Mass Cytometry Reveals Global Immune Remodeling with Multi-lineage Hypersensitivity to Type I Interferon in Down Syndrome

Abstract

People with Down syndrome (DS; trisomy 21) display a different disease spectrum relative to the general population, including lower rates of solid malignancies and higher incidence of neurological and autoimmune conditions. However, the mechanisms driving this unique clinical profile await elucidation. We completed a deep mapping of the immune system in adults with DS using mass cytometry to evaluate 100 immune cell types, which revealed global immune dysregulation consistent with chronic inflammation, including key changes in the myeloid and lymphoid cell compartments. Furthermore, measurement of interferon-inducible phosphorylation events revealed widespread hypersensitivity to interferon-α in DS, with cell-type-specific variations in downstream intracellular signaling. Mechanistically, this could be explained by overexpression of the interferon receptors encoded on chromosome 21, as demonstrated by increased IFNAR1 surface expression in all immune lineages tested. These results point to interferon-driven immune dysregulation as a likely contributor to the developmental and clinical hallmarks of DS.

Trial registration: ClinicalTrials.gov NCT02864108.

Keywords: CyTOF; Down syndrome; JAK/STAT; autoimmunity; immune dysregulation; inflammation; interferon; mass cytometry; single cell; trisomy 21.

Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.

Figures

Figure 1.. Mass Cytometry Reveals Global Immune Dysregulation in Adults with T21

Intact single events enriched for non-granulocytes of hematopoietic origin (CD45+CD66−) were run through viSNE and then compared between individuals with T21 (n = 18) and D21 controls (n = 18). (A) Similarities between overall shape of individual viSNE plots were quantified through pairwise comparisons by Jenson-Shannon (JS) scores, followed by hierarchical clustering. (B) PhenoGraph was used to resolve 31 clusters within viSNE plots. (C) PhenoGraph clusters were uniformly colored according to increased (red) or decreased (blue) frequency among total events in T21 viSNE plots compared to those of D21 controls. Significantly different clusters are labeled with numbers. (D) Box and whisker plots displaying the data for PhenoGraph Clusters 8 (a subset of B cells) and 25 (a subset of T cells expressing high levels of granzyme B). (E) Overlay of immune cell subsets defined by surface marker expression on events represented in viSNE plots. (F) A topographical analysis, kernel density estimate (KDE), was applied to viSNE plots to visualize areas with more (red) or less (blue) event density in samples with T21. Color is overlaid on a viSNE plot showing concatenated events from all T21 samples. (G) Manual gating of areas within viSNE plots highlighted by KDE. An area of CD4+ T cells (black) depleted of CD45RA+CD27+ events (blue) were interpreted as a subset of naive CD4+ T cells whereas an area enriched for CD45RA—CD27+ events (red) were interpreted as a subset of CD27+ memory CD4+ T cells. In all cases, statistical significance was determined by a Student’s t test (*p ≤ 0.05 and **p ≤ 0.01). See also Figure S1. All box and whisker plots denote the median within a box extending from the 25th to 75th percentiles and error bars span minimum to maximum values within the datasets.

Figure 2.. Hematopoietic Stem Cells, Progenitors, and Fibrocytes Are Significantly Dysregulated in Individuals with T21

(A and B) Hematopoietic stem and progenitor cells (HSPCs) were detected by CD34 and CD45 staining in the peripheral blood as depicted in Figures S2A and S2D then compared between individuals with T21 (n = 18) and D21 controls (n = 18). (B) Representative dot plots denoting frequency among the parent gate. (C and D) Among HSPCs, CD38 staining was used to delineate hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) from oligopotent progenitors (OPPs) in individuals with T21 (n = 18) and D21 controls (n = 18). (D) Representative dot plots denoting frequency among the parent gate. (E and F) Fibrocytes were detected by expression of COL1 among circulating HSPCs in individuals with T21 (n = 8) and D21 controls (n = 9), as depicted in Figure S2A, S2D, and S2F. See Figure S2G for COL1 metal minus one control. (F) Representative dot plots denoting frequency among the parent gate. (G–M) Among fibrocytes, the expression levels of CD45RO (G), COL1 (H and I), and HLA-DR (J–M) were compared by geometric mean metal intensity (gMMI). In (I) and (M), representative histograms are shown with numbers denoting gMMIs among the parent gate indicated in titles. Green represents an individual with T21 and gray a D21 control. (N) On a flow cytometer, positive staining of pro-collagen resolved fibrocytes among all hematopoietic (CD45+) events from white blood cells of children with T21 (n = 26) versus D21 controls (n = 7), as depicted in Figure S4A. (O) Lineage tree depicts subsets of HSPCs within the context of hematopoiesis and bone-marrow-derived fibrocytes. Color denotes fold-change where red is increased and blue is decreased frequency of subsets among HSPCs in individuals with T21 compared to D21 controls. In all cases, statistical significance was determined by a Student’s t test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001). All box and whisker plots denote the median within a box extending from the 25th to 75th percentiles and error bars span minimum to maximum values within the indicated datasets.

