Bisphosphonates Target B Cells to Enhance Humoral Immune Responses

Please cite this article in press as: Tonti et al., Bisphosphonates Target B Cells to Enhance Humoral Immune Responses, Cell Reports (2013), http://dx.doi.org/10.1016/j.celrep.2013.09.004

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Report

Bisphosphonates Target B Cellsto Enhance Humoral Immune ResponsesElena Tonti,1,2 Nereida Jimenez de Oya,1 Gabriele Galliverti,1 E. Ashley Moseman,2 Pietro Di Lucia,1 Angelo Amabile,1

Stefano Sammicheli,1 Marco De Giovanni,1 Laura Sironi,1,4 Nicolas Chevrier,3 Giovanni Sitia,1 Luigi Gennari,5

Luca G. Guidotti,1,6 Ulrich H. von Andrian,2,* and Matteo Iannacone1,2,7,*1Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, 20132 Milan, Italy2Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA3Harvard University, FAS Center for Systems Biology, Cambridge, MA 02138, USA4Department of Physics, University of Milano Bicocca, 20126 Milan, Italy5Department of Medicine, Surgery and Neurosciences, University of Siena, 53100 Siena, Italy6Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, CA 92037, USA7Vita-Salute San Raffaele University, 20132 Milan, Italy

*Correspondence: [email protected] (U.H.v.A.), [email protected] (M.I.)

http://dx.doi.org/10.1016/j.celrep.2013.09.004

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative WorksLicense, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are

credited.

SUMMARY

Bisphosphonates are a class of drugs that are widelyused to inhibit loss of bone mass in patients. Weshow here that the administration of clinically rele-vant doses of bisphosphonates in mice increasesantibody responses to live and inactive viruses, pro-teins, haptens, and existing commercial vaccine for-mulations. Bisphosphonates exert this adjuvant-likeactivity in the absence of CD4+ and gd T cells, neutro-phils, or dendritic cells, and their effect does not relyon local macrophage depletion, Toll-like receptorsignaling, or the inflammasome. Rather, bisphospho-nates target directly B cells and enhance B cellexpansion and antibody production upon antigenencounter. These data establish bisphosphonatesas an additional class of adjuvants that boosthumoral immune responses.

INTRODUCTION

Bisphosphonates (BPs) are small-molecule inhibitors of bone

resorption that are clinically approved for the treatment of skel-

etal diseases such as osteoporosis and Paget’s disease of

bone (Favus, 2010; Scott andGershon, 1970); these compounds

represent a large family of drugs that include first-generation

clodronate (CLD) and etidronate (ETD) and nitrogen-containing

alendronate (ALD), pamidronate (PMD), zoledronate (ZLD), and

neridronate (NRD). Upon liposome encapsulation, BPs like

CLD have been widely used to experimentally deplete tissue-

resident phagocytes in rodents (Moseman et al., 2012; Van

Rooijen and Sanders, 1994). A few studies in BP-treated mice

unexpectedly noted increased antigen (Ag)-specific humoral

immune responses (Gonzalez et al., 2010; Iannacone et al.,

2010; Norton et al., 2011); herein, we set out to systematically

dissect the mechanistic basis for this activity.

RESULTS AND DISCUSSION

As the majority of the above-mentioned studies utilized subcuta-

neously administered BP-encapsulated liposomes prior to local

viral challenge, we initially chose the same experimental setup to

ask whether liposome encapsulation is required to increase anti-

body (Ab) responses. To this end, footpads of C57BL/6 mice

were injected with PBS, PBS liposomes (PBS-Lip), CLD lipo-

somes (CLD-Lip), or CLD prior to infection in the same footpad

with vesicular stomatitis virus (VSV), a prototypic cytopathic

virus that induces an early T-independent immunoglobulin M

(IgM) response followed by a T-dependent immunoglobulin G

(IgG) response (Hangartner et al., 2006). When compared to

PBS-injected mice, mice that received CLD exhibited up to

100-fold higher neutralizing antibody (nAb) titers toward VSV

(Figure 1A), and this occurredwhenCLDwas administered either

prior to or concomitantly with the Ag (Figure S1A). Importantly,

free CLD was as effective as CLD-Lip (Figure 1A), it exhibited a

dose-dependent effect (Figure S1B), and its adjuvant activity

was shared by other BPs that are currently in clinical use,

including ETD, PMD, and ALD (Figure 1B).

Subcutaneously administered CLD also increased Ab titers

against inactive VSV, soluble proteins (ovalbumin [OVA]),

haptens (NP-CGG), and the adjuvant-containing formulation

Engerix-B (an approved vaccine against hepatitis B virus)

(Figures 1C–1F); similar results were observed when CLD

was administered intramuscularly along with the hemoaggluti-

nin/neuroaminidase subunits of the human influenza virus

A/NewCaledonia/20/99 (H1N1; Figure 1G).

CLD treatment increased both neutralizing IgM and IgG

responses against VSV (Figures S1C–S1E) without altering the

subtype of Ag-specific IgG induced upon immunization (Fig-

ure S1F), and this correlated with the total number of CD138+

Cell Reports 5, 1–8, October 31, 2013 ª2013 The Authors 1

mailto:[email protected]

mailto:[email protected]


https://www.researchgate.net/publication/7279897_Antiviral_antibody_responses_The_two_extremes_of_a_wide_spectrum?el=1_x_8&enrichId=rgreq-4560cf7d-976c-413d-a258-b8ea4ccafc75&enrichSource=Y292ZXJQYWdlOzI1Nzc1MzY2NTtBUzoxMDI4NzEyNDQ3Mzg1NjBAMTQwMTUzNzgyNDExNA==

https://www.researchgate.net/publication/47810767_Bisphosphonates_for_Osteoporosis_REPLY?el=1_x_8&enrichId=rgreq-4560cf7d-976c-413d-a258-b8ea4ccafc75&enrichSource=Y292ZXJQYWdlOzI1Nzc1MzY2NTtBUzoxMDI4NzEyNDQ3Mzg1NjBAMTQwMTUzNzgyNDExNA==

https://www.researchgate.net/publication/17733003_Determination_of_total_and_mercaptoethanol-resistant_antibody_in_the_serum_sample?el=1_x_8&enrichId=rgreq-4560cf7d-976c-413d-a258-b8ea4ccafc75&enrichSource=Y292ZXJQYWdlOzI1Nzc1MzY2NTtBUzoxMDI4NzEyNDQ3Mzg1NjBAMTQwMTUzNzgyNDExNA==

25Days after VSV

CLD CLD-Lip

PBS-Lip PBS

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D E F

G H I

Figure 1. Bisphosphonates Increase Antibody Responses to Live and Inactive Viruses, Proteins, Haptens, and Existing Commercial Vaccine

Formulations

(A) VSV nAb titers in the serum of C57BL/6mice that were footpad injectedwith PBS, PBS-liposomes (PBS-Lip), clodronate (CLD), or clodronate liposomes (CLD-

Lip) prior to VSV infection in the same footpad (see Experimental Procedures for details). n = 5 per group. Black asterisks, PBS versus CLD-Lip; red asterisks, PBS

versus CLD. Results are representative of more than ten independent experiments.

(B) VSV nAb titers 7 days p.i. in the serum of C57BL/6 mice that were footpad injected with CLD, etidronate (ETD), pamidronate (PMD), or alendronate (ALD) prior

to VSV infection in the same footpad. n = 5 per group; results are representative of three independent experiments.

(C) VSV nAb titers at day 14 postimmunization in the serum of C57BL/6 mice that were footpad injected with PBS or CLD prior to paraformaldehyde-inactivated

VSV (PFA-VSV) immunization in the same footpad. n = 10 per group; results are representative of three independent experiments.

(D) Ovalbumin (OVA)-specific IgG titers at day 14 postimmunization in the serum of C57BL/6 mice that were footpad injected with PBS or CLD prior to OVA

immunization in the same footpad. n = 4 per group; results are representative of three independent experiments.

(E) 4-Hydroxy-3-nitrophenylacetyl (NP)-specific IgG titers at day 14 postimmunization in the serum of C57BL/6 mice that were footpad injected with PBS

or CLD prior to NP-chicken gamma globulin (CGG) immunization in the same footpad. n = 5 per group; results are representative of two independent

experiments.

