Methods for detection, isolation and culture of mouse and human invariant NKT cells

Watarai, Hiroshi; Nakagawa, Ryusuke; Omori‐Miyake, Miyuki; Dashtsoodol, Nyambayar; Taniguchi, Masaru

doi:10.1038/nprot.2007.515

Cited by 128 publications

(117 citation statements)

References 31 publications

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“…Notably, the in vivo expansion of these cells peaked at day 3 postimmunization, compared with 7 d postimmunization for conventional αβ T cells (18). This speedy in vivo response is a signature of iNKT cells (17). These results indicate that the HSC-engineered iNKT cells are fully functional.…”

Section: Generation Of Clonal Inkt Cells Through Genetic Engineering supporting

confidence: 48%

“…Study of the iNKT cells from the B6-miNKT mice revealed that these iNKT cells displayed a typical phenotype of mouse iNKT cells in that they exhibited high expression of the NK1.1 marker, as well as a memory Tcell signature (CD62L lo CD44 hi ) and a CD4 + CD8 − or CD4 − CD8 − coreceptor expression pattern (Fig. 2F) (17). Almost all of these iNKT cells showed positive staining for TCR Vβ8, indicating that they expressed the transgenic clonal iNKT TCR and suggesting that they were derived from the miNKT-engineered HSCs (Fig.…”

Section: Generation Of Clonal Inkt Cells Through Genetic Engineering mentioning

confidence: 99%

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Genetic engineering of hematopoietic stem cells to generate invariant natural killer T cells

Smith

Liu

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Invariant natural killer T (iNKT) cells comprise a small population of αβ T lymphocytes. They bridge the innate and adaptive immune systems and mediate strong and rapid responses to many diseases, including cancer, infections, allergies, and autoimmunity. However, the study of iNKT cell biology and the therapeutic applications of these cells are greatly limited by their small numbers in vivo (∼0.01-1% in mouse and human blood). Here, we report a new method to generate large numbers of iNKT cells in mice through T-cell receptor (TCR) gene engineering of hematopoietic stem cells (HSCs). We showed that iNKT TCR-engineered HSCs could generate a clonal population of iNKT cells. These HSC-engineered iNKT cells displayed the typical iNKT cell phenotype and functionality. They followed a two-stage developmental path, first in thymus and then in the periphery, resembling that of endogenous iNKT cells. When tested in a mouse melanoma lung metastasis model, the HSC-engineered iNKT cells effectively protected mice from tumor metastasis. This method provides a powerful and high-throughput tool to investigate the in vivo development and functionality of clonal iNKT cells in mice. More importantly, this method takes advantage of the self-renewal and longevity of HSCs to generate a long-term supply of engineered iNKT cells, thus opening up a new avenue for iNKT cell-based immunotherapy. αβ T lymphocytes highly conserved from mice to humans. Like conventional αβ T cells, iNKT cells are derived from hematopoietic stem cells (HSCs) and develop in the thymus. However, they differ from conventional T cells in several important aspects, including their display of NK cell markers, their recognition of glycolipid antigens presented by the nonclassical monomorphic major histocompatibility complex (MHC) molecule CD1d, and their expression of semi-invariant T-cell receptors (identical α chains paired with a limited selection of β chains) (1, 2). Despite their small numbers in vivo (∼0.1-1% in mouse blood and ∼0.01-1% in human blood), iNKT cells have been suggested to play important roles in regulating many diseases, including cancer, infections, allergies, and autoimmunity (3). When stimulated, iNKT cells rapidly release a large amount of effector cytokines like IFN-γ and IL-4, both as a cell population and at the single-cell level. These cytokines then activate various immune effector cells, such as natural killer (NK) cells and dendritic cells (DCs) of the innate immune system, as well as CD4 helper and CD8 cytotoxic conventional αβ T cells of the adaptive immune system via activated DCs (3, 4). Because of their unique activation mechanism, iNKT cells can attack multiple diseases independent of antigen and MHC restrictions, making them attractive universal therapeutic agents (3, 4). Notably, because of the capacity of effector NK cells and conventional αβ T cells to specifically recognize diseased tissue cells, iNKT cell-induced immune reactions result in limited off-target side effects (3, 4).Restricted by their extremely low numbers, ...

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Section: Generation Of Clonal Inkt Cells Through Genetic Engineering supporting

confidence: 48%

Section: Generation Of Clonal Inkt Cells Through Genetic Engineering mentioning

confidence: 99%

Genetic engineering of hematopoietic stem cells to generate invariant natural killer T cells

Smith

Liu

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Hepatic mononuclear cells (MNCs) were isolated as described by Watarai et al [14] . Briefly, each mouse liver was first perfused with Hank's balanced salt solution to eliminate blood until the liver become pale.…”

