结核杆菌Hsp65与EGFP融合基因的构建及DC疫苗的制备

目的: 构建结核杆菌H37Rv株Hsp65与增强型绿色荧光蛋白 (EGFP)的融合基因pEGHsp65, 并以其转染小鼠的树突状细胞 (DC), 制备抗结核的DC疫苗。方法: 采用PCR技术, 从培养的结核杆菌H37Rv株中抽提Hsp65基因,克隆到含有EGFP基因的质粒pEGFP- C1中, 构建pEGHsp65融合基因。以其转染Hela细胞, 在共聚焦激光扫描荧光显微镜下观测不同时间荧光表达的强弱, 并用RT- PCR检测Hsp65mRNA的表达。以pEGHsp65融合基因转染小鼠骨髓细胞经GM- CSF和IL -4诱导分化的DC, 用MTT比色法检测DC疫苗刺激未致敏脾细胞的增殖。结果: 用EcoRⅠ和BglⅡ双酶切鉴定证实, H37Rv株Hsp65DNA已插入重组表达载体pEG -FP- C1中。将融合基因转染入Hela细胞, 48h转染率最高;用RT- PCR在mRNA水平上可检测到Hsp65mRNA的表达。用MTT比色法检测表明, 融合基因转染的DC能激活并引起未致敏的脾细胞增殖。结论: 成功地构建pEGHsp65融合基因和以其制备的DC疫苗, 为进一步观察其治疗结核病的效应奠定了基础。     

Immunomodulating activity of mycobacterial heat shock protein 65 in tumor cells

In recent years, heat shock proteins have been shown to be effective in enhancing the immunogenicity of tumors. In this study, we examined the effect of mycobacterial hsp65 gene transfection in a non-immunogenic and aggressive tumor cell-line in order to understand the factors that could contribute to the increase in immunogenicity mediated by Hsp65. The transfected cells were found to have indeed lost their tumorigenenicity and increased their immunogenicity. Tumor-specific cytotoxic T cells were present only in mice immunized with the Hsp65-expressing cells. Furthermore, endogenous Hsp70 was significantly increased in irradiated Hsp65-expressing cells and recombinant Hsp65 protein was able to stimulate the mRNA expression of various T helper 1 (Th1) and pro-inflammatory cytokines in splenocyte cultures, as well as a modest expansion of CD4 T cells. These results provide further evidence of the immunomodulating properties of Hsp65, which could be exploited for the treatment of cancer.     

Regulation of Th1/Th2 development by antigen-presenting cells in vivo

The aim of this work was to test whether the nature of the antigen-presenting cell influences the Th1/Th2 balance in vivo. Adoptive transfer of dendritic cells or macrophages, pulsed extracorporeally with antigen, results in antigen-specific T cell priming. Of note, macrophages and dendritic cells appear to differentially regulate the development of T lymphocytes. In addition, the population of splenic dendritic cells appears heterogeneous and includes the CD8a⁺ and CD8a⁻ subclasses which direct the differentiation of Th1 and Th2 cells, respectively. Using neutralizing antibodies and mice genetically deficient for various cytokines, we evaluated the role of several molecules in the development of Tcells in vivo. Our observations emphasize the role of CD86 and IL-6 for Th2 priming by macrophages, CD86 for Th1 priming by dendritic cells and IL-12 for Th1 priming by dendritic cells.     

