Cutting Edge: IL-2−Induced Expression of

the Amino Acid Transporters SLC1A5 and

This information is current as
of October 4, 2019.
CD98 Is a Prerequisite for NKG2D-Mediated
Activation of Human NK Cells
Helle Jensen, Marc Potempa, Dagmar Gotthardt and Lewis
L. Lanier
J Immunol 2017; 199:1967-1972; Prepublished online 7
August 2017;
doi: 10.4049/jimmunol.1700497

Downloaded from http://www.jimmunol.org/ by guest on October 4, 2019

http://www.jimmunol.org/content/199/6/1967

Supplementary http://www.jimmunol.org/content/suppl/2017/08/05/jimmunol.170049
Material 7.DCSupplemental
References This article cites 22 articles, 6 of which you can access for free at:
http://www.jimmunol.org/content/199/6/1967.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision
• No Triage! Every submission reviewed by practicing scientists
• Fast Publication! 4 weeks from acceptance to publication
*average

Subscription Information about subscribing to The Journal of Immunology is online at:

http://jimmunol.org/subscription
Permissions Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Email Alerts Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by

The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2017 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.

Cutting Edge
Cutting Edge: IL-2–Induced Expression of the Amino Acid
Transporters SLC1A5 and CD98 Is a Prerequisite for
NKG2D-Mediated Activation of Human NK Cells
Helle Jensen,* Marc Potempa,* Dagmar Gotthardt,*,† and Lewis L. Lanier*,†
Priming of human NK cells with IL-2 is necessary to
render them functionally competent upon NKG2D en-
gagement. We examined the underlying mechanisms
that control NKG2D responsiveness in NK cells and
found that IL-2 upregulates expression of the amino
acid transporters SLC1A5 and CD98. Using specific
inhibitors to block SLC1A5 and CD98 function, we
found that production of IFN-g and degranulation by
CD56bright and CD56dim NK cells following NKG2D
stimulation were dependent on both transporters. IL-2
priming increased the activity of mTORC1, and inhi-
bition of mTORC1 abrogated the ability of the IL-2–
primed NK cells to produce IFN-g in response to
NKG2D-mediated stimulation. This study identifies
a series of IL-2–induced cellular changes that regulates
the NKG2D responsiveness in human NK cells. The
Journal of Immunology, 2017, 199: 1967–1972.
T
he activating receptor NKG2D, which is expressed
by NK cells, CD8+ T cells, subsets of CD4+ T cells,
invariant NKT cells, and gd T cells (1), recognizes a
diverse array of ligands belonging to the MHC class I chain–
related protein family (i.e., MICA and MICB) and the UL16-
binding protein family (i.e., ULBP1–6) (1). NKG2D ligands
are normally absent or expressed at low levels on healthy
resting cells, but they are induced in many stressed, virally
infected, or transformed cells (2). The importance of the
NKG2D receptor in immune surveillance is emphasized
by the evasion strategies cancers and viruses employ to prevent
the surface expression of NKG2D ligands (1). Moreover,
NKG2D is implicated in the development and/or progression
of autoimmune diseases, for example, rheumatoid arthritis,
diabetes, celiac disease, and Crohn’s disease (1). NKG2D
associates with the DAP10 adapter molecule, which is required
for cell surface expression of NKG2D and promotes signaling
through the PI3K and Grb2-Vav pathways to regulate NK cell–
*Department of Microbiology and Immunology, University of California, San Fran-
cisco, San Francisco, CA 94143; and †Parker Institute for Cancer Immunotherapy, San
Francisco, CA 94143
ORCIDs: 0000-0002-8416-1236 (D.G.); 0000-0003-1308-3952 (L.L.L).
Received for publication April 7, 2017. Accepted for publication July 20, 2017.
L.L.L. is an American Cancer Society Professor and is funded by National Institutes of
