International Journal of Hematology

, Volume 90, Issue 2, pp 137–142

Human invariant natural killer T cells: implications for immunotherapy

Authors

  • Tsuyoshi Takahashi
    • Department of Hematology and OncologyGraduate School of Medicine, University of Tokyo
    • Department of Hematology and OncologyGraduate School of Medicine, University of Tokyo
Review Article

DOI: 10.1007/s12185-009-0379-1

Cite this article as:
Takahashi, T. & Kurokawa, M. Int J Hematol (2009) 90: 137. doi:10.1007/s12185-009-0379-1
  • 81 Views

Abstract

Human invariant natural killer T cells are a unique lymphocyte population that have an invariant T-cell receptor and recognize glycolipids instead of peptides in the restriction of CD1d molecules. These natural killer T cells play important roles in anti-tumor immunity, transplantation immunity, allergy, autoimmunity and microbial immunity. Since human natural killer T cells show high-level biological activity such as cytokine production, an anti-tumor effect and regulatory T-cell control, they may be a useful tool in immune-cell therapy. In this review, we summarize the immune responses mediated by human natural killer T cells, especially in tumor and transplantation immunity, and discuss their potential in clinical applications.

Keywords

NKTImmunotherapyTumor immunityTransplantation immunity

1 Introduction

Natural killer T (NKT) cells were originally defined by their co-expression of the αβ T-cell receptor (TCR) and NK receptor. In humans, these NKT cells consist of around 25% blood T cells and predominantly comprise memory phenotype (CD45 RO+) T cells [1, 2]. Invariant NKT (iNKT) cells make up only a small minority of human NKT cells, because these cells represent no more than 1% of all T cells in the lymphoid tissues and liver [3, 4]. The characteristics of iNKT cells and conventional NKT cells (T cells that express NK receptors) are listed in Table 1. iNKT cells are a unique T-cell population capable of regulating tumor immunity, transplantation immunity, allergy, autoimmunity and microbial immunity [5, 6]. Furthermore, it has recently been reported that iNKT cells and CD4+CD25+ regulatory T (Tregs) cells regulate each other [7, 8]. iNKT cells show high-level biological activities, such as a pronounced production of cytokines and chemokines, and cytotoxic activity. In addition, they can be expanded like T cells in vitro [9]. Given that iNKT cells exhibit these characteristics, they are a good candidate to become a potent immunological tool to regulate immune responses in humans. In this study, we review the immune response of human iNKT cells, especially in tumor and stem cell transplantation immunity, and their application to immune-cell therapy.
Table 1

Characterization of human natural killer T cells

 

Invariant NKT

Conventional NKT

TCRα repertoire

Vα24-Jα18 invariant

Heterogeneous

TCR Vβ repertoire

Vβ11

Heterogeneous

CD4/CD8

CD4−CD8−, CD4+, CD8+

CD4+, CD8+

Antigen recognition

CD1d restricted

HLA restricted

Cytokine production

CD4−CD8−, CD8+ (mainly Th1)

CD8+ (Th1)

CD4+ (Th1, Th2)

CD4+ (Th1, Th2)