Cell-Mediated Immune Responses and Protective Efficacy against Infection with Mycobacterium tuberculosis Induced by Hsp65 and hIL-2 Fusion Protein in Mice

Heat shock protein 65 (Hsp65) is an important immunodominant antigen against tuberculosis (TB), and interleukin-2 (IL-2) plays an important role in the regulation of antimycobacteria immune responses. In order to further increase the immunogenicity of Hsp65 against infection caused by Mycobacterium tuberculosis (MTB), we expressed MTB Hsp65 and human IL-2 fusion protein, Hsp65-hIL-2, in Escherichia coli. The expression of Hsp65-hIL-2 was confirmed by Western blotting using anti-Hsp65 MoAb and anti-hIL-2 MoAb, respectively. Hsp65-IL-2 and Hsp65 were then purified by Ni-NTA affinity chromatography. Mice were immunized with purified Hsp65-hIL-2 or Hsp65 emulsified in the adjuvant combination dimethyl dioctadecylammonium bromide and monophosphoryl lipid A. Eight weeks after immunization, there was significant proliferation of spleen lymphocytes in response to both Hsp65 and Hsp65-hIL-2 proteins. Interestingly, Hsp65-hIL-2 fusion protein elicited significantly higher levels of IFN-γ and IL-2 in the lymphocytes culture supernatant than that of the BCG (Denmark strain) immunized group and Hsp65 group ( P  < 0.05). After challenging the immunized mice with MTB, the bacteria loads in the spleens and lungs of mice immunized with the fusion protein were significantly lower than Hsp65 alone group, reaching an equivalent level as BCG immunization group. Our results suggest that the Hsp65 and hIL-2 fusion protein may serve as an alternative vaccine candidate against MTB infection.     

Induction of interleukin-12 production in mouse macrophages by berberine,a benzodioxoloquinolizine alkaloid,deviates CD4+ T cells from a Th2 to a Th1 response

In this study we investigated whether berberine-mediated induction of interleukin-12 (IL-12) production in antigen-presenting cells could regulate a cytokine profile of antigen-primed CD4+ T helper (Th) cells. Pretreatment with berberine induced IL-12 production in both macrophages and dendritic cells, and significantly increased the levels of IL-12 production in lipopolysaccharide-stimulated macrophages and in CD40 ligand-stimulated dendritic cells. Importantly, berberine pretreatment of macrophages increased their ability to induce interferon-gamma (IFN-gamma) and reduced their ability to induce IL-4 in antigen-primed CD4+ T cells. Berberine did not influence the macrophage cell surface expression of the class II major histocompatibility complex molecule, the co-stimulatory molecules CD80 and CD86, and intracellular adhesion molecule-1. Addition of neutralizing anti-IL-12p40 monoclonal antibody to cultures of berberine-pretreated macrophages and CD4+ T cells restored IL-4 production in antigen-primed CD4+ T cells. The in vivo administration of berberine resulted in the enhanced induction of IL-12 production by macrophages when stimulated in vitro with lipopolysaccharide or heat-killed Listeria monocytogenes, leading to the inhibition of the Th type 2 cytokine profile (decreased IL-4 and increased IFN-gamma production) in antigen-primed CD4+ T cells. These findings may point to a possible therapeutic use of berberine or medicinal plants containing berberine in the Th type 2 cell-mediated immune diseases such as allergic diseases.     

Stimulation of dendritic cells via CD40 enhances immune responses to Mycobacterium tuberculosis infection

The resolution of pulmonary tuberculosis (TB) critically depends on the development of the Th1 type of immune responses, as exemplified by the exacerbation of TB in IL-12-deficient mice. Therefore, vaccination strategies optimizing IL-12 production by antigen-presenting cells (APC) in response to mycobacteria may have enhanced protective efficacy. Since dendritic cells (DC) are the critical APC for activation of CD4+ and CD8+ T cells, we examined whether stimulation of Mycobacterium bovis bacillus Calmette Guérin (BCG)-infected DC via CD40 increased their ability to generate Th1-oriented cellular immune responses. Incubation of DC with an agonistic anti-CD40 antibody activated CD40 signaling in DC, as shown by increased expression of major histocompatibility complex class II and costimulatory molecules, mRNA production for proinflammatory cytokines and interleukin 12 (IL-12) p40. This activation pattern was maintained when DC were stimulated with anti-CD40 antibody and infected with BCG. Importantly, CD40-stimulated BCG-infected DC displayed increased capacity to release bioactive IL-12 and to activate gamma interferon (IFN-gamma) producing T cells in vitro. Moreover, when C57BL/6 mice were immunized with these DC and challenged with aerosol Mycobacterium tuberculosis, increased levels of mRNA for IL-12 p40, IL-18, and IFN-gamma were present in the draining mediastinal lymph nodes. However, the mycobacterial burden in the lungs was not reduced compared to that in mice immunized with BCG-infected non-CD40-stimulated DC. Therefore, although the manipulation of DC via CD40 is effective for enhancing immune responses to mycobacteria in vivo, additional strategies are required to increase protection against virulent M. tuberculosis infection.     

