Invariant natural killer T cells: innate-like T cells with potent immunomodulatory activities

Tissue Antigens ISSN 0001-2815

REVIEW ARTICLE

Invariant natural killer T cells: innate-like T cells with potentimmunomodulatory activitiesL. Wu, C. L. Gabriel, V. V. Parekh & L. Van Kaer

Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA

Key words

a-galactosylceramide; adjuvant; CD1d;

glycolipids; immunomodulation;

immunotherapy; innate immunity; invariant

natural killer T cells

Correspondence

L. Van Kaer, PhD

Department of Microbiology and Immunology

Vanderbilt University School of Medicine

Medical Center North

Room A-5301

1161 21st Avenue South

Nashville

TN 37232-2363

USA

Tel: 11 615 343 2707

Fax: 11 615 343 2972

e-mail: [email protected]

Received 11 March 2009; accepted

11 March 2009

doi: 10.1111/j.1399-0039.2009.01256.x

Abstract

Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that react with

glycolipid antigens presented by the major histocompatibility complex class

I-related glycoprotein CD1d. Although iNKT cells express an antigen-specific

receptor of the adaptive immune system, they behave more like cells of the innate

immune system. A hallmark of iNKT cells is their capacity to produce copious

amounts of immunoregulatory cytokines quickly after activation. The cytokines

produced by iNKT cells can influence the level of activation ofmany cell types of the

innate and adaptive immune systems as well as the quality of an adaptive immune

response. As such, iNKT cells have emerged as important regulators of immune

responses, playing a role in microbial immunity, autoimmunity, tumor immunity,

and a variety of inflammatory conditions. Although several endogenous and

exogenous glycolipid antigens of iNKT cells have been identified, how these

glycolipids orchestrate iNKT-cell functions remains poorly understood. Neverthe-

less, iNKT cells hold substantial promise as targets for development of vaccine

adjuvants and immunotherapies. These properties of iNKT cells have been

investigated most extensively in mouse models of human disease using the marine

sponge-derived agent a-galactosylceramide (a-GalCer) and related iNKT-cell

antigens. While these preclinical studies have raised enthusiasm for developing

iNKT-cell-based immunotherapies, they also showed potential health risks

associated with iNKT cell activation. Although a-GalCer treatment in humans

was shown to be safe in the short term, further studies are needed to develop safe and

effective iNKT-cell-based therapies.

Introduction

Cells of the innate immune system can sense microbial

pathogens through germ line-encoded receptors, called

pattern recognition receptors, which interact with con-

served pathogen-associated molecular patterns. In turn, the

cytokines and costimulatory molecules elaborated by cells

of the innate immune system are critically important for

initiating an effective adaptive immune response. B and T

cells of the adaptive immune system recognize microbial

pathogens through diverse, antigen-specific receptors that

are generated by somatic DNA rearrangement. Invariant

natural killer T (iNKT) cells are a distinct subset of T

lymphocytes that express a receptor of the adaptive immune

system, yet their specificity and behavior are more

reminiscent of cells of the innate immune system. Here, we

will review the salient characteristics and functions of

these fascinating cells and discuss their potential appli-

cations in the development of vaccine adjuvants and

immunotherapies.

iNKT cells and their cousins

Nearly two decades ago, it was reported that a small subset

of murine T lymphocytes expressed NK1.1 (also called

CD161), a marker that was previously considered to be

uniquely expressed by natural killer (NK) cells [reviewed in

(1)]. This finding, together with parallel studies from other

investigators characterizing a T-cell subset expressing

ª 2009 John Wiley & Sons A/S � Tissue Antigens 73, 535–545 535

intermediate T-cell receptor (TCR) surface levels with

a repertoire skewed toward Vb8 and a subset of

CD42CD82 (double-negative) T cells producing immuno-

regulatory cytokines, indicated the existence of a unique

subpopulation of T cells defined by NK1.1 expression.

Subsequent studies showed that the majority of these cells

were reactive with the major histocompatibility complex

(MHC) class I-related protein CD1d, and these cells were

called natural killer T (NKT) cells to reflect their hybrid

surface expression phenotype of conventional T cells and

NK cells. However, it soon became clear that not all NK1.1-

expressing cells recognized CD1d and that some CD1d-

restricted T cells failed to express the NK1.1 marker. The

need for a redefinition of these cells was further com-

pounded by the realization that the NK1.1 marker

(recognized by the PK136 antibody) is only expressed in

mouse strains such as C57BL/6 but not in many other

commonly used mouse strains (although these strains

express allelic variants of NK1.1 that are not recognized

by the PK136 antibody). These lymphocytes have now been

further categorized into three distinct subsets (1):

(a) Type I NKT cells, also called iNKT cells or

conventional NKT cells, are CD1d-restricted T cells

that express a semi-invariant TCR formed by the

combination of Va14–Ja18 and Vb8.2/Vb7/Vb2chains in mice and homologous Va24–Ja18 and

Vb11 chains in humans. The majority of these cells

express NK1.1 (CD161 in humans) (Figure 1A).

(b) Type II NKT cells, also called nonclassical NKT

cells, are also CD1d restricted but have a more

diverse TCR repertoire, and many of these cells

express NK1.1.

