigen or peptide of a complex antigenic system, such as a virus, is used for immune monitoring, either false negative data will be obtained, or the magnitude of the antigen-specific T cell compartment will go largely underestimated. In this work, we show how selection of the “right” antigen or antigenic peptides is critical for successful T cell immune monitoring against human cytomegalovirus (HCMV). Specifically, we demonstrate that individual HCMV antigens, along with previously reported epitopes, frequently failed to detect CD8+ T cell immunity in test subjects. Through systematic assessment of T cell reactivity against individual nonamer peptides derived from the HCMVpp65 protein, our data clearly establish that (i) systematic testing against all potential epitopes encoded by the genome of the antigen of interest is required to reliably detect CD8+ T cell immunity, and (ii) genome-wide, large scale systematic testing of peptides has become feasible through high-throughput ELISPOT-based “brute force” epitope mapping.

The scope of immune monitoring is to define the existence, magnitude, and quality of immune mechanisms operational in a host. In clinical trials and praxis, the assessment of humoral immunity is commonly confined to measurements of serum antibody reactivity without accounting for the memory B cell potential. Relying on fundamentally different mechanisms, however, passive immunity conveyed by pre-existing antibodies needs to be distinguished from active B cell memory. Here, we tested whether, in healthy human individuals, the antibody titers to SARS-CoV-2, seasonal influenza, or Epstein–Barr virus antigens correlated with the frequency of recirculating memory B cells reactive with the respective antigens. Weak correlations were found. The data suggest that the assessment of humoral immunity by measurement of antibody levels does not reflect on memory B cell frequencies and thus an individual’s potential to engage in an anamnestic antibody response against the same or an antigenically related virus. Direct monitoring of the antigen-reactive memory B cell compartment is both required and feasible towards that goal.

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.