Health Grants AI066897 and AI068129 and by the Parker Institute for Cancer Immu-
notherapy. H.J. is supported by a Lundbeck Foundation postdoctoral fellowship.
Address correspondence and reprint requests to Dr. Lewis L. Lanier, Department of
Microbiology and Immunology, University of California, San Francisco, 513 Parnassus
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700497
The Journal of
Immunology
mediated cytotoxicity and cytokine production (2). However,
the functional outcome of NKG2D stimulation depends on the
activation state of the cells. NKG2D stimulation of freshly
isolated human peripheral blood NK cells alone is insufficient
to induce cytotoxicity and cytokine production even though the
surface expression of NKG2D and intracellular signaling ma-
chinery are present (3, 4). In contrast, NK cells primed by IL-2
Downloaded from http://www.jimmunol.org/ by guest on October 4, 2019
efficiently kill NKG2D ligand–expressing target cells and pro-
duce cytokines after NKG2D stimulation (5, 6). The mecha-
nisms responsible for the IL-2–mediated change in NKG2D
responsiveness remain undetermined.
Human NK cells are subdivided into two major subsets.
CD56bright NK cells are immature, whereas CD56dim NK cells
comprise a mature NK cell subset. CD56bright NK cells express the
high-affinity IL-2 receptor, IL-2Rabg, whereas resting CD56dim
NK cells lack IL-2Ra (7). When IL-2 binds to its receptor, JAK1
and JAK3 are phosphorylated, which recruit and activate STAT1,
STAT3, and STAT5. The IL-2R also signals through PI3K,
AKT, and the MAPK pathways (8). Gene expression profiling of
resting and IL-2–primed human NK cells has shown that IL-2
treatment upregulates expression of a wide range of genes (9). In
this study, we found that IL-2 priming of human NK cells leads
to a JAK3-dependent upregulation of the glutamine transporter
solute carrier family 1 member 5 (SLC1A5) (ASCT2) and the
amino acid transporter SLC3A2/SLC7A5 (CD98). Furthermore,
we show that the activity of these transporters and mammalian
target of rapamycin complex 1 (mTORC1) is essential for func-
tional activation of both CD56bright and CD56dim NK cells in
response to NKG2D stimulation.
Materials and Methods
Cells
PBMCs were isolated from blood obtained from the Blood Centers of the
Pacific under an Institutional Review Board–approved protocol (no. 10-
00265) by density gradient centrifugation using Ficoll-Paque Plus (GE
Healthcare Bio-Sciences). NK cells were purified (.90–95%) using EasySep
NK cell enrichment kits (Stemcell Technologies). NK cells were cultured in
RPMI 1640 medium (Corning Cellgro; Mediatech) containing 10% FBS
(Thermo Scientific), 13 MEM nonessential amino acids solution (Life
Avenue, HSE 1001G, Box 0414, San Francisco, CA 94143-0414. E-mail address: lewis.
lanier@ucsf.edu
The online version of this article contains supplemental material.
Abbreviations used in this article: CT, cycle threshold; GPNA, L-glutamic acid g-(p-
nitroanilide) hydrochloride; HPRT, hypoxanthine phosphoribosyltransferase; mTORC1,
mammalian target of rapamycin complex 1; RT, room temperature; SLC1A5, solute carrier
family 1 member 5.
Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00
1968 CUTTING EDGE: AMINO ACID TRANSPORTERS REGULATE NKG2D RESPONSIVENESS
Technologies), 1 mM sodium pyruvate (University of California San Fran-
cisco Cell Culture Facility), 2 mM L-glutamine (University of California San
Francisco Cell Culture Facility), penicillin (100 IU/ml), streptomycin (100
mg/ml) (Corning Cellgro), and 200 U/ml human rIL-2 (provided by Pro-
metheus Laboratories).
Plate-bound Ab stimulation of NK cells
NK cells were cultured in medium with or without 200 U/ml rIL-2 for 5, 10,
15, 20, or 24 h at 37˚C and 5% CO2 prior to stimulation. Where indicated,
the following inhibitors were added to cells 1 h prior to stimulation: 10 mM
