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1154 NATURE MEDICINE • VOLUME 6 • NUMBER 10 • OCTOBER 2000

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Dendritic cells (DCs) are very specialized antigen-presenting cellsthat capture antigens, move from the periphery to lymphoid or-gans and present the processed antigens to resting, naive CD4+ Tlymphocytes1. After the DC–CD4+ T-cell interaction through theCD4+ T-cell antigen receptor, the CD4+ T cell becomes activatedand expresses many surface activation proteins1. Among these, themost important is CD40 ligand (CD40L, or CD154, a 33-kDa type IImembrane protein and a member of the tumor necrosis factor genefamily), which serves as a ligand for the CD40 receptor on the DC(refs. 2,3). After CD40L triggers CD40, the DC is enabled to directlyinteract with CD8+ cytotoxic T cells1,2. There has been considerableinterest in using DCs to help induce anti-tumor immunity1,4,5. Bcells also express CD40, and interaction of DCs with CD4+ T cells,through CD40L and CD40, is essential in enabling B cells to gener-ate antigen-specific antibodies3,6,7.

The requirement for CD4+ T cells as intermediates in the interac-tion of DCs with other components of the immune system is usefulin providing control of immune responses1,3,6,8. However, the con-sequences of having CD4+ T cells as the intermediates in control-ling immune responses result in inadequate host responses againstinfectious agents when there is an insufficient number of CD4+ Tcells, as in immunodeficiency disorders like after infection with thehuman immunodeficiency virus9.

Here we sought to determine if it is possible to geneticallychange DCs to take on the function of both DCs and CD4+ T cellsby genetically modifying the DCs to express CD40L. This shouldenable the DCs to interact with microbe-specific antigens and di-rectly activate B cells, thus generating microbe-specific protectiveantibodies without CD4+ T-cell help. We used an E1 adenovirus(Ad) gene transfer vector expressing mouse CD40L cDNA(AdmCD40L) to genetically modify DCs, pulsed the modified DCswith heat-killed Pseudomonas aeruginosa, and administered the

modified DCs to syngeneic hosts. Immunization of naive mice inthis way led to considerable protection against a lethal challengewith P. aeruginosa, mediated by Pseudomonas-specific humoral im-munity elicited without CD4+ T-cell help.

Cytokine production of AdmCD40L-modified DCsWe confirmed the activation of DCs after adenovirus-vector-medi-ated transfer of the CD40L cDNA by assessing the genetically mod-ified DCs for interleukin (IL)-12 and macrophage inflammatoryprotein (MIP)-1α secretion1,2 (Fig. 1). Infection of DCs withAdmCD40L resulted in an increase of 4,000% in IL-12 in the su-pernatant compared with infection with AdNull (a vector identi-cal to AdmCD40L, containing no transgene) or no infection (P <0.0001, for each comparison). Modification of DCs withAdmCD40L also stimulated the secretion of MIP-1α (400% greaterthan that with AdNull or no modification; P < 0.0001, for eachcomparison). The enhanced secretion of IL-12 and MIP-1α was ab-rogated by the addition of monoclonal antibody against mCD40L(MR1) , compared with the addition of control immunoglobulin(Ig) G (IL-12, P < 0.0001; MIP-1α, P < 0.01), indicating that infec-tion with AdmCD40L mediated functional CD40L expression onthe DC surface.

Interaction between DCs and B cells in vitroCo-culture of irradiated, AdmCD40L-modified DCs with syngenicB cells resulted in the proliferation of the B cells, which peaked 5days after initiation of the culture (Fig. 2). In contrast, co-culture ofirradiated AdNull-modified DCs or naive DCs with B cells inducedno B-cell proliferation.

AdmCD40L-modified DCs pulsed with P. aeruginosa processedand presented Pseudomonas antigens to co-cultured B cells, re-sulting in the stimulation of IgM and IgA production specific for

Dendritic cells genetically modified to express CD40 ligand andpulsed with antigen can initiate antigen-specific humoral

immunity independent of CD4+ T cells

TOSHIAKI KIKUCHI1, STEFAN WORGALL1,2, RAVI SINGH3,MALCOLM A.S. MOORE5 & RONALD G CRYSTAL1,3,4

1Division of Pulmonary and Critical Care Medicine of the Department of Medicine, 2Department of Pediatrics, 3Belfer Gene Therapy Core Facility and 4Institute of Genetic Medicine, Weill Medical College of Cornell University,

New York, New York, USA 5James Ewing Laboratory of Developmental Hematopoiesis Memorial Sloan-Kettering Cancer Center, New York, New York, USA