Interleukin-12 production is required for chlamydial antigen-pulsed dendritic cells to induce protection against live Chlamydia trachomatis infection

Immunization with dendritic cells pulsed ex vivo with antigens has been successfully used to elicit primary antigen-specific immune responses. We report that mouse bone marrow-derived dendritic cells pulsed with inactivated chlamydial organisms induced strong protection against live chlamydial infection in a mouse lung infection model. Either the dendritic cells or chlamydial organisms alone or macrophages similarly pulsed with chlamydial organisms failed to induce any significant protection. These observations suggest that dendritic cells can efficiently process and present chlamydial antigens to naive T cells in vivo. Mice immunized with the chlamydia-pulsed dendritic cells preferentially developed a Th1 cell-dominant response while mice immunized with the other immunogens did not, suggesting a correlation between a Th1 cell-dominant response and protection against chlamydial infection. We further found that dendritic cells produced a large amount of interleukin 12 (IL-12) upon ex vivo pulsing with inactivated chlamydial organisms, which may allow the dendritic cells to direct a Th1 cell-dominant response. Dendritic cells from mice deficient in the IL-12 p40 gene failed to produce IL-12 after a similar ex vivo pulse with chlamydial organisms, and more importantly, immunization with these dendritic cells failed to induce a Th1 cell-dominant response and did not induce strong protection against chlamydial infection. Thus, the ability of dendritic cells to efficiently process and present chlamydial antigens and to produce IL-12 upon chlamydial-organism stimulation are both required for the induction of protection against chlamydial infection. This information may be useful for the further design of effective chlamydial vaccines.     

Induction of cytotoxic T-lymphocyte responses using dendritic cells transfected with hepatocellular carcinoma mRNA

This study aims to induce an efficient expansion of cytotoxic T-lymphocytes (CTL) from peripheral blood mononuclear cells (PBMCs) using dendritic cells (DC) transfected with hepatocellular carcinoma (HCC) messenger RNA (mRNA) for adoptive immunotherapy of HCC. Dendritic cells are generated from PBMCs. HCC mRNA is isolated either from HepG-2 cells or from tumour tissue from three HCC patients, and then amplified using the polymerase chain reaction (PCR). Expansion of CTLs is achieved from PBMCs induced by DCs transfected with HCC mRNA and cytotoxicity is measured using a crystal violet staining assay. The proportion of CD3+, CD4+ and CD8+ cells is determined using flow cytometry. Dendritic cells transfected with the total HCC mRNA stimulated antigen-specific cytotoxic T-cell responses that are capable of recognising and killing autologous tumour cells in vitro. The cytotoxic activity was inhibited by treatment with anti-CD3, anti-CD8 and anti-MHC class I monoclonal antibodies, but not with anti-CD4 and MHC class II antibodies. In conclusion, HCC mRNA-transfected DCs may represent a broadly applicable vaccine strategy to induce potentially therapeutic CTL responses in HCC.     

