iNKT B Cells IL21

8/3/2019 iNKT B Cells IL21

1/8

2011

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nature immunology aDVaNCE ONLINE PUBLICaTION

A r t i c l e s

B cell responses fall into two general categories, T cell dependentand T cell independent. T celldependent 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 IIrestricted antigen presentation. B cell activationin 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 redpulp of the spleen, and although some class-switch recombination may

occur, these cells do not undergo affinity maturation. In contrast, GCreactions that occur in the follicles involve class-switch recombina-

tion, somatic hypermutation and affinity maturation, which producesplasma and memory cells of higher affinity2. Both memory B cells and

plasma cells are important for an enhanced memory response aftersubsequent reexposure to antigen.

T cellindependent responses by a B cell do not require any directinteraction with a helper T cell and can be one of two subtypes: type 1

or type 2. Type 1 T cellindependent 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 cellindependent 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 ofT cellindependent ligands stimulate an innate-like response that is

more transient than the T celldependent response and does not leadto an enhanced recall response. T cellindependent responses gener-

ally stimulate extrafollicular foci rather than GCs, do not generateantibodies with enhanced affinity and produce few plasma cells and

atypical memory cells1.

Well-characterized T celldependent B cell responses to proteinantigen depend on conventional CD4+ T cells. However, invariant

natural killer T cells (iNKT cells) also provide help for B cells3,4. MouseiNKT cells express a restricted T cell antigen receptor (TCR) repertoire

composed of the -chain variable region 14-chain joining region18 (V14-J18) paired with V8.2, V7 or V2 TCR -chains

5. The

iNKT cell TCR recognizes CD1d, a 2-microglobulin-associated non-polymorphic antigen-presenting molecule expressed mainly on pro-

fessional antigen-presenting cells such as dendritic cells, monocytesand B cells but also on other cells such as T cells and hepatocytes 6,7.

The CD1 family of antigen-presenting molecules is unique in that itsmembers have deep hydrophobic channels on their surfaces that are

able to bind and present lipid molecules to T cells. Many bacterialCD1d ligands have been identified8, but the most-studied ligand is

-galactosylceramide (-GalCer), a glycosphingolipid isolated frommarine sponges that is now available in synthetic form. It is known that

-GalCer binds CD1d with high affinity and rapidly activates nearlyall iNKT cells to proliferate and simultaneously secrete large amounts

of T helper type 1 and T helper type 2 cytokines. Like other innate-typecells, iNKT cells exist in a preactivated state with higher expression of

the activation markers CD44, CD69 and CD25 on their surface andhave 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) earlyduring infection and have an important role in defense against many

bacterial, parasitic and autoimmune diseases8. A role for iNKT cells inthe production of antibodies important for defense against infection

is most commonly demonstrated through comparison of infection ofintact versus CD1d- or iNKT celldeficient mice with live organisms.

This approach has characterized a role for iNKT cells in the production

1Trudeau Institute, Saranac Lake, New York, USA. 2Division o Rheumatology, Brigham & Womens Hospital, Harvard Medical School, Boston, Massachusetts, USA.3School o Biosciences, University o Birmingham, Edgbaston, Birmingham, UK. 4Department o Laboratory Medicine, Yale University School o Medicine, New Haven,

Connecticut, USA. Correspondence should be addressed to E.A.L. ([email protected]).

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 tocognate lipid antigen in an IL-21-dependent manner

Irah L King1, Anne Frtier1, Mihael Tighe1, Jhn Dibble1, Gerald F M Watts2, Nataha Veerapen3,Ann M Haberman4, Gurdyal S Besra3, Markus Mhrs1, Mihael 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 responsethat was uniquely dependent on iNKT cellderived 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 celldependent and T cellindependent type 2 B cell responses.
http://www.nature.com/doifinder/10.1038/ni.2172http://www.nature.com/doifinder/10.1038/ni.2172


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aDVaNCE ONLINE PUBLI CaTION nature immunology

