Flow Cytometry-Based Protocols for the Analysis of Human Plasma Cell Differentiation

Abstract

Humoral immunity is established after differentiation of antigen-specific B cells into plasma cells (PCs) that produce antibodies of relevant specificities. Defects in the development, activation, or differentiation of B cells severely compromises the immune response. Primary immunodeficiencies are often characterized by hypogammaglobulinemia and the inability to mount effective antigen-specific antibody responses, resulting in increased susceptibility to infections. After IgA deficiency, which is most often asymptomatic, common variable immunodeficiency (CVID) is the most prevalent symptomatic primary immunodeficiency, but in most cases the underlying genetic causes are unknown or their roles in disease pathogenesis are poorly understood. In this study, we developed a protocol for in vitro stimulation of primary human B cells for subsequent analyses of PC differentiation and antibody production. With this approach, we were able to detect a population of CD38 + IRF4 + Blimp-1 + cells committed to PC fate and IgG production, including when starting from cryopreserved samples. The application of functional assays to characterize PC differentiation and possible defects therein in B cells from patients suffering from primary antibody deficiencies with late B cell defects could increase our understanding of the disease pathophysiology and underlying mechanisms.

The Immune Response to SARS-CoV-2 Vaccination: Insights Learned From Adult Patients With Common Variable Immune Deficiency

“…Pathogenic loss-of-function or gain-of-function heterozygous variants have been reported to be associated with CVID. However, their functional relevance for susceptibility to infection and response to vaccination remains to be clarified ( 8 ). Unfortunately, genetic causes of most CVID cases remain undefined, and the diagnosis is predominantly based on hypogammaglobulinemia with impairment of antibody response to vaccine or natural antigen and reduced memory B cells (MBCs) frequency.…”

Comparison of the oxidative respiratory burst and mitogen-induced leukocyte responses of camels, goats, sheep, and cows

“…The mean stimulation index of camel PBML (10.43 ± 0.92) was significantly higher (p < 0.05) than those of goats (5.03 ± 0.90), sheep (5.62 ± 0.62), and cows (5.53 ± 0.78), in the presence of PWM. Although it cannot be determined from the present study whether a specific subpopulation of lymphocytes from camels is stimulated by PWM, it has been demonstrated in cows [53], humans [54], and mice [55] that PWM is capable of stimulating both T and B cells. Indeed, when T-cells were removed from lymphocytes by treating the cells with anti-CD4 antibody and depleted by a fluorescent activated cell sorter, there was a lowering of PWM-induced activity [53].…”

“…Indeed, when T-cells were removed from lymphocytes by treating the cells with anti-CD4 antibody and depleted by a fluorescent activated cell sorter, there was a lowering of PWM-induced activity [53]. Other studies have demonstrated that the number of T-cells presented in the culture stimulated with PWM increased the number of intracytoplasmic immunoglobulins in murine and human B-lymphocytes [54,55].…”

Tyrosine Kinase Inhibitors Target B Lymphocytes

“…Both ceritinib and entrectinib inhibited mature B cell populations in multiple experiments (summarized in Figure 1 ), with ceritinib showing lower EC50 concentrations and more consistent numbers ( Figure 1 ). We then focused on live cell populations of these CD19-positive B cells and analyzed the presence of mature BLIMP1/IRF4 double-positive lymphocytes, i.e., plasma cells [ 26 , 33 ] ( Figure 2 ). We found that the majority of live CD19-positive lymphocytes in our experiments with tyrosine kinase inhibitors were BLIMP1/IRF4 double-positive (62 ± 7.7%, Figure 2 ).…”