Inflammation-associated lysophospholipids as ligands for CD1d-restricted T cells in human cancer

doi:10.1182/blood-2008-04-149831Prepublished online June 5, 2008;

Amitabha Mazumder, David H Vesole, Sundar Jagannath and Madhav V DhodapkarDavid H Chang, Haiteng Deng, Phillip Matthews, Joseph Krasovsky, Govind Ragupathi, Radek Spisek, T cells in human cancerInflammation associated lysophospholipids as ligands for CD1d restricted

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Inflammation associated lysophospholipids as ligands for CD1d restricted T cells in human cancer David H. Chang1, Haiteng Deng2, Phillip Matthews1, Joseph Krasovsky1, Govind Ragupathi4, Radek Spisek1, Amitabha Mazumder5, David H. Vesole5, Sundar Jagannath5, Madhav V. Dhodapkar1,3,6,7 1Laboratory of Tumor Immunology and Immunotherapy, 2Proteomics Resource Center, 3Chris Browne Center for Immunologic Diseases, The Rockefeller University; 4Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, 5St. Vincent’s Cancer Center, New York, NY; 6Section of Hematology, Yale University; 7Program in Hematologic Malignancies, Yale Cancer Center, New Haven, CT 06520 Section: Immunobiology Key words: Glycolipids, inflammation, innate immunity, multiple myeloma, natural killer T cells Correspondence: Madhav V. Dhodapkar, MD; Section of Hematology; Yale University 333 Cedar Street; New Haven, CT 06510 Email: [email protected]

Blood First Edition Paper, prepublished online June 5, 2008; DOI 10.1182/blood-2008-04-149831

Copyright © 2008 American Society of Hematology




Abstract:

CD1d restricted T cells have been implicated in the pathogenesis of several chronic

inflammatory states. However the nature of the specific ligands recognized by these

cells in vivo in patients with inflammatory or malignant diseases remains unknown. We

took a biochemical approach to directly isolate and characterize the nature of CD1d

binding ligands from the plasma of myeloma patients. Characterization of these ligands

revealed several lysophosphatidylcholine (LPC) species. Human LPC-CD1d dimer

binding cells are TCRαβ+ T cells but predominantly Vα24-Vβ11-. Cytokine secretion by

LPC specific T cells is skewed towards IL13 secretion; and the frequencies of these

cells are increased in myeloma patients relative to healthy donors. These data identify a

distinct population of human CD1d-restricted T cells specific for inflammation associated

lysolipids and suggest a novel mechanism for inflammation mediated immune regulation

in human cancer.




Introduction

In contrast to CD4 and CD8+ T cells that recognize peptide ligands in the context of

major histocompatibility complex (MHC) class I and II molecules, a distinct subset of T

cells recognize lipid antigens in the context of CD1d molecules on antigen presenting

cells1,2. Broadly, two distinct types of CD1d restricted T cells have been identified3. One

subset of CD1d-restricted T cells, also known as invariant or type I natural killer T (NKT)

cells, express an invariant T cell receptor (iTCR: Vα24/Vβ11 in humans and Vα14 in

mice) and NK markers such as CD1612,4. However particularly in humans, many CD1d

restricted T cells lack iTCR and have been termed type II NKT cells5,6. Most of the early

work on CD1d restricted T cells was based on the recognition of a synthetic ligand α-

galactosyl ceramide (α-GalCer), by iNKT cells 4. Recent studies have shown that type I

NKT cells can recognize some microbial glycolipids, and self antigens such as

isoglobotrihexosylceramide (iGb3) (reviewed in Brutkiewicz)7. The nature of antigens

specifically recognized by type II NKT cells is less clear and limited to sulfatide and non-

lipidic small molecules 8,9. Binding of CD1d molecules to phospholipids has also been

demonstrated10-13. However, whether these molecules are commonly recognized by

populations of human T cells is not known.

Several studies have suggested an important role for CD1d restricted T cells in the

regulation of chronic inflammatory states. For example, type I NKT cells can promote

allergen induced asthma and atherosclerosis, and can mediate protection against some

autoimmune states in mice 1. Cancer is intricately linked to inflammation. NKT cells




have been implicated in both immune surveillance and immune regulation in cancer,

attributed to type I and II NKT cells respectively 1,14. In some setting, GD3 and tumor

derived glycosphingolipids have been implicated as ligands for iNKT cells15,16. However

the nature of the specific ligands recognized by either type I or II NKT cells in

inflammation or cancer in humans remains unclear.

Multiple myeloma (MM) is a plasma cell tumor wherein both the innate and adaptive

limbs of the immune response are able to recognize cancer cells17. In prior studies, we

have shown that clinical progression in myeloma is associated with dysfunction of type I

NKT cells 18. In order to identify the nature of CD1d binding ligands in human myeloma,

we took a biochemical approach to directly isolate and characterize the CD1d binding

lipids from the plasma of these patients. Here we show that CD1d binding ligands

isolated from myeloma patients that are recognized by human T cells are inflammation

associated lysophospholipids.




Materials and Methods

Plasma and cells from patient and healthy donors

Peripheral blood mononuclear cells (PBMC) were obtained from healthy donors (buffy

coats from New York Blood Center) and from myeloma patients following informed

consent approved by the institutional review board of the Rockefeller University and St.

Vincent’s Cancer Center. Blood mononuclear cells were isolated by density gradient

centrifugation using Ficoll-Paque (Amersham Biosciences, Upsala, Sweden). Plasma

was harvested from the top of the gradient and cryopreserved until use for lipid

isolation. Samples from myeloma patients were collected under the auspices of a

protocol for collection of samples for research approved by IRB at St. Vincents and

Rockefeller.