L-glutamic acid g-(p-nitroanilide) hydrochloride (GPNA) (Santa Cruz Bio-
technology) (1 M GPNA solution was prepared in DMSO and kept at 37˚C
immediately prior to its addition), 100 mM D-phenylalanine (Sigma-Aldrich)
(dissolved in medium for 1 h at 37˚C by vortexing every 5–10 min prior to
addition), 100 nM rapamycin (Calbiochem) (109.4 mM rapamycin in
DMSO stock was stored at 220˚C, thawed, and diluted in medium prior to
addition), and 100 nM Torin-1 (ApexBio) (1.645 mM Torin-1 in DMSO
stock was stored at 280˚C, thawed, and diluted in medium before addition).
Nunc MaxiSorp ELISA plates (Thermo Fisher Scientific) were washed twice
with PBS and then coated with 5 mg/ml anti-NKG2D mAb (1D11; Bio-
Legend) or 5 mg/ml control mouse IgG1 (MOPC-21; University of California
San Francisco mAb Core Facility) in PBS for 24 h at 4˚C. After coating, the
plates were washed twice with PBS and blocked in complete medium for 10
min at room temperature (RT). For IFN-g measurements, GolgiSTOP was
added to cells immediately prior to stimulation. Stimulation of 1.5 3 105
cells per well proceeded for 5 h at 37˚C and 5% CO2.
Real-time PCR
NK cells were cultured in medium with or without 200 U/ml human rIL-2,
10 ng/ml human rIL-15 (R&D Systems), or 10 ng/ml human rIL-12 for
6 h. Where indicated, 0.05% DMSO or 0.5 mM Jak3i (provided by Dr. J.
Taunton, Dr. A. Weiss, and Dr. G. Smith) (1 mM Jak3i in DMSO
was stored at 220˚C, thawed, and diluted in medium prior to addition)
was added to the cells 2 h prior to treatment with cytokines. Total RNA
was purified using an RNeasy mini kit (Qiagen), and cDNA was generated
from 0.05 mg of RNA using the SuperScript III first-strand synthesis
system (Invitrogen). Real-time PCR was performed with the SYBR Green
master mix reagent (Invitrogen) using standard conditions (primer melting
temperature used, 55˚C) and the following primer pairs: SLC1A5 forward,
59-GTGTCCTCACTCTGGCCATC-39, reverse, 59-TACAGGACCG-
GTCGACTAGC-39; SLC7A5 forward, 59-TCATCATCCGGCCTTCA-
TCG-39, reverse, 59-GAGCAGCAGCACGCAGA-39; SLC3A2 forward,
59-AGCTGGAGTTTGTCTCAGGC-39, reverse, 59-GGCCAATCTCA-
TCCCCGTAG-39; and hypoxanthine phosphoribosyltransferase (HPRT)
forward, 59-GACCAGTCAACAGGGGACAT-39, reverse, 59-CTTG-
CGACCTTGACCATCTT-39. Samples were normalized to HPRT and
the relative expression of the glutamine transporters was determined using
the comparative cycle threshold (CT) (i.e., 22DDCT) method, using the
media sample as the reference sample.
Flow cytometry
mAbs used for cell surface staining were: FITC–anti-CD98 (MEM-108;
BioLegend), FITC–anti-CD56 (HCD56; BioLegend), PerCP-Cy5.5–anti-
CD56 (HCD56; BioLegend), Alexa Fluor 700–anti-CD3 (HIT3a; Bio-
Legend), and allophycocyanin–anti-NKG2D (149810; R&D Systems). mAbs
used for intracellular staining were: PE–anti-pS6 (D57.2.2E; Cell Signaling
Technology), Alexa Fluor 647–anti-pSTAT5 [47/Stat5(pY694); BD Biosci-
ences], Alexa Fluor 647–anti-IFN-g, anti-ASCT2 (SLC1A5) (D7C12; Cell
Signaling Technology), and Alexa Fluor 647–conjugated goat anti-rabbit IgG
(A-21245; Invitrogen). For surface staining, cells were incubated with the
indicated mAbs or isotype-matched control Abs (BioLegend) for 20 min on
ice. During the pSTAT5 and pS6 measurements, the Alexa Fluor 700–anti-
CD3 and FITC–anti-CD56 were added to the cell culture 15 min prior to
fixation in 1.5% paraformaldehyde in PBS. For intracellular IFN-g staining,
cells were fixed and permeabilized using the Cytofix/Cytoperm kit (BD
Biosciences) and subsequently stained with Abs for 20 min on ice. For in-
tracellular staining of pS6 and pSTAT5, cells were fixed in 1.5% parafor-
maldehyde in PBS for 10 min at RT and permeabilized in cold 100%
methanol for 20 min on ice. Samples were either stored overnight at 220˚C
in methanol before staining or stained immediately following permeabiliza-
