The third dimension of ELISPOTs: Quantifying antibody secretion from individual plasma cells

Bromage, Erin; Stephens, Rebecca; Hassoun, Lama

doi:10.1016/j.jim.2009.05.005

Cited by 28 publications

(22 citation statements)

References 17 publications

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“…However, the ELISPOT assay does not measure the frequency of cells that actually interact with the antigen (which can for instance elicit an immune synapse for T cells or directly bind to antigen for B cells) but measures biological events such as cytokine release [28] or production of immunoglobulin after differentiation in vitro [29], [30], events that result from the interaction of the cells with the antigen. In this context, the estimated frequency is restricted to the cells that are able to be selectively stimulated by the antigen depending on read-out and thus may lead to an underestimation of the actual frequency of committed cells.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Antigen-Specific B Cells Using Nominal Antigen-Coated Flow-Beads

et al. 2013

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In order to characterize the reactivity of B cells against nominal antigens, a method based on the coupling of antigens onto the surface of fluorescent core polystyrene beads was developed. We first demonstrate that murine B cells with a human MOG-specific BCR are able to interact with MOG-coated beads and do not recognize beads coated with human albumin or pp65. B cells purified from human healthy volunteer blood or immunized individuals were tested for their ability to interact with various nominal antigens, including viral, vaccine, self and alloantigens, chosen for their usefulness in studying a variety of pathological processes. A substantial amount of B cells binding self-antigen MOG-coated beads can be detected in normal blood. Furthermore, greater frequencies of B cell against anti-Tetanic Toxin or anti-EBNA1 were observed in primed individuals. This method can reveal increased frequencies of anti-HLA committed B cells in patients with circulating anti-HLA antibodies compared to unsensitized patients and normal individuals. Of interest, those specific CD19 cells were preferentially identified within CD27−IgD+ (i-e naïve) subset. These observations suggest that a broad range of medical situations could benefit from a tool that allows the detection, the quantification and the characterization of antigen-specific blood B cells.

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Section: Discussionmentioning

confidence: 99%

Characterization of Antigen-Specific B Cells Using Nominal Antigen-Coated Flow-Beads

et al. 2013

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“…The size of spots in the ELISpot assay is thought to reflect the amount of antibody secreted by each cell. 9 Surprisingly given the differences in absolute ASC numbers and antibody levels outlined above, the ASCs and memory ASCs generated by the HD-unadjuvanted group tended to have larger spot sizes than the LD-adjuvanted groups after 12 weeks pb. On the other hand, at the earlier timepoints (up to 6 weeks pb), the LD-adjuvanted groups tended to have larger spot sizes but significant differences were not observed.…”

Section: Discussionmentioning

confidence: 94%

“…9 Although HD-unadjuvanted vaccine tended to generate fewer plasma cells and memory ASCs, this group tended to have larger spot sizes at later time-points, particularly in the BM (Figs. 2D, 3B, D; Fig.…”

Section: Serum Antibody Levels Are Maintained Long-term Following Ld-mentioning

confidence: 96%

Low hemagglutinin antigen dose influenza vaccines adjuvanted with AS03 alter the long-term immune responses in BALB/c mice

Yam

Brewer

Bleau

et al. 2016

Human Vaccines & Immunotherapeutics

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We investigated the long-term immune profiles of dose-sparing, AS03-adjuvanted vaccines compared to a traditional high-dose, unadjuvated influenza vaccine formulation. BALB/c mice received 2 IM injections of influenza A/Uruguay/716/2007 (H3N2) split vaccine antigen: high-dose (HD) (3 mg hemagglutinin (HA)/ dose) or low-dose (LD) formulations (0.03 mg or 0.003 mg HA) with AS03 and were followed to 34 weeks post-boost (pb). We examined serologic responses, spleen and bone marrow (BM) HA-specific antibodysecreting cells (ASCs) by ELISpot, influenza-specific cytokine/chemokine production in re-stimulated splenocytes by multiplex ELISA, and antigen-specific CD4C T cells that express cytokines (IL-2, IFNg, TNFa and IL-5) by flow cytometry. All formulations elicited robust serum antibody titers that persisted for at least 34 weeks. The number of antigen-specific ASCs in the spleen and BM were higher in the 2 LD CAS03 groups, but despite having fewer ASCs, the average spot size in the HD-unadjuvanted group was larger at later time-points, suggesting greater antibody production per cell. Striking differences in the long-term profiles induced by the different vaccine formulations may contribute to these different ASC profiles. The HD-unadjuvanted vaccine elicited strong Th2 cytokines during the first 6 weeks pb but LDCAS03 groups generated broader, more durable responses at later timepoints. Finally, the 0.03 mg HACAS03 group generated the greatest number of antigen-specific CD4C T cells and the highest percentage of polyfunctional cells that expressed 2 or more cytokines. Although all of the tested vaccines induced durable antibody responses, we show that different vaccine formulations (dose-sparing, adjuvant) generate distinct long-term immune profiles. Furthermore, our data suggest that the different profiles may be generated through unique mechanisms.

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“…The Krebs cycle and the consequent oxidative phosphorylation are more effective than aerobic glycolysis in ATP production. Every 12 h, a blood-derived plasma cell can produce up to 1.7 ng of antibodies (34), whereas it has been calculated that for each peptide bond formation 7.52 molecules of ATP are required (35). It is of note that at least in T cells, the Krebs cycle is not totally inhibited by LW6 (22), possibly due to the pyruvate-malate cycle and increased GLS1 expression (11,23).…”

Section: Discussionmentioning

confidence: 99%

Comparison of the effect of the aerobic glycolysis inhibitor dichloroacetate and of the Krebs cycle inhibitor LW6 on cellular and humoral alloimmunity

et al. 2017

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Abstract. Cell metabolism is altered during T-cell and B-cell activation and differentiation. Clarifying the exact metabolic shifts may lead to the development of new immunosuppressive medications. In this study, the effect of the aerobic glycolysis inhibitor dichloroacetate (DCA) and of the Krebs cycle enzyme malate dehydrogenase 2 (MDH2) inhibitor LW6 on T-cell alloimmune clonal expansion and on alloantibody production, was evaluated. T-cell clonal expansion was assessed in two-way mixed lymphocyte reaction (MLR). Humoral alloimmunity was evaluated by the alloantibody production in one-way MLR. For this purpose, an antibody-mediated complement-dependent cytotoxicity assay was developed in which the supernatants from one-way MLRs were used against resting peripheral blood mononuclear cells (PBMCs) derived from the same individual that contributed the stimulator cells for the respective MLR. DCA had a minimum effect on alloimmune T-cell clonal expansion, whereas it increased humoral immunity significantly. LW6 decreased both alloimmune T-cell proliferation and alloantibody production. The results indicate that MDH2 may be a perfect target for the development of new immunosuppressive medications, especially when inhibition of both cellular and humoral alloimmunity is desirable.

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The third dimension of ELISPOTs: Quantifying antibody secretion from individual plasma cells

Cited by 28 publications

References 17 publications

Characterization of Antigen-Specific B Cells Using Nominal Antigen-Coated Flow-Beads

Characterization of Antigen-Specific B Cells Using Nominal Antigen-Coated Flow-Beads

Low hemagglutinin antigen dose influenza vaccines adjuvanted with AS03 alter the long-term immune responses in BALB/c mice

Comparison of the effect of the aerobic glycolysis inhibitor dichloroacetate and of the Krebs cycle inhibitor LW6 on cellular and humoral alloimmunity

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