A r t i c l e s

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 inthe 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 ofadministering-GalCer together with haptenated proteins or influ-

enza virus peptides17,18. The provision of noncognate help byiNKT

cells to protein-reactive B cells has been shown to lead to humoralmemory responses, plasma-cell development, affinity maturation and

long-term maintenance of antibody responses17,18. Although cognateiNKT cell help has been demonstrated for B cells3, the outcome for

B cells after cognate help is unknown at present. Here we found thatcognate iNKT cell help for lipid antigenspecific 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 cellderived inter-

leukin 21 (IL-21). However, cognate iNKT cell help failed to driveclassical T celldependent aspects of humoral responses, includingthe humoral memory response and population expansion of antigen-

specific antibody-forming cells. We propose that the provision ofcognate iNKT cell help to B cells induces a constellation of traits that

is representative of a previously unknown class of B cell response: thetype 2 T celldependent 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, inwhich ~5% of B cells express a transgene encoding a BCR specific

for NP. We visualized antigen-specific extrafollicular foci in the redpulp and bridging channels of the spleen by confocal microscopy.

We identified these splenic architectural structures as clusters ofcells 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-GalCer

(Supplementary Fig. 1) or with haptenated protein antigen mixed

with lipid (NP l inked to keyhole limpet hemocyanin (NP-KLH) plus-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 cytometry analysis of

spleens from C57BL/6 wild-type mice showed that the NP-specificIgDB220loCD138+ plasmablast B cell population had notablyexpanded in all groups, but this population was much larger in

the group immunized with NP-GalCer (Fig. 1e,f). In addition,enzyme-linked immunospot analysis showed that spleens from mice

immunized with NP-GalCer, but not those from mice immunizedwith NP-KLH plus -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). Theseresults indicated that cognate iNKT cell help to B cells resulted in a

robust early plasmablast population expansion typical of the splenicresponse to T cellindependent antigens such as NP-Ficoll.

NP-GalCer

NP-KLH + alum PBS-BSA-DMSO

NP-KLH + -GalCera

c

b

d

NP-GC

15

10

5

0

TCR+C

D1d-tet+

iNKT

cells(106)

****

***

NP-KLH

+

alum DM

SO

NP-KLH

+

-GC

h

NP-GC

0

**

**

*

IgG+a

nti-NP

spots

(per106

splenocytes)

1

2

3

4

5

NP-KLH

+

alum DM

SO

NP-KLH

+

-GC

gNP

-G

C

12.5

10.0

7.5

5.0

2.5

0

** ****

**

NP-specificCD138+

B

cells(105)

NP-KLH

+

alum DMS

O

NP-KLH

+

-GC

f

NP-GC

0.57

0.16

0.31

105

104

103

102

0 0.02

0 103

102

104

105

10.9

2.3

CD138NP-APC

16.7

17.2

NP-KLH +

-GC

NP-KLH +

alum

DMSO

lgD

B220

e

Figure 1 Stimulation o B cells with cognate antigen (lipid) or

noncognate antigen (lipid plus protein) induces splenic extraollicular

oci. (ad) Conocal microscopy o spleens obtained rom B1-8 mice

5 d ater immunization with 5 g NP-GalCer (a), 100 g NP-KLH plus 5 g -GalCer (b), 100 g

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 identiy CD138+ NP-speciic plasmablasts. Scale bars, 100 m.(e) Flow cytometry o splenic plasmablast B cells rom wild-type C57BL/6 mice immunized as in ad.

Numbers below outlined (gated) areas indicate percent NP-speciic IgD cells (let) or B220loCD138+

cells (right). (f) Summary o results in e. (g) ELISPOT analysis o NP-speciic IgG-secreting splenic

B cells rom mice immunized as in ad. (h) Flow cytometry o TCR+iNKT cells binding the CD1d tetramer

(CD1d-tet+), rom the mice in f. Each symbol represents an individual mouse; small horizontal lines indicate the

mean (fh). *P 0.05 and **P 0.001 (unpaired two-tailed t-test (f,h) or Mann-Whitney test (g)). Data are

representative o two independent experiments with three to our mice per group ( ad) or are representative

o (e,g) or pooled rom (f,h) two independent experiments with ive mice per group.