(c) NKT-like cells consist of a diverse range of cell

types, including CD1d-independent, NK1.1-expressing

T cells and multiple other T-cell subsets that express

semi-invariant TCRs. To avoid confusion with type I

and type II NKT cells, and because these cells are

CD1d independent, it has been recommended that

these cell types should not be called NKT cells (1).

This review paper focuses on type I NKT cells, which we

will call iNKT cells. A key issue in studying iNKT cells is to

reliably identify these cells, which can be accomplished in

mice and humans by usingmultimeric forms of CD1d (most

commonly CD1d tetramers) loaded with the prototypic

iNKT-cell antigen a-galactosylceramide (a-GalCer, see

below). Functional studies of iNKT cells in vivo can be

performed most reliably with TCR gene Ja18-deficientmice. Analysis of CD1d-deficient mice, together with Ja18-deficient mice, can be used to distinguish between type I and

type II NKT-cell functions.

iNKT cells are found with the highest frequency in the

liver and the bone marrow of mice and at significant

numbers in the thymus, spleen, and peripheral blood [for

reviews regarding iNKT cells, see (2–5)]. In humans, the

frequency of NKT cells is usually much lower and with

a high degree of variability between individuals. Most

murine iNKT cells express the coreceptor CD4, and the

remaining cells express neither CD4 nor CD8, whereas

a significant proportion of iNKT cells in humans express

CD8a. In addition to NK1.1 (or CD161), iNKT cells

express a variety of other receptors that are characteristic of

the NK-cell lineage, including NKG2D, CD94 and mem-

bers of the Ly49 family of NK-cell receptors. iNKT cells

also constitutively express a variety of markers such as

CD44, CD69 and CD122 that are typical of activated or

memory T cells.

iNKT-cell antigens

Because of its limited diversity, the TCRof iNKT cells more

closely resembles the pattern recognition receptors ex-

pressed by cells of the innate immune system than the

diverse antigen-specific receptors expressed by cells of the

adaptive immune system. Because of their propensity to

react with autologous cells, it has been generally assumed

that iNKT cells recognize both endogenous and exogenous

glycolipid antigens. All iNKT cells from mice and humans

react with a-GalCer (6) (Figure 1B), a glycosphingolipid

that was isolated from the marine spongeAgelas mauritianus

during a screen of natural products with antimetastatic

activities in mice. a-GalCer is a very potent iNKT-cell

agonist and has been extensively studied with regard to its

interaction with CD1d and the invariant TCR of iNKT

cells, its immunomodulatory activities, and its therapeutic

properties. The trimolecular complex formed between

a-GalCer, CD1d and the invariant TCR has been investi-

gated at the atomic level and has shown an unusual and

unexpected recognition mode (7, 8). Numerous structural

variants of a-GalCer have been synthesized and analyzed

(see below and Figure 1B). Several of the known naturally

occurring exogenous iNKT-cell ligands are pathogen-

derived glycolipid antigens, such as glycosylceramides from

the cell wall of gram-negativeNovosphingobium (previously

called Sphingomonas) species (9–11) and diacylglycerols

from Lyme disease-causing Borrelia burgdorferi bacteria

(12) (Figure 1B). Interestingly, because the Novosphin-

gobium glycolipids are structurally similar to a-GalCer,

and because marine sponges are commonly colonized by

bacteria, includingNovosphingobium species, it is likely that

a-GalCer was isolated from Novosphingobium bacteria that

colonized the marine sponge. Several studies have also

shown reactivity of iNKT cells with select cellular, tumor-

derived, and other microbial glycolipids, but these reagents

were only recognized by small subsets of iNKT cells. An

endogenous, lysosomal glycosphingolipid, isoglobotrihex-

osylceramide (iGb3, Figure 1B) is capable of activating

murine and human iNKT cells (10), albeit at lower levels

iNKT cells L. Wu et al.

536 ª 2009 John Wiley & Sons A/S � Tissue Antigens 73, 535–545

than a-GalCer or the bacterial glycolipids, as would be

expected for an endogenous, physiological ligand. The role

of iGb3 in the development and function of iNKT cells is

still under investigation.

Presentation of glycolipid antigens to iNKTcells

CD1d molecules are expressed by professional antigen-

presenting cells (APCs), including dendritic cells (DCs),

macrophages, and B cells (CD1d is particularly abundant

on marginal zone B cells) as well as by double-positive

thymocytes and hepatocytes. Soon after synthesis in the

endoplasmic reticulum (ER), CD1d molecules bind with

endogenous phospholipids that facilitate the exit of CD1d

from the ER and its transport to the cell surface (13–15)

(Figure 2). Current evidence indicates that the loading of

phospholipids onto CD1d in the ER involves the action of

microsomal triglyceride transfer protein, which is a lipid

transfer protein (LTP) that plays an important role in the

assembly of apolipoprotein (apo) B with lipids in the ER.

