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Conventional PKC (cPKC)-alpha regulates TRIF-dependent IFN response factor 3 (IRF3)-mediated gene transcription, but its role in MyD88-dependent TLR signaling remains unknown. Herein, we demonstrate that PKC-alpha is induced by several MyD88-dependent TLR/IL-1R ligands and regulates cytokine expression in human and murine DC. First, inhibition of cPKC activity in human DC by cPKC-specific inhibitors, Gö6976 or HBDDe, downregulated the production of classical inflammatory/immunomodulatory cytokines induced by TLR2, TLR5 or IL-1R but not by TLR3 stimulation. Similarly, dominant negative PKC-alpha repressed Pam(3)CSK(4) induced NF-kappaB- and AP-1-driven promoter activities in TLR2-expressing human embryonic kidney 293 T cells. Dominant negative PKC-alpha inhibited NF-kappaB reporter activity mediated by overexpression of MyD88 but not TRIF. Unexpectedly, BM-derived DC from PKC-alpha(-/-) mice exhibited decreased TNF-alpha and IL-12p40 production induced by both MyD88- and TRIF-dependent ligands. Furthermore, PKC-alpha is coupled to TLR2 signaling proximal to MyD88 since MAPK and IkappaB kinase-alpha/beta phosphorylations and IkappaBalpha degradation were inhibited in PKC-alpha(-/-) BM-derived DC. Finally, co-immunoprecipitation assays revealed that PKC-alpha physically interacts with Pam(3)CSK(4) activated TLR2 in WT but not in MyD88(-/-) DC. Collectively this study identifies a species-specific role of PKC-alpha as a key component that controls MyD88-dependent cytokine gene expression in human and mouse but differentially regulates production of TRIF-dependent cytokines.

Background: Monoamine oxidase A (MAO-A), a mitochondrial enzyme that degrades monoamines including neurotransmitters, is highly expressed in basal cells of the normal human prostatic epithelium and in poorly differentiated (Gleason grades 4 and 5), aggressive prostate cancer (PCa). Clorgyline, an MAO-A inhibitor, induces secretory differentiation of normal prostate cells. We examined the effects of clorgyline on the transcriptional program of epithelial cells cultured from high grade PCa (E-CA)., Methods: We systematically assessed gene expression changes induced by clorgyline in E-CA cells using high-density oligonucleotide microarrays. Genes differentially expressed in treated and control cells were identified by Significance Analysis of Microarrays. Expression of genes of interest was validated by quantitative real-time polymerase chain reaction., Results: The expression of 156 genes was significantly increased by clorgyline at all time points over the time course of 6 - 96 hr identified by Significance Analysis of Microarrays (SAM). The list is enriched with genes repressed in 7 of 12 oncogenic pathway signatures compiled from the literature. In addition, genes downregulated >or= 2-fold by clorgyline were significantly enriched with those upregulated by key oncogenes including beta-catenin and ERBB2, indicating an anti-oncogenic effect of clorgyline. Another striking effect of clorgyline was the induction of androgen receptor (AR) and classic AR target genes such as prostate-specific antigen together with other secretory epithelial cell-specific genes, suggesting that clorgyline promotes differentiation of cancer cells. Moreover, clorgyline downregulated EZH2, a critical component of the Polycomb Group (PcG) complex that represses the expression of differentiation-related genes. Indeed, many genes in the PcG repression signature that predicts PCa outcome were upregulated by clorgyline, suggesting that the differentiation-promoting effect of clorgyline may be mediated by its downregulation of EZH2., Conclusion: Our results suggest that inhibitors of MAO-A, already in clinical use to treat depression, may have potential application as therapeutic PCa drugs by inhibiting oncogenic pathway activity and promoting differentiation.

Overexpression of CD95 (Fas/Apo-1) ligand (CD95L) has been shown to induce T cell tolerance but also, neutrophilic inflammation and rejection of allogeneic tissue. We explored the capacity of dendritic cells (DCs) genetically engineered to overexpress CD95L to induce an antitumor response. We first found that DCs overexpressing CD95L, in addition to MHC class I-restricted OVA peptides (CD95L-OVA-DCs), induced increased antigen-specific CD8(+) T cell responses as compared with DCs overexpressing OVA peptides alone. The enhanced T cell responses were associated with improved regression of a tumor expressing OVA, allowing survival of all animals. When DCs overexpressing CD95L (CD95L-DCs) were injected with the tumor expressing OVA, in vivo tumor proliferation was strikingly inhibited. A strong cellular apoptosis and a massive neutrophilic infiltrate developed in this setting. Neutrophil depletion prevented tumor regression as well as enhanced IFN-gamma production induced by CD95L-OVA-DCs. Furthermore, the CD8(+) T cell response induced by the coadministration of tumor cells and CD95L-DCs led to rejection of a tumor implanted at a distance from the DC injection site. In summary, DCs expressing CD95L promote tumor rejection involving neutrophil-mediated innate immunity and CD8(+) T cell-dependent adaptative immune responses.

Inflammatory responses mounted to eliminate parasites can be lethal if not counterbalanced by regulatory responses protecting the host from collateral tissue damage. Here, we show that the maintained inflammation associated with tissue damage, anemia, and reduced survival of Trypanosoma brucei-infected mice correlates with the absence of the expansion of the regulatory T (T(reg)) cell population. Induction of T(reg) cell expansion via CD28 superagonist antibody treatment in these mice down-regulated interferon-gamma production by T cells and tumor necrosis factor-alpha and reactive oxygen species production by classically activated macrophages, triggered the development of alternatively activated macrophages, delayed the onset of liver injury, diminished the anemia burden, and prolonged the survival of infected animals. Thus, triggering the expansion of the T(reg) cell population coupled with the induction of alternatively activated macrophages can restore the balance between pro- and anti-inflammatory signals and thereby limit the pathogenicity of African trypanosomiasis.

