A DNA adjuvant encoding a fusion protein between anti-CD3 single-chain Fv and AIMP1 enhances T helper type 1 cell-mediated immune responses in antigen-sensitized mice

T helper type 1 (Th1) cell-mediated immune responses contribute to host defences against intracellular pathogen infections and cancer. Previously, we found that aminoacyl tRNA synthetase-interacting multifunctional protein 1 (AIMP1) activated macrophages and dendritic cells to enhance Th1 responses. Herein, we manipulated this property to improve Th1 immune responses in vivo by constructing a mammalian expression plasmid (pAnti-CD3sFv/AIMP1) encoding AIMP1 fused to the anti-CD3 single-chain Fv (sFv), the smallest unit of the antibody that interacts with the CD3epsilon region of the T-cell receptor. Intramuscular injection of ovalbumin (OVA)-sensitized BALB/c mice with pAnti-CD3sFv/AIMP1 DNA adjuvant increased the OVA-specific, interferon-gamma production by their CD4(+) T cells and the levels of anti-OVA immunoglobulin G2a (IgG2a) isotype in their sera. Furthermore, the pAnti-CD3sFv/AIMP1 DNA adjuvant decreased interleukin-4 production and anti-OVA IgE levels in the OVA-injected mice. Importantly, the pAnti-CD3sFv/AIMP1 was more efficient than a mixture of pAnti-CD3sFv and pAIMP1 in inducing OVA-specific Th1 immune responses and also in inhibiting OVA-specific Th2 responses during antigen priming. These studies indicated that the pAnti-CD3sFv/AIMP1 fusion DNA adjuvant enhanced Th1 immune responses in antigen-sensitized mice.     

Efficient induction of T helper type 1-mediated immune responses in antigen-primed mice by anti-CD3 single-chain Fv/interleukin-18 fusion DNA

Two types of T helper (Th) cells - Th1 and Th2 - play different roles in protection and immunopathology. The Th1 cell-mediated immune response plays an important role in inducing the host defence against intracellular bacteria and also in cancer immunotherapy. To effectively induce Th1 immune responses, we constructed a mammalian expression plasmid (pAnti-CD3sFv/IL-18) carrying a fusion gene in which anti-CD3 single-chain Fv (sFv) cDNA, the smallest unit of antibody recognizing the CD3 epsilon moiety of the T-cell receptor, was covalently linked to mature interleukin (IL)-18 cDNA. Intramuscular injection of ovalbumin (OVA)-sensitized BALB/c mice with pAnti-CD3sFv/IL-18 DNA efficiently increased the production of both OVA-specific interferon-gamma and anti-OVA immunoglobulin G2a, compared to injection with pAnti-CD3sFv DNA. In addition, pAnti-CD3sFv/IL-18 was more efficient than a mixture of pAnti-CD3sFv + pIL-18 in inducing OVA-specific, Th1 immune responses and also in inhibiting OVA-specific, IL-4 production. These studies indicate that vaccination with pAnti-CD3sFv/IL-18 fusion DNA efficiently induces the Th1 immune response in antigen-sensitized mice.     

Interleukin-10-treated dendritic cells do not inhibit Th2 immune responses in ovalbumin/alum-sensitized mice

BACKGROUND: It is well known that the immunoregulatory cytokine interleukin (IL)-10 inhibits the accessory function of human dendritic cells (DC) in vitro. Recently, we have shown that these IL-10 DC inhibit the production of T helper cell 1 (Th1) and T helper cell 2 (Th2) cytokines by T cells from atopic individuals in vitro. The current study was set out to analyze whether IL-10 DC also exert inhibitory effects in vivo in a murine model of allergy to ovalbumin adsorbed to the adjuvant aluminium hydroxide (OVA/alum). METHODS: OVA-pulsed or unpulsed bone marrow-derived DC, treated with IL-10 or left untreated during generation, were injected intravenously into BALB/c mice prior to and during OVA/alum sensitization, and sera and immune responses of mesenterial lymph node cells were analyzed. Additionally, bronchoalveolar lavage was performed after intranasal challenge with OVA. RESULTS: Treatment of BALB/c mice with OVA-pulsed DC led to a significantly enhanced proliferation as well as Th2 (IL-4, IL-5), Th1 (interferon-gamma) and IL-10 cytokine production after restimulation of lymph node cells with OVA in vitro compared with OVA immunization alone. In contrast, using OVA-pulsed IL-10 DC for transfer, proliferation and cytokine production by lymph node cells were not enhanced. OVA-specific immunoglobulin G1 (IgG1) and IgG2a production were significantly increased after transfer of OVA-pulsed DC and OVA-pulsed IL-10 DC, respectively, whereas anti-OVA IgE production and airway eosinophilia remained unchanged. CONCLUSIONS: Our data indicate that IL-10 treatment of DC decreases the Th1 and Th2 stimulatory capacity of DC but does not actually inhibit systemic (IgE) and local (airway inflammation) allergen-specific immune responses in a murine model of allergy.     

