Type II NKT cells facilitate Alum-sensing and
Hemangi B. Shah, T. Scott Devera, Pragya Rampuria, Gillian A. Lang, and Mark L. Lang1
Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City,
RECEIVED APRIL 3, 2012; REVISED JUNE 7, 2012; ACCEPTED JUNE 26, 2012. DOI: 10.1189/jlb.0412177
Alum-based adjuvants facilitate vaccine-driven humoral
immunity, but their mechanism of action remains poorly
understood. Herein, we report that lack of type II NKT
cells is associated with intact, mature B cells but
dampened humoral immunity following immunization
with Alum-adsorbed T-dependent antigen. Type II
NKT cells facilitated production of IL-4, IL-5, IL-10, IL-
13, and antibody by LN and splenocyte cultures fol-
lowing Alum/antigen administration in vivo and anti-
gen restimulation in vitro. Addition of IL-4 and IL-5 to
type II NKT-deficient cultures restored in vitro anti-
body production. Intracellular staining revealed that
Alum-primed type II NKT cells coordinated IL-4 secre-
tion by T cells. Alum did not significantly affect CD1d
expression in vivo, but addition of CD1d-blocking mAb
diminished cytokine production and in vitro antibody
production. Type II NKT cells therefore function as
part of the Alum-sensing apparatus and in a CD1d-de-
pendent manner, facilitate TH2-driven humoral immu-
nity. This may have important consequences for un-
derstanding the mechanism of action of Alum-con-
taining vaccines. J. Leukoc. Biol. 92: 883–893; 2012.
The adjuvant properties of aluminum salts (referred to herein
as Alum) were first discovered in 1926, when it was demon-
strated that antigen precipitated onto aluminum potassium
sulfate produced better antibody responses than antigen alone
. Following this discovery, Alum has been used successfully
in several vaccines.
Alum was originally proposed to function as an antigenic
depot, slowly releasing antigen from the immunization site
and prolonging stimulation of the immune system. However,
removal of the Alum nodule did not compromise immunity
. More recently, it was reported that Alum-induced anti-
body responses required caspase 1-dependent activation of the
inflammasome . Direct inflammasome activation required
engulfment of Alum by phagocytic cells, whereas indirect acti-
vation required phagocytosis of DAMPs released by Alum-dam-
aged cells . Indeed, DAMPs, such as uric acid , which
when coinjected with T-dependent antigen, significantly en-
hanced adaptive immunity, including antigen-specific antibody
responses [6, 7]. Inflammasome-independent immune re-
sponses may occur when DAMPs stimulate signaling in DCs,
which enhances their interaction with Th cells [8, 9]. Thus,
results published so far strongly indicate that Alum-driven anti-
body responses operate through inflammasome- and uric acid-
dependent mechanisms [4, 8].
Alum administration enhances antigen presentation and up-
regulates expression of costimulatory molecules on APCs .
Immunization with Alum results in rapid recruitment of sev-
eral cell types, including monocytes, eosinophils, neutrophils,
NK cells, NKT cells, and DCs, to the site of immunization
. Recruited monocytes acquire antigen, traffic to the
draining LNs, and differentiate into CD11c?DCs that prime
naïve CD4?T cells, resulting in a TH2 response . Recruited
myeloid cells can also secrete IL-4 and promote B cell activa-
tion [11, 12]. These effects ultimately lead to a TH2 response,
which includes IL-4 secretion, B cell differentiation, and a ro-
bust antibody response .
As NKT cells accumulate at the site of Alum immunization
, and activated NKT cells can promote TH2 responses, we
hypothesized that NKT cells potentiate the adjuvant properties
of Alum. Type I NKT cells express a semi-invariant TCR, com-
bining a canonical V?14-J?18 rearrangement  with the
V?8.2, -7, or -2 ?-chains in mice [15, 16]. The TCR on type I
1. Correspondence: Dept. of Microbiology and Immunology, OUHSC, 940
Stanton L. Young Blvd., BMSB 1019, Oklahoma City, OK 73104, USA.
