Dendritic cells genetically modified to express CD40 ligand ... - Nature

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Dendritic cells genetically modified to express CD40 ligand and pulsed 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 1

Division of Pulmonary and Critical Care Medicine of the Department of Medicine, 2Department of Pediatrics, Belfer 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]


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We have investigated whether dendritic cells genetically modified to express CD40 ligand and pulsed with antigen can trigger B cells to produce antigen-specific antibodies without CD4+ T-cell help. Dendritic cells modified with a recombinant adenovirus vector to express CD40 ligand and pulsed with heat-killed Pseudomonas induced naive B cells to produce antibodies against Pseudomonas in the absence of CD4+ T cells in vitro, initiated Pseudomonas-specific humoral immune responses in vivo in wild-type and CD4–/– mice, and protected immunized wild-type and CD4–/–, but not B-cell–/– mice, from lethal intrapulmonary challenge with Pseudomonas. Thus, genetic modification of dendritic cells with CD40 ligand enables them to present a complex mixture of microbial antigens and establish CD4+ T cell-independent, B cell-mediated protective immunity against a specific microbe. Dendritic cells (DCs) are very specialized antigen-presenting cells that capture antigens, move from the periphery to lymphoid organs and present the processed antigens to resting, naive CD4+ T lymphocytes1. After the DC–CD4+ T-cell interaction through the CD4+ T-cell antigen receptor, the CD4+ T cell becomes activated and expresses many surface activation proteins1. Among these, the most important is CD40 ligand (CD40L, or CD154, a 33-kDa type II membrane protein and a member of the tumor necrosis factor gene family), which serves as a ligand for the CD40 receptor on the DC (refs. 2,3). After CD40L triggers CD40, the DC is enabled to directly interact with CD8+ cytotoxic T cells1,2. There has been considerable interest in using DCs to help induce anti-tumor immunity1,4,5. B cells also express CD40, and interaction of DCs with CD4+ T cells, through CD40L and CD40, is essential in enabling B cells to generate antigen-specific antibodies3,6,7. The requirement for CD4+ T cells as intermediates in the interaction of DCs with other components of the immune system is useful in providing control of immune responses1,3,6,8. However, the consequences of having CD4+ T cells as the intermediates in controlling immune responses result in inadequate host responses against infectious agents when there is an insufficient number of CD4+ T cells, as in immunodeficiency disorders like after infection with the human immunodeficiency virus9. Here we sought to determine if it is possible to genetically change DCs to take on the function of both DCs and CD4+ T cells by genetically modifying the DCs to express CD40L. This should enable the DCs to interact with microbe-specific antigens and directly activate B cells, thus generating microbe-specific protective antibodies 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 DCs with heat-killed Pseudomonas aeruginosa, and administered the 1154

modified DCs to syngeneic hosts. Immunization of naive mice in this way led to considerable protection against a lethal challenge with P. aeruginosa, mediated by Pseudomonas-specific humoral immunity elicited without CD4+ T-cell help. Cytokine production of AdmCD40L-modified DCs We confirmed the activation of DCs after adenovirus-vector-mediated transfer of the CD40L cDNA by assessing the genetically modified DCs for interleukin (IL)-12 and macrophage inflammatory protein (MIP)-1α secretion1,2 (Fig. 1). Infection of DCs with AdmCD40L resulted in an increase of 4,000% in IL-12 in the supernatant compared with infection with AdNull (a vector identical to AdmCD40L, containing no transgene) or no infection (P < 0.0001, for each comparison). Modification of DCs with AdmCD40L also stimulated the secretion of MIP-1α (400% greater than that with AdNull or no modification; P < 0.0001, for each comparison). The enhanced secretion of IL-12 and MIP-1α was abrogated 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 infection with AdmCD40L mediated functional CD40L expression on the DC surface. Interaction between DCs and B cells in vitro Co-culture of irradiated, AdmCD40L-modified DCs with syngenic B cells resulted in the proliferation of the B cells, which peaked 5 days after initiation of the culture (Fig. 2). In contrast, co-culture of irradiated AdNull-modified DCs or naive DCs with B cells induced no B-cell proliferation. AdmCD40L-modified DCs pulsed with P. aeruginosa processed and presented Pseudomonas antigens to co-cultured B cells, resulting in the stimulation of IgM and IgA production specific for NATURE MEDICINE • VOLUME 6 • NUMBER 10 • OCTOBER 2000