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Next we compared GC formation in the spleen after immunization

with lipid alone or lipid plus protein. Immunofluorescence labelingshowed that at 12 d into the response, B1-8 mice with a greater fre-

quency of NP-specific B cells, immunized with either NP-GalCer orNP-KLH plus -GalCer, developed frequent cell clusters positive for

GL7, an antibody clone specific for an as-yet-unidentified T cell andB cellactivation 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-KLHplus -GalCer and 21 GCs per spleen section in mice immunized with

NP-GalCer (Fig. 2e), a notable but not significant difference. Wealso counted, by flow cytometry, splenic B cell and iNKT cell popula-

tions 12 d after immunization (Fig. 2fi). All groups of immunizedC57BL/6 wild-type mice had a greater number and frequency of NP-

specific (B220+CD95+GL7+) GC B cells than did mice immunized withvehicle, but the GC B cells were significantly more numerous in mice

immunized with NP-KLH and-GalCer than in mice immunized with

NP-GalCer (Fig. 2f). The number of splenic iNKT cells positivelyidentified by the CD1d tetramer was similarly higher, showing greaterpopulation expansion after immunization with lipid plus protein but

not after immunization with lipid only (Fig. 2i). These results suggestedthat when iNKT cells recognizing the lipid component of NP-GalCer

provided cognate help to NP-specific B cells, the B cells were inducedto produce GCs, although they were smaller than the GCs derived after

noncognate iNKT cell help. Consistent with those data, the numberof NP-specific IgG-producing cells was significantly greater in both

groups of protein-immunized mice than in lipid-immunized mice atthis 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. Incontrast, cognate iNKT cell help recruited by a lipid-only immunization

strategy induced smaller GCs and was unable to sustain antigen-specificB 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-GalCera c NP-KLH + alum d PBS-BSA-DMSOb NP-KLH + -GalCer

e50 *

40

30

20

GCspersp

leensec

tion

10

0

NP-GC

NP-KLH

+

alum DM

SO

NP-KLH

+

-GC

f

8 ****

** **6

4

2

0

B220+CD95+GL7+NP-spec

ific

germ

ina

lcen

ter

Bce

lls

(10

4)

NP-GC

NP-KLH

+

alum DM

SO

NP-KLH

+

-GC

g

****

**

lgDCD38+

NP-spec

ific

Bce

lls

(10

4)

40

30

20

10

0

NP-GC

NP-KLH

+

alum DM

SO

NP-KLH

+

-GC

h

**

**

**

lgG+

an

ti-N

Pspo

ts

per

10

6s

plenocy

tes

100

80

60

40

20

0

NP-GC

NP-KLH

+

alum DM

SO

NP-KLH

+

-GC

i**

*

CD1d-te

t+TCR+

iNKTc

ells

(10

6)

4

3

2

1

0

NP-GC

NP-KLH

+

alum DM

SO

NP-KLH

+

-GC

Figure 2 Stimulation o B cells with cognate antigen (lipid) or noncognate antigen (lipid plus protein) leads

to the development o splenic GCs. (ad) Conocal microscopy o spleens obtained rom B1-8 mice 12 d

ater immunization with 5 g NP-GalCer (a), 100 g NP-KLH plus 5 g -GalCer (b), 100 g NP-KLH plus

alum (c) or PBS-BSA-DMSO (d), and labeled with GL7 (green), anti-CD3 (blue) and anti-B220 (red). Scalebars, 500 m. (e) Total GL7+ GCs per spleen section o the mice as in ad. (f,g) Flow cytometry o B220+

CD95+GL7+ GC cells (f) and IgDCD38+ NP-speciic B cells (g) rom spleens o C57BL/6 wild-type mice at

day 12 ater immunization as in ad. (h,i) ELISPOT analysis o NP-speciic IgG-secreting splenic B cells (h)

and low cytometry o TCR+CD1d tetramerpositive iNKT cells (i) rom wild-type C57BL/6 mice immunized

as in ad. Each symbol represents an individual mouse; small horizontal lines indicate the mean (ei).

*P 0.05 and **P 0.001 (Mann-Whitney test). Data are representative o two to three independent experiments with duplicate sections rom our

mice per group (ad) or are representative o (f,h,i) or pooled rom (e,g) two to three independent experiments with our to ive mice per group.

