Invariant natural killer T cells direct B cell responses to cognate lipid antigen in an IL-21-dependent manner

Abstract

Mouse invariant natural killer T cells (iNKT cells) provide cognate and noncognate help for lipid and protein-specific B cells, respectively. However, the long-term outcome for B cells after cognate help is provided by iNKT cells is unknown at present. Here we found that cognate iNKT cell help resulted in a B cell differentiation program characterized by extrafollicular plasmablasts, germinal-center formation, affinity maturation and a robust primary immunoglobulin G (IgG) antibody response that was uniquely dependent on iNKT cell-derived interleukin 21 (IL-21). However, cognate help from iNKT cells did not generate an enhanced humoral memory response. Thus, cognate iNKT cell help for lipid-specific B cells induces a unique signature that is a hybrid of classic T cell-dependent and T cell-independent type 2 B cell responses.

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44 VOLUME 13 NUMBER 1 JANUARY 2012 nature immunology
ARTICLES
B cell responses fall into two general categories, T cell dependent
and T cell independent. T cell–dependent responses require the
engagement of antigen through the B cell antigen receptor (BCR)
and cognate help from CD4+ T cells via major histocompatibility
complex class II–restricted antigen presentation. B cell activation
in this context results in either the extrafollicular proliferation of
B cells as plasmablasts or the entry of B cells into germinal centers
(GCs) for the subsequent development of memory or plasma cells1.
Extrafollicular plasmablasts cluster in the bridging channels and red
pulp of the spleen, and although some class-switch recombination may
occur, these cells do not undergo affinity maturation. In contrast, GC
reactions that occur in the follicles involve class-switch recombina-
tion, somatic hypermutation and affinity maturation, which produces
plasma and memory cells of higher affinity2. Both memory B cells and
plasma cells are important for an enhanced memory response after
subsequent reexposure to antigen.
T cell–independent responses by a B cell do not require any direct
interaction with a helper T cell and can be one of two subtypes: type 1
or type 2. Type 1 T cell–independent responses result from the stimu-
lation of B cells by ligands that activate without engaging the BCR,
such as the Toll-like receptor ligands lipopolysaccharide and CpG.
Type 2 T cell–independent responses involve ligands that engage the
BCR with multivalent epitopes such as polysaccharides or 4-hydroxy-
3-nitrophenylacetyl (NP) bound to Ficoll (NP-Ficoll). Both types of
T cell–independent ligands stimulate an innate-like response that is
more transient than the T cell–dependent response and does not lead
to an enhanced recall response. T cell–independent responses gener-
ally stimulate extrafollicular foci rather than GCs, do not generate
antibodies with enhanced affinity and produce few plasma cells and
atypical memory cells1.
Well-characterized T cell–dependent B cell responses to protein
antigen depend on conventional CD4+ T cells. However, invariant
natural killer T cells (iNKT cells) also provide help for B cells3,4. Mouse
iNKT cells express a restricted T cell antigen receptor (TCR) repertoire
composed of the α-chain variable region 14–α-chain joining region
18 (Vα14-Jα18) paired with Vβ8.2, Vβ7 or Vβ2 TCR β-chains5. The
iNKT cell TCR recognizes CD1d, a β2-microglobulin-associated non-
polymorphic antigen-presenting molecule expressed mainly on pro-
fessional antigen-presenting cells such as dendritic cells, monocytes
and B cells but also on other cells such as T cells and hepatocytes6,7.
The CD1 family of antigen-presenting molecules is unique in that its
members have deep hydrophobic channels on their surfaces that are
able to bind and present lipid molecules to T cells. Many bacterial
CD1d ligands have been identified8, but the most-studied ligand is
α-galactosylceramide (α-GalCer), a glycosphingolipid isolated from
marine sponges that is now available in synthetic form. It is known that
α-GalCer binds CD1d with high affinity and rapidly activates nearly
all iNKT cells to proliferate and simultaneously secrete large amounts
of T helper type 1 and T helper type 2 cytokines. Like other innate-type
cells, iNKT cells exist in a preactivated state with higher expression of
the activation markers CD44, CD69 and CD25 on their surface and
have a lower activation threshold than that of naive adaptive CD4+
T cells9,10. Thus, iNKT cells can regulate and activate myriad differ-
ent cell types (macrophages, dendritic cells, B cells and T cells) early
during infection and have an important role in defense against many
bacterial, parasitic and autoimmune diseases8. A role for iNKT cells in
the production of antibodies important for defense against infection
is most commonly demonstrated through comparison of infection of
intact versus CD1d- or iNKT cell–deficient mice with live organisms.
This approach has characterized a role for iNKT cells in the production
1Trudeau Institute, Saranac Lake, New York, USA. 2Division of Rheumatology, Brigham & Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
3School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK. 4Department of Laboratory Medicine, Yale University School of Medicine, New Haven,
Connecticut, USA. Correspondence should be addressed to E.A.L. (eleadbetter@trudeauinstitute.org).
Received 5 July; accepted 25 October; published online 27 November 2011; doi:10.1038/ni.2172
Invariant natural killer T cells direct B cell responses to
cognate lipid antigen in an IL-21-dependent manner
Irah L King1, Anne Fortier1, Michael Tighe1, John Dibble1, Gerald F M Watts2, Natacha Veerapen3,
Ann M Haberman4, Gurdyal S Besra3, Markus Mohrs1, Michael B Brenner2 & Elizabeth A Leadbetter1
Mouse invariant natural killer T cells (iNKT cells) provide cognate and noncognate help for lipid and protein-specific B cells,
respectively. However, the long-term outcome for B cells after cognate help is provided by iNKT cells is unknown at present.
Here we found that cognate iNKT cell help resulted in a B cell differentiation program characterized by extrafollicular
plasmablasts, germinal-center formation, affinity maturation and a robust primary immunoglobulin G (IgG) antibody response
that was uniquely dependent on iNKT cell–derived interleukin 21 (IL-21). However, cognate help from iNKT cells did not
generate an enhanced humoral memory response. Thus, cognate iNKT cell help for lipid-specific B cells induces a unique
signature that is a hybrid of classic T cell–dependent and T cell–independent type 2 B cell responses.