Objective: The aim of this work is to study variations of prostate cancer incidence by stage as a function of time and place in a region of France., Material and Methods: Retrospective observational survey conducted in five private and public urology centres representative of the various demographic features of the Nord-Pas-de-Calais region. In each centre, the medical records of the first 25 cases of prostate cancer diagnosed in 1998, 2002 and 2004, identified from histology laboratory data, were studied by means of a case report form evaluating the circumstances of diagnosis, PSA level, grade, stage (TNM 97, classification) and initial management., Results: This analysis was based on 123, 124 and 125 patients in five centres in 1998, 2002 and 2004, respectively. The age at diagnosis ranged from 71.14 to 68.9 years between 1998 and 2004 (p=0.054). Median PSA decreased over this six-year period from 18 to 10.8 ng/ml. Between 1998 and 2004, the percentage of patients with localized cancer (PSA<20 ng/ml) increased from 44.8 to 66.4% (p<0.05), the percentage of patients with locally advanced cancer (PSA between 20 and 50 ng/ml) decreased from 17 to 9.6% (p<0.05), the percentage of patients with regional or distant metastatic disease (N1 and/or M1 and/or PSA>50 ng/ml) decreased from 29.4 to 22.4% (p<0.05) and the percentage of patients receiving curative treatment increased from 30 to 54.4% (p<0.005)., Conclusion: The prostate cancer incidence by stage varied between 1998 and 2004, with a significantly higher proportion of localized stages, which can be explained by the increased use of screening and diagnostic tests. Routine surveys can measure trends and the amplitude of incidence variations in the population of a region. A representative survey conducted in centres throughout France would allow evaluation of national trends between two publications of incidence by stage results in French registries.

Tolerance to African trypanosomes requires the production of IFN-gamma in the early stage of infection that triggers the development of classically activated macrophages controlling parasite growth. However, once the first peak of parasitemia has been controlled, down-regulation of the type 1 immune response has been described. In this study, we have evaluated whether regulatory T cells (Tregs) contribute to the limitation of the immune response occurring during Trypanosoma congolense infection and hereby influence the outcome of the disease in trypanotolerant C57BL/6 host. Our data show that Foxp3+ Tregs originating from the naturally occurring Treg pool expanded in the spleen and the liver of infected mice. These cells produced IL-10 and limited the production of IFN-gamma by CD4+ and CD8+ effector T cells. Tregs also down-regulated classical activation of macrophages resulting in reduced TNF-alpha production. The Treg-mediated suppression of the type 1 inflammatory immune response did not hamper parasite clearance, but was beneficial for the host survival by limiting the tissue damages, including liver injury. Collectively, these data suggest a cardinal role for naturally occurring Tregs in the development of a trypanotolerant phenotype during African trypanosomiasis.

IL-27 is a heterodimeric cytokine composed of EBV-induced gene 3 and p28. Produced by dendritic cells (DCs) in response to TLR ligands, IL-27 recently emerged as a key regulator of inflammatory responses. In this study, we first demonstrate that Toll/IL-1R-containing adaptor inducing IFN-beta and its associated IFN regulatory factor (IRF) 3 transcription factor are critically involved in IL-27p28 expression in mouse DCs stimulated by TLR ligands. We then show that IL-27 serum levels are dramatically reduced in IRF3(-/-) upon LPS injection, indicating a critical role for IRF3 in TLR4-mediated IL-27 production in vivo. We identified an IRF3-binding site within the IL-27p28 promoter region which is required for IL-27p28 gene activation in reporter gene assays. In human DCs, IL-27p28 mRNA was preferentially induced by Toll/IL-1R-containing adaptor inducing IFN-beta-coupled TLR ligands and following CMV infection. Furthermore, chromatin immunoprecipitation studies demonstrate that IRF3 is recruited to the endogenous p28 promoter in TLR4-stimulated human DCs. We conclude that IRF3 activation is a master switch for IL-27 synthesis.

Maturation of dendritic cells (DC) is a critical step in the induction of T cell responses and depends on the activation of NF-kappaB transcription factors. Therefore, inhibition of NF-kappaB activation has been proposed as a strategy to maintain DC in an immature stage and to promote immune tolerance. Herein, we generated murine myeloid DC expressing a mutated IkappaBalpha acting as a superrepressor of the classical NF-kappaB pathway (s-rIkappaB DC) to investigate the consequences of NF-kappaB inhibition on the ability of DC to prime T cell responses. Upon in vitro LPS activation, maturation of s-rIkappaB DC was profoundly impaired as indicated by defective up-regulation of MHC class II and costimulatory molecules and reduced secretion of IL-12 p70 and TNF-alpha. In contrast, after injection, s-rIkappaB DC had the same capacity as control DC to migrate to draining lymph node and to induce Th1- and Th2-type cytokine production in a MHC class II-incompatible host mice. Likewise, s-rIkappaB DC pulsed with OVA were as efficient as control DC to induce Ag-specific T cell responses in vivo. Indeed, further in vitro experiments established that s-rIkappaB DC undergo efficient maturation upon prolonged contact with activated T cells via the alternative pathway of NF-kappaB activation triggered at least partly by lymphotoxin beta receptor ligation and involving processing of p100/RelB complexes.

Background: Whereas the participation of alloreactive T cells sensitized by indirect allorecognition in graft rejection is well documented, the nature of recipient antigen presenting cells recognized by indirect pathway CD4+ T cells within the graft has yet to be identified. The purpose of this study was to determine the role played by graft endothelium replacement in the immune recognition of cardiac allografts rejected by indirect pathway CD4+ T cells., Methods: Transgenic RAG2-/- mice expressing I-Ab-restricted male antigen H-Y-specific TcR were studied for their capacity to reject H-2k male cardiac allografts. Chronic vascular rejection in this model was due to the indirect recognition of H-Y antigen shed from H-2k male allograft and presented by the recipient's own I-Ab APC to transgenic T cells., Results: Immunohistochemical analysis of rejected grafts revealed the presence of numerous microvascular endothelial cells (EC) that expressed recipient's I-Ab MHC class II molecules. This observation suggested that graft endothelium replacement by I-Ab-positive cells of recipient origin could stimulate the rejection of male H-2k graft by I-Ab-restricted H-Y-specific T cells. To investigate further this possibility, hearts from H-2b-into-H-2k irradiation bone marrow (BM) chimera were transplanted in transgenic recipients. A direct correlation was observed between the presence of I-Ab-positive EC within myocardial microvessels and the induction of acute rejection of chimeric H-2k male cardiac allografts transplanted in transgenic recipients., Conclusions: We conclude that graft endothelium replacement by recipient-type cells is required for the rejection of cardiac allograft mediated by indirect pathway alloreactive CD4+ T cells.

