Distinct and Overlapping Effector Functions of Expanded Human CD4+, CD8α+ and CD4-CD8α- Invariant Natural Killer T Cells

Department of Immunology and Institute of Molecular Medicine, Trinity College Dublin, St James's Hospital, Dublin, Ireland.
PLoS ONE (Impact Factor: 3.23). 12/2011; 6(12):e28648. DOI: 10.1371/journal.pone.0028648
Source: PubMed


CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4(+), CD8α(+) and CD4(-)CD8α(-) double-negative (DN) subsets. CD4(+) iNKT cells expanded more readily than CD8α(+) and DN iNKT cells upon mitogen stimulation. CD8α(+) and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d(+) cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4(+) and CD8α(+) fractions, respectively. Only CD4(+) iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α(+), DN or CD4(+) iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.

Download full-text


Available from: Vincent O'Reilly,
  • Source
    • "pVOC significantly increased the percentage of iNKT cells expressing high levels of both cytokines (Figure 1B). These findings confirm a characteristic feature of iNKT cells that distinguishes them from most conventional T cells; they produce both Th1 cytokines such as IFN-gamma, and Th2 cytokines such as IL-4 [17]. The findings demonstrate that in 8 of 8 patients examined acute pVOC was accompanied by a rapid increase in the percentage of iNKT cells in the circulation that are activated to produce cytokines. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Reperfusion injury following tissue ischemia occurs as a consequence of vaso-occlusion that is initiated by activation of invariant natural killer T (iNKT) cells. Sickle cell disease (SDC) results in widely disseminated microvascular ischemia and reperfusion injury as a result of vaso-occlusion by rigid and adhesive sickle red blood cells. In mice, iNKT cell activation requires NF-κB signaling and can be inhibited by the activation of anti-inflammatory adenosine A2A receptors (A2ARs). Human iNKT cells are divided into subsets of CD4+ and CD4- cells. In this study we found that human CD4+ iNKT cells, but not CD4- cells undergo rapid NF-κB activation (phosphorylation of NF-κB on p65) and induction of A2ARs (detected with a monoclonal antibody 7F6-G5-A2) during SCD painful vaso-occlusive crises. These findings indicate that SCD primarily activates the CD4+ subset of iNKT cells. Activation of NF-κB and induction of A2ARs is concordant, i.e. only CD4+ iNKT cells with activated NF-κB expressed high levels of A2ARs. iNKT cells that are not activated during pVOC express low levels of A2AR immunoreactivity. These finding suggest that A2AR transcription may be induced in CD4+ iNKT cells as a result of NF-κB activation in SCD. In order to test this hypothesis further we examined cultured human iNKT cells. In cultured cells, blockade of NF-κB with Bay 11-7082 or IKK inhibitor VII prevented rapid induction of A2AR mRNA and protein upon iNKT activation. In conclusion, NF-κB-mediated induction of A2ARs in iNKT cells may serve as a counter-regulatory mechanism to limit the extent and duration of inflammatory immune responses. As activated iNKT cells express high levels of A2ARs following their activation, they may become highly sensitive to inhibition by A2AR agonists.
    PLoS ONE 10/2013; 8(10):e74664. DOI:10.1371/journal.pone.0074664 · 3.23 Impact Factor
  • Source
    • "Regarding the functionality of iNKT cells, human iNKT cells activated by α-GalCer are known to secrete a myriad of cytokines, with CD8+ iNKT cells biased toward a Th1 phenotype, CD4+ iNKT cells predominantly secreting Th2 cytokines, and DN iNKT cells exhibiting an intermediate Th1/Th2 phenotype [40]. Non-human primate iNKT cells have been shown to display a similar function to human iNKT cells, but there are some differences among different species. "
    [Show abstract] [Hide abstract]
    ABSTRACT: In humans, invariant natural killer T (iNKT) cells represent a small but significant population of peripheral blood mononuclear cells (PBMCs) with a high degree of variability. In this study, pursuant to our goal of identifying an appropriate non-human primate model suitable for pre-clinical glycolipid testing, we evaluated the percentage and function of iNKT cells in the peripheral blood of pig-tailed macaques. First, using a human CD1d-tetramer loaded with α-GalCer (α-GalCer-CD1d-Tet), we found that α-GalCer-CD1d-Tet+ CD3+ iNKT cells make up 0.13% to 0.4% of pig-tailed macaque PBMCs, which are comparable to the percentage of iNKT cells found in human PBMCs. Second, we observed that a large proportion of Vα24+CD3+ cells are α-GalCer-CD1d-Tet+CD3+ iNKT cells, which primarily consist of either the CD4+ or CD8+ subpopulation. Third, we found that pig-tailed macaque iNKT cells produce IFN-γ in response to α-GalCer, as shown by ELISpot assay and intracellular cytokine staining (ICCS), as well as TNF-α, as shown by ICCS, indicating that these iNKT cells are fully functional. Interestingly, the majority of pig-tailed macaque iNKT cells that secrete IFN-γ are CD8+ iNKT cells. Based on these findings, we conclude that the pig-tailed macaques exhibit potential as a non-human animal model for the pre-clinical testing of iNKT-stimulating glycolipids.
    PLoS ONE 10/2012; 7(10):e48166. DOI:10.1371/journal.pone.0048166 · 3.23 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Background Invariant natural killer T cells recognise glycolipid antigens such as α- galactosylceramide presented by CD1d. In preclinical models of B cell malignancies, α-galactosylceramide is an adjuvant to tumor vaccination, enhancing tumor-specific T cell responses and prolonging survival. However, numerical and functional invariant natural killer T cell defects exist in patients with some cancers. We aimed to assess this axis in patients with chronic lymphocytic leukemia. Design and methods Circulating invariant natural killer T cell numbers and antigen presenting cell CD1d expression were measured in patients with chronic lymphocytic leukemia and age-matched controls. Cytokine profile and in vitro proliferative capacity were determined. Patient- and control-derived invariant natural killer T cell lines were generated and characterized, and allogeneic and autologous responses to α-galactosylceramide-treated leukemia cells were assessed. Results Absolute numbers and phenotype of invariant natural killer T cells were normal in untreated chronic lymphocytic leukemia, and cytokine profile and proliferative capacity were intact. Chemotherapy-treated patients had reduced invariant natural killer T and myeloid dendritic cell numbers, but α- galactosylceramide-induced proliferation was preserved. Invariant natural killer T cell lines from patients lysed CD1d-expressing targets. Irradiated α-galactosylceramide- treated leukemic cells elicited allogeneic and autologous invariant natural killer T cell proliferation, and α-galactosylceramide treatment led to increased proliferation of conventional T cells in response to tumor. Conclusions The invariant natural killer T cell and CD1d axis is fundamentally intact in patients with early-stage chronic lymphocytic leukemia, and despite reduced circulating numbers, function is retained in fludarabine-treated patients. Immunotherapies exploiting the adjuvant effect of α-galactosylceramide may be feasible.
    Haematologica 10/2012; 98(3). DOI:10.3324/haematol.2012.072835 · 5.81 Impact Factor
Show more