© 2012 Nature America, Inc. All rights reserved.

nature immunology VOLUME 13 NUMBER 1 JANUARY 2012 4 5
ARTICLES
of antipathogen responses during infection with Borellia hermsii11,12,
Streptoccocus pneumoniae13 or Plasmodium falciparum14 and has
indicated marginal zone B cells are a likely partner for iNKT cells in
the spleen3,12,15,16.
Activated iNKT cells are appreciated as having a role as both cog-
nate and noncognate helpers of lipid and peptide-specific B cells.
Noncognate studies have characterized an adjuvant-like effect of
administering α-GalCer together with haptenated proteins or influ-
enza virus peptides17,18. The provision of noncognate help by iNKT
cells to protein-reactive B cells has been shown to lead to humoral
memory responses, plasma-cell development, affinity maturation and
long-term maintenance of antibody responses17,18. Although cognate
iNKT cell help has been demonstrated for B cells3, the outcome for
B cells after cognate help is unknown at present. Here we found that
cognate iNKT cell help for lipid antigen–specific B cells induced a
robust primary immunoglobulin G (IgG) antibody response charac-
terized by early extrafollicular plasmablast formation, GCs, antibody
affinity maturation and a dependence on iNKT cell–derived inter-
leukin 21 (IL-21). However, cognate iNKT cell help failed to drive
classical T cell–dependent aspects of humoral responses, including
the humoral memory response and population expansion of antigen-
specific antibody-forming cells. We propose that the provision of
cognate iNKT cell help to B cells induces a constellation of traits that
is representative of a previously unknown class of B cell response: the
type 2 T cell–dependent response.
RESULTS
Induction of extrafollicular foci and GCs
To determine whether the help provided by iNKT cells for lipid
and protein antigens induces similar B cell differentiation patterns,
we first assessed the extrafollicular plasmablast response at 5 d after
immunization of mice with antigens. For this we used B1-8 mice, in
which ~5% of B cells express a transgene encoding a BCR specific
for NP. We visualized antigen-specific extrafollicular foci in the red
pulp and bridging channels of the spleen by confocal microscopy.
We identified these splenic architectural structures as clusters of
cells that bound NP tagged with the fluorescent label allophyco-
cyanin (NP-APC) and expressed the plasmablast marker CD138.
Mice immunized with the haptenated lipid antigen NP–α-GalCer
(Supplementary Fig. 1) or with haptenated protein antigen mixed
with lipid (NP linked to keyhole limpet hemocyanin (NP-KLH) plus
α-GalCer) developed numerous CD138+NP-APC+ cells clustered in
small groups in extrafollicular T cell areas of the spleen (Fig. 1a,b).
The mice developed only a few NP-specific CD138+ foci in their
red pulp or bridging channels after immunization with NP-
KLH with aluminum hydroxide (alum) as the adjuvant (Fig. 1c),
whereas no NP-APC+ foci developed when we administered the
vehicle PBS-BSA-DMSO alone (0.1% BSA in PBS containing
≤0.25% dimethyl sulfoxide; Fig. 1d). Flow cytometr y analysis of
spleens from C57BL/6 wild-type mice showed that the NP-specific
IgD−B220loCD138+ plasmablast B cell population had notably
expanded in all groups, but this population was much larger in
the group immunized with NP–α-GalCer (Fig. 1e,f). In addition,
enzyme-linked immunospot analysis showed that spleens from mice
immunized with NP–α-GalCer, but not those from mice immunized
with NP-KLH plus α-GalCer, contained significantly more B cells
that produced NP-specific IgG than did those from mice immu -
nized with vehicle (Fig. 1g) despite having similarly greater iNKT
cell numbers than mice immunized with vehicle (Fig. 1h). These
results indicated that cognate iNKT cell help to B cells resulted in a
robust early plasmablast population expansion typical of the splenic
response to T cell–independent antigens such as NP-Ficoll.
NP–α-GalCer
NP-KLH + alum PBS-BSA-DMSO
NP-KLH + α-GalCer
a
c
b
d
NP–α-GC
15
10
5
0
TCRβ+ CD1d-tet+
iNKT cells (×106)
** ** ***
NP-KLH +
alum
DMSO
NP-KLH +
α-GC
h
NP–α-GC
0
**
***
IgG+ anti-NP spots
(per 106 splenocytes)
1
2
3
4
5
NP-KLH +
alum
DMSO
NP-KLH +
α-GC
g
NP–α-GC
12.5
10.0
7.5
5.0
2.5
0
** **
**
**
NP-specific CD138+
B cells (×105)
NP-KLH +
alum
DMSO
NP-KLH +
α-GC
f
NP–α-GC
0.57
0.16
0.31
10
5
10
4
10
3
10
2
0
0.02
010
3
10
2
10
4
10
5
10.9
2.3
CD138NP-APC
16.7
17.2
NP-KLH +
α-GC
NP-KLH +
alum
DMSO
lgD
B220
e
Figure 1 Stimulation of B cells with cognate antigen (lipid) or
noncognate antigen (lipid plus protein) induces splenic extrafollicular
foci. (a–d) Confocal microscopy of spleens obtained from B1-8 mice
5 d after immunization with 5 µg NP–α-GalCer (a), 100 µg NP-KLH plus 5 µg α-GalCer (b), 100 µg
NP-KLH plus alum (c) or PBS-BSA-DMSO (d) and labeled with antibody to B220 (anti-B220; green),
anti-CD138 (red) and NP-APC (blue) to identify CD138+ NP-specific plasmablasts. Scale bars, 100 µm.
(e) Flow cytometry of splenic plasmablast B cells from wild-type C57BL/6 mice immunized as in a–d.