Correspondence should be addressed to R.G.C.; email: [email protected]

We have investigated whether dendritic cells genetically modified to express CD40 ligand andpulsed with antigen can trigger B cells to produce antigen-specific antibodies without CD4+ T-cellhelp. Dendritic cells modified with a recombinant adenovirus vector to express CD40 ligand andpulsed with heat-killed Pseudomonas induced naive B cells to produce antibodies againstPseudomonas in the absence of CD4+ T cells in vitro, initiated Pseudomonas-specific humoral immuneresponses in vivo in wild-type and CD4–/– mice, and protected immunized wild-type and CD4–/–, butnot B-cell–/– mice, from lethal intrapulmonary challenge with Pseudomonas. Thus, genetic modifica-tion of dendritic cells with CD40 ligand enables them to present a complex mixture of microbialantigens and establish CD4+ T cell-independent, B cell-mediated protective immunity against a spe-cific microbe.

© 2000 Nature America Inc. • http://medicine.nature.com©

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NATURE MEDICINE • VOLUME 6 • NUMBER 10 • OCTOBER 2000 1155

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P. aeruginosa in the absence of CD4+ T cells (Fig. 3). To investi-gate the effect of AdmCD40L-modified DCs on the differentia-tion of CD19+ B cells into specific immunoglobulin-secretingcells, we co-cultured CD19+ B cells separated from the spleens ofnaive mice with adenovirus-vector-modified DCs pulsed with orwithout P. aeruginosa. When co-cultured with CD19+ B cells,AdmCD40L-modified DCs pulsed with P. aeruginosa inducedthe production of significant amounts of Pseudomonas-specificIgM and IgA compared with that induced by AdNull-modifiedDCs pulsed with P. aeruginosa, AdmCD40L-modified DCs aloneor naive DCs (IgM, 300–900% increase, P < 0.0001; IgA,300–800% increase, P < 0.0001). There was no stimulation inthe production of Pseudomonas-specific immunoglobulin sub-types IgG1, IgG2a, IgG2b or IgG3 (data not shown). The possi-bility that the presentation of the Pseudomonas antigens by DCswas responsible for the induced production of Pseudomonas-spe-cific antibodies was indicated by the use of three types of phar-macologic inhibitors on antigen-processing pathways,including brefeldin A (inhibition of endoplasmicreticulum/Golgi transport), cytochalasin D (suppression ofactin-dependent phagocytosis) and ammonium chloride (inhi-bition of acid pH-dependent degradation)8. Pseudomonas-spe-cific IgM and IgA secretion in the co-culture of B cells andAdmCD40L-modified DCs pulsed with P. aeruginosa was abro-gated significantly when DCs were primed with Pseudomonas inthe presence of any one of these inhibitors (IgM, P < 0.0001;IgA, P < 0.0001). Although these inhibitors blocked the process-ing of the bacterial antigens for presentation in the DCs, theDCs modified with AdmCD40L still expressed CD40L aftertreatment with brefeldin A, cytochalasin D or ammonium chlo-ride (that is, those treatments did not adversely modify the DCs;data not shown). To eliminate the possibility that CD4+ T cellscontaminating the DC or B-cell preparations could be responsi-ble for the observation of the in vitro generation of Pseudomonas-specific antibodies, we used DCs and B cells prepared fromCD4–/– mice in similar co-culture experiments. Despite this ab-solutely CD4-deficient condition, the results obtained forPseudomonas-specific IgM and IgA production from B cells co-cultured with AdmCD40L-modified DCs pulsed withPseudomonas were similar to those obtained with componentsfrom wild-type mice. Consistent with this, CD11c+ DCs purifiedfrom the DC culture and sorted by magnetic cell sorting, modi-fied with AdmCD40L and pulsed with P. aeruginosa also inducedPseudomonas-specific IgM and IgA secretion from B cells.

