Isolation and Functional Use of Human NKT Cells

Abstract

Isolation and Functional Use of Human NK T Cells (Mark A. Exley andSteven P. Balk, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts; S. Brian Wilson, Massachusetts General Hospital, Harvard Medical School, Cambridge, Massachusetts). This unit describes methods for the isolation and functional analysis of human invariant NK T cells. The first protocol uses standard multicolor flow cytometry methods and staining reagents specific for V to generate invariant NK T cell clones. Support protocols for secondary stimulations and for rapid expansion of invariant NK T cell clones and functional analysis of these cells are also included.

Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis

“…We thus cocultured LPMCs from the various patient groups with an EBV-transformed B cell line that expressed high levels of CD1d as a result of transfection with a CD1d construct (the 721.221 cell line, referred to here as 721 cells) and then subjected the cells to flowcytometric analysis to determine IL-13/IFN-γ secretion as described above. It should be noted that such cocultures were carried out in the presence of subactivating concentrations of PMA/IL-2, which has previously been shown to be required for such stimulation (18,21). As shown in Figure 3 Finally, the studies of UC LPMC IL-13 production resulting from stimulation by 721 cells measured by flow-cytometric analysis were fully corroborated by studies of IL-13 secretion measured by ELISA.…”

“…This line was derived from peripheral blood cells obtained from a healthy donor that had been purified by a single-step sorting technique using an antiinvariant TCR mAb (6B11) specific for the complementary determining region 3 (CDR3) of the Vα24/JαQ TCR. The sorted cells were then expanded and maintained in the presence of anti-CD3 (OKT3) (10 μg/ml) and IL-2 (100 U/ml) (21).…”

“…Assay of NKT cell function following stimulation by CD1d-transfected, EBV-transformed B cells was performed as described previously (18,21). In brief, 5 × 10 5 LPMCs from each patient population were cultured with equal numbers of mitomycin-pretreated CD1d-transfected cells (721.722 B cells) or identical nontransfected cells for 48 hours.…”

Heterogeneity in phenotype and function of CD8+ and CD4/CD8 double‐negative Natural Killer T cell subsets in sooty mangabeys

“…To further compare the functional characteristics of sooty mangabey NKT lymphocyte subsets, the cytokine profile of NKT clones following stimulation with CD1d‐transfected cell lines pulsed with α‐GalCer (C1R.d/αGC) was analyzed as previously described [26]. In vitro stimulation of NKT clones with C1R.d/αGC resulted in production of the Th1 cytokines TNF‐α, IFN‐γ, and IL‐2 by both subsets as observed by intracellular cytokine flow cytometry (Fig.…”

“…Positive NKT clones were maintained in complete media supplemented with 50 IU/ml IL‐2. For functional analyses, the clones were gradually switched to resting stage (10 IU/ml IL‐2) 48 hours prior to the assay as previously described [26].…”