Chapter 8 The Role of NKT Cells in Tumor Immunity

NKT cells are a relatively newly recognized member of the immune community, with profound effects on the rest of the immune system despite their small numbers. They are true T cells with a T cell receptor (TCR), but unlike conventional T cells that detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d, a nonclassical MHC molecule. As members of both the innate and adaptive immune systems, they bridge the gap between these, and respond rapidly to set the tone for subsequent immune responses. They fill a unique niche in providing the immune system a cellular arm to recognize lipid antigens. They play both effector and regulatory roles in infectious and autoimmune diseases. Furthermore, subsets of NKT cells can play distinct and sometimes opposing roles. In cancer, type I NKT cells, defined by their invariant TCR using Vα14Jα18 in mice and Vα24Jα18 in humans, are mostly protective, by producing interferon‐γ to activate NK and CD8⁺ T cells and by activating dendritic cells to make IL‐12. In contrast, type II NKT cells, characterized by more diverse TCRs recognizing lipids presented by CD1d, primarily inhibit tumor immunity. Moreover, type I and type II NKT cells counter‐regulate each other, forming a new immunoregulatory axis. Because NKT cells respond rapidly, the balance along this axis can greatly influence other immune responses that follow. Therefore, learning to manipulate the balance along the NKT regulatory axis may be critical to devising successful immunotherapies for cancer.

Introduction

NKT cells are a unique small subpopulation of true T cells, not NK cells, that nevertheless play a major role in regulating immune responses by bridging the innate and adaptive immune systems. Like cells of the innate immune system, NKT cells are among the first responders on the scene in a variety of infectious and inflammatory responses, and they set the stage and the tone for the subsequent adaptive immune response. They are pre‐armed with cytokine mRNA, and can produce cytokines very quickly on activation. These cytokines can then determine the nature and quality of the antigen‐specific T cell response that ensues. Like true T cells, they have an antigen‐specific T cell receptor (TCR) that allows them to recognize both self antigens and foreign antigens, and they provide the immune system with a mechanism for detecting lipid antigens not detected by conventional T cells. Their name was based on the original observation of expression of NK cell markers not present on conventional T cells except when activated, but these NK markers are no longer a pre‐requisite for defining NKT cells as we currently understand them. They have been found to play an important role in regulating transplantation tolerance, autoimmune disease, allergic disease and asthma, inflammatory responses, and infectious diseases ranging from bacteria and viruses to fungi and parasites.

In cancer, NKT cells were originally found in mostly a protective role, but more recently they have been found to also inhibit tumor immunosurveillance as well as cancer immunotherapy. This paradoxical behavior has now been found to be due to the presence of different subsets of NKT cells that mediate different functions. In one dichotomy between NKT cells with an invariant TCR (Type I) and those with more variable TCRs (Type II), these types have been found recently to cross‐regulate each other and thus to form a new immunoregulatory axis that can modulate subsequent immune responses. NKT cells can also regulate other innate immune cells such as dendritic cells (DCs), myeloid‐derived suppressor cells, and NK cells. In this review, we will focus on the role of different subsets of NKT cells in the immune response to cancer, both natural and induced, while also briefly reviewing their role in other disease processes to illustrate their potential activities where these have been better described in those diseases. We will first discuss each type of NKT cell and its function, and then discuss their interactions with each other and other cells and the potential translational/clinical applications of this knowledge. We conclude that learning to manipulate NKT cell function may lead to novel methods to treat or prevent cancer, or to synergize with other immunotherapies of cancer.

The discovery of NKT cells was not a single “Eureka moment” that occurred at one point in time, but rather a gradual process in which at least three developing independent lines of evidence from different subfields of immunology converged to lead to the definition of the NKT cell and the subsequent evolution of that concept to the understanding that we have today(Bendelac et al., 1997, Bendelac et al., 2007, Godfrey, et al., 2004, Macdonald, 2007, Taniguchi et al., 1996). It is of interest to note that one of the first lines of evidence was from an unexpected finding in examining the TCR repertoire of a series of suppressor T cell hybridomas, which were all found to use the same Vα chain (Vα14), the same Jα segment (Jα281, now called Jα18), and the same single N‐region glycine residue (Imai et al., 1986, Koseki et al., 1989). Thus, one of the earliest lines of evidence for the existence of NKT cells also indicated a potential regulatory role for these cells. Cells with this unique TCR were subsequently found at a surprisingly high frequency for a single TCR chain, at 1–2% of mouse spleen cells, 10–20% of liver hematopoietic mononuclear cells, and 40% of CD3⁺ T cells in the bone marrow (Cui et al., 1997, Koseki et al., 1989, Lantz and Bendelac, 1994, Makino et al., 1995). The fact that these cells used the same TCRα chain in mice of multiple MHC types suggested that they may be recognizing a monomorphic MHC molecule rather than the conventional polymorphic ones that differed among mouse strains.

