Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The VSports app下载. gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure V体育官网. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. .

. 2020 Mar 12;135(11):814-825.
doi: 10.1182/blood.2019001903.

High-parametric evaluation of human invariant natural killer T cells to delineate heterogeneity in allo- and autoimmunity

Tom Erkers  1   2 , Bryan J Xie  1 , Laura J Kenyon  1   3 , Brian Smith  1 , Mary Rieck  4   5 , Kent P Jensen  6   7 , Xuhuai Ji  3 , Marina Basina  8 , Samuel Strober  6 , Robert S Negrin  1 , Holden T Maecker  3   9 , Everett H Meyer  1   7
Affiliations

High-parametric evaluation of human invariant natural killer T cells to delineate heterogeneity in allo- and autoimmunity

Tom Erkers et al. Blood. .

Abstract

Human invariant natural killer T (iNKT) cells are a rare innate-like lymphocyte population that recognizes glycolipids presented on CD1d VSports手机版. Studies in mice have shown that these cells are heterogeneous and are capable of enacting diverse functions, and the composition of iNKT cell subsets can alter disease outcomes. In contrast, far less is known about how heterogeneity in human iNKT cells relates to disease. To address this, we used a high-dimensional, data-driven approach to devise a framework for parsing human iNKT heterogeneity. Our data revealed novel and previously described iNKT cell phenotypes with distinct functions. In particular, we found 2 phenotypes of interest: (1) a population with T helper 1 function that was increased with iNKT activation characterized by HLA-II+CD161- expression, and (2) a population with enhanced cytotoxic function characterized by CD4-CD94+ expression. These populations correlate with acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation and with new onset type 1 diabetes, respectively. Our study identifies human iNKT cell phenotypes associated with human disease that could aid in the development of biomarkers or therapeutics targeting iNKT cells. .

