Identification of a potent microbial lipid antigen for diverse Natural Killer T cells

Abstract

Invariant Natural Killer T (iNKT) cells are a well-characterized CD1d-restricted T cell subset. The availability of potent antigens and tetramers for iNKT cells has allowed this population to be extensively studied and has revealed their central roles in infection, autoimmunity, and tumor immunity. In contrast, diverse Natural Killer T (dNKT) cells are poorly understood because the lipid antigens they recognize are largely unknown. We sought to identify dNKT cell lipid antigen(s) by interrogating a panel of dNKT mouse cell hybridomas with lipid extracts from the pathogen Listeria monocytogenes. We identified Listeria phosphatidylglycerol (PG) as a microbial antigen that was significantly more potent than a previously characterized dNKT cell antigen, mammalian PG. Further, while mammalian PG loaded CD1d tetramers did not stain dNKT cells, the Listeria-derived PG loaded tetramers did. The structure of Listeria PG was distinct from mammalian PG since it contained shorter, fully-saturated anteiso fatty acid lipid tails. CD1d binding lipid displacement studies revealed that the microbial PG antigen binds significantly better to CD1d than counterparts with the same headgroup. These data reveal a highly-potent microbial lipid antigen for a subset of dNKT cells and provide an explanation for its increased antigen potency compared to the mammalian counterpart.

INTRODUCTION

Natural Killer T (NKT) cells are a subset of αβ TCR+ T cells that recognize lipids presented by the MHC class I-like molecule CD1d (1). These cells are further divided into two categories based upon TCR usage: semi-invariant/type I NKT (iNKT) cells and diverse/type II NKT (dNKT) cells. iNKT cells mostly express an invariant TCRα chain (Vα24-Jα18 in human, Vα14-Jα18 in mice) complexed with a limited repertoire of TCRβ chains, while dNKT cells typically express diverse TCRα and TCRβ chain sequences (1). For the past two decades, much of the work in the field has focused on iNKT cells due to the ability of α-Galactosylceramide (α-GalCer)-loaded CD1d tetramers to specifically identify these cells (2).

iNKT cells and dNKT cells are physiologically distinct cell populations. Not only do these two cell populations recognize different lipids bound within CD1d molecules, but even the topology of how their TCRs recognize the CD1d-lipid antigen complex can be clearly different (3). For iNKT cells, the orientation between the iNKT TCR and the CD1d-α-GalCer complex is parallel and focused over the F’ pocket of CD1d, biasing the majority of the TCR-CD1d interaction towards the invariant TCRα, with CDR1α and CDR3α accounting for all interactions with the α-GalCer antigen headgroup (4, 5). In contrast, two recent studies described the crystal structures of dNKT (clone XV19) derived TCRs in ternary complexes with the glycolipids sulfatide or lysosulfatide bound to CD1d (6, 7). They revealed that these TCRs bound in a manner more analogous to MHC-restricted TCRs, with an orthogonal orientation in which both TCRα and TCRβ’s CDR1 and CDR2 loops bind, perched over the A’ pocket, to CD1d, and the CDR3β loop provided the major contact with the bound sulfatide headgroup. Whether this is typical of all dNKT TCR-CD1d-antigen interactions remains to be determined, although recent crystallographic studies of a human γδ TCR, and a hybrid ‘δαβ’ TCR, interacting with lipid antigens α-GalCer and sulfatide, presented by CD1d, also showed orthogonal docking over the A’ pocket of CD1d (7–9). The fact that dNKT TCRs utilize diverse TCR α- and β-chains, and that the XV19 CD1d-dNKT TCR structural studies revealed that the variable CDR3 loops can dominate in lipid antigen recognition, suggests that dNKT cells may possess the capacity to recognize a great range of self- and foreign-lipid antigens.

One of the key distinguishing features of dNKT cells is that, unlike iNKT cells, they do not respond to α-GalCer and therefore are not identified by CD1d-α-GalCer tetramers. With the findings that dNKT cells may be present in humans at higher levels than iNKT cells, there is great interest in identifying physiologically relevant lipid antigens for dNKT cells (6, 10). To date, many of the identified dNKT cell lipid antigens have been either identified or confirmed by screening a panel of dNKT cell hybridomas. Using these T-T hybridomas several endogenous mammalian lipid antigens (e.g. sulfatide, phosphatidylglycerol, lysophosphatidylcholine, lysophosphatidylethanolamine, and diphosphatidylglycerol) have been confirmed as dNKT cell antigens (11–18).

With the notable exceptions of sulfatide-reactive and Gaucher lipid-reactive dNKT cells (12, 19), no other dNKT cell population has been directly identified in vivo because of the failure of tetramers to bind. Instead, the role of dNKT cells has been inferred indirectly by comparing mice lacking iNKT cells (Jα18 KO mice) to mice lacking both dNKT and iNKT cells (CD1d KO mice) (20, 21). Studies with these knockout mice have demonstrated a protective role for dNKT cells in a variety of pathogenic states, including: type 1 diabetes, concanavalin A-induced hepatitis, and murine infection with Schistosoma mansoni or Listeria monocytogenes (1, 10). However, these studies are also confounded by the fact that Jα18 KO mice have additional TCR Jα defects resulting in a limited TCRα repertoire that has only recently been appreciated (22).

Previously, we identified the first microbial dNKT cell lipid antigens (13). Using dNKT cell hybridomas, we found that phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) derived from Mycobacterium tuberculosis, the related Corynebacterium glutamicum, and mammalian PG are antigens for a subset of dNKT cells. The microbe-derived PGs and DPGs contained the same head group as their mammalian counterparts, but differed in their dominant fatty acid tail structure. The Corynebacterium derived PG/DPG variants were weak antigens and equivalent to the similarly weak mammalian PG/DPG in their ability to activate dNKT cells. We reasoned that microbial lipid antigens for dNKTs that are distinctly more active than mammalian PG might exist. Therefore, we designed an independent and unbiased search for potential dNKT lipid antigens from other microbes, such as Listeria monocytogenes.

Listeria is a Gram-positive facultative intracellular bacterium that infects and resides within the cytosol of macrophages, dendritic cells, hepatocytes, and epithelial cells (23–25). This pathogen is a common food contaminant and causes significant mortality in immunocompromised individuals and spontaneous abortions in pregnant women (26). There are three key reasons for investigating Listeria for lipid antigens. First, as an intracellular pathogen the Listeria-derived lipids would be likely to access the intracellular CD1d antigen presentation system in vivo. Second, data from comparing bacterial burdens in Jα18 KO mice to CD1d KO mice suggest a role for dNKT cells in clearing this pathogen (27, 28). Third, Listeria has been subjected to lipidomics analysis, wherein Fischer and Leopold identified many unique Listeria lipids that do not have mammalian homologues and thus might be recognized as foreign (29). Furthermore, our analysis of the lipidomics data revealed that a number of these lipids are capable of binding into CD1d, making them potential dNKT lipid antigens.