Induction of antibodies against Pseudomonas in vivoWe assessed the activation of B cells by immunization withCD40L/DCs/Pseudomonas in vivo by determining serum level ofPseudomonas-specific antibodies. C57Bl/6 mice immunized withAdmCD40L-modified DCs pulsed with P. aeruginosa producedsignificant amounts of serum antibodies against Pseudomonas(end-point titers: IgM, 300–700%, P < 0.0001; IgG1, 300–400%, P< 0.0001; IgG2b, 200–400%, P < 0.0001; and IgG3, 300–500%, P< 0.02), compared with amounts produced by mice immunizedwith AdNull-modified DCs pulsed with P. aeruginosa orAdmCD40L-modified DCs alone or non-immunized mice (Fig.4a–e). There was an insignificant increase in IgG2a levels (P >0.2). As a control for the specificity of the antibodies againstPseudomonas detected in vivo, serum of mice immunized withAdmCD40L-modified DCs pulsed with Escherichia coli was nega-tive for antibodies against Pseudomonas IgM, IgG and IgA (datanot shown). Like the increase in serum IgM and IgG antibodiesagainst Pseudomonas, there was a significant increase in serumIgA levels in the mice immunized with CD40L/DCs/Pseudomonascompared with that in control mice (P < 0.0001; Fig. 4f). Therewas no significant difference of respiratory mucosal IgA antibod-ies against Pseudomonas in epithelial lining fluid in the mice im-munized with CD40L/DCs/Pseudomonas (P > 0.1, for allcomparisons), except for the small difference betweenAdmCD40L-modified DCs pulsed with P. aeruginosa andAdmCD40L-modified DCs alone (P < 0.05; Fig. 4g). No other iso-types of antibodies against P. aeruginosa were detected in epithe-lial lining fluid. Although this study cannot prove thatPseudomonas-specific mucosal antibodies below the level of de-tection were responsible for the protective immunity seen, it ispossible that circulating specific immunoglobulins diffusingacross the mucosa afford the relevant protection, similar to thatin experimental infection with chimeric human immunodefi-ciency virus-1/simian immunodeficiency virus10,11.

To demonstrate that the in vivo generation of Pseudomonas-specific antibodies by CD40L modified DCs pulsed with heat-killed Pseudomonas could occur independently of CD4+ T cells,we repeated the experiment using CD4–/– mice (Fig. 5). The serumlevels of Pseudomonas-specific antibodies generated in the CD4–/–

mice paralleled those in the wild-type mice.

AdmCD40L/DCs induce protection against Pseudomonas AdmCD40L-modified DCs pulsed with heat-killed P. aeruginosa in-duced protective immunity, lasting at least 3 months, against alethal challenge with Pseudomonas in vivo (Fig. 6a and b).Immunization of C57Bl/6 mice with 5 × 104 AdmCD40L-modified

Fig. 1 Activation of dendritic cells by genetic modification with the mouseCD40L cDNA. a, IL-12 secretion by AdmCD40L-modified mouse DCs. b,MIP-1α production by AdmCD40L-modified mouse DCs. media, ¤ ; mono-clonal antibody against mCD40L, n ; control IgG, . Data represent mean ±standard error (n = 4 per data point).

a b

Fig. 2 Nonspecific B-cell proliferation induced by co-culture of mCD40L-modified DCs and syngeneic B cells. AdmCD40L-modified DCs (n ), AdNull-modified DCs (¡ ) and naive DCs (�) were co-cultured with B cells. Datarepresent means of duplicate cells.


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DCs pulsed with heat-killed P. aeruginosa 3 weeks before a lethalchallenge with 2 x 105 colony-forming units (CFU) P. aeruginosa en-meshed in agar beads resulted in 90% survival in of mice (P <0.0005, compared with all other groups; Fig. 6a). In contrast, im-munization with 5 × 104 AdNull-modified DCs pulsed with heat-killed P. aeruginosa or 5 × 104 AdmCD40L-modified DCs alone, orno immunization, led to survival of 10% or less in the infectedmice. The protective effect elicited by AdmCD40L-modified DCspulsed with heat-killed Pseudomonas also occurred when CD11c+

DCs used for immunization were purified by magnetic cell sorting(data not shown). We obtained similar results using mice immu-nized 3 months before the Pseudomonas challenge: 80% of mice im-munized with AdmCD40L-modified DCs pulsed with heat-killed P.aeruginosa survived at least 14 days after the lethal challenge with P.aeruginosa (Fig. 6b; P < 0.0001, compared with all other groups). Incontrast, the control groups of mice receiving AdNull-modifiedDCs pulsed with heat-killed P. aeruginosa or AdmCD40L-modifiedDCs alone 3 months before the instillation died within 5 days.