tion with the anti-pS6 and anti-pSTAT5 for 30 min at RT. For intracellular
staining of SLC1A5, cells were fixed in 1.5% paraformaldehyde in PBS for 10
min at RT, permeabilized in cold 100% methanol for 20 min on ice, and
then stained with anti-SLC1A5 for 45 min at RT, followed by staining with
anti-rabbit IgG for 20 min at RT. Samples were acquired on an LSR II (BD
Biosciences) and analyzed with FlowJo software (Tree Star). Live single cells
were gated based on forward and side light scatter profiles. NK cells were
gated as CD32CD56bright or CD32CD56dim.
Results and Discussion
Maximal IFN-g production by NK cells in response to NKG2D
stimulation requires IL-2 priming
IL-2 priming renders human NK cells able to produce IFN-g
in response to NKG2D stimulation (5, 6). To determine the
kinetics of IL-2 priming needed to induce NKG2D respon-
siveness, we cultured freshly purified peripheral blood NK
cells in medium with or without IL-2 for 5, 10, 15, 20, or
24 h. Cells were then stimulated with plate-bound anti-
NKG2D for 5 h and IFN-g production was measured by
flow cytometry. Few CD56bright NK cells and even fewer
CD56dim NK cells produced IFN-g after only 10 h of IL-2
priming (Fig. 1A). After 15 h of IL-2 priming, intermediate
levels of IFN-g+ cells were detected from both subsets,
whereas peak IFN-g production required 20–24 h priming
(Fig. 1A). Although the relationship between priming time
Downloaded from http://www.jimmunol.org/ by guest on October 4, 2019
and IFN-g production was consistent between donors, donor
variability was observed in the percentages of IFN-g+ cells at
24 h, ranging from 21 to 35% in CD56bright NK cells and
from 8 to 33% in CD56dim NK cells (Fig. 1B). These results
indicate that extended IL-2 priming of human NK cells is
required for robust IFN-g production in response to NKG2D
stimulation, suggesting that IL-2–induced synthesis of new
proteins might be necessary.
IL-2 priming increases the expression of SLC1A5 and CD98 in
NK cells
Searching the National Center for Biotechnology Information
Gene Expression Omnibus database (10) accession number
GSE8059 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?
acc=GSE8059) to identify genes differentially expressed in
human NK cells after IL-2 priming (9), we found three genes
of interest: SLC1A5, which encodes a high affinity L-gluta-
mine transporter (also designated ASCT2) (11), and SLC7A5,
which together with SLC3A2 encodes a heterodimeric bidi-
rectional antiporter (collectively designated CD98) that reg-
ulates the exchange of intracellular L -glutamine with
extracellular amino acids such as L-leucine, L-phenylalanine,
and L-tryptophan (11, 12). Consistent with the published
dataset (9), we observed an increase in the SLC1A5 mRNA
levels in NK cells after IL-2 priming (Fig. 2A). We also ob-
served an increase in expression level of SLC7A5 after IL-2
priming, whereas no change was observed for SLC3A2 (Fig.
2A). However, SLC3A2 was expressed at a high level in
resting NK cells compared with the housekeeping gene
HPRT, in contrast to the low basal levels of SLC1A5 and
SLC7A5 (Fig. 2B). Treatment with IL-15, which signals
through components of the IL-2 receptor (13), similarly in-
creased the expression of SLC1A5 and SLC7A5, and to a
lower extent SLC3A2 (Supplemental Fig. 1A). Treatment of
NK cells with IL-12, which stimulates IFN-g production by
NK cells (14), also increased the expression of SLC1A5,
SLC7A5, and SLC3A2 (Supplemental Fig. 1A), although to a
lesser degree than IL-15 and IL-2. IL-2 treatment leads to
phosphorylation of STAT5 (pSTAT5), and the level of
phosphorylation remained high throughout the course of IL-2
treatment (Supplemental Fig. 1B). We used a selective cova-
lent inhibitor of JAK3 (15), which completely blocks the
The Journal of Immunology 1969