Pertussis toxin (PTX) is known to be mitogenic for T lymphocytes, but its direct action on naive human T cells has not been specified. Herein, we show that PTX induces the proliferation of purified adult CD45RA(+)CD4(+) T cells independently of its ADP-ribosyltransferase activity. PTX directly induces TNF-alpha and IL-2 mRNA expression, modulates the level of several cell surface receptors and induces Forkhead box p3 (Foxp3) protein accumulation in naive CD4(+) T cells. Addition of autologous dendritic cells was found to be required for the production of high levels of IFN-gamma by PTX-stimulated naive T cells. These effects of PTX occurred in conjunction with activation of NF-kappaB and NFAT transcription factors. Overall, responses of neonatal CD4(+) T cells to PTX were similar to those of adult CD45RA(+)CD4(+) naive T cells except for their blunted CD40 ligand up-regulation. We suggest that the adjuvant properties of PTX during primary cell-mediated immune responses involve a direct action on naive T lymphocytes in addition to activation of antigen-presenting cells.

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by dendritic cells (DCs) in response to Toll-like receptor (TLR) ligation. While the mechanisms regulating IL-12p40 chain gene expression are well characterized, molecular events involved in IL-12p35 chain gene activation remain to be clarified. Since IL-12p35 mRNA was induced in human DCs activated through TLR3 or TLR4 but not TLR2, we investigated the potential role of interferon regulatory factor 3 (IRF-3) in IL-12p35 gene transactivation. First, a binding site for IRF-3 named interferon-stimulated response element-1 (ISRE-1) was identified in the human IL-12p35 promoter region between nucleotides -251 and -240. The ISRE-1 site was required for IL-12p35 gene activation in RAW 264.7 cells stimulated by lipopolysaccharide (LPS) or PolyI:C. Ectopic expression of IRF-3 was found to up-regulate IL-12p35 gene activation in the same system. Furthermore, chromatin immunoprecipitation (ChIP) studies demonstrated that IRF-3 is recruited to ISRE-1 site in TLR4- or TLR3-stimulated human DCs. Finally, experiments on DCs from IRF-3-deficient mice established that TLR4-induced IL-12p35 mRNA and IL-12p70 synthesis are impaired in absence of IRF-3. We conclude that IRF-3 binds to a critical cis-acting element in the IL-12p35 gene promoter and thereby represents a key factor for the induction of IL-12p70 synthesis in DCs.

Background: Acute rejection of MHC class II-disparate bm12 skin grafts by C57BL/6 recipient mice is characterized by massive graft infiltration by eosinophils, together with increased intragraft amounts of IL-4 and IL-5 mRNA. IL-5 blockade prevents the intragraft eosinophil infiltration and prolongs the survival of skin allografts. As the differentiation of T cell precursors into Th2 cells is largely driven by IL-4, we investigated the role of IL-4 in MHC class II-disparate allograft rejection., Methods: We performed skin grafts from MHC class II incompatible bm12 mice into wild-type C57BL/6 mice (IL-4) or C57BL/6 IL-4 deficient mice (IL-4). Graft survival, in vitro T cell reactivity, and histology were compared., Results: We observed that 50% of IL-4 mice rapidly rejected their bm12 allograft, whereas the other 50% retained their graft 60 days after transplantation. Histological examination of bm12 allografts retained by IL-4 mice showed a normal appearance with no inflammatory infiltrate and no eosinophils. Among IL-4 mice that acutely rejected their bm12 skin graft, we observed a dense polymorphonuclear infiltrate. The depletion of neutrophils significantly prolonged bm12 graft survival., Conclusions: Eosinophil infiltrates, typical of MHC class II disparate acute skin graft rejection, are critically dependent on the availability of IL-4. IL-4 mice reject MHC class II disparate skin grafts by a pathway of rejection where neutrophils play a direct causal role.

Background: CD8+ T cells are known to regulate type 2 helper T cell (Th2) alloreactive immune responses but their mode of activation is unclear. We investigated the role of host CD8+ T cells in experimental Th2-type graft-versus-host disease (GVHD) where donor/recipient disparity is restricted to a single major histocompatibility complex (MHC) class II antigen., Methods: Immunoglobulin (Ig) E serum levels, eosinophilia and lymphoid tissue hyperplasia were compared after injection of bm12 CD4+ T cells in either wild-type or CD8+ T cell-deficient (CD8-/-) C57BL/6 mice. In vitro, we explored effects of the addition of CD8+ T cells from wild-type or IFN-gamma-/- mice in mixed leukocyte cultures prepared with beta2 microglobulin-deficient (beta2m-/-) CD4+ T cells as responders or beta2m dendritic cells as stimulators., Results: HyperIgE resolved after 3 weeks in wild-type hosts whereas it persisted for 6 weeks in CD8-/- hosts. Eosinophil infiltrates in lymph nodes were significantly enhanced in CD8-/- hosts. Increased serum levels of IL-5 and IL-13 in CD8-/- hosts confirmed the enhancement of Th2-type responses in the context of recipient CD8+ T cell deficiency. Hyperplasia of lymph nodes and spleen were similar in both groups, as well as in vivo proliferation of donor CD4+ T cells. In vitro, CD8+ T cell regulation of the alloreactive Th2 response depended on their production of IFN-gamma and did not require expression of beta2m on CD4+ T cells or antigen-presenting cells., Conclusions: Host CD8+ T cells regulate alloreactive Th2 responses during graft-versus-host disease through an IFN-gamma dependent pathway, independently of the recognition of beta2m-associated MHC class I molecules.

Because rejection of allografts is primarily caused by T and B lymphocyte responses to allogeneic histocompatibility molecules, the role of innate immunity in organ transplant rejection is often overlooked. However, the very first damages to vascularized organ allografts are caused by ischemia-reperfusion, an inflammatory reaction involving activation of vascular endothelial cells and release of neutrophil chemoattractants. Herein, we review experimental observations suggesting that the early neutrophil influx in organ transplants favors T cell-mediated rejection.