PubMed Disclaimer

Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

V体育官网 - Figures

None
Graphical abstract
Figure 1.
Figure 1.
Gene signatures in patients after allogeneic HSCT with and without acute GVHD and in individuals who did not receive a transplant. (A) Isolation of iNKT cells. Representative plot showing the results of the purification procedure for all sequencing experiments. (B) Concatenated t-distributed stochastic neighbor embedding (tSNE) plot of single-cell messenger RNA-sequencing of iNKT cells from 1 healthy individual who did not receive a transplant (green), 1 patient after HSCT without GVHD (blue), and 1 patient after HSCT with onset of gastrointestinal GVHD (dark red). A heatmap shows selected differential gene expression (log10 scale of unique molecular identifier [UMI] counts) across the 3 cell clusters detected in the tSNE plots, and violin plots show the normalized distribution of expression of genes between the 3 individuals (bottom right). APC, antigen-presenting cell; FITC, fluorescein isothiocyanate.
Figure 2.
Figure 2.
Short-term kinetic assessment of primary iNKT cells with a targeted scRNA-seq approach. (A) Sequencing of 4000 iNKT cells targeting RNA expression of 312 T-cell–associated genes from 2 healthy donors. The cells from each donor were experimentally divided into 2 fractions: primary cells that received no in vitro activation and cells that were activated in vitro with CD3/CD28 Dynabeads overnight. The data have been dimensionally reduced and displayed as a tSNE plot. (B-D) Combined expression of (B) genes associated with an NK-cell phenotype, (C) genes associated with memory T cells (Tcmem), and (D) distribution of gene expression of immunologically relevant effector molecules across the clusters.
Figure 3.
Figure 3.
Characterization of iNKT cells in healthy individuals, in patients after receiving allogeneic HSCT, and in T1D patients. (A) iNKT cell phenotypes in peripheral blood were assessed using multiparametric flow cytometry in healthy individuals and in patients after allogeneic HSCT. The markers used for flow cytometry were derived from our scRNA-seq data and from those previously reported in the literature. Subpopulations were manually gated, and principal component analysis (PCA) was performed on the data set. Each dot represents 1 patient and is colored according to that patient’s clinical status. (B) Representative tSNE contour plots in 3 flow cytometric data sets consisting of 3 healthy individuals, 11 patients after HSCT without GVHD, and 3 patients at GVHD diagnosis (Dx). (C) Four parameters were identified from PCA that accounted for most of the variation among the 3 groups, and these parameters were manually assessed and presented in 2D dot plots showing the frequency of expression of each marker in each patient. (D) Representative tSNE contour plots in 1 flow cytometric data set consisting of 3 T1D patients. The parameters here are the same as in panel B. (E) Phenotypic expression of iNKT cells in T1D (n = 11) vs healthy individuals (n = 16). Means ± standard error of the mean (C,E). *P = .05-.01; **P = .009-.001; ***P < .001; ****P < .0001. ns, not significant.
Figure 4.
Figure 4.
Assessment of iNKT phenotypes that distinguishes treatment responders from nonresponders in acute GVHD patients, and GVHD onset from healthy immune reconstitution in allogeneic HSCT patients. (A) Representative visualization of tSNE (viSNE) plot from patients after HSCT (n = 11) and patients at GVHD diagnosis that later were corticosteroid (CS) responsive (n = 1) or refractory (n = 3). Each column shows the marker expression of a user-defined channel range. The cell number has been normalized to include the same number of cells from each group. (B) Summary of HLA-II expression in iNKT cells (n = 18) at GVHD diagnosis. The patients were retrospectively stratified by corticosteroid response after GVHD diagnosis. (C) Frequency of CXCR3 cells within the CD161HLA-II population in patients at GVHD diagnosis who were retrospectively stratified by corticosteroid response. (D) Phenotypic expression of iNKT cells from 34 patients on day 30 after HSCT with myeloablative conditioning (MAC). The patients analyzed did not display any clinical manifestations of GVHD. After day 30, 10 of the patients would develop GVHD (red) but 24 would not (blue). Tx, Transplantation. Means ± standard error of the mean (B,D). *P = .05-.01. ns, not significant.
Figure 5.
Figure 5.
Function of CD94+ iNKT cells. (A) Representative plot of CD4 and CD94 expression in primary iNKT cells (n = 23). (B) Representative plots from experiment showing mutually exclusive expression of CD4 and CD94. Primary iNKT cells were purity sorted by flow cytometry based on the highlighted populations CD4+CD94– and CD4–CD94–. The cells were then cultured for 7 days with or without stimulation with CD3/CD28 Dynabeads and IL-2. Plots on the right show the expression of CD94 gated on the CD4+ and the CD4– population on day 7. (C) Cytotoxicity of iNKT cells. CD1d+ Jurkat cells were incubated with α-galactosylceramide (αGC) overnight (far left). Bulk iNKT cells (left) or CD94-depleted iNKT cells (right) were then added in a 1:1 ratio. CD94-depleted iNKT cells induced significantly less apoptosis in target cells compared with bulk iNKT cells (far right, n = 8). (D) Allogeneic cytotoxicity toward APCs. Allo-myeloid dendritic cells (mDCs) were pulsed with αGC and mixed with iNKT cells sorted by FACS in a 1:1 ratio (n = 6). Cell death was measured using 7-aminoactinomycin D (7AAD) and Annexin V (AnV). The plot (far right) shows frequency of 7AAD+AnnexinV+ mDCs (blue) and 7AAD–AnnexinV+ mDCs (red). CD94+CD4– iNKT cells induced significantly more mDC death than CD94–CD4– and CD94–CD4+ iNKT cells. (E) Immune regulatory function of CD94+ iNKT cells. Purified CD3+ T cells were stimulated with CD3/CD28 Dynabeads for 72 hours. Addition of CD94-depleted iNKT cells in a dose-dependent manner decreased the proliferation of the T cells (n = 6). Means ± standard error of the mean (A,C-E).*P = .05-.01; ****P < .0001.
Figure 6.
Figure 6.
Kinetics of chronically activated iNKT cells. (A) Surface expression of HLA-II (top) and CD161 (bottom) measured on days 0, 3, and 10 after activation with 3 different stimuli (n = 12). (B) Expression frequency fold change of HLA-II (top) and CD161 (bottom) using reducing ratios of stimulation of iNKT cells with CD3/CD28 Dynabeads and 100 IU IL-2 for 3 days (n = 13). Data are normalized against a condition with no CD3/CD28 Dynabeads added. (C) Expression frequency fold change of HLA-II (top) and CD161 (bottom) with 1:10 CD3/CD28 Dynabeads:iNKT stimulation and increasing concentrations of IL-2 for 3 days. Data are normalized against a condition with no IL-2 added (n = 6). Means ± standard error of the mean (A-C). *P = .05-.01; ****P < .0001.
Figure 7.
Figure 7.
Intracellular expression of cytotoxic effector molecules and expression of exhaustion markers in chronically activated iNKT cells. (A) Representative histograms of IFN-γ or IL-4 expression based on CD161 and HLA-II expression (left) and a summary of the ratio of the frequency of IFN-γ to IL-4–positive cells based on CD161 and HLA-II expression (right; n = 7). (B) Mean fluorescence intensity of expression of perforin (Per), granzyme B (Gzm B), and granulysin based on CD161 and HLA-II expression in in vitro expanded iNKT cells (n = 7). (C) Frequency of expression of Tim-3, PD-1, and NKG2D based on CD161 and HLA-II expression in in vitro expanded iNKT cells (n = 7). Means ± standard error of the mean (A-C). *P = .05-.01; **P = .009-.001; ****P < .0001.
See this image and copyright information in PMC

V体育平台登录 - References

    1. Porcelli S, Yockey CE, Brenner MB, Balk SP. Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4-8- alpha/beta T cells demonstrates preferential use of several V beta genes and an invariant TCR alpha chain. J Exp Med. 1993;178(1):1-16. - VSports注册入口 - PMC - PubMed
    1. Dellabona P, Padovan E, Casorati G, Brockhaus M, Lanzavecchia A. An invariant V alpha 24-J alpha Q/V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4-8- T cells. J Exp Med. 1994;180(3):1171-1176. - PMC - PubMed
    1. Gutierrez-Arcelus M, Teslovich N, Mola AR, et al. . Lymphocyte innateness defined by transcriptional states reflects a balance between proliferation and effector functions. Nat Commun. 2019;10(1):687. - VSports注册入口 - PMC - PubMed
    1. Lynch L, O’Shea D, Winter DC, Geoghegan J, Doherty DG, O’Farrelly C. Invariant NKT cells and CD1d(+) cells amass in human omentum and are depleted in patients with cancer and obesity. Eur J Immunol. 2009;39(7):1893-1901. - PubMed
    1. Engel I, Seumois G, Chavez L, et al. . Innate-like functions of natural killer T cell subsets result from highly divergent gene programs. Nat Immunol. 2016;17(6):728-739. - PMC - PubMed

Publication types (VSports)

MeSH terms