7 61 7 61 7 61 7 610

0.5

1.0

1.5

2.0*

NP-KLH +alum

NP

-GalCer

NP-KLH +

-GalCerNP68-Ficoll

* *

NP4/NP25

Time (d)

Figure 3 Cognate and noncognate iNKT cell help induces antigen-speciic

antibody ainity maturation. ELISA o ainity maturation in serum rom

C57BL/6 wild-type mice immunized with 100 g NP-KLH plus alum,

0.5 g NP-GalCer, 100 g NP-KLH plus 0.5 g -GalCer, or 30 g

NP68-Ficoll, collected 7 d ater the primary challenge (day 7) and

7 d ater the secondary boost (day 61) and assessed on plates coated

with BSA conjugated to NP (our molecules (NP4-BSA) or twenty-ive

molecules (NP25-BSA)), presented as the ratio o 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 o three independent

experiments with eight to ten mice per group.


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A r t i c l e s

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-linkedimmunosorbent 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 primaryimmunization (day 7) with serum from the same mice collected

7 d after a secondary boost (day 61), we found that both cognate andnoncognate lipid antigens induced a significant increase in antibody

affinity after a boost immunization (Fig. 3). As expected, the knownT celldependent antigen NP-KLH in alum induced antibodies of

higher affinity after a secondary boost, whereas the well-describedT cellindependent 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 af finity 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-specificB cells21,22. Notably, mature iNKT cells are reported to share many

of the characteristics of traditional protein-specific TFH cells23; that

is, they migrate in response to the chemokine CXCL13 (via the

chemokine receptor CXCR5) but not CCL21 (via CCR7) 24, expressICOS (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 lipidspecific and noncognate lipidenhanced

antibody responses in this system. We immunized IL-21R-

deficient mice and wild-type mice with lipid antigen (NP-

GalCer), protein antigen (NP-KLH in alum) or protein plus lipidantigens (NP-KLH plus -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 cellindep endent antigen

NP-Ficoll induced IL-21-independent IgG, with no differencesbetween IL-21R-deficient and wild-type mice. In all groups tested,

there were no consistent differences between IL-21R-deficient andwild-type mice in anti-NP IgM titers (Fig. 4b). These data sug-

gested that IL-21 was required for antibody class switching, notmerely for antibody production. We confirmed IL-21 expression

byiNKT cells by real-time RT-PCR at early time points (days 57)and later time points (days 1113) after the administration of

0.5 g NP-GalCer (Fig. 4c).Next we sought to determine whether iNKT cells were the critical

source of IL-21. To address this, we generated mixedbone marrowchimeras in which we selectively deleted Il21 in iNKT cells. We created

these chimeras by reconstituting irradiated J18-deficient hosts with a

mixture of 25% IL-21-deficient bone marrow and 75% J18-deficientbone marrow. We created controls with IL-21-sufficient iNKT cells

through the use of J18-deficient hosts reconstituted with a mixtureof 25% wild-type bone marrow and 75% J18-deficient bone marrow.

Immunizing these mice with NP-KLH in alum, NP-KLH plus -GalCer or the lipid NP-GalCer showed that only cognate iNKT

cell help depended entirely on iNKT cellderived 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-K

LH+

alum

NP6

8-Ficoll

100

101

102

103

104

WT (day 7)

IL-21R-KO (day 7)

* ** **

IgGa

nti-NIP(g/ml)

NP

-GC

NP-KL

H+

-G

C10

0

101

102

103

104 WT (day 7)

IL-21R-KO (day 7)

**

IgMa

nti-NIP(g/ml)

NP-KL

H+

alum

NP6

8-Ficoll

NP

-GC

NP-KL

H+

-G

C10

0

101

102

103

104 WT (day 14)

IL-21R-KO (day 14)

* * *

NP-KL

H+

alum

NP6

8-Ficoll

NP

-GC

NP-KL

H+

-G

C

IgGa

nti-NIP(g/ml)

100

101

102

103

104 WT (day 21)

IL-21R-KO (day 21)

