scholarly journals Affinity Tag Coating Enables Reliable Detection of Antigen-Specific B Cells in Immunospot Assays

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1843
Author(s):  
Sebastian Köppert ◽  
Carla Wolf ◽  
Noémi Becza ◽  
Giuseppe A. Sautto ◽  
Fridolin Franke ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Immune Monitoring ◽  
Affinity Tag ◽  
Cell Compartment ◽  
Assay Sensitivity ◽  
Memory B Cell ◽  
Affinity Capture ◽  
Coating Efficiency ◽  
Assay Performance

Assessment of humoral immunity to SARS-CoV-2 and other infectious agents is typically restricted to detecting antigen-specific antibodies in the serum. Rarely does immune monitoring entail assessment of the memory B-cell compartment itself, although it is these cells that engage in secondary antibody responses capable of mediating immune protection when pre-existing antibodies fail to prevent re-infection. There are few techniques that are capable of detecting rare antigen-specific B cells while also providing information regarding their relative abundance, class/subclass usage and functional affinity. In theory, the ELISPOT/FluoroSpot (collectively ImmunoSpot) assay platform is ideally suited for antigen-specific B-cell assessments since it provides this information at single-cell resolution for individual antibody-secreting cells (ASC). Here, we tested the hypothesis that antigen-coating efficiency could be universally improved across a diverse set of viral antigens if the standard direct (non-specific, low affinity) antigen absorption to the membrane was substituted by high-affinity capture. Specifically, we report an enhancement in assay sensitivity and a reduction in required protein concentrations through the capture of recombinant proteins via their encoded hexahistidine (6XHis) affinity tag. Affinity tag antigen coating enabled detection of SARS-CoV-2 Spike receptor binding domain (RBD)-reactive ASC, and also significantly improved assay performance using additional control antigens. Collectively, establishment of a universal antigen-coating approach streamlines characterization of the memory B-cell compartment after SARS-CoV-2 infection or COVID-19 vaccinations, and facilitates high-throughput immune-monitoring efforts of large donor cohorts in general.

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

2021 ◽  
Author(s):  
Carla Wolf ◽  
Sebastian Koppert ◽  
Noemi Becza ◽  
Giuseppe A Sautto ◽  
Fridolin Franke ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Immune Monitoring ◽  
Affinity Tag ◽  
Cell Compartment ◽  
Assay Sensitivity ◽  
Memory B Cell ◽  
Affinity Capture ◽  
Coating Efficiency ◽  
Assay Performance

Assessment of humoral immunity to SARS-CoV-2 and other infectious agents is typically restricted to detecting antigen-specific antibody in the serum. Rarely does immune monitoring entail assessment of the memory B cell compartment itself, although it is these cells that engage in secondary antibody responses capable of mediating immune protection when pre-existing antibodies fail to prevent re-infection. There are few techniques that are capable of detecting rare antigen-specific B cells while also providing information regarding their precursory frequency, class/subclass usage and functional affinity. In theory, the ELISPOT/FluoroSpot (collectively ImmunoSpot) assay platform is ideally-suited for antigen-specific B cell assessments since it provides this information at single-cell resolution for individual antibody-secreting cells (ASC). Here, we tested the hypothesis that antigen coating efficiency could be universally improved across a diverse set of viral antigens if the standard direct (non-specific, low affinity) antigen absorption to the membrane was substituted by high affinity capture. Specifically, we report an enhancement in assay sensitivity and a reduction in required protein concentrations through the capture of recombinant proteins via their encoded hexahistidine (6XHis) affinity tag. Affinity tag antigen coating enabled detection of SARS-CoV-2 Spike receptor binding domain (RBD)-reactive ASC, and also significantly improved assay performance using additional control antigens. Collectively, establishment of a universal antigen coating approach streamlines characterization of the memory B cell compartment after SARS-CoV-2 infection or COVID-19 vaccinations, and facilitates high-throughput immune monitoring efforts of large donor cohorts in general.

scholarly journals Complexity of the human memory B-cell compartment is determined by the versatility of clonal diversification in germinal centers