(F) HBsAb titers (mIU/ml) 21 days p.i. in the serum of C57BL/6 mice that were injected in the footpad with PBS or CLD prior to Engerix-B (an approved vaccine

against hepatitis B virus) immunization in the same footpad. n = 5 per group; results are representative of five independent experiments.

(G) H1N1-specific IgG titers in the serum of C57BL/6mice that were injected intramuscularly with PBS or CLD prior to intramuscular immunization with H1N1. n = 5

per group; results are representative of two independent experiments.

(legend continued on next page)


2 Cell Reports 5, 1–8, October 31, 2013 ª2013 The Authors

A

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CD40L-/-+PBS CD40L-/-+CLD

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D.L.

C

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WT+PBS WT+CLD TCRδ-/-+PBSTCRδ-/-+ CLD

E

PBS CLD DT+PBSDT+CLD


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* ***

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***

MHC-II-/-+PBS MHC-II-/-+CLD

Figure 2. Bisphosphonates Increase Anti-

body Responses in the Absence of CD4+

and gd T Cells, Neutrophils, or Dendritic

Cells and Their Effect Does Not Require

Local Macrophage Depletion

(A) VSV neutralizing IgM titers in the serum of

MHC-II�/� mice that were footpad injected with

PBS or CLD immediately prior to VSV infection in

the same footpad. n = 5 per group; results are

representative of three independent experiments.

(B) VSV neutralizing IgM titers in the serum of

CD40L�/� mice that were footpad injected with


the same footpad. n = 5 per group; results are

representative of two independent experiments.

(C) VSV nAb titers in the serum of C57BL/6 mice

that were injected (or not) with Gr-1 depleting Ab

(a�Gr-1; see Experimental Procedures for details)

and were subsequently footpad injected with PBS

or CLD immediately prior to VSV infection in the

same footpad. n = 6 per group; black asterisks,

PBS versus CLD; red asterisks, aGr-1+PBS versus

aGr-1+CLD; results are representative of two in-

dependent experiments.

(D) VSV nAb titers in the serum of C57BL/6 (WT)

or TCRd�/� mice that were footpad injected with


the same footpad. n = 10 per group; black aster-

isks, WT+PBS versus WT+CLD; red asterisks,

TCRd�/�+PBS versus TCRd�/�+CLD; results are


(E) VSV nAb titers in the serum of CD11c-DTR-GFP

mice that were treated (or not) with diphtheria toxin

(see Experimental Procedures for details) and

were subsequently footpad injected with PBS or

CLD immediately prior to VSV infection in the same

footpad. n = 6 per group; black asterisks, PBS

versus CLD; red asterisks DT+PBS versus

DT+CLD. Results are representative of three in-

dependent experiments.

(F) VSV nAb titers at day 14 p.i. in the serum of

C57BL/6 mice that were footpad injected with

PBS, CLD, dextran sulfate or carrageenan prior to

VSV infection in the same footpad (see Experi-

mental Procedures for details). n = 3 per group;

results are representative of three independent

experiments.

Results are expressed as mean ± SEM. ***p <

0.001; **p < 0.01; *p < 0.05; D.L., detection limit.

See also Figures S2, S3, and S4.


plasma cells recovered from draining LNs (Figure S1G). In

combination with inactivated VSV, CLD boosted the partial

protection afforded by immunization with inactivated virus

alone (Figure 1H), and its adjuvant effect lasted for at least

4 months after a single administration (Figure 1I). CLD treatment

also increased neutralizing IgM titers upon VSV infection in

MHC-II�/� mice (which lack CD4+ T cells) and in CD40L�/�

(H) Kaplan-Meier survival curves of C57BL/6 mice injected in the right footpad

equivalents of PFA-inactivated VSV and clodronate (PFA-VSV+CLD) 3 weeks prio

VSV+CLD versus PFA-VSV: p < 0.05, log-rank (Mantel-Cox) test. Results are rep

(I) VSV nAb titers 125 days p.i. in the serum of C57BL/6 mice that were footpad i

group; results are representative of two independent experiments.

Results are expressed as mean ± SEM. ***p < 0.001; **p < 0.01; *p < 0.05. See a

mice, in which T cell help for B cells is known to be compro-

mised (Renshaw et al., 1994) (Figures 2A and 2B). While

these data do not rule out a possible effect of CLD on T cells,

they indicate that CLD adjuvant activity can occur indepen-

dently of CD4+ T cell help and raise mechanistic questions

about how this adjuvant effect is mediated. To address

this issue, we systematically analyzed cellular and molecular

with PBS, 106 pfu equivalents of PFA-inactivated VSV (PFA-VSV), or 106 pfu

r to infection in the left footpad with 3.53 108 pfu of VSV. n = 12 per group; PFA-

resentative of two independent experiments.

njected with PBS or CLD prior to VSV infection in the same footpad. n = 5 per

lso Figure S1.


https://www.researchgate.net/publication/15233705_Humoral_immune_responses_in_CD40_ligand-deficient_mice?el=1_x_8&enrichId=rgreq-4560cf7d-976c-413d-a258-b8ea4ccafc75&enrichSource=Y292ZXJQYWdlOzI1Nzc1MzY2NTtBUzoxMDI4NzEyNDQ3Mzg1NjBAMTQwMTUzNzgyNDExNA==

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Days after VSV

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*

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an Ig

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Vehicle CLD CT CT+CLD

0 2 6 8 10Weeks after immunization

4101

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OVA

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J

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* *

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an Ig

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Gene sets up-regulated in CLD

p-value 10-5 10-6

Extracellular region (GO:0005576)Immunoglobulin complex, circulating (GO:0042571)Humoral immune response (GO:0006959)Extracellular space (GO:0005615)Extracellular region part (GO:0044421)

0h 8h TERM

Figure 3. Bisphosphonates Directly Target B Cells and Enhance B Cell Expansion and Antibody Production upon Antigen EncounterIndependently of Toll-like Receptor Signaling or the Inflammasome

(A) Gene enrichment analysis of clodronate-dependent genes from microarray measurements of whole lymph node. Shown are the Gene Ontology (GO) terms

(rows) statistically enriched among the 156 genes with at least a 2-fold increase in expression at 0 and 8 hr (columns) after VSV infection in clodronate (CLD)-

treatedmice compared to control (PBS). Enriched GO term categories include BP (biological process) and CC (cellular component). Green: p value < 10�6; white:

p value < 10�4; gray: no enrichment. See Experimental Procedures for details.

(B) VSV nAb titers in the serum of DHLMP2A mice that were intravenously transferred with PBS- or CLD-treated VI10Yen B cells 24 hr prior to footpad VSV

infection. n = 6 per group; results are representative of two independent experiments.

(C) Quantification of the total number of VI10Yen B cells recovered from the spleen of the mice described in (B) 8 days after VSV.

(D) Quantification of the total number of cells recovered 48 hr upon in vitro culture of purified VI10Yen B cells with PBS, VSV, or 10 nM clodronate and VSV.

(E) VSV nAb titers in the serum of C57BL/6 (WT) or MyD88�/�/Trif�/� mice that were footpad injected with PBS or CLD immediately prior to VSV infection in the

same footpad. n = 4 per group; black asterisks, WT + PBS versus WT + CLD; red asterisks MyD88�/�/Trif�/�+PBS versus MyD88�/�/Trif�/�+CLD; results are


(legend continued on next page)




changes induced by BPs at the site of injection and at the level

of the draining LN.

First, we examined the BP injection site. In agreement with

previously published data (Iannacone et al., 2010; Norton et al.,

2011), footpad injection of CLD alone induced a local inflamma-

tory infiltrate composed mostly of Gr-1+ neutrophils and inflam-

matory monocytes (Figure S2A). Elimination of this infiltrate by

systemic anti-Gr-1 treatment (Figures S2A and S2B), however,

did not prevent CLD from increasing nAb titers upon VSV infec-

tion (Figure 2C), indicating that CLD adjuvant activity does not

require Gr-1+ leukocytes.