Interferon-γ- and interleukin-4-producing T cells can be primed on dendritic cells in vivo and do not require the presence of B cells

The antigen-presenting cell (APC) requirements for the in vivo induction of Th1-and Th2-type responses were investigated using a severe combined immunodeficiency (SCID)mouse chimera model. SCID mice adoptively transferred with either T cells [SCID(T)] or T + B cells [SCID(T + B)] and immunized with antigen in adjuvant were able to generate antigen-specific T cells which could produce both interferon (IFN)-γ and interleukin (IL)-4 upon in vitro restimulation. This suggests that B cell APC are not necessary for the priming of either IFN-γ- or IL-4-producing T cells in vivo. The ability of different APC to activate Th2-dependent effector mechanisms was also investigated. SCID(T) and SCID(T + B) mice were infected with the nematode parasite Nippostrongylus brasiliensis and analyzed for the development of IL-5-dependent peripheral blood eosinophilia. Following infection both SCID(T) and SCID(T + B) mice generated similar numbers of peripheral blood eosilnophils, suggesting that similar amounts of IL-5 had been produced. Therefore, B cell APC are also not required for the in vivo activation of Th2 cells to lymphokine production. To establish more precisely which APC prime T cells to produce IFN-γ and IL-4, normal mice were immunized by injection of syngeneic splenic dendritic cells which had been pulsed with antigen in vitro. T cells from these immunized mice were able to produce good IFN-γ and IL-4 responses upon in vitro restimulation with specific antigen; therefore, dendritic cells appear to be sufficient APC for the in vivo priming of both IFN-γ- and IL-4-producing T cells.     

Alternative versus classical activation of macrophages.

In parallel to the Th1/Th2 paradigm, antigen-presenting cells (APC) are divided into classically activated APC (dendritic cells/effector macrophages) and alternatively activated APC (IL-4-induced, alternatively activated macrophages/IL-10-induced, immature dendritic cells). Alternatively activated APC share a special molecular repertoire including receptors of innate immunity with broad specificity for foreign antigen and anti-inflammatory cytokines such as IL-1Ra and alternative macrophage activation-associated CC-chemokine-1. Alternatively activated APC mediated tolerance and downregulated inflammation. Abuse of alternatively activated APC in support of infectious susceptibility or tumor immune escape is counteracted by the classical pathway. Thus, classically and alternatively activated APC secure the balance between proinflammatory and anti-inflammatory immune reactions.     

Efficient induction of a Her2-specific anti-tumor response by dendritic cells pulsed with a Hsp70L1-Her2(341-456) fusion protein

Heat shock proteins (HSPs) have been shown to interact with antigen-presenting cells (APCs), especially dendritic cells (DCs). HSPs act as potent adjuvants, inducing a Th1 response, as well as antigen-specific CD8(+) cytotoxic T lymphocytes (CTL) via cross-presentation. Our previous work has demonstrated that Hsp70-like protein 1 (Hsp70L1), a new member of the Hsp70 subfamily, can act as a powerful Th1 adjuvant in a DC-based vaccine. Here we report the efficient induction of tumor antigen-specific T cell immune response by DCs pulsed with recombinant fusion protein of Hsp70L1 and Her2(341-456), the latter of which is a fragment of Her2/neu (Her2) containing E75 (a HLA-A2 restricted CTL epitope). The fusion protein Hsp70L1-Her2(341-456) promotes the maturation of DCs and activates them to produce cytokines, such as IL-12 and TNF-α, and chemokines, such as MIP-1α, MIP-1β and RANTES. Taken together, these results indicate that the adjuvant activity of Hsp70L1 is maintained in the fusion protein. Her2-specific HLA-A2.1-restricted CD8(+) CTLs can be generated efficiently either from the Peripheral blood lymphocytes (PBL) of healthy donors or from the splenocytes of immunized HLA-A2.1/K(b) transgenic mice by in vitro stimulation or immunization with DCs pulsed with the Hsp70L1-Her2(341-456) fusion protein. This results in more potent target cell killing in an antigen-specific and HLA-A2.1-restricted manner. Adoptive transfer of splenocytes from transgenic mice immunized with Hsp70L1-Her2(341-456)-pulsed DCs can markedly inhibit tumor growth and prolong the survival of nude mice with Her2(+)/HLA-A2.1(+) human carcinomas. These results suggest that Hsp70L1-Her2(341-456)-pulsed DCs could be a new therapeutic vaccine for patients with Her2(+) cancer.     