Microbe-specific effects of AdmCD40L-modified DCsWe next determined whether immunization using AdmCD40L-modified DCs generated specific protective immunity against the

microbe with which the DCs had been primed(Fig. 6c and d). Groups of C57Bl/6 mice re-ceived vaccinations of 5 × 104 AdmCD40L-modified DCs pulsed with either heat-killed P.aeruginosa or E. coli, or no vaccination. After 3weeks, the mice were challenged with intratra-cheal administration of 2 × 105 CFU P. aerugi-nosa or 1 × 108 CFU E. coli. Mice receivingimmunization of P. aeruginosa-pulsed CD40L-activated DCs were protected fromPseudomonas challenge, but immunizationwith E. coli-pulsed CD40L-activated DCs didnot protect the mice, nor did no immunization(P < 0.0001, CD40L-activated DCs pulsed withheat-killed P. aeruginosa compared with allother groups; Fig. 6c). In contrast, 60% of miceimmunized with E. coli-pulsed CD40L-acti-vated DCs were protected against subsequentE. coli challenge, whereas all non-immunizedmice or mice immunized with P. aeruginosa-pulsed CD40L-activated DCs died after infec-

tion with E. coli (P < 0.0005, DCs pulsed with heat-killed E. colicompared with all other groups; Fig. 6d).

Transferable anti-Pseudomonas immunityThe protection against Pseudomonas challenge provided by theCD40L/DC/Pseudomonas vaccine resided in the splenocytes of im-munized mice, as demonstrated by adoptive transfer of spleen cellsfrom immunized donor mice to naive recipient mice (Fig. 6e).Adoptive transfer of 5 × 107 spleen cells isolated 2 weeks after im-munization of AdmCD40L-modified DCs pulsed with P. aeruginosaprovided 80% protection against lethal challenge of P. aeruginosa (P< 0.001, AdmCD40L-modified DCs pulsed with P. aeruginosa com-pared with all other groups; Fig. 6e). No protection was provided bycontrol splenocytes from mice immunized with AdNull-modifiedDCs pulsed with P. aeruginosa or AdmCD40L-modified DCs aloneor with no immunization.

This transferable immunity depended on CD19+ B lymphocyte,as shown by transfer of CD19+ or CD19– splenocytes to the naivemice (Fig. 6f). To determine the capacity of B cells to preserve theinduced immunity, we separated splenocytes from mice immu-nized with AdmCD40L-modified DCs pulsed with P. aeruginosainto B cells or non-B cells using CD19 as a marker of B cells, andevaluated each cell fraction for protective ability against lethalchallenge of P. aeruginosa after transfer to naive recipient mice.Mice injected with CD19+ or total spleen cells experienced a signif-icant improvement in survival, with eight or nine of ten mice aliveat the end of the experiment on day 14, respectively (P < 0.01,CD19+ cells compared with CD19– cells). In contrast, only 20% of

a b

Fig. 3 Ability of AdmCD40L-modified DCs pulsed with P. aeruginosa to directly induce naive B cellsto secrete P. aeruginosa-specific antibody in vitro in the absence of CD4+ T cells. a, IgM levels. b, IgAlevels. Titers, the inverse of the dilution giving A415 ≤ 0.1; values represent means ± standard error oftriplicate cultures. Vertical axis, modifications to the DCs. AdmCD40L+PA (CD4–/–), all cell compo-nents from syngeneic CD4–/– mice; AdmCD40L+PA (CD11c+ DCs), CD11c+ DCs purified before mod-ification and co-culture with syngeneic B cells from wild-type mice; NH4Cl, ammonium chloride.

a b c d

e f gFig. 4 P. aeruginosa-specific antibodies generated in vivo in wild-typemice immunized with AdmCD40L-modified DCs pulsed withPseudomonas. a–f, Titers in serum samples: a, IgM. b, IgG1. c, IgG2a. d,IgG2b. e, IgG3. f, IgA. g, IgA titers in respiratory epithelial lining fluid.Titers, the inverse of the dilution giving A415≤0.1 values representmeans ± standard error (n = 3 mice per data point). AdmCD40L-modi-fied DCs pulsed with heat-killed P. aeruginosa, �; AdNull-modified DCspulsed with heat-killed P. aeruginosa, ; AdmCD40L-modified DCs, ;no immunization, �.


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mice were protected after intravenous injection of CD19– spleno-cytes, and no naive mice without any vaccination survived beyond5 days after Pseudomonas challenge.

The levels of serum antibodies against Pseudomonas measuredby enzyme-linked immunosorbent assay (ELISA) (Figs. 4 and 5)were relevant in vivo, because passive transfer of serum protectednaive recipient mice against subsequent lethal challenge with P.aeruginosa (P < 0.0001, AdmCD40L-modified DCs pulsed withheat-killed P. aeruginosa compared with all other groups; Fig. 6g).Naive mice receiving 100 µl of serum obtained from mice 3 weeksafter immunization of AdmCD40L-modified DCs pulsed with P.aeruginosa were completely protected against subsequentPseudomonas challenge. In contrast, control serum from mice im-munized with either AdNull-modified DCs pulsed with P. aerugi-nosa or AdmCD40L-modified DCs alone afforded only 10%protection of recipients, and naive mice without any vaccinationwere highly susceptible to Pseudomonas challenge, with no micesurviving beyond day 5.