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Fig. 1 Activation of dendritic cells by genetic modification with the mouse CD40L cDNA. a, IL-12 secretion by AdmCD40L-modified mouse DCs. b, MIP-1α production by AdmCD40L-modified mouse DCs. media, ¤ ; monoclonal antibody against mCD40L, n ; control IgG, . Data represent mean ± standard error (n = 4 per data point).

P. aeruginosa in the absence of CD4+ T cells (Fig. 3). To investigate the effect of AdmCD40L-modified DCs on the differentiation of CD19+ B cells into specific immunoglobulin-secreting cells, we co-cultured CD19+ B cells separated from the spleens of naive mice with adenovirus-vector-modified DCs pulsed with or without P. aeruginosa. When co-cultured with CD19+ B cells, AdmCD40L-modified DCs pulsed with P. aeruginosa induced the production of significant amounts of Pseudomonas-specific IgM and IgA compared with that induced by AdNull-modified DCs pulsed with P. aeruginosa, AdmCD40L-modified DCs alone or naive DCs (IgM, 300–900% increase, P < 0.0001; IgA, 300–800% increase, P < 0.0001). There was no stimulation in the production of Pseudomonas-specific immunoglobulin subtypes IgG1, IgG2a, IgG2b or IgG3 (data not shown). The possibility that the presentation of the Pseudomonas antigens by DCs was responsible for the induced production of Pseudomonas-specific antibodies was indicated by the use of three types of pharmacologic inhibitors on antigen-processing pathways, including brefeldin A (inhibition of endoplasmic reticulum/Golgi transport), cytochalasin D (suppression of actin-dependent phagocytosis) and ammonium chloride (inhibition of acid pH-dependent degradation)8. Pseudomonas-specific IgM and IgA secretion in the co-culture of B cells and AdmCD40L-modified DCs pulsed with P. aeruginosa was abrogated significantly when DCs were primed with Pseudomonas in the presence of any one of these inhibitors (IgM, P < 0.0001; IgA, P < 0.0001). Although these inhibitors blocked the processing of the bacterial antigens for presentation in the DCs, the DCs modified with AdmCD40L still expressed CD40L after treatment with brefeldin A, cytochalasin D or ammonium chloride (that is, those treatments did not adversely modify the DCs; data not shown). To eliminate the possibility that CD4+ T cells contaminating the DC or B-cell preparations could be responsible for the observation of the in vitro generation of Pseudomonasspecific antibodies, we used DCs and B cells prepared from CD4–/– mice in similar co-culture experiments. Despite this absolutely CD4-deficient condition, the results obtained for Pseudomonas-specific IgM and IgA production from B cells cocultured with AdmCD40L-modified DCs pulsed with Pseudomonas were similar to those obtained with components from wild-type mice. Consistent with this, CD11c+ DCs purified from the DC culture and sorted by magnetic cell sorting, modified with AdmCD40L and pulsed with P. aeruginosa also induced Pseudomonas-specific IgM and IgA secretion from B cells. NATURE MEDICINE • VOLUME 6 • NUMBER 10 • OCTOBER 2000