FIGURE 1. Maximal IFN-g production by NK cells in response to NKG2D stimulation requires IL-2 priming. NK cells were enriched from PBMCs and
cultured in medium with or without 200 U/ml IL-2 for the indicated times. Cells were stimulated with plate-bound Abs for 5 h and IFN-g production was
measured in CD56bright and CD56dim NK cells by flow cytometry. (A) Results are shown for one donor and are representative of four donors. (B) Data show
mean 6 SD from four donors. Statistical analysis was performed by a two-tailed unpaired Student t test. *p , 0.05, ***p , 0.001.

phosphorylation of STAT5 in IL-2–primed NK cells
(Supplemental Fig. 1C), to examine its role in the IL-2–in-
duced expression of SLC1A5 and SLC7A5. The induction of
SLC1A5 and SLC7A5 expression in the IL-2–primed NK
whereas the high basal expression of SLC3A2 was unaffected
(Fig. 2A). Expression of both SLC1A5 and CD98 protein was
increased in CD56bright and CD56dim NK cells after IL-2
priming (Fig. 2C–F), with the highest increase being ob-
served in CD56bright NK cells. Thus, IL-2 priming of human

Downloaded from http://www.jimmunol.org/ by guest on October 4, 2019

cells was abrogated after treatment with the JAK3 inhibitor,

FIGURE 2. IL-2 priming increases the expression of SLC1A5 and CD98 in NK cells. (A and B) NK cells were enriched from PBMCs and treated with DMSO
(0.05%) or 500 nM Jak3i for 2 h. Cells were then cultured in medium with or without 200 U/ml IL-2 for an additional 6 h. RNA was isolated and mRNA
expression was analyzed by real-time PCR. (A) Fold change of mRNA expression relative to the DMSO sample of the medium treated cells, calculated by DDCT
method, from three donors is shown. (B) Fold change of mRNA expression relative to the HPRT sample from three donors is shown. (C–F) Enriched NK cells
were cultured in medium with or without 200 U/ml IL-2 for 24 h. (C and D) The intracellular SLC1A5 protein level and (E and F) surface expression of CD98 on
CD56bright and CD56dim NK cells was measured by flow cytometry. (C and E) Results are shown for one donor and are representative of four donors. (D and F)
Data show fold change in mean fluorescence intensity (MFI) 6 SD relative to the average MFI of the medium-treated samples from four donors. Statistical
analysis was performed by a two-tailed unpaired Student t test. *p , 0.05, **p , 0.01, ***p , 0.001.
1970 CUTTING EDGE: AMINO ACID TRANSPORTERS REGULATE NKG2D RESPONSIVENESS

FIGURE 3. Inhibition of SLC1A5 and CD98 abrogates the NKG2D-mediated effector functions of IL-2–primed NK cells. NK cells enriched from PBMCs
were primed with 200 U/ml IL-2 for 24 h. Cells were treated with medium, DMSO (1%), 100 mM D-phenylalanine, or 10 mM GPNA for 1 h prior to
stimulation with plate-bound anti-NKG2D Abs for 5 h. IFN-g production was measured in CD56bright and CD56dim NK cells by flow cytometry. (A) Results are
shown for one donor and are representative of four donors. (B) Data show mean 6 SD from four donors. Statistical analysis was performed by a two-tailed

Downloaded from http://www.jimmunol.org/ by guest on October 4, 2019

unpaired Student t test. *p , 0.05, **p , 0.01.

NK cells induces expression of the amino acid transporters
SLC1A5 and CD98 at the mRNA and protein level.
Inhibition of SLC1A5 and CD98 abrogates the NKG2D-mediated
effector functions of IL-2–primed NK cells
We treated IL-2–primed NK cells with inhibitors against
SLC1A5 (i.e., GPNA) (12) or CD98 (i.e., D-phenylalanine)
(12) for 1 h prior to NKG2D stimulation to determine their
involvement in the NKG2D-induced production of IFN-g.
Pretreatment of IL-2–primed CD56bright and CD56dim NK
cells with either inhibitor completely blocked the NK cells
ability to produce IFN-g after NKG2D stimulation (Fig. 3).
Furthermore, blocking of SLC1A5 and CD98 activity de-
creased the NKG2D-induced degranulation by the IL-2–
primed NK cells (Supplemental Fig. 2A). The lack of re-
sponsiveness was not due to a change in the surface level of
NKG2D, as comparable levels were observed between the
control-treated and inhibitor-treated cells (Supplemental Fig.
2B). These results indicate that the activities of SLC1A5 and
CD98 play an important role in NKG2D-mediated IFN-g
production and degranulation.
IL-2 priming of NK cells increases S6 phosphorylation
Uptake of L-glutamine by SLC1A5 closely followed by export
of that L-glutamine in exchange for essential amino acids by
CD98 activates mTORC1 (12). To determine whether in-
creased expression of SLC1A5 and CD98 activates mTORC1
in the IL-2–primed NK cells, we measured the phosphory-
lation of S6, which is a downstream target of the mTORC1
pathway (16), by flow cytometry. pS6 was detected in both
CD56bright and CD56dim NK cells early after IL-2 treatment,
and the pS6 levels continued to increase during the 24-h
treatment period (Fig. 4). Treatment of the IL-2–primed
(24 h) CD56bright and CD56dim NK cells with GPNA or
D-phenylalanine almost completely abrogated phosphoryla-
tion of S6, whereas it only minimally affected phosphoryla-
tion of STAT5 (Supplemental Fig. 2C), confirming the
essential roles of SLC1A5 and CD98 in S6 phosphorylation at
this latter time point.
Inhibition of mTORC1 activity abrogates NKG2D-induced
IFN-g production
mTORC1 is a critical regulator of mRNA translation (16) and
controls IFN-g production by mouse NK cells in vivo after
polyinosinic-polycytidylic acid injection (17) and in vitro by
IL-15–primed mouse NK cells stimulated with IL-12 or
plate-bound Abs against the Ly49H (18). To examine its role
in NKG2D-mediated IFN-g production, we treated IL-2–
primed human NK cells with inhibitors against mTORC1
(i.e., Torin-1 and rapamycin) (19) for 1 h prior to stimula-
tion. Treatment of the IL-2–primed CD56bright and CD56dim
NK cells with either inhibitor significantly decreased IFN-g
production (Fig. 5). However, inhibition of mTORC1 did
not affect NK cell degranulation after NKG2D stimulation