* *

NP-KL

H+

alum

NP6

8-Ficoll

NP

-GC

NP-KL

H+

-GC

IgGa

nti-NIP(g/ml)

c

Ctrl 1

57

111

30

10

20

30

40 **

*

Time (d)

IL-21mRNA(relative)

100

101

102

103

104

100

101

102

103

104WT (day 14)

IL-21R-KO (day 14)

WT (day 21)

IL-21R-KO (day 21)

*

NP-KL

H+

alum

NP6

8-Ficoll

NP

-GC

NP-KL

H+

-GC

NP-KL

H+

alum

NP6

8-Ficoll

NP

-GC

NP-KL

H+

-G

C

IgMa

nti-NIP(g/ml)

IgMa

nti-NIP(g/ml)

Figure 4 IL-21R signaling is required or cognate iNKT cellmediated anti-NP responses. (a,b) Titers o NP-speciic IgG (a) and

IgM (b) in serum obtained rom C57BL/6 wild-type (WT) mice and IL-21R-deicient (IL-21R-KO) mice on days 0, 7, 14 and

21 ater immunization with 0.5 g NP-GalCer, 100 g NP-KLH plus 0.5 g -GalCer, 100 g NP-KLH plus alum, or 30 g

NP68-Ficoll. *P 0.05 and **P 0.001, wild-type versus IL-21R-deicient (Mann-Whitney test). (c) Real-time RT-PCR analysis

o IL-21 mRNA expression in iNKT cells sorted by low cytometry (as TCR+CD19 CD1d tetramerpositive iNKT cells) rom

mice transgenic or expression o V14, at 1 d, 1 week (days 57) and 2 weeks (days 1113) ater intraperitoneal immunization

with NP-GalCer (0.5 g per mouse) or PBS-BSA-DMSO; results are presented relative to the expression o mRNA rom the

housekeeping gene GAPDH. *P 0.001 (Mann-Whitney test). Data are rom two to ive independent experiments with three to

ive mice per group in each (a,b) or are pooled rom two to three experiments with 511 mice per group (c; mean and s.e.m.).

NP

-GC

NP-KL

H+

-GC

NP-KL

H+

alum

NP

-GC

NP-KL

H+

-GC

NP-KL

H+

alum

NP

-GC

NP-KL

H+

-GC

NP-KL

H+

alum

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104WT (day 8)

IL-21-KO (day 8)

*

IgGa

nti-NIP(g/ml)

WT (day 14)

IL-21-KO (day 14)

WT (day 21)

IL-21-KO (day 21)ba c

*

IgGa

nti-NIP(g/ml)

IgGa

nti-NIP(g/ml)

Figure 5 IL-21 produced by iNKT cells is

required or cognate lipid antigen help.

(ac) NP-speciic IgG titers in blood rom mixed

bone marrow chimeras with an iNKT cell compart-

ment able (wild-type) or unable (IL-21-deicient)

to produce IL-21, obtained on day 8 (a), day 14

(b) or day 21 (c) ater immunization with 0.5 g

NP-GalCer, 100 g NP-KLH plus 0.5 g

-GalCer, or 100 g NP-KLH plus alum.

*P 0.05, wild-type versus IL-21-deicient

(Mann-Whitney test). Data are rom two

independent experiments with three to ive mice

per group in each experiment (mean and s.e.m.).


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A r t i c l e s

cells only after immunization with the cognate iNKT cell antigenNP-GalCer (Fig. 5). Thus, noncognate iNKT cell help elicited by

NP-KLH plus -GalCer required IL-21, but it did not need to comefrom 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 generateB cell memory responses similar to noncognate iNKT cell help. To

address this, we immunized wild-type C57BL/6 mice with eitherNP-GalCer or NP-KLH plus -GalCer. Using classical CD4+

T cell help as a positive control, we immunized some mice withNP-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 secondaryintraperitoneal challenge of lipid antigen or protein antigen in PBS.