Induction of antibodies against Pseudomonas in vivo We assessed the activation of B cells by immunization with CD40L/DCs/Pseudomonas in vivo by determining serum level of Pseudomonas-specific antibodies. C57Bl/6 mice immunized with AdmCD40L-modified DCs pulsed with P. aeruginosa produced significant 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 immunized with AdNull-modified DCs pulsed with P. aeruginosa or AdmCD40L-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 against Pseudomonas detected in vivo, serum of mice immunized with AdmCD40L-modified DCs pulsed with Escherichia coli was negative for antibodies against Pseudomonas IgM, IgG and IgA (data not shown). Like the increase in serum IgM and IgG antibodies against Pseudomonas, there was a significant increase in serum IgA levels in the mice immunized with CD40L/DCs/Pseudomonas compared with that in control mice (P < 0.0001; Fig. 4f). There was no significant difference of respiratory mucosal IgA antibodies against Pseudomonas in epithelial lining fluid in the mice immunized with CD40L/DCs/Pseudomonas (P > 0.1, for all comparisons), except for the small difference between AdmCD40L-modified DCs pulsed with P. aeruginosa and AdmCD40L-modified DCs alone (P < 0.05; Fig. 4g). No other isotypes of antibodies against P. aeruginosa were detected in epithelial lining fluid. Although this study cannot prove that Pseudomonas-specific mucosal antibodies below the level of detection were responsible for the protective immunity seen, it is possible that circulating specific immunoglobulins diffusing across the mucosa afford the relevant protection, similar to that in experimental infection with chimeric human immunodeficiency virus-1/simian immunodeficiency virus10,11. To demonstrate that the in vivo generation of Pseudomonasspecific antibodies by CD40L modified DCs pulsed with heatkilled Pseudomonas could occur independently of CD4+ T cells, we repeated the experiment using CD4–/– mice (Fig. 5). The serum levels 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 induced protective immunity, lasting at least 3 months, against a lethal challenge with Pseudomonas in vivo (Fig. 6a and b). Immunization of C57Bl/6 mice with 5 × 104 AdmCD40L-modified

Fig. 2 Nonspecific B-cell proliferation induced by co-culture of mCD40Lmodified DCs and syngeneic B cells. AdmCD40L-modified DCs (n ), AdNullmodified DCs (¡ ) and naive DCs () were co-cultured with B cells. Data represent means of duplicate cells. 1155