Surface CD1d is then internalized by clathrin-mediated

endocytosis that depends on a tyrosine-based motif in the

cytoplasmic tail of CD1d. In addition to this pathway, it has

been reported that a subset of CD1d molecules reaches

Figure 1 Specificity of iNKT cells. (A) iNKT cells

express a semi-invariant T-cell receptor (TCR) that is

specific for glycolipid or lipid antigens presented by

CD1d on antigen-presenting cells (APCs). Most iNKT

cells also express the natural killer cell marker CD161

(also called NK1.1 in mouse). Activated iNKT cells can

produce a variety of cytokines and chemokines. (B)

iNKT cells can react with a variety of glycolipid and

lipid antigens, including endogenously derived iso-

globotrihexosylceramide (iGb3), Novosphingobium-

derived glycolipids such as a-galacturonosylceramide

(a-GalA-Cer), Borrelia burgdorferi-derived diacylgly-

cerols such as BbGL-IIc, and the marine sponge

Agelas mauritianus-derived glycolipid a-galactosyl-ceramide (a-GalCer). Several structural analogs of

a-GalCer, including OCH, C20:2, a-C-GalCer, and

threitolceramide (ThrCer), which have altered capac-

ity to activate iNKT cells and to promote the

immunomodulatory and immunotherapeutic proper-

ties of these cells, are also depicted. Key structural

differences between these antigens are indicated in

red.GM-CSF, granulocytemonocytecolony-stimulating

factor; IL, interleukin; IFN, interferon; INKT, invariant

natural killer T; TGF, transforming growth factor; TNF,

tumor necrosis factor.


L. Wu et al. iNKT cells

endosomal compartments after association with the MHC

class II-associated invariant chain in the ER. Within late

endosomes, theER-loaded phospholipids are exchanged for

antigenic lipids that can activate iNKT cells. These

glycolipids can be derived from endogenous sources, such

as lysosomal iGb3, or from exogenous sources. Multiple

pathwaysmight be engaged to deliver exogenous glycolipids

to the CD1d-loading compartment: (a) phagocytosis of

microbes or their products, (b) engagement of mannose

within glycolipids by mannose receptors, (c) engagement

of modified low-density lipoprotein (LDL) by scavenger

receptors, and (d) internalization of apoE-containing lipid

complexes through the LDL receptor or related lipoprotein

receptors. After their arrival in late endosomal compart-

ments, endogenous or exogenous glycolipidsmight undergo

processing (e.g. by carbohydrate hydrolases), followed by

loading onto CD1d with the assistance of a variety of LTPs,

including saposins, GM2 activator and Niemann–Pick C2

protein. These antigenic CD1d–glycolipid complexes are

then displayed at the cell surface for recognition by

iNKT cells.

In recent years, it has become clear that multiple viral

pathogens, including Kaposi sarcoma-associated herpes

virus, HIV-1, herpes simplex virus-1, vesicular stomatitis

virus, and vaccinia virus, can interfere with CD1d traffick-

ing and, thus, impede glycolipid antigen presentation to

iNKT cells (16). Furthermore, Chlamydia trachomatis

downregulates CD1d expression on human epithelial cells

by targeting its degradation by both cellular and chlamydial

proteasomes (17).

iNKT-cell development

Like conventional T cells, iNKT cells develop in the thymus

(18). iNKT cells develop relatively late during T-cell

ontogeny, owing to structural constraints in the recombi-

nation of TCRVa14 and Ja18 gene segments.Generation of

the canonical Va14–Ja18 rearrangement is a random event.

After expression of surface TCRs at the double-positive

thymocyte stage, iNKT cells diverge from conventional

T cells. iNKT cells require positive selection by CD1d–

glycolipid complexes in the thymus. However, in sharp

contrast with the positive selection of conventional T cells,

which is dependent on cortical thymic epithelial cells, iNKT

cells are selected by CD1d-expressing, double-positive

thymocytes. This selection likely involves endogenous

iNKT-cell antigens, and iGb3 has been suggested to play

such a role (10). This selection process also involves

homotypic interactions between members of the signaling

lymphocyte activation molecule (SLAM) family of recep-

tors (19). These receptors signal through the adaptor protein

SLAM-associated protein, the Src kinase Fyn, and the

downstreamtranscription factor nuclear factor (NF)-kB (20).

The selected iNKT cells subsequently expand and undergo

multiple maturation steps that ultimately result in the

characteristic surface phenotype and innate-like effector

functions of iNKT cells. These maturation steps involve

a variety of signal transducers, transcription factors, and

costimulatory molecules. Importantly, recent studies have

showna critical role of the transcription factor promyelocytic

leukemia zinc finger (PLZF) in directing the innate-like

effector differentiation of iNKT cells during thymic develop-

ment (21, 22). Furthermore, the licensing of mature iNKT

cells for cytokine production requires granulocyte monocyte

colony-stimulating factor (GM-CSF) signaling (23). After

Figure 2 The CD1d antigen processing and presentation pathway. After

its synthesis in the endoplasmic reticulum (ER), the CD1d heavy chain

associates with b2-microglobulin (b2m). This complex is loaded with ER-

resident phospholipids with the assistance of lipid transfer proteins

(LTPs) such asmicrosomal triglyceride transfer protein (MTP). These lipid

chaperones facilitate transport of CD1d to the cell surface. Surface CD1d

molecules are then endocytosed and enter endosomal and lysosomal

compartments, where the ER-derived lipids are replaced with endoge-

nous or exogenous glycolipids, with the assistance of LTPs such as the

saposin proteins (SAPs). Exogenous glycolipids can reach the CD1d-

loading compartment by multiple pathways, including carbohydrate

receptors, scavenger receptors, or lipoprotein receptors, or following

phagocytosis of microorganisms. Some glycolipid antigens might

undergo carbohydrate processing by carbohydrate hydrolases (Hy).

