natural killer t ( nkt ) cells are innate - like lymphocytes typified by coexpression of receptors characteristic of natural killer and conventional t cells . as such , murine nkt cells generally bear ly49 receptors , nkg2 family of receptors , cd94 , and nk1.1 ( the latter only being expressed in specific strains , including the commonly used c57bl/6 ) .
human nkt cells often express similar surface molecules including cd56 , cd161 , cd94 , nkg2d , and nkg2a .
both human and mouse nkt cells display a variety of stimulatory and inhibitory t cell - associated receptors and ligands ( e.g. , cd28 and cd154 ) , whose expression depends on the activation status of the cell .
finally , both human and murine nkt populations include cd4 and cd4cd8 ( double negative ; dn ) subpopulations ; while cd8 nkt cells are found in humans , they are rare in mice . the t cell receptors ( tcrs ) expressed by nkt cells recognize the conserved and nonpolymorphic mhc class i - like molecule , cd1d . unlike classical mhc class i - like molecules , the expression of cd1d
is largely restricted to cells of bone marrow origin including antigen presenting cells ( apcs ) such as dendritic cells ( dcs ) , macrophages , and b cells .
furthermore , the cd1d molecule ( via heterodimerization with 2-microglobulin ) specializes in displaying lipid moieties rather than protein polypeptides .
importantly , intact expression of cd1d is critical for the development of nkt cell populations , as cd1d/ mice are devoid of these cells .
nkt cells are further subclassified into type i or ii lineages , depending on the composition of their tcr and the cd1d - presented glycolipid antigens to which they respond .
type i or invariant nkt ( inkt ) cells express canonical tcr chains comprised of specific gene segments ( v14-j18 in mice and v24-j18 in humans ) that preferentially pair with specific tcr chains ( v8 , v7 , or v2 in mice and v11 in humans ) .
these invariant tcr pairings confer reactivity to cd1d and a restricted array of presented glycolipid antigens .
the dependence of inkt cells on the v14-j18-comprised tcr is demonstrated by v14 tcr transgenic mice , in which a higher frequency and number of inkt cells are observed , and also j18/ mice , in which no mature inkt cells develop . despite the conserved use of the invariant tcr , inkt cell populations are phenotypically ( e.g. , presence or absence of cd4 expression ) and functionally ( e.g. , preferential production of certain cytokines , such as il-17 ) diverse .
the prototypical ( and first discovered ) inkt cell stimulatory glycolipid , alpha - galactosylceramide ( -galcer ) , was identified during a screening for compounds from marine sponges ( agelas species ) with antitumor activity . since this initial discovery ,
a number of naturally occurring and synthetic lipid antigens have been described to bind cd1d and activate inkt cells .
these cells are now typically identified using cd1d tetramers loaded with -galcer or its synthetic analogs ( e.g. , pbs-57 ; ) .
in contrast , type ii or variant nkt ( vnkt ) cells bear a more diverse array of tcr and  chains and have been shown to recognize sulfatide moieties presented by cd1d .
more recently , type ii nkt cells have also begun to be better characterized through development of cd1d tetramers loaded with sulfatide [ 9 , 10 ] , but these cells are still less well characterized than their invariant brethren . given
that far more is known regarding the antitumor activity of inkt cells , we will predominantly focus our attention on these cells .
inkt cells develop in the thymus , by originating from cd4cd8 double positive ( dp ) thymocytes .
positive selection of inkt cells is mediated by homotypic interactions of dp cells and recognition of glycolipid antigen - cd1d complexes [ 1114 ] ; however , the nature of the self - antigens involved in this process remains somewhat elusive
. like conventional t cells , maturation of inkt cells at the dp stage and beyond depends on the ability to construct a functional tcr and intact signaling . as such ,
inkt cells are profoundly diminished or absent in mice lacking expression of rag , cd3 , lck , zap-70 , slp-76 , itk , lat , or vav [ 1521 ] .
transcriptionally , development of inkt cells at the dp stage is regulated by the transcription factor rort , which prolongs the survival of dp thymocytes by upregulating bcl - xl , to allow sufficient time for distal tcr gene segment rearrangements to occur [ 22 , 23 ] .