Numbers below outlined (gated) areas indicate percent NP-specific IgD− cells (left) or B220loCD138+
cells (right). (f) Summary of results in e. (g) ELISPOT analysis of NP-specific IgG-secreting splenic
B cells from mice immunized as in a–d. (h) Flow cytometry of TCRβ+ iNKT cells binding the CD1d tetramer
(CD1d-tet+), from the mice in f. Each symbol represents an individual mouse; small horizontal lines indicate the
mean (f–h). *P ≤ 0.05 and **P ≤ 0.001 (unpaired two-tailed t-test (f,h) or Mann-Whitney test (g)). Data are
representative of two independent experiments with three to four mice per group ( a–d) or are representative
of (e,g) or pooled from (f,h) two independent experiments with five mice per group.
© 2012 Nature America, Inc. All rights reserved.

46 VOLUME 13 NUMBER 1 JANUARY 2012 nature immunology
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Next we compared GC formation in the spleen after immunization
with lipid alone or lipid plus protein. Immunofluorescence labeling
showed that at 12 d into the response, B1-8 mice with a greater fre-
quency of NP-specific B cells, immunized with either NP–α-GalCer or
NP-KLH plus α-GalCer, developed frequent cell clusters positive for
GL7, an antibody clone specific for an as-yet-unidentified T cell– and
B cell–activation antigen that labels GC B cells (Fig. 2a,b), in a manner
similar to that of mice immunized with NP-KLH plus alum (Fig. 2c).
Mice immunized with vehicle had low background number of GCs of
approximately 10 per spleen section (Fig. 2d,e). There was an aver-
age of 24 GCs per spleen section in mice immunized with NP-KLH
plus α-GalCer and 21 GCs per spleen section in mice immunized with
NP–α-GalCer (Fig. 2e), a notable but not significant difference. We
also counted, by flow cytometry, splenic B cell and iNKT cell popula-
tions 12 d after immunization (Fig. 2f–i). All groups of immunized
C57BL/6 wild-type mice had a greater number and frequency of NP-
specific (B220+CD95+GL7+) GC B cells than did mice immunized with
vehicle, but the GC B cells were significantly more numerous in mice
immunized with NP-KLH and α-GalCer than in mice immunized with
NP–α-GalCer (Fig. 2f). The number of splenic iNKT cells positively
identified by the CD1d tetramer was similarly higher, showing greater
population expansion after immunization with lipid plus protein but
not after immunization with lipid only (Fig. 2i). These results suggested
that when iNKT cells recognizing the lipid component of NP–α-GalCer
provided cognate help to NP-specific B cells, the B cells were induced
to produce GCs, although they were smaller than the GCs derived after
noncognate iNKT cell help. Consistent with those data, the number
of NP-specific IgG-producing cells was significantly greater in both
groups of protein-immunized mice than in lipid-immunized mice at
this later time point (Fig. 2h). Of note, we also observed more NP-
specific CD38+IgD− memory-phenotype B cells in all groups of antigen-
immunized mice at day 12 (Fig. 2g), but differences between the groups
were not significant. Thus, noncognate iNKT cell help seemed to induce
and maintain conventional GCs containing protein-specific B cells. In
contrast, cognate iNKT cell help recruited by a lipid-only immunization
strategy induced smaller GCs and was unable to sustain antigen-specific
B cell population expansion and antibody production.
Induction of BCR affinity maturation
GCs provide an environment for B cell maturation that enables selec-
tion for BCRs of higher affinity19. Given that both noncognate and
NP–α-GalCer
acNP-KLH + alum dPBS-BSA-DMSO
bNP-KLH + α-GalCer
e
50 *
40
30
20
GCs per spleen section
10
0
NP–α-GC
NP-KLH +
alum
DMSO
NP-KLH +
α-GC
f
8** **
** **
6
4
2
0
B220+CD95+GL7+ NP-specific
germinal center B cells (×104)
NP–α-GC
NP-KLH +
alum
DMSO
NP-KLH +
α-GC
g
** ** **
lgD–CD38+ NP-specific
B cells (×104)
40
30
20
10
0
NP–α-GC
NP-KLH +
alum
DMSO
NP-KLH +
α-GC
h
**
***
*
lgG+ anti-NP spots
per 106 splenocytes
100
80
60
40
20
0
NP–α-GC
NP-KLH +
alum
DMSO
NP-KLH +
α-GC
i**
*
CD1d-tet+TCRβ+
iNKT cells (×106)
4
3
2
1
0
NP–α-GC
NP-KLH +
alum
DMSO
NP-KLH +
α-GC
Figure 2 Stimulation of B cells with cognate antigen (lipid) or noncognate antigen (lipid plus protein) leads
to the development of splenic GCs. (a–d) Confocal microscopy of spleens obtained from B1-8 mice 12 d
after immunization with 5 µg NP–α-GalCer (a), 100 µg NP-KLH plus 5 µg α-GalCer (b), 100 µg NP-KLH plus
alum (c) or PBS-BSA-DMSO (d), and labeled with GL7 (green), anti-CD3 (blue) and anti-B220 (red). Scale
bars, 500 µm. (e) Total GL7+ GCs per spleen section of the mice as in a–d. (f,g) Flow cytometry of B220+
CD95+GL7+ GC cells (f) and IgD−CD38+ NP-specific B cells (g) from spleens of C57BL/6 wild-type mice at
day 12 after immunization as in a–d. (h,i) ELISPOT analysis of NP-specific IgG-secreting splenic B cells (h)
and flow cytometry of TCRβ+CD1d tetramer–positive iNKT cells (i) from wild-type C57BL/6 mice immunized
as in a–d. Each symbol represents an individual mouse; small horizontal lines indicate the mean (e–i).
*P ≤ 0.05 and **P ≤ 0.001 (Mann-Whitney test). Data are representative of two to three independent experiments with duplicate sections from four
mice per group (a–d) or are representative of (f,h,i) or pooled from (e,g) two to three independent experiments with four to five mice per group.