As expected, boosting of mice with NP-KLH in PBS after previousimmunization with NP-KLH in alum resulted in a distinct memory

response (Fig. 6a); that is, the anti-NP titers after the boost weremuch higher than the antibody titers that resulted from the initial

primary challenge. Titers after the boost were also much higher thanthe antibody titers in age-matched mice that received their primary

protein antigen in alum challenge on day 177 (Fig. 6a). Mice that

received NP-GalCer in vehicle as a primary challenge and again asa secondary boost developed the same antibody titers after the boostas those of mice that received a primary challenge with NP-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 anintravenous route (Fig. 7), a protocol more commonly used to

demonstrate anti-protein memory responses. The response to lipidantigen was most similar to the anti-NP response generated by the

T cellindependent antigen NP-Ficoll (Fig. 7b). We obtained similarresults after challenge and boost with higher doses of lipid antigen

(5 g per mouse; Supplementary Fig. 2), which indicated that antigenavailability 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 samewhether the adjuvant used is a lum or the lipid -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 demonstratedthat 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 viaIL-21R. However, B cells that received noncognate iNKT cell help

made a greater humoral memory response after rechallenge, whereasthose B cells that received cognate iNKT cell help made a secondary

response of the same magnitude as the primary response. Our resultsare consistent with other studies of noncognate iNKT cell help17,18

and support the proposal that iNKT cells are memory-like innatelymphocytes 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 specializesin helping lipid-specific B cells to generate GC responses. In response

to immunization with protein antigens, IL-21 from conventional TFHcells acts directly on GC B cells to support plasma-cell differentia-

tion2628. Data from our bone marrowchimera studies demonstratedthat iNKT cells provided cognate lipidspecific T cell help through the

production of IL-21. Thus, iNKT cells are able to function in part asiNKTFH 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-GalCer and the noncognate mixture of NP-KLH plus-GalCer stimulated similar antigen-specific extrafollicular foci and

50 60 70 80

0

2

4

6

PBS; PBSNP-KLH + alum; NP-KLH

NP-KLH + -GalCer; NP-KLHPBS; NP-KLH + alum

PBS; NP-KLH + -GalCer

**

**

** *

*

** **

Time (d)

IgGa

nti-NIP(mg/ml)

a b

50 60 70 80

0

0.2

0.4

0.6

0.8

1.0

PBS; PBS

NP-GalCer; NP-GalCer

NP68-Ficoll; NP68-FicollPBS; NP-GalCer

PBS; NP68-Ficoll

Time (d)

IgGa

nti-NIP(mg/ml)

Figure 7 Only noncognate iNKT cell help induces an antibody memory

response ater rechallenge on day 46. Anti-hapten ELISA o NP-speciic

IgG in blood obtained (periodically up to day 45) rom C57BL/6

wild-type mice immunized intraperitoneally on day 0 with PBS, 2.2 g

NP-KLH in alum or 2.2 g NP-KLH plus 0.5 g -GalCer (a), or with PBS,

0.5 g NP-GalCer, or 30 g NP68-Ficoll (b), then given a secondary

intravenous boost on day 46 with PBS or the same dose o NP-KLH or

NP-KLH plus -GalCer, or intraperitoneal boost o NP-KLH plus alum (a),

or intravenous boost o PBS, NP-GalCer or NP68-Ficoll (b), ollowed by

additional sampling o blood on days 3, 7, 14 and 29 ater the boost

(key: primary challenge; secondary boost); ull time course, Supplementary

Figure 4. *P 0.05 and **P 0.001, versus the corresponding primary

immunization group (Mann-Whitney test). Data are rom one experiment

with eight mice per group (mean s.e.m.).

b

170 180 190 200

0

50

100

150

200

PBS + alum; PBS + alumNP-GalCer; NP-GalCer

Time (d)

IgGa

nti-NIP(g/ml)

1

2

PBS; NP-GalCer

170 180 190 200

0

0.5

1.0

1.5

2.0

PBS + alum; PBS + alumNP-KLH + alum; NP-KLHPBS; NP-KLH + alum

*

**

Time (d)