Reagents

All lipid products were purchased from Avanti lipids (Alabaster, AL), dissolved in

chloroform at 10 mg/ml and stored at -20ºC as storage stock. To prepare 1 mg/ml

working stock, phospholipids were first diluted 1:5 with 100% DMSO, then 1:2 with

water. All phospholipid compounds were stored in either amber glass vials or

siliconized Eppendorf tubes covered with aluminum foil (to minimized light exposure).

Anti-Vα24-FITC and anti-Vβ11-PE antibodies were purchased from Immunotech

(Fullerton, CA). CD1d-DimerX and most fluorochrome-conjugated antibodies were

purchased from BD Biosciences (San Jose, CA). Anti-CD8-PE-Texas-Red, anti-CD19-




PE-Cy5.5, and anti-CD4-PacificBlue antibodies were purchased from Invitrogen

(Carlsbad, CA). The α-galactosyl-ceramide (α-GalCer) was kindly provided by Kirin

Breweries (Tokyo, Japan).

Isolation of bulk lipids from plasma:

Extraction of bulk lipids was performed based on the method of Bligh and Dyer 19.

Plasma (50 ml) was mixed 1:1 with PBS, stirred in a beaker while adding ammonium

sulfate (Fisher) slowly until saturation at room temperature, and transferred and stirred

continuously overnight at 4ºC for protein precipitation. The mixture was centrifuged at

1000g for 1 hour at 4ºC to pellet the solid precipitates, and liquid mixture was then

collected, frozen, and lyophilized using freeze-dry system (Labconco, Kansas City, MO).

The lyophilized powder was then extracted sequentially with three combinations of

organic solvents (chloroform:methanol:water at 2:1:0, 1:2:0, 10:10:1). The three extracts

were pooled and organic solvents removed using rotary evaporator. The extract was

then redissolved in 5 ml of water and stored frozen in aliquots at -20ºC until use.

Mass Spectrometry Analysis

Lipid samples from extracts and elutes were desalted, cleaned and concentrated using

micro-C18 ZipTip (Millipore). The lipids were eluted from ZipTip with 2 µl aqueous

acetonitrile solution containing 50% acetonitrile (v/v) and 0.1% formic acid, and loaded

into a GlassTip (New Objective). The analysis was performed using an Applied

Biosystems QSTAR QqTOF mass spectrometer operated in both positive and negative




modes. Ions of interest were selected and subsequently dissociated by collision for

analysis of the ionic fragments.

Cross-linking CD1d-DimerX to beads and elution of lipids

The crosslinking of CD1d-DimerX to magnetic beads was done based on

manufacturer’s protocol. In brief, Dynabeads M-280 (sheep anti-mouse IgG from

Invitrogen) were washed twice in PBS, resuspended in PBS/BSA at 1 μg Dimer to

1~2x107 beads ratio, and incubated overnight at 4ºC with constant rotation. Then,

Dimer-Beads were washed twice with 0.2 M triethanolamine, pH 8.2, and crosslinked

with freshly prepared 5.4 mg/ml dimethyl pimelimidate dihydrochloride (Sigma) at room

temperature with constant rotation for 30 min. The crosslinking was stopped by

resuspending in 1 ml of 50 mM Tris, pH 7.5 and incubated for 15 min with rotational

mixing, and washed thrice with 1 PBS/BSA before stored at the original volume of

beads. As a control, some Dynabeads were processed through the same procedure as

above but without CD1d-DimerX. For ligand loading, 200 μl of crosslinked beads were

washed thrice with PBS while bulk extract was diluted 60:40 with DMSO. At the end of

wash, beads were mixed with 125 μl of bulk lipid extract and incubated overnight at

37ºC. For elution, ligand-loaded beads were washed once with ice-cold PBS, then

mixed with 0.1 M citrate at pH 2.0, incubated for 10 min at 37ºC. CD1d conjugated

beads not loaded with lipid ligands were used as additional controls. The lipid elute was

then collected after magnetic separation and saved for analysis. The beads were

washed thrice in PBS and saved for next use.




Loading of CD1d dimers and detection of CD1d-lipid reactive T cells

The loading and staining using CD1d DimerX was done based on manufacturer’s

protocol. For loading with α-GalCer (as positive control), 1 μl of DimerX (0.5 mg/ml)

was mixed with 1 μl of 10 molar excess α-GalCer (20 μg/ml). For loading with

lysophosphatidylcholine (LPC), 1 mg/ml LPC was first diluted to 80:20 with water (0.8

mg/ml), then mixed 1:1 with CD1d-DimerX for the final concentration of 50-400 μM. The

loading of all other phospholipids was done in equimolar ratio to LPC. The unloaded

dimer control was prepared by mixing CD1d-DimerX with the carrier only. The dimer-

mix was incubating overnight at 37ºC and stored at 4ºC before use. For some

experiments, mixtures of defined lipids at different ratios (e.g. LPC and PC) were

concurrently loaded onto CD1d. For some experiments, bulk lipids from myeloma or

healthy donor plasma were directly used to load CD1d dimers. For some experiments,

CD1d dimerX was preincubated with defined lipid ligands (1:1 ratio) for 4 hours at 37ºC

as described above. After 4 hours, 10 molar excess of α-GalCer or carrier control was

added and mixed before incubating overnight at 37ºC.

For staining human T cells with loaded CD1d dimer, T cells were first washed twice in

PBS then stained with dead cell exclusion dye (Live/Dead Aqua dead cell stain kit,

Invitrogen) for 15 min as recommended by the manufacturer. Cells were then washed

twice in wash buffer (PBS with 0.5% BSA), blocked with blocking buffer (wash buffer

with 0.1 mg/ml human IgG whole molecules (Jackson lab) at a concentration of about

1~3 million cells per 40 μl for 10 min at room temp. Dimer-staining mix was then

prepared by first diluting CD1d-Dimer-mix at 0.5 μg of CD1d-DimerX to 10 μl of blocking




buffer, then added to blocked cells at 1:5 ratio. Cells were then stained for about 30

minutes to 1 hour at room temperature in the dark. After dimer staining, cells were

washed twice with wash buffer, blocked with 10% rat serum in PBS for 5 min at room

temp (1~3 million in 25 μl) and then stained with anti-mouse-IgG1-APC or PE (clone

X56 from BD) at 1:1 ratio for the final dilution of 1:500 of Ab for 15-30 minutes on ice.