more recent studies have shown that heb , the e protein family of basic helix - loop - helix transcription factors , regulates inkt cell development by regulating rort and bcl - xl mrna .
finally , the absence of the transcription factor runx1 also blocks inkt cell development at the earliest detectable inkt cell - committed subset .
inkt cell development at the dp stage also critically depends on the signals generated by engagement of the signaling lymphocyte activation molecule ( slam ) family of surface receptors , which are expressed on developing inkt cells , as well as conventional dp thymocytes .
slam family receptor signaling is transduced by the adaptor molecule sap ( slam - associated protein ) , which in turn binds to the tyrosine kinase fyn , and results in propagation of a phosphorylation signal [ 25 , 26 ] . accordingly , inkt cells fail to develop in mice and humans bearing mutations in the gene that encodes for sap [ 2729 ] , in mice lacking fyn or expressing a mutant version of sap that can not bind fyn [ 23 , 30 ] , in mice in which both ly108 and slam signaling are simultaneously disrupted , and in those lacking the transcription factor cmyb ( which is necessary for appropriate expression of sap and certain slam family members ) . taken together
, these studies establish the importance of the slam - sap - fyn signaling axis in inkt cell development .
following positive selection , inkt cells undergo distinct stages of maturation that are characterized by the sequential acquisition of cd24 , cd44 , and nk1.1 : cd24cd44nk1.1 ( stage 0 ) , cd24cd44nk1.1 ( stage 1 ) , cd24cd44nk1.1 ( stage 2 ) , and finally cd24cd44nk1.1 ( stage 3 ) . as these cells progress through these developmental stages
, they begin to upregulate nk cell markers ( e.g. , nkg2d and ly49 receptors ) , cd69 , and cd122 and acquire distinct effector functions ( e.g. , production of il-4 , ifn- , perforin , and granzymes ) .
one of the key regulators of inkt cell development and acquisition of an effector / memory phenotype and functions is the broad complex tramtrack bric - a - brac - zinc finger transcription factor plzf , whose expression is highest in stage 0 and 1 populations [ 36 , 37 ] .
plzf - deficient animals exhibit a severe reduction in inkt cell number and plzf - deficient inkt cells fail to cosecrete th1 and th2 cytokines upon stimulation [ 36 , 37 ] .
recently , it was demonstrated that the lethal-7 microrna posttranscriptionally regulate plzf expression and inkt cell effector functions .
the transcription factor t - bet is indispensable for the final maturation stages of inkt cells [ 39 , 40 ] and absence of this transcription factor results in reduced inkt cell numbers due to developmental blockade at stage 2 .
t - bet - deficient inkt cells fail to proliferate in response to il-15 as they lack surface expression of cd122 , a component of the il-15 receptor .
in addition , t - bet - deficient inkt cells fail to produce ifn- in response to tcr stimulation and exhibit defective cytolytic activity [ 39 , 40 ] as t - bet directly regulates the activation of genes associated with mature inkt cell functions , such as perforin , cd178 , and ifn- . as inkt cells progress to stage 1 , a proportion of cells downregulate cd4 , giving rise to dn inkt cells .
generation of the cd4 inkt cell lineage and production of th2-type cytokines is critically regulated by the transcription factor gata-3 .
similar to plzf - deficient inkt cells , gata-3 deficient inkt cells fail to produce th1 or th2 cytokines in response to -galcer .
recent studies have identified a unique subpopulation of nk1.1cd4 inkt cells that are transcriptionally regulated by rort and capable of producing large quantities of il-17 upon stimulation . as such ,
inkt cells are also sometimes classified into nkt1 , nkt2 , and nkt17 based on their cytokine production profiles and respective expression of t - bet , gata-3 , and rort [ 43 , 44 ] .
finally , mechanistic target of rapamycin ( mtor ) signaling has also been shown to be important for inkt cell lineage diversification and acquisition of effector functions [ 4548 ] , and loss of mtor2 may result in loss of nkt17 cells . taken together , these recent studies provide new insights into the transcriptional regulation of inkt cell maturation and functional differentiation .
the importance of inkt cells in mediating protection against tumors is highlighted by several findings .
