Hepatocellular carcinoma (HCC) can have viral or non-viral causes
. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but ...biomarker-based stratification of patients for optimal response to therapy is an unmet need
. Here we report the progressive accumulation of exhausted, unconventionally activated CD8
PD1
T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8
PD1
T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8
PD1
CXCR6
, TOX
, and TNF
T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8
T cells or TNF neutralization, suggesting that CD8
T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8
PD1
T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.
Mass cytometry is developing as a means of multiparametric single-cell analysis. In this study, we present an approach to barcoding separate live human PBMC samples for combined preparation and ...acquisition on a cytometry by time of flight instrument. Using six different anti-CD45 Ab conjugates labeled with Pd104, Pd106, Pd108, Pd110, In113, and In115, respectively, we barcoded up to 20 samples with unique combinations of exactly three different CD45 Ab tags. Cell events carrying more than or less than three different tags were excluded from analyses during Boolean data deconvolution, allowing for precise sample assignment and the electronic removal of cell aggregates. Data from barcoded samples matched data from corresponding individually stained and acquired samples, at cell event recoveries similar to individual sample analyses. The approach greatly reduced technical noise and minimizes unwanted cell doublet events in mass cytometry data, and it reduces wet work and Ab consumption. It also eliminates sample-to-sample carryover and the requirement of instrument cleaning between samples, thereby effectively reducing overall instrument runtime. Hence, CD45 barcoding facilitates accuracy of mass cytometric immunophenotyping studies, thus supporting biomarker discovery efforts, and it should be applicable to fluorescence flow cytometry as well.
Daratumumab, a human monoclonal antibody that targets CD38, depletes plasma cells and is approved for the treatment of multiple myeloma. Long-lived plasma cells are implicated in the pathogenesis of ...systemic lupus erythematosus because they secrete autoantibodies, but they are unresponsive to standard immunosuppression. We describe the use of daratumumab that induced substantial clinical responses in two patients with life-threatening lupus, with the clinical responses sustained by maintenance therapy with belimumab, an antibody to B-cell activating factor. Significant depletion of long-lived plasma cells, reduction of interferon type I activity, and down-regulation of T-cell transcripts associated with chronic inflammation were documented. (Supported by the Deutsche Forschungsgemeinschaft and others.).
Sample barcoding is a powerful method for harmonizing mass cytometry data. By assigning a unique combination of barcode labels to each cell sample, a set of individual samples can be pooled and ...further processed and acquired as a large, single sample. For assays that require uncompromised profiling of cell-surface markers on live cells, barcoding by metal-labeled antibodies targeting cell-surface epitopes is the barcoding approach of choice. Here we provide an optimized and validated protocol for cell-surface barcoding of ten PBMC samples with palladium-labeled β2-microglobulin (B2M) antibodies used in a 5-choose-2 barcoding scheme, for subsequent immune phenotyping by mass cytometry. We further provide details on the generation of palladium-labeled antibodies utilizing amine-reactive isothiocyanobenzyl-EDTA (ITCB-EDTA) that permits the implementation of antibody-based barcoding not interfering with lanthanide channels typically used for analyte detection in mass cytometry assays.
COVID-19 can cause severe neurological symptoms, but the underlying pathophysiological mechanisms are unclear. Here, we interrogated the brain stems and olfactory bulbs in postmortem patients who had ...COVID-19 using imaging mass cytometry to understand the local immune response at a spatially resolved, high-dimensional, single-cell level and compared their immune map to non-COVID respiratory failure, multiple sclerosis, and control patients. We observed substantial immune activation in the central nervous system with pronounced neuropathology (astrocytosis, axonal damage, and blood-brain-barrier leakage) and detected viral antigen in ACE2-receptor-positive cells enriched in the vascular compartment. Microglial nodules and the perivascular compartment represented COVID-19-specific, microanatomic-immune niches with context-specific cellular interactions enriched for activated CD8+ T cells. Altered brain T-cell-microglial interactions were linked to clinical measures of systemic inflammation and disturbed hemostasis. This study identifies profound neuroinflammation with activation of innate and adaptive immune cells as correlates of COVID-19 neuropathology, with implications for potential therapeutic strategies.