2015 ◽  
Vol 112 (38) ◽  
pp. E5281-E5289 ◽  
Author(s):  
Bettina Budeus ◽  
Stefanie Schweigle de Reynoso ◽  
Martina Przekopowitz ◽  
Daniel Hoffmann ◽  
Marc Seifert ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Clone ◽  
Human Memory ◽  
Memory B Cells ◽  
Sequencing Analysis ◽  
Cell Compartment ◽  
Memory B Cell ◽  
Cell Clones ◽  
B Cell Subsets

Our knowledge about the clonal composition and intraclonal diversity of the human memory B-cell compartment and the relationship between memory B-cell subsets is still limited, although these are central issues for our understanding of adaptive immunity. We performed a deep sequencing analysis of rearranged immunoglobulin (Ig) heavy chain genes from biological replicates, covering more than 100,000 memory B lymphocytes from two healthy adults. We reveal a highly similar B-cell receptor repertoire among the four main human IgM+ and IgG+ memory B-cell subsets. Strikingly, in both donors, 45% of sequences could be assigned to expanded clones, demonstrating that the human memory B-cell compartment is characterized by many, often very large, B-cell clones. Twenty percent of the clones consisted of class switched and IgM+(IgD+) members, a feature that correlated significantly with clone size. Hence, we provide strong evidence that the vast majority of Ig mutated B cells—including IgM+IgD+CD27+ B cells—are post-germinal center (GC) memory B cells. Clone members showed high intraclonal sequence diversity and high intraclonal versatility in Ig class and IgG subclass composition, with particular patterns of memory B-cell clone generation in GC reactions. In conclusion, GC produce amazingly large, complex, and diverse memory B-cell clones, equipping the human immune system with a versatile and highly diverse compartment of IgM+(IgD+) and class-switched memory B cells.

scholarly journals Exclusion of Natural Autoantibody-Producing B Cells from IgG Memory B Cell Compartment during T Cell-Dependent Immune Responses

2009 ◽  
Vol 182 (12) ◽  
pp. 7634-7643 ◽  
Author(s):  
Agata Matejuk ◽  
Michael Beardall ◽  
Yang Xu ◽  
Qi Tian ◽  
Daniel Phillips ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Immune Responses ◽  
B Cell ◽  
Cell Compartment ◽  
Memory B Cell ◽  
Natural Autoantibody

scholarly journals Immature and Transitional B Cells Are Latency Reservoirs for a Gammaherpesvirus

2010 ◽  
Vol 84 (24) ◽  
pp. 13045-13052 ◽  
Author(s):  
Carrie B. Coleman ◽  
Michael S. Nealy ◽  
Scott A. Tibbetts
Keyword(s):  
B Cells ◽  
B Cell ◽  
Human Herpesvirus ◽  
Nuclear Antigen ◽  
Cell Compartment ◽  
Memory B Cell ◽  
Murine Gammaherpesvirus ◽  
Transitional B Cells ◽  
Immature B Cells ◽  
Gammaherpesvirus 68

ABSTRACT Gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8 [HHV-8]), Epstein-Barr virus (EBV), and murine gammaherpesvirus 68 (MHV68; also known as gammaherpesvirus 68 [γHV68] or murine herpesvirus 4 [MuHV-4]), establish lifelong latency in the resting memory B cell compartment. However, little is known about how this reservoir of infected mature B cells is maintained for the life of the host. In the context of a normal immune system, the mature B cell pool is naturally maintained by the renewable populations of developing B cells that arise from hematopoiesis. Thus, recurrent infection of these developing B cell populations could allow the virus continual access to the B cell lineage and, subsequent to differentiation, the memory B cell compartment. To begin to address this hypothesis, we examined whether MHV68 establishes latency in developing B cells during a normal course of infection. In work described here, we demonstrate the presence of viral genome in bone marrow pro-pre-B cells and immature B cells during early latency and immature B cells during long-term latency. Further, we show that transitional B cells in the spleen are latently infected and express the latency-associated nuclear antigen (LANA) throughout chronic infection. Because developing B cells normally exhibit a short life span and a high rate of turnover, these findings suggest a model in which gammaherpesviruses may gain access to the mature B cell compartment by recurrent seeding of developing B cells.

