Differential proliferative response of NKT cell subpopulations to in vitro stimulation in presence...

Differential proliferative response of NKT cellsubpopulations to in vitro stimulation in presence ofdifferent cytokines

Henry Lin1,2, Mie Nieda3 and Andrew J. Nicol1,2

1 Queensland Institute of Medical Research, Brisbane, Australia2 Department of Medicine, University of Queensland, Brisbane, Australia3 School of Medicine, Yokohama City University, Yokohama, Japan

Human Va24+Vb11+ NKT (NKT) cells have immune regulatory activities associated with

rejection of tumors, infections and control of autoimmune diseases. They can be stimulated

to proliferate using a-galactosylceramide (KRN7000) and have the potential for therapeutic

manipulation. Subpopulations of NKT cells (CD4+CD8–, CD4–CD8+ and CD4–CD8–) have

functionally distinctive Th1/Th2 cytokine profiles and their relative numbers following

stimulation may influence the Th1/Th2 balance, which may result in or prevent disease. We

aimed to determine the effect of different cytokines in culture during stimulation of NKT cells

on the relative proportions of NKT cell subpopulations. Our results show that all NKT cell

subpopulations expanded following stimulation with KRN7000 and IL-2, IL-7, IL-12 or IL-15.

Expansion capacity differed between subpopulations, resulting in different relative

proportions of CD4+ and CD4– NKT cell subpopulations, and this was influenced by the

cytokine used for stimulation. A Th1-biased environment was observed after stimulation of

NKT cells. NKT cells expanded under all conditions evaluated demonstrated significant

cytotoxicity against U937 tumor cells. In view of the potential for NKT cell subsets to alter the

balance of Th1 and Th2 environment, these data provide insights into the effects of NKT cell

manipulation for possible therapeutic applications in different disease settings.

Key words: NKT cells / Cytokines / a-Galactosylceramide / KRN7000

1 Introduction

Natural killer T (NKT) cells are a unique class of

T lymphocytes characterized by the expression of an

invariant T cell receptor (TCR) that recognizes glycolipids

presented by the MHC-like molecule CD1d. Many

studies have demonstrated the potential role of NKT cells

as an important immune regulator involved in tumor

surveillance, immunity against a range of infections and

control of autoimmune diseases [1–10]. Specific activa-

tion and potent expansion of NKT cells can be readily

achieved using a-galactosylceramide (a-GalCer,

KRN7000), providing a potential avenue for manipulation

of NKT cells for therapeutic purposes [2, 11–13].

Upon TCR activation, NKT cells rapidly produce large

amount of both Th1 and Th2 cytokines [1, 9]. This rapid

cytokine production, enabling NKT cells to control the

balance of Th1/Th2 cytokines in the microenvironment,

may underpin a potential therapeutic role in a number of

disease settings. Of particular interest is the substantial

evidence supporting the anti-tumor role of NKT cell-

derived IFN-c in both human and murine models [14].

Production of IFN-c by NKT cells directly induces anti-

proliferative responses against human tumor cells [1] and

has the potential to initiate anti-tumor activity indirectly

through the activation of T cells [15] and NK cells [16–19].

In contrast to these data supporting a beneficial,

protective role for NKT cells, other studies have shown

that Th2 cytokines produced by NKT cells inhibit Th1-like

responses such as those involved in immune rejection of

tumor [20, 21]. Clearly, clinical strategies in which

NKT cell function or activation are to be manipulated

require an understanding of the factors that influence

Th1/Th2 balance before and after manipulation to ensure,

for example, that desired Th1 responses are not negated

by unwanted Th2 responses.