Another line of evidence also related to TCR repertoire. Two groups reported the presence of a small number (0.4%) of mouse thymocytes that expressed Vβ8 despite being CD4⁻CD8⁻ double negative (DN), a population that had been thought to be too immature to express a TCR, and these were found to be CD44⁺, CD5⁺, and NK1.1⁺ (Ballas and Rasmussen, 1990, Budd et al., 1987, Fowlkes et al., 1987, Sykes, 1990). These cells were also characterized in almost every case as producing copious amounts of cytokines. A similar population was identified among mature CD4⁺ T cells (Arase et al., 1992, Bendelac et al., 1994a, Hayakawa et al., 1992, Takahama and Singer, 1992, Takahama, et al., 1991). These cells were also found to express lower levels of CD3 (Arase et al., 1992, Arase et al., 1993, Bendelac et al., 1994a, Levitsky et al., 1991).

The third line of evidence helped to tie these two lines together and explain the invariant TCR. Lantz and Bendelac were able to produce thymocyte hybridomas that were Vβ8⁺, CD5 high, and CD44⁺, and these were found to have mRNA for the Vα14 TCR chain (Lantz and Bendelac, 1994). Similarly, the Vα14⁺ cells in peripheral tissues were found to also express predominantly Vβ8 and NK1.1 (Makino et al., 1995, Taniguchi et al., 1996). Thus, the unusual Vβ8⁺ cells in the thymus turned out to be the same cells (or precursors) as the invariant TCR Vα14⁺ cells originally detected as suppressor T cell hybridomas. Further, these Vα14/Vβ8 thymocyte hybridomas were found to recognize a relatively monomorphic nonclassical class I‐like MHC molecule, CD1d, explaining their ability to use a nearly identical TCR in mice of different MHC types (Bendelac, 1995, Bendelac et al., 1995). This explained the finding that NK1.1⁺ TCRαβ T cells in liver, whether CD4⁺ or CD4⁻CD8⁻ DN, required β2‐microglobulin and thus a molecule in the class I MHC family (Ohteki and MacDonald, 1994). Thus, the concept gradually emerged of a specialized subset of TCRαβ⁺ T cells that recognized antigens presented by the class I‐like MHC molecule CD1d, expressed NK1.1, high levels of CD5 and CD44, and usually expressed a particular semi‐invariant TCR using the Vα14Jα18 TCRα chain and most often a Vβ8 (and more recently also Vβ2 or Vβ7) TCRβ chains. The concept was further solidified when it was discovered that these cells responded to a glycolipid antigen, α‐Galactosylceramide (α‐GalCer), derived from a marine sponge or microorganisms symbiotic with the sponge (Kawano et al., 1997). These cells were termed NKT cells (Godfrey, et al., 2004, Makino et al., 1995) because of their NK cell‐like expression of NK1.1 despite being true CD3⁺ T cells. Similar Vα24Jα18⁺Vβ11⁺ NKT cells were found in humans, in which Vα24 and Vβ11 are the human homologs of Vα14 and Vβ8 (Dellabona et al., 1994, Porcelli et al., 1993).

As noted above, NKT cells were originally defined based on their expression of NK‐like markers such as NK1.1 (CD161) even though they were true CD3⁺ T cells. However, confusion arose early on about what cells really constituted the NKT population (Godfrey et al., 2004). NKT was interpreted to mean natural killer T cells even though these cells were not particularly characterized by NK‐like functions, although they were part of the innate immune system. Also, NK1.1 was not expressed in many mouse strains, and T cells that otherwise fit the definition of NKT cells, including expression of the invariant Vα14Jα18⁺Vβ8⁺ TCR and recognition of CD1d, were sometimes found to be NK1.1 negative. Also, conventional T cells, when activated, express NK1.1 as an activation marker, so this was not a reliable marker to distinguish NKT cells from conventional class I or class II MHC‐restricted T cells (Assarsson et al., 2000, Slifka et al., 2000, Terabe et al., 2008). For all these reasons, the definition of NKT cells was modified to encompass all true TCRαβ T cells that were restricted by the nonclassical class I MHC molecule CD1d (Godfrey et al., 2004).