Dependence on CD4+ T cells or B cellsImmunization with Pseudomonas-pulsed naive DCs (pulsed withPseudomonas without CD40L modification) induces weak immu-nity against P. aeruginosa in wild-type and CD8–/– mice, but not in

CD4–/– mice12. The considerably enhanced immunity againstPseudomonas induced by CD40L-modified DCs pulsed withPseudomonas can be achieved in the absence of CD4 T cells; that is,CD40L genetic modification of DCs not only augments the anti-Pseudomonas immunity but also affects the antigen-specific immu-nity independent of T-cell help. Here, studies with knockout miceshowed that B cells were required for anti-Pseudomonas immunityinduced by vaccination with CD40L/DCs/Pseudomonas, but CD4+ Tcells were not required (Fig. 6h). To evaluate the contribution oflymphocyte subpopulations to the protective immunity affordedby immunization with CD40L/DCs/Pseudomonas, we immunizedgroups of CD4+ T cell-deficient, B cell-deficient or wild-type micewith or without AdmCD40L-modified DCs pulsed with P. aerugi-nosa, then challenged them 3 weeks later by intratracheal injectionof P. aeruginosa. CD4–/– immunized mice were completely protectedfrom lethal challenge with P. aeruginosa, as were wild-type immu-nized mice. In contrast, there was no protective immunity in B cell-deficient immunized mice compared with wild-type mice withoutimmunization; all B cell-deficient mice died within 3 days ofPseudomonas instillation (P < 0.001, CD4+ T cell-deficient mice com-pared with B cell-deficient mice; Fig. 6h).

DiscussionOur results are consistent with the idea that antigen-pulsed DCstransduced to express CD40L efficiently generate antigen-specificprotective humoral immunity in vivo. The receptor for CD40L isCD40, a 40-kDa type I transmembrane protein found on antigen-presenting cells, including B cells, DCs and activatedmacrophages2,3. Humoral (antibody) immune responses require theproduction of antigen-specific antibodies generated after prolifera-tion and differentiation of B cells; proliferation results in expansionof B-cell clones specific for the antigen, and differentiation resultsin heavy-chain isotype switching, affinity maturation of antibod-ies, and memory B-cell generation7. For B cells to proliferate anddifferentiate, two distinct types of signals are required: the antigen,which interacts with membrane immunoglobulin molecules (B-cell receptors) on specific B cells; and triggering of CD40 on the B-cell surface by CD40L expressed on activated CD4+ helper Tlymphocytes3,6,7. Mutations in the CD40L gene cause the X-linkedhyper-IgM syndrome, characterized by normal-to-high levels ofIgM with absence of IgG, IgA and IgE classes of immunoglobulinsin serum, and susceptibility to bacterial infections2. CD40L expres-

Fig. 5 P. aeruginosa-specific antibodies generated in CD4–/– C57Bl/6 miceimmunized with AdmCD40L-modified DCs pulsed with Pseudomonas.im-munized CD4–/– mice, n ; immunized wild-type mice, ¤ ; immunized wild-type mice, non-immunized wild-type mice, . Data represent titers(theinverse of the dilution giving A415 ≤ 0.1) in serum samples, as means ±standard error (n = 3 mice per data point) .

Fig. 6 Mice immunized with AdmCD40L-modified DCs pulsed with P.aeruginosa develop Pseudomonas-specific, CD4+ T cell-independent, B cell-dependent protection against lethal bronchopulmonary infection of P.aeruginosa. Survival (vertical axes), percentage of surviving mice (n = 10mice per group). All mice were infected with 2 × 105 CFU P. aeruginosa as achallenge, except in d. a and b, AdmCD40L-modified DCs protect immu-nized mice against Pseudomonas challenge 3 weeks (a) and 3 months (b)after immunization. c and d, AdmCD40L-modified DCs induce specific im-munity against microbes with which DCs have been pulsed. Challenge: P.

a b c d e f g h

aeruginosa (c) and 1 × 108 CFU E. coli (d). e and f, Induced immunity is trans-ferable by splenocytes. e, Transfer of total splenocytes from immunizedmice. f, Transfer of CD19+ or CD19– splenocytes. Two weeks after immuniza-tion, each cell fraction from the spleen (2 × 107 CD19+ cells, 3 × 107 CD19–

cells or 5 × 107 total splenocytes) was transferred intravenously to recipients.Recipient mice were challenged 7 d after the transfer (day 0). g, Passivetransfer of serum from immunized mice. Recipients were challenged 6 hafter the transfer of heat-inactivated serum from immunized mice (day 0). h,Requirement for CD4+ T cells or B cells to establish protective immunity.