FIGURE 4. IL-2 priming of NK cells in-

creases S6 phosphorylation. NK cells enriched
from PBMCs were cultured in medium with
or without 200 U/ml IL-2 for the indicated
times. The intracellular level of pS6 was
measured in CD56bright and CD56dim NK
cells by flow cytometry. Results are shown for
one donor and are representative of three
donors.
The Journal of Immunology
FIGURE 5. Inhibition of mTORC1 activity abrogates NKG2D-induced IFN-g production. NK cells enriched from human PBMCs were primed with 200 U/
ml IL-2 for 24 h. Cells were treated with medium, 100 nM rapamycin, or 100 nM Torin-1 for 1 h prior to stimulation with plate-bound anti-NKG2D Abs for
5 h. IFN-g production was measured in CD56bright and CD56dim NK cells by flow cytometry. (A) Results are shown for one donor and are representative of three
donors. (B) Data show mean 6 SD from three donors. Statistical analysis was performed by a two-tailed unpaired Student t test. *p , 0.05.
(Supplemental Fig. 2A). The lack of responsiveness was again
not due to a change in the surface level of NKG2D, as
comparable levels were observed between the control-treated
and inhibitor-treated cells (Supplemental Fig. 2B). Taken to-
gether, these results highlight the essential role of mTORC1
in the NKG2D-mediated IFN-g production by IL-2–primed
NK cells, but not the degranulation.
In this study we show that IL-2 priming of CD56bright and
CD56 dim NK cells leads to an upregulation of the amino
acid transporters SLC1A5 and CD98 and an increase in
mTORC1 activity. The rate-limiting step in mTORC1 acti-
vation by the bidirectional transport of L-glutamine has been
shown to be the import of L-glutamine through SLC1A5
(12). The transport of L-glutamine out of the cells through
CD98 appears to occur rapidly in the presence of essential
amino acids (12), which makes it difficult to the measure the
intracellular levels of L-glutamine in IL-2–primed NK cells
under physiological conditions. Thus, it remains to be de-
termined whether the import of L-glutamine by SLC1A5 or
its export by CD98 prevails in regulating the mTORC1 ac-
tivation in IL-2–primed NK cells.
In summary, our findings reveal a crucial role for SLC1A5
and CD98 in controlling NKG2D responsiveness in human
NK cells. Blocking either of the transporters completely ab-
rogated the NKG2D-mediated IFN-g production by the IL-
2–primed NK cells. Furthermore, activation of mTORC1 by
the transporters was found to be critical for the ability of the
IL-2–primed NK cells to produce IFN-g after NKG2D
stimulation. In contrast to IFN-g production, mTORC1
activity was not essential for NKG2D-mediated degranula-
tion. The presence of lytic granules in many human NK cells
is independent of the activation status (20), likely accounting
for the continued ability of IL-2–primed NK cells to degra-
nulate in the absence of mTORC1-promoted protein syn-
thesis. Higher intracellular concentrations of L-leucine or L-
arginine has been shown to cause the exchange of GDP for
GTP among Rag GTPases, thereby promoting translocation
of mTORC1 to the lysosomal membranes and subsequent
activation by the GTPase Rheb (21). Further characterization
of the precise mechanism that regulates the mTORC1 activity
1971
Downloaded from http://www.jimmunol.org/ by guest on October 4, 2019
in the IL-2–primed NK cells and whether additional
glutamine-dependent pathways are involved will guide us
toward a more comprehensive understanding of how amino
acid transport regulates the NKG2D responsiveness in NK
cells. A recent study by Nakaya et al. (22) reported that
SLC1A5 and CD98 activity was required for activation of
mTORC1 in mouse T cells following stimulation, and the
absence of SLC1A5 resulted in fewer IFN-g–producing
T cells after Listeria monocytogenes infection (22). Our data
suggest a similar linkage between L-glutamine transport,
mTORC1 activation, and IFN-g production in human NK
cells and thus provide novel insight into the cellular changes
caused by IL-2 priming that controls the NKG2D respon-
siveness in NK cells.
Acknowledgments
We thank the Lanier laboratory for comments and discussions, Prome-
theus Laboratories for providing human IL-2, and Dr. Jack Taunton,
Dr. Art Weiss, and Dr. Geoff Smith for providing the Jak3 inhibitor.
Disclosures
L.L.L. and the University of California, San Francisco have licensed in-
tellectual property rights regarding NKG2D for commercial applications.