Interleukin-12-secreting fibroblasts are more efficient than free recombinant interleukin-12 in inducing the persistent resistance to Mycobacterium avium complex infection

To determine whether the paracrine secretion of interleukin-12 (IL-12) can efficiently stimulate the resistance to Mycobacterium avium complex (MAC) infection, 3T3 fibroblasts were stably transfected to secrete IL-12 (480 U/106 cells/48 hr) and their effect on MAC infection was investigated in genetically susceptible BALB/c mice, compared with that of free recombinant IL-12 (rIL-12). Injection with IL-12-secreting fibroblasts (3T3-IL-12) during intranasal infection with MAC resulted in a significant decrease in the bacterial load of the lung during the entire 10-week observation period, while rIL-12 reduced the bacterial load initially, at 2 weeks, but not by 10 weeks postinfection. Lung CD4+ T cells in mice injected with the 3T3-IL-12 cells showed a persistent T helper type 1 (Th1) response throughout the 10-week period. Furthermore, immunization with the 3T3-IL-12 cells induced and maintained significantly higher levels of cytotoxic activity and nitric oxide production by lung cells than did rIL-12 immunization. This work suggests that IL-12-secreting fibroblasts may serve as a vehicle for paracrine secretion of IL-12 for immunotherapy of MAC infection.     

Potentiation of antigen-specific, Th1 immune responses by multiple DNA vaccination with an ovalbumin/interferon-gamma hybrid construct.

The preferential differentiation of T helper (Th) cells to Th1 or Th2 subsets is important with respect to susceptibility or resistance to particular infections, or to autoimmune diseases and allergic diseases. To more effectively drive immune responses toward antigen-specific Th1 responses, we constructed a mammalian expression vector (pOVA/IFN-gamma) carrying a hybrid gene in which the ovalbumin (OVA) (a model antigen) cDNA was covalently linked to murine interferon-gamma (IFN-gamma) cDNA. Intramuscular injection of BALB/c mice with the pOVA/IFN-gamma DNA increased both the production of OVA-specific IFN-gamma by CD4+ T cells and the ratio of anti-OVA immunoglobulin G (IgG) 2a to IgG1 isotypes, while the injection with the pOVA alone, or with the mixture of the pOVA and pIFN-gamma, caused no or little increase. Furthermore, the OVA-specific, Th1 immune responses were dramatically augmented by multiple injections with the pOVA/IFN-gamma DNA. These studies indicate that the direct linkage of an OVA gene to an IFN-gamma gene in the expression plasmid is required for efficiently confining the Th1 effects of IFN-gamma to the OVA-specific cells, and the linkage effect of the OVA/IFN-gamma DNA can be potentiated by multiple vaccination.     

Failure of rIL-12 administration to inhibit established IgE responses in vivo is associated with enhanced IL-4 synthesis by non-B/non-T cells