Next, we considered the role of two additional footpad-resi-

dent cell types: gd T cells and conventional dendritic cells

(DCs). gd T cells can provide help to B cells (Pao et al., 1996;

Wen et al., 1996) and are reportedly activated by CLD (Schilbach

et al., 2001) or ALD (Thompson et al., 2010). Footpad CLD or ALD

injection did not increase the local number of gd T cells (Fig-

ure S2C), and VSV infection of TCRd�/� mice (Itohara et al.,

1993) revealed that CLD and ALD adjuvant activity is maintained

in the absence of these cells (Figures 2D and S2D). We then

reasoned that BPs might act on DCs, because DCs can present

Ag to B cells (Qi et al., 2006) and most known adjuvants are

thought to function by activating DCs (Coffman et al., 2010).

Incubating DCs with CLD in vitro did not alter expression of acti-

vation markers, such as CD86 and CD40 (Figures S3A and S3B),

and the systemic elimination of these cells in vivo (Figure S3C)

did not significantly inhibit CLD adjuvant activity. Indeed, CLD

administration in DC-depleted mice challenged with VSV re-

sulted in nAb titers that, by day 7 postinfection (p.i.), were com-

parable to those observed in DC-competent animals subjected

to the same procedures (Figure 2E). It is worth noting that, inde-

pendently of CLD treatment, DC depletion caused a transient

reduction in nAb titers at day 4 p.i., consistent with a role of

DC in early B cell activation (Qi et al., 2006; Scandella et al.,

2007). Overall, these data indicate that CLD adjuvant activity

does not require conventional DCs.

We next analyzed the draining LN. The only quantitative

change in LN cellular composition detectable upon CLD treat-

ment was the depletion of CD169+ macrophages lining the sub-

capsular and medullary sinuses (Figures S4A and S4B). We

found it surprising that BPs increase Ab responses despite

depleting subcapsular sinus macrophages, because these cells

were recently identified as critical Ag-presenting cells for B cell

responses (Carrasco and Batista, 2007; Junt et al., 2007; Phan

et al., 2007). To clarify these seemingly contradictory observa-

tions, we sought alternative strategies to deplete LN macro-

phages independently of BP administration. These strategies

(F) VSV nAb titers in the serum of C57BL/6 (WT) or caspase-1�/�mice that were in

same footpad. n = 10 per group; black asterisks, WT+PBS versus WT+CLD; red

sentative of three independent experiments.

(G) Ovalbumin (OVA)-specific IgG titers in the serum of C57BL/6mice that were im

(CT), or OVA + cholera toxin + CLD (CT + CLD). n = 5 per group; results are repr

(H) OVA-specific IgA titers in the feces of the mice described in (G).

(I) Total IgG in the sera of patients immediately prior to or at the indicated time poin

Table S2 for patient characteristics.

(J) Total IgG in the sera of patients immediately prior to or at the indicated time po

for patient characteristics.

Results are expressed as mean ± SEM. ***p < 0.001; **p < 0.01; *p < 0.05. D.L.,

(footpad dextran sulfate or carrageenan injections in wild-type

[WT] mice or footpad diphtheria toxin injection in CD11c-DTR

mice) effectively depleted LN macrophages (Figure S4C; Ianna-

cone et al., 2010), but they failed to increase nAb titers upon VSV

infection (Figures 2F and S4D). This indicates that LN macro-

phage depletion per se does not increase humoral immune

responses and suggests that CLD adjuvant activity does not

rely on LN macrophages.

To investigate the molecular basis for CLD adjuvant effect, we

next performed genome-wide mRNA profile analysis of draining

LNs from mice treated with either PBS or CLD and sacrificed

before (0 hr) or 8 hr after VSV infection (Figure S5; Table S1).

This analysis showed that a relatively small number of genes

were regulated by CLD treatment (Figures 3A, S6, S7, S8, S9,

and S10; Table S1). Most of the downregulated genes upon

CLD treatment were macrophage specific (Figures S6, S7, and

S8; Table S1), in line with the observation that CLD treatment

depleted LN macrophages (Figure S4). CLD treatment also

downregulated type I interferon (IFN-I) genes induced upon

VSV infection (Figures S7 and S8; Table S1). This is consistent

with previously published data showing that LN subcapsular

sinus macrophages are a major source of IFN-I during this infec-

tion and their depletion inhibits IFN-I gene expression (Ianna-

cone et al., 2010). Reduced IFN-I gene expression could have

also resulted from plasmacytoid DC (pDC) dysfunction, as these

cells represent an additional source of IFN-I in this system and

they are not depleted upon BP treatment (Iannacone et al.,

2010). The CLD-dependent reduced expression of the pDC-spe-

cific marker Siglec-H (Blasius et al., 2006) is also suggestive of

pDC dysfunction (Figures S6 and S7; Table S1). It is noteworthy,

however, that depletion of pDCs (which reduced IFN-I by�50%;

Iannacone et al., 2010) failed to increase Ab responses (Fig-

ure S11A), suggesting that CLD adjuvant activity does not rely

on pDCs.We also noted that some natural killer (NK) cell-specific

transcripts were reduced upon CLD treatment (Figures S6 and

S7; Table S1). Depleting these cells did not alter the capacity

of CLD to increase nAb responses upon VSV infection (Fig-

ure S11B), arguing against a role for NK cells in the adjuvant

activity of BPs.

Next, we examined genes that were upregulated by CLD

treatment. CLD induced several neutrophil-specific transcripts

(Figures 3A, S9, and S10; Table S1), in keeping with the ability

of BPs to recruit and activate these cells (Norton et al., 2011;

2012). Data previously described in Figure 2C, however,

ruled out a role for neutrophils in BP adjuvant activity. Of note,

CLD upregulated a number of B cell-specific transcripts and

gene sets associated with B cell function (Figures 3A, S9,

jected in the footpad with PBS or CLD immediately prior to VSV infection in the

asterisks, caspase-1�/�+PBS versus caspase-1�/�+CLD; results are repre-

munized orally with OVA alone (vehicle), OVA + CLD (CLD), OVA + cholera toxin

esentative of two independent experiments.

ts after a single intravenous infusion of zoledronate (ZLD) is shown. n = 11; see

ints after a single intravenous infusion of neridronate (NRD). n = 9; see Table S2

detection limit. See also Figures S5, S6, S7, S8, S9, S10, and S11.



and S10; Table S1). When compared to controls, these B cell-

related transcripts were higher in CLD-treated animals before

(Figure S9) and after (Figure S10) VSV infection, suggesting

that CLD might directly impact B cells. To directly test this pos-

sibility, we isolated B cells from VI10Yen mice (which express a

VSV-specific B cell receptor), and treated them with PBS or

CLD in vitro prior to adoptive transfer into DHLMP2A mice

(which are devoid of surface-expressed and secreted Abs). In

this experimental setup - where Abs can be produced only by

the transferred B cells—VSV infection led to nAb titers that

were higher in mice that received CLD-treated, rather than

PBS-treated, VI10Yen B cells (Figure 3B); this correlated with

the capacity of CLD to promote Ag-specific B cell expansion

both in vivo and in vitro (Figures 3C and 3D). Taken together,

these results indicate that CLD directly targets B cells to

enhance their expansion and their Ab production upon Ag

encounter. Although the molecular pathways by which CLD

and other BPs impact B cell function remain to be determined,

it is worth noting that CLD adjuvanticity did not involve the two

known signaling pathways through which most other adjuvants

are thought to act (Coffman et al., 2010) (the Toll-like receptors

and the inflammasome pathway, which rely on Myd88/TRIF and

caspase-1 for signaling, respectively). Indeed, VSV nAb titers in

CLD-treated Myd88�/�/TRIF�/� and caspase-1�/� mice were

indistinguishable from those of similarly treated WT mice (Fig-

ures 3E and 3F).

Since BPs are often taken orally by patients, we next sought

to evaluate whether these small molecules act as adjuvants

upon oral immunization in mice. As expected (Pierre et al.,

1992), the oral administration of OVA alone failed to induce a

detectable OVA-specific Ab response (Figures 3G and 3H).