Here, we used a panel of iNKT and dNKT hybridomas to screen fractionated Listeria lipids for CD1d-restricted antigens. Interestingly, this unbiased screen revealed reactivity in some of the same chemical classes of lipids identified previously, namely PG, but with critically important differences. Listeria-derived PG and DPG differed significantly in their fatty acid architecture compared to the mammalian/Corynebacterium variants. Importantly, Listeria-derived PG is a strikingly more potent antigen for dNKT cells. By performing lipid antigen CD1d binding assays and tetramer staining we provide insights into the structural basis for the high potency of microbial compared to mammalian lipid antigens that share identical lipid headgroups.

MATERIALS AND METHODS

Growth and lipid extraction of Listeria monocytogenes

Wild-type Listeria monocytogenes (strain 10403S, a gift from H. Shen, University of Pennsylvania) was grown in brain-heart infusion broth (BD Biosciences) supplemented with 200 μg/ml streptomycin (Sigma-Aldrich) (BHI-STR) overnight to stationary phase at 37°C and 225 RPM. The following day, flasks containing pre-warmed BHI-STR broth were inoculated at ~1:420 v/v and grown until mid-log phase (OD600 ≥ 0.4). Once at mid-log phase, bacteria were pelleted by centrifugation, washed with PBS, and then lyophilized. After up to 48 h of lyophilization, Listeria pellets were processed for extraction of crude polar lipids as previously described (30). Once isolated, lipids were weighed, resuspended in 2:1 v/v chloroform:methanol (2:1 C:M) and then stored in glass 15 ml tubes at −20°C until used.

Cell lines

The following mouse NKT hybridomas were tested for reactivity against Listeria lipids: 24.9, DN32, 14S.6, 14S.10, 14S.15, 431.A11, TBA7, VII68, VIII24.1.D, and XV19.2 (31–34). The hybridomas not generated in the Brenner lab were kindly provided by S. Cardell (Göteborgs Universitet, Sweden) and A. Bendelac (University of Chicago). Hybridoma cells were maintained in NKT Growth Media [RPMI (Gibco) supplemented with 10% v/v FBS (Gemini), 10 mM Hepes (Gibco), 2 mM L-glutamine (Gibco), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco), and 55 nM 2-mercaptoethanol (Gibco)]. RAW.267 (RAW) and RAW cells stably transfected with mouse CD1d (RAW-CD1d) were maintained in DMEM Complete [DMEM supplemented with 10% v/v FBS (Gemini), 2 mM L-glutamine (Gibco), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco)]. When hybridomas were co-cultured with either RAW or RAW-CD1d cells or with plate-bound recombinant CD1d, cells were incubated overnight in RPMI Complete [RPMI (Gibco) supplemented with 10% v/v FBS (Gemini), 2 mM L-glutamine (Gibco), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco)]. For tetramer and dextramer experiments, the TCR-deficient T cell hybridoma BW58 (35) was stably transfected with CD3, TCRα, and TCRβ from one of four hybridomas [Vβ8.2 (an iNKT cell hybridoma), TBA7 (“TBA7-High”), 14S.6, or XV19]. BW58 and TCR transfected clones were maintained in DMEM-NKT media [DMEM (Gibco) supplemented with 10% v/v FBS (Gemini), 15 mM Hepes (Gibco), 2 mM L-glutamine (Gibco), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco), 55 nM 2-mercaptoethanol (Gibco), 1 mM sodium pyruvate (Gibco), and 1X non-essential amino acids (Gibco). All cells were maintained in 37°C incubators at 5% (RPMI media) or 10% (DMEM media) CO₂.

LC-MS fractionation of polar Listeria lipids

Preparative HPLC experiments were carried out using a custom Waters (Milford, MA) Autopurification HPLC system comprised of a Waters 2767 one-bed injection-collection sample manager, a 2545 quaternary gradient module that can pump up to 150 ml/min, a System Fluidic Organizer (SFO) coupled to a single-quadrupole Waters 3100 Mass Detector (MS) equipped with Z Spray^™ API ion source, a Waters 2424 Evaporative Light Scattering detector (ELSD), and a Waters 2998 Photodiode Array Detector (PDA). In addition, during preparative mode, the system is coupled to two Waters 515 HPLC pumps used for make-up liquid delivery as well as a 1000:1 splitter that can tolerate a flow rate of 8–30 ml/min. During fractionation, 99.9% of the lipid is sent to a fraction collector for use in future bioassays. A small percentage (0.1%) of the polar lipid is sent to the detectors to identify the lipid-containing fractions to help with fractionation. The entire system was controlled by MassLynx 4.1 software. Chromatographic analyses and separation of crude polar extracts was performed based on the eluent method on a chemically bonded polyvinyl alcohol-silica (PVA) stationary phase (36). The total LC-MS run was pooled into 19 fractions using time and ELSD to designate fractions. Pooled fractions were weighed, resuspended in 2:1 C:M, and stored at −20°C until ready for use.

Analytical and preparative thin layer chromatography (TLC)

For analytical TLCs, lipids were spotted onto aluminum-backed silica TLC plates (EMD Chemicals Inc.) and then dried under low pressure. Next, lipids were resolved in a solvent system designed for the optimal separation of phospholipids (chloroform:acetic acid:methanol:water at a ratio of 40:25:3:6 v/v/v/v). Plates were then dried under low pressure, cut (if appropriate) and sprayed with one of four TLC stains [Dittmer-Lester reagent (phosphate stain), α-naphthol (sugar stain), molybdophosphoric acid (MPA, a general stain), and ninhydrin (an amino group stain)] (29, 30). Charring of sugar stained, MPA stained, and amino group stained plates was used to develop those plates. Preparative TLCs were performed by first spotting the lipid across the origin of a plastic-backed TLC plate (EMD Chemicals Inc.). The plates were then dried under low pressure, resolved in the 40:25:3:6 system, and dried as above. Next, a small segment of the TLC plate was cut off for staining with one of the above TLC stains, which was used to mark the bands of lipid in the unstained section of the plate. The marked silica regions for each lipid band was scraped off the plastic, moved to a glass 15 ml tubes, and the lipid extracted from the silica with 3 sequential washes of 2:1 C:M. After drying, lipids were weighed, resuspended in 2:1 C:M, and stored at −20°C until used. It is critical to note that the amount of TLC purified lipid(s) in the tube cannot be determined solely by measuring weight, as the measured weight includes some transferred silica. When the identity of the purified lipid was unknown (Fig. 3B), lipids were resuspended in 2:1 C:M based on the total weight of the tube and an estimated expected yield. Because the weights for resuspended lipids were approximated, the amount loaded is shown as the fold-dilution of the stock lipid. To determine the relative concentration for identified lipids, concentrations were measured by spotting both the TLC purified lipid and a relevant standard (either Corynebacterium PG, DPG, or Streptococcus DGDG) onto an analytical TLC plate at varying concentrations in order to generate a standard curve. The plates were then dried, resolved with the 40:25:3:6 system, re-dried, stained with MPA, and charred to develop. The plates were then scanned at ≥600 dpi and densitometry was preformed with ImageJ software (U. S. National Institutes of Health) and quantification determined by comparison to known standards.

