α-Glucuronosyl and α-glucosyl diacylglycerides, natural killer T cell-activating lipids from bacteria and fungi

Article abstract of DOI:10.1039/c9sc05248h

Natural killer T cells express T cell receptors (TCRs) that recognize glycolipid antigens in association with the antigen-presenting molecule CD1d. Here, we report the concise chemical synthesis of a range of saturated and unsaturated α-glucosyl and α-glucuronosyl diacylglycerides of bacterial and fungal origins from allyl α-glucoside with Jacobsen kinetic resolution as a key step. These glycolipids are recognized by a classical type I NKT TCR that uses an invariant Vα14-Jα18 TCR α-chain, but also by an atypical NKT TCR that uses a different TCR α-chain (Vα10-Jα50). In both cases, recognition is sensitive to the lipid fine structure, and includes recognition of glycosyl diacylglycerides bearing branched (R- and S-tuberculostearic acid) and unsaturated (oleic and vaccenic) acids. The TCR footprints on CD1d loaded with a mycobacterial α-glucuronosyl diacylglyceride were assessed using mutant CD1d molecules and, while similar to that for α-GalCer recognition by a type I NKT TCR, were more sensitive to mutations when α-glucuronosyl diacylglyceride was the antigen. In summary, we provide an efficient approach for synthesis of a broad class of bacterial and fungal α-glycosyl diacylglyceride antigens and demonstrate that they can be recognised by TCRs derived from type I and atypical NKT cells.

Full text of DOI:10.1039/c9sc05248h

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a-Glucuronosyl and a-glucosyl diacylglycerides,
natural killer T cell-activating lipids from bacteria
and fungi†
Cite this: Chem. Sci., 2020, 11, 2161
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Satvika Burugupalli,‡a Catarina F. Almeida,‡bc Dylan G. M. Smith,‡a Sayali Shah,a
bc
Onisha Patel,d Jamie Rossjohn,def Adam P. Uldrich,bc Dale I. Godfrey
*
ab
*
and Spencer J. Williams
Natural killer T cells express T cell receptors (TCRs) that recognize glycolipid antigens in association with the
antigen-presenting molecule CD1d. Here, we report the concise chemical synthesis of a range of saturated
and unsaturated a-glucosyl and a-glucuronosyl diacylglycerides of bacterial and fungal origins from allyl a-
glucoside with Jacobsen kinetic resolution as a key step. These glycolipids are recognized by a classical type
I NKT TCR that uses an invariant Va14-Ja18 TCR a-chain, but also by an atypical NKT TCR that uses
a different TCR a-chain (Va10-Ja50). In both cases, recognition is sensitive to the lipid fine structure, and
includes recognition of glycosyl diacylglycerides bearing branched (R- and S-tuberculostearic acid) and
unsaturated (oleic and vaccenic) acids. The TCR footprints on CD1d loaded with a mycobacterial a-
glucuronosyl diacylglyceride were assessed using mutant CD1d molecules and, while similar to that for
a-GalCer recognition by a type I NKT TCR, were more sensitive to mutations when a-glucuronosyl
diacylglyceride was the antigen. In summary, we provide an efficient approach for synthesis of a broad
class of bacterial and fungal a-glycosyl diacylglyceride antigens and demonstrate that they can be
recognised by TCRs derived from type I and atypical NKT cells.