A protocol for generation of clinical grade mRNA-transfected monocyte-derived dendritic cells for cancer vaccines

With the aim of producing large quantities of mRNA-transfected monocyte-derived dendritic cells (DCs) to be used as cancer vaccines, a new clinical grade procedure has been developed. Peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis were enriched for monocytes by immunomagnetic depletion of CD19+ B cells and CD2+ T cells employing the ISOLEX 300i device. After 5 days of culture of enriched monocytes in gas permeable Teflon bags, using serum-free medium supplemented with granulocyte/macrophage-colony stimulating factor and interleukin-4 (IL-4), immature DCs were generated. Following transfection with mRNA from three human prostate cancer cell lines (DU145, LNCaP and PC-3), employing a newly developed square wave electroporation procedure, the immature DCs were immediately transferred to Teflon bags and matured for 48 h, using serum-free medium supplemented with IL-1alpha, IL-6, tumour necrosis factor-alpha and PGE2. The electroporation procedure efficiently transferred mRNA into the DCs with minor effect on the viability of the cells. The generated matured transfected DCs show high expression of the antigens CD83, CD80, CD86 and human leucocyte antigen-DR. Freezing and thawing of the transfected matured DCs had minor effect on cell viability and the phenotype. From 4 x 109 PBMCs, about 1 x 108 transfected matured DCs are produced. The thawed transfected DCs were able to elicit primary T-cell responses in vitro against antigens encoded by the prostate cancer mRNA as shown by enzyme-linked immunospot assay using mock-transfected DCs as control. Based on these results, clinical trials in cancer patients have been initiated.     

Regulation of T helper cell differentiation in vivo by soluble and membrane proteins provided by antigen-presenting cells

The aim of this study was to test whether the nature of the antigen-presenting cell (APC) can influence the Th1 / Th2 balance in vivo. Our data show that dendritic cells (DC), pulsed extra corporeally with antigen, induced the development of cells secreting IL-2, IFN-γ and IL-4 upon antigen rechallenge in vitro. Priming with peritoneal macrophages sensitized cells that produced IL-4 but not IFN-γ. To identify the factors involved in T helper development, mice were primed with APC with or without treatment with neutralizing antibodies to co-stimulatory molecules or cytokines. Our results indicate that priming with DC or macrophages is strictly dependent on the CD28-CTLA4/ B7 interaction. Of note, CD86 provides the initial signal to induce naive T cells to become IL-4 producers, whereas CD80 is a more neutral differentiation signal. IL-12, released by the DC, appears as a potent and obligatory inducer of differentiation for IFN-γ-producing cells. IL-6, although produced by both APC populations, is necessary to direct activation of the Th2-type response by macrophages but not by DC.     

Albumin fusion with granulocyte-macrophage colony-stimulating factor acts as an immunotherapy against chronic tuberculosis