Figure 3. TLC purification of LC-MS fraction C identifies 6 active bands.

A. Listeria LC-MS fraction C was spotted at 150 μg/spot on a TLC plate and ran in the 40:25:3:6 system. The plate was then cut into strips and each individual strip was stained with one of four stains: Dittmer-Lester reagent (‘Phosphate’, to stain phosphate groups), α-naphthol (‘Sugar’, to stain sugar groups), MPA (‘General’, a general fatty acid tail stain), and ninhydrin (‘Amino’, to stain amino groups). These stains were used to identify 12 lipid bands for extraction B. The TLC plate-eluted lipid bands were incubated with 14S.6 cells and RAW (gray) or RAW-CD1d (black) cells in triplicate. Because of the silica plate extraction, weights of TLC bands reflected both silica and lipid weight. Therefore, the lipid bands were resuspended based on total weight and fold-dilutions of the stock lipids are displayed. IL-2 ELISAs were performed on culture supernatants after 16–18 h of co-culture. C and D. Collision MS/MS spectra for TLC Bands 1–3 were all identified as diphosphatidylglycerol (DPG) (C) and TLC band 4 was identified as phosphatidylglycerol (PG). E to H. Structures of the dominant DPG (E) and PG (F) lipids in the TLC bands, with comparison to mammalian (G) and Corynebacterium (H) PG demonstrating the different fatty acid tails found in these species. Panel B is representative of two independent experiments and presented as mean ± SEM.

Measuring reactivity to lipids by ELISA

RAW or RAW-CD1d cells were resuspended in RPMI Complete and 5 × 10⁴ cells were plated onto 96-well round-bottom plates (Falcon). Lipids were transferred to 10 ml conical glass tubes (Kimble Chase), dried in a Genevac EZ-2 personal evaporator, resuspended in RPMI Complete, and then serially diluted and added to each well as appropriate. No lipid control wells were given RPMI Complete from 10 ml glass tubes dried with an equivalent volume of the lipid vehicle (2:1 C:M). After a pre-incubation at 37°C and 5% CO₂ for at least 30 min, 5 × 10⁴ hybridoma cells were added to each well and cultured for 16 to 18 h. Supernatants were then removed and analyzed for the presence of IL-2 by sandwich ELISA with matched anti-mouse IL-2 antibody pairs (BD Pharmingen). IL-2 protein for ELISA standards were from R&D Systems.

Plate-bound presentation of lipids in CD1d

Lipids or 2:1 C:M vehicle were dried in a Genevac EZ-2 personal evaporator and then resuspended in 50 mM pH 6 citrate buffer supplemented with 0.25% CHAPS (Citrate-CHAPS, both from Sigma-Aldrich). The lipid was then mixed with biotinylated CD1d (provided by the NIH Tetramer Facility) that was also diluted into Citrate-CHAPS at a 20:1 w/w ratio in glass HPLC insert tubes (Supelco). Lipids were then bound to CD1d for 24 to 48 h, after which the pH was adjusted to 7.4 with 1M Tris pH 9. Finally, PBS was added to double the initial loading volume and stored at 4°C until use. For binding CD1d onto plates, 0.05, 0.2, or 0.25 μg of CD1d was added per well of a streptavidin-coated plate (Thermo Scientific Pierce) for 1 h at room temperature. Plates were then extensively washed with sterile PBS before addition of 5 × 10⁴ hybridoma cells per well in RPMI Complete. After 16 to 18 h at 37°C and 5% CO₂, supernatants were then collected and analyzed for IL-2 by ELISA.

Tetramers, dextramers, and flow cytometry

Biotinylated mouse CD1d (provided by the NIH Tetramer Facility) was diluted into TBS pH 8 to 0.4 mg/ml and lipids were dried as above then resuspended in TBS pH 8 supplemented with 0.05% tyloxapol (“TBS-Tyl”, Acros Organics) at 1 mg/ml. Lipids or TBS-Tyl vehicle (“Mock”) was mixed with CD1d in HPLC insert tubes at a molar ratio of 35:1 lipid:CD1d molecules. After a 20–24 h incubation at 37°C, CD1d was formed into tetramers by incubation with streptavidin-APC (Invitrogen) or diluted with PBS to 0.1 mg/ml of CD1d and stored at 4°C for future loading into dextramers. Dextramer-APC backbone (a gift from Immudex, Copenhagen, Denmark) was mixed with CD1d at a ratio of 4 CD1d molecules per 1 streptavidin binding site on the Dextramer backbone at least 30 mins before use.

For flow cytometry, 5 × 10⁴ hybridoma cells per well were first pre-incubated with brilliant-violet 421 labeled PBS-57 (an α-GalCer analogue) CD1d tetramers (provided by the NIH Tetramer Facility) then stained with APC-labeled ‘mock’ or loaded tetramers (0.8 μg of CD1d per well) or dextramers (3 μl of Dextramer-APC backbone plus 1.15 μg CD1d per well). Finally, cells were stained with PE labeled anti-TCR-β (clone H57-597, Biolegend) and analyzed on a BD FACSCanto™ II flow cytometer. Data was analyzed with FlowJo (Treestar). For analysis, samples were first gated by FSC-A & SSC-A to identify ‘live’ cells, followed by singlet gating (FSC-A by FSC-H). Next, cells were gated for TCR expression. Tetramer or dextramer (mean fluorescence intensity of APC-A) levels were then determined.