Administration of rIL-12 offers a widely successful tactic for preferential induction of type 1 immune responses in vivo. Its use to modulate ongoing cytokine or effector responses has proven to be substantially more difficult. Immediate hypersensitivity is the most common human immunologic disease. Here, rIL-12 was administered to C57Bl/6 and outbred CD1 mice with ongoing ovalbumin (OVA)-specific IgE responses in an attempt to redirect established type 2 cytokine and antibody production. Despite use of a broad range of treatment protocols for >4 months following initial immunization, recall IgE responses were consistently unaffected. rIL-12-treated mice exhibited strong in vivo and in vitro IFN-gamma responses, increased approximately 40-fold relative to controls, but also markedly enhanced (15- to 20-fold) OVA-specific IL-4 production. CD4 T cell function was successfully transformed from a type 2- to a type 1-dominated pattern following long-term IL-12 administration in vivo, as measured by strongly reduced IL-4 and IL-10 responses in antigen-stimulated primary culture, and 5-fold reductions in the frequencies of IL-4- and IL-10-producing OVA-specific CD4 T cells. However, chronically rIL-12-treated mice exhibited increased numbers of non-B/non-T cells that when re-stimulated with specific allergen, produce IL-4 at levels 20-fold higher than did CD4 T cells while IL-13 responses are unaffected. Collectively, the data indicate that even effectively shifting CD4 T cell activation from a type 2- to a type 1-dominated response does not in itself lead to altered effector (IgE) responses upon antigen re-exposure.     

Influence of the polyclonal activation induced by Plasmodium chabaudi on ongoing OVA-specific B- and T-cell responses

Infection by Plasmodium chabaudi results in polyclonal activation, massive proliferation and differentiation of lymphocytes with parasite-unrelated specificities. To verify if polyclonal activation includes experienced B and T lymphocytes and if it modifies pre-established cytokine and Ig-isotype patterns, mice were immunized with ovalbumin (OVA) in alum, a condition that favours T helper 2/immunoglobulin G1 (Th2/IgG1) responses, and infected with P. chabaudi 7 or 80 days later. Polyclonal activation markedly increased the number of anti-OVA Ig-secreting cells in the spleen, an effect more patent in mice infected 7 days after OVA immunization, but also evident in mice infected after 80 days. The Ig-isotype profile predefined by immunization was not qualitatively modified by polyclonal activation. Thus, although P. chabaudi infection preferentially induces IgG2a, the expanded anti-OVA response is dominated by IgG1. Polyclonal expansion of the anti-OVA response did not yield an enlarged memory B-cell pool that could be recalled months later by OVA boosting. Moreover, polyclonal activation of anti-OVA IgG1-secreting cells did not increase this antibody in serum, a probable consequence of the high Ig turnover observed during infection. When OVA-specific T-cell cytokines were evaluated, we observed an increase of both interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) in mice infected 7 days after immunization, whereas in those infected after 80 days, only IL-4 was augmented. These results suggest that polyclonal activation expands experienced B- and T-cell compartments, preserving their antibody and cytokine patterns.     

Immunostimulatory oligodeoxynucleotide from Bifidobacterium longum suppresses Th2 immune responses in a murine model

We have reported previously that novel immunostimulatory sequence (ISS) oligodeoxynucleotide (ODN) BL07S from a probiotic strain of Bifidobacterium longum inhibited immunoglobulin (Ig) E production in vitro. However, whether ISS-ODNs from probiotics regulate T helper type 2 (Th2)-polarized immune reactions in vivo remains unclear. To evaluate the inhibitory effects of ODN BL07S on type I allergic response, BALB/c mice were injected with or without ODN BL07S in the presence of ovalbumin (OVA) on days 0 and 14. Serum Ig levels (IgE, IgG1 and IgG2a) and cytokine levels (interferon (IFN)-gamma, interleukin (IL)-12, IL-4, IL-5, IL-10 and IL-13) were investigated in splenocyte cultures from days 14-28. Production of OVA-specific and total IgE were significantly suppressed by administration of ODN BL07S, but not by ODN BL06S, a non-ISS-ODN. Compared to controls, ODN BL07S induced significantly lower levels of Th2 cytokines (IL-4 and IL-5) in splenocyte cultures, and significantly higher levels of serum OVA-specific IgG2a. These effects of ODN BL07S on modulation of Th2 immune response were dose-dependent. The present results demonstrate that ODN BL07S from genomic DNA of B. longum BB536 prevents antigen-induced Th2 immune responses in vivo, suggesting that ISS-ODNs from probiotics might be useful in preventing allergic disease.     