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microbe with which the DCs had been primed (Fig. 6c and d). Groups of C57Bl/6 mice received vaccinations of 5 × 104 AdmCD40Lmodified DCs pulsed with either heat-killed P. aeruginosa or E. coli, or no vaccination. After 3 weeks, the mice were challenged with intratracheal administration of 2 × 105 CFU P. aeruginosa or 1 × 108 CFU E. coli. Mice receiving immunization of P. aeruginosa-pulsed CD40Lactivated DCs were protected from Pseudomonas challenge, but immunization with E. coli-pulsed CD40L-activated DCs did not protect the mice, nor did no immunization (P < 0.0001, CD40L-activated DCs pulsed with Fig. 3 Ability of AdmCD40L-modified DCs pulsed with P. aeruginosa to directly induce naive B cells to secrete P. aeruginosa-specific antibody in vitro in the absence of CD4+ T cells. a, IgM levels. b, IgA heat-killed P. aeruginosa compared with all levels. Titers, the inverse of the dilution giving A415 ≤ 0.1; values represent means ± standard error of other groups; Fig. 6c). In contrast, 60% of mice triplicate cultures. Vertical axis, modifications to the DCs. AdmCD40L+PA (CD4–/–), all cell compoimmunized with E. coli-pulsed CD40L-actinents from syngeneic CD4–/– mice; AdmCD40L+PA (CD11c+ DCs), CD11c+ DCs purified before modvated DCs were protected against subsequent ification and co-culture with syngeneic B cells from wild-type mice; NH4Cl, ammonium chloride. E. coli challenge, whereas all non-immunized mice or mice immunized with P. aeruginosapulsed CD40L-activated DCs died after infecDCs pulsed with heat-killed P. aeruginosa 3 weeks before a lethal tion with E. coli (P < 0.0005, DCs pulsed with heat-killed E. coli challenge with 2 x 105 colony-forming units (CFU) P. aeruginosa en- compared with all other groups; Fig. 6d). meshed in agar beads resulted in 90% survival in of mice (P < 0.0005, compared with all other groups; Fig. 6a). In contrast, im- Transferable anti-Pseudomonas immunity munization with 5 × 104 AdNull-modified DCs pulsed with heat- The protection against Pseudomonas challenge provided by the killed P. aeruginosa or 5 × 104 AdmCD40L-modified DCs alone, or CD40L/DC/Pseudomonas vaccine resided in the splenocytes of imno immunization, led to survival of 10% or less in the infected munized mice, as demonstrated by adoptive transfer of spleen cells mice. The protective effect elicited by AdmCD40L-modified DCs from immunized donor mice to naive recipient mice (Fig. 6e). pulsed with heat-killed Pseudomonas also occurred when CD11c+ Adoptive transfer of 5 × 107 spleen cells isolated 2 weeks after imDCs used for immunization were purified by magnetic cell sorting munization of AdmCD40L-modified DCs pulsed with P. aeruginosa (data not shown). We obtained similar results using mice immu- provided 80% protection against lethal challenge of P. aeruginosa (P nized 3 months before the Pseudomonas challenge: 80% of mice im- < 0.001, AdmCD40L-modified DCs pulsed with P. aeruginosa communized with AdmCD40L-modified DCs pulsed with heat-killed P. pared with all other groups; Fig. 6e). No protection was provided by aeruginosa survived at least 14 days after the lethal challenge with P. control splenocytes from mice immunized with AdNull-modified aeruginosa (Fig. 6b; P < 0.0001, compared with all other groups). In DCs pulsed with P. aeruginosa or AdmCD40L-modified DCs alone contrast, the control groups of mice receiving AdNull-modified or with no immunization. This transferable immunity depended on CD19+ B lymphocyte, DCs pulsed with heat-killed P. aeruginosa or AdmCD40L-modified DCs alone 3 months before the instillation died within 5 days. as shown by transfer of CD19+ or CD19– splenocytes to the naive mice (Fig. 6f). To determine the capacity of B cells to preserve the Microbe-specific effects of AdmCD40L-modified DCs induced immunity, we separated splenocytes from mice immuWe next determined whether immunization using AdmCD40L- nized with AdmCD40L-modified DCs pulsed with P. aeruginosa modified DCs generated specific protective immunity against the into B cells or non-B cells using CD19 as a marker of B cells, and evaluated each cell fraction for protective ability against lethal challenge of P. aeruginosa after transfer to naive recipient mice. Mice injected with CD19+ or total spleen cells experienced a significant improvement in survival, with eight or nine of ten mice alive at the end of the experiment on day 14, respectively (P < 0.01, CD19+ cells compared with CD19– cells). In contrast, only 20% of

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Fig. 4 P. aeruginosa-specific antibodies generated in vivo in wild-type mice immunized with AdmCD40L-modified DCs pulsed with Pseudomonas. 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 represent means ± standard error (n = 3 mice per data point). AdmCD40L-modified DCs pulsed with heat-killed P. aeruginosa, ; AdNull-modified DCs pulsed with heat-killed P. aeruginosa, ; AdmCD40L-modified DCs, ; no immunization, . NATURE MEDICINE • VOLUME 6 • NUMBER 10 • OCTOBER 2000


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

mice were protected after intravenous injection of CD19– splenocytes, and no naive mice without any vaccination survived beyond 5 days after Pseudomonas challenge. The levels of serum antibodies against Pseudomonas measured by enzyme-linked immunosorbent assay (ELISA) (Figs. 4 and 5) were relevant in vivo, because passive transfer of serum protected naive recipient mice against subsequent lethal challenge with P. aeruginosa (P < 0.0001, AdmCD40L-modified DCs pulsed with heat-killed P. aeruginosa compared with all other groups; Fig. 6g). Naive mice receiving 100 µl of serum obtained from mice 3 weeks after immunization of AdmCD40L-modified DCs pulsed with P. aeruginosa were completely protected against subsequent Pseudomonas challenge. In contrast, control serum from mice immunized with either AdNull-modified DCs pulsed with P. aeruginosa or AdmCD40L-modified DCs alone afforded only 10% protection of recipients, and naive mice without any vaccination were highly susceptible to Pseudomonas challenge, with no mice surviving beyond day 5. Dependence on CD4+ T cells or B cells Immunization with Pseudomonas-pulsed naive DCs (pulsed with Pseudomonas without CD40L modification) induces weak immunity against P. aeruginosa in wild-type and CD8–/– mice, but not in

