The Biology of NKT Cells Albert Bendelac - part 3 pptx

On the left, infection by Gram-negative, LPS-negative Sphingomonas induces direct activation of NKT cells through recognition of microbial cell wall α-glycuronylceramide.. In light of th

pathogen and member of the Rickettsiales that

is of widespread significance for mammals,

including wild and domesticated ruminants,

dogs, and humans from some regions of the

world such as Africa and East Asia Ehrlichia

muris activates NKT cells independently of

iGb3, and its clearance was profoundly altered

in CD1d- or Jα18-deficient animals (23)

Ehrlichia is a Gram-negative, LPS-negative

obligate intracellular bacterium, whose cell

wall composition has not been elucidated

Interestingly, many other bacteria,

particu-larly the Gram-negative, LPS-positive ones,

can activate NKT cells However, rather than

provide their own NKT ligands like

Sphin-gomonas or Ehrlichia, these bacteria appear

to trigger autoreactive NKT cell responses

(23, 60) In the case of Salmonella, this is

suggested by the abrogation of NKT cell

activation in the presence of DCs lacking

β-hexosaminidase B, the enzyme responsible

for the generation of iGb3 from iGb4 in the

lysosome, and by blocking experiments with

the lectin Griffonia simplicifolia IB4, which

recognizes the terminal sugar of the Galα1-3Gal epitope of iGb3 bound to CD1d and blocks NKT cell activation (23) Strikingly, NKT cell activation by Gram-negative,

LPS-positive Salmonella is absolutely dependent

upon TLR signaling through the adaptors MyD88 and Trif, and upon IL-12 release by the APC, although the precise TLR combi-nation and the corresponding microbial struc-tures involved remain to be determined Thus, the proposed scenario suggests that TLR sig-naling leading, but not limited, to IL-12 secre-tion enhances the ability of DCs to stimulate NKT cells through presentation of

endoge-nous ligands (Figure 7).

Whether TLR signaling induces an up-regulation of iGb3 or changes in the expres-sion of other factors such as, for example, NK receptor ligands is unclear Contrary to an early report (195), NKT cells do not usually

Late endosome/lysosome

TLR4

IL-12p40

LPS Gram-negative,

LPS-negative

bacteria

Gram-negative bacteria

?

NKT cell

iGb3 Bacterial Ag

Figure 7

Dual recognition of self and microbial glycosphingolipids during microbial infections On the left,

infection by Gram-negative, LPS-negative Sphingomonas induces direct activation of NKT cells through

recognition of microbial cell wall α-glycuronylceramide On the right, infection by Gram-negative,

LPS-positive Salmonella activates TLR4 through LPS and induces IL-12, revealing constitutive

autoreactive recognition of iGb3 through the secretion of IFN-γ (indirect microbial recognition).

constitute the predominant cell type that pro-duces IFN-γ in response to IL-12 in vivo (60, 196) This explains why they generally

do not appear to be essential in fighting Gram-negative, LPS-positive bacteria How-ever, an impact on bacterial clearance has been observed in the case of lung infection

with Pseudomonas aeruginosa, where

bacterial count in the lung within 6–24 h postinoculation and an approximately three-fold decrease in MIP-2 and neutrophils in the bronchoalveolar lavage (197) This may not

be the case at other sites of infection (198)

Variations have been noted as well in reports assessing the role of NKT cells versus NK cells in LPS-induced toxic shock in vivo (199, 200)

Primary Biliary Cirrhosis and

Sphingomonas

An intriguing connection between primary

biliary cirrhosis (PBC), Sphingomonas, and

NKT cells has emerged recently PBC is a disease characterized by the presence of an-timitochondrial antibodies, liver lymphocytic infiltrates, and the chronic destruction of the biliary epithelium, which leads to cirrhosis (201) Interestingly, the autoantibodies recog-nize an epitope of the mitochondrial PDC-E2 enzyme that is particularly well conserved

in Novosphingobium aromaticivorans, a strain

of Sphingomonas Furthermore, PBC patients,

including those lacking antimitochondrial an-tibodies, were specifically seropositive against

Sphingomonas, which was detected by PCR in

stool samples of 25% of diseased or healthy individuals, suggesting that PBC may be in-duced by aberrant host reactivity to this bac-terium (202) PBC patients also showed an en-richment of Vα24 NKT cells in liver biopsies, but a depletion in blood (203) In light of the

recent finding that Sphingomonas cell wall

gly-colipids specifically activate NKT cells, these studies suggest that NKT cells may play a key role in the pathogeny of PBC by promoting

aberrant responses to Sphingomonas.