By contrast, the concomitant oral administration of OVA

and CLD induced OVA-specific serum IgG and fecal immuno-

globulin A (IgA) responses (Figures 3G and 3H). Furthermore,

CLD was able to increase the OVA-specific Ab response

afforded when OVA was administered together with the

mucosal adjuvant cholera toxin (Pierre et al., 1992) (Figures

3G and 3H). These data indicate that CLD is an effective oral

adjuvant capable of increasing both systemic and mucosal

Ab responses.

Finally, we tested whether the nitrogen-containing BPs ZLD

and NRD increase Ab responses in a cohort of patients affected

by osteoporosis or Paget disease of bone (Table S2). Notably, a

transient but significant increase in total serum IgG levels was

detected for up to 3 months upon a single intravenous infusion

of BPs (Figures 3I and 3J). Although the impact of BPs on Ag-

specific Ab responses remains to be determined, this observa-

tion suggests that these drugs enhance B cell responses in

humans.

In conclusion, these data establish BPs as adjuvants that

target B cells to increase humoral immune responses. Transla-

tional evaluation of these compounds in humans should be rela-

tively straightforward, because BPs, unlike all other adjuvants,

are already widely used in the clinic as stand-alone drugs with

excellent safety profiles. Thus, BPs could be readily combined

with both existing and newly developed vaccines, especially in

settings where immune responses to a vaccine alone are weak

or where Ags are in short supply.


EXPERIMENTAL PROCEDURES

Mice

C57BL/6 and Balb/c mice were purchased from Charles River or The Jackson

Laboratory. CD11c- GFP-DTR [B6.FVB-Tg(Itgax-DTR/EGFP)57Lan/J] mice

were purchased from The Jackson Laboratory. MHC-II�/� (B6.129-H2-

Ab1tm1Doi/DoiOrl) mice were provided by P. Dellabona (San Raffaele

Scientific Institute). TCRd�/� (B6.129P2-Tcrdtm1Mom/J) mice were provided

by W. Havran (The Scripps Research Institute). MyD88�/�/TrifLps2/Lps2 mice

were provided by B. Beutler (The Scripps Research Institute). Caspase-1�/�

(B6.129S2-Casp1tm1sesh) and CD40L�/� (B6.129S2-Cd40tm1Imx/J) mice were

obtained through the Swiss Immunological Mutant Mouse Repository (Zurich,

Switzerland). DHLMP2A mice (Casola et al., 2004) were originally provided by

K. Rajewsky (Harvard Medical School) and bred five generations against

C57BL/6 mice. VI10Yenmice (Hangartner et al., 2003) were originally provided

by R. M. Zinkernagel and H. Hengartner. Bone marrow chimeras were gener-

ated by irradiation of C57BL/6 mice with 1,300 rad in split doses and reconsti-

tution with CD11c-DTR-GFP bone marrow; mice were allowed to reconstitute

for at least 8 weeks prior to use. Mice were housed under specific pathogen-

free conditions and used at 6–8 weeks of age. All experimental animal proce-

dures were approved by the Institutional Animal Committees of San Raffaele

Scientific Institute, Harvard Medical School, and The Scripps Research

Institute.

Bisphosphonate Treatment

CLD, ALD, PMD, and ETD were obtained from Sigma, dissolved in PBS, and

injected in the footpad in a volume of 20 ml. The injected amount for each

bisphosphonate was 2 mg, unless otherwise indicated. In the experiments

described in Figure 1A, 30 ml of clodronate liposomes (CLD-Lip) or PBS lipo-

somes (PBS-Lip, both provided by N. Van Rooijen) were injected in the footpad

7 days before antigen administration.

Infections and Immunizations

Mice were infected with 104 plaque-forming units (pfu) of VSV serotype Indiana

(VSV-IND), 104 pfu of VSV serotype New Jersey (VSV-NJ), or 106 pfu of

VSVeGFP (Iannacone et al., 2010). Alternatively, mice were immunized with

106 pfu equivalent of PFA-inactivated VSV (Bachmann et al., 1993). Viruses

were dissolved in 20 ml of PBS and injected into the footpad. In survival exper-

iments, mice were challenged with 3.5 3 108 pfu of VSV in the left footpad

3 weeks after PFA-VSV immunization in the right footpad. Other antigens

used for footpad immunization experiments included 4-hydroxy-3-nitro-

phenyl-chicken gamma globulin (NP-CGG, 50 mg/dose; Biosearch Technolo-

gies), ovalbumin (OVA, 100 mg/dose; Sigma), and Engerix-B (20 ml/dose given

twice 2 weeks apart; GlaxoSmithKline). Hemoagglutinin/neuroaminidase sub-

units from the human influenza virus A/NewCaledonia/20/99 (H1N1, 3 mg/dose;

provided by P. Dellabona) was injected in the anterior tibialis muscle.

For experiments involving oral immunization, mice were given vehicle or

10 mg of CLD 24 hr prior to receiving 1 mg of OVA with or without 10 mg of

cholera toxin. All treatments were administered via gavage in 0.2 ml of antacid

buffer (3% sodium bicarbonate) on days 0, 7, and 21. For the measurement of

mucosal IgA, fresh fecal pellets were collected and immediately frozen

at �20�C. Before analysis, fecal pellets were weighed and dissolved in prote-

ase inhibitor solution at a concentration of 200 mg/ml, as described previously

(Lauterslager et al., 2001).

All infectious work was performed in designated BL-2 workspaces in accor-

dance with institutional guidelines. Mice were retro-orbitally bled at the indi-

cated time points for Ag-specific Abs and measured by endpoint ELISA (Galli

et al., 2007; Tonti et al., 2012), VSV neutralization assay (Iannacone et al.,

2010), or with an HBsAb detection kit (Diagnostic Bioprobes) in accordance

with the manufacturer’s instructions. For determination of VSV-neutralizing

IgG titers, serawere incubatedwith equal volumes of 0.1M2-mercaptoethanol

in PBS for 1 hr at room temperature before dilution (Scott and Gershon, 1970).

Confocal Microscopy

Confocal microscopy analysis of popliteal LNs was performed as described

previously (Moseman et al., 2012). Sections were stained with eFluor450-

conjugated anti-B220 (RA3-6B2, eBioscience), FITC-conjugated anti-CD169


(3D6.112, AbD Serotec), and Alexa Fluor 647-conjugated anti-TCRb (H57-597,

BioLegend).

In Vivo Depletion of Neutrophils, DCs, LN Macrophages, pDCs, and

NK Cells

Neutrophils were depleted by intraperitoneal injection of 100 mg of Gr-1 Abs

(RB6-8C5, BioXCell) every 2 days, beginning 3 days prior to immunization.

DCs were depleted from CD11c-GFP-DTR / C57BL/6 bone marrow chi-

meric mice by injecting diphtheria toxin (DT, Sigma) in the footpad (50 ng) and

in the peritoneum (500 ng) every 2 days, starting the day prior to immunization.

Popliteal LN macrophages were depleted by injecting 2 mg of carrageenan

(Sigma) in the footpad every 2 days, starting 5 days prior to immunization.

Alternatively, LN macrophages were depleted by footpad injection of 1 mg

of dextran sulfate (Sigma) 5 days prior to immunization. Serum from CD11c-

DTR-GFP mice (described in Figure S3 in Iannacone et al., 2010), where LN

macrophages are depleted by a single footpad injection of DT (4 ng) 6 days

before VSV infection, were assessed for VSV neutralizing Ab titers 4 days after

VSV infection.

Serum from the mice described in Figures 3J and S8b of Iannacone et al.

(2010) (where pDCs were depleted by intravenous injection of 500 mg of

PDCA-1 depleting Ab [JF05-1C2.4.1, Miltenyi Biotec] 24 hr prior to VSV infec-

tion) were assessed for VSV neutralizing Ab titers 7 days after VSV infection.

NK cells were depleted by intravenous injection of Asialo-GM1 Abs

(Cedarlane) every 2 days beginning 3 days prior to VSV infection, as described

previously (Tonti et al., 2012).

All depletions were confirmed by flow cytometry and/or confocal

microscopy.