IgGa

nti-NIP(mg/ml)

a

Figure 6 Only noncognate iNKT cell help induces antibody memory

response ater day 177 rechallenge. Anti-hapten ELISA o NP-speciic

IgG in blood (obtained periodically up to day 166) rom C57BL/6

wild-type mice immunized (1) intraperitoneally on day 0 with PBS plus

alum, 2.2 g NP-KLH plus alum or PBS alone (a), or PBS plus alum,

0.5 g NP-GalCer or PBS alone (b), then given a secondary (2)

intraperitoneal boost on day 177 (arrow) o PBS plus alum, 2.2 g

NP-KLH, or NP-KLH plus alum (a), or an intraperitoneal boost o PBS

plus alum or 0.5 g NP-GalCer, ollowed by additional sampling o

blood on days 3, 7, and 14 ater the boost (key: primary challenge;

secondary boost); ull 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 rom one experiment

with nine mice per group (mean s.e.m.).


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A r t i c l e s

numbers of GCs. However, the B cell outcome of cognate help from

iNKT cells was notably different from the outcome after help fromconventional CD4+ T cells that benefited from enhanced antigen-

presenting function secondary to iNKT cell activation. In part, iNKTcell help for cognate haptenated lipid did not entirely mimic extrafol-

licular focidominated responses to T cellindependent antigens butinstead reflected a response based more on T celldependent extra-

follicular foci and GCs. The noncognate-immunization approachwith NP-KLH plus -GalCer did stimulate more GC B cells than did

strictly lipid immunization but resulted in similar numbers of totalGCs. That finding was consistent with our observation that spleens

from mice immunized with NP-KLH plus -GalCer had relatively

larger GCs, which suggested that in this case, activation of iNKTcells functioned more as an adjuvant than stimulating a lipid-specific

response in parallel. Given that mice immunized with NP-GalCeror with NP-KLH plus -GalCer induce affinity maturation (probably

via GCs) and have similar numbers of memory-phenotype CD38+antigen-specific B cells31, it is possible that such GCs are present

but inadequate or prematurely involute. Many elements have beenidentified as being critical for sustaining a T celldependent 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 criticalaspect of GC persistence, particularly later in a GC response when

the way in which antigen is presented, such as in the form of immunecomplexes, may be different33. Expression of the costimulatory mol-

ecule PD-1 on T cells has also been shown to be important for thesurvival of B cells in GCs and subsequent long-lived plasma cell out-

put without affecting affinity maturation34. Heterogeneity in memoryB cell development may also affect the humoral recall response 35.

The contribution of these factors to iNKT cellB cell interactionsand their relevance to iNKT cellinduced formation of GCs remain

to be determined.Finally, iNKT cells have been proposed to localize outside of the splenic

white pulp under homeostatic conditions

36

. Thus, how iNKT cellB cellinteractions 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 2T cellindependent antigens38. Marginal zone B cells express abundant

CD1d and secrete mainly IgM and IgG3 (ref. 39), the antibody isotypesproduced in response to the pure synthetic lipid antigen NP-GalCer3.

Thus, they may also be one of the main B cell subpopulations to receiveboth cognate and noncognate iNKT cell help16.

In conclusion, we propose lipid-specific, type 2 T celldependentresponses 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 2T celldependent responses include the formation of extrafollicularfoci 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 whenrapid iNKT cell help could provide a unique advantage. Future studies

should investigate humoral immunity to live pathogens induced bycognate and noncognate lipid antigens.

METHODS

Methods and any associated references are available in the online versionof the paper at http://www.nature.com/natureimmunology/.

Note: Supplementary information is available on the Nature Immunology website.

AcKNoWLEDGMENTSWe thank M. Nussenzweig (Rockefeller University) for B6.SJL B1-8hi mice;M. Exley (Dana Farber Cancer Institute) for C57BL/6 V14-transgenic mice andC57BL/6 J18-deficient mice; K. Rajewsky (Center for Blood Research) forB1-8f mice; M. Rincon (University of Vermont) for IL-21-deficient mice; andthe US National Institutes of Health Tetramer Core for mouse CD1d-PBS57tetramers 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 RoyalSociety (G.S.B.), The Wellcome Trust (084923/B/08/Z to G.S.B.) and the MedicalResearch Council (G.S.B.).

AUTHoR coNTRIBUTIoNSI.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. didexperiments, 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 conceptualadvice and E.A.L. initiated and directed the research, did experiments, analyzed thedata and wrote the manuscript.

coMPETING FINANcIAL INTERESTSThe authors declare no competing financial interests.