Background: Allografts are occasionally accepted in the absence of immunosuppression. Because naturally occurring CD4(+)CD25(+) regulatory T cells (natural CD25(+) Treg cells) have been shown to inhibit allograft rejection, we investigated their influence on the outcome of allografts in nonimmunosuppressed mouse recipients., Methods: We compared survival times of male CBA/Ca skin grafts in female CBA/Ca recipients expressing a transgenic anti-HY T-cell receptor on a RAG-1(+/+) (A1[M]RAG+) or a RAG-1(-/-) (A1[M]RAG-) background. Depletion of natural CD25(+) Treg cells in A1[M]RAG+ mice was achieved by in vivo administration of the PC61 monoclonal antibody. The influence of natural CD25(+) Treg cells on the fate of major histocompatibility complex class II-mismatched (C57BL/6X bm12)F1 skin or bm12 heart transplants in C57BL/6 recipients was also assessed. Finally, we investigated the impact of natural CD25(+) Treg cells on the production of T-helper (Th)1 and Th2 cytokines in mixed lymphocyte cultures between C57BL/6 CD4(+) CD25(-) T cells as responders and bm12 or (C57BL/6X bm12)F1 antigen-presenting cells as stimulators., Results: Male allografts were spontaneously accepted by female A1(M)RAG+ mice but readily rejected by female A1(M)RAG+ mice depleted of natural CD25(+) Treg cells by pretreatment with the PC61 monoclonal antibody. Depletion of CD25(+) Treg cells also enhanced eosinophil-determined rejection of (C57BL/6X bm12)F1 skin grafts or bm12 cardiac grafts in C57BL/6 recipients. Finally, natural CD25(+) Treg cells inhibited the production of interleukin (IL)-2, interferon-gamma, IL-5, and IL-13 in mixed lymphocyte culture in a dose-dependent manner., Conclusion: Natural CD25(+) Treg cells control Th1- and Th2-type allohelper T-cell responses and thereby influence the fate of allografts in nonimmunosuppressed recipients.

Viral interleukin (IL)-10 (vIL-10) has been widely described as an immunoregulatory cytokine that does not possess the T-cell costimulatory activities of cellular IL-10; it was therefore believed to be a more potent tolerogenic mediator. The immunosuppressive properties of this cytokine are partly attributed to its capacity to render dendritic cells (DCs) unable to undergo full maturation and to activate T cells. We reported here that myeloid DCs retrovirally transduced with vIL-10 had an impaired production of IL-12 and a decreased expression of MHC class II molecules but had minor defects in costimulatory molecule expression and no alteration on CCR5 and CCR7 expression. In mixed leukocyte reaction, vIL-10-transduced C57BL/6 bm12 (MHC class II mismatch) DCs had a reduced capacity to stimulate C57BL/6 wild-type CD4+ T-cell proliferation. We show that bm12 vIL-10-transduced DC administration in CD8-/- C57BL/6 mice promoted IFN-gamma production, down-regulated TH2-type cytokine production, and did not induce skin graft tolerance. These findings suggest that vIL-10-transduced DC may surprisingly facilitate Th1-type inflammatory responses in vivo.

Biological effects of two structurally contrasting PCB congeners (coplanar 77 and non coplanar 153) were investigated by measuring the induction of CYP1A immunopositive protein (CYP1A IPP) in the pyloric caeca and the production of reactive oxygen species (ROS) by amoebocytes in the common sea star Asterias rubens. CYP1A IPP was quantified using a specially designed ELISA which uses competitive binding between sea stars and trout CYP1A IPPs. Only the coplanar congener had a significant effect on the two considered biological responses. Intensity of the effects was dose-dependent. However, the highest dose of PCB 77 induced a dramatic decrease of ROS production. It is concluded that coplanar PCBs straightforwardly affect key biological processes such as the immune system and mixed-function oxidase (MFO) system.

Because rejection of allografts is primarily caused by T and B lymphocyte responses to allogenic histocompatibility molecules, the role of innate immunity in organ transplant rejection is often overlooked. However, the very first damages to vascularized organ allografts are caused by ischemia-reperfusion, an inflammatory reaction involving activation of vascular endothelial cells and release of neutrophil chemoattractants. Herein, we review experimental observations suggesting that the early neutrophil influx in organ transplants favors T cell-mediated rejection.

Dendritic cells (DC) genetically engineered to express Fas (CD95) ligand (FasL-DC) have been proposed as immunotherapeutic tools to induce tolerance to allografts. However, we and others recently showed that FasL-DC elicit a vigorous inflammatory response involving granulocytes and can promote Th1-type CD4+ and cytotoxic CD8+ T lymphocytes. This prompted us to evaluate the pathology induced by intravenous injection of FasL-DC in mice. We observed that FasL-DC obtained after retroviral gene transfer of bone marrow precursors derived from Fas-deficient C57Bl/6 mice induce massive pulmonary inflammation and pleuritis one day after a single intravenous injection in C57Bl/6 mice. Two months later, all mice presented granulomatous vasculitis of small to medium sized vessels, alveolar haemorrhage and pleuritis. In these lesions, apoptotic bodies were found in large number. Anti-neutrophilic cytoplasmic and anti-myeloperoxidase autoantibodies were not detected. This study documents that intravenous injection of FasL-DC causes severe lung granulomatous vasculitis. This new animal model for vasculitis is inducible, highly reproducible and shares many features with human Wegener granulomatosis. This model may be an appropriate tool to further investigate the pathogenesis of vasculitis and test new therapeutic strategies. Moreover, our findings highlight the potential severe complications of FasL-DC-based immunotherapy.

Ischemia/reperfusion during liver transplantation triggers a complex cascade of inflammatory events that may lead to organ dysfunction. Herein, we investigated the consequences of hepatic ischemia/reperfusion on liver dendritic cells. Liver damage was documented by increased levels of serum alanine aminotransferase and by histopathology showing large areas of hepatocyte cytolysis. MHC class II+ CD45-B220 F4/80 dendritic cells were detected in necrotic areas 20 hours after reperfusion. Dendritic cells freshly isolated from reperfused livers displayed a mature phenotype characterized by upregulated expression of B7 costimulatory molecules; MHC-class II, and CD1d molecules. As shown by real-time PCR, IL-10, and TGF-beta mRNA accumulated in liver dendritic cells isolated after reperfusion, whereas IL-12p40 mRNA levels were decreased and IFN-gamma mRNA levels were unchanged. These results suggest that hepatic ischemia/reperfusion results in maturation of dendritic cells, which preferentially produce inhibitory cytokines., (Copyright 2004 Elsevier Inc.)