Parasitic Infections

Shofield and colleagues (204) suggested that the production of IgG antibodies to the malaria circumsporozoite antigen, a key com-ponent of protective immune responses in humans, depended on NKT cell recogni-tion of malarial glycosylphosphatidylinosi-tol antigens in a mouse model However, additional experiments failed to detect a CD1d-dependent component to this antibody response, and glycosylphosphatidylinositols have not been identified as NKT cell anti-gens in other reports (205, 206) In the con-text of helminth infection, DCs pulsed with

Schistosoma mansoni eggs activated NKT cells

to secrete Th1 and Th2 cytokines in vitro in a β-hexosaminidase-B-dependent but MyD88-independent manner, suggesting recognition

of the self ligand iGb3 in the absence of TLR signaling (207)

Viral Infections

Relatively modest defects in the clearance of some viruses have been reported in CD1d-deficient mice infected with encephalomy-ocarditis virus (208) or coxsackie B3 (209), but these defects were not observed in Jα18-deficient mice, ruling out a specific role

of Vα14 NKT cells Infections with lym-phocytic choriomeningitis virus, mouse cy-tomegalovirus, vaccinia virus, and coronavirus were unaffected Studies in humans have sug-gested a profibrotic role of Vα24 NKT cells

in hepatitis C (85) and the accumulation of non-Vα24 CD1d-restricted T cells (210) Al-though a specific role of Vα14 NKT cells

in HSV infection remains controversial (211, 212), recent studies have suggested that vi-ral invasion may be associated with counter-measures against CD1d or NKT cells For example, HSV-1 drastically and specifically impaired CD1d recycling from the lysosome

to the plasma membrane, an essential pathway for glycolipid antigen presentation to NKT cells (96) Kaposi sarcoma–associated herpes virus encodes two modulators of immune

recognition, MIR1 and MIR2, that

downreg-ulated CD1d in addition to other

immunolog-ically relevant molecules such as MHC class

I, CD86, and intracellular adhesion molecule

(ICAM)-1 through ubiquitination of lysine

residues in their cytoplasmic tail (95) The

lethal outcome of infections with

Epstein-Barr virus in patients with X-linked

lympho-proliferative (XLP) immunodeficiency

syn-drome due to SAP mutations was

hypothe-sized to result from the absence of NKT cells

(144) Which of these effects or associations

reflect a specific viral evasion/immune defense

strategy and the nature of the NKT ligands

in-volved in these infectious conditions remain

to be determined

NKT Cells in Noninfectious

Diseases

A role of NKT cells has been suggested in a

wide variety of disease conditions At present,

however, many reports, lacking a detailed

mechanistic understanding, remain isolated

or are based merely on the analysis of NKT

cell–deficient mice Rather than compiling an

exhaustive list of the published claims, this

re-view provides a critical appraisal of selected

reports carrying important conceptual or

clin-ical implications One frequently overlooked

but recurrent methodological issue inherent

in the use of CD1d- or Jα18-deficient mice

is the extent to which gene-deficient mice

are matched with littermate controls with

re-spect to genetic background and

environmen-tal factors This is particularly important in

studies of complex multigenetic diseases such

as diabetes, lupus, cancer, or asthma In

ad-dition, the injection of αGalCer as a

gain-of-function experiment should be interpreted

with caution because the massive release of

cy-tokines induced by this procedure is unlikely

to model chronic diseases It may not be

sur-prising, therefore, that some claims have

be-come controversial or will need to be

rein-terpreted, complicating the task of drawing a

clear picture of the involvement of NKT cells

in noninfectious diseases

Type I diabetes. The relative deficiency of NKT cells in NOD mice (36, 37), combined with the notion that these cells represent a po-tent source of Th2 cytokines, prompted the original speculation of a causal relationship with diabetes Early claims that humans with type I diabetes exhibited severe NKT cell de-fects and that their sera had less IL-4 than controls (213, 214) were not confirmed when more specific methodologies became available (38, 215) Researchers interpreted reports of aggravated disease in CD1d-deficient NOD mice (216, 217) as suggesting that, although defective, the residual NKT cells in NOD mice still suppressed autoimmunity How-ever, independent studies in different colonies

of CD1d-deficient and Jα18-deficient mice failed to support these claims (218), and par-tial reconstitution of NKT cells in NOD

mice carrying the B6 Nkt1 locus did not

pro-tect against diabetes (34) Transgenic expres-sion of the Vα14-Jα18 TCRα chain in NOD mice prevented diabetes, but this could be explained by the reduced frequency of islet-specific T cells and the general Th2 bias of these mice (219) Likewise, the suppression

of diabetes by αGalCer multi-injection regi-mens could be the mere consequence of mas-sive cytokine release (220, 221) More di-rect transfer experiments using diabetogenic