Cells were then washed twice with wash buffer, and stained with additional markers.

Typically, the following antibodies were used for staining: anti-Vα24-FITC, anti-Vβ11-

PE, anti-CD3-PE, anti-CD3-Alexa700, anti-CD56-PECy7, anti-CD8-PE-TexasRed, anti-

CD4-PacBlue, anti-CD19-PECy5.5. For some experiments, cells were also stained with

anti-TCRαβ-PE and anti-TCRγδ-PE. For acquisition using BD-LSR II, the cells were

resuspended in wash buffer and data acquired directly. For acquisition using BD-

Calibur, staining with 7-AAD was utilized for excluding dying cells.

Expansion of Lyso-PC specific T cells

PBMC were separated into CD14+ and CD14- fractions using CD14 magnetic beads

(Miltenyi Biotec). CD14+ monocytes were used for dendritic cell culture and cultured in

either 1% plasma or in serum-free Aim-V media (Invitrogen) in the presence of GMCSF

and IL4 as previously described20. CD14- PBMC were similarly cultured in either RPMI

1640 (Cellgro) with 5% pooled human serum (Labquip, NY) or in Aim-V medium. For

some experiments, expansion of invariant NKT cells using α-GalCer loaded DCs was

performed as previously described21. For loading LysoPC, immature DCs on day 5 of

culture were cultured for 48 hours in the presence of 20 μg/ml of LPC (LPC-C18-1). For

the expansion of LPC specific T cells, DCs were cultured with responder CD14- cells




(DC:responder ratio of 1:10 to 1:20) in media supplemented weekly with 50 U/ml of IL-2

(Chiron). For some experiments, the cocultures were setup in serum free conditions,

without or with supplementation with 20 μg/ml of LPC. After 1-2 weeks of coculture, the

presence of LPC specific T cells was monitored by flow cytometry, as described above.

Activation of human invariant NKT (iNKT) cells by immobilized CD1d-DimerX:

The loading of CD1d dimers with defined lipids or α-GalCer, was performed as specified

above. After overnight incubation, lipid loaded CD1d dimer, or unloaded CD1d dimer as

control, was diluted in PBS at a ratio of 1 μg of CD1d dimer to 100 μl of PBS, added to a

96-well U-bottom plate, centrifuged at 300g at room temperature and incubated at 37ºC

for about 4 to 6 hours. The Dimer-PBS mixture was removed just before adding purified

iNKT cells (0.5-1 x106 cells / well). Culture supernatants were harvested after 40~48

hours of culture and analyzed for the presence of interferon-γ by ELISA following the

manufacturer’s protocol (Biosource International).

Detection of cytokine production by CD1d-LysoPC+ T cells

CD3+ CD1d-LPC+Va24- T cells (> 95% purity) were obtained from cultures of human T

cells described above, by flow sorting using BD FACSAria cell-sorting system. As a

control, CD3+ CD1d-LysoPC- Vα24- T cells were also sorted. For stimulation with PMA

and ionomycin, sorted cells (CD1d-LysoPC+ or CD1d-LysoPC-) were plated at

concentration of 5-10 x 104 cells in 100 μl of fresh culture media (RPMI with 5% pooled

human serum), and stimulated with PMA (500 ng/ml) and ionomycin (1 μM) in a 96-well

U bottom plate. For stimulation with immobilized anti-CD3, CD1d-LPC+ sorted cells




were plated at 104 cells/ 100 μl of fresh culture media in anti-human-CD3 antibody-

coated or control plate (BD Biosciences) as suggested by the manufacturer. For some

experiments, T cells were depleted of CD1d-LPC+ cells or mock-depleted by sorting,

prior to plating at 1x106 cells per 200 μl in anti-human-CD3 antibody-coated or control

plates. For some experiments, sorted 5 x 104 LysoPC-CD1d dimer+ T cells were

cultured with CD1d expressing cell line (C1rd cells; kindly provided by Dr Mark Exley,

Boston), or mock transfected control cells at a T cell: APC ratio of 1:10, as described6,

with or without prior pulsing with 40 μg/ml of LPC-C181, with 5 ng/ml PMA and 5 U/ml

IL2 in Aim-V media . After 40-48 hours of co-culture, supernatants were harvested and

the presence of cytokines was measured by Luminex analysis with human cytokine

detection kits from Upstate-Millipore, as described 22. In some experiments, IL-17 was

detected by an ELISA kit following manufacturer’s protocol (eBioscience).

Statistics:

Comparison of data between groups was performed using Mann Whitney test.

Significance was analyzed with two sided p values <0.05.