Received 17th October 2019
Accepted 9th January 2020
DOI: 10.1039/c9sc05248h
rsc.li/chemical-science
(Va24-Ja18 in humans), and can recognize the prototypical
glycolipid antigen a-galactosyl ceramide (a-GalCer). Conversely,
Introduction
CD1d-restricted type II NKT cells express a range of TCR a-and
b-chains and do not recognize a-GalCer presented by CD1d. In
an earlier study,3 we discovered a population of NKT cells in
mice that responded to a-GalCer, yet utilised a different TCR a-
chain (Va10-Ja50) to that of type I NKT cells. These cells
therefore share some characteristics of type I and type II NKT
cells and were dened as ‘atypical’ NKT cells.4 Atypical NKT cells
are present in most human individuals (11/19 human subjects),
and in most mice.5 Atypical NKT cells appear to represent up to
10% of the number of CD1d-a-GalCer reactive NKT cells in
human blood, and roughly 1% of CD1d-a-GalCer reactive NKT
cells in mouse thymus (approx. 104 cells per thymus).5
While atypical NKT cells can recognize a-GalCer when pre-
sented by CD1d, this synthetic antigen is of uncertain biological
relevance. We demonstrated that atypical NKT cells exhibit
unique reactivity to an a-glucuronosyl diacylglyceride (a-GlcA-
DAG) from Mycobacterium smegmatis termed Gl-A (1), contain-
ing R-tuberculostearic acid (R-TBSA) at the sn-1 position and
palmitic acid at the sn-2 position, hereaer referred to as a-
GlcADAG-(R-C19:0/C16:0) 1 (Fig. 1).3 Recently, CD1d tetramers
loaded with 1 were used to identify a polyclonal subset of NKT
cells, including populations of a-GalCer-reactive type I NKT
cells, atypical Va10Ja50 NKT cells and a-GlcADAG reactive type
II NKT T cells.6 A 3D structure of a type II-NKT TCR–CD1d-1
An important branch of cellular immunity involves specialized
T cell populations that can recognize assorted lipid-based
antigens when displayed on antigen-presenting cells. Natural
killer T (NKT) cells are unconventional T cells with the ability to
recognize glycolipid antigens presented by the MHC-class I-like
molecule CD1d.1 NKT cells are traditionally classied into two
main groups: type I NKT cells and type II NKT cells.2 Type I NKT
cells express an invariant TCR a-chain (Va14-Ja18) in mice
aSchool of Chemistry, Bio21 Molecular Science and Biotechnology Institute, University
of Melbourne, Parkville, Victoria, 3010, Australia. E-mail: sjwill@unimelb.edu.au
bDepartment of Microbiology and Immunology, Peter Doherty Institute for Infection
and Immunity, University of Melbourne, Parkville, Victoria 3010, Australia
cAustralian Research Council Centre of Excellence for Advanced Molecular Imaging,
University of Melbourne, Parkville, Victoria 3010, Australia. E-mail: godfrey@
unimelb.edu.au
dInfection and Immunity Program, Department of Biochemistry and Molecular Biology,
Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia
eAustralian Research Council Centre of Excellence for Advanced Molecular Imaging,
University of Monash, Monash, Victoria 3010, Australia
fInstitute of Infection and Immunity, Cardiff University School of Medicine, Cardiff
CF14 4XN, UK
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9sc05248h
‡ These authors contributed equally.
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glucopyranoside involving epoxidation and hydrolytic kinetic
resolution to build a diastereomerically pure glycidyl a-D-glu-
copyranoside that could be elaborated to a-GlcDAG deriva-
tives.13 The use of methoxyacetyl groups enabled the
preparation of the unsaturated variant Streptococcus pneumo-
niae a-GlcDAG-(C16:0/D11-C18:1; also known as SPN-GlcDAG).14
This new approach provided the opportunity to develop
a concise synthesis of compound 1 and analogues. We synthe-
sized S-TBSA from (S)-citronellyl bromide, which was converted
to alkene 9 with C6H13MgBr and Li2CuCl4,15 subjected to
a tandem cross-metathesis/reduction to afford the ethyl ester