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Fig. 6 Mice immunized with AdmCD40L-modified DCs pulsed with P. aeruginosa develop Pseudomonas-specific, CD4+ T cell-independent, B celldependent protection against lethal bronchopulmonary infection of P. aeruginosa. Survival (vertical axes), percentage of surviving mice (n = 10 mice per group). All mice were infected with 2 × 105 CFU P. aeruginosa as a challenge, except in d. a and b, AdmCD40L-modified DCs protect immunized mice against Pseudomonas challenge 3 weeks (a) and 3 months (b) after immunization. c and d, AdmCD40L-modified DCs induce specific immunity against microbes with which DCs have been pulsed. Challenge: P. NATURE MEDICINE • VOLUME 6 • NUMBER 10 • OCTOBER 2000

CD4–/– mice12. The considerably enhanced immunity against Pseudomonas induced by CD40L-modified DCs pulsed with Pseudomonas can be achieved in the absence of CD4 T cells; that is, CD40L genetic modification of DCs not only augments the antiPseudomonas immunity but also affects the antigen-specific immunity independent of T-cell help. Here, studies with knockout mice showed that B cells were required for anti-Pseudomonas immunity induced by vaccination with CD40L/DCs/Pseudomonas, but CD4+ T cells were not required (Fig. 6h). To evaluate the contribution of lymphocyte subpopulations to the protective immunity afforded by immunization with CD40L/DCs/Pseudomonas, we immunized groups of CD4+ T cell-deficient, B cell-deficient or wild-type mice with or without AdmCD40L-modified DCs pulsed with P. aeruginosa, then challenged them 3 weeks later by intratracheal injection of P. aeruginosa. CD4–/– immunized mice were completely protected from lethal challenge with P. aeruginosa, as were wild-type immunized mice. In contrast, there was no protective immunity in B celldeficient immunized mice compared with wild-type mice without immunization; all B cell-deficient mice died within 3 days of Pseudomonas instillation (P < 0.001, CD4+ T cell-deficient mice compared with B cell-deficient mice; Fig. 6h). Discussion Our results are consistent with the idea that antigen-pulsed DCs transduced to express CD40L efficiently generate antigen-specific protective humoral immunity in vivo. The receptor for CD40L is CD40, a 40-kDa type I transmembrane protein found on antigenpresenting cells, including B cells, DCs and activated macrophages2,3. Humoral (antibody) immune responses require the production of antigen-specific antibodies generated after proliferation and differentiation of B cells; proliferation results in expansion of B-cell clones specific for the antigen, and differentiation results in heavy-chain isotype switching, affinity maturation of antibodies, and memory B-cell generation7. For B cells to proliferate and differentiate, two distinct types of signals are required: the antigen, which interacts with membrane immunoglobulin molecules (Bcell receptors) on specific B cells; and triggering of CD40 on the Bcell surface by CD40L expressed on activated CD4+ helper T lymphocytes3,6,7. Mutations in the CD40L gene cause the X-linked hyper-IgM syndrome, characterized by normal-to-high levels of IgM with absence of IgG, IgA and IgE classes of immunoglobulins in serum, and susceptibility to bacterial infections2. CD40L expres-