Results:

In order to identify the nature of CD1d binding lipids in the plasma of myeloma patients,

bulk lipids from these samples were obtained by chloroform/methanol (C/M)

extraction19. Analysis of bulk lipids by electrospray ionization mass spectrometry (ESI-

MS) revealed the presence of many species in the extracts from both patients tested

(Figure 1a). In order to identify the CD1d binding fraction, we first loaded the bulk lipids

onto CD1d dimer conjugated to magnetic beads and then eluted the bound fragments

as described under methods. Magnetic beads not conjugated with CD1d were used as

a control. Another control was CD1d conjugated beads not treated with lipids that did

not yield any contaminating lipids in the beads themselves (data not shown). Although

the mass spectra in bulk lipids from both patients differed and included several lipid

species as expected, the eluates from CD1d conjugated beads in both patients were

remarkably similar (Figure 1b). The most abundant ions were observed at m/z 496,

520, 522, 524 and 544. To further validate the enrichment of these ions, the samples

eluted from beads alone or CD1d-conjugated beads were spiked with C14-LysoPC (m/z

468) as an internal standard, and analyzed by ESI-MS. These data again confirmed the

relative enrichment of previously identified species in the elutes from CD1d-conjugated

beads (Figure 1c). The MS/MS spectrum of ions at m/z 496.33 is displayed in Figure

1d. It shows a major fragment at m/z 184. The m/z of this fragment corresponds to the

head group of phosphatidylcholine. Indeed, the other fragments observed at m/z 60, 86,

and 104 are also typical dissociation products of phosphatidylcholine23,24. The same

samples were also analyzed in ESI-negative mode. An ion at m/z 480.28 was observed




that corresponds to the [M-15]- species observed at m/z 496.33 in positive mode. The

[M-15]- species was formed by the loss of the methyl group from phosphatidylcholine.

The MS/MS of 480.28 is displayed in Figure 1e. A major fragment was observed at m/z

255 that matches to one of the fatty acid anions in phosphatidylcholine according to the

general rules of fragmentation of phopholipid in negative mode. The molecular weight of

palmitic acid is 256, suggesting that one side chain of phosphatidylcholine at m/z 496.3

for [M+H]+ or 480.28 for [M-15]- is palmitic acid. Therefore, we conclude that the major

ion binding to CD1d molecule is hexadecanoly-sn-glycero-3-phosphocholine (Figure

1f). To determine the regioisomer of this lysophospholipid species, we examined the

MS/MS of the sodiated hexadecanoly-sn-glycero-3-phosphocholine at m/z 518.32 and

found that the intensity of a fragment at m/z 104 is more abundant than that at m/z 147

demonstrating that species we identified in this work is 1-hexadecanoly-2-hydroxy-sn-

glycero-3-phosphocholine (LPC)24. The analysis of other species identified in these

studies revealed that they were all LPC species with different acyl chains (16:0, 18:0,

18:1, 18:2, 20:4) (Table 1). To further confirm our results, authentic 1-hexadecanoly-2-

hydroxy-sn-glycero-3-phosphocholine was purchased and analyzed using tandem mass

spectrometry. Fragmentation patterns of authentic 1-hexadecanoly-2-hydroxy-sn-

glycero-3-phosphocholine were identical to what we observed in the present work (not

shown). Therefore the great majority of CD1d binding lipids isolated from the plasma of

myeloma patients were LPC species, which are structurally distinct from ceramides. The

isolation of LPC from myeloma plasma is consistent with prior studies showing that

levels of this lipid in myeloma is much higher than in healthy donors25. Consistent with




this, when bulk lipids from myeloma patients were loaded onto CD1d dimer, the staining

for human T cells was better than that seen with lipids from healthy donors (Figure 1g).

Several species of LPC exist in vivo depending on the properties of the acyl chain26. We

focused on two of the species (C16:0 and C18:1) identified in our experiments. In order

to confirm the findings with elution experiments, we loaded equimolar quantities of LPC

(C16:0 and C18:1) and PC on CD1d dimer and compared their subsequent binding to

cultures of human T cells (Figure 2a). CD1d dimers loaded with LPC showed clear

staining for human T cells. Staining with C18:1 LPC was generally superior to that with

C16:0 (Figure 2a), and this species was used in subsequent experiments. The

efficiency of staining with LPC loaded CD1d dimers was dependent on the dose of LPC

used to load the dimer and evident at a dose of 50-100 μM (Figure 2b). As an irrelevant

MHC molecule, MHCI tetramer coincubated with LPC or PC did not stain these T cells.

In cultures of human T cells, the predominant population stained with LPC-CD1d was

TCRαβ+ and a subset of Vα24-Vβ11- CD3+ T cells, which consisted predominantly of

CD4+, but also CD8+ T cells (Figure 2c). In contrast to CD1d-LPC, CD1d dimer loaded

with α-GalCer clearly stained Vα24+ T cells in these cultures. Equimolar loading of

CD1d with the parent lipid, PC led to minimal staining. However the addition of

increasing concentration of PC to LPC led to dose dependent inhibition of staining of T

cells, which is consistent with the capacity of PC to bind CD1d 12,13 and/or interfere with

the capacity of LPC to load CD1d and stain type II NKT cells (Figure 2d). Together

these data show that LPC loaded onto CD1d can bind a distinct population of human T

cells.




In order to further evaluate the ability of LPC to bind CD1d, we took advantage of the

ability of α-GalCer to load CD1d and of the loaded CD1d dimer to stain Vα24+Vβ11+ T

cells (also termed type I or iNKT cells) with high mean fluorescence intensity (MFI)

(Figure 2e). When LPC (400 μM) was coincubated with α-GalCer (20 μM) for loading

CD1d dimer, there was mild reduction in the intensity of staining, but iNKT cells could

still be stained (Figure 2e). However when CD1d dimers were preincubated with LPC

prior to loading of α-GalCer, it clearly reduced the intensity of staining for iNKT cells,

suggesting that under these conditions, LPC could competitively inhibit the loading of α-

GalCer on CD1d (Figure 2f). Culture of purified human iNKT cells with immobilized

CD1d-dimer-α-GalCer normally leads to a rapid release of cytokines like interferon-γ21.