The exact mechanisms determining whether NKT cells

promote Th1 or Th2 immune response in different

settings remain unclear. Subpopulations of NKT cells,

characterized according to expression of CD4 and CD8

[CD4+, CD8+ and CD4–CD8– (double-negative, DN)

[DOI 10.1002/eji.200324834]

Received 17/12/03Revised 17/6/04Accepted 12/7/04

Abbreviations: a-GalCer: a-Galactosylceramide DN: Dou-ble-negative Mo-DC: Monocyte-derived dendritic cells

2664 H. Lin et al. Eur. J. Immunol. 2004. 34: 2664–2671

f 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji.de

NKT cells] may be crucial to these different responses

[22–26]. These NKT cell subsets all recognize a-GalCer

and related glycolipids presented by CD1d but have

distinct cytokine expression profiles, providing an avenue

through which NKT cells, as a family, can initiate different

immune responses. The CD8+ [22, 23, 25] and DN [22, 24]

NKT cell subpopulations predominantly produce Th1-like

cytokines. CD4+ NKT cells also produce Th1 cytokines,

but in addition, these cells produce significantly higher

levels of Th2 cytokines than other NKT subsets [22–26].

Activation of the Th2 cytokine-producing potential of

CD4+ NKT cells may be unwanted in conditions demand-

ing a Th1 response, for example malignancy, and

beneficial in diseases where a Th2-type response is

desired. An observed inhibitory role of NKT cells, leading

to suppression of anti-tumor response in some circum-

stance, is attributable to CD4+ NKT cells [20, 21].

Subpopulations of NKT cells may be differentially acti-

vated, depending on the local environment, to initiate the

required immune response in different settings. Factors

of importance within the environment are likely to include

the specific characteristics of the antigen-presenting

cells (APC) involved, relative and absolute amounts of

various cytokines and the presence or absence of other

immune cells [11, 12, 25, 27, 28]. The role of various

cytokines, including IL-2, IL-7, IL-12 and IL-15, in

conjunction with APC and a-GalCer, in the proliferation

of human NKT cells has previously been reported [12, 27,

29]. However, these studies did not specifically address

whether different environments alter the proportion and

function of NKT cell subpopulations. In order to deter-

mine whether manipulation of the NKT cell environment

may allow tailoring of NKT cell responses to a given

therapeutic situation, we investigated the effect of IL-2,

IL-7, IL-12 and IL-15 on the proliferative responses and

relative proportions of NKT cell subpopulations. The

impact of these cytokines on the cytotoxic function of

NKT cells and the potential to skew immune response

toward either Th1 or Th2 type was evaluated.

2 Results

2.1 Effects of cytokines on the proliferativeresponse of NKT cells

PBMC co-cultured with autologous monocyte-derived

dendritic cells (Mo-DC) in the presence of a-GalCer and

IL-2, IL-7, IL-12 or IL-15 resulted in 500- to 900-fold

expansion of NKT cells after 7 days of culture. Prolifera-

tion of NKT cells was greatest in culture conditions

containing IL-2 or IL-15 (Fig. 1). Analysis of proliferative

response of specific NKT cell subpopulations showed

that subsets of NKT cells responded differently to

different cytokines (Fig. 1), although these differences

were not statistically significant, possibly due to small

sample size. When IL-2 was present during in vitro

stimulation of NKT cells, the greatest proliferative re-

sponses were seen in CD4+ NKT cells. In contrast, when

IL-7 or IL-15 was present in culture, the greatest

proliferative responses were seen in DN NKT cells. The

CD8+ NKT cell subset proliferated most in conditions

including IL-15. In all culture conditions analyzed,

proliferation of CD8+ NKT cells was significantly less

than that of CD4+ and DN NKT cells.

2.2 Effects of cytokines on the relativeproportions of NKT cell subpopulations

Assessment of CD4 and CD8 surface phenotype of

NKT cells among different human subjects demonstrated

DN NKT cells to be the predominant population before in

vitro stimulation, followed by CD8+ and CD4+ NKT cells,

although these differences were not statistically sig-

nificant (Table 1). The observed dominance of the DN

NKT cell population was consistent with previous studies

[22, 27]. CD8+ NKT cells were found in greater proportion

than CD4+ NKT cells, contrary to other studies, but

differences were not statistically significant.