7 61 7 61 7 61 7 61
0
0.5
1.0
1.5
2.0 *
NP-KLH +
alum
NP–
α
-GalCer
NP-KLH +
α-GalCer
NP68-Ficoll
* *
NP
4
/NP
25
Time (d)
Figure 3 Cognate and noncognate iNKT cell help induces antigen-specific
antibody affinity maturation. ELISA of affinity maturation in serum from
C57BL/6 wild-type mice immunized with 100 µg NP-KLH plus alum,
0.5 µg NP–α-GalCer, 100 µg NP-KLH plus 0.5 µg α-GalCer, or 30 µg
NP68-Ficoll, collected 7 d after the primary challenge (day 7) and
7 d after the secondary boost (day 61) and assessed on plates coated
with BSA conjugated to NP (four molecules (NP4-BSA) or twenty-five
molecules (NP25-BSA)), presented as the ratio of binding to NP4 to
binding to NP25 (NP4/NP25). Each symbol represents an individual
mouse; small horizontal lines indicate the mean. *P ≤ 0.0001
(unpaired t-test). Data are representative of three independent
experiments with eight to ten mice per group.
© 2012 Nature America, Inc. All rights reserved.

nature immunology VOLUME 13 NUMBER 1 JANUARY 2012 4 7
ARTICLES
cognate lipid antigens stimulated the formation of GCs, we next
investigated antibody affinity maturation driven by immunization
with each form of lipid antigen. We used a standard enzyme-linked
immunosorbent assay (ELISA) to assess the binding of serum anti-
body to sparsely haptenated proteins versus highly haptenated pro-
teins20. In comparing serum from mice collected 7 d after primary
immunization (day 7) with serum from the same mice collected
7 d after a secondary boost (day 61), we found that both cognate and
noncognate lipid antigens induced a significant increase in antibody
affinity after a boost immunization (Fig. 3). As expected, the known
T cell–dependent antigen NP-KLH in alum induced antibodies of
higher affinity after a secondary boost, whereas the well-described
T cell–independent antigen of Ficoll haptenated with 68 molecules
of NP (NP68-Ficoll) failed to induce significant affinity maturation
(Fig. 3). Thus, just as both forms of iNKT cell help (cognate and
noncognate) stimulated GCs, they also both induced affinity matu-
ration of BCRs.
IL-21 is critical component of iNKT cell help for B cells
Follicular helper T cells (TFH cells) have been reported to enter the
B cell follicle specifically to provide cognate help for protein-specific
B cells21,22. Notably, mature iNKT cells are reported to share many
of the characteristics of traditional protein-specific TFH cells2 3; that
is, they migrate in response to the chemokine CXCL13 (via the
chemokine receptor CXCR5) but not CCL21 (via CCR7)24, express
ICOS (data not shown) and secrete IL-21 (ref. 25). To determine if
iNKT cells provide B cell help similarly to protein-specific TFH cells,
we assessed the importance of signaling via IL-21 and its receptor,
IL-21R, for cognate lipid–specific and noncognate lipid–enhanced
antibo dy resp ons es in this system. We immunized IL-21R-
deficient mice and wild-type mice with lipid antigen (NP–α-
GalCer), protein antigen (NP-KLH in alum) or protein plus lipid
antigens (NP-KLH plus α-GalCer). In all three cases, early NP-
specific IgG antibodies were less abundant in IL-21R-deficient
mice than in wild-type mice (Fig. 4a). As a negative control,
intraperitoneal administration of the T cell–independent antigen
NP-Ficoll induced IL-21-independent IgG, with no differences
between IL-21R-deficient and wild-type mice. In all groups tested,
there were no consistent differences between IL-21R-deficient and
wild-type mice in anti-NP IgM titers (Fig. 4b). These data sug-
gested that IL-21 was required for antibody class switching, not
merely for antib ody production. We confirmed IL-21 expression
by iNKT cells by real-time RT-PCR at early time points (days 5–7)
and later time points (days 11–13) after the administration of
0.5 µg NP–α-GalCer (Fig. 4c).
Next we sought to determine whether iNKT cells were the critical
source of IL-21. To address this, we generated mixed–bone marrow
chimeras in which we selectively deleted Il21 in iNKT cells. We created
these chimeras by reconstituting irradiated Jα18-deficient hosts with a
mixture of 25% IL-21-deficient bone marrow and 75% Jα18-deficient
bone marrow. We created controls with IL-21-sufficient iNKT cells
through the use of Jα18-deficient hosts reconstituted with a mixture
of 25% wild-type bone marrow and 75% Jα18-deficient bone marrow.