However, when these CD1d molecules were preincubated with LPC prior to α-GalCer,

their ability to induce interferon-γ from purified iNKT cells was inhibited, which further

supported the observed capacity of preloading with LPC to inhibit α−GalCer dependent

activation of iNKT cells (Figure 2g). Notably, immobilized CD1d dimer loaded with LPC

alone elicited little IFNγ from Vα24+Vβ11+ T cells, which is consistent with Vα24-Vβ11- T

cells being the predominant population stained with these reagents. Interestingly,

preloading with PC under these conditions did not alter GalCer mediated iNKT staining

and activation. This is somewhat surprising in view of the interference of LysoPC

dependent staining of a different type of NKT cells (type II NKT cells) by concurrent

addition of PC (Figure 2d). However it is consistent with the possibility that the

recognition of CD1d by type I versus type II NKT cells may differ, and/ or that the




structural aspects of LPC loading into CD1d (which has not yet been examined) may

differ from that of PC27.

Next we examined whether human LPC specific T cells could be expanded in culture.

Culture of human T cells in 5% pooled human serum with both unpulsed DCs, as well

as those pulsed with LPC led to expansion of LPC specific T cells in culture (Figure 3a,

3b). Although this was initially surprising, we realized that several studies have shown

the presence of LPC in normal human serum 25,28,29. The presence of LPC in lipids from

5% PHS was confirmed by MALDI analysis (data not shown), which is consistent with

prior studies 25,28,29. Therefore we repeated these co-cultures in serum free media. In

this setting, the expansion of LPC specific T cells in the presence of unpulsed DCs was

abrogated and specific expansion of LPC specific T cells could be demonstrated only in

the presence of the specific ligand (Figure 3c, 3d). These data demonstrate the

feasibility of ligand dependent expansion of human LPC specific T cells in culture.

In order to characterize the functional properties of these cells, we sorted CD1d-LPC+

CD3+ T cells and compared their cytokine profile in response to stimulation by

PMA/ionomycin, relative to CD1d-LPC- CD3+ T cells (Figure 4a). Vα24- CD1d-LPC+ T

cells secreted higher levels of several cytokines including IL13, IL5, IL8 and IFNγ in

response to PMA/ionomycin, relative to CD1d-LPC- control CD3+ T cells (Figure 4a).

Next, we compared the capacity of these sorted T cells to secrete IL13 and IFNγ in

response to plate bound anti-CD3. Vα24- CD1d-LPC+ T cells secreted more IL13

relative to CD1d-LPC- control T cells under these conditions (Figure 4b). Interestingly,




depletion of this subpopulation from these cultures led to significant decline in the anti-

CD3 mediated secretion of IL13, compared to mock sorted or undepleted cells,

suggesting that this subpopulation of T cells may be a major producer of IL13 in these

cultures (Figure 4c). LPC specific T cells also secreted IL13 in response to LPC loaded

CD1d expressing antigen presenting cells (APCs), suggesting that they can respond to

ligand specific stimulation by secretion of this cytokine (Figure 4d).

Prior studies have documented that sera from myeloma patients contain much higher

levels of LPC, as well as higher palmitic / linoleic acid ratio, relative to healthy controls25.

This may help explain the facile loading of bulk lipids from myeloma patients but not

healthy donors, on CD1d dimer that we observed. Therefore we tested whether the

frequency of LPC specific T cells was altered in human myeloma. CD1d multimers

loaded with LPC (versus PC or unloaded CD1d dimer as a control) were used to stain

fresh human PBMCs from healthy donors or myeloma patients (Figure 5a, 5b). The

frequency of CD1d-LPC+ T cells was increased in myeloma patients relative to healthy

controls. These cells lack the expression of invariant T cell receptor. Therefore MM

patients have an increase in a distinct population of CD1d restricted T cells relative to

healthy controls.




Discussion:

In this study we have identified a novel subset of human CD1d restricted T cells that

recognize lysophosphatidylcholine (LPC). LPC is generated by the action of

phospholipase A2 on phosphatidylcholine (PC). It is a major constituent of oxidized low

density lipoprotein (LDL) in atherosclerotic plaques, and elevated levels of

lysophospholipids have been observed in allergic and autoimmune inflammation,

asthma and human cancer, including in multiple myeloma 25,26,30,31. Therefore the

findings made in this study may apply to the putative role for CD1d restricted T cells in

diverse inflammatory states.

Staining of human T cells with LPC loaded CD1d dimers was inhibited in the presence

of PC. However, in contrast to LPC, CD1d dimers loaded with PC did not stain human T

cells. Although binding of some T cell lines to murine CD1d molecules with PC or PI

species has been observed, these ligands have not been shown to be recognized by

bulk populations of human or murine T cells 10-13,32-34. These lipids may however play

important chaperone like roles in intracellular assembly of CD1 molecules 13. Our data

are consistent with a model wherein the recognition of CD1d by these T cells may

depend on the balance between PC and LPC, suggested to be a sensitive indicator of

inflammation35. These studies therefore support the hypothesis that some CD1d

restricted cells may serve as a cellular sensor system for the detection of inflammation

in tissues. In this regard, it is notable that several species of LPC exist in vivo 26, and the




differences in acyl chains may lead to different functional consequences36. Inflammation

is a common feature of many human cancers and can lead to high levels of several

species of LPC or related ligands in the malignant tissue 26. Therefore the generation of

LPC may be a mechanism by which inflammation might paralyze innate immunity

against cancer18.

Majority of cells that bind LPC loaded CD1d dimers in humans were Vα24-Vβ11- T cells

3. Such CD1d restricted T cells have been termed as Type II NKT cells. It is of interest

that the cytokine profile of these cells after TCR based stimulation is somewhat skewed

towards IL13. Indeed, IL13 was the only cytokine detected after ligand specific

stimulation in culture. Type II NKT cells have been implicated in suppressing tumor

immune surveillance in mice in an IL13 dependent manner in settings where the release

of such ligands (e.g. release of platelet activating factor, a structurally similar lipid, after

UV induced injury) might occur in vivo 14,37. IL13 producing type II NKT cells have also

been observed in the involved tissues in patients with ulcerative colitis38. The

preferential production of IL13 by LPC specific T cells is also of interest, because these

cytokines have been implicated in promoting tumor growth, fibrosis and angiogenesis in

malignant tissues and inflammation39,40. Specific targeting of these cells may therefore

provide novel approaches to regulate inflammation and innate immunity in the clinic.