Fig. 1. Mean � SE fold expansion of total NKT cells and NKT cell subsets (n=7) following 7 days of stimulation of PBMC with a-GalCer, autologous Mo-DC and cytokines.

Eur. J. Immunol. 2004. 34: 2664–2671 Differential proliferative response of NKT cell subpopulations 2665


Since NKT cells can be classed into two functionally

distinct groups (i.e. CD4– or CD4+ NKT cells) based on

their Th1 or Th2 cytokine expression profiles, the relative

CD4–:CD4+ ratio of NKT cells was assessed to determine

the potential of NKT cells to induce Th1 or Th2 immune

responses. Before in vitro stimulation there was a

predominance of CD4– NKT cells [CD4–:CD4+ NKT cell

ratio = 4.5�1.4 (mean � SE)]. After stimulation the

CD4–:CD4+ NKT cell ratio was reduced in all culture

conditions analyzed with a percent reduction ranged

from approximately 25% to 50% in culture conditions

containing IL-7 and IL-2, respectively (Fig. 2). Among the

culture conditions analyzed, the post-culture CD4–:CD4+

NKT cell ratio was highest in samples stimulated in the

presence of IL-7 or IL-15 and lowest in samples

stimulated in the presence of IL-2 or IL-12. Despite

these differences, CD4– NKT cells remained the domi-

nant population in all culture conditions analyzed.

2.3 Th1/Th2 cytokine production following in vitrostimulation of NKT cells

Supernatants obtained following in vitro stimulation of

NKT cells demonstrated an environment that significantly

favors Th1-type immune response (Fig. 3A). The

production of IFN-c was found to be considerably higher

than that of all other cytokines analyzed (TNF-a, IL-2, IL-4, IL-6, IL-10). When Th2 cytokines were analyzed

separately without comparison with IFN-c, the produc-

tion of the Th2 cytokines IL-6 and IL-10 was highest in

culture conditions that contained IL-2 (Fig. 3B). Assess-

ment of cytoplasmic IFN-c expression showed that a

significantly greater proportion of NKT cells expressed

IFN-c than NK and T cell populations (Fig. 4). However,

depletion of NKT cells from PBMC cultured in presence

of IL-12 produced a relatively small decrease in the

release of IFN-c into the culture supernatant (data not

shown).

2.4 Cytotoxic function of NKT cells followingin vitro stimulation

Following in vitro stimulation, NKT cells demonstrated

significant cytotoxicity against the tumor target U937

cells in a dose-dependent manner (Fig. 5). This

observation was consistent with previous studies [8,

23, 26, 30]. Of interest, different cytokines differed in their

capacity to induce NKT cell cytotoxic activity against the

target, U937. Greatest killing activity of NKT cells was

seen in NKT cells stimulated in the presence of IL-15.

3 Discussion

3.1 Differential proliferative response of NKT cellsubpopulations to stimulation with differentcytokines

The data described here demonstrate that NKT cell

subpopulations differ in their proliferative responses to

different cytokines. Previous studies suggested that

greatest expansion of NKT cells in response to specific

stimulation by a-GalCer presented by APC occurred in

the presence of IL-15 [11, 12]. Our data demonstrate that

this IL-15-induced cell expansion is mostly attributable to

proliferation of CD4– NKT cells. Both IL-7 and IL-15

preferentially expanded DN NKT cells. In contrast, the

presence of IL-2 during stimulation resulted in prefer-

ential expansion of CD4+ NKT cells. These findings are

consistent with data showing that CD4+ NKT cells

exclusively express high-affinity IL-2Ra [24, 25], and

with previous reports that high concentrations of IL-2

during in vitro stimulation of NKT cells preferentially

expand CD4+ NKT cells [5]. The preferential expansion of

Table 1. Mean � SE percentage of NKT cell subpopulations (n=7) before and after in vitro stimulation in presence of different

cytokines as indicated

D0 D7 IL-2 D7 IL-7 D7 IL-12 D7 IL-15

%CD4+ NKT 28�8% 42�11% 38�11% 42�11% 37�11%

%CD8+ NKT 34�3% 13�3% 18�3% 16�3% 16�3%

%DN NKT 38�7% 38�10% 38�10% 36�8% 42�9%

Fig. 2. Mean � SE ratio of CD4–:CD4+ NKT cells (n=7)

following 7 days of stimulation with a-GalCer, autologous

Mo-DC and cytokines. The proportion of CD4– NKT cells was

calculated by addition of the percentage of CD8+ and DN

NKT cells.