CD1d complexes loaded with lysosomal or exogenously derived

glycolipids are subsequently displayed at the cell surface.



acquisition of their innate-like phenotype, iNKT cells egress

from the thymus in amechanism that involves lymphotoxin-breceptor signaling. Nevertheless, in mouse, a significant

proportion of iNKT cells remain in the thymus, where they

become long-lived residents. Recruitment of iNKT cells to

peripheral tissues requires expression of the sphingosine

1-phosphate 1 receptor, and entry into the liver requires

expression of the chemokine receptor CXCR6.

Pathways of iNKT-cell activation

In most immune responses where iNKT cells have been

implicated, the mechanism of iNKT-cell activation remains

poorly understood. Nevertheless, studies with microbial

pathogens have shown two general pathways of iNKT-cell

activation (14, 24) (Figure 3). In the direct pathway of

iNKT-cell activation (Figure 1A), which is engaged by

Novosphingobium species and B. burgdorferi, cognate gly-

colipids bind CD1d and activate iNKT cells directly.

However, iNKT cells also become activated to many

microbes that lack cognate iNKT-cell antigens (25).

iNKT-cell activation during infection with such micro-

organisms is driven by cytokines. In this indirect pathway of

iNKT-cell activation (Figure 3B), engagement of signaling

pattern recognition receptors, most notably toll-like recep-

tors, with microbial products leads to the production of

cytokines such as interleukin (IL)-12, IL-18, and/or type I

interferons (IFNs) that can activate iNKT cells. The latter

mechanism has been implicated in the activation of iNKT

cells by several bacterial and viral microorganisms and their

products. For some microorganisms, iNKT-cell activation

also requires CD1d expression on the APCs, in which case

cytokines might function to amplify low-affinity interac-

tions of the iNKT-cell receptor with complexes between

CD1d and endogenous glycolipids such as iGb3. Similar

mechanisms might be involved in the activation of iNKT

cells during acute and chronic inflammatory conditions.

iNKT-cell functions

A hallmark of iNKT cells is to rapidly produce copious

amounts of cytokines quickly after TCR engagement (2–5).

Interestingly, however, iNKT cells are capable of producing

a wide array of cytokines (Figure 1A), including IL-2, IL-4,

IL-6, IL-10, IL-13, transforming growth factor (TGF)-b,IFN-g, and tumor necrosis factor (TNF)-a, and some iNKT

cells can also produce IL-17. iNKT cells also produce

growth factors for hematopoietic cells such asGM-CSF and

multiple chemokines such as macrophage inflammatory

protein (MIP)-1a and RANTES. In addition, these cells

have cytolytic activity, owing to high levels of granzyme B,

perforin, FasL, and TNF-related apoptosis-inducing

ligand. The effector functions exhibited by iNKT cells are

strongly influenced by the nature, strength, and context of

the stimulus as well as by the APC type and activation

status. For example, activation by cognate iNKT antigens

typically results in IFN-g and IL-4 production by iNKT

cells, whereas the cytokine-driven pathway of iNKT-cell

activation typically fails to induce substantial amounts of

IL-4, suggesting that IL-4 production by these cells largely

depends on TCR engagement. DCs appear to be the most

effective APCs in presenting glycolipids and activating

iNKT cells in vivo. Furthermore, although CD1d was

thought to lack significant polymorphism, a recent study

has identified severalmurineCD1d alleles that can affect the

presentation of endogenous and exogenous antigens to

iNKT cells (26).

iNKT cells can be divided into subsets based on CD4 and

NK1.1 (in mice) expression (27, 28). These distinct subsets of

iNKT cells show substantial diversity in their ability to

produce cytokines (29), which might explain their functional

differences. In humans, CD41 iNKT cells produce both Th1

and Th2 cytokines, whereas CD42 iNKT cells primarily

produce Th1 cytokines. Although differences in cytokine

production are less dramatic in mouse iNKT-cell subsets,

differences in the capacity of CD41 and CD42 iNKT cells to

regulate immune responses have been observed (28). IL-17

production by murine iNKT cells appears to be specific to

a subpopulation of CD42NK1.12 cells (29).

iNKT cells are capable of extensive cross talk with

a variety of other cell types, including DCs, macrophages,

neutrophils, NK cells, and conventional B and T cells, but

also B-1 B cells, marginal zone B cells, gd T cells, different

subsets of regulatory T (Treg) cells, type II NKT cells, and

myeloid-derived suppressor cells. In addition, because of

their capacity to promote cytokines that can bias adaptive

immune responses toward T helper type 1 (Th1), Th2, Th17,

or Treg-cell differentiation, iNKT cells can have a profound

impact on the quality of an immune response.

iNKT cells play important roles in a variety of immune

responses, as shown in studies with iNKT-cell-deficient

mice (because of CD1d or Ja18 gene disruption), Va14–Ja18 transgenic animals, and/orCD1d-blocking antibodies.