A long duration of treatment and emerging drug resistance pose significant challenges for global tuberculosis (TB) eradication efforts. Therefore, there is an urgent need to develop novel strategies to shorten TB treatment regimens and to treat drug-resistant TB. Using an albumin-fusion strategy, we created a novel albumin-fused granulocyte-macrophage colony-stimulating factor (albGM-CSF) molecule that harnesses albumin’s long half-life and targeting abilities to enhance the biostability of GM-CSF and direct it to the lymph nodes, where the effects of GM-CSF can increase dendritic cell populations crucial for eliciting a potent immune response. In this study, we demonstrate that albGM-CSF serves as a novel immunotherapy for chronic Mycobacterium tuberculosis (Mtb) infections by enhancing GM-CSF biostability in serum. Specifically, albumin is very safe, stable, and has a long half-life, thereby enhancing the biostability of GM-CSF. In the lungs and draining lymph nodes, albGM-CSF is able to increase the numbers of dendritic cells, which are crucial for the activation of naive T cells and for eliciting potent immune responses. Subcutaneous administration of albGM-CSF alone reduced the mean lung bacillary burden in mice with chronic tuberculosis infection. While GM-CSF administration was associated with IL-1β release from Mtb-infected dendritic cells and macrophages, higher IL-1β levels were observed in albGM-CSF-treated mice with chronic tuberculosis infection than in mice receiving GM-CSF. Albumin fusion with GM-CSF represents a promising strategy for the control of chronic lung tuberculosis infections and serves as a novel therapeutic vaccination platform for other infectious diseases and malignancies.     

Regulation of T helper cell differentiation in vivo by GP43 from Paracoccidioides brasiliensis provided by different antigen-presenting cells

Paracoccidioidomycosis, endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis. The infection can evolve into different clinical forms that are associated with various degrees of suppressed cell-mediated immunity. Assuming that the effector immune response is a consequence of the preferential activation of either Th1 or Th2 subsets, in the present work we evaluated whether the nature of antigen-presenting cells (APCs) can influence the Th1/Th2 balance in vivo. It was observed that the injection of mature dendritic cells (DCs), macrophages and B cells primed the mice and induced a proliferation of T cells in vitro. It was seen that DCs from resistant mice stimulated predominantly interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), whereas macrophages activated IL-10, IL-4 and IFN-gamma-secreting T cells and B cells IL-4 and IL-10 only. Results presented here clearly demonstrate that DC drives the development of cells secreting Th1-derived cytokines, whereas B cells induce the differentiation of a Th2 phenotype in vivo.     

Mycobacterium leprae 65hsp antigen expressed from a retroviral vector in a macrophage cell line is presented to T cells in association with MHC class II in addition to MHC class I.

Mycobacterium leprae lives free in the cytoplasm in infected macrophages. To test if an M. leprae antigen released into the cytoplasm would associate with major histocompatibility complex (MHC) class II we introduced the gene encoding the 65 kDa heat-shock protein (ML65hsp) into a retroviral shuttle vector (pZIPNeoSV(X)) and transfected the murine macrophage cell line J774G8. S1 nuclease mapping and Western blot analysis of the transfected cell line (CJ11) showed that specific messenger RNA and ML65hsp antigen were stably expressed. Presence of antigen at the cell surface was demonstrated by flow cytometric analysis with specific monoclonal antibodies (mAb). Antigen-specific T lymphocytes were stimulated by CJ11 cells to proliferate and release interleukins (IL-2 and IL-3). These responses were blocked by mAbs specific for either MHC class II or for the mycobacterial antigen. The endogenous antigen was also recognised by MHC class I-dependent cytotoxic T cells; cytotoxicity was inhibited by mAbs against either MHC class I molecules or ML65hsp. Thus, production of ML65hsp within the host cytoplasm resulted in association of the antigen with both MHC class I and MHC class II antigen-presenting structures and evoked both lymphocyte proliferation and cytotoxicity towards the antigen-presenting cell. These findings may be relevant to the development of recombinant subunit vaccines against intracellular pathogens.     

Presentation of mycobacterial antigens by human dendritic cells: lack of transfer from infected macrophages.

When exposed to a challenge of 10 Mycobacterium bovis BCG cells per antigen-presenting cell, most human monocytes engulf several organisms. In contrast, blood dendritic cells which are potent antigen-presenting cells for several antigens are not detectably phagocytic for mycobacteria. We investigated the possibility that infected macrophages might regurgitate antigens for presentation by populations of human blood dendritic cells. Macrophages were infected with M. bovis BCG, mixed with uninfected dendritic cells, and added to immune T cells, either bulk T cells or cloned populations from BCG vaccinees or patients recovering from tuberculosis. The macrophages were from donors who were mismatched to the T cells so that transfer of antigen to major histocompatibility complex-matched dendritic cells could be evaluated. As we describe, there was no evidence for the transfer of mycobacterial antigens from macrophages to dendritic cells in a form that was stimulatory for the T cells.     