CD4+ NKT cells in the presence of IL-2 was not altered by

the addition of IL-7 or IL-15 (data not shown).

3.2 Dominance of CD4– NKT cells followingin vitro stimulation

Previous studies have suggested potentially undesirable

effects of CD4+ NKT cells for anti-tumor therapy. Our

results suggest that the use of IL-7 and IL-15 in

conjunction with a-GalCer may produce amore favorable

bias of NKT cell populations toward CD4– cells, particu-

larly when IL-2 is not used. The relative proportions of

different NKT cell subsets following in vitro expansion

was heavily influenced by their relative number prior to

stimulation. No single cytokine has been shown to

sufficiently skew NKT cell populations toward any

particular subtype for cytokine manipulation alone to

be sufficient for obtaining defined populations for clinical

use. If in vitro expanded NKT cells are to have a clinical

role in the treatment of malignancy, procedures to enrich

for specific, desired NKT cell subtypes according to

surface marker expression are likely to be required.

3.3 Th1-biased cytokine environment followingstimulation of NKT cells

Following in vitro stimulation of NKT cells, a significantly

Th1-biased culture environment, containing primarily

Fig. 3. (A) Mean � SE concentrations (pg/ml) of Th1/Th2 cytokines (n=7) in culture supernatants of PBMC after 7 days of

stimulation with a-GalCer, autologousMo-DC and the cytokines indicated. (B) Mean� SE concentrations (pg/ml) of Th2 cytokines

(n=7) in culture supernatants of PBMC after 7 days of stimulation with a-GalCer, autologous Mo-DC and the cytokines indicated.

Fig. 4.Representative data showing the percentage of IFN-c+ cells within NKT, NK and T cell population following 7 days of culture

with a-GalCer, autologousMo-DC and IL-12. Cells used for positive control were additionally stimulated with PMA and ionomycin.



IFN-c with low or undetectable levels of Th2-type

cytokines, was observed. This was true in all culture

conditions regardless of the specific cytokines used and

independent of NKT cell subpopulations. Whilst remain-

ing Th1-biased, the level of Th2 cytokines was highest in

the presence of IL-2. Since IL-2 preferentially expanded

CD4+ NKT cells, the only subset having a significant

capacity to produce Th2 cytokines, it is likely that

increased production of Th2 cytokines observed with IL-

2 treatment is related to expansion of this subset.

The high concentration of IFN-c observed after stimula-

tion of NKT cells was in agreement with previous studies

[1, 23, 26, 31] highlighting the important role of NKT cells

in IFN-c production, particularly when IL-12was used [27,

32–34]. To evaluate the role of NKT cells in the production

of IFN-c under our culture condition, cytoplasmic IFN-canalysis and IFN-c enzyme-linked immunosorbent as-

says (ELISA) after NKT cell depletion was performed.

Intracellular assessments showed that the NKT cell

population has the highest IFN-c production after

stimulation.However, depletion ofNKT cells fromcultures

of PBMC resulted in only marginal reduction of IFN-c in

culture supernatant. The discrepancy between high IFN-cexpression of NKT cells and the lack of IFN-c reduction

after NKT cell depletion most likely occurred because in

our culture condition the number of NKT cells was far less

than that of T cells. Therefore, although NKT cells

contribute to IFN-c production, the amount of IFN-c in

culture was maintained by the greater number of T cells.