Mass spectrometric identification of lipid structure

Both high-resolution (R=100,000 at m/z 400) and low-energy CAD tandem mass spectrometry were performed as previously described, with the exceptions that samples were dissolved into methanol instead of 2:1 C:M, the automatic gain control of the ion trap was set to 5 × 10⁴, and the ES needle was set to 4.0 kV. (13). For structural analysis of fatty acids, TLC-purified lipids were first treated via alkaline hydrolysis to liberate fatty acids, which were then isolated and derivatized with N-(4-aminomethylphenyl) pyridinium (AMPP), and subjected to MS as described (37).

Synthesis of lipid standards

Synthesis of mammalian (18:1/16:0) or Listeria (15:0/17:0) PGs were made in a stepwise fashion starting with a glycerol backbone as described in Supplemental Figure 3.

Generation of soluble TCR and CD1d

Mouse CD1d/β2m expression vector with a BirA and 6-Histidine Tag (construct originally provided by Mitchell Kronenberg, La Jolla institute of Allergy and Immunology, CA, USA) was expressed, purified and biotinylated in house as previously described (38). Soluble mouse TCR production was achieved by using chimeric mouse-variable – human-constant domains as previously described (38). Individual TCR chains were cloned into pET-30 (Novagen) vectors for the TCRβ chain or pET-28 (Novagen) for the TCRα chain, and expressed in BL-21 E. coli (DE3)pLysS. Inclusion body protein preparations were isolated and refolded as previously described (39) except for the addition of 5M urea into the refold buffer. TCRs were purified by anion exchange chromatography, immobilized metal affinity chromatography and gel filtration. TCR purity was assessed by gel electrophoresis and predicted mass confirmed by time-of-flight mass spectrometry. TCR refolding was confirmed by ELISA using an antibody reactive against a conformational epitope for the TCR constant domain (clone 12H8 produced in house) and anti-V_8.1/8.2 (clone KJ16-133 eBioscience).

GD3 lipid displacement assay

GD3 ganglioside (Matreya #1504) was suspended in TBS-Tyl at 1 mg/mL and mixed at 3:1 molar ratio with in-house generated biotinylated mouse CD1d (CD1d-bio) at 1 mg/mL in TBS pH 8 for 20 h at room temperature. GD3 loaded CD1d-bio was purified by MonoQ anion exchange chromatography. Listeria PG or Corynebacterium PG resuspended at 1 mg/mL in TBS-Tyl were incubated at a 30:1 molar ratio with purified GD3 loaded CD1d-bio. PG loaded fractions were then purified using MonoQ anion exchange chromatography. These fractions were then used in affinity measures with TBA7 by Surface Plasmon Resonance (SPR). Excess lipid and detergent were removed prior to each chromatography run using a PD10 desalting column (Amersham Biosciences).

Surface plasmon resonance analysis

The SPR experiments were operated at 25°C on a BIAcore 3000 instrument and conducted in HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl). 1% bovine serum albumin was added to prevent any non-specific binding. Four thousand response units (RUs) of CD1d-bio loaded with the lipid antigens Corynebacterium PG or Listeria PG – LC-MS E were coupled onto the streptavidin sensor chip. An HLA class I molecule was immobilized on one flow cell for reference subtraction. Biotin was then injected to block the free streptavidin sites. Nine serial dilutions of TBA7 from 200 μM to 0.78 μM were passed through as analyte. BIAevaluation software was used for data analysis.

Data Presentation and Statistical Analysis

All IL-2 ELISA graphs, fold-change graphs, and percent-displaced graphs were generated in GraphPad Prism 5.0b. Statistics (one-way ANOVAs) were performed with GraphPad Prism 5.0b.

RESULTS

We isolated polar Listeria lipids from mid-log phase bacteria cultures and co-cultured the crude polar lipid mixture with two iNKT cell hybridomas (Fig. 1A and B) and eight different dNKT cell hybridomas (Fig. 1C–J). To be considered activated, the following requirements were necessary: (i) a dose response between lipid antigen concentration and hybridoma IL-2 production, (ii) IL-2 production when co-cultured with lipid antigen and RAW 264.7 cells transfected to express high levels of CD1d (RAW-CD1d) is significantly higher than when co-cultured with no exogenously added lipid, and (iii), little to no response when co-cultured with lipid antigen and untransfected RAW 264.7 (RAW) cells. By these requirements, both iNKT cell hybridomas (Fig. 1A and B) failed to be activated by crude Listeria polar lipids, as also noted by others (31–34, 36, 40). In contrast, six of the eight dNKT cell hybridomas were positively activated by Listeria polar lipids in a CD1d-dependent manner. Based upon these results, we chose two dNKT hybridomas, 14S.6 and TBA7, that gave strong responses to the polar lipid extract for further study to identify the relevant Listeria lipid antigens present in the crude extract.

Figure 1. Listeria monocytogenes polar lipids activate diverse but not invariant NKT hybridomas.

Polar lipids derived from the bacterium Listeria monocytogenes were incubated at various concentrations in triplicate with a panel of NKT cell hybridomas in the presence of RAW 264.7 cells (RAW, gray circles) or RAW cells stably transfected with CD1d (RAW-CD1d, black circles). Hybridomas were also co-cultured with RAW or RAW-CD1d cells without adding exogenous lipid in duplicate (gray and black asterisks). After 16–18 h of co-culture, supernatants were assayed for the presence of IL-2 by ELISA. Each graph is presented as mean ± SEM and is representative of four (A, B, C), three (C, D, E, F, G, J) or two (H, I) independent experiments.

We fractionated the Listeria polar lipid extract by preparative HPLC. This preparative liquid chromatography-mass spectrometry (LC-MS) system first involves using 3 isocratic solvent mixtures on a PVC silica column to facilitate separation of lipids based on head group structure. Four different detectors are used to reveal lipids, including a UV detector, a single quadrupole mass spectrometer, an evaporative light scattering detector (ELSD), and a photodiode array detector. We used ELSD (measured in light scattering units, LSU) and retention time as the criteria for separating the total LC-MS run into 19 fractions. These 19 fractions were then tested for biological activity. Although fractions encompassing the whole run were tested for activity, the major antigenic fractions correlated with the presence of detectable LSU signals (Fig. 2A and data not shown). The two major LSU peaks appeared at approximately 10 min and between 38 and 48 min elution time. The peak at 10 min (“Peak 1”) was identified by TLC to consist of free fatty acids and roughly equal amounts of 1,2- and 1,3-diacylglycerol (DAG) (data not shown). The stimulatory Peak 1 fraction was weaker than those collected between 38 and 48 min and was not further characterized here. The 6 LC-MS fractions collected between 38 and 48 min activated the diverse hybridomas 14S.6 and TBA7 in a CD1d-dependent manner (Fig. 2B–E).