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aeruginosa (c) and 1 × 108 CFU E. coli (d). e and f, Induced immunity is transferable by splenocytes. e, Transfer of total splenocytes from immunized mice. f, Transfer of CD19+ or CD19– splenocytes. Two weeks after immunization, 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, Passive transfer of serum from immunized mice. Recipients were challenged 6 h after the transfer of heat-inactivated serum from immunized mice (day 0). h, Requirement for CD4+ T cells or B cells to establish protective immunity. 1157



ARTICLES sion is restricted almost exclusively to activated helper T cells, and depends on antigen-mediated stimulation of T cells by antigen-presenting cells, especially DCs, thus maintaining the specificity of the immune response2. Here we have shown that genetic modification of DCs to express CD40L accomplishes the goal of directly activating B cells to induce functionally relevant antigen-specific humoral immune responses that lead to protection against the lethal infection in a microbe-specific way. After adenovirus-mediated gene transfer of the CD40L cDNA to a DC to express CD40L, the genetically modified DC was capable of directly activating B cells and presenting antigens with which the modified DC had been loaded. Irradiated CD40L-modified DCs induced nonspecific proliferation of B cells. Moreover, Pseudomonas-specific IgM and IgA antibodies were detected in the supernatants of in vitro co-cultures of naive B cells and AdmCD40L-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 administered to naive mice, the CD40L-transduced DCs induced the production of Pseudomonas-specific IgM, IgG and IgA antibodies, indicating that the B cells underwent an isotype switch in vivo. As with the in vitro generation of Pseudomonas-specific antibodies, in vivo administration of CD40L-modified DCs to CD4–/– mice induced Pseudomonas-specific IgM, IgG, and IgA antibodies in the same pattern as in wild-type mice; that is, in the conditions absolute CD4 deficiency, genetic modification of DCs with the T-cell activation gene CD40L enabled the host defense system to generate antigen-specific antibodies in vivo as well as in vitro. Whereas a function for DCs in humoral immune responses is well established in the context of DC-mediated activation of CD4+ helper T cells, it is generally believed that B cells cannot be directly stimulated by DCs to produce antigen-specific antibodies. Nonspecific activation of B cells by DCs has been found when DCs are activated. For example, DCs incubated with B cells that have been pre-activated with CD40L-transfected fibroblasts induce the proliferation of B cells by soluble mediators, enhance the amount of secretion of IgM, IgG and IgA, and CD40 ligation of DCs augments IgM production from co-cultured CD40-activated B cells13,14. Here we extended this idea by using CD40L-modified DCs to permit the direct interaction between DCs and B cells, resulting in the direct induction of naive B cells to secrete antigen-specific antibodies. 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 CD40Lmodified DCs spontaneously secreted cytokines; moreover, DCs genetically modified with a recombinant adenovirus to express CD40L become self-activated through CD40 on their surface16. As CD4+ helper T cells are essential in specific antimicrobial immunity, agonistic monoclonal antibody against mouse CD40, plasmid DNA expressing soluble trimeric CD40L and recombinant trimeric CD40L protein have been evaluated in an attempt to circumvent CD4+ T-cell help in competent immune responses to pathogenic microorganisms. Intraperitoneal administration of monoclonal antibody against mouse CD40 to BALB/c mice with Streptococcus pneumoniae or Salmonella typhimurium antigens can generate strong, isotype-switched antibody responses that are protective against subsequent S. pneumoniae or S. typhimurium infections, respectively17,18. BALB/c mice are protected from the footpad infection of intracellular parasite Leishmania major when given monoclonal antibody against CD40 intraperitoneally or trimeric CD40L-expressing DNA and leishmanial protein into the footpad19,20. Finally, intraperitoneal administration of recombinant 1158