Transplantation tolerance induced by neonatal injection of semiallogeneic spleen cells is associated in several strain combinations with a pathological syndrome caused by Th2 differentiation of donor-specific CD4(+) T lymphocytes. We investigated the role of host CD8(+) T cells in the regulation of this Th2 pathology. IgE serum levels and eosinophilia significantly increased in BALB/c mice neonatally injected with (A/J x BALB/c)F(1) spleen cells when CD8(+) T cells were depleted by administration of anti-CD8 mAb or when beta(2)-microglobulin-deficient mice were used as recipients. In parallel, increased serum levels of IL-5 and IL-13 were measured in blood of tolerant CD8(+) T cell-deficient mice. Whereas neonatally injected mice were unable to generate anti-donor cytotoxic effectors, their CD8(+) T cells were as efficient as control CD8(+) T cells in reducing the severity of Th2 pathology and in restoring donor-specific cytotoxicity in vitro after in vivo transfer in beta(2)-microglobulin-deficient mice. Likewise, CD8(+) T cells from control and tolerant mice equally down-regulated the production of Th2 cytokines by donor-specific CD4(+) T cells in vitro. The regulatory activity of CD8(+) T cells depended on their secretion of IFN-gamma for the control of IL-5 production but not for IL-4 or IL-13. Finally, we found that CD8(+) T cells from 3-day-old mice were already able to down-regulate IL-4, IL-5, and IL-13 production by CD4(+) T cells. We conclude that regulatory CD8(+) T cells controlling Th2 responses are functional in early life and escape neonatal tolerization.

Background: Dendritic cells (DC) at the immature state express low levels of major histocompatibility complex and costimulatory molecules and are poor stimulators of primary T-cell response in vitro. Injection of immature bone marrow-derived DC, however, was shown to prime in vivo alloreactive CD4 T lymphocytes toward type 2 cytokine-producing cells in the absence of CD8 T-cell activation., Methods: We undertook the present study to determine whether Th2-immunization by immature DC could lead to allograft rejection. We first analyzed, in the major histocompatibility complex class II antigen-disparate B6-anti-bm12 combination, the capacity of immature DC to regulate the activity of alloreactive CD4 T cells. We then determined, in this model of weak antigenicity, whether injection of bm12 DC in B6 recipients before transplantation could modify the survival of vascularized bm12 cardiac allografts., Results: We confirmed that in vitro immature DC are poor stimulators of T-cell alloresponse. However, when given in vivo, immature bm12 DC primed anti-bm12 T cells for the production of interleukin (IL)-4. Moreover, they induced the acute rejection of bm12 cardiac allograft. The process of rejection was dependent on IL-4 because immunization of IL-4-deficient mice did not trigger rejection., Conclusions: Allogeneic immature DC generated with granulocyte-macrophage colony-stimulating factor are potent stimulators of primary alloreactive response in vivo and prime for transplant rejection. Our results indicate that strategies based on immature DC for the induction of transplantation tolerance should be considered with caution.

Dendritic cells (DCs) genetically engineered to overexpress CD95 (Fas) ligand (CD95L-DC) were proposed as tools to induce peripheral tolerance to alloantigens. Herein, we observed that CD95L-DC obtained after retroviral gene transfer in bone marrow (BM) precursors derived from CD95-deficient (lpr/lpr) mice elicit much stronger allospecific type 1 helper T-cell and cytotoxic T-cell activities than control DCs upon injection in vivo, although they induce lower T-cell responses in vitro. Indeed, a single injection of CD95L-DC prepared from C57BL/6 mice was sufficient to prime bm13 recipients for acute rejection of C57BL/6 skin allografts that were otherwise tolerated in the context of this single weak major histocompatibility complex (MHC) class I incompatibility. Massive neutrophil infiltrates depending on interleukin (IL)-1 signaling were observed at sites of CD95L-DC injection. Experiments in IL-1 receptor-deficient mice or in animals injected with depleting anti-Gr1 monoclonal antibody (mAb) established that neutrophil recruitment is required for the development of vigorous T-cell responses after injection of CD95L-DC in vivo.

In this report, we show that intravenous (i.v.) injection into mice of a complex made of the cationic lipid diC14-amidine and the luciferase reporter plasmid (pCMV-luc) results in efficient gene expression in several organs but elicits an inflammatory response characterized by a release of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) into the serum of treated animals. A single preinjection of free diC14-amidine liposomes improves the i.v. transfection efficiency of the diC14-amidine/protamine/pCMV-luc complex as much as 40 times. This improvement is correlated with the ability of free liposomes to inhibit TNF-alpha but not IFN-gamma production resulting from complex injection. TNF-alpha-rich serum obtained from mice injected with diC14-amidine/protamine/pCMV-luc complex inhibits luciferase expression in transfected mouse lung endothelial (MLE) cells cultured in vitro, whereas IFN-gamma has no effect. This inhibitory effect can be partly abolished by treating the mouse serum with a specific anti-TNF-alpha antibody. These data point out that cationic lipids are potent inhibitors of the inflammatory response to the CpG motifs in plasmid DNA. This property is shown to enhance the in vivo transfection efficiency.

Beta(2)-microglobulin (beta(2)m)-derived peptides are minor transplantation Ags in mice as beta(2)m-positive skin grafts (beta(2)m(+/+)) are rejected by genetically beta(2)m-deficient recipient mice (beta(2)m(-/-)). We studied the effector pathways responsible for the rejection induced by beta(2)-microglobulin-derived minor transplantation Ags. The rejection of beta(2)m(+/+) skin grafts by naive beta(2)m(-/-) mice was dependent on both CD4 and CD8 T cells as shown by administration of depleting mAbs. Experiments performed with beta(2)m(-/-)CD8(-/-) double knockout mice grafted with a beta(2)m(+/+) MHC class I-deficient skin showed that sensitized CD4 T cells directed at beta(2)m peptides-MHC class II complexes are sufficient to trigger rapid rejection. Rejection of beta(2)m(+/+) grafts was associated with the production of IL-5 in vitro, the expression of IL-4 and IL-5 mRNAs in the grafted tissue, and the presence within rejected grafts of a considerable eosinophil infiltrate. Blocking IL-4 and IL-5 in vivo and depleting eosinophils with an anti-CCR3 mAb prevented graft eosinophil infiltration and prolonged beta(2)m(+/+) skin graft survival. Lymphocytes from rejecting beta(2)m(-/-) mice also displayed an increased production of IFN-gamma after culture with beta(2)m(+/+) minor alloantigens. In vivo neutralization of IFN-gamma inhibited skin graft rejection. Finally, beta(2)m(+/+) skin grafts harvested from B6(lpr/lpr) donor mice, which lack a functional Fas molecule, survived longer than wild-type beta(2)m(+/+) skin grafts, showing that Fas-Fas ligand interactions are involved in the rejection process. We conclude that IL-4- and IL-5-dependent eosinophilic rejection, IFN-gamma-dependent mechanisms, and Fas-Fas ligand interactions are effector pathways in the acute rejection of minor transplantation Ags.