Figure 3.. Monocyte and Dendritic Cell Homeostasis Are Disrupted in the Peripheral Blood of Individuals with T21

(A and B) Within mass cytometry data, monocyte subsets were delineated by expression of canonical markers as depicted in Figure S2 and then compared between individuals with T21 (n = 18) and D21 controls (n = 18). (B) Representative dot plots are shown with numbers denoting frequency among monocytes. (C and D) Flow cytometry was used to evaluate the frequency (C) and number (D) of classical and intermediate monocytes within the peripheral blood of individuals with T21 (n = 37 for frequency and n = 31 for relative cell number) and D21 controls (n = 45 for frequency and n = 41 for relative cell number), as depicted in Figure S4C. (E–K) Phenotypic characterization of myeloid subsets by mass cytometry. In (E) and (F), CD1c expression was used to resolve a subset of conventional dendritic cells (cDCs) among myeloid DCs (mDCs) in individuals with T21 (n = 18) and D21 controls (n = 18). In (G) and (I), the levels of CD16 or CD56 protein expression were compared between individuals with T21 (n = 18) and D21 controls (n = 18) by geometric mean metal intensity (gMMI). In (H) and (J), representative histograms are shown with numbers denoting gMMIs among the parent gate indicated in titles. Green represents an individual with T21 and gray a D21 control. Plasmacytoid DCs are denoted as pDCs. (K) Lineage tree depicts subsets of the myeloid lineage within the monocyte and mDC compartments of individuals with T21 compared to D21 controls. Color denotes fold change, where red is increased and blue is decreased frequency of subsets among monocyte or mDC compartments in T21 compared to D21 controls. In all cases, statistical significance was determined by a Student’s t test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001). All box and whisker plots denote the median within a box extending from the 25th to 75th percentiles and error bars span minimum to maximum values within the indicated datasets.

Figure 4.. NK and CD8+ T Cells in Adults with T21 Display Signs of Heightened Activation and Increased Cytotoxic Potential

(A–C) Mass cytometry was used to identify (A) bulk NK cells and (B and C) NK cell subsets, as described in Figures S2 and Table S5, within the peripheral blood of individuals with T21 (n = 18) and D21 controls (n = 18). (D) Flow cytometry was used to determine the frequency of CD56++CD16— and CD56+CD16+ NK cell subsets among single events within the peripheral blood of individuals with T21 (n = 38) and D21 controls (n = 51), as depicted in Figure S4C. (E) Within mass cytometry data, the level of protein expression indicated in heatmap titles was compared between individuals with T21 (n = 18) and D21 controls (n = 18) by geometric mean metal intensity (gMMI) for the indicated NK cell populations. Heatmaps display average gMMIs (left) or Arcsinh ratios (T21 gMMIs — D21 gMMIs, right) for the listed cell types. (F) A representative histogram, with numbers denoting gMMIs among the parent gate indicated in the title. Green represents an individual with T21 and gray a D21 control. (G) Mass cytometry was used to discriminate different subsets of T cells within the peripheral blood of individuals with T21 (n = 18) and D21 controls (n = 18) as described in Figure S2 and Table S5. Heatmaps indicate the log2 fold change (FC; T21 over D21) in frequency within all T cells for each of the indicated subsets. (H) Box and whisker plot showing the depletion of naive CD8+ T cells in individuals with T21. (I) Representative example of CD8+ T cell subset distribution in D21 versus T21 samples. (J) Mass cytometry was used as in (G) to delineate subsets of CD8+ T cells that were negative or positive for granzyme B (GZMB) and/or PD1. Heatmaps indicate the log2 fold change (FC, T21 over D21) in frequency within all T cells for each of the indicated subsets. (K) Box and whisker plots showing data for naive CD8+ T cells divided by GZMB and PD1 expression status. (L) Representative example of GZMB and PD1 expression among CD8+ T cells in D21 versus T21 samples. All statistical significance was determined by a Student’s t test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001). All box and whisker plots denote the median within a box extending from the 25th to 75th percentiles and error bars span minimum to maximum values within the indicated datasets.