LPC is also known to be a major target of naturally occurring antibodies that recognize

oxidized LDL and molecular motifs on dying cells41. Interestingly, recent studies have

begun to implicate such autoreactive B cells as preferred targets of transformation in




human B cell tumors42. Therefore the detection of increased levels of LPC specific T

cells in myeloma patients may be of additional and more direct relevance to the

pathogenesis of myeloma. Further studies are needed to evaluate the potential role of

lipid antigens in driving the origin of B cell tumors, and to evaluate lipid reactive T cells

in related B cell tumors, particularly chronic lymphocytic leukemia.




Acknowledgments:

This work is supported in part by funds from the National Institutes of Health

(CA106802, CA109465, Rockefeller CTSA), Damon Runyon Cancer Research Fund,

and Dana Foundation. DHC is a Human Immunology Fellow of the Dana Foundation /

CRI-Irvington Institute. We thank Ralph M. Steinman for his mentorship, Philip

Livingston and Kavita M. Dhodapkar for discussions about this paper, Judy Adams for

help with figures; Arlene Hurley, RN, for help with clinical aspects; John Gathuru for

technical assistance; and members of Dhodapkar lab for many helpful discussions. The

authors report no financial conflicts of interest.




Author Contributions:

DHC performed research, analyzed data, helped write and prepare manuscript.

HD, PM, JK, GR, RS performed some aspects of research.

AM, DV, SJ performed clinical research.

MVD designed research, analyzed data, wrote paper.




References:

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https://www.researchgate.net/publication/10673838_Cross-presentation_of_Disialoganglioside_GD3_to_Natural_Killer_T_Cells?el=1_x_8&enrichId=rgreq-149443dbc708faf0eaedcc5228d058da-XXX&enrichSource=Y292ZXJQYWdlOzUzMjA3OTA7QVM6OTk4NjY4MDYwNjMxMTNAMTQwMDgyMTUxMDYyMg==

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35. Fuchs B, Schiller J, Wagner U, Hantzschel H, Arnold K. The phosphatidylcholine/lysophosphatidylcholine ratio in human plasma is an indicator of the severity of rheumatoid arthritis: investigations by 31P NMR and MALDI-TOF MS. Clin Biochem. 2005;38:925-933. 36. McCarthy C, Shepherd D, Fleire S, et al. The length of lipids bound to human CD1d molecules modulates the affinity of NKT cell TCR and the threshold of NKT cell activation. J Exp Med. 2007;204:1131-1144. 37. Moodycliffe AM, Nghiem D, Clydesdale G, Ullrich SE. Immune suppression and skin cancer development: regulation by NKT cells. Nat Immunol. 2000;1:521-525. 38. Fuss IJ, Heller F, Boirivant M, et al. Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis. J Clin Invest. 2004;113:1490-1497. 39. Wynn TA. IL-13 effector functions. Annu Rev Immunol. 2003;21:425-456. 40. Aspord C, Pedroza-Gonzalez A, Gallegos M, et al. Breast cancer instructs dendritic cells to prime interleukin 13-secreting CD4+ T cells that facilitate tumor development. J Exp Med. 2007;204:1037-1047. 41. Binder CJ, Silverman GJ. Natural antibodies and the autoimmunity of atherosclerosis. Springer Semin Immunopathol. 2005;26:385-404. 42. Lanemo Myhrinder A, Hellqvist E, Sidorova E, et al. A new perspective: molecular motifs on oxidized LDL, apoptotic cells, and bacteria are targets for chronic lymphocytic leukemia antibodies. Blood. 2008;111:3838-3848.




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https://www.researchgate.net/publication/7702733_The_phosphatidylcholinelysophosphatidylcholine_ratio_in_human_plasma_is_an_indicator_of_the_severity_of_rheumatoid_arthritis_Investigations_by_P-31_NMR_and_MALDI-TOF_MS?el=1_x_8&enrichId=rgreq-149443dbc708faf0eaedcc5228d058da-XXX&enrichSource=Y292ZXJQYWdlOzUzMjA3OTA7QVM6OTk4NjY4MDYwNjMxMTNAMTQwMDgyMTUxMDYyMg==




Figure Legends:

Figure 1. Isolation and identification of CD1d binding lipids from myeloma patient plasma 1a and 1b– ESI-Mass spectra of lipid species from bulk extracts (1a) and CD1d-conjugated bead eluents (1b) from plasma of two myeloma patients (MM1 and MM2). 1c – ESI-Mass spectra of eluents from beads only control and CD1d-beads, spiked with a LPC species (LPC-C14, m/z 368, arrow) as an internal reference. Data shown are representative of findings on 4 separate patients. 1d and 1e – Tandem mass spectra (MS/MS) of major species observed for eluents of myeloma patient studied in figure 1c. Figure 1f is MS/MS of 496.3 of [M+H]+ and figure 1g is MS/MS of 480.3 of [M-15]-. 1f– Structure of 1-hexadecanoly-2-hydroxy-sn-glycero-3-phosphocholine, or Lysophosphatidylcholine (LPC) – C16:0. 1g. Bulk lipids isolated from myeloma patients or healthy donors were used to load CD1d dimers and stain cultures of human T cells as described under methods. Numbers represent percent cells in the quadrant. Figure 2. Binding of CD1d-dimer molecules loaded with lysophosphatidylcholine (LysoPC) to a subpopulation of CD3+ T cells. 2a – CD1d-dimers loaded with two species of LPC (LPC-C16 and LPC-C18:1) stained a unique population of CD3+ T cells: FACS dot plots of CD1d-dimers loaded with vehicle control (-), 400 μM of two species of LPC-(C16 and C18:1) or PC, or 20 μM of α-GalCer, and used to stain cultured human PBMC. Numbers in the quadrant correspond to percent cells out of total CD3+ cells and data shown are gated for live cells. The staining is representative of 4 individual donors. 2b. CD1d dimers were loaded with different concentrations of C18:1 LPC, prior to staining of human T cell cultures as described above. As controls, T cells were also stained with MHC-I tetramer cultured with 400 μM of LPC or PC. Numbers in the quadrant correspond to percent cells out of total CD3+ cells and data shown are gated for live cells. 2c – Phenotype of CD1d-LPC-C18:1+ cells: FACS dot plots of staining of CD1d-LPC-C181 (as in fig 2a) but plotted against different markers – TCRαβ, Vα24, and Vβ11 as indicated. Data for the staining of Vα24+ cells in these cultures with CD1d-αGalCer is