scholarly journals Enforced Expression of Bcl-2 Selectively Perturbs Negative Selection of Dual Reactive Antibodies

2001 ◽  
Vol 8 (3-4) ◽  
pp. 223-234 ◽  
Author(s):  
Evangelia Notidis ◽  
Shailaja Hande ◽  
Tim Manser
Keyword(s):  
B Cells ◽  
B Cell ◽  
Negative Selection ◽  
Somatic Hypermutation ◽  
Serum Antibody ◽  
Affinity Maturation ◽  
Peripheral Tolerance ◽  
Cell Compartment ◽  
Memory B Cell ◽  
Selection Of

We investigated the role of apoptosis in the development of B cell memory by analyzing the (p-azophenylarsonate) Ars response in a line of A strain mice in which expression of human Bcl-2 was enforced in the B cell compartment. Previous studies of the Ars immune response in these A. Bcl-2 mice, demonstrated that a large percentage of the antibodies expressed by the Ars induced memory B cell compartment had accumulated point mutations via somatic hypermutation that increased their affinity for both Ars and the autoantigen DNA (“dual reactive” antibodies). This was in sharp contrast to normal A strain mice which displayed no dual reactive B cells in their Ars induced memory B cell compartment. These data suggested that interference with apoptotic pathways regulated by Bcl-2 allows developing memory B cells that have acquired autoreactivity to bypass a peripheral tolerance checkpoint. Further studies of these mice, reported here, demonstrate that enforced expression of Bcl-2 does not alter serum antibody affinity maturation nor positive selection of B cells expressing somatically mutated antibody with an increased affinity for Ars. Moreover, the somatic hypermutation process was unaffected in A. Bcl-2 mice. Thus, enforced expression of Bcl-2 in A. Bcl-2 mice appears to selectively alter a negative selection process that operates during memory B cell differentiation.

scholarly journals A checkpoint for autoreactivity in human IgM+ memory B cell development

2006 ◽  
Vol 203 (2) ◽  
pp. 393-400 ◽  
Author(s):  
Makoto Tsuiji ◽  
Sergey Yurasov ◽  
Klara Velinzon ◽  
Saskia Thomas ◽  
Michel C. Nussenzweig ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Somatic Hypermutation ◽  
Cell Development ◽  
B Cell Development ◽  
Cell Selection ◽  
Cell Compartment ◽  
Memory B Cell ◽  
Healthy Humans ◽  
Low Levels

Autoantibodies are removed from the repertoire at two checkpoints during B cell development in the bone marrow and the periphery. Despite these checkpoints, up to 20% of the antibodies expressed by mature naive B cells in healthy humans show low levels of self-reactivity. To determine whether self-reactive antibodies are also part of the antigen-experienced memory B cell compartment, we analyzed recombinant antibodies cloned from single circulating human IgM+ memory B cells. Cells expressing antibodies specific for individual bacterial polysaccharides were expanded in the IgM+ memory compartment. In contrast, B cells expressing self-reactive and broadly bacterially reactive antibodies were removed from the repertoire in the transition from naive to IgM+ memory B cell. Selection against self-reactive antibodies was implemented before the onset of somatic hypermutation. We conclude that a third checkpoint selects against self-reactivity during IgM+ memory B cell development in humans.

scholarly journals Depletion of circulating IgM memory B cells predicts unfavourable outcome in COVID-19

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Marco Vincenzo Lenti ◽  
Nicola Aronico ◽  
Ivan Pellegrino ◽  
Emanuela Boveri ◽  
Paolo Giuffrida ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Incidence Rate ◽  
Cumulative Incidence ◽  
Memory B Cells ◽  
Cell Depletion ◽  
B Cell Depletion ◽  
Cell Compartment ◽  
Memory B Cell ◽  
Cumulative Incidence Rate