Immunizing these mice with NP-KLH in alum, NP-KLH plus α-
GalCer or the lipid NP–α-GalCer showed that only cognate iNKT
cell help depended entirely on iNKT cell–derived IL-21. Specifically,
chimeric mice with IL-21-deficient iNKT cells had less NP-specific
IgG at all time points than did chimeras with IL-21-sufficient iNKT
a b
NP-KLH +
alum
NP
68
-Ficoll
10
0
10
1
10
2
10
3
10
4
WT (day 7)
IL-21R-KO (day 7)
* ** **
IgG anti-NIP (µg/ml)
NP–α-GC
NP-KLH +
α-GC
10
0
10
1
10
2
10
3
10
4
WT (day 7)
IL-21R-KO (day 7)
**
IgM anti-NIP (µg/ml)
NP-KLH +
alum
NP
68
-Ficoll
NP–
α-GC
NP-KLH +
α-GC
10
0
10
1
10
2
10
3
10
4
WT (day 14)
IL-21R-KO (day 14)
* * *
NP-KLH +
alum
NP
68
-Ficoll
NP–
α-GC
NP-KLH +
α-GC
IgG anti-NIP (µg/ml)
10
0
10
1
10
2
10
3
10
4
WT (day 21)
IL-21R-KO (day 21)
* *
NP-KLH +
alum
NP
68
-Ficoll
NP–
α-GC
NP-KLH +
α-GC
IgG anti-NIP (µg/ml)
c
Ctrl
1
5–7
11–13
0
10
20
30
40
**
*
Time (d)
IL-21 mRNA (relative)
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
WT (day 14)
IL-21R-KO (day 14)
WT (day 21)
IL-21R-KO (day 21)
*
NP-KLH +
alum
NP
68
-Ficoll
NP–
α-GC
NP-KLH +
α-GC
NP-KLH +
alum
NP68
-Ficoll
NP–
α-GC
NP-KLH +
α-GC
IgM anti-NIP (µg/ml)
IgM anti-NIP (µg/ml)
Figure 4 IL-21R signaling is required for cognate iNKT cell–mediated anti-NP responses. (a,b) Titers of NP-specific IgG (a) and
IgM (b) in serum obtained from C57BL/6 wild-type (WT) mice and IL-21R-deficient (IL-21R-KO) mice on days 0, 7, 14 and
21 after immunization with 0.5 µg NP–α-GalCer, 100 µg NP-KLH plus 0.5 µg α-GalCer, 100 µg NP-KLH plus alum, or 30 µg
NP68-Ficoll. *P ≤ 0.05 and **P ≤ 0.001, wild-type versus IL-21R-deficient (Mann-Whitney test). (c) Real-time RT-PCR analysis
of IL-21 mRNA expression in iNKT cells sorted by flow cytometry (as TCRβ+CD19− CD1d tetramer–positive iNKT cells) from
mice transgenic for expression of Vα14, at 1 d, 1 week (days 5–7) and 2 weeks (days 11–13) after intraperitoneal immunization
with NP–α-GalCer (0.5 µg per mouse) or PBS-BSA-DMSO; results are presented relative to the expression of mRNA from the
housekeeping gene GAPDH. *P ≤ 0.001 (Mann-Whitney test). Data are from two to five independent experiments with three to
five mice per group in each (a,b) or are pooled from two to three experiments with 5–11 mice per group ( c; mean and s.e.m.).
NP–α-GC
NP-KLH +
α-GC
NP-KLH +
alum
NP–α-GC
NP-KLH +
α-GC
NP-KLH +
alum
NP–α-GC
NP-KLH +
α-GC
NP-KLH +
alum
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
WT (day 8)
IL-21-KO (day 8)
*
IgG anti-NIP (µg/ml)
WT (day 14)
IL-21-KO (day 14)
WT (day 21)
IL-21-KO (day 21)
ba c
*
IgG anti-NIP (µg/ml)
IgG anti-NIP (µg/ml)
Figure 5 IL-21 produced by iNKT cells is
required for cognate lipid antigen help.
(a–c) NP-specific IgG titers in blood from mixed–
bone marrow chimeras with an iNKT cell compart-
ment able (wild-type) or unable (IL-21-deficient)
to produce IL-21, obtained on day 8 (a), day 14
(b) or day 21 (c) after immunization with 0.5 µg
NP–α-GalCer, 100 µg NP-KLH plus 0.5 µg
α-GalCer, or 100 µg NP-KLH plus alum.
*P ≤ 0.05, wild-type versus IL-21-deficient
(Mann-Whitney test). Data are from two
independent experiments with three to five mice
per group in each experiment (mean and s.e.m.).
© 2012 Nature America, Inc. All rights reserved.

48 VOLUME 13 NUMBER 1 JANUARY 2012 nature immunology
ARTICLES
cells only after immunization with the cognate iNKT cell antigen
NP–α-GalCer (Fig. 5). Thus, noncognate iNKT cell help elicited by
NP-KLH plus α-GalCer required IL-21, but it did not need to come
from iNKT cells.
Cognate versus noncognate humoral memory responses
Given the finding that cognate iNKT cell help resulted in GC for-
mation and affinity-matured antibody responses, we next sought
to determine whether cognate iNKT cell help could generate
B cell memory responses similar to noncognate iNKT cell help. To
address this, we immunized wild-type C57BL/6 mice with either
NP–α-GalCer or NP-KLH plus α-GalCer. Using classical CD4+
T cell help as a positive control, we immunized some mice with
NP-KLH in alum. After that primary intraperitoneal immunization,
we allowed the mice to rest for 177 d to let the initial hapten-specific
antibody response wane, then boosted the mice with a secondary
intraperitoneal challenge of lipid antigen or protein antigen in PBS.
As expected, boosting of mice with NP-KLH in PBS after previous
immunization with NP-KLH in alum resulted in a distinct memor y
response (Fig. 6a); that is, the anti-NP titers after the boost were
much higher than the antibody titers that resulted from the initial
primary challenge. Titers after the boost were also much higher than
the antibody titers in age-matched mice that received their primary
protein antigen in alum challenge on day 177 (Fig. 6a). Mice that
received NP–α-GalCer in vehicle as a primary challenge and again as
a secondary boost developed the same antibody titers after the boost
as those of mice that received a primary challenge with NP–α-GalCer
at day 177 (Fig. 6b).
We obtained results similar to those above with a shorter delay
between challenges (46 d rather than 177 d) and boosting via an
intravenous route (Fig. 7), a protocol more commonly used to
demonstrate anti-protein memory responses. The response to lipid
antigen was most similar to the anti-NP response generated by the
T cell–independent antigen NP-Ficoll (Fig. 7b). We obtained similar
results after challenge and boost with higher doses of lipid antigen
(5 µg per mouse; Supplementary Fig. 2), which indicated that antigen
availability was not a confounding factor in our studies. Together these
data are consistent with published reports demonstrating that the
humoral memory response to protein immunization is the same
whether the adjuvant used is alum or the lipid α-GalCer. However,
we found that responses to a haptenated lipid antigen, despite elicit-
ing a robust primary antibody response, failed to generate a memory
B cell antibody response.