shown as a control. Bottom panel shows FACS dot plot gated for CD1d-LPC-C18:1+ T cell population for the proportion of CD4+/CD8+/double-negative subpopulations. 2d. CD1d dimers were loaded with either 400 μM of LysoPC or PC alone, or with a mixture of LysoPC and PC as indicated, prior to use for staining T cells as in Fig 2a. 2e. Histogram plots of staining of iNKT cells by CD1d dimers loaded with α-GalCer (20 μM), LPC, PC or the combination of α-GalCer with PC or LPC. Numbers represent mean fluorescent intensity of staining. 2f Competition between LPC and α-GalCer for binding to iNKT cells: Histogram overlay plots comparing mean fluorescence shifts between CD1d-dimer loaded with vehicle control (light gray solid), positive control (α-GalCer, dark gray solid), and LPC- C18:1, or PC (open curves). Competition was setup by first loading CD1d dimer with vehicle control, LPC-C18:1, or PC, for 4 hours, then loading with vehicle control or α-GalCer. Ligand loaded CD1d-dimers were then used to stain iNKT cells. Data are representative of 4 similar experiments. 2g – Differential iNKT stimulation by ligand-loaded and immobilized CD1d-dimer: Polyclonal populations of Vα24+ T cells were isolated using magnetic beads from cultures expanded in vitro using α-GalCer. Purified Vα24+ T cells were cultured for 40 to 48 hours in plates with immobilized CD1d dimer, loaded with vehicle control or α-GalCer with or without preincubation with control, PC or LPC as competing ligands as described under methods. PHA was used as positive control. The level of IFN-γ in the supernatant was monitored using by ELISA. * p<0.05. Data are representative of 3 similar experiments. Figure 3 Expansion of human CD1d-LPC+ T cells in culture 3a – Expansion of CD1d-LPC+ cells in serum-containing media: Representative FACS plot showing the presence of LPC specific T cells in freshly isolated PBMCs or after co-culture with DC pulsed with or without LPC in culture media with 5% pooled human serum. Numbers in the quadrant correspond to percent cells out of total CD3+ cells, and data shown are gated for live cells. 3b – Summary of experiments (n=8) with unpulsed-DC or LPC-pulsed DC in serum-containing culture media. Each dot corresponds to percent of CD1d-LPC+ cells per total CD3+ cells, and each line corresponds to data before and after two-week expansion. 3c – Expansion of CD1d-LPC+ cells in serum-free media: Representative FACS plot showing the presence of LPC specific T cells in freshly isolated PBMCs or after coculture with DC pulsed with or without LPC in serum free media. Numbers in the quadrant correspond to percent cells out of total CD3+ cells and data shown are gated for live cells.




3d – Summary of experiments (n=4) with unpulsed DC or LPC pulsed DCs in serum-free media. Each dot corresponds to percent of CD1d-LPC+ cells per total CD3+ cells, and each line corresponds to data before and after one-week expansion. Figure 4. Cytokine secretion by CD1d-LPC+ T cells. 4a – T cell subsets were flow-sorted as indicated for CD1d-LPC+ and CD1d-LPC- cell population and stimulated with PMA and ionomycin. The supernatant was harvested and analyzed for the presence of IL13, IL5, IL8, IL17, IFN-γ, and IL4 by Luminex, IL17 by ELISA. The data shown are for each donor (n=8) for each cytokine. Significant p values: IL13-0.001; IL5-0.003; IL8-0.02; IFNγ-0.049. 4b– Differential expression of IL13 and IFN-γ by CD1d-LysoPC+ T cells following anti-CD3 stimulation: CD3+CD1d-LysoPC+ and CD3+CD1d-LPC- were flow sorted as indicated in fig 3e and stimulated by plate immobilized anti-CD3 antibodies or control. Secretion of IL-13 and IFN-γ were monitored by Luminex after 40-48 hours of culture. Data shown are average (+/- SD) of 4 experiments. * p<0.05 4c – Decline in anti-CD3 stimulated IL13 secretion in human PBMCs following the depletion of CD1d-LysoPC+ cells: Cultured human PBMC were either mock-depleted or depleted of CD1d-LysoPC+ cells by flow sorting. The cells were then stimulated with anti-CD3 , and the production of IL13 monitored by Luminex. Data shown are average (+/- SD) of 3 experiments. * p<0.05 4d – Secretion of IL13 in response to LysoPC stimulation. Cultured PBMC were flow sorted as indicated in Fig 3e. CD1d-LysoPC+ and CD1d-LysoPC- T cells were then stimulated with LysoPC pulsed CD1d-expressing cell line (or unpulsed cells, as well as CD1d negative control cells as a control) in serum-free media for 40-48 hours. Secretion of IL13 was monitored by Luminex. Data shown are average (+/- SD) of 3 similar experiments. * p<0.05 Figure 5 Detection of CD1d-LPC+ T cells in multiple myeloma patients. 5a – Representative FACS dot plots of a healthy donor and a myeloma patient stained with CD1d-dimer loaded with vehicle control or LPC. The number in the quadrant corresponds with the percent of cells out of total CD3+ cells. Data shown are gated for live cells. 5b – Summary of percent of CD1d-LPC+ cells out of total CD3+ for healthy donors (n=22) and multiple myeloma patients (n=11). Each dot corresponds to one donor. Horizontal bars represent means. * p<0.05