trimeric CD40L protein induces specific antibody responses that protect recipients of allogeneic mouse bone marrow transplant from herpes simplex virus 1 infection21. All these strategies must be used cautiously, as the expression of CD40L is normally restricted almost exclusively to activated CD4+ helper T cells under exquisite regulation, and because in mouse models it has been shown that inappropriate presence of the CD40L may bring about adverse consequences such as inflammation or abnormal lymphoproliferation22,23. The use of adenovirus vectors for the transfer of CD40L cDNA to DCs may have a benefit for the purpose of stimulating host CD40, because adenovirus-mediated expression of the transgene is locally confined to the transduced cells without induction of systemic adverse effects, and is limited in duration of expression24–26. The strategy of pulsing CD40L-modified DCs with inactivated microbes should enlist the powerful capacity of DCs to present antigens to host immune systems and elicit pathogen-specific immunity. Methods Adenovirus vectors. AdmCD40L is an E1−E3-replication-deficient recombinant Ad5-based vector containing an expression cassette with the cytomegalovirus early/immediate promoter/enhancer ‘driving’ the mouse CD40L cDNA (ref. 27). The control AdNull vector is identical to this but contains no transgene28. Amplification, purification and titration of the vectors were done as described29,30. All vectors were free of replication-competent adenovirus31. Preparation and activation of DCs. Bone marrow-derived DCs were grown in complete RPMI 1640 medium (10% FBS, 2 mM L-glutamine, 100 µg/ml streptomycin and 100 units/ml penicillin) supplemented with 10 ng/ml recombinant mouse granulocyte–macrophage colony-stimulating factor and 2 ng/ml recombinant mouse IL-4 (both from R & D Systems, Minneapolis, Minnesota)16,32. To demonstrate that adenovirus-mediated transfer of CD40L resulted in activation of DCs, DCs purified from bone marrow were transduced for 4 h with AdmCD40L or AdNull, at a multiplicity of infection of 40, or with PBS, 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 IL12 p40 or mouse MIP-1α in the culture medium were determined by ELISA (R&D Systems, Minneapolis, Minnesota). An antibody against mCD40L antibody 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 additional 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+ B cells were isolated from the spleens of naive C57Bl/6 mice using anti-CD19 microbeads (Miltenyi Biotech, Auburn, California), and were then cultured in 96well culture plates with 2 × 104 DCs in complete RPMI 1640 medium supplemented with 20 ng/ml recombinant mouse IL-4 (R & D Systems, Minneapolis, Minnesota). Before the co-culture, DCs were modified with adenovirus vectors (AdmCD40L or AdNull, at a multiplicity of infection of 100) and were irradiated (3,000 rad). The number of viable cells was measured as the absorbance at 490 nm using an MTS colorimetric assay kit (Promega). The percentage of proliferation was calculated as 100 × ([experimental absorbance] – [background absorbance]) / ([absorbance at start of the co-culture on day 0] – [background absorbance]). To assess the ability of AdmCD40L-modified DCs pulsed with Pseudomonas to directly induce naive B cells to secrete Pseudomonas-specific antibody in vitro, 1 × 105 CD19+ B lymphocytes/ml from a naive C57Bl/6 mouse, purified using 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 heatkilled P. aeruginosa, AdmCD40L-modified DCs or DCs with no treatment in the presence of 20 ng/ml IL-4 (R&D Systems, Minneapolis, Minnesota). To demonstrate in vitro processing was involved, AdmCD40L-modified DCs pulsed with heat-killed P. aeruginosa were treated with 5 µg/ml brefeldin A, NATURE MEDICINE • VOLUME 6 • NUMBER 10 • OCTOBER 2000