iNKT cells play a protective role in infections with a variety

of pathogens (25), including Pseudomonas aeruginosa,

B. burgdorferi, Ehrlichia muris, Chlamydia pneumoniae,

Novosphingobium capsulatum, Streptococcus pneumoniae,

Leishmania major, Schistosoma mansoni, Cryptococcus neo-

formans, influenza virus, Theiler’s murine encephalomyeli-

tis virus, and herpes simplex viruses 1 and 2. However,

iNKT cells can also play a pathogenic role during microbial

infection, such as during the immune response toChlamydia

muridarum. These studies also showed that the genetic

background of the animals used can have a significant

impact on the contribution of iNKT cells to antimicrobial

immunity. For example, iNKT cells protected against

cerebral malaria in BALB/c mice but exacerbated disease

in C57BL/6 mice. Likewise, iNKT cells played a protective



role in the immune response to respiratory syncytial virus

(RSV) in BALB/cmice, but exacerbated disease in C57BL/6

mice. In the case of infection with lymphocytic choriome-

ningitis virus, iNKT cells inhibited viral replication in the

pancreas and liver but not in the spleen (30), suggesting that

these cells can induce tissue-specific antiviral effects.

Because a significant proportion of human iNKT cells

express CD4 and the chemokine receptors CCR5 and

CXCR6, these cells are effective targets for HIV-1 infection

andmight serve as an early reservoir of HIV-1 replication in

infected individuals (31). iNKT cells play a protective role in

host immunity to tumors (32), which has been shown in

transplantable, chemically induced, and genetic tumor

models. iNKT cells generally also play a protective role in

autoimmunity (33), such as during the progression of

diabetes in nonobese diabetic (NOD) mice and in some

mouse models of multiple sclerosis (MS) and systemic lupus

erythematosus. However, iNKT cells can also contribute to

pathology in autoimmunity, as shown in models of

collagen-induced arthritis and antibody-mediated arthritis,

and during the development of primary biliary cirrhosis in

transgenic mice expressing a dominant-negative TGF-breceptor or in mice infected with Novosphingobium aroma-

ticivorans (34). iNKT cells also make an important

contribution to the development of tolerance in a variety

of experimental models, including anterior chamber-

associated immune deviation in the eye, anti-CD4-mediated

and costimulatory blockade in transplant tolerance, ultra-

violet- and burn-wound-induced immune suppression, fetal

tolerance, and graft-vs-host disease. iNKT cells play an

important pathogenic role in the development of a variety of

inflammatory diseases and hypersensitivities in mice, includ-

ing atherosclerosis, contact hypersensitivity, transplant

rejection, oxazolone-induced colitis, concanavalin-A-

induced hepatitis, lipopolysaccharide-induced inflamma-

tion and shock, hepatic ischemia–reperfusion injury, and

allergen-, ozone-, and virus-induced airway hyperreactivity.

Finally, iNKT cells, through their capacity to produce

colony-stimulating factors such as GM-CSF, also contrib-

ute to hematopoiesis during steady-state conditions and

situations of immune activation.

While these studies have shown critical roles of iNKT

cells in both health and disease, iNKT-cell-deficient mice do

not develop any obvious spontaneous pathologies, except

for lupus-like nephritis in aged Ja18 knockout mice (35). In

addition, iNKT cells appear to be absent in ruminants (36),

suggesting functional redundancy with other innate-like or

CD1-restricted T-cell subsets.

Response of iNKT cells to glycolipid antigens

The in vivo response of iNKT cells to glycolipid antigen

stimulation has been explored most extensively with

a-GalCer (37). Within hours after a-GalCer treatment,

iNKT cells produce copious amounts of cytokines, includ-

ing IL-2, IL-4, and IFN-g. Activated iNKT cells produce

IL-4 more rapidly than most other cytokines, and cytokine

production is short lived, lasting only a few days. iNKT cells

Figure 3 Two ways to activate an iNKT cell. (A) In the direct pathway of iNKT-cell activation, microbial antigens that reach CD1d-containing endosomal

compartments through any of the pathways depicted in Figure 2 are loaded onto CD1dwith the assistance of lipid transfer proteins such as the saposins

(SAPs). These complexes are then displayed at the cell surface for recognition by the invariant T-cell receptor (TCR) on iNKT cells. (B) In the indirect

pathway of iNKT-cell activation,microbial products engageextracellular or intracellular toll-like receptors (TLRs),which induce the production of cytokines

such as interleukin (IL)-12, IL-18, and/or type I interferons (IFN-a and IFN-b). These cytokines subsequently engage cytokine receptors on iNKT cells,

resulting in iNKT-cell activation. Different cytokines have been implicated in the activation of iNKT cells in response to distinct microbial products. For

somemicrobial products, iNKT-cell activation through this cytokine-drivenmechanism also requires engagement of the invariant TCR on iNKT cells with

CD1d molecules bound with endogenous antigens. INKT, invariant natural killer T.



activated in thismanner also profoundly downregulate their

surface TCRs, which become nearly undetectable by 8–12 h

but are mostly restored by 24 h after a-GalCer injection.

This phenomenon is largely responsible for the transient

‘disappearance’ of iNKT cells following a-GalCer injection

when these cells are identified with reagents that bind with

surface TCRs (anti-TCR-b antibodies or CD1d tetramers).