The other authors have no financial conflicts of interest.
References
1. Lanier, L. L. 2015. NKG2D receptor and its ligands in host defense. Cancer
Immunol. Res. 3: 575–582.
2. Champsaur, M., and L. L. Lanier. 2010. Effect of NKG2D ligand expression on
host immune responses. Immunol. Rev. 235: 267–285.
3. Bryceson, Y. T., M. E. March, H.G. Ljunggren, and E. O. Long. 2006. Synergy
among receptors on resting NK cells for the activation of natural cytotoxicity and
cytokine secretion. Blood 107: 159–166.
4. Fauriat, C., E. O. Long, H.G. Ljunggren, and Y. T. Bryceson. 2010. Regulation of
human NK-cell cytokine and chemokine production by target cell recognition.
Blood 115: 2167–2176.
5. Bauer, S., V. Groh, J. Wu, A. Steinle, J. H. Phillips, L. L. Lanier, and T. Spies.
1999. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible
MICA. Science 285: 727–729.
6. Al-Hubeshy, Z. B., A. Coleman, M. Nelson, and M. R. Goodier. 2011. A rapid
method for assessment of natural killer cell function after multiple receptor cross-
linking. J. Immunol. Methods 366: 52–59.
7. Cooper, M. A., T. A. Fehniger, and M. A. Caligiuri. 2001. The biology of human
natural killer-cell subsets. Trends Immunol. 22: 633–640.
1972 CUTTING EDGE: AMINO ACID TRANSPORTERS REGULATE NKG2D RESPONSIVENESS
8. Boyman, O., and J. Sprent. 2012. The role of interleukin-2 during homeostasis and
activation of the immune system. Nat. Rev. Immunol. 12: 180–190.
9. Dybkaer, K., J. Iqbal, G. Zhou, H. Geng, L. Xiao, A. Schmitz, F. d’Amore, and
W. C. Chan. 2007. Genome wide transcriptional analysis of resting and IL2 acti-
vated human natural killer cells: gene expression signatures indicative of novel
molecular signaling pathways. BMC Genomics 8: 230.
10. Edgar, R., M. Domrachev, and A. E. Lash. 2002. Gene expression omnibus: NCBI
gene expression and hybridization array data repository. Nucleic Acids Res. 30: 207–
210.
11. Pochini, L., M. Scalise, M. Galluccio, and C. Indiveri. 2014. Membrane trans-
porters for the special amino acid glutamine: structure/function relationships and
relevance to human health. Front Chem. 2: 61.
12. Nicklin, P., P. Bergman, B. Zhang, E. Triantafellow, H. Wang, B. Nyfeler,
H. Yang, M. Hild, C. Kung, C. Wilson, et al. 2009. Bidirectional transport of
amino acids regulates mTOR and autophagy. Cell 136: 521–534.
13. Carson, W. E., J. G. Giri, M. J. Lindemann, M. L. Linett, M. Ahdieh, R. Paxton,
D. Anderson, J. Eisenmann, K. Grabstein, and M. A. Caligiuri. 1994. Interleukin
(IL) 15 is a novel cytokine that activates human natural killer cells via components
of the IL-2 receptor. J. Exp. Med. 180: 1395–1403.
14. Gately, M. K., L. M. Renzetti, J. Magram, A. S. Stern, L. Adorini, U. Gubler, and
D. H. Presky. 1998. The interleukin-12/interleukin-12-receptor system: role in
normal and pathologic immune responses. Annu. Rev. Immunol. 16: 495–521.
15. Smith, G. A., K. Uchida, A. Weiss, and J. Taunton. 2016. Essential biphasic role for
JAK3 catalytic activity in IL-2 receptor signaling. Nat. Chem. Biol. 12: 373–379.
16. Holz, M. K., B. A. Ballif, S. P. Gygi, and J. Blenis. 2005. mTOR and S6K1 mediate
assembly of the translation preinitiation complex through dynamic protein inter-
change and ordered phosphorylation events. Cell 123: 569–580.
17. Donnelly, R. P., R. M. Loftus, S. E. Keating, K. T. Liou, C. A. Biron,
C. M. Gardiner, and D. K. Finlay. 2014. mTORC1-dependent metabolic
reprogramming is a prerequisite for NK cell effector function. J. Immunol. 193:
4477–4484.
18. Nandagopal, N., A. K. Ali, A. K. Komal, and S.H. Lee. 2014. The critical role of
IL-15–PI3K–mTOR pathway in natural killer cell effector functions. Front.
Immunol. 5: 187.
19. Thoreen, C. C., and D. M. Sabatini. 2009. Rapamycin inhibits mTORC1, but not
completely. Autophagy 5: 725–726.
20. Orange, J. S. 2008. Formation and function of the lytic NK-cell immunological
synapse. Nat. Rev. Immunol. 8: 713–725.
21. Dibble, C. C., and B. D. Manning. 2013. Signal integration by mTORC1 coor-
dinates nutrient input with biosynthetic output. Nat. Cell Biol. 15: 555–564.
22. Nakaya, M., Y. Xiao, X. Zhou, J.H. Chang, M. Chang, X. Cheng, M. Blonska,
X. Lin, and S.C. Sun. 2014. Inflammatory T cell responses rely on amino acid
transporter ASCT2 facilitation of glutamine uptake and mTORC1 kinase activa-
tion. Immunity 40: 692–705.
Downloaded from http://www.jimmunol.org/ by guest on October 4, 2019
Figure S1
A SLC1A5 SLC7A5 SLC3A2
15 5 **** 2.0 ***
mRNA expression