10, and then saponied to give S-TBSA 11 (Fig. 2A). R-TBSA was
prepared analogously by tandem alkene cross-metathesis of (S)-
2,6-dimethyltetradecene (prepared in one step from (R)-cit-
ronellyl bromide15) with ethyl 6-heptenoate using Hoveyda–
Grubbs II catalyst and in situ reduction using hydrogen, fol-
lowed by saponication.16
As reported by Shah et al.17 allyl a-D-glucopyranoside was
converted to the methoxyacetate by treatment with MeOAcCl/
pyr, treated with mCPBA to obtain a 1:0.95 mixture of 20R/20S
epoxides, then subjected to hydrolytic kinetic resolution using
S,S-Jacobsen's catalyst to provide the stereopure 20R-epoxide
(Fig. 2B). Regioselective brominolysis with Li2NiBr4 afforded
bromohydrin 12, a key intermediate allowing introduction of an
acyl group at the sn-2 position by direct acylation, followed by
introduction of a second acyl group at the sn-1 position by
nucleophilic substitution. We have previously used quantitative
13C NMR and 1-13C-labelled fatty acids to show that while
stepwise acylation processes that proceed via hydroxyl-ester
intermediates give imperfect regioselectivity (owing to intra-
molecular acyl transfer), this acylation/substitution strategy
gives exquisite regioselectivity (R > 99%) at the limit of detec-
tion.14 While this analytical approach is limited to 1-13C-labelled
fatty acids it is presumed that the regioselectivity observed in
the published example extends to the present case. Thus, acyl-
ation of 12 with palmitoyl chloride gave monoester 13, and
substitution with the tetrabutylammonium salt of R-TBSA
afforded the diester 15 (Fig. 2C). The methoxyacetyl groups were
removed by treatment with t-BuNH2 in MeOH to afford a-
GlcDAG-(R-C19:0/C16:0) 12, and a-GlcADAG-(R-C19:0/C16:0) 1
was obtained by oxidation with TEMPO/BAIB in MeCN/H2O.
Application of the same approach to monoester 13 but with S-
TBSA afforded a-GlcDAG-(S-C19:0/C16:0) 6 and a-GlcADAG-(S-
C19:0/C16:0) 2.
Fig. 1 Structure of a-glucuronosyl diacylglyceride Gl-A (1, a-GlcA-
DAG (R-C19:0/C16:0)) from Mycobacterium smegmatis.
complex revealed how this microbial ligand is presented by
CD1d to an NKT TCR.6
Brennan and coworkers reported the isolation of 1 from M.
smegmatis MNC strain 13.7 In order to conrm its structural
assignment, and to explore its biosynthesis and immunological
activity, we completed its total synthesis.8 This synthetic route
enabled the preparation of two analogues that exposed an
intriguing structure–activity relationship for activation of NKT
cells. Thus, while compound 1 stimulated the proliferation of
Va10Ja50 NKT cells from mouse thymus, the C16:0/R-C19:0
regioisomer did not, and a C18:0/C16:0 analogue lacking the
distinguishing methyl branch of TBSA was also inactive.3 These
results suggest that the methyl branch is critical for loading
and/or correct presentation of 1 to the Va10Ja50 NKT TCR.
Brennan and coworkers also reported the isolation from M.
smegmatis of the lipoform a-GlcADAG-(C18:1/C16:0), in which
a double bond is present between C9-10 of the sn-1 fatty acid
ester;7 an identical compound is present in C. glutamicum.8,9
Closely related compounds have been isolated from the path-
ogenic fungus Aspergillus fumigatus,10 Pseudomonas diminuta11
and plants.12 Here we report the details of the approach used to
obtain these compounds, a second generation synthesis of a-
glucuronosyl diacylglycerides that dramatically shortens and
simplies the preparation of this class of compounds, and
allows for the rst time the synthesis of unsaturated members
of this family. Using a range of analogues varying in the
stereochemistry and nature of the lipid, and the carbohydrate
head-group, we explore the molecular basis for NKT cell
recognition of this class of molecule, and identify atypical TCRs
with the ability to recognize a family of a-glucosyl and a-glu-
curonosyl diacylglycerides of bacterial and fungal origins.