Activated iNKT cells also downregulate the surface

expression of the NK1.1 marker, and this downregulation

is long lasting (up to 6 months). a-GalCer-activated iNKT

cells also rapidly proliferate in vivo, resulting in profound

expansion of the iNKT-cell population in multiple organs,

which peaks around day 3 after a-GalCer treatment. In

some studies, the iNKT-cell population was shown to

expand up to 10- to 15-fold in the spleen and 2- to 3-fold in

liver. After this expansion phase, the iNKT-cell population

contracts through homeostatic mechanisms that involve the

proapoptotic Bcl-2 family member Bim, reaching pre-

treatment levels around 10–15 days.

A cardinal feature of adaptive immunity is the develop-

ment of immunological memory. However, this is not the

case for iNKT cells, which develop an anergic phenotype

after a-GalCer treatment (37). This anergic phenotype

persists for up to 6 weeks after the initial a-GalCer

treatment. A similar anergic phenotype is seen following

challenge of mice with multiple bacterial pathogens or their

products (38–40) and following injection of sulfatide (41),

a ligand of type II NKT cells. Thus, the development of

anergy might be a common outcome of iNKT-cell

activation. Acquisition of an anergic phenotype by acti-

vated iNKT cells likely provides a means to avoid chronic

cytokine production by these cells that could cause

uncontrolled inflammation. The anergic phenotype induced

by a-GalCerwas intrinsic to these cells, and the proliferative

defect could be overcome by culture in the presence of IL-2.

iNKT-cell anergy was associated with sustained expression

of the inhibitory, costimulatory receptor programmed

death-1 (PD-1) (42, 43). Blockade of PD-1 interactions with

its ligands, PD-L1 and PD-L2, was able to prevent the

generation of iNKT-cell anergy by a-GalCer but not by

bacteria or sulfatide. The critical role of the PD-1/PD-L

pathway in the induction of iNKT-cell anergy was further

shown in PD-1 knockout animals, which are resistant to

a-GalCer-induced iNKT-cell anergy (43). Furthermore,

certain glycolipid treatment modalities can effectively

activate iNKT cells while avoiding the induction of anergy.

This includes the delivery ofa-GalCer in the context ofDCs,

loaded onto a recombinant CD1d molecule, formulated as

a nanoparticle, or injected subcutaneously at low doses.

Numerous structural analogs of a-GalCer have been

synthesized and analyzed (37). Some reagents, such as the

sphingosine-truncated a-GalCer analog called ‘OCH’

(Figure 1B) and the C20:2 variant containing a diunsatu-

rated N-acyl chain (Figure 1B), induce a Th2-biased

cytokine production profile by iNKT cells. Conversely,

the C-glycoside analog a-C-GalCer (Figure 1B) promotes

a Th1 bias in the cytokine production profile of iNKT cells.

Furthermore, iNKT cells stimulated with threitolceramide

(Figure 1B), a nonglycosidic a-GalCer analog, produce

a cytokine profile similar to that of a-GalCer but recover

more quickly from activation-induced anergy (44).

Immunomodulatory activities ofiNKT-cell ligands

The cytokines secreted by a-GalCer-activated iNKT cells

can rapidly activate a variety of other cell types, including

DCs, macrophages, NK cells, B cells, and conventional

T cells (37, 45). This is evidenced by expression of activation

markers such as CD69 on NK cells, B cells, and T cells and

induction of costimulatory molecules on DCs, macro-

phages, and B cells. Activation of DCs involves interactions

between CD40 and OX40 ligand on these cells with CD40

ligand and OX40, respectively, on iNKT cells. Activated

DCs subsequently produce proinflammatory cytokines

such as IL-12 and TNF-a, which further contribute to the

cytokine storm. IL-12 derived fromDCs and IFN-g derivedfrom iNKT cells also induce NK cells to produce IFN-g.Furthermore, a-GalCer-activated iNKT cells enhance the

immune responses mediated by cytotoxic T cells (CTL),

CD41 T cells, and B cells. a-GalCer promotes not only

T-cell-dependent but also T-cell-independent antibody

responses, probably owing to the capacity of iNKT cells

to interact withCD1donB cells, includingB-1 andmarginal

zone B cells. These characteristics of activated iNKT cells

form the basis for the adjuvant activities of iNKT-cell

ligands (46).

a-GalCer also influences the quality of an adaptive

immune response (37, 45). A single a-GalCer injection

induces a substantial rise in serum immunoglobulin E levels,

suggesting Th2 deviation of the adaptive immune response.

Th2 deviation is characteristically observed in chronic

a-GalCer injection protocols, which might be associated

with the capacity of anergic iNKT cells to produce residual

amounts of Th2 but not of Th1 cytokines.

The cytokine storm that ensues following a-GalCer

treatment can also lead to significant pathology (47).

Because many iNKT cells reside in the liver, a single

a-GalCer treatment induces transient hepatitis in mice.

a-GalCer treatment in some mouse strains can also induce

abortion, probably because a substantial number of iNKT

cells reside in the uterus. Furthermore, a single intranasal

administration of a-GalCer sensitizes mice to the develop-

ment of airway hypersensitivity. Nevertheless, a-GalCer

treatment in humans, who have fewer iNKT cells thanmice,

has proven to be safe, at least in the short term (48).