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sion is restricted almost exclusively to activated helper T cells, anddepends on antigen-mediated stimulation of T cells by antigen-pre-senting cells, especially DCs, thus maintaining the specificity of theimmune response2.

Here we have shown that genetic modification of DCs to expressCD40L accomplishes the goal of directly activating B cells to in-duce functionally relevant antigen-specific humoral immune re-sponses that lead to protection against the lethal infection in amicrobe-specific way. After adenovirus-mediated gene transfer ofthe CD40L cDNA to a DC to express CD40L, the genetically modi-fied DC was capable of directly activating B cells and presentingantigens with which the modified DC had been loaded. IrradiatedCD40L-modified DCs induced nonspecific proliferation of B cells.Moreover, Pseudomonas-specific IgM and IgA antibodies were de-tected in the supernatants of in vitro co-cultures of naive B cells andAdmCD40L-modified DCs pulsed with heat-killed Pseudomonas.This occurred with DCs and B cells obtained from CD4–/– mice.Furthermore, when pulsed with heat-killed Pseudomonas and ad-ministered to naive mice, the CD40L-transduced DCs induced theproduction of Pseudomonas-specific IgM, IgG and IgA antibodies,indicating that the B cells underwent an isotype switch in vivo. Aswith the in vitro generation of Pseudomonas-specific antibodies, invivo administration of CD40L-modified DCs to CD4–/– mice in-duced Pseudomonas-specific IgM, IgG, and IgA antibodies in thesame pattern as in wild-type mice; that is, in the conditions ab-solute CD4 deficiency, genetic modification of DCs with the T-cellactivation gene CD40L enabled the host defense system to gener-ate antigen-specific antibodies in vivo as well as in vitro.

Whereas a function for DCs in humoral immune responses iswell established in the context of DC-mediated activation of CD4+

helper T cells, it is generally believed that B cells cannot be directlystimulated by DCs to produce antigen-specific antibodies.Nonspecific activation of B cells by DCs has been found when DCsare activated. For example, DCs incubated with B cells that havebeen pre-activated with CD40L-transfected fibroblasts induce theproliferation of B cells by soluble mediators, enhance the amountof secretion of IgM, IgG and IgA, and CD40 ligation of DCs aug-ments IgM production from co-cultured CD40-activated B cells13,14.Here we extended this idea by using CD40L-modified DCs to per-mit the direct interaction between DCs and B cells, resulting in thedirect induction of naive B cells to secrete antigen-specific antibod-ies. The data indicate this is through CD40 activation of the B cells,but do not exclude involvement of soluble mediators such as IL-12,which stimulate the B cell13,15. Our study has shown that CD40L-modified DCs spontaneously secreted cytokines; moreover, DCsgenetically modified with a recombinant adenovirus to expressCD40L become self-activated through CD40 on their surface16.

As CD4+ helper T cells are essential in specific antimicrobial im-munity, agonistic monoclonal antibody against mouse CD40, plas-mid DNA expressing soluble trimeric CD40L and recombinanttrimeric CD40L protein have been evaluated in an attempt to cir-cumvent CD4+ T-cell help in competent immune responses topathogenic microorganisms. Intraperitoneal administration ofmonoclonal antibody against mouse CD40 to BALB/c mice withStreptococcus pneumoniae or Salmonella typhimurium antigens cangenerate strong, isotype-switched antibody responses that are pro-tective against subsequent S. pneumoniae or S. typhimurium infec-tions, respectively17,18. BALB/c mice are protected from the footpadinfection of intracellular parasite Leishmania major when givenmonoclonal antibody against CD40 intraperitoneally or trimericCD40L-expressing DNA and leishmanial protein into the foot-pad19,20. Finally, intraperitoneal administration of recombinant

trimeric CD40L protein induces specific antibody responses thatprotect recipients of allogeneic mouse bone marrow transplantfrom herpes simplex virus 1 infection21. All these strategies must beused cautiously, as the expression of CD40L is normally restrictedalmost exclusively to activated CD4+ helper T cells under exquisiteregulation, and because in mouse models it has been shown thatinappropriate presence of the CD40L may bring about adverse con-sequences such as inflammation or abnormal lymphoprolifera-tion22,23. The use of adenovirus vectors for the transfer of CD40LcDNA to DCs may have a benefit for the purpose of stimulatinghost CD40, because adenovirus-mediated expression of the trans-gene is locally confined to the transduced cells without inductionof systemic adverse effects, and is limited in duration of expres-sion24–26. The strategy of pulsing CD40L-modified DCs with inacti-vated microbes should enlist the powerful capacity of DCs topresent antigens to host immune systems and elicit pathogen-spe-cific immunity.