Figure 2. LC-MS fractionation of Listeria polar lipids.

A. Listeria polar lipids were subjected to LC-MS fractionation by head group. B. Inset from panel A, highlighting the light scattering peaks collected for LC-MS fractions A–F. C–E. 14S.6 (left) and TBA7 (right) hybridomas were incubated with LC-MS fractions A or B (panel C) fractions C or D (panel D) or with fractions E or F (Panel E) in the presence of RAW (grey lines) or RAW-CD1d (black lines) cells in triplicate. Hybridomas were also co-cultured with RAW or RAW-CD1d cells without adding exogenous lipid in duplicate (gray and black asterisks). After 16–18 h of co-culture, supernatants were assayed for the presence of IL-2 by ELISA. Each graph is representative of three independent experiments and presented as mean ± SEM.

The fact that all of these fractions are running closely together suggested that they may share common polar head groups. Further, we have previously noted that in our HPLC system, LSU peaks in this time range typically indicate the presence of phospholipids. By analyzing migration on TLC plates in a solvent system designed for resolving phospholipids, we found that fractions A-F all included the presence of phosphate-containing lipids that migrated to similar heights, suggesting they all shared similar structures in different ratios (Supplemental Fig. 1). At the concentrations tested, LC-MS fraction C showed a clear dose-response across concentrations and was available in high enough quantities for further analysis (Fig. 2D). After further separation by TLC, we subjected LC-MS fraction C to a variety of stains (Fig. 3A) (13). Dittmer-Lester (‘Phosphate’) stains phosphate-containing molecules with a blue color on a white background. Alpha-naphthol (‘Sugar’) stains lipids containing carbohydrate groups with a dark purple color. Molybdophosphoric acid (MPA, ‘General’) is a general lipid stain that is thought to stain fatty acid tails. Finally, LC-MS fraction C was also stained with ninhydrin (‘Amino’), which stains amino groups reddish pink but can also non-specifically mark some lipids with a brown color.

Based on these TLC stains, we identified 12 different lipid bands from LC-MS fraction C and then isolated each by preparative TLC. These bands were then tested for activity with hybridoma 14S.6 (Fig. 3B). The top 6 bands (TLC 1–6), which contained all the phosphate or sugar positive lipid bands, activated hybridoma 14S.6 but the bottom 6 bands did not. Tandem Mass Spectrometry (MS/MS) on the 6 active TLC bands identified these as lipid species with glycerol backbones and each lipid was dominated by fatty acid tails of length 17:0/15:0 in the sn1 and sn2 positions, respectively. By MS/MS analysis of the TLC-purified lipids, TLC bands 1–3 consisted of diphosphatidylglycerol (DPG) (Fig. 3C), TLC band 4 was identified as phosphatidylglycerol (PG) (Fig. 3D), and TLC band 5 was digalactosyldiacylglycerol (DGDG) (Supplemental Fig. 2C). However, further TLC-based analysis of TLC-purified DGDG from Listeria determined that this activity was likely due to a co-migrating ultra-violet (UV) light positive molecule. DGDG from either Streptococcus pneumoniae or LC-MS fraction B (that did not contain the co-migrating UV+ band) was inactive (Supplemental Fig. 2B). TLC band 6 was not visible by any of the 4 TLC stains and MS/MS data was inconclusive. We next generated N-(4-aminomethylphenyl) pyridinium (AMPP) derivatives of the fatty acid tails for further analysis of the fatty acid structure by Gas Chromatography-MS (GC-MS) (37). GC-MS on AMPP derivatives from DPG, PG, and DGDG revealed that the fatty acids are predominately anteiso methyl-branched fatty acids (Fig. 3E and F), a structure that is not found in mammals (35, 41).

LC-MS fractions A–F all contain phospholipids that resolve on TLC plates with similar mobilities, suggesting that these fractions contain lipids with the same head groups (Supplemental Fig. 1). Next, we TLC-isolated putative DPG and PG bands from the other LC-MS fractions that contained enough materials for further study (fractions BE), confirmed by MS/MS that these were DPG or PG, and that the major fatty acid tails are anteiso isomers by GC-MS on AMPP derivatives of the fatty acid tails. In total, we isolated DPGs from LC-MS fractions B–E and PGs from LC-MS fractions C–E. Although the same lipid was found throughout multiple fractions, we did identify differences in the fatty acid substituent compositions. These differences included the presence or absence of plasmenyl PG (fatty acids with a sn1 ether linker and sn2 ester linkage) (37) and different ratios of PG species (e.g., the ratio of 15:0/15:0 to 17:0/16:0 and 17:0/17:0) (Fig. 3D and data not shown).

Recently, we reported that PG and DPG were dNKT cell antigens derived either from mammals or Corynebacterium glutamicum (13). However, the two species had very similar lipid structures; notably the same dominant fatty acid tails (16:0 and 18:1) were present but opposite in sn1 and sn2 orientation (Fig. 3G and H). Importantly, when Corynebacterium PG or DPG was compared with mammalian PG or DPG, there was no difference in their potency in activating dNKT cells (13). Unlike the previously described PG and DPG lipids from mammals, Listeria PG and DPG have a distinct fatty acid architecture, prompting us to ask if Listeria PG and DPG are more or less stimulatory than the corresponding mammalian (or Corynebacterium) sources. When comparing the ability to activate hybridomas 14S.6 and TBA7, Listeria DPG was an equally potent antigen to Corynebacterium DPG (Supplemental Fig. 2A). However, we found that Listeria-derived PGs were strikingly more potent antigens than Corynebacterium-derived PG, as measured by their ability to activate the dNKT hybridomas at lower lipid concentrations (Fig. 4A). Next, we calculated the Listeria PG concentration needed to obtain an equivalent level of IL-2 production as the first Corynebacterium concentration to be clearly above background levels (Table I). When we calculated the fold-change in concentration needed to get the same level of activity, we found that the Listeria PGs were between 10 and 100-fold more potent than Corynebacterium-derived PG, which is itself similar in potency to PG from mammals (Table II and Fig. 4B).

Figure 4. Listeria-derived PG’s are 10 to 100-fold more antigenic than Corynebacterium or mammalian PG.