Results
Concise synthesis of a-glucosyl and a-glucuronosyl
diacylglycerides
The compatibility of methoxyacetyl groups with alkene-
containing fatty acids allowed the total synthesis of unsatu-
We have reported that a-GlcADAG-(R-C19:0/C16:0) 1 is pre- rated variants. Thus by a similar approach involving substitu-
sented by mouse CD1d and recognised by populations of NKT tion of monoester 13 with the tetrabutylammonium salt of oleic
cells expressing the atypical Va10Ja50 TCR a-chain, the classical acid (/17), and cleavage of methoxyacetyl groups we obtained
type I NKT TCR a-chain and also certain type II NKT TCRs.3 The a-GlcDAG-(C18:1/C16:0) 7 (Fig. 2C). While catalytic TEMPO/
original approach to synthesising 1 required 27 total steps BAIB could be applied to oxidize the primary alcohol of 7 to
involving preparation of three fragments with a longest linear the acid, better results were obtained using stoichiometric
route of 23 steps.8 This included an 8-step synthesis of R-TBSA, Bobbitt's
salt
(4-(acetylamino)-2,2,6,6-tetramethyl-1-oxo-
and involved 5 types of protecting groups, including the use of piperidinium tetrauoroborate)18 affording a-GlcADAG-(C18:1/
benzyl ethers as protecting groups which prevented the C16:0) 3. By a similar approach (12/14/18) the regioisom-
synthesis of unsaturated variants. We recently developed ers a-GlcDAG-(C16:0/C18:1) 8 and a-GlcADAG-(C16:0/C18:1) 4
a concise approach to a-glucosyl diacylglycerides from allyl a-D- were also prepared. Collision-induced dissociation, multistage
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Fig. 2 (A) Synthesis of S-TBSA. (B) Previously reported synthesis of the key bromohydrin (ref. 17). (C) Synthesis of a-glucosyl and a-glucuronosyl
diacylglycerides.
mass spectrometry was used to show that the synthetic mate- Va14Ja18+ Vb8.2+ NKT TCR (clone VB8-STD20), or two atypical
rials had the indicated arrangement of fatty acyl groups. In Va10Ja50+ NKT TCRs (clones A10B8.13 and A10B8.36) with
particular, consistent with previously established rules,8,19 loss different TCR-b chains. These assays directly measured binding
of the fatty acyl group at the sn-1 glycerol position produces of CD1d–antigen complex with the TCR-transduced cell lines
a more intense peak than that derived from the loss of the sn-2- using CD1d-tetramer staining and ow cytometry (Fig. 3A), and
linked acyl chain.
their ability to activate these cells as measured by interleukin
(IL)-2 production (Fig. 3B).
a-GalCer-loaded CD1d tetramers strongly stained all three
NKT TCR+ BW58 cells (Fig. 3A) and plate-bound a-GalCer-
loaded CD1d strongly activated these cells (Fig. 3B). Notably,
while the VB8-STD type I NKT TCR+ cell line was also mildly
reactive to vehicle-loaded CD1d tetramers (which contain
Glycolipid recognition by type I and atypical NKT TCRs
We explored the ability of the various a-GlcADAGs and a-
GlcDAGs produced in this study to be presented in the context
of CD1d molecules to both type I and atypical NKT cell-derived
TCRs. We used BW58 cell lines transduced with either a type I
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Fig. 3 Reactivity of CD1d-restricted TCR+ cell clones to glycolipids. (A) (i) Representative plots of flow cytometry of type I (VB8-STD) and atypical
(A10B8.1 and A10B8.3) TCR-transduced BW58 cells labelled with CD1d tetramers loaded with lipids. Numbers in the top left corners of
representative plots indicate mean fluorescence intensity (MFI) for each tetramer. (ii) Graphs display MFI for CD1d-tetramers loaded with the
indicated lipids (x-axes) for each cell line. Data are representative of 4 independent experiments. One of the 4 datapoints for the CD1d-a-GalCer,
-Endo and -a-GlcADAG controls for A10B8.1, 8.3 and VB8-STD TCRs were part of a larger experiment with additional TCRs, previously published
in ref. 6 (B) TCR+ cell lines were co-cultured overnight in the presence of plate-bound CD1d–glycolipid–Ag complex (pre-loaded at 10 mg mLꢀ1).