Pubhd onn a hp://www.nau.om/naummunoogy/.

rpn and pmon nfomaon avaab onn a hp://www.nau.om/pn/ndx.hm.

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nature immunology doi:10.1038/ni.2172

ONLINE METHODSMice. 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 V14-transgenic mice with a greater

frequency of iNKT cells 41 and C57BL/6 J18-deficient mice lacking J18+

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 specificfor 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 450IgD (11-26c.2a; BD), phycoerythrin-indotricarbocyanine

anti-B220 (RA3-6B2; Biolegend), phycoerythrinanti-Fas (Jo2; BD), fluores-

cein isothiocyanateGL7 (GL7; BD), fluorescein isothiocyanateanti-CD38

(90; BD), phycoerythrinanti-CD138 (281-2; BD), biotin-IgG1 (A85-1;BD), streptavidin-allophycocyanin-indotricarbocyanine (BD) and Pacific

Blueanti-TCR (H57-597; Biolegend). The iNKT cells were identified with

tetramers of mouse CD1d-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 J18-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% J18-deficient bone marrow mixed with

25% wild-type bone marrow, or 75% J18-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 ofiNKT cell, T cell and B cell lineages in the

spleen and liver were confirmed by flow cytometry after 7 weeks.

Antigens, immunization and serum collection. NP-KLH, NP-BSA, KLH,

NP68-Ficoll (Biosearch Technologies), NP-GalCer and -GalCer (synthe-

sized by N.V. and G.S.B. as described3) were administered intraperitoneally

in a volume 200 l unless noted otherwise. Immunizations included 2.2 g or

100 g protein precipitated in alum or suspended in PBS plus 0.1% (wt/vol)

BSA, or 0.55 g 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-GalCer lipid contained a single

NP hapten per -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 g NP-KLH is the molar equivalent

of 0.5 g NP-GalCer divided by the number of haptens on the KLH protein

(20). Serum was collected retro-orbitally or submandibularly and was storedat 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 cell suspen-

sions from C57BL/6 mice were cultured in duplicate overnight at 37 C

on MultiScreen-HA ELISPOT plates (Millipore) coated with NIP 15-

BSA (Biosearch Technologies), and nonspecific binding was blocked by

incubation with a solution of 1% (wt/vol) BSA in PBS. Spots were detectedwith horseradish peroxidaseconjugated anti-mouse IgG (1030-05;

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. TCR+CD19 CD1d tetramerpositive iNKT cells were

isolated from V14-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 manufacturers 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 m in

thickness and embedded in optimum cutting temperature compound were

labeled with fluorescein isothiocyanateanti-B220 (RA3-6B2; BD) plus anti-

body to f luorescein isothiocyanateAlexa Fluor 488 (A11096; Invitrogen),

phycoerythrinanti-CD138 (281-2; BD), rabbit anti-CD3 (145-2C11; BD)

plus Alexa Fluor 647anti-rabbit (A21245; Invitrogen), phycoerythrin

anti-B220 (RA3-6B2; eBioscience), fluorescein isothiocyanateGL7 (GL7;

BD) plus antibody to f luorescein isothiocyanateAlexa 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 o antigen-receptor afnity in

T cell-independent antibody responses in vivo. Nat. Immunol.3, 399406 (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,

12851293 (1996).

42. Cui, J. et al. Requirement or V14 NKT cells in IL-12-mediated rejection o tumors.

Science28, 16231626 (1997).

43. Lam, KP., Kuhn, R. & Rajewsky, K. In vivoablation o surace immunoglobulin on

mature B cells by inducible gene targeting results in rapid cell death. Cell 90,

10731083 (1997).

44. Ozaki, K. et al. A critical role or IL-21 in regulating immunoglobulin production.

Science298, 16301634 (2002).

45. Eaton, S.M., Burns, E.M., Kusser, K., Randall, T.D. & Haynes, L. Age-related deects

in CD4 T cell cognate helper unction lead to reductions in humoral responses.

J. Exp. Med.200, 16131622 (2004).

Documents

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