first , a number of independent studies have shown a decrease in the number of inkt cells in the peripheral blood of patients with a variety of cancers and even precancerous myelodysplastic syndromes [ 4951 ] . moreover ,
the inkt cells that persist appear to have decreased proliferative and functional responses [ 5254 ] .
interestingly , an increased frequency of peripheral blood inkt cells in cancer patients portends a more favorable response to therapy [ 55 , 56 ] . while these observations identify an association between inkt cell numbers and/or function and development of malignancy , they do not provide a direct causal link
this link has been established in a number of mouse studies in which the biology of the host and initiation of tumors can be more systematically manipulated via gene knockouts , antibody depletion strategies , and adoptive transfer of various lymphocyte populations into cancer - predisposed or tumor - challenged hosts . in mice that are prone to development of tumors due to loss of one allele of a tumor suppressor ( p53+/ ) , absence of inkt cells ( by virtue of genetic knockout of the j18 gene segment or cd1d ) results in earlier and more frequent development of tumors and thus shorter survival , when compared to inkt - sufficient littermates .
similarly , treatment of cd1d/ and j18/ mice with a carcinogen resulted in increased incidence and earlier onset of tumors in comparison to treated wild type mice .
conversely , administration of -galcer to mice controlled the growth and metastasis of adoptively transferred [ 59 , 60 ] or carcinogen - induced [ 61 , 62 ] or spontaneous   tumors .
moreover , adoptive transfer of inkt cells into j18/ inkt cell - deficient mice prevented the growth of subcutaneous sarcomas .
finally , adoptive transfer of small numbers of purified inkt cells into lymphocyte - deficient nod - scid - il2r/ ( nsg ) mice was sufficient to protect mice from challenge with a cd1d tumor .
these findings collectively argue that inkt cells play a central and nonredundant role in the response to tumors .
further studies would shed light on the mechanisms by which inkt cells exert these antitumor effects .
engagement of the invariant tcr by cd1d / glycolipid antigen complexes results in inkt cell activation , an event that is typified by rapid and robust production of a variety of cytokines and chemokines , including  but not limited to  il-2 , il-4 , il-10 , il-13 il-17 , ifn- , tnf , tgf , gm - csf , rantes , eotaxin , mip-1 , and mip-1 [ 65 , 66 ] . the nature and magnitude of the inkt cell cytokine response
depend on the glycolipid antigen ; for example , -galcer - mediated inkt cell activation elicits a strong ifn--dominated cytokine response , while och ( a synthetic analog of -galcer with a truncated lipid chain ) elicits a response with significantly higher level of il-4 production .
the rapidity of this cytokine response is attributed to the semiactivated state of inkt cells and the presence of preformed cytosolic mrna for a variety of cytokines .
indeed , administration of -galcer to inkt cell - sufficient , but not inkt cell - deficient , mice results in polyclonal activation of conventional t , b , and nk cells within 3 - 4 hours   and also eventually leads to the mobilization of macrophages and neutrophils .
intriguingly , it was previously believed that mammalian species are incapable of producing glycolipids ( such as -galcer ) , in which the sugar moiety is attached via an o - linkage to the ceramide backbone in an alpha - anomeric configuration . despite the absence of -glucosyl or -galactosyl transferases in mammals , recent findings indicate that a small percentage of the glycolipids that are constitutively presented by mammalian cd1d are indeed -anomeric . whether the percentage of cd1d - presented -anomeric glycolipids is altered in tumor tissues
nonetheless , following encounter with cd1d / antigen complexes displayed by apcs , inkt cells not only produce cytokines but also upregulate surface expression of cd154 ( see figure 1(a ) ) .
ligation of apc - expressed cd40 is especially important for mediating subsequent maturation and functional activation of dcs , subsequent upregulation of cd80 and cd86 , and amplified production of ifn- [ 72 , 73 ] .
in addition , the ligation of the chemokine receptor cxcr6 on inkt cells by cxcl16 expressed on apcs also provides costimulatory signals resulting in robust -galcer - induced inkt cell activation .
importantly , matured dcs are potent producers of il-12 , which induces sustained ifn- production by inkt cells [ 7577 ] .
the importance of inkt cells in il-12-mediated tumor rejection was effectively demonstrated by the defective clearance of a variety of tumors in j18/ mice .
