The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional ...assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers.
Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathology, and it remains unclear whether T cells contribute to disease pathology. Here, we combined single-cell ...transcriptomics and single-cell proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune-complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Increased generation of C3a in severe COVID-19 induced activated CD16+ cytotoxic T cells. Proportions of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a were associated with fatal outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.
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•Severe COVID-19 is marked by activated, highly cytotoxic CD16+ T cells•Immune-complex-mediated degranulation of CD16+ T cells causes endothelial cell injury•C3a-rich environment in severe COVID-19 promotes differentiation of CD16+ T cells•Activated CD16+ T cells and complement proteins are associated with fatal outcome
Generation of the C3a complement protein fragment by SARS-CoV-2 infection drives differentiation of a CD16-expressing T cell population that associates with severe COVID-19 disease outcomes.
Mass cytometry permits the high dimensional analysis of cellular systems at single-cell resolution with high throughput in various areas of biomedical research. Here, we provide a state-of-the-art ...protocol for the analysis of human peripheral blood mononuclear cells (PBMC) by mass cytometry. We focus on the implementation of measures promoting the harmonization of large and complex studies to aid robustness and reproducibility of immune phenotyping data.
Given its uniformly high expression on plasma cells, CD38 has been considered as a therapeutic target in patients with systemic lupus erythematosus (SLE). Herein, we investigate the distribution of ...CD38 expression by peripheral blood leukocyte lineages to evaluate the potential therapeutic effect of CD38-targeting antibodies on these immune cell subsets and to delineate the use of CD38 as a biomarker in SLE. We analyzed the expression of CD38 on peripheral blood leukocyte subsets by flow and mass cytometry in two different cohorts, comprising a total of 56 SLE patients. The CD38 expression levels were subsequently correlated across immune cell lineages and subsets, and with clinical and serologic disease parameters of SLE. Compared to healthy controls (HC), CD38 expression levels in SLE were significantly increased on circulating plasmacytoid dendritic cells, CD14
CD16
monocytes, CD56
CD16
natural killer cells, marginal zone-like IgD
CD27
B cells, and on CD4
and CD8
memory T cells. Correlation analyses revealed coordinated CD38 expression between individual innate and memory T cell subsets in SLE but not HC. However, CD38 expression levels were heterogeneous across patients, and no correlation was found between CD38 expression on immune cell subsets and the disease activity index SLEDAI-2K or established serologic and immunological markers of disease activity. In conclusion, we identified widespread changes in CD38 expression on SLE immune cells that highly correlated over different leukocyte subsets within individual patients, but was heterogenous within the population of SLE patients, regardless of disease severity or clinical manifestations. As anti-CD38 treatment is being investigated in SLE, our results may have important implications for the personalized targeting of pathogenic leukocytes by anti-CD38 monoclonal antibodies.
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from lymphoblasts of T-cell origin. While TALL accounts for only 15% of childhood and 25% of adult ALL, 30% of patients ...relapse with a poor outcome. Targeted therapy of resistant and high-risk pediatric T-ALL is therefore urgently needed, together with precision medicine tools allowing the testing of efficacy in patient samples. Furthermore, leukemic cell heterogeneity requires drug response assessment at the single-cell level. Here we used single-cell mass cytometry to study signal transduction pathways such as JAK-STAT, PI3K-AKT-mTOR and MEK-ERK in 16 diagnostic and five relapsed T-ALL primary samples, and investigated the in vitro response of cells to Interleukin-7 (IL-7) and the inhibitor BEZ-235. T-ALL cells showed upregulated activity of the PI3K-AKT-mTOR and MEK-ERK pathways and increased expression of proliferation and translation markers. We found that perturbation induced by the ex vivo administration of either IL-7 or BEZ-235 reveals a high degree of exclusivity with respect to the phospho-protein responsiveness to these agents. Notably, these response signatures were maintained from diagnosis to relapse in individual patients. In conclusion, we demonstrated the power of mass cytometry single-cell profiling of signal transduction pathways in T-ALL. Taking advantage of this advanced approach, we were able to identify distinct clusters with different responsiveness to IL-7 and BEZ-235 that can persist at relapse. Collectively our observations can contribute to a better understanding of the complex signaling network governing T-ALL behavior and its correlation with influence on the response to therapy.