DISCUSSION
Our studies here have shown that cognate and noncognate iNKT cell
help for B cells led to very different B cell outcomes. We demonstrated
that after immunization with either cognate or noncognate lipid, mice
generated strong primary anti-NP IgG responses characterized by
early extrafollicular foci and, later, GCs dependent on signals via
IL-21R. However, B cells that received noncognate iNKT cell help
made a greater humoral memory response after rechallenge, whereas
those B cells that received cognate iNKT cell help made a secondary
response of the same magnitude as the primary response. Our results
are consistent with other studies of noncognate iNKT cell help17,18
and support the proposal that iNKT cells are memory-like innate
lymphocytes able to stimulate a rapid, robust response from the time
of their initial activation.
In the context of the cognate-help studies, iNKT cells may be func-
tioning as a previously unknown TFH cell population that specializes
in helping lipid-specific B cells to generate GC responses. In response
to immunization with protein antigens, IL-21 from conventional TFH
cells acts directly on GC B cells to support plasma-cell differentia-
tion26–28. Data from our bone marrow–chimera studies demonstrated
that iNKT cells provided cognate lipid–specific T cell help through the
production of IL-21. Thus, iNKT cells are able to function in part as
‘iNKTFH cells’. It is known that iNKT cells express many of the same
surface costimulatory molecules that TFH cells express (for example,
CD40L and ICOS)29,30 but, as we have shown here, differ from TFH
cells in their ability to generate a memory B cell population.
Our imaging studies showed that both the cognate iNKT anti-
gen NP–α-GalCer and the noncognate mixture of NP-KLH plus
α-GalCer stimulated similar antigen-specific extrafollicular foci and
50 60 70 80
0
2
4
6
PBS; PBS
NP-KLH + alum; NP-KLH
NP-KLH + α-GalCer; NP-KLH
PBS; NP-KLH + alum
PBS; NP-KLH + α-GalCer
**
**
** *
*
** **
Time (d)
IgG anti-NIP (mg/ml)
a b
50 60 70 80
0
0.2
0.4
0.6
0.8
1.0
PBS; PBS
NP–α-GalCer; NP–α-GalCer
NP68-Ficoll; NP68-Ficoll
PBS; NP–α-GalCer
PBS; NP68-Ficoll
Time (d)
IgG anti-NIP (mg/ml)
Figure 7 Only noncognate iNKT cell help induces an antibody memory
response after rechallenge on day 46. Anti-hapten ELISA of NP-specific
IgG in blood obtained (periodically up to day 45) from C57BL/6
wild-type mice immunized intraperitoneally on day 0 with PBS, 2.2 µg
NP-KLH in alum or 2.2 µg NP-KLH plus 0.5 µg α-GalCer (a), or with PBS,
0.5 µg NP–α-GalCer, or 30 µg NP68-Ficoll (b), then given a secondary
intravenous boost on day 46 with PBS or the same dose of NP-KLH or
NP-KLH plus α-GalCer, or intraperitoneal boost of NP-KLH plus alum (a),
or intravenous boost of PBS, NP–α-GalCer or NP68-Ficoll (b), followed by
additional sampling of blood on days 3, 7, 14 and 29 after the boost
(key: primary challenge; secondary boost); full time course, Supplementary
Figure 4. *P ≤ 0.05 and **P ≤ 0.001, versus the corresponding primary
immunization group (Mann-Whitney test). Data are from one experiment
with eight mice per group (mean ± s.e.m.).
b
170 180 190 200
0
50
100
150
200
PBS + alum; PBS + alum
NP–α-GalCer; NP–α-GalCer
Time (d)
IgG anti-NIP (µg/ml)
1°
2°
PBS; NP–α-GalCer
170 180 190 200
0
0.5
1.0
1.5
2.0
PBS + alum; PBS + alum
NP-KLH + alum; NP-KLH
PBS; NP-KLH + alum
*
**
Time (d)
IgG anti-NIP (mg/ml)
a
Figure 6 Only noncognate iNKT cell help induces antibody memory
response after day 177 rechallenge. Anti-hapten ELISA of NP-specific
IgG in blood (obtained periodically up to day 166) from C57BL/6
wild-type mice immunized (1°) intraperitoneally on day 0 with PBS plus
alum, 2.2 µg NP-KLH plus alum or PBS alone (a), or PBS plus alum,
0.5 µg NP–α-GalCer or PBS alone (b), then given a secondary (2°)
intraperitoneal boost on day 177 (arrow) of PBS plus alum, 2.2 µg
NP-KLH, or NP-KLH plus alum (a), or an intraperitoneal boost of PBS
plus alum or 0.5 µg NP–α-GalCer, followed by additional sampling of
blood on days 3, 7, and 14 after the boost (key: primary challenge;
secondary boost); full time course, Supplementary Figure 3. *P ≤ 0.05
and **P ≤ 0.001, versus mice immunized with PBS and boosted with
NP-KLH plus alum (Mann-Whitney test). Data are from one experiment
with nine mice per group (mean ± s.e.m.).
© 2012 Nature America, Inc. All rights reserved.

nature immunology VOLUME 13 NUMBER 1 JANUARY 2012 4 9
ARTICLES
numbers of GCs. However, the B cell outcome of cognate help from
iNKT cells was notably different from the outcome after help from
conventional CD4+ T cells that benefited from enhanced antigen-
presenting function secondary to iNKT cell activation. In part, iNKT
cell help for cognate haptenated lipid did not entirely mimic extrafol-
licular foci–dominated responses to T cell–independent antigens but
instead reflected a response based more on T cell–dependent extra-
follicular foci and GCs. The noncognate-immunization approach
with NP-KLH plus α-GalCer did stimulate more GC B cells than did
strictly lipid immunization but resulted in similar numbers of total
GCs. That finding was consistent with our observation that spleens
from mice immunized with NP-KLH plus α-GalCer had relatively
larger GCs, which suggested that in this case, activation of iNKT
cells functioned more as an adjuvant than stimulating a lipid-specific
response in parallel. Given that mice immunized with NP–α-GalCer
or with NP-KLH plus α-GalCer induce affinity maturation (probably
via GCs) and have similar numbers of memor y-phenotype CD38+
antigen-specific B cells31, it is possible that such GCs are present
but inadequate or prematurely involute. Many elements have been
identified as being critical for sustaining a T cell–dependent B cell
GC response, including Dock8 expression in B cells, the formation
of immune synapses between B cells and T cells, and integrin signal-
ing32. The quality and/or quantity of a BCR signal can be a critical
aspect of GC persistence, particularly later in a GC response when
the way in which antigen is presented, such as in the form of immune
complexes, may be different33. Expression of the costimulatory mol-
ecule PD-1 on T cells has also been shown to be important for the
survival of B cells in GCs and subsequent long-lived plasma cell out-
put without affecting affinity maturation34. Heterogeneity in memory
B cell development may also affect the humoral recall response35.