After 16 hours, the supernatants were collected and the presence of IL-2 measured by cytometric bead array. Graphs show IL-2 detected in the
supernatant. Data is derived from 3 independent experiments.
endogenous (Endo) lipids incorporated during CD1d synthesis), higher than Endo- or vehicle-loaded CD1d tetramers, indicating
the two atypical NKT TCR+ cell lines were not, indicating that that all of the glycolipid lipoforms synthesized in this study can
they are more dependent on the lipid antigen presented by the be loaded and presented by CD1d (Fig. 3A). Furthermore, IL-2
CD1d–lipid complex. Similarly, plate-bound vehicle-loaded or was detected in the supernatants of A10B8.3 cells stimulated
unloaded CD1d elicited measurable responses (IL-2 secretion) with plate-bound CD1d in the presence of each synthetic variant
by the VB8-STD type I NKT TCR+ cell line but not by either of the at levels above that of Endo- or vehicle-loaded CD1d (Fig. 3B).
atypical NKT TCR+ cell lines (Fig. 3B). Importantly, the A10B8.3 This indicates that all the synthetic glycolipids are NKT cell
TCR+ cells were clearly stained by CD1d tetramers loaded with agonists. Conversely A10B8.1 TCR+ cells were not stained by
all of the a-GlcDAG and a-GlcADAG variants examined, at levels CD1d tetramers loaded with any of the synthetic a-GlcDAGs and
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a-GlcADAGs, indicating that this TCR is highly selective for a- residues were important for TCR binding. The results were used
GalCer (Fig. 3A).3 Whilst both the A10B8.3 and VB8-STD TCR+ to generate surface-impact maps displayed on the structure of
cell lines were stained by a-GlcADAG-loaded CD1d tetramers, CD1d, which provide insight into the TCR binding footprint on
and stimulated by a-GlcADAG-loaded CD1d, both cell lines gave CD1d.
lower mean uorescent intensities (MFIs) and IL-2 levels than
For the type I TCR (VB8-STD), the TCR footprint data shows
with CD1d-a-GalCer (Fig. 3A and B) indicating that they are that the CD1d residues required for optimal activation are
weaker agonists than a-GalCer. CD1d tetramers loaded with a- skewed towards the F0 pocket of CD1d (Fig. 4), which is
GlcADAG-(S-C19:0/C16:0), -(R-C19:0/C16:0) or -(C16:0/C18:1) consistent with the type I NKT TCR-CD1d-a-GalCer crystal
displayed the greatest abilities to stain and activate A10B8.3 structure.20 Moreover, the footprints are broadly similar, irre-
TCR+ cells (Fig. 3A). Of note, the VB8-STD TCR+ cell line showed spective of whether CD1d was loaded with a-GlcADAG-(R-C19:0/
a different tetramer staining pattern across the glycolipids, with C16:0), a-GalCer or endogenous ligands (Endo). The footprint
a-GlcADAG-(C18:1/C16:0) showing the highest (14-fold higher for CD1d-a-GalCer differed from the other two, through
than CD1d–Endo) followed by a-GlcADAG-(C18:1/C16:0), -(R- involvement of L145 and no involvement of K86. The binding
C19:0/C16:0), -(S-C19:0/C16:0) and -(C16:0/C18:1) (Fig. 3A).
footprint for a-GalCer-loaded CD1d mutants undergoes at most
Our studies also included a range of a-glucosyl diac- only minor to moderate changes in TCR binding, whereas those
ylglycerides (Fig. 3). In addition to those synthesized herein, we for the other two ligands are perturbed to a greater degree. This
also studied SPN-GlcDAG, a cis-vaccenic acid-containing a- suggests that contacts provided by a high affinity antigen such
GlcDAG from Streptococcus pneumoniae (Fig. S1†).14 CD1d–SPN- as a-GalCer can compensate for the loss of certain interactions
GlcDAG tetramers provided moderate staining of the VB8STD between CD1d and the type I NKT TCR.