mature dcs also support the priming and activation of cd8 t cells , culminating in optimal effector and memory cell formation [ 72 , 78 ] .
finally , the sustained release of ifn- by inkt cells leads to activation and proliferation of nk cells and nk cell secretion of ifn-. the combination of cytokines ( e.g. , il-2 , il-12 , and ifn- ) as a result of inkt cell activation also leads to upregulation of death - inducing ligands ( e.g. , cd178 or cd253 ) on nk cells and cd8 t cells [ 79 , 80 ] .
these sequential activation events are believed to be critical for the -galcer - induced inkt cell - mediated antitumor effects [ 76 , 81 , 82 ] . as such , inkt cells not only bridge the activation of innate and adaptive immunity , but also indirectly potentiate the antitumor activity of other cytotoxic effector lymphocytes .
tumor establishment and growth are believed to be intricately modulated by a myriad of soluble and contact - derived signals obtained from the tumor microenvironment ( tme ) , which consists of the tumor cells themselves , tumor - infiltrating lymphocytes ( tils ) , and stromal cells that communicate in a dynamic and bidirectional manner .
in addition to their indirect modulation of other effector lymphocyte populations , inkt cells may also regulate tumor growth via their effects on the tme ( see figure 1(b ) ) . indeed following intravenous administration ,
inkt cells were shown to represent a significant percentage of the tils in patients with head and neck carcinomas [ 83 , 84 ] .
importantly , higher frequency of tumor - infiltrating inkt cells correlated with overall and disease - free survival as an independent prognostic factor in primary colorectal cancer patients   and with tumor regression in head and neck carcinomas .
conversely , in patients with primary hepatocellular or metastatic cancer , cd4 inkt cells that produced high levels of th2-type cytokines and had low cytolytic activity were enriched within the tumor and appeared to inhibit the expansion of antigen - specific cd8 t cells , suggesting that these particular inkt cells may contribute to generate an immunosuppressive microenvironment . in experimental studies ,
cotransfer of human monocytes and inkt cells to tumor - bearing nod - scid mice suppressed tumor growth when compared with mice that received monocytes alone .
importantly , inkt cells can target tumor supportive cells such as tumor - associated macrophages ( tams ) , a highly plastic monocyte - derived subset of inflammatory cells that can exert immunosuppressive functions , and promote tumor proliferation and matrix turnover [ 88 , 89 ] .
indeed tams are known to produce il-6 , a cytokine that appears to promote the proliferation of many solid tumors , including neuroblastomas and breast and prostate carcinomas .
found that macrophage density correlated positively with microvessel counts and negatively with patient relapse - free survival .
since tams cross - present neuroblastomaderived endogenous cd1d ligand(s ) , they can be specifically recognized and killed by inkt cells in an il-15-dependent process .
other potential inkt cell tme targets include myeloid - derived suppressor cells ( mdscs ) .
mdscs have been found to accumulate in the blood , lymph nodes , and bone marrow and at tumor sites in most patients and experimental animals with cancer and inhibit both adaptive and innate immunity .
the absence of inkt cells in mice during influenza virus infection resulted in the expansion of mdscs , high viral titer , and increased mortality .
the adoptive transfer of inkt cells abolished the suppressive activity of mdscs and restored virus - specific immune responses , resulting in reduced viral titers and increased rates of host survival .
thus , certain populations of inkt cells may help alter the tme via their effects on tams and mdscs , to help create a tumor - suppressive or immune - permissive milieu .
in addition to their indirect control of tumor growth , inkt cells can mediate direct killing of tumor targets ( see figure 1(c ) ) .
inkt cells alone , or in combination with nk cells , have been shown to kill a variety of tumor targets in vitro [ 6 , 93 , 94 ] .
while this mechanism of killing appears to be dependent on the presence of stimulatory glycolipids and cd1d [ 95 , 96 ] , inkt cell cytotoxicity also appears to be triggered via ligation of nkg2d by target - expressed stress ligands .
it remains to be seen whether mult1 , the newly identified shed form of high affinity nkg2d ligand that triggers nk - mediated tumor rejection in mice , also activates inkt cells .