Tissue Digestion and Flow Cytometry

Single-cell suspensions of LNs, spleens, and footpads were generated as

described in Iannacone et al. (2010). All flow cytometry analyses were per-

formed in fluorescence-activated cell sorting buffer containing PBS with

2 mM EDTA and 2% FBS on a FACS CANTO (BD Pharmingen) and analyzed

with FlowJo software (Tree Star).

Abs used included FITC-conjugated anti-CD169 (3D6.112, AbD Serotec),

Alexa Fluor 488-conjugated and PE-Cy7-conjugated anti-B220 (RA3-6B2,

BioLegend), PE-conjugated anti-TCRd (GL3, BioLegend), and anti-CD86

(GL-1, BioLegend), PE-Cy7-conjugated anti-CD45 (30-F11, BioLegend), PB-

conjugated anti-Ly6C (HK1.4, BioLegend), APC-Cy7-conjugated anti-F4/80

(BM8, BioLegend), Alexa Fluor 647-conjugated anti-CD8 (53-6.7, BioLegend),

biotinylated anti-CD11b (M1/70, BioLegend), PE-conjugated anti-CD138

(281-2, BioLegend), PE-Cy7-conjugated anti-CD3 (145-2C11, BioLegend),

PE-Cy5-conjugated anti-NK1.1 (PK136, BioLegend), PerCP-Cy5.5-conju-

gated anti-Gr1 (RB6-8C5), APC-conjugated anti-CD40 (HM40-3, BD

Pharmingen), Alexa Fluor 488-conjugated anti-igG (A-11023, Invitrogen), PE-

Cy7-conjugated anti-CD11c (N418, eBioscience), eFluor450-conjugated

anti-CD4 (RM4-5, eBioscience). The idiotypic Ab 35.61 for detection of the

VI10 BCR in VI10Yenmice (Hangartner et al., 2003) was produced from hybrid-

oma supernatants in accordance with standard methods.

Isolation and Activation of DCs and B Cells

Splenic DCs were isolated from C57BL/6 mice that were injected subcutane-

ously 11–14 days earlier with 43 106 Flt3-ligand-secreting B16 tumor cells, as

described previously (Cavanagh et al., 2005). CD11c+ DCs were purified by

positive selection with anti-CD11c microbeads (>95% CD11c+, Miltenyi) and

cultured in the presence of 1 mg/ml LPS (E. coli 0.26:B6, Sigma) or with the indi-

cated concentrations of CLD for 48 hr prior to flow cytometry analysis.

Naive B cells from spleens of VI10Yen mice were negatively selected by

magnetic isolation with CD43 beads (Miltenyi) as described previously (Junt

et al., 2007). The purity was 98% as determined by CD19 surface staining.

B cells (106 cells/ml) were cultured in RPMI 1640 media (Lonza) supplemented

with 10% fetal bovine serum, 50 mM 2-ME (Sigma), 10 mM HEPES (Lonza),

2 mM L-glutamine (Lonza), and 100 U/ml penicillin/streptomycin with or

without 10 nM CLD. After 4 hr, VI10Yen B cells were harvested, washed three

times, and either intravenously injected into DHLMP2A recipients (107 cells/

mouse) 24 hr prior to footpad VSV infection (Figures 3B and 3C) or cultured

for 48 hr in the presence of VSV at a multiplicity of infection of 1 (Figure 3D).

Gene Expression Profiling

Whole lymph nodes were lysed in 600 ml of QIAzol (QIAGEN) reagent using the

TissueLyser II. Total RNA was extracted following the miRNeasy kit’s proce-

dure (QIAGEN), and sample quality was tested on a 2100 Bioanalyzer (Agilent).

RNAwas reverse transcribedwith the High Capacity cDNAReverse Transcrip-

tion kit (Applied Biosystems). Real time quantitative PCR reactions were per-

formed on the LightCycler 480 system (Roche) with FastStart Universal

SYBR Green Master Mix (Roche). Every reaction was run in triplicate and

GAPDH levels were used as an endogenous control for normalization. For

oligonucleotide microarray hybridization, 1 mg of RNA were labeled, frag-

mented, and hybridized to an Affymetrix Mouse Genome 430A 2.0 Array. After

scanning, the expression value for each gene was calculated with RMA

(Robust Multi-Array) normalization using R. The average intensity difference

values were normalized across the sample set. Probe sets that were absent

in all samples according to Affymetrix flags were removed. All values below

40 were floored to 40. Only probe sets that changed in two biological dupli-

cates by 2-fold or more were analyzed further in this study: (1) regulated genes

were hierarchically clustered using the software Gene-E (http://www.

broadinstitute.org/cancer/software/GENE-E/), and (2) functional enrichment

of GO terms (BP, biological processes; CC, cellular components) and KEGG

pathway enrichment analysis were performed using DAVID (http://david.

abcc.ncifcrf.gov) (Huang et al., 2009).

Patients

Total serum IgG levels were assessed in 20 patients affected by osteoporosis

or Paget disease of bone (age range 52–78 years; Table S2) that received a sin-

gle intravenous injection of 5 mg zoledronate (Aclasta, Novartis Pharmaceuti-

cals) or 200 mg neridronate (Nerixia, Abiogen Pharma). Serum samples were

obtained before and 1, 30, 90, 180, and 360 days after BP infusion. IgG levels

were quantified from stored samples by an immunodiffusion technique using

NOR Partigen immunoplates purchased from Siemens (Siemens Healthcare

Diagnostics). All experiments involving human subjects were approved by

the institutional review board of the University of Siena and performed after

obtaining informed consent from all subjects.

Statistical Analyses

Results are expressed as mean ± SEM. All statistical analyses were performed

in Prism (GraphPad Software). Means between two groups were compared

with a two-tailed t test. Means among three or more groups were compared

with one-way or two-way analysis of variance with Bonferroni’s post hoc

test. Kaplan-Meier survival curves were compared with the log-rank (Mantel-

Cox) test.

ACCESSION NUMBERS

The microarray data discussed in this publication have been deposited in

NCBI’s Gene Expression Omnibus and are accessible through GEO Series

accession number GSE50403.

SUPPLEMENTAL INFORMATION

Supplemental Information includes eleven figures and two tables and can be

foundwith this article online at http://dx.doi.org/10.1016/j.celrep.2013.09.004.

AUTHOR CONTRIBUTIONS

E.T., L.G.G., U.H.v.A., andM.I. designed the study; E.T., N.J.d.O, G.G., E.A.M.,

P.D.L., A.A., S.S., M.D.G., L.S., and M.I. performed experiments; E.T. and M.I.

analyzed the data; N.C. performed and analyzed themicroarray data; L.G. per-

formed measurements in human subjects; G.S. gave conceptual advice; and

E.T., L.G.G., U.H.v.A. and M.I. wrote the manuscript.

ACKNOWLEDGMENTS

We thank T. Cataudella, A. Fiocchi, B. Fiore, D. Covarello, and M. Mainetti for

technical support; R. Serra for secretarial assistance; P. Dellabona (San


http://www.broadinstitute.org/cancer/software/GENE-E/

http://www.broadinstitute.org/cancer/software/GENE-E/

http://david.abcc.ncifcrf.gov

http://david.abcc.ncifcrf.gov



Raffaele Scientific Institute) for providing MHC-II�/� mice; W. Havran (The

Scripps Research Institute) for providing TCRd�/� mice; B. Beutler (The

Scripps Research Institute) for providing MyD88�/�/TrifLps2/Lps2 mice; S. Whe-

lan (Harvard Medical School) for providing VSV and VSVeGFP; N. van Rooijen

for providing clodronate liposomes; F. Benvenuti (International Centre for Ge-

netic Engineering and Biotechnology) for providing Flt3-ligand-secreting B16

tumor cells; S. Cenci, C. Scielzo, P. Ghia, and the members of the Iannacone,

Guidotti and von Andrian laboratories for helpful discussions; and F.V. Chisari,

A. Mondino, P. Dellabona, R. Pardi, and Z.M. Ruggeri for critical reading of the

manuscript. This work was supported by ERC grants 281648 (to M.I.) and

250219 (to L.G.G.); NIH grants AI40696 (to L.G.G.) and AI078897 and

AI069259 (to U.H.v.A.); Italian Association for Cancer Research (AIRC) grant

9965 (to M.I.); and a career development award from the Giovanni Armen-

ise-Harvard Foundation (to M.I.).