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•Performed detailed molecular and spatial analysis of the COVID-19 brain immune response•Pathognomonic microglial nodules and T cell infiltration are present in COVID-19 brains•Altered microglia-T-cell interactions correlate with systemic measures of inflammation•Vascular leakage is linked with immune activation, ACE2 expression, and viral antigen
COVID-19 can cause severe neurological symptoms. By deep spatial analysis of postmortem brain tissue, Schwabenland et al. identify accumulation of distinct microglial and T cell subsets in microglial nodules and the perivasculature. They observe neuroinflammation with axonal damage, virus-associated perivascular inflammation, and compromised blood-brain barrier. This profound neuroinflammation highlights the need for better strategies against this COVID-19 CNS manifestation.
Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective ...immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.
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•SARS-CoV-2 infection induces profound alterations of the myeloid compartment•Mild COVID-19 is marked by inflammatory HLA-DRhiCD11chi CD14+ monocytes•Dysfunctional HLA-DRloCD163hi and HLA-DRloS100Ahi CD14+ monocytes in severe COVID-19•Emergency myelopoiesis with immature and dysfunctional neutrophils in severe COVID-19
Analysis of patients with mild and severe COVID-19 reveals the presence of dysfunctional neutrophils in the latter that is linked to emergency myelopoiesis.
Anti-CD20 therapy using rituximab directly targeting B cells has been approved for treatment of non-Hodgkin lymphoma, rheumatoid arthritis and anti-neutrophil cytoplasmic antibody-associated ...vasculitides and has led to reappreciation of B-lineage cells for anti-rheumatic treatment strategies. Moreover, blocking B-cell activating factor with belimumab, a drug that is licensed for treatment of active, seropositive systemic lupus erythematosus (SLE), represents an alternative, indirect anti-B-cell approach interfering with proper B-cell development. While these approaches apparently have no substantial impact on antibody-secreting plasma cells, challenges to improve the treatment of difficult-to-treat patients with SLE remain. In this context, anti-CD19 antibodies have the promise to directly target autoantibody-secreting plasmablasts and plasma cells as well as early B-cell differentiation stages not covered by anti-CD20 therapy. Currently known distinct expression profiles of CD19 by human plasma cell subsets, experiences with anti-CD19 therapies in malignant conditions as well as the rationale of targeting autoreactive plasma cells in patients with SLE are discussed in this review.
Microglia, the specialized innate immune cells of the CNS, play crucial roles in neural development and function. Different phenotypes and functions have been ascribed to rodent microglia, but little ...is known about human microglia (huMG) heterogeneity. Difficulties in procuring huMG and their susceptibility to cryopreservation damage have limited large-scale studies. Here we applied multiplexed mass cytometry for a comprehensive characterization of postmortem huMG (10
- 10
cells). We determined expression levels of 57 markers on huMG isolated from up to five different brain regions of nine donors. We identified the phenotypic signature of huMG, which was distinct from peripheral myeloid cells but was comparable to fresh huMG. We detected microglia regional heterogeneity using a hybrid workflow combining Cytobank and R/Bioconductor for multidimensional data analysis. Together, these methodologies allowed us to perform high-dimensional, large-scale immunophenotyping of huMG at the single-cell level, which facilitates their unambiguous profiling in health and disease.
Emerging data indicate that SARS-CoV-2-specific CD8
T cells targeting different viral proteins are detectable in up to 70% of convalescent individuals
. However, very little information is currently ...available about the abundance, phenotype, functional capacity and fate of pre-existing and induced SARS-CoV-2-specific CD8
T cell responses during the natural course of SARS-CoV-2 infection. Here, we define a set of optimal and dominant SARS-CoV-2-specific CD8
T cell epitopes. We also perform a high-resolution ex vivo analysis of pre-existing and induced SARS-CoV-2-specific CD8
T cells, applying peptide-loaded major histocompatibility complex class I (pMHCI) tetramer technology. We observe rapid induction, prolonged contraction and emergence of heterogeneous and functionally competent cross-reactive and induced memory CD8
T cell responses in cross-sectionally analyzed individuals with mild disease following SARS-CoV-2 infection and three individuals longitudinally assessed for their T cells pre- and post-SARS-CoV-2 infection. SARS-CoV-2-specific memory CD8
T cells exhibited functional characteristics comparable to influenza-specific CD8
T cells and were detectable in SARS-CoV-2 convalescent individuals who were seronegative for anti-SARS-CoV-2 antibodies targeting spike (S) and nucleoprotein (N). These results define cross-reactive and induced SARS-CoV-2-specific CD8
T cell responses as potentially important determinants of immune protection in mild SARS-CoV-2 infection.
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