iNKT Cell Overview

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• What are iNKT cells?
• iNKT cell development
• iNKT activation and repression
• Repression of iNKT cells
• iNKT cell markers
• iNKT cell isolation

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Invariant natural killer T (iNKT) cells belong to the heterogenous group of natural killer T (NKT) cells, which display properties of both T cells and natural killer (NK) cells and help to bridge the innate and adaptive immune responses [4]. iNKT cells are CD1d-restricted T lymphocytes that employ an invariant T cell receptor (TCR) alpha chain plus a limited repertoire of TCR beta chains to recognize specific lipid antigens (Figure 1) [1,2,3]. They have been observed to respond quickly (within minutes) to lipid antigenic stimulation, secreting different types of cytokines depending on the stimuli [5,6]. iNKT cells are among the first cell type to respond to a pathogenic stimulus, and their cytokine production helps to determine the course of the immune response [6]. Due to their antitumor efficacy, iNKT cells have recently been investigated as potential targets for cancer immunotherapy [4,5].

In human peripheral blood, iNKT cells represent ~1% of circulating T cells, while in human adipose tissue this percentage increases to ~10–25% [5]. In human liver, iNKT cells are found in much lower frequencies; non-invariant or diverse NKT (dNKT) cells predominate [5]. As in human adipose tissue, iNKT cells are found at a frequency of ~10–25% in mouse adipose tissue. However, iNKT cells represent a much greater percentage (~20–50%) of T cells in mouse liver, as compared with human liver [5]. In mouse thymus, spleen, and blood, the frequency of iNKT cells is low, ~0.5–2% of circulating T cells [5].

Like conventional T cells, iNKT cells develop in the thymus [2,3,7]. The double-positive (DP; CD4⁺ CD8⁺) thymocytes act as progenitor cells for all lymphocytes that belong to the αβ T cell lineage [7,8]. iNKT cells belong to this subset and therefore also have DP thymocytes as progenitors, although a small proportion utilize an alternate pathway (see below) [2,3,8]. Like conventional T cells, iNKT cells develop through four double-negative (DN) stages (DN1–DN4), with a β-chain rearrangement occurring at the DN3 stage [3,8]. While a small subset can mature directly after the DN4 stage through an alternative pathway; the majority of DN iNKT cells transition into the four DP stages of development (S0–S3) [7,8].

After completing the four DN stages, the DP cells go through four additional stages of development, starting with the random rearrangement of their TCRα loci resulting in the invariant α-chains (Table 1) [1,2,3,8]. Unlike DP precursors of conventional T cells, which are selected by thymic epithelial cells, iNKT DP precursors are positively selected when their TCR recognizes self-lipid antigens on CD1d-expressing DP thymocytes [4,7,8]. With this unique DP-DP interaction, the positively selected thymocytes are imparted with the developmental program of iNKT cells during S0 [9,10,11]. This process generates secondary signals and co-stimulation through members of the signal lymphocytic-activated molecules (SLAM) family of receptors [7,8,9]. The engagements through the TCR and the SLAM receptors result in downstream expression of the Egr2 transcription factor in the iNKT precursors. During S1, this Egr2 transcription factor is then recruited to the promoter site of the Zbtb16 gene, which encodes the master iNKT transcription factor PLZF [2,3,7,8,9,10,11]. Expression of the PLZF transcription factor is required for guiding the effector properties of the developing iNKT cells [2,3,7]. In particular, PLZF is responsible for specifying the migration patterns of iNKT cells to peripheral organs, as well as for the ability to produce cytokines when iNKT cells are stimulated [2,3].

iNKT cells migrate out of the thymus at S2 or S3. The NKT2 and NKT17 subsets refer to S2 iNKT-migrated cells, whereas the NKT1 subset develops through S3 [2,3,8]. These three subsets have lineage-specific transcription factors and produce different combinations of cytokines (Table 2) [2,3,8]. NKT1 cells express T-box 21 transcription factor and can release interferon gamma (IFNγ). NKT2 cells have GATA binding protein 3 (GATA3) expression and release interleukin (IL)-4 cytokine at steady state. NKT17 cells express the retinoic acid receptor–related orphan nuclear receptor gamma (RORγt) transcription factor and IL-17 cytokine.

The alternative pathway of a small subset of iNKT precursors includes the omission of development through the DP stages [2,3,8]. In this alternative development process, precursors from the DN4 stage contain a population that expresses Vα14Jα18 mRNA, which allows for production of mature DN iNKT cells [2,3]. Thus, based on CD4 and CD8 expression, mature iNKT cells can be divided into three functionally distinct subsets: CD4⁺ CD8^-, CD4^- CD8^-, and CD4^- CD8⁺ [12].