However, at present, it is unclear whether a-GalCer

treatment in humans can elicit sufficiently strong biological



responses to bring forth its adjuvant and therapeutic

activities.

iNKT-cell antigens as vaccine adjuvants

The capacity of iNKT-cell ligands to enhance the efficacy of

vaccines against multiple microbial pathogens has been

tested (46, 49). Inclusion of a-GalCer in a malaria vaccine

resulted in enhanced antimalarial immunity, in a manner

that was dependent on IFN-g production by iNKT cells.

a-C-GalCer, which induces a Th1-biased cytokine pro-

duction profile in iNKT cells, showed profoundly enhanced

adjuvant activity in the malaria vaccine compared with

a-GalCer. a-GalCer was also effective as a mucosal

adjuvant in different vaccine formulations directed against

influenza virus. Likewise, a-GalCer enhanced the immuno-

genicity of an HIV-1 DNA vaccine, enhancing CD4 and

CD8 T-cell responses as well as humoral immunity.

The adjuvant activities of iNKT cells have also been

tested in multiple cancer vaccines (46, 49). a-GalCer

substantially enhanced the priming and boosting of CD8

T cells directed against ovalbumin antigens or the human

cancer/testis antigen NY-ESO-1, resulting in the eradica-

tion of established tumors bearing these antigens. Coad-

ministration of a-GalCer with irradiated plasmacytoma

cells also enhanced tumor immunity, in a manner that

involved DC maturation. Similarly, injection of a-GalCer-

loaded and CD1d-transfected B16melanoma cells provided

protection against rechallenge of the melanoma cells.

iNKT cells as targets for immunotherapy

a-GalCer was originally discovered by Kirin Brewery

Company, Ltd. (Gunma, Japan) as a natural product with

antimetastatic activities in mice (50). The antitumor

activities of a-GalCer have been shown in a variety of

transplantable, chemically induced, and genetic tumor

models (48). Although iNKT cells themselves have cytolytic

activity, the tumor target cells do not need to express CD1d

for therapeutic efficacy, suggesting that iNKT cells are not

the relevant effector cells. Instead, NK cells and antigen-

specific CTLs appear to be the critical effector cells through

their cytolytic activities and their capacity to produce

IFN-g, which has antiangiogenic activities. Delivery of

a-GalCer in the context of DCs provided superior anti-

metastatic activities, in part because this treatment avoids

the induction of iNKT-cell anergy. Likewise, combined

treatment with a-GalCer and antibodies that block the

PD-1/PD-L pathway, thus avoiding anergy induction, was

superior to a-GalCer treatment alone (42, 43). Further-

more, a-C-GalCer wasmore effective than a-GalCer, which

is probably because of its superior capacity to induce IFN-gproduction by iNKT cells, to prime DCs, and to trans-

activate NK cells and CD8 T cells. These preclinical studies

have paved the way for phase I and phase II trials in cancer

patients (48). These studies used free a-GalCer, a-GalCer

presented byDCs, or transfer of cells expanded in vitro from

peripheral bloodmononuclear cells culturedwith a-GalCer.

Although these treatments were safe, it has generally been

challenging to obtain strong biological responses, which is

likely because of the low frequency of iNKT cells in humans,

as well as the iNKT-cell dysfunction that has been observed

in cancer patients. The most encouraging trial to date

involved a study where patients with non-small cell lung

cancer were administered four times with peripheral blood

mononuclear cells cultured in the presence of a-GalCer, IL-2,

and GM-CSF (51). This treatment was well tolerated and

was accompanied by the induction of iNKT-cell-dependent

responses. The patients with increased IFN-g-producingcells, compared with poor responders, had an increased

median survival time (responders: 31.9 months, n ¼ 10;

poor responders: 9.7 months, n ¼ 7; P ¼ 0.0015).

The capacity of a-GalCer tomodulatemicrobial infection

has also been investigated (52). a-GalCer treatment was

generally associated with enhanced pathogen clearance and

an improved disease course, as shown for Mycobacterium

tuberculosis, S. pneumoniae, P. aeruginosa, Trypanosoma

cruzi, C. neoformans, hepatitis B virus, and influenza virus.

However, for C. muridarum infection, delayed rather than

enhanced clearance was observed. Mechanisms of action

appeared to be diverse. In some cases, divergent results were

obtainedwith regard to pathogen clearance and disease. For

example, a-GalCer treatment of RSV-infected mice delayed

viral clearance but improved weight loss. For Plasmodium

species, a-GalCer treatment promoted the clearance of

organisms from mice infected with the sporozoite stages of

the parasite but delayed clearance of mice infected with

erythrocyte stages. This is likely because of the critical role

of IFN-g and CD81 T cells in clearing the liver but not

blood stages of the organism, which is consistent with the

enhanced capacity of a-C-GalCer to clear malaria parasites

from sporozoite-infected mice. Importantly, the time

window in which a-GalCer treatment was effective is rather

narrow, typically within a few days of infection. As such,

iNKT-cell activation will likely provide little benefit to

established infections. No benefit of a-GalCer treatment

was observed in patients chronically infected with hepatitis

C virus (53).