T cells and NKT cells have suggested sup-pressive or enhancing roles of NKT cells

in different experimental systems (222, 223) Although other more circumstantial studies have suggested a role of NKT cells in this disease, it seems reasonable to conclude at this point that there is no decisive evidence for a substantial or specific role of NKT cells in mouse or human type I diabetes

shown to accumulate in aging NZB/W mice (224) and suggested to help B cells produce anti-DNA antibodies (225) However, studies

of CD1d-deficient lupus-prone mice have not yielded concordant results (226–228), and injections of αGalCer ameliorated or

aggravated disease, depending on the mouse strain (229)

Cancer. Similar to the general immune sup-pression of T cells commonly encountered in cancerous states, NKT cells were decreased or functionally hyporeactive in cancer-bearing mice and humans (230, 231) One tumor shed glycosphingolipids that could inhibit the stim-ulation of NKT cells in vitro (232) How-ever, multiple mechanisms are likely to con-tribute to the deficiency of both T and NKT cells In one report, the frequency of sarcomas six months after intramuscular injection of the chemical carcinogen methylcholantrene (MCA) decreased two- to threefold in Jα18 knockout NKT cell–deficient mice (233)

This observation, which suggested that NKT cells, similar to γδ T cells and NK cells, may

be agents of immune surveillance against pri-mary cancers has remained isolated

In a tumor transplant model, subcutaneous injection of a fibrosarcoma tumor line derived from MCA-inoculated Jα18-deficient mice produced tumors that grew faster in Jα18-deficient compared with wild-type mice and were prevented by transfers of purified NKT cells into Jα18-deficient hosts (234) CD1d expression and the presence of CD8 T cells

in the host were required for tumor rejection, implying ligand recognition on host-derived cells, presumably APCs, rather than on tu-mor cells The nature of the tutu-mor-associated NKT ligands has not been identified These experiments also revealed a specialized func-tion of liver DN—as compared with CD4—

NKT cells in this Th1-mediated response (128)

In apparent contrast with this fibrosar-coma model, CD1d-deficient mice controlled the growth of otherwise relapsing subcuta-neous transplants of the 15-12RM tumor line, suggesting that a natural CD1d-dependent mechanism suppressed tumor rejection (235)

Further studies dissected a complex cellu-lar network that involved IL-13-producing CD1d-restricted CD4 suppressors interact-ing with TGF-β-producinteract-ing myeloid cells to

suppress antitumor CTL responses Because Jα18-deficient mice did not share the pheno-type of CD1d-deficient mice, the study con-cluded that other less well-known types of CD1d-restricted T cells might be involved (236) As in the MCA-induced tumor trans-plants, these tumors did not express CD1d, yet CD1d expression by host cells, presumably APCs, was required to observe the NKT cell effects In contrast, the growth of the CD1d-transfected RMA/S tumor cell line cells was inhibited by Vα14 NKT cells (237) In con-clusion, the notion that mVα14 and hVα24 NKT cells regulate cancer rejection is based largely on tumor transplant models, and the relevance to natural clinical conditions re-mains to be determined

were reported to exhibit decreased allergen-induced airway hyperreactivity in the alum-ovalbumin model of asthma, where mice are intraperitoneally sensitized with ovalbumin mixed in alum and subsequently challenged with ovalbumin inhalation (238, 239) How-ever, similar studies in another laboratory have failed to observe differences between CD1d-deficient and wild-type mice (R Lock-sley, personal communication) In humans with persistent, moderate-to-severe asthma, Vα24 NKT cells dominated the bronchial Th2 infiltrate (240) The extent of this NKT cell expansion has been disputed, however, perhaps reflecting differences in the cohorts

of asthma patients examined or the methods for identifying NKT cells (241)

de-creased the level of atherosclerosis in

apoE-or LDL receptapoE-or–deficient mice, although the effects observed were only mild and transient in some studies (241, 242)

