Extensive flow cytometric immunophenotyping of human PBMC incorporating detection of chemokine receptors, cytokines and tetramers

Characterization of immune cells is essential to advance our understanding of immunology and flow cytometry is an important tool in this context. Addressing both cellular phenotype and antigen-specific functional responses of the same cells is valuable to achieve a more integrated understanding of immune cell behavior and maximizes information obtained from precious samples. Until recently, panel size was limiting, resulting in panels generally focused on either deep immunophenotyping or functional readouts. Ongoing developments in the field of (spectral) flow cytometry have made panels of 30(+) markers more accessible, opening up possibilities for advanced integrated analyses. Here, we optimized immune phenotyping by co-detection of markers covering chemokine receptors, cytokines and specific T cell/peptide tetramer interaction using a 32-color panel. Such panels enable integrated analysis of cellular phenotypes and markers assessing the quality of immune responses and will contribute to our understanding of the immune system.Immunogenetics and cellular immunology of bacterial infectious disease

Detailed characterization of human Mycobacterium tuberculosis specific HLA-E restricted CD8(+) T cells

Immunogenetics and cellular immunology of bacterial infectious disease

Differences in IgG responses against infection phase related Mycobacterium tuberculosis (Mtb) specific antigens in individuals exposed or not to Mtb correlate with control of TB infection and progression

Immunogenetics and cellular immunology of bacterial infectious disease

Circulating Mycobacterium tuberculosis DosR latency antigen-specific, polyfunctional, regulatory IL10(+) Th17 CD4 T-cells differentiate latent from active tuberculosis

Immunogenetics and cellular immunology of bacterial infectious disease

BCG vaccination-induced acquired control of mycobacterial growth differs from growth control preexisting to BCG vaccination

Bacillus Calmette-Guèrin - vaccination induces not only protection in infants and young children against severe forms of tuberculosis, but also against nontuberculosis related all-cause mortality. To delineate different factors influencing mycobacterial growth control, here we first investigate the effects of BCG-vaccination in healthy Dutch adults. About a quarter of individuals already control BCG-growth prior to vaccination, whereas a quarter of the vaccinees acquires the capacity to control BCG upon vaccination. This leaves half of the population incapable to control BCG-growth. Single cell RNA sequencing identifies multiple processes associated with mycobacterial growth control. These data suggest (i) that already controllers employ different mechanisms to control BCG-growth than acquired controllers, and (ii) that half of the individuals fail to develop measurable growth control irrespective of BCG-vaccination. These results shed important new light on the variable immune responses to mycobacteria in humans and may impact on improved vaccination against tuberculosis and other diseases.Immunogenetics and cellular immunology of bacterial infectious disease

CD39 is involved in mediating suppression by Mycobacterium bovis BCG-activated human CD8(+) CD39(+) regulatory T cells

Regulatory T (Treg) cells can balance normal tissue homeostasis by limiting inflammatory tissue damage, e.g. during pathogen infection, but on the other hand can also limit protective immunity induced during natural infection or following vaccination. Because most studies have focused on the role of CD4(+) Treg cells, relatively little is known about the phenotype and function of CD8(+) Treg cells, particularly in infectious diseases. Here, we describe for the first time the expression of CD39 (E-NTPDase1) on Mycobacterium-activated human CD8(+) T cells. These CD8(+) CD39(+) T cells significantly co-expressed the Treg markers CD25, Foxp3, lymphocyte activation gene-3 (LAG-3), and CC chemokine ligand 4 (CCL4), and suppressed the proliferative response of antigen-specific CD4(+) T helper-1 (Th1) cells. Pharmacological or antibody mediated blocking of CD39 function resulted in partial reversal of suppression. These data identify CD39 as a novel marker of human regulatory CD8(+) T cells and indicate that CD39 is functionally involved in suppression by CD8(+) Treg cells.Immunogenetics and cellular immunology of bacterial infectious disease

A multistage-polyepitope vaccine protects against Mycobacterium tuberculosis infection in HLA-DR3 transgenic mice

Immunogenetics and cellular immunology of bacterial infectious disease

BCG-vaccination induces divergent pro-inflammatory or regulatory T-cell responses in adults: Variability in adult BCG-induced T-cell responses

Mycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG), the only currently available vaccine against tuberculosis, induces variable protection in adults. Immune correlates of protection are lacking, and analyses on cytokine-producing T-cell subsets in protected vs. non-protected cohorts have yielded inconsistent results. We studied the primary T-cell response, both pro-inflammatory and regulatory T-cell responses, induced by BCG-vaccination in adults. Twelve healthy adult volunteers, who were Tuberculin skin test (TST) -negative, QuantiFERON test (QFT) -negative, and BCG-naïve, were vaccinated with BCG and followed-up prospectively. BCG-vaccination induced an unexpectedly dichotomous immune response in this small, BCG-naive young adult cohort: BCG-vaccination induced either IFNγ+IL2+TNFa+ polyfunctional CD4+ T-cells concurrent with CD4+IL17A+ and CD8+IFNγ+ T-cells, or, in contrast, virtually absent cytokine responses with induction of CD8+ regulatory T-cells. Significant induction of polyfunctional CD4+IFNγ+IL2+TNFa+ T-cells and IFNγ production by PBMCs was confined to individuals with strong immunization-induced local skin inflammation and increased serum C-reactive protein (CRP). Conversely, in individuals with mild inflammation, regulatory-like CD8+ T-cells were uniquely induced. Thus, BCG-vaccination either induced a broad pro-inflammatory T-cell response with local inflammatory reactogenicity, or in contrast a predominant CD8+ regulatory T-cell response with mild local inflammation, poor cytokine induction and absent polyfunctional CD4+ T-cells. Further detailed fine mapping of the heterogeneous host response to BCG-vaccination using classical and non-classical immune markers will enhance our understanding of the mechanisms and determinants that underlie the induction of apparently opposite immune responses, and how these impact on BCGs ability to induce protective immunity to TB.Immunogenetics and cellular immunology of bacterial infectious disease

Human CD8(+) T-cells recognizing peptides from Mycobacterium tuberculosis presented by HLA-E have an unorthodox Th2-like, multifunctional, Mtb-inhibitory phenotype and represent a novel human T-cell subset

Immunogenetics and cellular immunology of bacterial infectious disease