B Cells and B Cell Blasts Withstand Cryopreservation While Retaining Their Functionality for Producing Antibody

Abstract

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.

Interferon α Enhances B Cell Activation Associated With FOXM1 Induction: Potential Novel Therapeutic Strategy for Targeting the Plasmablasts of Systemic Lupus Erythematosus

“…Although three B cell subclusters and one plasma cell subcluster were shown to express ISGs, the distinct expression of cell-cycle-related genes among the subclusters was not identified. This inconsistency with the results of the present study may be caused by the loss of highly proliferative plasmablasts in the cryopreservation and thawing processes, although it is reported that cryopreservation does not lose the antibody-secreting capacity of plasmablasts in PBMC ( 35 , 36 ). The present study is the first to report that cell-cycle-related genes, FOXM1, and its downstream genes, as well as ISGs, were elevated to a higher degree in freshly isolated plasmablasts of SLE patients compared with those of healthy donors.…”

Analysis of Antigen‐Specific Human Memory B Cell Populations Based on In Vitro Polyclonal Stimulation

“…A commonly held view is that cryopreservation reduces the number and functionality of MBCs. However, a number of studies of in vitro MBC stimulation with a range of stimulation cocktails have reported similar results for fresh and frozen PBMCs (Buisman, de Rond, Öztürk, ten Hulscher, & van Binnendijk, 2009;Crotty et al, 2004;Fecher et al, 2018;Pinna et al, 2009;Weiss et al, 2012).…”

“…It is easy to prepare and requires no more than standard consideration of the quality of individual components. The combination of just R848 and IL‐2 is also a frequently used and highly effective stimulation cocktail (Fecher et al., 2018; Jahnmatz et al., 2013; Pinna et al., 2009). We have found that the addition of IL‐10 results in a small but consistent 3‐ to 4‐fold increase in the yield of IgG MASCs and MPAbs from PBMCs stimulated as described in Basic Protocol 2.…”

Affinity tag coating enables reliable detection of antigen-specific B cells in ImmunoSpot assays

“…In contrast the memory B cell component, consisting of resting lymphocytes, can be detected via its ASC activity only following in vitro differentiation. Moreover, ongoing optimizations in cryopreservation of resting B cells and B cell blasts [66,90], along with the high-throughput capability of the ImmunoSpot approach, also enable simultaneous assessments of these respective compartments and detailed comparisons; such as Ig class usage and relative precursory frequency. In this context, it is important to note that acutely following infection or vaccination, the majority of circulating plasmablasts will be endowed with high-affinity, antigenspecific BCR since they were successfully recruited into the T cell-dependent immune response [4,91].…”

“…Thawing, washing and counting of PBMC was performed according to previously described protocols [65,66] using CTL's Live/Dead cell counting suite on an ImmunoSpot® S6 Ultimate Analyzer (CTL). Subsequently, cells were resuspended at 2 x 10 6 cells/mL in complete B cell medium (BCM) containing RPMI 1640 (Lonza, Walkersville, MD) supplemented with 10% fetal bovine serum (Gemini Bioproducts, West Sacramento, CA, USA), 100U/mL penicillin, 100μg/mL streptomycin, 2mM L-glutamine, 1mM sodium pyruvate, 8mM HEPES (all obtained from Life Technologies, Grand Island, NY, USA) and 50μM beta-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) containing B-Poly-S TM reagent (from CTL).…”