A. Listeria PG from fractions C, D, and E were compared in activity to Corynebacterium (Cg) PG when co-cultured in triplicate with either 14S.6 or TBA7 hybridomas in the presence of RAW-CD1d or RAW (not shown) cells. B. The fold-difference in concentration required for similar activity to Corynebacterium PG was calculated. C. Synthetic Listeria and mammalian PGs were assayed for activity as in A. The fold-difference in concentration required for similar activity to synthetic mammalian PG was assayed. Panel A is representative of three independent experiments, panel B is a combination of three experiments, and panel C is representative (left panel) or a combination (right panel) of two independent experiments. *p<0.05, **p<0.01, ***p<0.05 by one-way ANOVA. All graphs are presented as mean ± SEM.

Table I.

Concentration of Listeria PGs for equivalent activity to Corynebacterium PG.

14S.6
Replicate	Corynebacterium	LC-MS C	LC-MS D	LC-MS E
1	2.5 μg/mL	0.1 μg/mL	0.15 μg/mL	0.03 μg/mL
2	2.5 μg/mL	0.2 μg/mL	0.8 μg/mL	0.02 μg/mL
3	2.5 μg/mL	0.2 μg/mL	0.45 μg/mL	0.03 μg/mL

TBA7
Replicate	Corynebacterium	LC-MS C	LC-MS D	LC-MS E
1	10 μg/mL	0.2 μg/mL	1.0 μg/mL	0.06 μg/mL
2	10 μg/mL	0.6 μg/mL	1.5 μg/mL	0.15 μg/mL
3	2.5 μg/mL	0.08 μg/mL	0.2 μg/mL	0.03 μg/mL

Table II.

Replicates for the fold-difference in the activity of PG.

14S.6
Replicate	Corynebacterium	LC-MS C	LC-MS D	LC-MS E
1	1	25	16.7	83.3
2	1	12.5	3.1	125
3	1	12.5	5.6	83.3
Average:	1	16.7	8.4	97.2

TBA7
Replicate	Corynebacterium	LC-MS C	LC-MS D	LC-MS E
1	1	50	10	166.7
2	1	16.7	6.7	66.7
3	1	16.7	12.5	83.3
Average:	1	32.6	9.7	105.6

To confirm these findings, we synthesized the dominant Listeria PG variant (a17:0/a15:0) and compared its ability to activate TBA7 cells to that of synthetic Mammalian PG (16:0/18:1). These results supported our previous observations that Listeria PG was a more potent antigen than mammalian PG, and was similar in fold-difference in activity (~13-fold) to Listeria LC-MS C & D PGs (Fig. 4C).

To determine if cellular processing of Listeria PG was required for presentation to dNKT cells, we tested activity using an antigen presenting cell (APC)-free system. We loaded the most active Listeria PG, LC-MS fraction E PG (PG - LC-MS E), or the prototypical iNKT cell antigen α-galactosylceramide (α-GalCer) onto biotinylated CD1d, and then bound 0.2 μg of CD1d per well to streptavidin-coated plates. Different NKT hybridomas were then incubated with the plate-bound CD1d (Fig. 5A). As expected, α-GalCer-loaded CD1d activated the iNKT DN32 hybridoma but did not activate the dNKT TBA7 hybridoma. Importantly, PG - LC-MS E-loaded CD1d activated the dNKT TBA7 hybridoma but not the iNKT DN32 hybridoma. Mock-loaded CD1d did not activate DN32 cells, but weakly activated the TBA7 cells, reflecting the known CD1d autoreactivity seen with many dNKT hybridomas. Next, we determined if the different antigenic properties of the various PGs would be reflected in this APC-free system. Indeed, when we loaded Corynebacterium PG or the various TLC-purified Listeria PGs into plate-bound CD1d, we measured a dose-dependent increase in IL-2 production (Fig. 5B, compare left and right panels). The amount of IL-2 produced at 0.25 μg CD1d/well (Fig. 5B, right panel) closely mimicked the fold-difference in activity seen in the system using live RAW-CD1d APCs (Fig. 4B, right panel), suggesting that the difference in activity detected is not due to processing of the LC-MS fractions within RAW-CD1d cells.

Figure 5. TBA7 hybridoma cells recognize Listeria-derived PGs in cell free systems.

A. Biotinylated murine CD1d molecules were mock loaded, loaded with α-GalCer, or loaded with PG - LC-MS E. The loaded CD1d molecules were then bound to streptavidin-coated plates at 0.2 μg CD1d per well and washed before adding either DN32 or TBA7 hybridomas overnight. The next day, supernatants were analyzed for production of IL-2 by ELISA. B. CD1d molecules that were mock loaded or loaded with various PGs were bound to streptavidin-coated plates at 0.05 μg CD1d or 0.25 μg CD1d per well and incubated overnight with TBA7 hybridomas. The next day, supernatants were analyzed for production of IL-2 by ELISA. C. TBA7-High or Vβ8.2 (iNKT) hybridoma cells were stained with PBS-57 (an α-GalCer analogue) loaded CD1d tetramer at room temperature followed by staining with mock or lipid loaded CD1d tetramers at 37°C. Finally, cells were stained with an anti-TCRβ mAb, washed, and then analyzed by flow cytometry. Left histograms are example staining patterns with different CD1d tetramers (solid light grey = mock loaded, black line = PG – LC-MS E loaded, dashed grey line = DGDG-Sp loaded). Right graphs are measuring the fold-increase in MFI as compared to the mock-loaded CD1d MFI. D. TBA7-High or Vβ8.2 TCR transduced cells were first stained with PBS-57 loaded CD1d tetramer followed by staining with mock or lipid-loaded CD1d dextramers. After staining with an anti-TCRβ mAb, cells were analyzed by flow cytometry. Left histograms are example staining patterns with different CD1d dextramers (solid light grey = mock loaded, black line = PG – LC-MS E loaded, dashed grey line = DGDG-Sp loaded, dashed black line = PG – Cg loaded). Right graphs are measuring the fold-increase in MFI as compared to the mock loaded CD1d MFI. Panels A & B are representative of 3 independent experiments, with each independent experiment consisting of three technical replicates while panels C & D are representative histograms (left panels) or combinations of (right graphs) 2 independent experiments. *p<0.05, **p<0.01 by one-way ANOVA. All graphs are presented as mean ± SEM.