consistent with their direct cytotoxic capacity , inkt cells express perforin and granzymes , as well as cd178 [ 34 , 96 , 99 , 100 ] . in our hands ,
blockade of cd1d - mediated lipid antigen presentation , disruption of t cell receptor ( tcr ) signaling , or loss of perforin expression was found to significantly reduce inkt cell killing in vitro .
moreover , we demonstrated that inkt cells alone were sufficient for control of the growth of a t cell lymphoma in vivo that preferentially relies on perforin and the adaptor protein sap [ 64 , 69 ] .
mechanistically , inkt cells rely on sap for formation of stable conjugates with the tumor targets as well as proper orientation of the lytic machinery at the immunological synapse . despite the majority of studies implicating inkt cells as having an antitumor role , a limited number of studies also implicate inkt cells as suppressing antitumor responses , but
these paradoxic responses may be related to the level of tumor cd1d expression [ 102 , 103 ] .
alternatively , these differences may stem from the fact that  contrary to the use of c57bl/6 mice in the previously discussed studies 
these last two studies were performed in balb / c mice , in which there is a predominance of il-4-producing th2 phenotype inkt cells .
interestingly , the antitumor responses of inkt cells may be regulated by the activity of type ii nkt cells .
demonstrated that type ii variant nkt ( vnkt ) cells were sufficient for the downregulation of tumor immunosurveillance and relapse growth of a model fibrosarcoma in an antigen - dependent manner , while a second study found that activation of vnkt cells with sulfatide antigen could suppress the activation of inkt cells .
conversely , type ii vnkt cells were , in at least one study , suggested to promote the antitumor activity of cpg oligodeoxynucleotides .
this suppression appears to be mediated through a contact- and il-10-dependent mechanism [ 109 , 110 ] .
indeed , induction of treg cells suppressed the protective effect of adoptive transfer of inkt cells into j18/ mice .
consistent with these findings , depletion of treg cells or short - term elimination of their suppressive activity results in enhanced inkt cell - mediated antitumor responses and increased nk and cd8 t cell activation and ifn- production .
interestingly , the ability of treg cells to suppress inkt cell proliferation depends on the degree of invariant tcr agonism , such that responses to weak ( e.g. , och ) , but not strong ( e.g. , -galcer ) , agonists were effectively suppressed . when viewed collectively , these findings suggest that inkt cells possess inherent capacity for direct cytotoxicity but their antigenic exposure may modulate whether their antitumor effects can be suppressed by treg and vnkt cells .
given the preponderance of evidence suggesting that the activation of inkt cells provides protection against the growth and metastasis of a variety of tumors , safety of -galcer administration was examined in a phase i trial .
while administration of -galcer was well tolerated at a range of doses , no clinical responses were observed in patients with advanced solid tumors . on the heels of this study ,
nieda et al . showed that treatment of metastatic cancer patients with -galcer - pulsed immature monocyte - derived dcs resulted in dramatic increases in serum ifn- and il-12 and activation of nk and t cells in the majority of subjects .
importantly , this phase i trial also documented reduction in tumor biomarkers and tumor necrosis in several patients .
these findings were extended in a study of a small number of patients , in which the -galcer - pulsed dcs were matured prior to adoptive transfer .
this study demonstrated a > 100-fold increase in blood inkt cell numbers in all patients , and this increase was long - lived ( > 6 months ) .
a number of subsequent clinical trials , all with limited number of patients with advanced head and neck or non - small cell lung cancers , have since employed similar strategies of adoptive transfer of -galcer - pulsed apcs [ 115118 ] .
collectively , these studies demonstrate increases in blood ifn- levels and inkt cells in some but not all patients , stabilization of disease in a few of the subjects , and absence of severe treatment - related toxicities . in a different approach ,
chemotherapy - refractory 5 lymphoma patients were treated with autologous peripheral blood mononuclear cells ( pbmcs ) stimulated with anti - cd3 , il-2 , and ifn-. this ex vivo stimulation resulted in enrichment of nkt cells ( to ~20% on average ) , and this cell fraction was shown to possess the highest cytotoxic capacity in vitro .
of the nine patients who received adoptive transfer of these cells , two showed partial responses and two others had stabilization of disease .