The contribution of these factors to iNKT cell–B cell interactions
and their relevance to iNKT cell–induced formation of GCs remain
to be determined.
Finally, iNKT cells have been proposed to localize outside of the splenic
white pulp under homeostatic conditions36. Thus, how iNKT cell–B cell
interactions occur in situ is not clear. One possibility supported by a
published study may be that iNKT cells ‘preferentially’ interact with mar-
ginal zone B cells37. These B cells are situated along the marginal sinus
at the primary point for the entry of blood-borne particulate antigens
into the spleen. This positions them to specialize in responding to type 2
T cell–independent antigens38. Marginal zone B cells express abundant
CD1d and secrete mainly IgM and IgG3 (ref. 39), the antibody isotypes
produced in response to the pure synthetic lipid antigen NP–α-GalCer3.
Thus, they may also be one of the main B cell subpopulations to receive
both cognate and noncognate iNKT cell help16.
In conclusion, we propose lipid-specific, type 2 T cell–dependent
responses as a new subcategory of B cell antigen-specific responses
that are a hybrid of the other three established categories of B cell
antigens. According to our assessment, the characteristics of type 2
T cell–dependent responses include the formation of extrafollicular
foci and GCs accompanied by affinity maturation and the absence
of functional humoral memory responses. This category of antigen-
specific responses remains to be characterized in the context of live
infection but may have an important role early in infection when
rapid iNKT cell help could provide a unique advantage. Future studies
should investigate humoral immunity to live pathogens induced by
cognate and noncognate lipid antigens.
METHODS
Methods and any associated references are available in the online version
of the paper at http://www.nature.com/natureimmunology/.
Note: Supplementary information is available on the Nature Immunology website.
ACKNOWLEDGMENTS
We thank M. Nussenzweig (Rockefeller University) for B6.SJL B1-8hi mice;
M. Exley (Dana Farber Cancer Institute) for C57BL/6 Vα14-transgenic mice and
C57BL/6 Jα18-deficient mice; K. Rajewsky (Center for Blood Research) for
B1-8f mice; M. Rincon (University of Vermont) for IL-21-deficient mice; and
the US National Institutes of Health Tetramer Core for mouse CD1d-PBS57
tetramers and unloaded CD1d tetramers. Supported by the Trudeau Institute
(E.A.L.), the US National Institutes of Health (AI028973-23 and AI063428-
06 to M.B.B. and T32 A1049823-10 to I.L.K.), J. Bardrick (G.S.B.), the Royal
Society (G.S.B.), The Wellcome Trust (084923/B/08/Z to G.S.B.) and the Medical
Research Council (G.S.B.).
AUTHOR CONTRIBUTIONS
I.L.K. designed and did experiments, analyzed data and edited the manuscript;
A.F., M.T., J.D. and G.F.M.W. designed and did experiments; A.M.H. did
experiments, edited the manuscript and provided technical advice; N.V. and G.S.B.
synthesized and provided lipid antigens; M.M. and M.B.B. provided conceptual
advice and E.A.L. initiated and directed the research, did experiments, analyzed the
data and wrote the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/natureimmunology/.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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ARTICLES
© 2012 Nature America, Inc. All rights reserved.

nature immunology
doi:10.1038/ni.2172
ONLINE METHODS
Mice. C57BL/6 wild-type mice, B6.SJL B1-8hi mice40 (heterozygous for targeted
insertion of a mutated variable 186.2 immunoglobulin heavy chain bearing
specificity for the hapten NP), C57BL/6 Vα14-transgenic mice with a greater
frequency of iNKT cells 41 and C57BL/6 Jα18-deficient mice lacking Jα18+
iNKT cells42 were housed and bred at the Dana-Farber Cancer Institute and
the Trudeau Institute according to standards of the animal care and use com-
mittees of each institution. C57BL/6 B1-8f mice43 (heterozygous for targeted
insertion of variable region 186.2 of the immunoglobulin heavy chain specific
for NP) were bred and housed at Yale University School of Medicine accord-
ing to standards of the institutional animal care and use committee. C57BL/6
IL-21R-deficient mice were generated as described44, and IL-21-deficient mice
came from the Mutant Mouse Regional Resource Center at the University
of California, Davis, and were provided by M. Rincon. All live animal
experimental protocols were approved by the Dana-Farber Cancer Institute
Institutional Animal Care and Use Committee or the Trudeau Institute Animal
Care and Use Committee.
Flow cytometry. Single-cell suspensions were prepared from the spleen
and stained with the following monoclonal antibodies for flow cytometry :
Alexa Fluor 450–IgD (11-26c.2a; BD), phycoerythrin-indotricarbocyanine–
anti-B220 (RA3-6B2; Biolegend), phycoerythrin–anti-Fas (Jo2; BD), fluores-
cein isothiocyanate–GL7 (GL7; BD), fluorescein isothiocyanate–anti-CD38
(90; BD), phycoerythrin–anti-CD138 (281-2; BD), biotin-IgG1 (A85-1;
BD), streptavidin-allophycocyanin-indotricarbocyanine (BD) and Pacific
Blue–anti-TCRβ (H57-597; Biolegend). The iNKT cells were identified with
tetramers of mouse CD1d–α-GalCer (PBS57; US National Institutes of Health)
conjugated to allophycocyanin. Samples were acquired on a FACSCanto II
(BD) and were analyzed with FlowJo software (TreeStar).