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10 µg/ml cytochalasin D or 50 mM ammonium chloride (all from Sigma) for 30 min before, as well as during, the Pseudomonas pulse. To demonstrate CD4+ 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 from the DC culture with the magnetic cell sorting system (Miltenyi Biotech, Auburn, California) before modification of DCs with AdmCD40L and Pseudomonas and subsequent co-culture with naive B cells. After 14 d, the titer of various isotypes of P. aeruginosa-specific antibody in culture supernatants (200 µl) was determined by ELISA using heat-killed P. aeruginosa as the antigen33. End-point titers were determined as the reciprocal of the dilution at or below a fixed absorbance value of 0.1; negative results were given a titer of the lowest dilution. As controls for the specificity of the ELISA, no significant IgM, IgG or IgA antibodies against Pseudomonas were detected in antisera obtained from mice immunized against E. coli, but positive IgM, IgG and IgA antibodies against Pseudomonas were detected in sera from mice immunized against Pseudomonas. In vivo generation of Pseudomonas-specific antibodies. Female C57Bl/6 mice 6–8 weeks old, from the Jackson Laboratories (Bar Harbor, Maine), were housed in specific pathogen-free conditions. To immunize the mice, AdmCD40L-modified DCs (multiplicity of infection, 100; 4 h at 37 °C) were incubated with heat-killed (1 h at 56 °C) Pseudomonas (PAO1) for 4 h at a ratio of ten bacterial equivalents to one DC. The adenovirus was added first, immediately followed by the bacteria. Gentamicin sulfate (Sigma) was then added to a concentration of 200 µg/ml, and the cell suspension was incubated for another 30 min to kill the remaining bacteria. The cells were extensively washed twice with PBS, and 5 × 104 DCs in 100 µl PBS were injected intravenously into the tail vein. Controls included AdNull-modified DCs pulsed with heat-killed P. aeruginosa, AdmCD40L-modified DCs alone, and naive mice without any immunization. Two weeks after immunization, antibodies against Pseudomonas were assessed in serum by ELISA. To assess the titer of respiratory mucosal antibodies against Pseudomonas, respiratory epithelial lining fluid was prepared by instillation of 1.5 ml PBS to mouse lungs and withdrawal of the fluid. After centrifugation, the supernatant was collected and assayed for end-point titers of antibodies against Pseudomonas by ELISA. Protection against lethal respiratory infection with Pseudomonas. To assess the ability of AdmCD40L-modified DCs pulsed with Pseudomonas to develop Pseudomonas-specific, CD4+ T cell-independent, B cell-dependent protection against lethal bronchopulmonary infection with Pseudomonas, C57Bl/6 mice were immunized with AdmCD40L-modified DCs pulsed with heat-killed P. aeruginosa or heat-killed E. coli. AdNull-modified DCs pulsed with heat-killed P. aeruginosa, or AdmCD40L-modified DCs alone were used as controls. All DC immunizations used 5 × 104 DCs per mouse. Additional controls included naive mice without any immunization. Female C57Bl/6 mice, 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 the C57Bl/6 background were obtained from the Jackson Laboratories (Bar Harbor, Maine), and the mice were immunized as described above. The PAO1 strain of P. aeruginosa enmeshed in agar beads was prepared based on a published method34. The density of viable P. aeruginosa enmeshed in agar beads was determined by plating serial dilutions of homogenized bead suspension onto MacConkey agar plates. Pseudomonas agar beads (50 µl) were instilled slowly through a catheter into the lungs of anesthetized mice. All mice were checked daily for 14 d for symptoms and mortality. Obviously moribund mice were killed, and this was considered the date of death. For the E. coli infection of mice, E. coli (25922 strain; Amercican Type Culture Collection, Rockville, Maryland) were grown to log phase, washed three times with PBS and resuspended in PBS at the desired concentration as determined by spectrophotometry. Numbers of bacteria were confirmed by determining the CFU of diluted aliquots on LB agar plates. An inoculum (50 µl) of 1 × 108 CFU E. coli was implanted into the lungs of anesthetized mice, but without agar beads. Survival was assessed by Kaplan-Meier analysis. Acknowledgments We thank N. Mohamed for help in preparing this manuscript. These studies were supported, in part, by National Institutes of Health grant P01 HL51746-06A1, the Will Rogers Memorial Fund (Los Angeles, California), the Cystic Fibrosis Foundation (Bethesda, Maryland) and GenVec (Gaithersburg, Maryland). NATURE MEDICINE • VOLUME 6 • NUMBER 10 • OCTOBER 2000

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