AbstractImpaired immune responses have been hypothesised to be a possible trigger of unfavourable outcomes in coronavirus disease 2019 (COVID-19). We aimed to characterise IgM memory B cells in patients with COVID-19 admitted to an internal medicine ward in Northern Italy. Overall, 66 COVID-19 patients (mean age 74 ± 16.6 years; 29 females) were enrolled. Three patients (4.5%; 1 female) had been splenectomised and were excluded from further analyses. Fifty-five patients (87.3%) had IgM memory B cell depletion, and 18 (28.6%) died during hospitalisation (cumulative incidence rate 9.26/100 person-week; 5.8–14.7 95% CI). All patients who died had IgM memory B cell depletion. A superimposed infection was found in 6 patients (9.5%), all of them having IgM memory B cell depletion (cumulative incidence rate 3.08/100 person-week; 1.3–6.8 95% CI). At bivariable analyses, older age, sex, number of comorbidities, and peripheral blood lymphocyte count < 1500/µl were not correlated with IgM memory B cell depletion. A discrete-to-marked reduction of the B-cell compartment was also noticed in autoptic spleen specimens of two COVID-19 patients. We conclude that IgM memory B cells are commonly depleted in COVID-19 patients and this correlates with increased mortality and superimposed infections.

High Level EBV Persistence in the IgM+ IgD+ CD27+ B Cell Subset in Patients with X-Linked Lymphoproliferative Disease Lacking Isotype-Switched Memory B Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2276-2276
Author(s):  
Sridhar Chaganti ◽  
Cindy Ma ◽  
Andrew Bell ◽  
Debbie Croom-Carter ◽  
Andrew Hislop ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Lymphoproliferative Disease ◽  
Small Population ◽  
Memory B Cells ◽  
Germinal Centre ◽  
Memory Cells ◽  
Cell Compartment ◽  
Memory B Cell ◽  
Virus Load

Abstract Epstein-Barr virus (EBV) infects >90% of population world wide and, in healthy virus carriers, establishes life long persistence in the immunoglobulin (Ig)Dneg, CD27+ (“class-switched”) memory B cell compartment normally produced by antigen stimulation and transit through germinal centres. Patients with the X-linked lymphoproliferative disease (XLP) cannot make such class-switched memory B cells due to an inherited mutation in the slam-associated protein (SAP) gene involved in the maturation of antibody responses. Interestingly, XLP patients are highly susceptible to severe primary EBV infection and develop a fulminant infectious mononucleosis (IM) which is often fatal and where the symptoms progress to resemble those of a different (non-familial) disease, EBV-associated haemophagocytosis syndrome (EBV-AHS), caused by virus entry into the NK or T cell system. Some XLP patients survive their primary infection, but the nature of EBV carriage in these individuals (lacking conventional memory B cells) remains unresolved. To investigate this further we obtained blood samples from 8 such XLP patients. EBV load in total peripheral blood mononuclear cells (PBMC), determined by quantitative PCR, occupied a broad range but on average was 2 to 3-fold fold higher than that of healthy controls. The virus was concentrated within the B cell (CD19+) compartment, as in healthy carriers, and not within T or NK cells, as typically seen in EBV-AHS. We then confirmed that these XLP patients indeed lacked conventional class-switched memory B cells but did carry a small population of IgM+, IgD+, CD27+ (“non-switched”) memory cells; however their circulating B cell pool was dominated by naïve (IgM+, IgD+, CD27neg) cells and by expanded numbers of immature “transitional” (CD10+ CD27neg) cells. In each of 4 cases studied by cell sorting, EBV was concentrated in this small subset of “non-switched” memory B cells. To see if the high virus load detected in these patients indicated true virus persistence as opposed to recent or recurrent infection, serial samples obtained over a 3 year period from two XLP patients were assayed. Virus load was stable and, in one case with the highest load, screening with markers of virus polymorphism detected the same resident strain over time. Our results in XLP patients make it clear that EBV can persist in the absence of a conventional class-switched memory B cell compartment. Instead, the virus is sequestered in a small population of “non-class-switched memory” cells with Ig gene mutations. The origin of such cells, which are also detectable in the blood of normal donors, is uncertain; however their existence in XLP patients suggests that such cells arise independently of germinal centre activity and hence that EBV may be able to colonise its host without exploiting germinal centre transit.

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