Received: July 18, 2013

Revised: September 3, 2013

Accepted: September 4, 2013

Published: October 10, 2013

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Supplemental Information

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Figure S1. Bisphosphonates Increase Antibody Responses to Live and Inactive Viruses, Proteins, Haptens and Existing Commercial Vaccine

Formulations, Related to Figure 1

(A) VSV nAb titers in the serum of C57BL/6mice that were footpad injected with CLD 3 days and 1 day (day�3,�1) or immediately (day 0) prior to VSV infection in

the same footpad. n = 5 per group. Black asterisks, PBS versus CLD day�3,�1; red asterisks PBS versus CLD day 0; results are representative of 3 independent

experiments.

(B) VSV nAb titers 7 days p.i. in the serum of C57BL/6 mice that were footpad injected with PBS or with the indicated doses of CLD prior to VSV infection in the

same footpad. n = 5 per group; results are representative of 2 independent experiments.

(C) VSV nAb titers in the serum of C57BL/6 mice that were footpad injected with PBS or CLD prior to infection in the same footpad with VSV. n = 5 per group;

results are representative of 3 independent experiments.

(D) Neutralizing IgM titers in the serum of the same mice described in (a).

(E) Neutralizing IgG titers of the same mice described in (a).

(F) VSV-specific IgG1 and IgG2c titers 7 day p.i. in the serum of C57BL/6 mice that were footpad injected with PBS or CLD prior to VSV infection in the same

footpad. n = 5 per group; results are representative of 2 independent experiments.

(G) Total number of CD138+ cells recovered from draining popliteal lymph nodes (Pop LN) that were pooled from 3 C57BL/6 mice footpad injected with PBS or

CLD prior to VSV infection in the same footpad. Results are representative of 2 independent experiments.

Results are expressed as mean ± SEM. ***: p < 0.001; **: p < 0.01; *: p < 0.05; D.L.: detection limit.

Cell Reports 5, 1–8, October 31, 2013 ª2013 The Authors S1

CD11b

Gr-

1

2 3 4

PBS 1.03

0

2

3

4

5

CLD 32.6

2 3 4 5

PBS+α-Gr-10

2 3

CLD+α-Gr-10.12

A

0

10

20

30

40

%fo

otpa

d G

r1hi

ghC

D11

b+

(of C

D45

+ ce

lls)

**

** *

B

PBS

CLD ALD

PBS

4.864.9

0.0740.01

TCRδ-/- WT

10.9

13

C

CD3

TCR

δ

WT WT

Neu

traliz

ing

Ab

titer

(d14

)

103

104

102

105

101

PBS CLD ALD PBS CLD ALD

WT TCRδ-/-

n.s. n.s.

n.s.

D

Figure S2. Neutrophils and gd T Cells Are Not Required for Bisphosphonate Adjuvant Activity, Related to Figures 2C and 2D

(A and B) Representative FACS plots (A) and quantification (B) of the number of Gr-1highCD11b+ cells present in digested footpads from the mice described in

Figure 2C. Numbers show the percentages of CD45+ cells within each gate. n = 4 per group; results are representative of 2 independent experiments.

(C) Representative FACS plots of digested footpads fromPBS-injected TCRd�/�mice or PBS-, CLD- or alendronate (ALD)-injected C57BL/6 (WT)mice. Numbers

show the percentages of CD45+ cells within each gate. Results are representative of 2 independent experiments (n = 3 per experiment).

(D) VSV nAb titers 14 days p.i. in the serum of C57BL/6 (WT) or TCRd�/� mice that were footpad injected with PBS, CLD or ALD prior to VSV infection in the same

footpad. n = 5 per group; results are representative of 3 independent experiments.

**: p < 0.01; *: p < 0.05; n.s.: non statistically significant.

S2 Cell Reports 5, 1–8, October 31, 2013 ª2013 The Authors

20000

15000

10000

5000

0

MFI

CD

86 400

300

200

100

0

MFI

CD

40

500

600

CLD CLD

Control

1.78 0.0146 0.0114 0.0013

DT

Day 0 p.i. Day 4 p.i. Day 14 p.i.

Spleen

0.13 0.0204 0.0043 0.0043

Pop LN

CD11c

GFP

A B

C

Figure S3. Bisphosphonate Adjuvant Activity Does Not Require Conventional Dendritic Cells, Related to Figure 2E

(A and B) CD86 (A) or CD40 (B) expression on purified CD11c+ DC that were cultured in the presence of 1 mg/ml LPS or with the indicated concentrations of CLD

for 48 hr prior to flow cytometry analysis.

(C) Representative FACS plots of digested spleens or popliteal lymph nodes (Pop LN) from CD11c-GFP-DTR/ C57BL/6 bone marrow chimeric mice that were

left untreated (control) or injected with diphtheria toxin (DT). Numbers show the percentage of cells within each gate. Results are representative of 2 independent

experiments (n = 3 per experiment). Results are expressed as mean ± SEM.


B PBS CLD B220 TCRβ CD169 B220 TCRβ CD169

100μm 100μm

Pop

LN

cel

ls (x

105 )

5

10

15

20

25

0 Total B T

A CLD

1

2

3

4

0

Pop

LN

cel

ls (x

105 )

* PBS

CD169

CD

11b

1.25 0.0866

0.0916 0.0966

PBS CLD

dextran sulfate carrageenan

1.25 0.0866

0.0916 0.0966

control DT

Neu

traliz

ing

Ab

titer

(d4)

103

104

102

105

101

n.s.

C D

Figure S4. Lymph Node Macrophage Depletion Per Se Does Not Increase Humoral Immune Responses, Related to Figure 2F

(A) Cellular subset frequency in popliteal lymph nodes (Pop LN) from C57BL/6 mice that were treated with PBS or CLD. n = 3 per group; results are representative

of 3 independent experiments.

(B) Representative confocal micrographs of Pop LN from C57BL/6 mice that were treated with PBS or CLD. Sections were stained with anti-B220 (gray), anti-

TCRb (red) and anti-CD169 (green) to identify B cells, T cells and macrophages, respectively. Scale bars represent 100 mm. Results are representative of 2

independent experiments (n = 3 per experiment).

(C) Representative FACS plots of digested popliteal lymph nodes from C57BL/6 mice that were treated with PBS, CLD, dextran sulfate or carrageenan. Numbers

show the percentage of cells within each gate. Results are representative of 2 independent experiments (n = 3 per experiment).

(D) VSV nAb titers 4 days p.i. in the serum of CD11c-GFP-DTR mice that were left untreated (control) or were depleted of CD169+ LN macrophages through a

single footpad injection of diphtheria toxin (DT) 6 days prior to VSV infection. These mice were described in Figure S3 from ref. (Iannacone et al., 2010).

Results are expressed as mean ± SEM. *: p < 0.05; n.s.: non statistically significant.


PBS PBS CLD CLD VSV 8h

Row max

Row min

Figure S5. mRNA Expression Profiles of Differentially Expressed Genes in Whole Lymph Node, Related to Figure 3A

Shown are expression profiles for 4761 differentially expressed genes (rows) at 0 and 8 hr after VSV infection inmice treatedwith CLD or PBS as control (columns).

Shown are genes with at least a 2-fold change in expression between treatments in both duplicate arrays (each column corresponds to a replicate). Values from

multiple probe sets targeting the same gene were collapsed and gene-expression profiles were hierarchically clustered. Results are expressed as mean ± SEM.


