The generation of the B cell response upon vaccination is characterized by the induction of different functional and phenotypic subpopulations and is strongly dependent on the vaccine formulation, including the adjuvant used. Here, we have profiled the different B cell subsets elicited upon vaccination, using machine learning methods for interpreting high‐dimensional flow cytometry data sets. The B cell response elicited by an adjuvanted vaccine formulation, compared to the antigen alone, was characterized using two automated methods based on clustering (FlowSOM) and dimensional reduction (t‐SNE) approaches. The clustering method identified, based on multiple marker expression, different B cell populations, including plasmablasts, plasma cells, germinal center B cells and their subsets, while this profiling was more difficult with t‐SNE analysis. When undefined phenotypes were detected, their characterization could be improved by integrating the t‐SNE spatial visualization of cells with the FlowSOM clusters. The frequency of some cellular subsets, in particular plasma cells, was significantly higher in lymph nodes of mice primed with the adjuvanted formulation compared to antigen alone. Thanks to this automatic data analysis it was possible to identify, in an unbiased way, different B cell populations and also intermediate stages of cell differentiation elicited by immunization, thus providing a signature of B cell recall response that can be hardly obtained with the classical bidimensional gating analysis. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
Analysis of multifunctional CD4+ T cells is fundamental for characterizing the immune responses to vaccination or infection. Major histocompatibility complex (MHC)/peptide tetramers represent a powerful technology for the detection of antigen-specific T cells by specific binding to their T-cell receptor, and their combination with functional assays is fundamental for characterizing the antigen-specific immune response. Here we optimized a protocol for the detection of multiple intracellular cytokines within epitope-specific CD4+ T cells identified by the MHC class II tetramer technology. The optimal procedure for assessing the functional activity of tetramer-binding CD4+ T cells was based on the simultaneous intracellular staining with both MHC tetramers and cytokine-specific antibodies upon in vitro restimulation of cells with the vaccine antigen. The protocol was selected among procedures that differently combine the steps of cellular restimulation and tetramer staining with intracellular cytokine labeling. This method can be applied to better understand the complex functional profile of CD4+ T-cell responses upon vaccination or infection.
Proteasomes are responsible for intracellular proteolysis and play an important role in cellular protein homeostasis. Cells of the immune system assemble a specialized form of proteasomes, known as immunoproteasomes, in which the constitutive catalytic sites are replaced for cytokine-inducible homologues. While immunoproteasomes may fulfill all standard proteasome’ functions, they seem specially adapted for a role in MHC class I antigen processing and CD8+ T-cell activation. In this way, they may contribute to CD8+ T-cell-mediated control of intracellular infections, but also to the immunopathogenesis of autoimmune diseases. Starting at the discovery of its catalytic subunits in the genome, here, we review the observations shaping our current understanding of immunoproteasome function, and the consequential novel opportunities for immune intervention.
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