Roles of CD4+ and CD8+ T cells in adjuvant activity of diesel exhaust particles in mice

Through an imbalance in Th1 and Th2 cytokine profiles, diesel exhaust particles (DEP) are thought to induce Th2-dominated IgE and IgG1 production. However, the roles of CD4+ and CD8+ T-cell subtypes in the increased immune responses to antigen in mice exposed to DEP are unclear. In the present study, we investigated whether treatment with anti-CD4 or anti-CD8 mAb abrogated the adjuvant activity of DEP. On day -1 and day 1, each group of mice was injected intraperitoneally with anti-CD4, anti-CD8, or rat IgG (vehicle). On day 0, the mice were immunized with ovalbumin (OVA) or OVA plus DEP. After 3 weeks, each mouse was boosted with 10 microg of OVA alone. On day 7 after the first injection with OVA+DEP or OVA alone, the numbers of total, IA+, CD80+/IA+ and CD86+/IA+ cells in peritoneal exudate cells (PEC) were higher in OVA+DEP-immunized mice than in OVA-immunized mice. Depletion of CD8+ cells resulted in a modulation of the production of granulocyte-macrophage colony-stimulating factor, IL-12 and PGE(2) in peritoneal exudate fluid from OVA+DEP-immunized mice. On day 28, DEP injection markedly increased IL-4 production in the culture supernatants of spleen cells from CD4+ or CD8+-depleted mice. Depletion of CD8+ cells in OVA+DEP-immunized mice resulted in a decrease in IFN-gamma production compared with that in OVA-immunized mice. Adjuvant activity of DEP was observed in anti-OVA IgE, anti-OVA IgG1, anti-OVA IgG3, and total IgE production. Depletion of CD4+ T cells abrogated the adjuvant effect of DEP on anti-OVA IgE, and anti-OVA IgG1 production in plasma. However, depletion of CD8+ T cell inhibited the upregulated anti-OVA IgG3 production. These findings suggest that DEP injection may affect not only the function of CD4+ cells but also that of CD8+ T-cell subsets to modulate the synthesis of proinflammatory cytokine in PEC and type-1 and type-2 cytokine production in spleens.     

Dietary nucleotides can up-regulate antigen-specific Th1 immune responses and suppress antigen-specific IgE responses in mice

BACKGROUND: It has been reported that dietary nucleotides enhance T helper cell activities. In this study, we have determined the effects of dietary nucleotides on antigen-specific Th1 and Th2 responses and IgE responses. METHODS: Ovalbumin (OVA)-specific T cell receptor (TCR) transgenic (OVA-TCR Tg) mice, 3 weeks old, were fed a nucleotide-free diet (NT(-) diet) or the NT(-) diet supplemented with dietary nucleotides (NT(+) diet) for 4 weeks. Cytokine production by spleen cells and macrophages obtained from these mice was measured in vitro. BALB/c mice, 3 weeks old, immunized intraperitoneally with OVA adsorbed onto alum, were fed the NT(-) diet or the NT(+) diet for 4 weeks. Serum levels of antigen-specific antibodies in the BALB/c mice were determined by ELISA. RESULTS: The level of production of antigen-specific interferon-gamma by spleen cells was significantly higher in the OVA-TCR Tg mice fed the NT(+) diet than in the control mice. The levels of secretion of bioactive IL-12 by spleen cells and peritoneal macrophages were also significantly increased in the NT(+) diet group. The serum OVA-specific IgE level was significantly decreased in BALB/c mice fed the NT(+) diet compared with those fed the NT(-) diet. CONCLUSION: These results show that dietary nucleotides up-regulate the antigen-specific Th1 immune response through the enhancement of IL-12 production and suppress the antigen-specific IgE response.     

Interleukin-18 plays a role in both the alum-induced T helper 2 response and the T helper 1 response induced by alum-adsorbed interleukin-12

Previous studies have shown that the antigen-specific T helper 2 (Th2) response induced by alum adjuvants is interleukin (IL)-4 independent. As a role for IL-18 in Th2 induction has recently been described, in addition to its role in enhancing Th1 responses, we have studied the Th2 response induced by ovalbumin (OVA) adsorbed to alum in wild-type and IL-18-deficient mice. Our results indicate that while endogenous IL-18 facilitates alum-induced IL-4 production, OVA-specific immunoglobulin G1 (IgG1) and IgE production remain unaffected. Furthermore, antigen-specific Th1 responses induced with alum/IL-12-adsorbed OVA were demonstrated to be highly IL-18 dependent. Despite these observations, injection of BALB/c mice with exogenous IL-18 adsorbed to alum/OVA did not alter IL-4 or interferon-gamma production by T cells and had little effect on the relative production of IgG1/IgG2a antibody subclasses compared with alum/OVA inoculated mice. However, the previously described synergism between IL-12 and IL-18 in Th1 induction was evident as the Th1-promoting activity of alum/IL-12 against adsorbed OVA was greatly augmented by the coadministration of IL-18. These results indicate that while alum-induced IL-18 can facilitate Th2 induction, the addition of exogenous IL-18 cannot further enhance the alum-induced Th2 response.     