MethodsAdenovirus vectors. AdmCD40L is an E1−E3-replication-deficient recombi-nant Ad5-based vector containing an expression cassette with the cytomegalovirus early/immediate promoter/enhancer ‘driving’ the mouseCD40L cDNA (ref. 27). The control AdNull vector is identical to this but contains no transgene28. Amplification, purification and titration of the vectorswere done as described29,30. All vectors were free of replication-competent adenovirus31.

Preparation and activation of DCs. Bone marrow-derived DCs were grown incomplete RPMI 1640 medium (10% FBS, 2 mM L-glutamine, 100 µg/ml strep-tomycin and 100 units/ml penicillin) supplemented with 10 ng/ml recombi-nant mouse granulocyte–macrophage colony-stimulating factor and 2 ng/mlrecombinant mouse IL-4 (both from R & D Systems, Minneapolis,Minnesota)16,32. To demonstrate that adenovirus-mediated transfer of CD40Lresulted in activation of DCs, DCs purified from bone marrow were transducedfor 4 h with AdmCD40L or AdNull, at a multiplicity of infection of 40, or withPBS, pH 7, alone (naive), and were cultured at a density of 5 × 106 cells/ml.Culture medium (400 µl) was collected after 72 h, and the levels of mouse IL-12 p40 or mouse MIP-1α in the culture medium were determined by ELISA(R&D Systems, Minneapolis, Minnesota). An antibody against mCD40L anti-body or control IgG (both at a concentration of 10 µg/ml; PharMingen, San Diego, California) added at the initiation of the culture served as an addi-tional control.

In vitro activation of B cells. To assess the ability of CD40L-modified,Pseudomonas-pulsed DCs to induce the proliferation of B cells, 2 × 104 CD19+ Bcells were isolated from the spleens of naive C57Bl/6 mice using anti-CD19 mi-crobeads (Miltenyi Biotech, Auburn, California), and were then cultured in 96-well culture plates with 2 × 104 DCs in complete RPMI 1640 mediumsupplemented with 20 ng/ml recombinant mouse IL-4 (R & D Systems,Minneapolis, Minnesota). Before the co-culture, DCs were modified with ade-novirus vectors (AdmCD40L or AdNull, at a multiplicity of infection of 100)and were irradiated (3,000 rad). The number of viable cells was measured asthe absorbance at 490 nm using an MTS colorimetric assay kit (Promega). Thepercentage of proliferation was calculated as 100 × ([experimental ab-sorbance] – [background absorbance]) / ([absorbance at start of the co-cultureon day 0] – [background absorbance]).

To assess the ability of AdmCD40L-modified DCs pulsed with Pseudomonasto directly induce naive B cells to secrete Pseudomonas-specific antibody invitro, 1 × 105 CD19+ B lymphocytes/ml from a naive C57Bl/6 mouse, purifiedusing a magnetic cell sorter system (Miltenyi Biotech, Auburn, California),were cultured for 14 d in a 96-well plate in a final volume of 200 µl with 1 ×105 cells/ml AdmCD40L-modified DCs pulsed with heat-killed P. aeruginosa(10 Pseudomonas per DC; PAO1 strain of Pseudomonas, provided by A. Prince,Columbia University, New York), AdNull-modified DCs pulsed with heat-killed P. aeruginosa, AdmCD40L-modified DCs or DCs with no treatment inthe presence of 20 ng/ml IL-4 (R&D Systems, Minneapolis, Minnesota). Todemonstrate in vitro processing was involved, AdmCD40L-modified DCspulsed with heat-killed P. aeruginosa were treated with 5 µg/ml brefeldin A,


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10 µg/ml cytochalasin D or 50 mM ammonium chloride (all from Sigma) for30 min before, as well as during, the Pseudomonas pulse. To demonstrateCD4+ T-cell independence, both DCs and B cells were prepared from CD4–/–

mice, or CD11c (αX integrin), one of the DC markers. DCs were purified fromthe DC culture with the magnetic cell sorting system (Miltenyi Biotech,Auburn, California) before modification of DCs with AdmCD40L andPseudomonas and subsequent co-culture with naive B cells. After 14 d, thetiter of various isotypes of P. aeruginosa-specific antibody in culture super-natants (200 µl) was determined by ELISA using heat-killed P. aeruginosa asthe antigen33. End-point titers were determined as the reciprocal of the dilu-tion at or below a fixed absorbance value of 0.1; negative results were given atiter of the lowest dilution. As controls for the specificity of the ELISA, no sig-nificant IgM, IgG or IgA antibodies against Pseudomonas were detected in an-tisera obtained from mice immunized against E. coli, but positive IgM, IgGand IgA antibodies against Pseudomonas were detected in sera from mice im-munized against Pseudomonas.