Gene Symbol Fold change ImmGenCcl8 6.55Cd209f 5.58Retn 4.49Rya3 4.16Angptl4 3.54LOC100048885 3.54Gpr34 3.23Wasf1 2.85Car3 2.68Thrsp 2.53Ccl12 2.58Eps8l1 2.39Adipoq 2.38Dab2 2.36Cfh 2.29Clec10a 2.27Fcna 2.18Col12a1 2.12Mcoln3 2.28 B cellMcoln2 2.89 B cellP2ry12 3.95 Dendritic cellCd209d 5.41 Dendritic cellP2ry14 2.36 Dendritic cellIl22ra2 8.31 Dendritic cellCd209e 2.91 Dendritic cellSiglech 2.74 Dendritic cellHpgd 2.12 Dendritic cellEtv5 3.41 γδ T cellCpa3 3.49 γδ T cellAbca9 2.36 Macroph/monoScarb1 2.01 Macroph/monoF13a1 8.11 Macroph/monoFcgr1 2.69 Macroph/monoCpt1a 2.01 Macroph/monoCd209b 14.24 Macroph/monoFolr2 14.13 Macroph/monoCbr2 9.34 Macroph/monoTimd4 4.44 Macroph/monoMrc1 4.09 Macroph/monoMcpt4 3.55 Macroph/monoSlc15a2 3.44 Macroph/monoMarco 2.43 Macroph/monoHmox1 2.42 Macroph/monoCsf1r 2.34 Macroph/monoEdnrb 2.36 Macroph/monoFrmd4b 2.22 Macroph/monoMs4a8a 2.28 Macroph/monoTrf 2.24 Macroph/monoLyve1 2.15 Macroph/monoP2ry13 2.15 Macroph/monoCadm1 2.1 Macroph/monoRgl1 2.09 Macroph/monoGfra2 2.09 Macroph/monoCysltr1 2.03 Macroph/monoFabp4 2.02 Macroph/monoNinj1 2 Macroph/monoC1qa 2 Macroph/monoCd300ld 2.23 NeutrophilItgam 2.21 NeutrophilDdx60 2.18 NeutrophilCma1 4.56 NK cell

PBS CLD

Row max

Row min

Figure S6. mRNA Expression Profiles of Genes Decreased upon CLD Treatment inWhole Lymph Node before Infection, Related to Figure 3A

Shown are expression profiles for 62 differentially expressed genes (rows) with at least a 2-fold decrease in expression in CLD-treatedmice compared to controls

(PBS). The right-most column indicates the cell types in which each gene is specifically expressed based on the Immunological Genome (ImmGen) database.

Results are expressed as mean ± SEM.


Gene Symbol Fold change ImmGenCcl8 16.05Ifna9 5.93Ccl12 5.86Sct 5.59Ifnab 5.3Nts 5.03Aplnr 3.69Ifna5 3.61Ifna4 3.53Ifna1 3.3Adamts4 2.48Klk1 3.07AI447881 3.13Tppp3 2.99Tifab 2.67Rdh10 2.76Ifna2 2.79Ddx4 2.61Clec10a 2.56Mthfr 2.56Astx 2.52Sco1 2.49Gm10471 2.45Ifnb1 2.44Rilpl1 2.3Irf7 2.37Herc5 2.18Mt2 2.3Fndc3a 2.26Ctsg 2.26Asb13 2.25Mpo 2.25LOC100045567 2.23Zbp1 2.23Cxcl11 2.23Stx3 2.2Gm12185 2.19Ccl11 2.19Tmem171 2.18Sirpb1a 2.17Adamts9 2.16Dcp2 2.16Dhx58 2.15Mt1 2.14Gm9706 2.14Rnf217 2.14Ptx3 2.14Armcx6 2.11Gla 2.11Uba7 2.09Sema3a 2.09Ccr5 2.08Lgals3 2.07AW011738 2.06Oas1a 2.05Slfn9 2.044933412E12Rik 2.03Slc38a4 2.03Gm9706 2.02Tbc1d13 2.01Sectm1a 2.01Akap12 2.12 B cellCar13 2.44 B cellGpsm2 2.23 B cellPpa1 2.11 B cellLOC100039614 2.1 B cellCd209d 4.48 Dendritic cellP2ry14 3.11 Dendritic cellBst2 2.58 Dendritic cellSgcb 2.1 Dendritic cellIl22ra2 6.22 Dendritic cellApod 3.72 Dendritic cellHpgd 3.14 Dendritic cellCd209e 2.75 Dendritic cellTimp1 2.69 Dendritic cell9030625A04Rik 2.64 Dendritic cellCd86 2.6 Dendritic cellSiglech 2.59 Dendritic cellSlamf9 2.48 Dendritic cellGatm 2.27 Dendritic cellCh25h 2.04 Dendritic cellEmp1 2 Dendritic cellCpa3 6.21 γδ T cellMefv 2.79 Macroph/monoFcgr1 3.94 Macroph/mono4933429F08Rik 3.93 Macroph/monoVcan 3.25 Macroph/monoMs4a6d 3.26 Macroph/monoOas2 2.75 Macroph/monoFcgr3 2.64 Macroph/monoXdh 2.63 Macroph/monoF13a1 2.61 Macroph/monoClec4a3 2.56 Macroph/monoCcr2 2.55 Macroph/monoPtpro 2.36 Macroph/monoTmem106a 2.32 Macroph/monoIfi204 2.28 Macroph/monoIl13ra1 2.24 Macroph/monoRasa4 2.17 Macroph/monoLilrb3 2.17 Macroph/monoMs4a6c 2.16 Macroph/monoHba-a1 2.12 Macroph/monoPde7b 2.11 Macroph/monoPpbp 2.11 Macroph/monoOasl2 2.08 Macroph/monoPlod3 2.08 Macroph/monoSamhd1 2.07 Macroph/monoFcer1g 2.06 Macroph/monoGpr35 2.04 Macroph/monoLy6i 2.03 Macroph/monoTnfrsf21 2.01 Macroph/monoAI607873 2 Macroph/monoUbe2l6 2 Macroph/monoMmp13 32.01 Macroph/monoCd209b 18.45 Macroph/monoTimd4 6.91 Macroph/monoMcpt4 5.89 Macroph/monoFolr2 5.85 Macroph/monoFcgr4 3.48 Macroph/monoTcfec 3.32 Macroph/monoRgl1 3.26 Macroph/monoMs4a8a 2.98 Macroph/monoAdap2 2.79 Macroph/monoCd5l 2.66 Macroph/monoMarco 2.25 Macroph/monoC3ar1 2.5 Macroph/monoC1qb 2.27 Macroph/monoGm11428 2.11 Macroph/monoFrmd4b 2.09 Macroph/monoCcl9 2.07 Macroph/monoEmilin2 2.06 Macroph/monoSdc3 2.04 Macroph/monoLipg 3.03 NeutrophilLOC100044115 2.89 NeutrophilIl1rn 2.54 NeutrophilOas3 2.6 NeutrophilHk3 2.39 NeutrophilAA467197 2.57 NeutrophilCarhsp1 2.07 NeutrophilMxd1 2.15 NeutrophilDdx60 2.12 NeutrophilRsad2 2.11 NeutrophilChi3l3 2.02 NeutrophilCma1 7.63 NK cellGzmb 3.16 NK cellGzma 3.1 NK cellKlrk1 2.73 NK cellEomes 2.44 NK cellIl12rb1 2.44 NK cellMlkl 2.13 NK cellIl18 2.07 NK cellEtv6 2.19 Stem cellAldh1b1 2.88 T cellAtp10a 2.13 T cell

PBSVSV 8h

CLDVSV 8hPBS CLD

VSV 8h

Row max

Row min

Figure S7. mRNA Expression Profiles of Genes Decreased upon CLD Treatment in Whole Lymph Node after Infection, Related to Figure 3A

Shown are expression profiles for 155 differentially expressed genes (rows) with at least a 2-fold decrease in expression in CLD-treated mice compared to

controls (PBS) at 8 hr after VSV infection. The right-most column indicates the cell types in which each gene is specifically expressed based on the Immunological

Genome (ImmGen) database. Results are expressed as mean ± SEM.