Antitumor activity

++

− ~ +

2 Phenotype and function

Mouse iNKT cells were originally defined as cells that express NK1.1 (NKR-P1B or NKR-P1C) and use an invariant TCR α-chain (Vα14-Jα18) that pairs preferentially with Vβ8.2, 7 or 2 and are restricted with the non-polymorphic, major histocompatibility complex (MHC) class I-like molecule, CD1d [1012]. However, it is problematic to define iNKT cells as NK1.1+ T cells because NK1.1 expression is restricted to some mouse strains such as C57BL/6 and other strains do not express it, although CD1d-dependent T cells exist in these strains. Furthermore, not all CD1d-dependent T cells that have invariant TCR express NK1.1 in C57BL/6 mice. Thus, mouse iNKT cells are defined more accurately as CD1d-dependent natural killer-like T cells [13, 14]. The human homologs of mouse iNKT cells are Vα24 TCR+ iNKT cells, because many features of mouse iNKT cells are conserved in human Vα24+ iNKT cells [1517]. Human iNKT cells express the NK receptor, CD161 (NKR-P1A), and the TCR of the Vα24-Jα18 invariant TCR α-chain that pairs preferentially with Vβ11 (human Vα24 and Vβ11 are homologs of mouse Vα14 and Vβ8, respectively [1821]). Thus, human iNKT cells can be identified as a Vα24/Vβ11 TCR double-positive or CD1d-tetramer-positive fraction. The frequency of human iNKT cells in each organ is largely different from that in mice. In mice, the frequency of iNKT cells among whole T cells in the bone marrow and liver are ~20 and ~30%, respectively, while in humans, they are both very low [4, 22]. The frequency of iNKT cells in healthy human peripheral blood is about 0.05% of CD3+ T cells, although wide variation is observed among individuals [23]. In C57BL/6 mice, more than half of NK1.1+ T cells are iNKT cells and vice versa [13], while, in humans, iNKT cells comprise less than 1% of CD161+ T cells, although most of the Vα24+ invariant T cells express CD161 [24]. Phenotypically, CD4−CD8−double-negative (DN) and CD4+ iNKT cells have been reported in mice, while DN, CD4+ and CD8+ iNKT cells have been reported in humans [3, 25, 26]. The functions of human are similar to those of mouse iNKT cells in many respects. Both have been shown to be activated by synthetic glycolipids such as α-galactosylceramide (α-GalCer) in a CD1d-restricted and invariant TCR-mediated manner. They produce many cytokines, such as IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IL-21, IFN-γ, TNF-α, TGF-β and GM-CSF, which can potentially influence immune regulation, such as the determination of the Th1/Th2 profile and/or activation or suppression of immune reactions [3, 27, 28]. Especially, they rapidly and abundantly produce large amounts of IFN-γ and IL-4 on stimulation. Interestingly, DN and CD8+ iNKT cells preferentially produce Th1-type cytokines, while CD4+ iNKT cells produce both Th1-and Th2-type cytokines in humans, although all of them show a similar direct cytotoxicity toward tumors [3, 25, 26]. iNKT cells also produce chemokines such as RANTES, Eotaxin, MIP-1α and MIP-1β [29]. In addition, iNKT cells express cytolytic molecules such as granzyme B, perforin and FasL and exhibit cytolytic activity against tumor cells, which might be important as an anti-tumor effect.

3 iNKT ligands

The first described iNKT ligand was KRN7000 derived from a marine sponge, which was identified by screening the anti-tumor activity of melanoma-transplanted mice [30]. Later, it was referred to as α-GalCer [31]. Subsequently, some synthetic derivatives were reported. OCH, which is a truncated type of α-GalCer in both the acyl and sphingosine chains, induces more IL-4 and less IFN-γ compared with α-GalCer [32]. α-C-GalCer is a carbon glycoside analog of α-GalCer, which induces more IFN-γ and less IL-4 compared with α-GalCer [33]. Regarding physiological ligands, isoglobotrihexosylceramide (iGb3) was reported as a self-ligand [34]. iGb3 appears to be an important self-ligand for the development of iNKT cells, because β-hexosaminidase-B-deficient mice, which lack the ability to degrade iGb4 into iGb3, exhibited a marked reduction of NKT cells production. However, this is now controversial because iGb3 could not be detected in mouse or human thymocytes and dendritic cells [35]. Furthermore, iNKT cells developed normally in mice lacking iGb3 synthase, the enzyme responsible for iGb3 synthesis [36]. Other possibly important physiological ligands for iNKT cells are microbial antigens, such as glycosylceramides from the cell wall of Gram-negative lipopolysaccharide (LPS)-negative Sphingomonas bacteria, which are usually found in the environment [3739].

4 iNKT and tumor immunity

The anti-tumor effect of iNKT cells has been well studied in the context of the α-GalCer-induced activation of these cells in mice. Actually, α-GalCer was originally screened due to its anti-melanoma effect, as mentioned above [30]. iNKT cells exhibit anti-tumor activity in many ways. They activate anti-tumor immune cells such as NK cells by IFN-γ secretion and also lead to the maturation and activation of dendritic cells (DCs) by CD40-CD40L interaction and cytokine production, such as IFN-γ and IL-4, and, in turn, DCs activate NK cells and CD8+ cytotoxic T lymphocytes (CTL) (Fig. 1) [4044]. Furthermore, recently there was a report that iNKT cells inhibited immunosuppressive myeloid-derived suppressor cells and elicited an anti-tumor effect [45]. However, the anti-tumor function of iNKT cells is still controversial [46, 47]. In humans, iNKT cells recognize tumor cells directly and exhibit cytotoxicity against them. Metelitsa et al. [48] reported that human iNKT cells directly recognized CD1d+ tumor cell lines and mediated anti-tumor cytotoxicity. They also observed that human CD1d+ myelomonocytic leukemia cells were effective, direct targets of iNKT cells [49]. We reported that many human leukemic T-cell lines expressed CD1d and could be directly killed by iNKT cells in a CD1d-dependent fashion [50]. This killing activity was observed in the absence of α-GalCer. However, the addition of α-GalCer to the assay system enhanced this killing activity. Moreover, some primary leukemic cells from T-cell acute lymphoblastic leukemia (T-ALL) patients expressed CD1d, and they were good, direct targets of iNKT cells. Thus, iNKT cells can exhibit potent anti-tumor activity, both directly by targeting CD1d and indirectly by activating immune cells such as NK cells and CTLs.
https://static-content.springer.com/image/art%3A10.1007%2Fs12185-009-0379-1/MediaObjects/12185_2009_379_Fig1_HTML.gif
Fig. 1