TCR+ cells, which reects moderate staining of type I NKT cells
As the atypical A10B8.1 TCR+ cell line could not be activated
in a previous study,21 and a similar level of staining was seen by CD1d–Endo or CD1d-a-GlcADAG-(C19:0/C16:0) 1, only the
with a-GlcDAG-(C18:1/C16:0); all other GlcDAGs exhibited little effect of CD1d mutants loaded with a-GalCer could be assessed.
to no staining enhancement over Endo. The A10B8.1 TCR+ cells Again, mutants with variations around the F0 pocket had the
did not exhibit measurable staining or activation by any of the most signicant loss of activation, although fewer of the
a-GlcDAGs when presented by CD1d. Conversely, A10B8.3 TCR+ mutated residues had an impact relative to the type I TCR with
cells were strongly stained (35-fold MFI, relative to Endo) by the same ligand (Fig. 4). Among all of the amino acid mutations
SPN-GlcDAG-loaded CD1d tetramers, with intermediate levels analysed, Glu83Ala resulted in a greater decrease in A10B8.1
of staining by a-GlcDAG-(S-C19:0/C16:0), -(R-C19:0/C16:0), TCR response than for the type I TCR, suggesting that this is
-(C18:1/C16:0) and -(C16:0/C18:1) loaded CD1d tetramers. IL-2 a pivotal residue for A10B8.1 TCR recognition of CD1d loaded
was detected in the supernatants of A10B8.3 TCR+ cells in with this antigen. These results are consistent with structural
response to plate-bound SPN-GlcDAG- and other GlcDAG- studies with the Va10Ja50 Vb8.1 TCR, in which all these resi-
loaded CD1d molecules, demonstrating that they act as NKT dues were involved in TCR contacts.3
cell agonists.
For the atypical A10B8.3 TCR+ cell line, the footprints were
Collectively, these data show that the a-glucuronosyl and a- investigated for both a-GlcADAG-(C19:0/C16:0) and a-GalCer
glucosyl diacylglycerides we have synthesised are capable of (Fig. 4). Several mutation positions impacted on A10B8.3 TCR
binding to CD1d and can stimulate certain NKT cells, including reactivity with the CD1d-a-GlcADAG complex, indicating
type I and atypical NKT cells. Furthermore, the reactivity of engagement with CD1d residues Glu83, Lys86, Met87, Thr159.
these cells can be inuenced by the nature of the lipids, and This suggests that the TCR footprint is positioned towards the
their arrangement at the sn-1 and sn-2 positions on the glycerol. F0 pocket of CD1d. Interestingly, the responses to Arg21Ala,
Arg79Ala, Ser76Ala, Met162Ala and Ala152Gly mutants were
greater (>150%) than those elicited by the control, suggesting
that these CD1d amino acid side chains might normally impede
interactions of the A10B8.3 TCR with CD1d. When a-GalCer was
CD1d binding footprints for glycolipid recognition by type I
and atypical NKT TCRs
Given the divergent TCR usage and different patterns of Ag loaded into the mutant CD1d molecules, the A10B8.3 TCR was
reactivity by type I and atypical TCRs, the binding footprints of less affected by mutations in the side chains of CD1d during a-
A10B8.3, A10B8.1 or VB8-STD TCRs on CD1d–lipid complexed GalCer recognition, with the Glu83Ala mutation being the sole
with a-GalCer, a-GlcADAG-(C19:0/C16:0), and endogenous lipid variant for which attenutated activation was observed. This
(Endo) were investigated. This was achieved using a panel of result may reect the tighter binding of the A10B8.3 TCR to
lipid-loaded mutant CD1d molecules in a plate-bound assay CD1d-a-GalCer, allowing for cellular activation even when
similar to that described above (Fig. 4). The CD1d molecules binding is partially perturbed at other sites.