3.4 Enhancement of NKT cell cytotoxicityby IL-15

Addition of IL-15 resulted in enhancement of NKT cell

cytotoxic activities against the U937 cell line. A significant

component of this cytotoxicity has previously been

shown to result from TNF-related apoptosis-inducing

ligand (TRAIL)/TRAIL-R interactions [30]. In the studies

described here, we did not determine whether this is a

general effect of IL-15 on all NKT cell subpopulations or a

result of IL-15-induced changes in the relative sizes of

NKT cell subpopulations. Previous studies have con-

firmed that all NKT subpopulations exert some cytotoxic

activity against U937 cells [8, 23, 26].

In summary, the use of IL-15 during in vitro stimulation of

NKT cells resulted in greatest proliferation and highest

cytotoxicity of NKT cells. The presence of IL-7 resulted in

the highest CD4–:CD4+ NKT cell ratio with the lowest Th2

cytokine production. A combination IL-7 and IL-15 in

combination with a-GalCer and Mo-DC may be optimal

for in vitro expansion of NKT cells for anti-tumor

applications, but additional purification procedures to

select for the desired subpopulation may be required.

Greater understanding of the effects of different cyto-

kines on NKT cell subpopulations is also likely to be

helpful in the design of vaccine or treatment strategies for

various diseases, utilizing the capacity of a-GalCer to

potently stimulate NKT cells.

4 Material and methods

4.1 NKT cell stimulation

Human adult PBMC were isolated from peripheral blood of

seven donors by density gradient centrifugation using Ficoll-

Paque (Amersham Biosciences). Informed consent was

obtained from donors before blood collection and the study

was approved by the Human Research Ethics Committee of

the Royal Brisbane Hospital and the Queensland Institute of

Medical Research. Monocytes from PBMC, obtained by 1-h

adherence in a tissue flask, were cultured in the presence of

500 U/ml of human recombinant IL-4 (R&D Systems) and

400 U/ml of human recombinant granulocyte-macrophage

colony stimulating factor (Schering-Plough) for 5 days to

produce Mo-DC used as CD1d-expressing APC. For

stimulation of NKT cells, PBMC were cultured with auto-

logous Mo-DC at a PBMC:DC ratio of 20:1 in AIM-V medium

(Gibco BRL) supplemented with 10% fetal calf serum (JRH

Biosciences) and 100 ng/ml a-GalCer (Kirin Brewery) for

7 days. To assess the effect of different cytokines on

NKT cells, human recombinant IL-2 (10 U/ml), IL-7 (10 ng/

ml), IL-12 (10 ng/ml) or IL-15 (10 ng/ml) (R&D Systems) was

added to the culture. A control without the addition of any

cytokines could not be established under our culture

condition as insufficient NKTexpansion occurred, precluding

accurate analysis of NKT cell subpopulations.

4.2 Flow cytometric analysis

Assessment of NKT cell subpopulations defined by CD4+ or

CD8+ surface molecule expression was performed using

Fig. 5. Cytotoxicity of NKT cells against U937 cells.

Representative data indicating the cytotoxicity of NKT cells

against U937 cells after stimulation with a-GalCer, auto-

logous Mo-DC and various cytokines as indicated. Percen-

tage of cell death was measured by Annexin V uptake of

U937 cells after 4-h incubation with NKT cells.



four-color flow cytometry (Coulter Epics XL) with the

following fluorochrome-conjugated monoclonal antibodies:

anti-Va24 (C15, IgG1), anti-Vb11 (C21, IgG2a), anti-CD4 (T4,

IgG1), anti-CD8 (B9.11, IgG1) (Beckman Coulter). Cytoplas-

mic IFN-c expression of NKT, NK and T cells was assessed

using five-color flow cytometry (Coulter Cytomics FC500)

with the following additional monoclonal antibodies: anti-

IFN-c (45.15, IgG1), anti-CD56 (N901, IgG1), anti-CD3

(UCHT1, IgG1). Since Va24+Vb11+ NKT cells represent a

very small fraction of PBMC, to ensure accuracy of flow

cytometric evaluation, up to 2�106 cells were assessed to

acquire >200 NKT cells. Mo-DC were identified as high-

forward, high-side scatter lineage-negative cells using anti-

CD3 (UCHT1, IgG1), anti-CD19 (B4, IgG1) and anti-CD14

(RMO52, IgG2a) (Beckman Coulter). These cells were then

assessed for CD40 (MAB89, IgG1), CD86 (HA5.2B7, IgG2b),

CD83 (HB15a, IgG2b) and CD1d (42.1) expression to confirm

their functional properties.

