Human Natural Killer T Cells Are Heterogeneous in Their Capacity to Reprogram Their Effector Functions

BACKGROUND: Natural killer T (NKT) cells are a subset of T cells that help potentiate and regulate immune responses. Although human NKT cell subsets with distinct effector functions have been identified, it is unclear whether the effector functions of these subsets are imprinted during development or can be selectively reprogrammed in the periphery. RESULTS: We found that neonatal NKT cells are predominantly CD4+ and express higher levels of CCR7 and CD62L and lower levels of CD94 and CD161 than adult CD4+ or CD4− NKT cell subsets. Accordingly, neonatal NKT cells were more flexible than adult CD4+ NKT cells in their capacity to acquire Th1- or Th2-like functions upon either cytokine-mediated polarization or ectopic expression of the Th1 or Th2 transcription factors T-bet and GATA-3, respectively. Consistent with their more differentiated phenotype, CD4- NKT cells were predominantly resistant to functional reprogramming and displayed higher cytotoxic function. In contrast to conventional T cells, neither the expression of CXCR3 nor the cytotoxic capacity of neonatal NKT cells could be reprogrammed. CONCLUSIONS AND SIGNIFICANCE: Together, these results suggest that neonatal CD4+, adult CD4+, and adult CD4− NKT may represent unique states of maturation and that some functions of human NKT cells may be developmentally imprinted, while others are acquired similar to conventional T cell subsets during peripheral maturation and differentiation. Given the potent immuno-regulatory functions of NKT cells, these findings have important implications for the development of novel NKT cell-based therapeutics and vaccines

GWAS of random glucose in 476,326 individuals provide insights into diabetes pathophysiology, complications and treatment stratification

Conventional measurements of fasting and postprandial blood glucose levels investigated in genome-wide association studies (GWAS) cannot capture the effects of DNA variability on ‘around the clock’ glucoregulatory processes. Here we show that GWAS meta-analysis of glucose measurements under nonstandardized conditions (random glucose (RG)) in 476,326 individuals of diverse ancestries and without diabetes enables locus discovery and innovative pathophysiological observations. We discovered 120 RG loci represented by 150 distinct signals, including 13 with sex-dimorphic effects, two cross-ancestry and seven rare frequency signals. Of these, 44 loci are new for glycemic traits. Regulatory, glycosylation and metagenomic annotations highlight ileum and colon tissues, indicating an underappreciated role of the gastrointestinal tract in controlling blood glucose. Functional follow-up and molecular dynamics simulations of lower frequency coding variants in glucagon-like peptide-1 receptor (GLP1R), a type 2 diabetes treatment target, reveal that optimal selection of GLP-1R agonist therapy will benefit from tailored genetic stratification. We also provide evidence from Mendelian randomization that lung function is modulated by blood glucose and that pulmonary dysfunction is a diabetes complication. Our investigation yields new insights into the biology of glucose regulation, diabetes complications and pathways for treatment stratification

Identification of NKT cell subsets in neonatal and adult blood PBMC.

<div><p>(A) NKT cells in either neonatal or adult PBMC were identified by staining for the invariant TCR using antibodies against Vα24 (FITC) and Vβ11 (unlabelled followed by goat anti-mouse APC).</p> <p>Double positive cells were identified as NKT cells and gated on for further analysis to determine CD4 (PE) and CD8 (biotin followed by strepavidin-PerCP-Cy5.5) expression.</p> <p>(B) NKT cell subsets were identified by staining for Vα24, Vβ11, and CD4 as in 1A and then gated on for further analysis. Expression of the indicated cell surface marker on these subsets was then determined.</p> <p>Combinations of the following antibodies were used, depending on the particular stain: Vα24 (FITC or unlabelled), Vβ11 (unlabelled or PE), CD4 (PE, biotin, or unlabelled), CCR7 (unlabelled), CD62L (PE), CD94 (unlabelled), CD161 (FITC), CD28 (biotin), goat anti-mouse (APC), and strepavidin (PerCP-Cy5.5). One representative donor is shown.</p> <p>(C) Summary of NKT cell phenotype data for all neonatal and adult donors. P values are indicated.</p> <p>For comparison of nCD4+ to either aCD4+ or aCD4− subsets, P values were generated using the Wilcoxon rank sum test.</p> <p>For comparison of aCD4+ to aCD4− subsets, P values were generated using the Wilcoxon signed rank test.</p> <p>(D) For CD4+ NKT cells of adult donors represented in 1C, the ratio of CD161 (% expression) to CD62L (% expression) was generated and compared to CCR7 expression using linear regression with a resulting equation of y = −19.277X+86.676 and p = 0.008.</p></div

Cytokine polarization of NKT cell subsets.

<div><p>Neonatal or adult NKT cells were purified and activated under conventional Th0 (medium alone; NKT0), Th1 (NKT1), or Th2 (NKT2) polarizing conditions as described in the methods and expanded in IL-2-containing media.</p> <p>These cells were re-stimulated for 6–8 hours using DCs pulsed with α-GalCer (100 ng/ml), or alternatively, anti-CD3 and anti-CD28, in the presence of GolgiStop (BD) and were then intracellularly stained with antibodies against IL-4 (PE) and IFN-γ (APC).</p> <p>NKT cells were also co-stained with anti-Vβ11 (FITC) to exclude contaminating non-NKT cells.</p> <p>For adult NKT cells, CD4 expression was also assessed by cell surface staining (CD4-biotin followed by strepavidin-PerCP-Cy5.5) to differentiate CD4+ from CD4− NKT cells.</p> <p>One representative donor out of 5 is shown.</p></div

Functional profile of NKT cell subsets ectopically expressing T-bet or GATA-3.