Adjuvant effect of zinc oxide on Th2 but not Th1 immune responses in mice

Background and aim: We investigated the effect of zinc oxide (ZnO) on Th1 and Th2 immune responses in mice.

Material and methods: Mice were intraperitoneally administered with ovalbumin (OVA) with or without varying doses of ZnO (day 0). On day 21, anti-OVA IgG, IgG2a, IgG1, and IgE antibodies in sera, OVA-specific proliferative responses of spleen cells, and production of Th1 cytokines including IFN-γ as well as Th2 cytokines such as IL-4 and IL-5 were measured.

Results: The results showed that administration of OVA with ZnO was followed by greater increases in anti-OVA IgG and the antigen-specific splenocyte proliferation compared to that of OVA alone. The production of anti-OVA IgG1 and IgE and secretion of IL-4 and IL-5 were markedly enhanced by ZnO. The enhancing effect of ZnO on these Th2 responses was as strong as aluminium hydroxide (Alum) that was widely used as an adjuvant. In contrast, treatment with OVA plus ZnO failed to affect production of anti-OVA IgG2a as well as IFN-γ. It was also observed that ZnO had a stimulating effect on the secretion of the proinflammatory cytokine IL-17 from a new lineage of effector Th cells.

Conclusion: These results suggest that ZnO appears to have an adjuvant effect on the immune system, especially Th2 but not Th1 immune responses.     

Effect of rolipram, a phosphodiesterase IV inhibitor, on allergic footpad swelling using various adjuvants in mice

We studied the effect of rolipram, a phosphodiesterase (PDE) IV inhibitor, on allergic footpad swelling in mice. For this study, varying adjuvants including complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and Imject Alum (Alum) were used because the extent of antigen-specifically induced T helper type 1 (Th1) and Th2 responses had been shown to depend on adjuvants used. To induce allergic footpad swelling, we immunized mice with ovalbumin (OVA) emulsified in either CFA or IFA, dissolved in Alum or in phosphate-buffered saline (PBS) as a control (day 0), followed by subcutaneous injection of the antigen into footpads on day 21. Rolipram was given orally to the animals daily from days 0-20. Results showed that treatment with rolipram was followed by an increase in early swelling at 0.5 h and a decrease in late swelling at 6 and 24 h in the CFA group. In the IFA group, rolipram significantly enhanced swelling at, but not after, 30 min. In the Alum and the PBS groups, the PDE inhibitor failed to affect the OVA-specific footpad reaction at all times examined. Treatment of the CFA and IFA groups with rolipram significantly inhibited the production of the Th1 antibody anti-OVA immunoglobulin G2a (IgG2a), and the drug enhanced Th2 cell-dependent anti-OVA IgE production. In both groups, rolipram also enhanced the secretion of Th2 cytokines including interleukin-4 (IL-4) and IL-10. These findings suggest that rolipram may facilitate early allergic footpad swelling mediated by Th2 immune responses, while the late phase of swelling associated with Th1 responses may be attenuated by the PDE IV inhibitor.     