4.3 Assessment of cytokine production after in vitro

stimulation of NKT cells

Supernatants of cell cultures were collected on day 7 after

stimulation and stored at -80�C until cytokine assessment.

Production of IL-2, IL-4, IL-6, IL-10, TNF-a and IFN-ccytokines was determined using the Human Th1/Th2

Cytokine CBA II kit (BD Biosciences) according to the

manufacturer’s protocols.

4.4 Cell preparation for cytoplasmic IFN-c assessment

For intracellular staining of IFN-c, 5 lg/ml Brefeldin A

(Sigma-Aldrich) was added to culture media that contained

IL-12 and incubated overnight on day 6 of culture. This

culture condition was chosen as it showed the greatest

production of IFN-c determined by CBA assay. A positive

control was established by addition of 1 ng/ml of phorbol

myristate acetate (PMA) and 1 lM of ionomycin (all from

Sigma-Aldrich) and incubated overnight on day 6 of culture.

On day 7, cells were harvested and then prepared using the

Intraprep-Permeabilization reagent (Beckman Coulter) ac-

cording to the manufacturer’s protocols. Cells were then

analyzed on flow cytometer.

4.5 Assessment of the effect of NKT cell depletion on

IFN-c production

NKT cell-depleted population was obtained by removal of

the positively selected Va24+ cells from PBMC using

miniMACS (Miltenyi Biotec). These cells were cultured for

7 days and the supernatants of cell culture were collected on

day 7 and stored at –80�C until cytokine assessment.

Production of IFN-c was assessed using Human IFN-cOptEIA ELISA set (BD Biosciences) according to the

manufacturer’s protocols.

4.6 Assessment of cytotoxicity of NKT cells

Cytotoxic activity of stimulated NKT cells against the

NKT cell-sensitive tumor cell line U937 [30] was assessed

by flow cytometry using the Annexin V/7-aminoactino-

mycin D (7-AAD) kit (Beckman Coulter), an assay which

correlates well with the Cr-release assay [35]. Purified

NKT cells were obtained by positive selection of Va24+ cells

usingminiMACS (Miltenyi Biotec) on day 7 of stimulation and

were used as effector cells. Prior to cytotoxicity assays, U937

cells were labeled with PKH26 dye (Sigma) according to the

manufacturer’s protocol to ensure distinction between target

and effector cells during flow cytometric analysis. U937 cells

were co-cultured with NKT cells and incubated at 37�C and

5% CO2 for 4 h at E/T ratios of 20:1, 10:1 and 5:1. A negative

control containing U937 cells without effector cells was set-

up to take into account any spontaneous cell death during

the assay period. Following incubation, the U937 cells were

examined for cell death by 7-AAD uptake and early apoptosis

by Annexin V binding using the Annexin V/7-AAD kit. The

percentage cytotoxicity of the PKH26-labeled U937 cells

was calculated by subtracting Annexin V+ or 7-AAD+ target

cells, measured in appropriate controls without effector cells.

Acknowledgements: We would like to thank Kirin Brewery

for providing a-GalCer (KRN7000) for this research. This

study was supported by the Queensland Cancer Fund,

Suncorp Metway, and the Leukemia Foundation of Queens-

land.

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Correspondence: Henry Lin, Clive Berghofer Cancer Re-

search Centre, Queensland Institute of Medical Research,

300 Herston Rd, Herston 4029, Brisbane, Australia

Fax: +61-7-3845-3774

e-mail: [email protected]



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