Background & Aims: Eosinophils are observed in several liver diseases, but their contribution in the pathogenesis of these disorders remains poorly investigated. Concanavalin A (Con A)-induced hepatitis is an experimental model of immune-mediated liver injury in which natural killer T (NKT) cells play a critical role through the production of interleukin (IL)-4 and the expression of Fas ligand (FasL). Because activated NKT cells also produce IL-5, a critical cytokine for eosinophil maturation and function, the role of IL-5 was investigated in this model., Methods: IL-5-deficient mice, eosinophil depletion in wild-type (WT) mice, and NKT cell transfer from WT- or IL-5-deficient mice into NKT cell-deficient mice were used to assess the role of IL-5 and eosinophils., Results: Liver eosinophil infiltrate and IL-5 production were observed after Con A challenge. Liver injury was dramatically reduced in IL-5-deficient or eosinophil-depleted mice. In addition, residual hepatitis observed in Fas-deficient mice was abolished after IL-5 neutralization. Finally, we showed that NKT cells constituted a critical source of IL-5. Indeed, transfer of WT NKT cells to mice lacking NKT cells restored liver injury, whereas transfer of IL-5-deficient NKT cells did not., Conclusions: These observations highlight the pathologic role of IL-5 and eosinophils in experimental immune-mediated hepatitis.

Dendritic cells (DC) are the most potent presenters of alloantigens and therefore are responsible for the induction of allograft rejection. Genetic modifications of DC allowing the expression of a tolerogenic molecule may render them immunosuppressive. We transduced bone marrow-derived DC with recombinant MFG retrovirus encoding either viral interleukin (vIL)-10 or Fas ligand (FasL) to induce transplantation tolerance. Up to 10 ng/ml of bioactive vIL-10 was produced by DC after transfer of the corresponding gene. Although the inhibitory properties of vIL-10-transduced DC were revealed in vitro in a mixed lymphocyte culture, no clear down-regulation of the allogeneic response was observed in vivo after single or multiple injections of those DC overexpressing vIL-10. When we transduced wild-type bone marrow-derived DC with recombinant MFG retrovirus encoding murine FasL, cells quickly died, probably because of suicidal or fratricidal Fas-dependent death. Indeed, only DC from Fas-deficient lpr mice survived to FasL gene transfer. Those FasL-transduced lpr DC exhibited a strong cytotoxic activity against Fas-positive targets in vitro. DC overexpressing FasL did not behave as immunosuppressive DC in vivo. The subcutaneous injection of FasL+ lpr DC in MHC class II-disparate mice hyperactivated the allospecific proliferation of T cells in the draining lymph nodes compared with mice treated with control-transduced DC. These results argue against the development of FasL+ DC or vIL-10-secreting DC as immunosuppressive tools in vivo. The alternative pathways of T-cell activation triggered by these genetically modified DC need to be investigated.

A significant proportion of patients with the hypereosinophilic syndrome suffer from oligoclonal expansion of type 2 helper T lymphocytes (Th2). Herein, we first provide evidence that mice immunized at birth against a single MHC class II alloantigen develop pathological features mimicking this variant of the hypereosinophilic syndrome. Indeed, C57BL / 6 mice injected at birth with (C57BL/ 6 x bm12)F1 spleen cells displayed T lymphocytes producing high levels of IL-5 and IL-13, increased blood eosinophil counts, eosinophilic infiltrates in various tissues, hyperplasia of lymphoid tissues, as well as serum hyperIgE. Moreover, eotaxin mRNA accumulated in the spleen of these animals. IL-4-deficient mice developed neither expansion of Th2 cells nor pathological changes except splenomegaly. Eotaxin mRNA accumulation was also prevented in these animals. We conclude that neonatal exposure to a single MHC class II alloantigen is sufficient to elicit an IL-4-dependent hypereosinophilic syndrome mimicking the lymphocytic variant of this disorder in humans.

Eosinophils release inflammatory mediators and cationic proteins that are instrumental in the pathogenesis of allergic diseases such as bronchial asthma. Here, we review experimental observations indicating that eosinophils are also involved in the rejection of allografts. We propose that their role as effectors of transplant damage becomes crucial when classical pathways of rejection are inhibited and T helper 2 (Th2) cells dominate the alloimmune response.

CD4 T cells play a crucial role in the acute rejection of MHC class II-disparate skin allografts, mainly by Fas/Fas ligand-mediated cytotoxicity. Because recent observations indicate that eosinophils may be found within allografts rejected by CD4 T cells, we evaluated the role played by IL-5, the main eosinophil growth factor, and by eosinophils in the rejection of MHC class II-disparate skin grafts. C57BL/6 mice rapidly rejected MHC class II-disparate bm12 skin grafts. Rejected skins contained a dense, aggressive eosinophil infiltrate. Lymphocytes isolated from lymph nodes draining rejected bm12 skin were primed for IL-5 secretion, and IL-5 mRNA was present within rejected grafts. The IL-5/eosinophil pathway played an effector role in allograft destruction, because the rejection of bm12 skin was significantly delayed in IL-5-deficient mice as compared with wild-type animals. The role of the IL-5/eosinophil pathway was further investigated in MHC class II-disparate donor-recipient strains unable to establish Fas/Fas ligand interactions. Fas ligand-deficient gld/gld mice rejected bm12 skins, and bm12 mice rejected Fas-deficient lpr/lpr C57BL/6 skins. Neutralization of IL-5 prevented acute rejection in both combinations. We conclude that MHC class II-disparate skin allografts trigger an IL-5-dependent infiltration of eosinophils that is sufficient to result in acute graft destruction.

C57BL/6 mice injected with the 145-2C11 anti-CD3 mAb and grafted with MHC class II disparate bm12 skin develop a chronic rejection characterized by interstitial dermal fibrosis, a marked eosinophil infiltrate, and an obliterative intimal vasculopathy. Because these changes occur in the absence of alloreactive antibodies, we examined the contribution of cytokines in their pathogenesis. Chronically rejected grafts showed a marked accumulation of both IL-4 and IL-5 mRNA. Mixed lymphocyte reaction experiments established that mice undergoing chronic rejection were primed for IL-4, IL-5, and IL-10 secretion. In vivo administration of anti-IL-4 mAb completely prevented allograft vasculopathy as well as graft eosinophil infiltration and dermal fibrosis. Injection of anti-IL-5 mAb or the use of IL-5-deficient mice as recipients also resulted in the lack of eosinophil infiltration or dermal fibrosis, but these mice did develop allograft vasculopathy. Administration of anti-IL-10 mAb did not influence any histologic parameter of chronic rejection. Thus, in this model, IL-4- and IL-5-mediated tissue allograft eosinophil infiltration is associated with interstitial fibrosis. IL-4, but not eosinophils, is also required for the development of obliterative graft arteriolopathy.