Abbreviations: ?-GC??-galactosylceramide, ASC?antibody-secreting cell,
CD1d?/?mice?cross between CD1d-deficient and C57Bl/6 mice,
associated molecular pattern, KLH?keyhole limpet hemocyanin,
NKT?NK T cell, NLRP3?nucleotide-binding domain and leucine-rich re-
peat-containing gene family, pyrin domain containing 3, NP?4-hydroxy-
hole limpet hemocyanin, adsorbed to Alum, OUHSC?The University of
Oklahoma Health Sciences Center, SNP?single nucleotide polymor-
phism, sulfatide?3-sulfated galactosylceramide, TIM?T cell Ig domain
and mucin domain
?/?/J?18?/?mice?CD1d/J?18 knockout mice, DAMP?damage-
The online version of this paper, found at www.jleukbio.org, includes
0741-5400/12/0092-883 © Society for Leukocyte Biology
Volume 92, October 2012
Journal of Leukocyte Biology 883
NKT cells recognizes a CD1d-binding ?-GC glycolipid mole-
cule expressed on APCs . CD1d/glycolipid and TCR inter-
actions facilitate activation of NKT cells leading to regulation
of antimicrobial and tumor immunity, autoimmunity, and self-
Type II NKT cells are CD1d-restricted, nonreactive with
?-GC, and express a variable TCR . A subset of type II
NKT cells recognizes lipids, including sulfatide from the my-
elin sheath . Type II NKT cells play a protective role in
autoimmune diabetes, experimental autoimmune encephalo-
myelitis, and Con A-induced hepatitis [20–22] and can sup-
press tumor immuno-surveillance . In contrast to type I
NKT cells, which are known to mediate enhanced antibody
responses against foreign antigen , no such role for type II
NKT cells has been described.
In this study, we used inbred mouse strains deficient in NKT
cells to determine the contribution of types I and II NKT cells
to Alum-induced humoral immune responses. In vivo and ex
vivo assays were used to demonstrate that type II rather than
type I NKT cells were required for optimal antibody responses
to Alum-adsorbed antigen. We have therefore uncovered an
important part of the mechanism by which the immune system
responds to Alum and believe that this will contribute to the
understanding of vaccine-induced immunity.
MATERIALS AND METHODS
Female C57Bl/6 mice and C57Bl/6-CD45.1 congenic mice were purchased
from the National Cancer Institute (Bethesda, MD, USA). CD1d?/?mice
 and J?18?/?mice  were provided by Dr. Mark Exley (Harvard
Medical School, Boston, MA, USA) and Dr. Mitchell Kronenberg (La Jolla
Institute for Allergy and Immunology, La Jolla, CA, USA), respectively, and
were bred in OUHSC (Oklahoma City, OK, USA)-specific pathogen-free
animal resources facility. CD1d?/?mice used in this study were the F1 lit-
ter, generated from a cross between CD1d?/?and C57Bl/6 mice.
CD1d?/?mice had been backcrossed on to the C57Bl/6 genetic back-
ground for at least 12 generations before receipt by our laboratory. This
was confirmed by independent genome-wide analysis of SNP (DartMouse;
Dartmouth Medical School, Lebanon, NH, USA). SNP analysis of J?18?/?
mice showed a predominant C57Bl/6 background (?82%) with the re-
mainder of the SNPs showing a 129 genetic background. This variation was
not associated with changes in the biological responses measured in the
current study (as compared with C57Bl/6 controls). Animals purchased
were shipped from the supplier’s specific pathogen-free facility directly to
the same room in the OUHSC facility as the in-house mice. Purchased
mice were allowed to acclimatize under the same environmental conditions
as the in-house mice for 1–2 weeks before starting experiments. All studies
were approved by the OUHSC Institutional Animal Care and Use Commit-
The following reagents were purchased: NP hapten-conjugated KLH and
NP-conjugated BSA (Biosearch Technologies, Novato, CA, USA); OVA
(ICN Biomedicals, Aurora, OH, USA); Imject-Alum (aluminum hydroxide
and magnesium hydroxide suspension; Pierce Biotechnology, Rockford, IL,
USA); FcR-blocking 2.4G2 mAb and rat IgG1 (Bio X Cell, West Lebanon,
NH, USA); fluorochrome-conjugated mAb (eBioscience, San Diego, CA,
USA, and BD Biosciences, San Diego, CA, USA); mouse Ig molecules and
HRP-conjugated anti-Ig mAb (Southern Biotech, Birmingham, AL, USA);
LPS (Invivogen, San Diego, CA, USA); and PMA, ionomycin, and Brefeldin
A (Sigma-Aldrich, St. Louis, MO, USA). The following mAb clones were
used in the study: allophycocyanin-conjugated anti-NK1.1 (PK136), PE-anti-
CD93 (AA4.1), FITC-anti-CD45R/B220 (RA3-6B2), and PE-anti-IL-4
(11B11) and PE-rat IgG1 mAb, purchased from eBioscience. FITC-anti-
TCR-? (H57-597) and PE-anti-IFN-? (XMG1.2) were purchased from BD
Biosciences. The CD1d-specific 20H2 mAb is nonagonistic and blocks
CD1d-dependent activation of NKT hybridomas . The 20H2 mAb was
purified from hybridoma supernatants as described previously .