Table 1 Various species of Lyso-phosphatidylcholine identified by MS/MS

Ion m/z Composition [M+H]+ 496.32 2-16:0-lyso

[M+Na]+ 518.32 2-16:0-lyso [M+H]+ 520.32 2-18:2-lyso [M+H]+ 522.34 2-18:1-lyso [M+H]+ 524.34 2-18:0-lyso

[M+Na]+ 542.31 2-18:2-lyso [M+H]+ 544.31 2-20:4-lyso

[M+Na]+ 566.33 2-20:4-lyso




Relative Intensity

m/z, amu

Relative Intensity

520

664

620

496

708

544

752

797

MM1

450 550 650 750 850

496

520

54 4

522

MM1

Relative Intensity

Relative Intensity

m/z, amu

450 550 650 750 850

758

520

652

627

544

659

496

576

674

464

512

684

730

838

889

MM2

450 550 650 750 850

496

520

544

522

MM2

1a

1b




m/z, amu100 200 300 400 500 600

255.21

480.28

191.00

224.05

Relative Intensity

100 200 300 400 500 600

184

104

496

478

86 313

258

60 166

m/z, amu

Relative Intensity

Beads only CD1d-Dimer-Beads

m/z, amu

2.0e4

1.0e5

2.0e5

460 500 540 580

520

496

468

544 4.0e4

1.0e5

2.0e5

460 500 540 580

520

544

496

568522

566

468

m/z, amu

Relative Intensity

1c

1d

1e

LysoPC-C16 OP

O

O-

ON+

O

OOH

1f




(-) HD1MM1 HD2MM20.00 0.04 0.022.772.06

CD3

CD1dDimer

1g




(-) LPC-C16 LPC-C18:1 PC0.01 0.21 0.51 0.01 0.10

0.03 0.46 0.53 0.00

αGC

CD1d-Dimer +

LPC

CD3

CD1dDimer

0.53 0.00

Vα24 Vβ11

62

29

8

CD4

CD8

TCRαβ

0.51

2a

2c

2b

CD3

CD1dDimer

(-) 50μM 100μM 200μM 400μMLPC

0.01 0.10 0.23 0.19 0.36

CD3

MHC ITetramer

0.01 0.01 0.01

CD1d-Dimer + αGalCer

Vα24

CD1d-Dimer +

LPC

CD3

(-) LPC PC




PC/ αGCLPC/ αGC

2e

CD1d-Dimer

count

2d

CD3

CD1dDimer

LPC +

0.150.19 0.12 0.040.08

50μM PC25μM PC 100μM PC 200μM PC 400μM PC

CD3

CD1dDimer

(-) LPC

0.170.01

PC

0.01

(-) GC LPC LPC+GC PC PC+GC

CD1d-Dimer

count

11.4 806 45.4 420 11.1 911

2f




2g

0

2

4

6

8

1st Ligand: - PC LPC - PC LPC2nd Ligand: - - - αGC αGC αGC PHA

Interferon-γSecretionBy iNKT (ng/ml)

*




CD3

CD1d(-)

CD3

CD1d-LPC

Fresh2-week culture

DC(-) DC(LPC)0.01 0.01 0.00

0.06 0.95 1.21

0.0

0.3

0.6

0.9

1.2

1.5

1.8

d 0 d 7 d 14 d 0 d 7 d 14 d 0 d 7 d 14 d 0 d 7 d 14

DC (-) DC LPC DC (-) DC LPC

CD1d-LPC iNKT

% of

CD3+

cells

3a

3b




3c

3d

0

0.2

0.4

0.6% of

CD3+

cells

Day 0 7 0 7

AimVDC(-) DC(LPC)

0.01

0.39

0.02

0.03CD3

CD1d(-)

CD3

CD1d-LPC

Fresh

0.00

0.03

1week-culture in AimVDC(-) DC(LPC)




4a

0

500

1000

1500

2000

2500

3000

CD1d-LPC - + - + - + - +Cytokines: IL8 IL5 IL17 IL4

0

2000

4000

6000

8000

10000

12000

CD1d-LPC - + - +Cytokines: IL13 IFN γ

cytokine

Secretion

(pg/ml)

cytokine

Secretion

(pg/ml)




4c

0

500

1000

1500

2000

CD1d-LPC + - + - + - + -anti-CD3 + + - - + + - -

IL13 IFN γ

4bcytokineSecretion(pg/ml)

*

0

2000

4000

6000

Mock-depleted

CD1d-LPC+depleted

α-CD3 Control

IL13Secretion(pg/ml)

*




4d

0

500

1000

1500

2000

2500

Antigen (-) LPC (-) LPC (-) LPC (-) LPC

Responder T cells: CD1d-LPC+ CD1d-LPC- CD1d-LPC+ CD1d-LPC-

IL13Secretion(pg/ml)

*

CD1d+ APC CD1d- Control APCAPC




CD3

CD1dDimer

HD(-) LPC

MM(-) LPC

0.01 0.07 0.03 0.65

0.0

0.2

0.4

0.6

0.8

CD1d-αGC+

Healthy Donors(n=22)

CD1d-LPC+ CD1d-αGC+

MM patients (n=11)

CD1d-LPC+

p < 0.001

% of

CD3+

5b

5a




Inflammation-associated lysophospholipids as ligands for CD1d-restricted T cells in human cancer

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Transcript of Inflammation-associated lysophospholipids as ligands for CD1d-restricted T cells in human cancer