Background: Chronic rejection remains a leading cause of allograft loss. Histologically, it is characterized by arterial intimal thickening and parenchymal fibrosis. The immune mechanisms triggering chronic rejection are still uncompletely understood., Methods: We performed major histocompatibility complex (MHC) class H-incompatible skin grafts from C-H2bm12 (bm12, H2bm12) into C57BL/6 (C57BL/6, H2b) recipients immunosuppressed with a short course of anti-CD3 monoclonal antibodies to prevent acute rejection., Results: More than 80% of grafts survived for prolonged periods, but eventually all displayed macroscopic and microscopic evidence of chronic rejection. At histology, there was a progressive arterial intimal thickening as well as intense dermal fibrosis. This was accompanied by an inflammatory infiltrate consisting of lymphocytes and macrophages, but also of a considerable number of eosinophils. Mice with chronic rejection were unable to generate anti-donor MHC class II cytotoxic T lymphocyte activity at either 20 or 60 days after transplant. Furthermore, transplantation of bm12 skins on C57BL/6-congenic, Ig knock-out mice was associated with the development of a chronic rejection that was identical to that occurring in wild-type C57BL/6 animals, indicating that alloantibodies are not necessary in this model., Conclusions: (1) Skin grafts may undergo chronic rejection with the characteristic lesions of vasculopathy and fibrosis; (2) chronic rejection of MHC class II-disparate skins may occur in the absence of direct cytotoxic T lymphocyte activity or alloantibodies.

To investigate the consequences of CD40 engagement on the neonatal induction of transplantation tolerance, BALB/c mice were injected at birth with (A/J x BALB/c) F1 spleen cells together with activating anti-CD40 mAb and grafted 4 wk later with A/J skin. Whereas A/J allografts were accepted in mice neonatally injected with F1 cells and control Ab, they were acutely rejected in mice injected with F1 cells and anti-CD40 mAb. Neonatal administration of anti-CD40 mAb resulted in enhanced anti-A/J CTL activity, increased IFN-gamma, and decreased IL-4 production by donor-specific T cells in vitro. Experiments using anti-cytokine mAb and IFN-gamma-deficient mice demonstrated that CD40 ligation prevents neonatal allotolerance through an IFN-gamma- and IL-12-dependent pathway. Finally, we found that newborn T cells express less CD40L than adult T cells upon TCR engagement. Taken together these data indicate that insufficiency of CD40/CD40L interactions contribute to neonatal transplantation tolerance.

We investigated the effect of IL-12 on the induction of transplantation tolerance by neonatal injection of allogenic cells. We first observed that injection of newborn BALB/c mice with IL-12 and (A/J x BALB/c)F1 spleen cells prevented the Th2 alloimmune response induced by neonatal inoculation of F1 cells alone and allowed the differentiation of T cells secreting high amounts of IL-2 and IFN-gamma in mixed lymphocyte cultures with donor-type stimulators. Furthermore, IL-12 administration resulted in the emergence of anti-donor cytotoxic T lymphocyte responses although at lower levels than in control uninjected mice. In parallel, we found that mice injected at birth with IL-12 and F1 cells did not develop chimerism and were able to reject a donor-type skin graft as efficiently as control mice. We conclude that IL-12 inhibits the Th2 polarization of the newborn response to alloantigens and prevents thereby the establishment of transplantation tolerance.

A role for the Fc receptor beta chain (FcRbeta) in the pathogenesis of allergy has been suggested by genetic studies. FcRbeta is a subunit common to the high-affinity IgE (FcepsilonRI) and low-affinity IgG (FcgammaRIII) receptors, both of which contribute to the initiation of allergic reactions. Current in vitro data suggest that FcRbeta can function as either a positive or negative regulator, leaving a mechanistic explanation for its association with the development of atopy unclear. To address this controversy, we have generated novel mouse models relevant to human Fc receptor function. Analysis of FcepsilonRI- and FcgammaRIII-dependent responses in these mice provides unequivocal genetic evidence that FcRbeta functions as an amplifier of early and late mast cell responses and, remarkably, in vivo anaphylactic responses.

Fc epsilonRI gamma (Fc gamma) is a member of the zeta family of signal transducing molecules that function as components of both the TCR and Fc receptors (FcR). While the majority of thymocytes and T cells express TCRs containing zeta-chain homodimers, certain unique populations of T cells express TCRs that contain both zeta and Fc gamma. To examine the ability of Fc gamma to substitute for zeta-chain in T cell development and function, we introduced a transgene encoding Fc gamma into mice made genetically deficient for zeta-chain (zeta(e)-/-). Analysis of thymocyte development in zeta(e)-/-;Fc gamma Tg mice demonstrated that Fc gamma was able to support the maturation of both gammadelta TCR+ and alphabeta TCR+ T cells. However, positive selection of alphabeta TCR+ thymocytes was less efficient in zeta(e)-/-;Fc gamma Tg mice than in zeta(e)-/- mice reconstituted with zeta-chain. This difference may be due to the fact that Fc gamma contains a single immunoreceptor tyrosine-based activation motif (ITAM) whereas zeta-chain contains three ITAMs. Interestingly, the peripheral T cells that develop in zeta(e)-/- mice reconstituted with Fc gamma are functional and respond to TCR-specific stimuli. These data suggest that Fc gamma and zeta are interchangeable in their ability to mediate T cell development and function, however zeta-chain is more efficient at promoting positive selection and T cell maturation. The difference in efficiency between zeta and Fc gamma may be responsible in part for the unusual developmental and functional properties of T cells that constitutively express Fc gamma as a signaling component of their TCRs.