<div><p>NKT cells were activated under non-polarizing conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000050#pone-0000050-g002" target="_blank">Figure 2</a>, transduced at the time of activation with either the control lentiviral vector or vectors expressing T-bet or GATA3, and expanded in IL-2-containing media.</p> <p>One representative donor out of 3 is shown for each.</p> <p>(A) Intracellular stain of nCD4+ NKT cells was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000050#pone-0000050-g002" target="_blank">Figure 2</a> and cells were co-stained with anti-mCD24 (FITC) to identify transduced cells.</p> <p>(B) Intracellular cytokine stains of aCD4+ or aCD4− NKT cells were performed as in 4A.</p> <p>CD4 expression was determined by co-staining with anti-CD4 (biotin followed with strepavidin PerCP-Cy 5.5).</p> <p>(C) CCR4 or CXCR3 expression on T-bet- or GATA-3-transduced nNKT cells was assayed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000050#pone-0000050-g003" target="_blank">Figures 3A and 3B</a>, and cells were co-stained with anti-mCD24 (FITC) as a marker for transduction.</p> <p>(D) CCR4 expression of T-bet or GATA-3-transduced aCD4+ or aCD4− NKT cell was examined as in 4C, and cells were stained with anti-CD4 (PE) to differentiate between CD4+ and CD4− NKT cells.</p></div

Chemokine receptor expression on polarized NKT cell subsets.

<div><p>One representative neonatal or adult sample of 5 is shown for each.</p> <p>(A) CCR4 expression on polarized NKT cell lines was assayed by staining with anti-CCR4 (unlabelled followed by goat anti-mouse APC) and anti-CD4 (PE) to differentiate between adult CD4+ and CD4− NKT cells and anti-Vβ11 (FITC) to exclude contaminating non-NKT cells.</p> <p>Isotype control is depicted in light gray.</p> <p>(B) CXCR3 (unlabelled followed by goat anti-mouse APC) expression on polarized NKT cell subsets were assayed as in 3A.</p></div

Cytotoxic capability of NKT cell subsets.

<div><p>Experiments were performed in triplicate per donor; standard deviation is depicted.</p> <p>For A, B, C, and D, one representative donor out of three independent experiments is shown. (A) NKT cells were added at an Effector:Target (NKT cell: 3T3 cell) ratio of 1∶1 to adherent 3T3 cells in the presence of different α-GalCer concentrations.</p> <p>After 18–20 hours, NKT cells were collected and stained with anti-CD25 (unlabelled followed by goat anti-mouse APC) to determine activation.</p> <p>(B) As in A, NKT cells at an NKT:3T3 of 1∶1 were activated in the presence of varying concentrations of α-GalCer.</p> <p>After 18–20 hours, cells were collected by trypsinization to remove any 3T3 cells that were still adherent and target 3T3 cells were stained with propidium iodide to determine their viability.</p> <p>(C) α-GalCer was added at a concentration of 100 ng/ml to adherent 3T3 cells.</p> <p>NKT cell subsets were then added at different NKT:3T3 ratios. Viability of 3T3 cells after an 18–20 hour incubation period was determined as in 5B.</p> <p>(D) Polarized nCD4+ NKT cells were generated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000050#pone-0000050-g002" target="_blank">Figure 2</a>.</p> <p>The cytotoxic capacity of these NKT0, NKT1, and NKT2 lines was assayed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000050#pone-0000050-g005" target="_blank">Figure 5C</a>.</p> <p>One NKT:3T3 ratio for each donor is shown.</p></div

Transcription factor GATA-1 potently represses the expression of the HIV-1 coreceptor CCR5 in human T cells and dendritic cells

CC chemokine receptor 5 (CCR5) is the major HIV-1 coreceptor and its expression levels are a critical determinant of HIV-1 infection. However, the molecular mechanisms of CCR5 regulation in primary targets of HIV-1 remain unknown. Despite binding to conserved DNA elements, we show that the transcription factors GATA binding protein 1 (GATA-1) and GATA-3 differentially suppress the expression of CCR5 in stem-cell–derived dendritic cells and primary human T-cell subsets. In addition, GATA-1 expression was also more potent than GATA-3 in suppressing T helper 1 (Th1)–associated genes, interferon-γ (IFNγ), and CXC chemokine receptor-3 (CXCR3). GATA-1, but not GATA-3, potently suppressed CCR5 transcription, thereby rendering human T cells resistant to CCR5-tropic HIV-1 infection. However, GATA-1 could also serve as a surrogate for GATA-3 in its canonic role of programming Th2 gene expression. These findings provide insight into GATA-3–mediated gene regulation during T-cell differentiation. Importantly, decoding the mechanisms of GATA-1–mediated repression of CCR5 may offer an opportunity to develop novel approaches to inhibit CCR5 expression in T cells