*** * ***
4
[fold change]

** 1.5
10
3
1.0
2
5
1 0.5

0 0 0.0
m

5
-1

-1

-1

-1

-1

-1
iu

iu

iu
IL

IL

IL

IL

IL

IL
ed

ed

ed
m

m
B
CD56bright CD56dim
14 14
[fold change in MFI]

[fold change in MFI]

12 *** 12
10 10
pSTAT5

pSTAT5

8 8 **
6 6
4 4
2 2
0 0
15 -2

30 in
in

15 -2

30 in
in
hr
hr

hr

hr

hr
hr

hr

hr
m

m
IL

IL
1
2

4
24

1
2

4
24
o

o
N

C
CD56 bright CD56 dim
Jak3i, IL-2
DMSO, IL-2
Jak3i
DMSO
Cell #

control stain

pSTAT5

Figure S1. IL-2 priming activates phosphorylation of

STAT5 in a Jak3 dependent manner. (A) NK cells were
enriched from PBMCs and cultured in medium with or without
10 ng/ml IL-12 or 10 ng/ml IL-15 for 6h. RNA was isolated from
the cells and used for real-time PCR analysis. The graphs show
fold change of mRNA expression relative to the medium-treated
cells, calculated by ΔΔCT method, from three donors. (B) Enriched
NK cells were cultured in medium or primed with 200 U/ml IL-2 for the
indicated time-points. The intracellular level of phosphorylated
STAT5 (pSTAT5) was measured in CD56 bright and CD56 dim NK
cells by flow cytometry. Data show fold change in MFI ± s.d.
relative to the medium-treated cells from the same time-point
from three donors. (C) Enriched NK cells were treated with DMSO
(0.05%) or 500 nM Jak3i for 2h. The cells were then kept in
culture medium with or without 200 U/ml IL-2 for an additional 15 min.
The intracellular level of pSTAT5 was measured in CD56 bright and
CD56 dim NK cells by flow cytometry. Data show results from
one donor and are representative of three donors. Statistical
analysis was performed by 2-tailed unpaired student’s t-test
(*p<0.05, **p<0.01, ***p< 0.001, ****p<0.0001).
Figure S2
A
medium DMSO GPNA D-phenylalanine Torin-1 rapamycin
77.7% 66.9% 16.3% 5.8% 67.0% 60.2%

bright
CD56

36.0% 24.1% 4.7% 1.4% 24.5% 24.9%

dim
CD56
CD107a

Ssc

B C CD56bright CD56dim
CD56bright CD56dim
medium
IL-2
IL-2 + D-phenylalanine
IL-2 + GPNA
Cell #
Cell #

NKG2D pS6

medium
DMSO
D-phenylalanine
Cell #

GPNA
rapamycin
Torin-1

pSTAT5

Figure S2. Inhibition of SLC1A5 or CD98 activity abrogates NKG2D-mediated degranulation by

IL-2 primed NK cells and alters the intracellular level of pS6, but not pSTAT5 or the cell surface
level of NKG2D. (A, B) NK cells were enriched from PBMCs and primed with 200 U/ml IL-2 for 24 h. The
cells were treated with medium, DMSO (1%), 100 mM D-phenylalanine, 10 mM GPNA, 100 nM
rapamycin, or 100 nM Torin-1 for 1 h prior to stimulation with plate-bound Abs for 5 h. (A) IFNγ production or
(B) NKG2D cell surface expression was measured on CD56 bright and CD56 dim NK cells by flow cytometry.
Data show results from one donor and are representative of (A) four or (B) two donors. (C) Enriched NK
cells were cultured in medium or primed with 200 U/ml IL-2 for 24 h. The cells were left untreated or treated
with 100 mM D-phenylalanine or 10 mM GPNA for 1 h and the intracellular level of phosphorylated S6 (pS6)
and STAT5 (pSTAT5) was measured in CD56 bright and CD56 dim NK cells by flow cytometry. Data show
results from one donor and are representative of two donors.