Because iNKT cells express the αβ TCR, it was assumed that they would recognize and respond to peptides expressed by major histocompatibility complex (MHC) antigen–presenting proteins, similar to conventional T cells [1,2]. However, it is now widely accepted that iNKT cells predominantly recognize and respond to glycolipids [13,14,15,16]. These lipid antigens are presented by the CD1d molecule, a nonpolymorphic MHC class I-like glycoprotein. In mice, the invariant or fixed Vα14Jα18 TCR α-chain is usually paired with a Vβ8.2, Vβ7, or Vβ2 TCR β-chain [13,14,15,16]. Human iNKT cells also have a similar complex in which the Vα24Jα18 TCR α-chain is paired with the Vβ11 TCR β-chain. The mouse and human TCR α-chains and the mouse and human TCR β-chains are homologs, and consequently the mouse and human iNKT cells have highly conserved antigen specificity [13,14,15,16].

In vivo stimulation of iNKT cells through their TCR induces a rapid inflammatory response. Once the TCR recognizes the Cd1d-expressed lipid antigens, iNKT cells release various cytokines, such as T-helper type 1 (Th1) and type 2 (Th2) cytokines [2,3,4,5,14]. Stimulation of iNKT cells induces the release of cytokines by other immune cells as well [13,14,15,16]. The nature of the iNKT cell responses depends on the structure of the lipid antigen that is identified. Some lipid antigens induce the release of Th1 cytokines such as IFNγ and tumor necrosis factor (TNFα), while other lipid antigens may cause release of Th2 cytokines including IL-4 and IL-13 (Table 2) [15,16]. The lipid antigen glycolipid α-galactosylceramide (αGalCer) can cause a Th0 skewing response, which stimulates the iNKT cells to release both Th1 (IFNγ) and Th2 (IL-4) type responses [17,18].

Biasi et al. (2016) investigated possible phenotypic changes of iNKT cells in patients with multiple sclerosis (MS) [19]. They studied a total of 165 patients at various stages of the disease and found that patients with progressive MS showed a higher ratio of CD4+ iNKT cells. They also found that patients with MS mainly produced Th1 and Th17 cytokines, such as TNFα, IFNγ, and IL-17 [19]. Their data suggest that the persistent inflammatory status in active and progressive MS disease is characterized by the presence of cytokine-producing CD4+ iNKT cells [19].

The rapidly expanding array of immunotherapies has led to increased interest in investigating iNKT cells for therapeutic purposes. It has been suggested that, in addition to cytotoxic T lymphocytes (CTLs) and NK cells, iNKT cells can also elicit antitumor responses [4,5,18]. iNKT cells can induce antitumor responses either by killing cancer cells through antigen recognition or by depleting the immunosuppressive tumor-associated macrophages (TAMs) [4,5,18]. This depletion of TAMs leads to downstream activation and increased trafficking of NK cells and CTLs. Studies done in mice showed that administration of αGalCer or αGalCer-pulsed dendritic cells leads to increased release of IFNγ and tumor inhibition [18,19]. Furthermore, iNKT cell activation increases lipid biosynthesis through promotion by PPARγ and PLZF transcription factors [18,20]. Lactic acid build-up in the tumor microenvironment may also be responsible for the reduction in expression of PPARγ in iNKT cells, leading to reduced lipid biosynthesis and downstream inhibition [18,20].

As mentioned earlier, iNKT cells recognize bacterial lipid antigens such as αGalCer. For flow cytometry, CD1d-based tetramers that are loaded with αGalCer analogs can be used to detect iNKT cells [21]. iNKT cells can be identified based on their CD3 expression and their binding of CD1d-based tetramers [21]. Subpopulations of iNKT cells can be identified using the expression profiles of other iNKT cell markers (Table 3) [21,22,23,24].

Table 2. Key cytokines secreted by iNKT cells.

Table 3. iNKT cell markers used for cell type identification [22,23,24].

iNKT cells are a heterogenous population of T cells that express NK cell markers, as well as a TCR [21,22]. These NK cell markers include CD161 and CD56 in humans, and NK1.1 and DX5 in mice [21,22]. Both human and mouse iNKT cells can be isolated from peripheral blood mononuclear cells (PBMCs). Through flow cytometry, PBMCs can be sorted with antibodies for TCR Vα24Jα18, CD3, CD4, CD8, and CD161 to gate and isolate iNKT cell populations [21,22].

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