Immunizations and bleeds
Mice were anaesthetized by inhalation using a 4% isoflurane/96% O2mixture.
Seven- to 8-week-old mice were immunized with 10 ?g NP-KLH/Alum. NP-KLH
(10 ?g) was mixed with 100 ?l PBS and 100 ?l Alum, stirred at room tempera-
ture for 30 min, and injected via the s.c. route (divided equally over both flanks).
A booster vaccine consisting of 10 ?g NP-KLH in 200 ?l PBS was administered
s.c., 28 days later. Heparinized capillary tubes were used to collect 100 ?l blood
retro-orbitally. Capillary tubes were transferred into microcentrifuge tubes. The
blood samples were allowed to clot and incubated overnight at 4°C. The samples
were then centrifuged at 13,000 g for 15 min, and sera were collected using pi-
pettes and stored in aliquots at ?20°C.
Ninety-six well plates (Nunc, Rochester, NY, USA) were coated with NP-
BSA at 10 ?g/mL or goat anti-mouse Ig at 5 ?g/mL in 0.1 M Na2HPO4
(pH 9.0), overnight at 4°C. The plates were washed four times with PBS/
0.05% v/v Tween 20 and blocked at room temperature for 2 h with 1%
BSA in PBS/0.05% Tween 20/0.05% NaN3.Plates loaded with diluted sera
or supernatant and mouse Ig (standards) as appropriate were incubated
overnight at 4°C. Plates were washed four times and incubated for 1 h at
room temperature with HRP-conjugated anti-mouse IgG1 (0.125 ?g/mL)
and for 2 h with HRP-conjugated anti-IgG, -IgG2b, -IgG2c, -IgG3, and -IgM
(0.5 ?g/mL). After four washes, plates were developed with 90 ?l/well col-
orimetric substrate 2,2=-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-
diammonium salt (ABTS; KPL, Gaithersburg, MD, USA) at room tempera-
ture for 4 min to detect the anti-NP antibody response and for 2 min to
detect the total anti-Ig response. The reaction was stopped by addition of
110 ?l 10% w/v SDS to each well. The absorbance of the samples at 405
nm was measured using a Dynex MRX Revelation plate reader (Dynex
Technologies, Chantilly, VA, USA). End-point anti-NP antibody titers were
determined at an absorbance of ?0.01.
Multiscreen high-throughput satellite (HTS) 96-well ELISPOT plates (Milli-
pore, Bedford, MA, USA) were pre-wet with 15 ?l/well 35% ethanol. Plates
were then washed twice with PBS and coated with NP-BSA, anti-Ig, or OVA
in PBS (10 ?g/mL), overnight at 4°C. After washing with PBS and blocking
with 10% FCS in RPMI 1640, 3 ? 106cells/well from bone marrow were
added in triplicate, a threefold serial dilution of the cells was performed,
and then, the plates were incubated at 37°C for 4.5 h. After three washes
with PBS/0.05% Tween 20, plates were incubated overnight at 4°C with
HRP-conjugated anti-IgM, IgG (0.5 ?g/mL), or IgG1 (0.125 ?g/mL) in
PBS with 5% FCS. Plates were washed three times with PBS/0.05% Tween
20 and developed with 100 ?l/well colorimetric solution [47.5 mL 0.0075 N acetic
acid/0.0175 M sodium acetate/2.5 mL dimethylformamide containing one tablet
of 3-amino-9-ethyl-carbazole and 0.0005% H2O2(Sigma-Aldrich)]. The plates were
allowed to develop for 10 min and then washed 20 times with double-distilled wa-
ter. Spots, corresponding to ASCs, on the dried plates were enumerated using KS
ELISPOT 4.10 software (Carl Zeiss, Thornwood, NY, USA).
Harvesting tissue and cell isolation
Spleen, bone marrow (femur and tibia), and LNs (axillary and inguinal)
were harvested, and cells were isolated using mechanical disruption and
erythrocyte lysis, as described previously . Cells were enumerated using
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adjuvant ? B cell ? T cell ? cytokine ? plasma cell
Shah et al.
Type II NKT cells and humoral immunity
Volume 92, October 2012
Journal of Leukocyte Biology 893