Bone marrow chimeras. Recipient C57BL/6 Jα18-deficient mice were irradi-
ated twice with 500 rads and were allowed to rest for a few hours or overnight.
Recipient mice were reconstituted with 5 × 106 bone marrow cells. Donor
bone marrow included either 75% Jα18-deficient bone marrow mixed with
25% wild-type bone marrow, or 75% Jα18-deficient bone marrow mixed with
25% IL-21-deficient bone marrow. These reconstitution mixtures resulted in
mice in which only the iNKT cells were deficient in IL-21 production or all
cells were normal. Reconstitution of iNKT cell, T cell and B cell lineages in the
spleen and liver were confirmed by flow cytometr y after 7 weeks.
Antigens, immunization and serum collection. NP-KLH, NP-BSA, KLH,
NP68-Ficoll (Biosearch Technologies), NP–α-GalCer and α-GalCer (synthe-
sized by N.V. and G.S.B. as described3) were administered intraperitoneally
in a volume 200 µl unless noted otherwise. Immunizations included 2.2 µg or
100 µg protein precipitated in alum or suspended in PBS plus 0.1% (wt/vol)
BSA, or 0.5–5 µg lipid antigen solubilized in ≤0.25% (vol/vol) DMSO, then
suspended in PBS with 0.1% (wt/vol) BSA. NP-KLH in these studies contained
20 NP haptens per KLH protein, and NP–α-GalCer lipid contained a single
NP hapten per α-GalCer molecule. In serum, the protein is most probably
monomeric, whereas the lipid is more probably micellular or bound to a lipid
binding protein, which makes equal comparisons of molar quantities or hapten
quantities challenging. The quantity 2.2 µg NP-KLH is the molar equivalent
of 0.5 µg NP–α-GalCer divided by the number of haptens on the KLH protein
(20). Serum was collected retro-orbitally or submandibularly and was stored
at –20 °C until assessment by ELISA.
ELISA. NP-specific IgG and IgM in serum was assessed by heteroclitic
ELISA specific for NIP (4-hydroxy-5-iodo-3-nitrophenyl) as described3.
Affinity was assessed by ELISA on plates simultaneously coated with
NP4-BSA or NP25-BSA (presented as a ratio of binding to NP4 to the
binding to NP25 ).
ELISPOT. Eight half-log serial dilutions of primary spleen c ell suspen-
sions from C57BL/6 mice were cultured in duplicate overnight at 37 °C
on Multi Screen-HA ELISPOT plates (Millipore) coated with NIP1 5-
BSA (Biosearch Technologies), and nonspecific binding was blocked by
incubation with a solution of 1% (wt/vol) BSA in PBS. Spots were detec ted
wit h horserad ish peroxidase–conjugate d anti-mouse IgG ( 103 0-0 5;
Southern Biotech) and plates were developed with an AEC staining kit
(Sigma). Spots were scanned and counted on an Immunospot analyzer
(CTL Analyzers).
Real-time RT-PCR. TCRβ+CD19− CD1d tetramer–positive iNKT cells were
isolated from Vα14-transgenic mice with a BD Influx high-speed cell sorter.
RNA was extracted from TRIzol-fixed cells with an RNeasy mini kit according
to the manufacturer’s instructions (Qiagen). A High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems) plus RNase inhibitors were used for
the production of cDNA. Primers and probes from Applied Biosystems and the
TaqMan 7500 Fast System and software (Applied Biosystems) were used for real-
time RT-PCR of cDNA samples; expression was calculated by the change-in-
threshold method (∆∆CT) with GAPDH mRNA (encoding glyceraldehyde
phosphate dehydrogenase) as a reference.
Confocal fluorescence microscopy. Frozen spleens cut into sections 7 µm in
thickness and embedded in optimum cutting temperature compound were
labeled with fluorescein isothiocyanate–anti-B220 (RA3-6B2; BD) plus anti-
body to f luorescein isothiocyanate–Alexa Fluor 488 (A11096; Invitrogen),
phycoerythrin–anti-CD138 (281-2; BD), rabbit anti-CD3 (145-2C11; BD)
plus Alexa Fluor 647–anti-rabbit (A21245; Invitrogen), phycoer ythrin–
anti-B220 (RA3-6B2; eBioscience), fluorescein isothiocyanate–GL7 (GL7;
BD) plus antibody to fluorescein isothiocyanate–Alexa Fluor 488 (A11096;
Invitrogen) and NP-APC. NP-APC was conjugated as reported45. Fluorescent
images were obtained with a TE2000-U inverted microscope with a C1
Plus Confocal System (Nikon; Partners Confocal Microscopy Core) and
an Axiovert 200M fluorescence microscope (Zeiss; Trudeau Institute).
Final ‘stitched’ high-resolution whole-spleen confocal images were obtained
with a TCS SP5 confocal microscope with LAS AF 2.2.1 software (Leica;
Trudeau Institute).
Statistics. GraphPad PRISM 5 software was used for nonparametric two-tailed
t-tests for normally distributed data sets with ten or more samples. The two-
tailed nonparametric Mann-Whitney test was used for smaller data sets for
which normality could not be determined.
40. Shih, T.-A.Y., Roederer, M. & Nussenzweig, M.C. Role of antigen-receptor affinity in
T cell-independent antibody responses in vivo. Nat. Immunol. 3, 399–406 (2002).
41. Bendelac, A., Hunziker, R.D. & Lantz, O. Increased interleukin 4 and immunoglobulin
E production in transgenic mice overexpressing NK1 T cells. J. Exp. Med. 184,
1285–1293 (1996).
42. Cui, J. et al. Requirement for Vα14 NKT cells in IL-12-mediated rejection of tumors.
Science 28, 1623–1626 (1997).
43. Lam, KP., Kuhn, R. & Rajewsky, K. In vivo ablation of surface immunoglobulin on
mature B cells by inducible gene targeting results in rapid cell death. Cell 90,
1073–1083 (1997).
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