ob-servations suggesting a suppressive role of NKT cells in some models of delayed-type hypersensitivity (242, 243), in anterior

chamber–associated immune deviation (244),

and in burn injury (245) have been reported

In summary, contrasting with numerous

reports suggesting a contribution of NKT

cells in a range of noninfectious diseases, a

convincing picture has not yet emerged as to

the strength or consistency of the observed

effects, their mechanisms, or their relevance

to physiological or clinical conditions Future

experiments are needed to define those

dis-eases and conditions that are regulated

specif-ically by mVα14 or hVα24 NKT cells and to

dissect the mechanisms involved

THE LARGER CD1 UNIVERSE

Although T cells recognizing lipids

pre-sented by other CD1 isotypes were the first

discovered (44), their study now represents

only a small fraction of the current

in-vestigations on CD1-mediated antigen

pre-sentation, which focus overwhelmingly on

the CD1d/NKT cell system CD1d is the

only representative in mouse and rat of a

larger family of β2-microglobulin-associated

MHC-like molecules that, in other

mam-malian species, comprises CD1a, -b, and -c,

as well as CD1e (44) CD1 and MHC are

en-coded in different loci, but recent genomic

studies in chicken suggest that they originated

from the same primordial MHC locus (246)

CD1a, -b, and -c differ in their location in

different endosomal compartments, in early

recycling to late endosome and lysosome, and

also in the architecture of their lipid-binding

grooves, which suggests that each is

special-ized to capture different lipids in different

en-dosomal compartments (44) Individual self

and microbial lipid-specific T cell clones have

been derived in vitro in humans, but relatively

little is known about the T cell types and TCR

repertoires associated with CD1a, -b, and -c

and about their function in the human system

With respect to CD1d, however, it is

well established that the major population of

CD1d-restricted T cells in mouse is the NKT

cell population that expresses semi-invariant

TCRs, predominantly Vα14-Jα18, and

per-forms innate-like functions (19) The pres-ence of a more diverse population has been suggested recently, more convincingly in hu-mans, indicating that an adaptive population

of lipid-specific CD1d-restricted T cells may

be available (210, 247, 248) The biology of these cells remains largely unexplored, and fu-ture studies in this area would resolve a fasci-nating and long-standing debate in the field

of T cell recognition Indeed, glycolipids are not easily mutated or modified, and although the potential theoretical combinations of car-bohydrates are extremely diverse, the universe

of microbial glycolipids is limited owing to en-zyme specificity for both donor and acceptor substrates in glycolipid synthesis Thus, the glycolipid-specific repertoire did not evolve under the same pressure that operated on the peptide-specific repertoire, where single mu-tations produce new T cell epitopes How diverse and specific this glycolipid-specific repertoire may be is an important question for future research because conserved glycol-ipids may represent ideal, fixed targets for vaccine development In addition, how cross-reactive the MHC- and CD1-restricted TCR repertoires are is a fundamental issue that remains to be investigated Given that the groove of CD1 molecules is significantly nar-rower than that of MHC proteins and that at least a proportion of the TCR repertoire ap-pears to be intrinsically MHC-restricted (249, 250), one would assume that the peptide-specific and glycolipid-peptide-specific TCR reper-toire should be essentially non-cross-reactive,

a prediction that remains to be tested

SUMMARY

Recent studies have elucidated novel and striking aspects of NKT cell development and of the cell and structural biology of lipid antigen processing and recognition Key candidate antigens have been identified that provide a framework for understanding the evolution and function of this innate-like lin-eage, particularly in microbial infections Fu-ture work will clarify the range and naFu-ture

of the most physiologically relevant ligands and the structural basis of their recognition

by the semi-invariant TCRs These solid ad-vances in fundamental biology should help

develop a mechanistic understanding of the broad and sometimes controversial array of diseases in which NKT cells are increasingly implicated

ACKNOWLEDGMENTS

We thank past and present members of our laboratories for their contributions to the un-derstanding of NKT cell biology; Seth Scanlon and Omita Trivedi, for help with the figures; and Richard Locksley, Diane Mathis, and Thomas Blankenstein for sharing unpublished

re-sults Dirk Zajonc generated the structural representation in Figure 2 This work is supported

by the Howard Hughes Medical Institute (A.B.) and by a program project grant from the National Institutes of Health (A.B., P.B.S., L.T.) No review on NKT cell biology can ade-quately describe every interesting paper, and we apologize to those investigators whose work could not be cited because of space limitations

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