Gene sets down-regulated in CLD

p-value

0h 8h Term Response to wounding (9611)Membrane invagination (10324)Endocytosis (6897)Immune response (6965)Defense response (6952)Cytokine-cytokine receptor interaction (4060)Response to virus (9615)Extracellular region (5576)Cytosolic DNA-sensing pathway (4623)RIG-I-like receptor signaling pathway (4622)Natural killer cell mediated cytotoxicity (4650)Immune effector process (2252)Positive regulation of phagocytosis (50766)Toll-like receptor signaling pathway (4620)Regulation of phagocytosis (50764)Inflammatory response (6954)Taxis (42730)Chemotaxis (6935)Extracellular region part (44421)Positive regulation of endocytosis (45807)Autoimmune thyroid disease (05320)Regulation of autophagy (4140)Jak-STAT signaling pathway (4630)Regulation of endocytosis (30100)Adaptive immune response based on somatic recombination of Ig superfamily domain immune receptors (2460)Adaptive immune response (2250)Extracellular space (5615)

10-5 10-15

Figure S8. Gene Enrichment Analysis of CLD-Dependent Genes fromMicroarrayMeasurements ofWhole Lymph Node, Related to Figure 3A

Shown are the Gene Ontology (GO) terms and canonical pathways (KEGG database) (rows) statistically enriched among the 217 genes with at least a 2-fold

decrease in expression at 0 and 8 hr (columns) after VSV infection in CLD-treated mice compared to control (PBS). Enriched GO term categories include BP:

biological process, and CC: cellular component. Green: p value < 10�15; white: p value < 10�4; gray: no enrichment. Results are expressed as mean ± SEM.


Gene Symbol Fold change ImmGen

Fpr2 2.38Slc4a1 2.44Camp 3.03Ahsp 3.45Ltf 4Rrm2 2.13 B cellCcnb1 2.08 B cellHbb-b1 3.45 B cellIgh-6 2.08 B cellIgkv14-111 2.27 B cellIghg1 2.56 B cellNgp 2.78 B cellIgh-3 5.56 B cellIghg 6.25 B cellHba-a1 2.78 Macroph/monoPpbp 3.57 Macroph/monoSlpi 2.33 NeutrophilS100a8 2 NeutrophilStfa2l1 2.04 NeutrophilS100a9 2.04 NeutrophilSlfn4 2.17 NeutrophilTrem3 2.22 NeutrophilMmp9 3.23 Neutrophil1100001G20Rik 3.13 NeutrophilI f i tm6 3.57 NeutrophilCd177 3.57 NeutrophilLcn2 3.85 NeutrophilMmp8 5 NeutrophilCar2 3.13 Stem cell

PBS CLD

Row max

Row min

Figure S9. mRNA Expression Profiles of Genes Increased upon CLD Treatment in Whole Lymph Node before Infection, Related to Figure 3A

Shown are expression profiles for 29 differentially expressed genes (rows) with at least a 2-fold increase in expression in CLD-treated mice compared to controls

(PBS). The right-most column indicates the cell types in which each gene is specifically expressed based on the Immunological Genome (ImmGen) database.

Results are expressed as mean ± SEM.


Gene Symbol Fold change ImmGen2310035P21Rik 2Selm 2Fhl1 2Chpt1 2.04Cilp 2.04Mmd 2.04Cdh2 2.08Cygb 2.083110080E11Rik 2.082510009E07Rik 2.08Higd1c 2.13Naa35 2.13Fmo1 2.13Prss23 2.13Cyp1b1 2.13Lum 2.17Vegfc 2.17Ebf3 2.17Maob 2.17Dclk1 2.179430047G12Rik 2.17Dsc2 2.17Evi5 2.17Parm1 2.17Il7 2.224930426D05Rik 2.22Fbxl7 2.22Nedd4 2.22Mxd4 2.27Fam168a 2.335830408C22Rik 2.33Irx3 2.332410066E13Rik 2.33Bnip3 2.33Synpo2 2.38Bok 2.38Arrb1 2.38Stard4 2.38AI256396 2.44Thrsp 3.334931406C07Rik 2.44Pdgfra 2.44LOC100047565 2.44Meox2 2.63Hist1h1c 2.5Plin4 2.63Timp4 2.63Vsig10 2.63Scara5 2.7Cyp26b1 2.86Aoc3 2.86Ghr 2.94Abca8a 2.94Odz3 3.03Retn 3.03Gdf10 3.33Inmt 3.45Atp1a2 3.45Cfd 3.7Car3 3.7Gm7202 3.85BB144871 3.85Adipoq 4.17Cxcl5 4.35Cyp2e1 7.14Lpl 2.22 B cellCcna2 2.27 B cellBmf 2.08 B cellNuak1 2.13 B cellCpm 2 B cellArhgef12 2.08 B cellCyp51 2.27 B cellFcer2a 2.38 B cellCiita 2.04 B cellCd55 2.17 B cellTmem204 2.08 B cellSrpk3 2.13 B cellCdc25b 2.13 B cellIgj 2.27 B cellProx1 2.44 B cellIgk-V28 2.63 B cellIgh-6 11.11 B cellIghg 7.69 B cellIghg1 8.33 B cellIgh-3 14.29 B cellZmynd15 2.17 Dendritic cellPard6g 2.08 Dendritic cellGria3 2.22 Dendritic cellPxdn 2 Dendritic cellCyp4b1 2 Dendritic cellGstm2 2.04 Dendritic cellAno1 2.04 Dendritic cellDsg2 2.04 Dendritic cellSlc36a2 3.13 Dendritic cellAdh1 2.04 γδ T cellC1ql3 2.17 γδ T cellSlc40a1 2.94 Macroph/monoB230120H23Rik 2.04 Macroph/monoIl13ra2 2 Macroph/monoMyl9 2.04 Macroph/monoNfia 2.08 Macroph/monoFbxo32 2.04 Macroph/monoKifap3 2.04 Macroph/monoSlco2b1 2.33 Macroph/monoTrim2 2.13 Macroph/monoTtc3 2.08 Macroph/monoDhrs3 2.22 Macroph/monoIdh1 2.27 Macroph/monoKitl 2.27 Macroph/monoCd36 2.56 Macroph/monoFabp4 2.56 Macroph/monoPon3 2.7 Macroph/monoSlc15a2 1 0 Macroph/monoGclm 2.04 NeutrophilSlpi 2.63 NeutrophilMmp9 3.33 NeutrophilAcsl1 2.56 NeutrophilCes3 4.17 NeutrophilNrarp 2.17 NK cellKlhl4 2.22 NK cellA930038C07Rik 2.63 NK cellMpdz 2.08 Stem cellMettl7a1 2.44 Stem cellSlc22a3 2.78 Stem cellSdpr 3.23 Stem cellSerpinf1 2.04 Stem cellHmgcs2 2.7 T cell

PBSVSV 8h

CLDVSV 8h

Row max

Row min

PBS CLD VSV 8h

Figure S10. mRNA Expression Profiles of Genes Increased upon CLD Treatment in Whole Lymph Node after Infection, Related to Figure 3A

Shown are expression profiles for 127 differentially expressed genes (rows) with at least a 2-fold increase in expression in CLD-treatedmice compared to controls

(PBS) at 8 hr after VSV infection. The right-most column indicates the cell types in which each gene is specifically expressed based on the Immunological Genome

(ImmGen) database. Results are expressed as mean ± SEM.


Neu

traliz

ing

Ab

titer

(d7)

103

104

102

105

101

**

* *

** B

Neu

traliz

ing

Ab

titer

(d7)

103

104

102

105

101

A

PBS α-PDCA-1

n.s.

Figure S11. CLD Increases Ab Responses Independently of pDCs or NK Cells, Related to Figure 3A(A) VSV nAb titers 7 days p.i. in the serum of C57BL/6 mice that received PBS or were depleted of pDCs via anti-PDCA-1 (a�PDCA1) injection 24 hr prior to VSV

infection. These mice were described in Figure 3j and Suppl. Figure 8b from ref. (Iannacone et al., 2010).

(B) VSV nAb titers 7 days p.i. in the serum of C57BL/6 mice that were injected (or not) with anti-Asialo-GM1 depleting Ab (a�Asialo-GM1, seeMethods for details)

and were subsequently footpad injected with PBS or CLD immediately prior to VSV infection in the same footpad. n = 5; results are representative of 2 inde-

pendent experiments.

Results are expressed as mean ± SEM. **: p < 0.01; *: p < 0.05; n.s.: non statistically significant.


Bisphosphonates Target B Cells to Enhance Humoral Immune Responses

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