These results prompted us to determine if Listeria PG was an antigen for the other 6 dNKT hybridomas. We performed both APC-containing (RAW-CD1d cells) and APC-free (plate-bound CD1d) experiments to determine reactivity to Listeria PG – LC-MS E (Table III). In the APC-free system, three of the hybridomas (14S.6, 14S.10, and TBA7) were found to be reactive to Listeria PG. These results match what was found for Corynebacterium PG by Tatituri et al (13). In the APC-containing system, an additional two dNKT hybridomas (VII68 and VIII24.1.D) were activated by Listeria PG. As we have generally found that the plate-bound CD1d system is less sensitive for weak antigens than the APC-containing system, this likely reflects a low affinity reactivity for Listeria PG by these two hybridomas.

Table III.

Reactivity of dNKT hybridomas to Listeria PG – LC-MS E.

Hybridoma	RAW-CD1d APCs	Plate-bound CD1d
14S.6	+	+
14S.10	+	+
14S.15	−	−
431.A11	−	−
TBA7	+	+
VII68	+	−
VIII24.1.D	+	−
XV19.2	−	−

Reactivity to Listeria PG LC-MS E was tested in both APC-containing (RAW-CD1d cells) and APC-free (plate-bound CD1d) systems. + is reactive, − is not reactive.

The high activity of Listeria PG in the CD1d plate-bound assay compared to other established PG sources prompted us to determine if Listeria PG-loaded CD1d tetramers would bind to TBA7 cells. In the CD1d plate-bound assay, excess detergent and lipid is washed away before adding hybridoma cells to the wells. To minimize excess detergent and lipid in the tetramer preparations, we first optimized the system by modifying our lipid-loading protocol. In addition, we generated hybridoma cell lines that expressed high levels of TCR. The TCR-negative immortalized T cell line BW58 was transfected with CD3 and the TBA7 TCR (“TBA7-High”) or a typical iNKT cell TCR (“Vβ8.2”) (35). As expected, CD1d tetramers loaded with PBS-57 (a synthetic α-GalCer analogue) stained Vβ8.2 cells but not TBA7-High cells (data not shown). Further, Listeria PG-loaded CD1d tetramers did not bind to Vβ8.2 cells (Fig. 5C), the parent BW58 hybridoma cells (data not shown), or BW58 cells transfected with the PG-nonreactive dNKT XV19 hybridoma cell TCR (data not shown). In contrast, we found that tetramers loaded with Listeria PG - LC-MS E stained TBA7-High cells, while mock-loaded CD1d tetramer or tetramers made of CD1d loaded with the irrelevant lipid DGDG from Streptococcus pneumoniae (DGDG – Sp) bound only at background levels (Fig. 5C, left panels). Together, these functional and tetramer staining experiments demonstrate that Listeria PG loaded CD1d is a cognate antigen for the dNKT TBA7 TCR.

Listeria PG – LC-MS E loaded tetramers gave a signal that was approximately 2-fold higher than for vehicle-loaded tetramers (Fig. 5C, right panels). We next attempted to increase the positive-negative signal separation by utilizing dextramer technology. Dextramers are dextran backbones containing multiple fluorophore and streptavidin binding sites per molecule (42). Because they contain 12 to 24 antigen-presenting molecules per dextramer backbone, they are useful for identifying rare primary T cell populations because of their increased TCR avidity and fluorescence compared to tetramers. Indeed, Kasmar et al. successfully utilized CD1a dextramers to identify dideoxymycobactin-restricted T cells ex vivo from human PBMC (43). We generated mock-loaded CD1d dextramers along with Listeria PG – LC-MS E, PG – Cg, or DGDG – Sp-loaded CD1d dextramers and used them to stain dNKT TBA7-High, BW58 cells transfected with the 14S.6 TCR (14S.6-High), and iNKT Vβ8.2 cells (Fig. 5D, left panel and data not shown). We found that PG LC-MS E loaded CD1d dextramers specifically bound to TBA7-High cells and gave a signal that was approximately 8-fold higher than for vehicle-loaded CD1d dextramers (Fig. 5D, right panel). The PG LC-MS E loaded CD1d dextramers did not bind to 14S.6-High cells (data not shown), suggesting that the 14S.6 TCR affinity for this complex is very low. Finally, we also tested the ability of the less active PG from Corynebacterium to identify TBA7 TCR-expressing T cells with this newly optimized system. PG – Cg loaded CD1d dextramers also consistently stained the TBA7-High cells, albeit to a much lower degree than Listeria PG – LC-MS E dextramers and did not reach statistical significance (~1.5-fold higher MFI than mock-loaded).

Previous studies on iNKT cell antigens have demonstrated that alterations in the fatty acid tails of lipids can dramatically alter their activity, either by modulating their binding to CD1d or by indirectly impacting on iNKT TCR recognition (44–46). Listeria PGs are dominated by short chain lengths and fully saturated anteiso methyl-branched fatty acids. In contrast, the less antigenic mammalian or Corynebacterium PG have longer acyl chains, an unsaturation, and lack anteiso branches. These differences in fatty acid tail composition could either (i) alter how well the lipid binds into CD1d, (ii) could alter the orientation of the PG head group into a more favorable position for TCR binding, or (iii) alter CD1d conformation that subsequently impacts on TCR binding. To test if the more potent Listeria PG – LC-MS E loads into CD1d more efficiently than Corynebacterium PG, we performed a disialo-ganglioside GD3 (GD3) lipid displacement assay. GD3 is a negatively charged lipid with a large sugar-based head group that can be displaced from CD1d by other lipids. We first loaded CD1d with GD3, purified the GD3-loaded CD1d complexes by MonoQ anion exchange chromatography, and then measured the ability of Listeria or Corynebacterium PG to displace GD3, which results in earlier elution relative to the CD1d-GD3 complex. We found that Listeria PG– LC-MS E displaced approximately 2-fold more GD3 than Corynebacterium PG under the same conditions (35% vs 19% loaded, Fig. 6B).

Figure 6. Listeria PG loads into CD1d more efficiently than Corynebacterium PG.

A. Mono-Q anion exchange purified GD3-loaded CD1d was incubated overnight with either Listeria or Corynebacterium PG at a 30:1 molar ratio of PG:GD3-CD1d. 0.05% tyloxapol vehicle control was also used to determine spontaneous GD3 displacement. The displacement of GD3 in each condition was then determined by MonoQ anion exchange chromatography using a FPLC. The conductivity (thin light-grey line) denotes the increasing salt concentration added to the column and is used to allow for overlaying different chromatograms. B. The peak height of the non-displaced and displaced peaks from three independent experiments were converted into percent displacement. C and D. Surface plasmon resonance (SPR) was performed with Listeria LC-MS E (C) or Corynebacterium (D) PG-loaded CD1d-biotin (purified as shown in A), immobilized to a streptavidin-coated SPR chip. Soluble TBA7 TCR was passed over the chip at various concentrations (200 μM to 0.78 μM). The left panels show TBA7 TCR binding (measured as response units, RU) over time at different concentrations. The right panels show the equilibrium affinity analysis used to determine the kinetics of dissociation (K_d). Panel A is representative of 3 independent experiments, with collective data from the three experiments depicted in panel B. Panels C and D show results from duplicate runs from one of two independent experiments ***P<0.001 by one-way ANOVA. Panel B and the right panels of C & D are presented as mean ± SEM.