iNKT cells activate many immune cells. iNKT cells and DCs interact with each other. iNKT cells are activated on recognizing the antigen presented by DCs and also by IL-12 from DCs. In turn, activated iNKT cells mature DCs through cytokine (IL-4, GM-CSF, TNF-α, etc.) and CD40-CD40L stimulation. Activated iNKT cells produce many cytokines such as IL-2 and IFN-γ, and activate numerous immune cells such as NK cells, B cells and T cells. Mature DCs also efficiently activate these immune cells

5 iNKT and stem cell transplantation

Allogeneic stem cell transplantation is a curative therapy for hematologic malignancies. However, graft-versus-host disease (GVHD) is one of the most serious complications. It has been indicated in a mouse acute GVHD model that NK1.1+ T cells obtained from donor bone marrow can suppress GVHD induced by peripheral blood T cells from the same donor [51]. It has also been shown that a selected conditioning regimen, which preserves more host-residual NK1.1+ or DX5+ T cells than other T cells, is advantageous for reducing acute GVHD [52]. Furthermore, the suppressive effect of α-GalCer on induced acute GVHD has been demonstrated in a mouse model [53, 54]. We and others provided direct evidence that iNKT cells reduced GVHD in a MHC-mismatched bone marrow transplantation model [54, 55]. In this model, host-residual iNKT cells played a crucial role in reducing the severity of GVHD, and this reduction was associated with a delayed increase in serum Th2 cytokine levels. In humans, the number of NKT cells was lower in patients with, than in those without, GVHD after hematopoietic stem cell transplantation [56]. Furthermore, a conditioning regimen involving fractionated total lymphoid irradiation (TLI) plus anti-thymocyte globulin, which is a strategy to increase the number of iNKT cells and protect against GVHD in a mouse GVHD model [52, 57], decreased the incidence of acute GVHD after hematopoietic stem cell transplantation in humans [58]. An in vitro study revealed that donor CD4+ T cells showed a marked increase in IL-4 production in transplant recipients and also showed a marked reduction in their proliferative response to alloantigenic stimulation in the mixed-lymphocyte reaction, although the precise underlying mechanisms for a reduction in acute GVHD through the use of TLI and ATG conditioning are as yet unknown in humans [58]. Given that iNKT cells can recognize and kill hematological tumors and also suppress GVHD, we may be able to separate GVHD and the graft versus leukemia/lymphoma (GVL) effect using iNKT cells. Morris et al. [59] reported that donor iNKT cells activated by G-CSF analogs elicited GVL activity, while they reduced GVHD in a murine model of allogeneic stem cell transplantation. Pillai et al. [60] also reported that host iNKT cells protected against GVHD and the progression of B-cell lymphoma after TLI and anti-thymocyte serum conditioning in a murine model. However, Morris et al. [61] recently reported that iNKT cells aggravated GVHD on administration of G-CSF after bone marrow transplantation in a murine model; thus, iNKT cells appear to be a double-edged sword in the allogeneic transplantation setting .