In vivo generation of Pseudomonas-specific antibodies. Female C57Bl/6mice 6–8 weeks old, from the Jackson Laboratories (Bar Harbor, Maine), werehoused in specific pathogen-free conditions. To immunize the mice,AdmCD40L-modified DCs (multiplicity of infection, 100; 4 h at 37 °C) were in-cubated with heat-killed (1 h at 56 °C) Pseudomonas (PAO1) for 4 h at a ratio often bacterial equivalents to one DC. The adenovirus was added first, immedi-ately followed by the bacteria. Gentamicin sulfate (Sigma) was then added to aconcentration of 200 µg/ml, and the cell suspension was incubated for an-other 30 min to kill the remaining bacteria. The cells were extensively washedtwice with PBS, and 5 × 104 DCs in 100 µl PBS were injected intravenously intothe tail vein. Controls included AdNull-modified DCs pulsed with heat-killed P.aeruginosa, AdmCD40L-modified DCs alone, and naive mice without any im-munization. Two weeks after immunization, antibodies against Pseudomonaswere assessed in serum by ELISA. To assess the titer of respiratory mucosal an-tibodies against Pseudomonas, respiratory epithelial lining fluid was preparedby instillation of 1.5 ml PBS to mouse lungs and withdrawal of the fluid. Aftercentrifugation, the supernatant was collected and assayed for end-point titersof antibodies against Pseudomonas by ELISA.

Protection against lethal respiratory infection with Pseudomonas. To as-sess the ability of AdmCD40L-modified DCs pulsed with Pseudomonas to de-velop Pseudomonas-specific, CD4+ T cell-independent, B cell-dependentprotection against lethal bronchopulmonary infection with Pseudomonas,C57Bl/6 mice were immunized with AdmCD40L-modified DCs pulsed withheat-killed P. aeruginosa or heat-killed E. coli. AdNull-modified DCs pulsedwith heat-killed P. aeruginosa, or AdmCD40L-modified DCs alone were usedas controls. All DC immunizations used 5 × 104 DCs per mouse. Additionalcontrols included naive mice without any immunization. Female C57Bl/6mice, CD4-deficient mice (C57Bl/6-CD4tm1Mak) and B cell-deficient mice(C57Bl/6-Igh-6tm1Cgn), 6–8 weeks old, that had been back-crossed to theC57Bl/6 background were obtained from the Jackson Laboratories (BarHarbor, Maine), and the mice were immunized as described above. ThePAO1 strain of P. aeruginosa enmeshed in agar beads was prepared based ona published method34. The density of viable P. aeruginosa enmeshed in agarbeads was determined by plating serial dilutions of homogenized bead sus-pension onto MacConkey agar plates. Pseudomonas agar beads (50 µl) wereinstilled slowly through a catheter into the lungs of anesthetized mice. Allmice were checked daily for 14 d for symptoms and mortality. Obviouslymoribund mice were killed, and this was considered the date of death. Forthe E. coli infection of mice, E. coli (25922 strain; Amercican Type CultureCollection, Rockville, Maryland) were grown to log phase, washed threetimes with PBS and resuspended in PBS at the desired concentration as deter-mined by spectrophotometry. Numbers of bacteria were confirmed by deter-mining the CFU of diluted aliquots on LB agar plates. An inoculum (50 µl) of1 × 108 CFU E. coli was implanted into the lungs of anesthetized mice, butwithout agar beads. Survival was assessed by Kaplan-Meier analysis.

AcknowledgmentsWe thank N. Mohamed for help in preparing this manuscript. These studies weresupported, in part, by National Institutes of Health grant P01 HL51746-06A1,the Will Rogers Memorial Fund (Los Angeles, California), the Cystic FibrosisFoundation (Bethesda, Maryland) and GenVec (Gaithersburg, Maryland).

RECEIVED 6 JULY; ACCEPTED 29 AUGUST 2000

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