Section: Isolation Of Hepatic Mononuclear Cellsmentioning

confidence: 99%

Betulin from Hedyotis hedyotidea ameliorates concanavalin A-induced and T cell-mediated autoimmune hepatitis in mice

et al. 2016

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Hedyotis hedyotidea has been used in traditional Chinese medicine for the treatment of autoimmune diseases. However, the mechanisms underlying for the effect remain unknown. We previously showed that, among 11 compounds extracted from H hedyotidea, betulin produced the strongest suppressive effect on T cell activation. Here, we examined the hepatoprotective effects of betulin against acute autoimmune hepatitis in mice and the mechanisms underlying the effects. Freshly isolated mouse splenocytes were stimulated with concanavalin A (Con A, 5 μg/mL) in the presence of betulin, the cell proliferation was assessed with CSFE-dilution assay. Mice were injected with betulin (10, 20 mg· kg -1 ·d -1 , ip) for 3 d. One hour after the last injection, the mice were injected with Con A (15 mg/kg, iv) to induce acute hepatitis. Blood samples and liver tissues were harvested at 10 h after Con A injection, and serum transaminase levels and liver histopathology were detected; serum levels of proinflammatory cytokines, hepatic T lymphocyte ratios, and functional statuses of conventional T and NKT cells were also analyzed. Betulin (16 and 32 μmol/L) dose-dependently suppressed the proliferation of Con A-stimulated mouse splenocytes in vitro. In Con A-challenged mice, preinjection with betulin (20 mg· kg -1 ·d -1 ) significantly decreased the levels of proinflammatory cytokines IFN-γ, TNF-α and IL-6, and ameliorated liver injury. Furthermore, pretreatment with betulin (20 mg· kg -1 ·d -1 ) significantly inhibited the Con A-induced activation of NKT and conventional T cells, and decreased production of proinflammatory cytokines IFN-γ, TNF-α and IL-6 in these two cell populations. Betulin has immunomodulatory effect on overly activated conventional T and NKT cells and exerts hepatoprotective action in mouse autoimmune hepatitis. The findings provide evidence for the use of H hedyotidea and its constituent betulin in the treatment of autoimmune diseases.

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“…Hepatic lymphocytes were isolated using a previously described protocol with minor modifications. 35 Briefly, mice were euthanized by intraperitoneal injection with pentobarbital sodium, and the liver was perfused with PBS via the portal vein until it became pale. The liver was then removed and gently pressed through a 200-gauge stainless steel mesh into PBS containing 2% FCS (Gibco, Carlsbad, CA, USA).…”

Section: Ethics Statementmentioning

confidence: 99%

Runt-related transcription factor 3 is involved in the altered phenotype and function in ThPok-deficient invariant natural killer T cells

et al. 2014

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The interplay between the CD4-lineage transcription factor ThPok and the CD8-lineage transcription factor, runt-related transcription factor 3 (Runx3), in T-cell development has been extensively documented. However, little is known about the roles of these transcription factors in invariant natural killer T (iNKT) cell development. CD1d-restricted iNKT cells are committed to the CD4 1 CD8 2 and CD4 2 CD8 2 sublineages, which respond to antigen stimulation with rapid and potent release of T helper (Th) 1 and Th2 cytokines. However, previous reports have demonstrated a new population of CD8 1 NKT cells in ThPok-deficient mice. In the current study, we sought to determine whether Runx3 was involved in the re-expression of CD8 and function of iNKT cells in the absence of ThPok. We used mice lacking Runx3, ThPok or both and verified that Runx3 was partially responsible for the appearance of CD8 1 iNKT cells in ThPok knockout mice. Additionally, Runx3 participated in the immune response mediated by iNKT cells in a model of a-galactosylceramide-induced acute hepatitis. These results indicate that Runx3 is crucial for the phenotypic and functional changes observed in ThPok-deficient iNKT cells. and the canonical T-cell receptor a (TCRa) chain (Va14-Ja18 in mice and V24-J18 in humans) coupled to specific Vb chains (Vb8, Vb7 and Vb2 in mice and Vb11 in humans). These receptors are responsible for the interaction of NKT cells with CD1d molecules expressed on CD4 1 CD8 1 double-positive (DP) thymocytes. 1 NKT cells are classified into type I (defined as invariant (iNKT) or classical NKT cells) and type II based on the antigens recognized by each and their TCR repertoires. 2 Our study focused on type I NKT cells (hereafter referred to as iNKT cells). iNKT cells recognize the glycosphingolipid antigen a-galactosylceramide (a-GalCer) and a-GalCer loading with the tetrameric form of CD1d is often used to define the iNKT cell subset because of the high affinity of CD1d for the iNKT cell TCR. 3 Although iNKT cells originate from CD4 1 CD8 1 (DP) thymocytes 4 and are selected by MHC class

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Methods for detection, isolation and culture of mouse and human invariant NKT cells

Cited by 128 publications

References 31 publications

Genetic engineering of hematopoietic stem cells to generate invariant natural killer T cells

Genetic engineering of hematopoietic stem cells to generate invariant natural killer T cells

Betulin from Hedyotis hedyotidea ameliorates concanavalin A-induced and T cell-mediated autoimmune hepatitis in mice

Runt-related transcription factor 3 is involved in the altered phenotype and function in ThPok-deficient invariant natural killer T cells

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