6 Implications for immunotherapy

As mentioned above, iNKT cells can play an important role in tumor immunity, transplantation immunity, and so on. Thus, iNKT cells might be a powerful tool for immune-cell therapy. Some clinical trials of NKT cell therapy have been reported. Table 2 summarizes these. Giaccone et al. [62] reported a phase I study involving the intravenous injection of α-GalCer in twenty-four advanced solid tumor patients. In that study, no dose-limiting toxicity was observed, and the therapy was well tolerated. Immuno-monitoring demonstrated that circulating iNKT cells decreased rapidly and serum levels of TNF-α and GM-CSF increased. However, no anti-tumor effect was observed clinically. Because the direct injection of glycolipid antigen has the potential to induce iNKT cell anergy, it might be better to use glycolipid-loaded DCs for therapy [63, 64]. In other clinical trials, α-GalCer-loaded DCs or in vitro-expanded iNKT cells were administrated [6569]. All therapies were well tolerated, and immune-monitoring revealed some immunological responses in each clinical study. However, the clinical responses were limited in all trials, and so further studies are needed to achieve better and reproducible clinical results.
Table 2

Clinical trials of iNKT-based immunotherapy

Report (reference)

Patients

Diagnosis

Methods

Toxicity ≥grade 3

Immunological response

Clinical response

Giaccone et al. [62]

24

Solid tumors

α-GalCer iv

Fever (1)

Increased serum TNF-α and GM-CSF

SD (7)

Nieda et al. [65]

12

Solid tumors

α-GalCer-loaded DCs iv

None

Increased iNKT and NK cells,

increased serum IFN-γ and IL-12

Decreased tumor marker (2),

necrosis of tumor (1)

Ishikawa et al. [66]

11

Lung cancer

α-GalCer-loaded DCs iv

None

Increased iNKT cells

SD (3)

Motohashi et al. [67]

6

Lung cancer

iNKT cells iv

None

Increased IFN-γ secreting MNCs

SD (2)

Chang et al. [68]

5

Myeloma,

solid tumors

α-GalCer-loaded DCs iv

None

Increased iNKT cells,

increased serum IL-12 and IP-10

Decreased serum M protein (1), decreased urine M protein,

SD (1)

Uchida et al. [69]

9

Head and neck cancer

α-GalCer-loaded APCs submucosa

None

Increased iNKT cells,

enhanced NK activity

PR (1), SD (5)

Numbers of patients are indicated in parenthesis

PR partial response, SD stable disease, MNCs mononuclear cells, IP-10 IFN-γ-inducible protein-10

It is difficult to predict whether iNKT cells enhance or attenuate anti-tumor effect, because iNKT cells produce both Th1-and Th2-type cytokines. Furthermore, iNKT cells can activate regulatory T cells and, in turn, regulatory T cells suppress immune responses (Fig. 2). However, there may be some ways to overcome this problem. One is to use subpopulations of iNKT cells. As mentioned above, CD4− iNKT cells preferentially produce Th1-type cytokines, while CD4+ iNKT cells produce both Th1- and Th2-type cytokines. Thus, the use of in vitro-expanded CD4− iNKT subpopulations may elicit a favorable anti-tumor response. The use of iNKT ligands that preferentially induce a Th1-deviated response such as α-C-GalCer in cancer cell therapy also may be effective. Another method is to select tumors that express CD1d. iNKT cells can directly recognize CD1d+ tumor cells such as T-ALL, myelomonocytic leukemia, and other hematological malignancies, and exhibit potent cytotoxicity. Thus, direct and indirect synergistic effects may be achieved using iNKT cells. One effective situation to perform iNKT cell therapy may be to apply it after stem cell transplantation in CD1d+ hematological malignancy patients, because iNKT cells suppress GVHD while preserving the GVL effect, as reported in a mouse model [60]. Recently, an excellent review, which focused on vaccination strategies involving iNKT cells, was reported [70].
https://static-content.springer.com/image/art%3A10.1007%2Fs12185-009-0379-1/MediaObjects/12185_2009_379_Fig2_HTML.gif
Fig. 2

iNKT cells are a double-edged sword in tumor immunity. iNKT cells activate or suppress the immune response depending on the conditions. Under some conditions, iNKT cells directly recognize the tumor and/or activate anti-tumor effector cells such as NK cells and CTL, and exhibit anti-tumor activity. In other conditions, however, iNKT cells suppress anti-tumor effector cells via Th2-type cytokines and/or the activation of regulatory T cells (Tregs). The open arrow represents activation and the filled arrow suppression

7 Conclusions

We have summarized the immune response of human iNKT cells and discussed the possibility of employing them in cancer cell therapy and immunotherapy after stem cell transplantation. Although there are still many unclarified areas regarding the biology of iNKT cells, they have the potential to become a very effective tool to regulate human immunity, such as tumor and transplantation immunity. We hope that iNKT cell therapy will be developed and established as a standard treatment in the near future.

Copyright information

© The Japanese Society of Hematology 2009