two subsequent studies by motohashi et al . evaluated the adoptive transfer of ex vivo expanded inkt cell - enriched cells to patients with advanced cancer . in the first ,
6 patients with advanced non - small cell lung cancer were treated with either a low or a high dose of ex vivo expanded inkt cells .
of the 3 patients treated with the high dose , all had an increase in the frequency of ifn--producing pbmcs and 2 showed expansion of inkt cells . although no clinical responses were observed in this study , a follow - up trial of 17 patients with advanced head and neck cancers treated with a high dose of inkt cell - enriched autologous pbmcs showed a significant increase in ifn--producing pbmcs in 10 of 17 patients .
importantly , while none of these patients displayed tumor regression , 5 had disease stabilization and the mean survival time for the subjects with higher frequencies of ifn--producing pbmcs was tripled above those with low percentages of ifn--producing pbmcs ( 29.3 versus 9.7 months ) .
administered both in vitro expanded inkt cells and -galcer - pulsed apcs to patients with advanced head and neck squamous cell carcinomas .
treatment increased the frequencies of inkt cells and ifn--producing pbmcs , and a partial clinical response or disease stabilization was observed in 7 of 8 patients . although the responses in these studies have not been profound , it must be noted that these inkt cell - based immunotherapies have all been conducted on patients with advanced malignancies  often those in whom standard chemotherapy , irradiation , and/or surgical excision treatments had failed .
future studies of inkt cell - based immunotherapy may be able to take advantage of two recent technologies .
as mentioned previously , many malignancies are associated with a decrease in the numbers and proliferative capacity of peripheral blood inkt cells . in order to circumvent the difficulty of being able to expand these infrequent and potentially defective cells from patients , watarai et al .
generated induced pluripotent stem ( ips ) cells from mature inkt cells and then expanded large numbers of inkt cells from these established ips cells .
ips - nkt cells generated in this fashion were demonstrated to be able to activate and expand antigen - specific cd8 t cell responses to limit the growth of leukemia in mice   without inducing graft versus host disease ( gvhd ) .
described inkt cells engineered to express cars bearing specificity for gd2 , a highly expressed moiety on neuroblastoma cells . in their studies , they showed that inkt cells expanded from the pbmcs of healthy human donors and transduced with retroviral car constructs could protect humanized nsg mice against metastatic neuroblastoma without inducing gvhd .
whether these two technologies could be combined to generate functional car - bearing ips - nkt cells remains to be seen .
inkt cells are innate - like effector lymphocytes that not only are directly cytotoxic , but also possess the unique ability to nucleate the antitumor responses of other effector lymphocytes and alter the cellular and angiogenic makeup of the tumor microenvironment . as such , the promise of an effective inkt cell - based immunotherapy can only be realized by devising and evaluating strategies that simultaneously maximize each of these antitumor effector mechanisms . the challenge for the future will thus be to identify these strategies and apply them to tumors against which inkt cells wield the most optimal responses .
 natural killer t ( nkt ) cells are innate - like lymphocytes that were first described in the late 1980s . since their initial description , 
 numerous studies have collectively shed light on their development and effector function . 
 these studies have highlighted the unique requirements for the activation of these lymphocytes and the functional responses that distinguish these cells from other effector lymphocyte populations such as conventional t cells and nk cells . 
 this body of literature suggests that nkt cells play diverse nonredundant roles in a number of disease processes , including the initiation and propagation of airway hyperreactivity , protection against a variety of pathogens , development of autoimmunity , and mediation of allograft responses . in this review , however , we focus on the role of a specific lineage of nkt cells in antitumor immunity . 
 specifically , we describe the development of invariant nkt ( inkt ) cells and the factors that are critical for their acquisition of effector function . 
 next , we delineate the mechanisms by which inkt cells influence and modulate the activity of other immune cells to directly or indirectly affect tumor growth 
 . finally , we review the successes and failures of clinical trials employing inkt cell - based immunotherapies and explore the future prospects for the use of such strategies . 
1. Introduction
2. iNKT Cell Development and Acquisition of Effector Function
3. iNKT Cells and Antitumor Immunity
4. iNKT Cell-Based Immunotherapy
5. Concluding Remarks