carry point mutations where individual solvent exposed amino
Taken together, these data suggest that the a-GlcADAG-(R-
acids have been converted to alanine at sites across the docking C19:0/C16:0) antigen can be recognised in the context of
face of the protein. These amino acids were selected based on a CD1d footprint skewed towards the F0 pocket of CD1d that is
previous studies that demonstrated their involvement in broadly similar for type I, type II,6 and atypical NKT cells, but
contacts with type I20 or type II NKT TCRs.22,23 The impact of that the individual amino acid TCR–CD1d contacts vary in their
these mutations on the activation of the A10B8.3, A10B8.1 or importance for cellular activation. Furthermore, in the case of
VB8-STD TCR cell lines allowed an assessment of which the atypical TCR, several of the TCR–CD1d contacts appear to
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Fig. 4 Determination of CD1d-binding patterns by different NKT cell TCRs. Soluble alanine mutants of mouse CD1d loaded with a-GalCer, a-
GlcADAG-(C19:0/C16:0) or unloaded (Endo), were immobilized in culture plates and investigated for their ability to stimulate NKT cell lines
expressing the A10B8.1 and A10B8.3 atypical NKT TCRs. The VB8-STD type I NKT TCR data referring to CD1d-a-GalCer and CD1d-Endo was
published in ref. 6 and is included here as a control. After 16 hours of stimulation, cells were harvested and analysed by flow cytometry for CD69
expression. The level of activation elicited by each mutant was assessed by CD69 upregulation, in comparison to non-activated samples. Data is
shown as percentage normalized to the response elicited by the control CD1d mutant (Asp226Ala); Asp226 is located distal to the antigen/TCR
binding interface within the a3 domain of CD1d. (A) Graphs depict mean ꢁ SEM derived from n ¼ 2 independent experiments for A10B8.1 or n ¼ 3
for VB8-STD and A10B8.3 TCRs. The data from each experiment was averaged from duplicate cultures. Responses to CD1d-a-GlcADAG (black),
CD1d-a-GalCer (grey), and CD1d–Endo (white), are shown for relevant TCRs. (B) Corresponding surfaces of CD1d (PDB code 1Z5L), with impact
maps, colour coded as per key.
normally interfere with recognition of a-GlcADAG, but not of a- fatty acids, and in addition provides the a-glucosyl analogues,
GalCer, possibly providing an explanation for the higher which are formed as the penultimate intermediates.
potency of the latter antigen.
Most studies of NKT cells have focussed on the classical
Va14Ja18+ type I NKT cells, which is partly because of the use of
the prototypic a-GalCer glycolipid antigen that uniformly
detects, and stimulates, these cells. The existence of other types
of CD1d-restricted NKT cells (broadly dened as type II NKT
cells) has been known for many years,24 but a lack of knowledge
of the specic lipid antigens to which they respond has limited
investigations into the broader NKT cell family. In some cases
lipid antigens such as sulfatide and phosphatidyl glycerol have
been helpful for studying discrete subsets of these cells and
more such studies are needed. Other studies have shown that
some lipid antigens, such as a-GlcDAG or a-GlcADAG, appear to
stimulate a subset of type-I NKT cells21 or a distinct population
of ‘atypical’ Va10Ja50+ canonical NKT cells.3,6 Atypical NKT cells
do not neatly t the mould of type I (a-GalCer specic,
Va14Ja18+) or type II (a-GalCer non-reactive, TCR-diverse)
because they fall partway between, using TCR-a chains other
than Va14Ja18 (Va24Ja18 in humans) but are still able to
respond to a-GalCer presented by CD1d.3,6 While atypical NKT
cells may seem like type I NKT cells by their common reactivity
to a-GalCer, their specicity for other glycolipid antigens can
differ substantially from type I NKT cells.3,5
Discussion
Here we present the total synthesis of important microbial
glycolipid antigens that can be targeted by CD1d-restricted
NKT cells. These antigens include a-GlcADAG 3 (from M.
smegmatis and C. glutamicum), and a second generation total
synthesis of a-GlcADAG 1 (from M. smegmatis), as well as
a panel of analogues that include molecules representative of
a-GlcADAGs from the fungus A. fumigatus. The present
approach affords a-GlcADAG 1 in 8 linear steps from allyl a-D-
glucopyranoside, and 10 steps in total including the prepa-
ration of R-TBSA, compared to the previously reported
approach which required 23 linear steps and 27 steps overall.