CpG-DNA stimulates cellular and humoral immunity and promotes Th1 differentiation in aged BALB/c mice

We examined whether CpG-DNA could be used as adjuvant to induce a T helper cell type-1 (Th1) immunity in aged BALB/c mice that showed a Th2 polarization. Bordetella pertussis and complete Freund's adjuvant (CFA) were used as well. Immunization with ovalbumin (OVA)/CpG-DNA showed that the immunoglobulin G (IgG)2a/IgG1 ratio and OVA-specific T cell response were similar in young and aged mice. OVA/CpG-DNA induced the secretion of interferon-gamma (IFN-gamma) and absence of interleukin (IL)-5. Similar results were found in mice immunized with OVA/CFA. When mice were immunized with OVA/B. pertussis, we found that the IgG2a/IgG1 ratio and OVA-specific T cell response were lower in aged mice and elicited IFN-gamma and IL-5. In vitro CpG-DNA stimulated antigen-presenting cells to display IL-12 and up-regulate the expression of major histocompatibility complex class II and B7-2 on B cells as efficiently in aged as in young mice, but the up-regulation of B7-1 was stronger in aged mice. The findings demonstrate that CpG-DNA is able to induce a young-like Th1 specific immune response in aged mice.     

Active CD4+ helper T cells directly stimulate CD8+ cytotoxic T lymphocyte responses in wild-type and MHC II gene knockout C57BL/6 mice and transgenic RIP-mOVA mice expressing islet beta-cell ovalbumin antigen leading to diabetes

CD4+ helper T (Th) cells play crucial role in priming, expansion and survival of CD8+ cytotoxic T lymphocytes (CTLs). However, how CD4+ Th cell's help is delivered to CD8+ T cells in vivo is still unclear. We previously demonstrated that CD4+ Th cells can acquire ovalbumin (OVA) peptide/major histocompatibility complex (pMHC I) and costimulatory CD80 by OVA-pulsed DC (DC(OVA)) stimulation, and then stimulate OVA-specific CD8+ CTL responses in C57BL/6 mice. In this study, we further investigated CD4+ Th cell's effect on stimulation of CD8 CTL responses in major histocompatibility complex (MHC II) gene knockout (KO) mice and transgenic rat insulin promoter (RIP)-mOVA mice with moderate expression of self OVA by using CD4+ Th cells or Th cells with various gene deficiency. We demonstrated that the in vitro DC(OVA)-activated CD4+ Th cells (3 x 10(6) cells/mouse) can directly stimulate OVA-specific CD8+ T-cell responses in wild-type C57BL/6 mice and MHC II gene KO mice lacking CD4+ T cells. A large amount of CD4+ Th cells (12 x 10(6) cells/mouse) can even overcome OVA-specific immune tolerance in transgenic RIP-mOVA mice, leading to CD8+ CTL-mediated mouse pancreatic islet destruction and diabetes. The stimulatory effect of CD4+ Th cells is mediated by its IL-2 secretion and CD40L and CD80 costimulations, and is specifically delivered to OVA-specific CD8+ T cells in vivo via its acquired pMHC I complexes. Therefore, the above elucidated principles for CD4+ Th cells will have substantial implications in autoimmunity and antitumor immunity, and regulatory T-cell-dependent immune suppression.     

Molecular differences between the divergent responses of ovalbumin-specific CD4 T cells to alum-precipitated ovalbumin compared to ovalbumin expressed by Salmonella

CD4 T helper (Th) cell differentiation defined by in vitro cytokine-directed culture systems leaves major gaps in our knowledge of the mechanisms driving divergent Th differentiation. This is evident from our analysis of the response of mouse ovalbumin-specific CD4 T cells to different forms of ovalbumin that induce markedly distinct responses in vivo. We show that live attenuated ovalbumin-expressing Salmonella (SalOVA) induce Th1-associated T-bet and IFN-gamma. Conversely, alum-precipitated ovalbumin (alumOVA) induces the Th2-associated GATA-3 and IL-4. The early diversity occurring within these CD4 T cells isolated 3 days after immunization was assessed using real-time RT-PCR microfluidic cards designed with 384 selected genes. The technique was validated both at the population and single cell levels at different stages of the responses, showing beta2-microglobulin to be a more stably expressed reference mRNA than either beta-actin or 18S RNA. SalOVA was then shown selectively to induce the OVA-specific CD4 T cells to produce many chemokines and pro-inflammatory cytokines, contrasting with alumOVA-induced cells that only produced a few Th2-associated cytokines. Several cytokines and features associated with follicular helper functions were induced in the OVA-specific CD4 T cells by both antigens. Finally, IL-17RB is strongly associated with OVA-specific CD4 T cells responding to alumOVA, suggesting that alum may promote Th2 immune response through a role for the IL-25/IL-17RB pathway.