Application of IL-5 ELISPOT assays to quantification of antigen-specific T helper responses

To be able to determine the frequencies of helper-type T lymphocyte (Th) precursors specific for viral or tumor-associated antigens, an ELISPOT assay for IL-5 production was developed. The assay reproducibility was first determined using fresh or cryopreserved peripheral blood mononuclear cells (PBMC) obtained from six normal donors and tested for IL-5 production after 48 h stimulation with phytohemagglutinin (PHA). Both inter-assay (within-subject CV=1.6%) and intra-assay (within-subject CV=0.8%) variabilities were found to be acceptable, and the frequencies of IL-5 secreting cells were comparable in cryopreserved and fresh PBMC of the same donors. The presence and frequency of Th precursor cells to viral capsid L1 protein (viral-like particles, VLP-L1) of human papillomavirus type 16 (HPV-16) in PBMC obtained from seven non-immunized donors and two volunteers immunized with VLP-L1 were then evaluated. Using autologous dendritic cells as antigen-presenting cells (APC) for VLP-L1 in direct 48-h ELISPOT assays, the mean frequency of IL-5 secreting CD4+ T cells was found to be <1/10(5) (negative) in normal donors but was 1/2, 436 and 1/3678 in the two volunteers immunized with VLP-L1. The assay is applicable to monitoring of the frequency of antigen-specific T cells in the peripheral circulation of individuals immunized with HPV-derived antigens. To test the assay utility in the assessment of Th tumor-reactive lymphocytes, we also used it to determine the frequency of the wild-type sequence (wt) p53(22-36) peptide-specific, HLA-DR4-restricted T cells in the bulk CD4+ T cell line. This frequency was 1/33. The ELISPOT assay for IL-5 production can be reliably used to measure Th-type responses in a variety of experimental settings.     

Dendritic cells retrovirally overexpressing IL-12 induce strong Th1 responses to inhaled antigen in the lung but fail to revert established Th2 sensitization

It has been postulated that low-level interleukin (IL)-12 production of antigen-presenting cells is associated with the risk of developing atopic asthma. To study the relationship between IL-12 production capacity of dendritic cells (DCs) and development of T helper type 2 (Th2) responses in the lung, we genetically engineered DCs to constutively overexpress bioactive IL-12. Retrovirally mediated overexpression of IL-12 in DCs strongly polarized naive ovalbumin (OVA)-specific CD4+ T cells toward Th1 effector cells in vitro. After intratracheal injection, OVA-pulsed IL-12-overexpressing DCs failed to induce Th2 responses in vivo and no longer primed mice for Th2-dependent eosinophilic airway inflammation upon OVA aerosol challenge, readily observed in mice immunized with sham-transfected, OVA-pulsed DCs. Analysis of a panel of cytokines and chemokines in the lung demonstrated that the lack of Th2 sensitization was accompanied by increased production of the Th1 cytokine interferon-gamma (IFN-gamma), chemokines induced by IFN-gamma, and the immunoregulatory cytokine IL-10. When Th2 priming was induced using OVA/alum prior to intratracheal DC administration, DCs constitutively expressing IL-12 were no longer capable of preventing eosinophilic airway inflammation and even enhanced it. These data show directly that high-level expression of IL-12 in DCs prevents the development of Th2 sensitization. Enhancing IL-12 production in DCs should be seen as a primary prevention strategy for atopic disorders. Enhancing IL-12 production in DCs is less likely to be of benefit in already Th2-sensitized individuals.