The step-change in efficiency arises from the use of a single
protecting group, the avoidance of technically demanding a-
glucosylation, the use of an acylation/substitution process to
introduce two distinct fatty acyl chains, and the late-stage
regioselective oxidation with TEMPO or, better still, Bob-
bitt's reagent. This approach is compatible with unsaturated
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We have demonstrated a role for TCR-b chain of atypical Gram-positive bacteria, and fungi. These glycolipids bind to
Va10Ja50+ NKT cell TCRs in modulating antigen reactivity of a- CD1d and stimulate type I and atypical Va10Ja50 NKT cells,
GlcADAG and the variants synthesized in this study. Thus, the with the ne structures of the fatty acid tails of the antigens, and
Va10Ja50+ VB8.3+ (A10B8.3) TCR recognized a range of a-glu- the TCR-b chains of the cells, acting as key determinants of
cosyl and a-glucuronosyl diacylglycerides whereas the Va10Ja50 antigen recognition. These ndings extend the range of
Vb8.1+ (A10B1.8) TCR did not recognise these a-glucosyl and a- microbial glycolipids that can be presented by CD1d and
glucuronosyl diacylglycerol lipids but still strongly reacted to emphasize that there is scope within the more diverse and less
the prototypic ligand a-GalCer, despite a conserved CD1d extensively studied atypical NKT cell repertoire for recognition
footprint over the F0 pocket of CD1d. This is reminiscent of how of these antigens.
TCR-b chain can regulate type I NKT cell recognition of different
glycolipid antigens.25 These ndings highlight the value in
investigating different types of glycolipid antigens in the context
Conflicts of interest
of subsets of NKT cells that express distinct TCR-a and TCR-
b chains. Through such studies, we are likely to see different
hierarchies of antigen recognition depending on the cell types
D. I. G. is a shareholder and member of the scientic advisory
board of Avalia Immunotherapies, a company working on NKT
cell-based vaccines. The other authors declare no conicts of
interest.
and TCR chains involved, some of which may be overlooked if
studies continue to focus exclusively on Va14Ja18+ type I NKT
cells.
An interesting feature of the A10B8.3 TCR is its reactivity to Acknowledgements
a-GlcADAG-(R-C19:0/C16:0) 1 and a-GlcADAG-(S-C19:0/C16:0) 2,
This work was supported by grants from the Australian
which contain branched lipids. CD1d can tolerate some varia-
Research Council (ARC) (DP160100597; CE140100011) and the
tion in lipid structure, including unsaturation and lipid chain
National Health and Medical Research Council of Australia
length.4 However, more complex lipids such SMC124 (a
(NHMRC) ( 1113293 & 1016629). APU was supported by an ARC
cyclopropane-modied a-galactosylceramide related to plako-
Future Fellowship (FT140100278); JR is supported by an
side A)26 and the terminal branch present within the aga-
Australian ARC Laureate Fellowship; DIG is supported by an
lasphins (the progenitors of a-GalCer)27 suggest that CD1d can
tolerate more complex lipid structures within their lipid
NHMRC Senior Principal Research Fellowships (1117766); and
SJW was supported by an ARC Future Fellowship
binding pockets. Indeed, another CD1 protein, CD1b, can load
(FT130100103).
and present methyl-branched and cyclopropanated mycolic
acids,28,29 and CD1c can present multiply methyl-branched
mannose polyprenolphosphate.30 While some differences are
Notes and references
seen between the R- and S-diastereoisomers of the TBSA-
containing a-GlcADAGs they are not signicant (see Fig. 3Aii),
suggesting that CD1d can accommodate these differences in
lipid structure without affecting TCR targeting of the CD1d–
glycolipid complex. This data complements a recent study that
showed a selection of the synthetic lipids described herein
could activate type II NKT cells.6
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glucosyl and a-glucuronosyl diacylglycerides from various
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