Because of its capacity to promote Th2 immune responses,

particularly when injected repeatedly, a-GalCer has been

tested in multiple mouse models of autoimmunity (33, 50).

Chronic a-GalCer treatment protected NOD mice against

diabetes, in a manner that involved Th2 deviation of auto-

antigen-specific immune responses, but the role of Th2 cyto-

kines in this process remains unproven. Foxp3-expressing

Treg cells, tolerogenic DCs, and anergy induction in

pathogenic T cells all have been implicated as factors



contributing to disease protection. OCH also protected

against disease, and C20:2 appeared to be more effective

than botha-GalCer andOCH.a-GalCer andOCHwere also

effective in protecting mice against the development of

experimental autoimmune encephalomyelitis (EAE) in mice,

a model forMS.However, these studies also showed that the

timing of iNKT-cell activation can significantly impact

treatment efficacy, with early treatment ameliorating disease

and late treatment exacerbating disease in some studies. In

these EAE models, disease amelioration was typically

associated with Th2 deviation and disease exacerbation was

associated with Th1 deviation. Nevertheless, both Th1 and

Th2 cytokines have been implicated in disease protection,

which is consistent with a pathogenic role of Th17 cells in

EAE. a-GalCer also protected mice against experimental

autoimmune myasthenia gravis, an antibody-dependent

disease, in a mechanism that appeared to rely on Foxp3-

expressing Treg cells. a-GalCer and OCH were able to

protectC57BL/6mice against collagen-induced arthritis, and

this was associated with Th2 deviation. a-GalCer also

attenuated collagen-induced arthritis in DBA/1 mice, with

a critical role for IL-10. Surprisingly, however, a single

injection of a-C-GalCer was also protective in the DBA/1

model of collagen-induced arthritis, and thiswas suggested to

be because of general suppression of T-cell responses. In

contrast with its effects on collagen-induced arthritis,

a-GalCer moderately enhanced joint inflammation in an

antibody-mediated arthritis model. a-GalCer, OCH, and

a-C-GalCer were all protective in an experimental model of

ocular autoimmunity, and surprisingly, a-C-GalCer was

most effective (54). This protective effect of a-C-GalCer was

associated with dampening of Th1 and Th17 effector

functions. a-GalCer was generally protective in chemically

induced and geneticmodels of lupus-like disease, but in some

mouse strains, or when administered late in the disease

process, it exacerbated disease (55). Disease protection was

typically associated with Th2 deviation. a-GalCer was also

protective inamodel for autoimmune thyroiditis, in amanner

that correlated with reduced IFN-g and reduced autoanti-

body production. Collectively, these studies have shown that

iNKT-cell activation typically protects against autoimmu-

nity but that treatment efficacy is influenced by a variety of

parameters, including the nature and dose of the iNKT-cell

antigens, the frequency and route of injections, the timing of

treatment relative to disease initiation and progression, the

particular animalmodel used, and the genetic background of

the animals (33, 50). Many of these issues pose problems for

translation to treatment of human autoimmunity.

A single injection of a-GalCer into recipient mice

significantly reduced morbidity and mortality of graft-vs-

host disease (56). This was dependent on stimulation of host

iNKT cells and Th2 deviation of donor T cells.

Although iNKT cells exhibit potent therapeutic proper-

ties in a number of diseases, iNKT-cell activation can also

exacerbate disease such as allergic reactions and atheroscle-

rosis in mice (33, 55).

Conclusions and outlook

iNKT cells are unique lymphocytes that bridge the innate

and adaptive immune systems. These cells acquire their

innate-like phenotype during thymic development by

interaction with CD1d–glycolipid complexes on double-

positive cells and under direction of the transcription factor

PLZF. iNKT cells can respond to endogenous and exo-

genous glycolipid antigens but can also become activated

indirectly by cytokine-driven mechanisms. Quickly after

activation, these cells produce a variety of cytokines with

potent immunomodulatory activities, but they then enter

a period of quiescence. iNKT cells regulate a variety of

immune responses. iNKT-cell ligands exhibit potent

immune-enhancing properties that are being exploited for

the development of vaccine adjuvants. iNKT-cell activation

also has therapeutic effects in mice, and clinical trials with

a-GalCer for cancer are underway. The preclinical studies

with a-GalCer and related iNKT-cell antigens have also

shown potential risks associated with iNKT-cell activation

that will need to be overcome in order to develop safe and

effective iNKT-cell-based vaccine adjuvants and therapies.

Future studies should focus on developing means to better

control the outcome of iNKT-cell activation on immune

responses and disease using diverse treatment modalities

and within genetically diverse hosts.

Acknowledgments

We apologize to colleagues whose work we did not cite

because of space constraints or omission. We thank many

colleagues, especially Dr Sebastian Joyce (Vanderbilt

University School of Medicine), for helpful discussions.

The authors’ work was supported by grants from the

National Institutes of Health (to LVK), a discovery grant

from the Diabetes Research and Training Center at

Vanderbilt University School of Medicine (to LW), a pre-

doctoral fellowship from the National Institutes of Health

(to CLG), and a postdoctoral fellowship from the National

Multiple Sclerosis Society (to VVP).

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Invariant natural killer T cells: innate-like T cells with potent immunomodulatory activities

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