We next utilized surface plasmon resonance (SPR) to determined if the TBA7 TCR affinity for Listeria PG – LC-MS E differed from that of Corynebacterium PG. CD1d was loaded with Listeria PG or Corynebacterium PG using the GD3 displacement approach described above to ensure optimal loading. CD1d with endogenous lipid antigen was used as a control. These preparations were then immobilized to the streptavidin SPR chips via their biotin tags. Purified and soluble TBA7 TCR was then passed over CD1d-Ag and the binding affinity (measured in Response Units, RU) was measured. The TCR affinity for Listeria PG – LC-MS E -loaded CD1d (K_d = 71 μM) was similar to CD1d loaded with Corynebacterium PG (K_d = 94 μM) (Fig. 6C and D). For comparison, these affinities are lower than the previously published TCR affinities for the dNKT hybridoma XV19 TCR binding to sulfatide + CD1d (K_d = 24 μM) or the nanomolar iNKT TCR affinity observed for α-GalCer-loaded CD1d (K_d = 0.07 μM) (6, 38). Accordingly our data suggest that the increased potency of Listeria PG is probably not due to higher affinity interactions with the TCRs, but rather, may be attributable to improved loading and/or binding to CD1d.

DISCUSSION

There is a growing appreciation that many T cells do not recognize peptides in the context of MHC-I or MHC-II molecules (47). Such non-MHC restricted T cells, which can recognize lipid antigens (48) or riboflavin metabolites (49), include NKT (CD1d restricted), mucosal-associated invariant T (MAIT, MR1 restricted), CD1a/CD1b/CD1c T cells, and TCR γδ T cells. Collectively, these cells can represent between 10% and 50% of circulating T lymphocytes depending on the human donor (10, 50–53). By employing the use of CD1 or MR1 multimers loaded with specific antigens, investigators have started to interrogate these non-MHC restricted T cell populations in vivo (43, 52, 54).

Primary dNKT cells are poorly understood because of the lack of tools for identifying these cells in vivo (55). To date, the only primary dNKT cell population that has been carefully studied ex vivo with CD1d tetramers are dNKT cells restricted against the mammalian lipid sulfatide (12, 56). However, because of the wide range of TCR Vα and Vβ chains used by dNKT cells, it is unlikely that the function(s) of sulfatide-restricted dNKT cells can be generalized to all dNKT cells. Indeed, the dNKT XV19 hybridoma, which recognizes sulfatide and was the basis for testing sulfatide-loaded CD1d tetramers, does not recognize the Listeria PG antigens studied here (Table III and data not shown). This finding highlights the need to identify more, high-potency, dNKT cell antigens for further interrogation of dNKT cells in vivo.

Microbial lipid antigens are ideal targets for dNKT cells. While mammalian self-lipids may be responsible for dNKT cell selection in the thymus, like self-peptides for MHC-restricted T cells, the most potent antigens recognized in the periphery may be of microbial origin. Because Listeria is an intracellular microbial pathogen, it was an attractive model organism for identifying antigens for dNKT cells (29, 57). By performing an unbiased search for Listeria lipid antigens, we identified the microbial versions of two known dNKT cell phospholipid antigens, PG and DPG, as dNKT cell antigens. Importantly, Listeria PG is 10 to 100-fold more potent antigen than mammalian or the structurally related Corynebacterium PG.

These results prompted us to consider why Listeria PG is a more potent antigen than the structurally similar Corynebacterium/mammalian PG. Because these two lipids share identical head groups, it was unsurprising to find that the dNKT TBA7 TCR bound to CD1d loaded with either Listeria or Corynebacterium PG with similar affinities. However, we found that Listeria PG loaded into CD1d (displacing the charged lipid GD3) approximately 2-fold more efficiently than does Corynebacterium PG. Because TCR activation leads to signaling cascades that can exponentially amplify the original signal, we attribute the higher potency of Listeria PG to its increased CD1d loading efficiency over Corynebacterium PG. However, we cannot discount the possibility that Listeria PG may also be more stably bound within CD1d over time.

There are 3 differences between the fatty acid tails found in Listeria that could be modulating its ability to load or stay within CD1d more efficiently than mammalian PG. One hypothesis is that the anteiso methyl fatty acid branches (found only in some microbes) acts as a ‘hook’ within the CD1d hydrophobic channels and increases the stability of the lipid-CD1d complex. Alternatively, it is possible that other aspects of Listeria PG, such as its shorter tails or lack of a double bond, make it easier to load or remain bound within CD1d molecules.

Previous attempts to generate Corynebacterium or mammalian PG CD1d tetramers were not successful in our group. However, identification of the more potent Listeria PG variant prompted us to see if CD1d tetramers loaded with Listeria PG could bind with high enough avidity to TBA7 cells to stain in flow cytometry. Indeed, Listeria PG – LC-MS E loaded CD1d tetramers specifically bound to TBA7 TCR-transduced cells but not to the same cells transduced with other (or no) TCRs (Fig. 5C, D and data not shown). By utilizing dextramers that contain ~12 CD1d molecules and multiple fluorophores per molecule we were able to further increase the signal-to-noise ratio so that the majority of the Listeria PG dextramer-stained cells could be separated from the mock or irrelevant lipid-loaded dextramer-stained cells. This increase in signal may be critical, as it provides a new reagent suitable to identify and interrogate dNKT cells in vivo. We are now optimizing this technology to stain primary human and mouse dNKT cells, including taking advantage of the recent insight that unlabeled anti-fluorophore antibodies can stabilize TCR-multimer complexes with affinities similar to those found here (58).

In summary, we have identified a new lipid antigen for dNKT cells with a distinctively microbial signature, namely short fully-saturated anteiso lipid tails. Notably, this microbial version of PG is much more active than the previously known PG antigens from mammals or Corynebacterium, which have structurally related lipid tails. Importantly, by identifying a high potency microbial dNKT cell antigens for different dNKT cell populations, we can begin to dissect the poorly understood nature of dNKT cells in vivo.