Even within this CD1d‐restricted population now defined as NKT cells, different subsets could be defined. The major distinction was between the classical NKT cells expressing the invariant Vα14Jα18 TCR in the mouse or Vα24Jα18 in the human, called type I NKT cells (also known as invariant NKT cells or iNKT cells), and another subset of cells that were also CD1d‐restricted but expressed more diverse TCRs, not employing the canonical Vα14Jα18 or Vα24Jα18, which were dubbed type II NKT cells (Godfrey et al., 2004) (Table I). The discovery and characterization of the latter subset will be discussed in Section III.A later. However, it was clear that the only common characteristic was the CD1d restriction, and therefore the absence of all NKT cells (both type I and type II) in CD1d deficient mice. Therefore, also, the development of CD1d‐α‐GalCer tetramers made it possible to uniquely and unambiguously identify type I NKT cells by flow cytometry (Benlagha et al., 2000, Karadimitris et al., 2001, Mars et al., 2002), but unfortunately, CD1d tetramers that identify all type II NKT cells are not available (although a subset may be identifiable by CD1d‐sulfatide tetramers (Jahng et al., 2004) as discussed in Section III.A). A consequence of this definition is that because CD1d was found to present lipids or glycolipids rather than peptides (Bendelac et al., 2007, Brutkiewicz, 2006, Tupin et al., 2007), NKT cells are the primary class of T cells that can provide the immune system with a mechanism of specific recognition of lipid antigens, whether from self or from microbial invaders. These are reviewed under the individual subsets. Other independent characteristics used to define subsets of NKT cells include CD4⁺ vs CD4⁻CD8⁻ DN populations, and NK1.1⁺ and NK1.1⁻ populations, as well as tissue origin (Ambrosino et al., 2007, Berzofsky and Terabe, 2008, Terabe and Berzofsky, 2007). These will be discussed below in the context of functional activities of type I or type II NKT cells.

NKT cells are positioned to play a pivotal role in the immune system, as they form a bridge between the innate and adaptive immune systems, having a foot in both camps. They have true TCRs and antigen specificity like conventional T cells, albeit for lipids rather than peptides, but they also have a more limited repertoire and rapid response characteristic of the innate immune system. Other true T cells that have such specialized function exist, such as γδ‐T cells and MAIT (mucosal associated invariant T) cells. Like NKT cells, MAIT cells express a canonical TCR, in this case using the Vα19Jα33 chain in mice and Vα7.2Jα33 in humans, and they also appear to play a regulatory role (Shimamura and Huang, 2002, Treiner et al., 2003). Unlike NKT cells, they depend on the gut flora and are absent in germ‐free mice. Some γδ‐T cells also express NK‐like markers and fill unique niches (Arase et al., 1995, Carding and Egan, 2002, Lees et al., 2001, Vicari et al., 1996). At various times, some of these have been included in the category of NKT cells but are now recognized to be distinct T cell subsets.

Like innate immune cells, NKT cells are rapid responders on the scene when the immune system is activated, and help to call into play other members, ranging from NK cells of the innate immune system to conventional CD4⁺ or CD8⁺ T cells of the adaptive immune system. Thus, their regulatory role can be pivotal in orchestrating other responses that come later. From the earliest studies on NKT cells as described above, even before the term NKT cells was applied, they were found to be copious producers of cytokines, both Th1 cytokines like interferon‐γ and Th2 cytokines like IL‐4 or IL‐13 (Table I). Two recent studies have found that an NK1.1‐negative subset of type I NKT cells can rapidly produce IL‐17, contributing to neutrophil recruitment (Michel et al., 2007, Rachitskaya et al., 2008). NKT cells can also make IL‐21, which can act back on NKT cells in an autocrine fashion (Coquet et al., 2007). It is now understood that the rapid cytokine response relates in part to the presence of preformed mRNA for cytokines such as interferon‐γ and IL‐4, allowing the cell to respond more quickly without the need for gene transcription (Matsuda et al., 2003, Stetson et al., 2003). The presence of the preformed mRNA in turn may relate to the recognition of self antigens that keep the NKT cells primed to respond, as suggested by their ability to respond to IL‐12 produced by macrophages or DCs stimulated by bacterial LPS, even in the absence of an exogenous CD1d‐presented specific antigen (Brigl et al., 2003). This very early production of cytokines by NKT cells was proposed as a potential solution to the dilemma that it takes IL‐4 to induce a Th2 cell to make IL‐4, so where does the initial IL‐4 come from to initiate the process? It was found that IL‐4 from NKT cells could promote Th2 responses and IgE production, and that defective IgE production in SJL mice was related to the absence of CD4⁺ NKT cells that made IL‐4 (Yoshimoto et al., 1995a, Yoshimoto et al., 1995b). Although NKT cells may not be the only such source of early IL‐4, their ability to respond first and steer subsequent adaptive responses makes their regulatory functions all the more influential throughout the immune system.

NKT cells also function as part of the adaptive immune system, in filling a void in the antigen repertoire of conventional T cells, which generally recognized only peptide fragments of proteins, not lipids. The ability of NKT cells to recognize self lipids, discussed below under the individual subsets, may be one reason they can have profound impact on autoimmune disease. On the other hand, their ability to recognize bacterial lipids, also outlined below, gives the adaptive T‐cell immune system another handle on invading microbes, by detecting their lipid content as well as their proteins (Bendelac et al., 2007, Brutkiewicz, 2006, Kinjo et al., 2005, Tupin et al., 2007). Thus, NKT cells serve as regulatory cells and potentially effector cells in responses ranging from autoimmune disease and allergy to infectious diseases and cancer.