In mouse mast cells, both Fc epsilonRI and Fc gammaRIII are alpha beta gamma2 tetrameric complexes in which different alpha chains confer IgE or IgG ligand recognition while the signaling FcR beta and gamma chains are identical. We used primarily noninvasive techniques (changes in body temperature, dye extravasation) to assess systemic anaphylactic responses in nonanesthetized wild-type, Fc epsilonRI alpha chain -/- and FcR gamma chain -/- mice. We confirm that systemic anaphylaxis in mice can be mediated largely through IgG1 and Fc gammaRIII and we provide direct evidence that these responses reflect activation of Fc gammaRIII rather than Fc gammaRI. Furthermore, we show that Fc gammaRIII-dependent responses are more intense in normal than in congenic mast cell-deficient KitW/KitW-v mice, indicating that Fc gammaRIII responses have mast cell-dependent and -independent components. Finally, we demonstrate that the upregulation of cell surface expression of Fc gammaRIII seen in Fc epsilonRI alpha chain -/- mice corresponds to an increased association of Fc gammaRIII alpha chains with FcR beta and gamma chains and is associated with enhanced Fc gammaRIII-dependent mast cell degranulation and systemic anaphylactic responses. Therefore, the phenotype of the Fc epsilonRI alpha chain -/- mice suggests that expression of Fc epsilonRI and Fc gammaRIII is limited by availability of the FcR beta and gamma chains and that, in normal mice, changes in the expression of one receptor (Fc epsilonRI) may influence the expression of functional responses dependent on the other (Fc gammaRIII).

Fc epsilon RI gamma (gamma) is a member of a group of related proteins (the zeta-family dimers) that function as signal-transducing components of both Fc receptors and the T cell antigen receptor (TCR). Analysis of gamma expression during fetal thymus ontogeny revealed that it is expressed in early thymocytes, before the initiation of clonotypic TCR-alpha and TCR-beta gene rearrangement but is down-regulated in most adult thymocytes. To explore a possible role for gamma in thymocyte development, we generated transgenic mice in which this protein was overexpressed at all stages of ontogeny. Overexpression of gamma inhibited the maturation of T cells as well as natural killer (NK) cells. The developmental effects were transgene dose related and correlated with markedly delayed maturation of fetal CD4-CD8- FcRII/III+ thymocytes, cells thought to include the progenitors of both T and NK cells. These results suggest that the zeta and gamma chains serve distinctive functions in thymocyte development and indicate that Fc receptor(s) may play an important role in regulating the differentiation of early progenitor cells within the thymus.

Mast cells and basophils, which are activated by IgE and allergens through the high affinity IgE receptor (Fc epsilon RI), play a prominent role in anaphylaxis in the mouse. Mice deficient in this receptor become resistant to passive anaphylaxis. As a first step in developing an in vivo model that more closely mimics the IgE-mediated responses in man, we used a combination of transgenic and embryonic stem cell technology to generate a mouse line in which the murine Fc epsilon RI alpha-chain has been replaced with its human homologue. We demonstrate here that these mice express a tetrameric high affinity IgE receptor, in which the human alpha-chain associates with the murine beta- and gamma-chains, and that upon triggering with relevant Ag, this receptor mediates the initiation of the expected intracellular events. In addition, we show that the human alpha-chain restores an anaphylactic response to the nonresponsive alpha-deficient parental mouse line. This "humanized" mouse represents a potentially important model system, not only for studying the role of IgE in human immune responses, but also for testing potential therapeutic reagents that can interfere with responses mediated through the human Fc epsilon RI receptor.

The use of anti-CD3 monoclonal antibodies (mAb) to treat allograft rejection has been complicated by the morbidity observed during the first days of treatment, secondary to T cell activation and cytokine release. Available evidence in a mouse model indicates that F(ab')2 fragments of an anti-CD3 mAb are not mitogenic in vitro and can be injected in vivo without apparent toxicity. However, their immunosuppressive capacity is dramatically reduced, suggesting that long-term immunosuppression mediated by anti-CD3 antibodies in vivo may be associated to their mitogenic capacity. This paper demonstrates that a poorly mitogenic anti-CD3 mAb is able to induce potent immunosuppression in vivo with reduced morbidity. This finding suggests that immunosuppression in vivo by anti-CD3 mAbs is not directly related to their activation properties but nevertheless requires signaling capacities. Therefore, immunosuppression in vivo may be best achieved by using antibodies able to deliver an incomplete activation signal to T cells (thus avoiding systemic cytokine release), possibly leading to anergy. The implications of this study for the development of immunosuppressive antibodies are discussed.

The aim of this work is to induce tumor resistance to a B cell lymphoma in BALB/c mice using elements of the immune system. It has indeed been shown by us and by others that antigen-presenting cells (APC) like dendritic cells can induce efficient immune responses and can even substitute for Freund's adjuvant. Here we show that mice immunized with syngeneic dendritic cells pulsed in vitro with tumor antigen (BCL1 idiotype expressed by lymphoma cells) are protected against a subsequent tumor inoculation. The in vivo resistance can be correlated with the induction of a humoral response specific for the idiotype expressed by the tumor. No such protection can be achieved when B cells are used as APC. These data show that effector cells in tumor-bearing animals can be recruited and activated using dendritic cells, providing long-lasting immune surveillance.

Mast cells and basophils, which are activated by immunoglobulin E (IgE) and allergen, play a prominent role in anaphylaxis. However, they express at least three types of IgE receptor, including the high affinity IgE receptor (Fc epsilon RI). The relative contribution of these IgE receptors, and possibly other receptors such as Fc epsilon RII/CD23 and Mac-2, to the genesis of in vivo anaphylaxis is still unclear. To address this question, we have generated Fc epsilon RI-deficient mice. These mice appear normal and express a normal number of mast cells, but they are resistant to cutaneous and systemic anaphylaxis. These data demonstrate that Fc epsilon RI is necessary for the initiation of IgE-dependent anaphylactic reactions. Therefore, interfering with its function should be an effective means of treating allergy, regardless of the allergen specificity.

The aim of this study was to develop an immunization procedure avoiding external adjuvant. Data are presented showing that syngeneic dendritic cells (DC), which have been pulsed in vitro with antigen, induce a strong antibody response in mice. By contrast, antigen (Ag)-pulsed low-density B cells, although equally able to induce interleukin 2 secretion by an Ag-specific T cell hybridoma in vitro, only weakly prime the mice in vivo. Moreover, we show that the injection of Ag-pulsed DC induces the synthesis of isotypes similar to the immunoglobulin classes detected after immunization with the same Ag in complete Freund's adjuvant. Importantly, high amounts of IgG2a antibodies are produced, suggesting that T helper type 1 cells are activated. Collectively, these data indicate that DC can initiate a primary humoral response and that they may be used as physiological adjuvant in vivo.