Immune-mediated inflammatory diseases (IMIDs) are typically characterised by relapsing and remitting flares of inflammation. However, the unpredictability of disease flares impedes their study. ...Addressing this critical knowledge gap, we use the experimental medicine approach of immunomodulatory drug withdrawal in rheumatoid arthritis (RA) remission to synchronise flare processes allowing detailed characterisation. Exploratory mass cytometry analyses reveal three circulating cellular subsets heralding the onset of arthritis flare - CD45RO
PD1
CD4
and CD8
T cells, and CD27
CD86
CD21
B cells - further characterised by single-cell sequencing. Distinct lymphocyte subsets including cytotoxic and exhausted CD4
memory T cells, memory CD8
CXCR5
T cells, and IGHA1+ plasma cells are primed for activation in flare patients. Regulatory memory CD4
T cells (Treg cells) increase at flare onset, but with dysfunctional regulatory marker expression compared to drug-free remission. Significant clonal expansion is observed in T cells, but not B cells, after drug cessation; this is widespread throughout memory CD8
T cell subsets but limited to the granzyme-expressing cytotoxic subset within CD4
memory T cells. Based on our observations, we suggest a model of immune dysregulation for understanding RA flare, with potential for further translational research towards novel avenues for its treatment and prevention.
Providing humoral immunity, antibody-secreting plasma cells and their immediate precursors, the plasmablasts, are generated in systemic and mucosal immune reactions. Despite their key role in ...maintaining immunity and immunopathology, little is known about their homeostasis. Here we show that plasmablasts and plasma cells are always detectable in human blood at low frequency in any unimmunized donor. In this steady state, 80% of plasmablasts and plasma cells express immunoglobulin A (IgA). Expression of a functional mucosal chemokine receptor, C-C motif receptor 10 (CCR10) and the adhesion molecule β7 integrin suggests that these cells come from mucosal immune reactions and can return to mucosal tissue. These blood-borne, CCR10+ plasmablasts also are attracted by CXCL12. Approximately 40% of plasma cells in human bone marrow are IgA+, nonmigratory, and express β7 integrin and CCR10, suggesting a substantial contribution of mucosal plasma cells to bone marrow resident, long-lived plasma cells. Six to 8 days after parenteral tetanus/diphtheria vaccination, intracellular IgG+ cells appear in blood, both CD62L+, β7 integrin−, dividing, vaccine-specific, migratory plasmablasts and nondividing, nonmigratory, CD62L− plasma cells of different specificities. Systemic vaccination does not impact on peripheral IgA+ plasmablast numbers, indicating that mucosal and systemic humoral immune responses are regulated independent of each other.
Aberrant production of autoantibodies by inappropriately self-reactive plasma cells is an inherent characteristic of autoimmune diseases. Several therapeutic strategies aim to deplete the plasma cell ...pool, or to prevent maturation of B cells into plasma cells. However, accepted views of B-cell biology are changing; recent findings show that long-lived plasma cells refractory to immunosuppressants and B-cell depletion therapies contribute to the maintenance of humoral memory and, in autoimmunity, to autoreactive memory. As a consequence of their longevity and persistence, long-lived plasma cells can support chronic inflammatory processes in autoimmune diseases by continuously secreting pathogenic antibodies, and they can contribute to flares of symptoms. As long-lived plasma cells are not sufficiently eliminated by current therapies, these findings are extremely relevant to the development of novel concepts for the treatment of autoimmune diseases. Thus, long-lived plasma cells appear to be a promising new therapeutic target.
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