Figure 5.. The B Cell Compartment of Adults with T21 Is Skewed toward More Differentiated Subsets Associated with Inflammatory and Autoimmune States

(A, B, and D–M) B cells were delineated among non-granulocytes by mass cytometry and manual gating as depicted in Figure S2 and then compared between individuals with T21 (n = 18) and D21 controls (n = 18). (B, E, F, H, and J) Representative dot plots of the indicated cell type labeled in green with numbers denoting frequency among the parent gate indicated in titles. BMEM, memory B cells; BNA, naive B cells; PBs, plasmablasts. (C) Flow cytometry was used to evaluate the number and frequency of circulating B cells among single events in individuals with T21 (n = 40 for frequency and n = 32 for relative cell number) and D21 controls (n = 53 for frequency and n = 45 for relative cell number), as depicted in Figure S4C. (D, G, and I) Within mass cytometry data, B cell subsets were resolved among B cells of individuals with T21 (n = 18) and D21 controls (n = 18), as depicted in Figure S2. (J and K) An alternative CD19HI gating strategy was used to identify CD11c+ B cells with numbers denoting frequency among the parent gate, B cells (left), or atypical/IgM IgD double-negative memory B cells (DN aBMEM). (J) Representative dot plots on both sides of the arrow correspond to the same individual with T21 or D21 control. (L) Representative histograms from an individual with T21 with numbers denoting geometric mean metal intensities (gMMIs) of PD1 expression among the indicated parent gate. (M) Lineage tree depicts B cell subsets of individuals with T21 compared to D21 controls. Color denotes fold change, where red is increased and blue is decreased frequency of subsets among the B cells in T21 compared to D21 controls. In all cases, statistical significance was determined by a Student’s t test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001). All box and whisker plots denote the median within a box extending from the 25th to 75th percentiles and error bars span minimum to maximum values within the indicated datasets.

Figure 6.. Global Upregulation of IFNAR1 Surface Protein Expression on Circulating Immune Cells of Individuals with T21

(A–C) A total of 100 immune cell types were resolved by manual gating on expression of canonical markers as depicted in Figure S2, and then levels of IFNAR1 surface protein expression were compared between individuals with T21 (n = 18) and D21 controls (n = 18) by geometric mean metal intensity (gMMI). (A) Each dot represents the mean gMMI for IFNAR1 among a gated cell type for all samples of a given karyotype. (B) Volcano plot showing statistical analysis of IFNAR1 expression. Horizontal dashed line indicates the statistical cut-off of *p ≤ 0.05 as defined by Student’s t test. See Table S4D for full statistical analysis. (C) Representative histograms of IFNAR1 protein expression with numbers denoting gMMIs. pDCs, plasmacytoid dendritic cells; mDCs, myeloid DCs; ILCs, innate lymphoid cells. (D) Scatterplots displaying IFNR mRNA expression as defined by RNA-seq of bulk white blood cells of individuals with T21 (n = 10) versus D21 controls (n = 9). Statistical significance was calculated using DESeq2. mRNA expression values are displayed in reads per kilobase per million (RPKM). See Table S4E for full statistical analysis.

Figure 7.. Global IFN-α Hypersensitivity among Circulating Immune Cells from Individuals with T21

Whole blood was incubated directly ex vivo for 30 min with (+IFN) or without (basal) IFN-α−2a before processing for mass cytometry. Phospho-epitopes among bulk immune subtypes were resolved by manual gating on expression of canonical markers, as depicted in Figure S2, and then compared between individuals with T21 (n = 8) and D21 controls (n = 8). (A–G) Arcsinh ratios shown in volcano plots and heatmaps were generated from geometric mean metal intensity (gMMIs) of each of the 8 phospho-epitopes among 9 immune subtypes. (H) Radar plots scaled to the minimum and maximum gMMIs for the indicated phospho-epitope, where each spoke represents a distinct immune subset, with cohort and stimulation status denoted by line and color. Dot plots represent fold changes in the indicated phospho-epitopes within the indicated immune subset from each biological replicate, with lines connecting samples from the same individual with and without IFN stimulation. In all cases, statistical significance was determined by a Student’s t test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001).