Oral immunization of interleukin-4 (IL-4) knockout mice with a recombinant Salmonella strain or cholera toxin reveals that CD4+ Th2 cells producing IL-6 and IL-10 are associated with mucosal immunoglobulin A responses.

Mucosal immunoglobulin A (IgA) responses are often associated with Th2-type cells and derived cytokines, and interleukin-4 (IL-4) knockout (IL-4-/-) mice with impaired Th2 cells respond poorly to oral antigens. However, we have noted that IL-4-/- mice have normal mucosal IgA levels, which led us to query whether different oral delivery systems could elicit mucosal immunity. Two oral regimens were used: (i) a live recombinant Salmonella strain which expresses fragment C (ToxC) of tetanus toxin, and (ii) soluble tetanus toxoid (TT) with cholera toxin (CT) as an adjuvant. Oral immunization of IL-4-/- mice with recombinant Salmonella vaccine expressing ToxC induced brisk mucosal IgA and serum IgG (mainly IgG2a) anti-TT antibody responses. TT-specific CD4+ T cells from spleen or Peyer's patches produced gamma interferon, indicative of Th1 responses; however, IL-6 and IL-10 were also seen. Oral immunization of IL-4-/- mice with TT and CT induced weak mucosal IgA to TT; however, brisk IgA anti-CT-B responses and CT-B-specific CD4+ T cells producing IL-6 and IL-10 were also noted. These results show that although IL-4-dependent antibody responses are impaired, mucosal IgA responses are induced in IL-4-/- mice. These result suggest that certain cytokines, i.e., IL-6 and IL-10 from Th2-type cells, play an important compensatory role in the induction and regulation of mucosal IgA responses.     

Interferon-gamma receptor-deficient mice exhibit impaired gut mucosal immune responses but intact oral tolerance.

Interferon-gamma (IFN-gamma) receptor knock-out (IFN-gamma R -/-) mice were used to analyse the role of IFN-gamma in mucosal immune responses following oral immunization. We found that the IFN-gamma R -/- mice demonstrated 50% reduced spot-forming cell (SFC) responses in the gut lamina propria and spleen after oral immunization with keyhold limpet haemocyanin (KLH) plus cholera toxin (CT) adjuvant. The IFN-gamma R -/- mice exhibited 10-fold reduced total serum KLH-specific antibody levels compared with wild-type mice after oral immunization, while after intravenous immunization, no such difference was seen, suggesting a selective impairment of mucosal immune responses. Moreover, oral immunizations resulted in impaired interleukin-4 (IL-4), IL-10 and IFN-gamma production by spleen T cells from IFN-gamma R -/- mice, indicating that no reciprocal up-regulation of Th2-activities had occurred despite the lack of IFN-gamma R function. No reduction in Th1 or Th2 cytokines was observed following systemic immunizations. Despite potentially strong modulating effects of IFN-gamma on epithelial cell IgA transcytosis and electrolyte barrier functions, CT-immunized IFN-gamma R -/- mice demonstrated unaltered protection against CT in ligated intestinal loops together with normal anti-CT IgA and total IgA levels in gut lavage. Oral feeding with KLH followed by parenteral immunization resulted in strongly suppressed SFC numbers and reduced cell-mediated immunity in both wild-type and IFN-gamma R -/- mice. CT-adjuvant abrogated induction of oral tolerance in both IFN-gamma R -/- and wild-type mice. Collectively, our data argue that the two major response patterns induced by oral administration of protein antigen, i.e. active IgA immunity and oral tolerance, are differently regulated. Thus, IFN-gamma R -/- mice have impaired mucosal immune responses while induction of oral tolerance appears to be unaffected by the lack of IFN-gamma functions.     

Mistletoe lectins enhance immune responses to intranasally co-administered herpes simplex virus glycoprotein D2

The mucosal adjuvant properties of the three type 2 ribosome-inactivating proteins (RIPs) from the European mistletoe, Viscum album L., were investigated. Mistletoe lectins were compared with cholera toxin (CT) as adjuvants when delivered nasotracheally together with herpes simplex virus glycoprotein D2 (gD2). All three mistletoe lectins (MLI, MLII, MLIII) were potent mucosal adjuvants. Co-administration of MLI, MLII or MLIII with gD2 led to significantly higher levels of gD2-specific mucosal immunoglobulin A (IgA) and systemic immunoglobulin G (IgG) antibody than when the antigen was delivered alone. The levels of antibodies induced were similar to those generated in mice immunized with gD2 and the potent mucosal adjuvant CT. Administration of ML1 with gD2 enhanced the antigen-specific splenic T-cell proliferative response. Interleukin-5 (IL-5), but not interferon-gamma (IFN-gamma), was detected in supernatants from splenocytes stimulated in vitro with gD2. This indicates that MLI enhanced type 2 T-helper cell (Th2) responses to the bystander antigen, gD2. Analysis of the gD2- and lectin-specific IgG subclass titres in mice immunized with gD2 and MLI, MLII or MLIII revealed a high ratio of IgG1 : IgG2a, which is compatible with the selective induction of Th2-type immune responses.     

Signaling via interleukin-4 receptor alpha chain is required for successful vaccination against schistosomiasis in BALB/c mice

Although protective immunity in C57BL/6 mice induced by a single dose of the radiation-attenuated schistosome vaccine is believed to be mediated by Th1-type immune responses, we here report that in BALB/c mice protection can also depend upon signaling via the interleukin-4 (IL-4) receptor which conventionally governs the development of Th2-type immune responses. We show that in BALB/c mice deficient for the IL-4 receptor alpha chain (IL-4Ralpha(-/-)), which are unresponsive to IL-4 and IL-13, vaccine-induced protection is abrogated compared with that in wild-type (WT) mice. In vaccinated IL-4Ralpha(-/-) mice, IL-12p40 production by cells from the skin exposure site was elevated, although gamma interferon (IFN-gamma) production in draining lymphoid tissues was similar in WT and IL-4Ralpha(-/-) mice. Nevertheless, the effector response in IL-4Ralpha(-/-) mice was Th1 biased with elevated IFN-gamma in the lungs and higher immunoglobulin G2a (IgG2a) and IgG2b titers but negligible quantities of Th2-associated IgG1 and IgE. Interestingly, levels of IL-4 were equivalent in WT and IL-4Ralpha(-/-) mice, indicating that Th2 responses were not dependent upon signaling by IL-4 or IL-13. No differences in the phenotype and composition of the pulmonary effector mechanism that might explain the failure to induce protection in IL-4Ralpha(-/-) mice were detected. However, passive transfer of partial protection to naive IL-4Ralpha(-/-) mice, using serum from vaccinated WT mice, indicates that Th2-associated antibodies such as IgG1 have a role in parasite elimination in BALB/c strain mice and that signaling via IL-4R can be an important factor in the generation of protection.     

Differential susceptibility of C57BL/6 and DBA/2 mice to ovalbumin-induced pulmonary eosinophilia regulated by Th1/Th2-type cytokines

To test the effect of genotype on immune response, C57BL/6 and DBA/2 mice were sensitized with aluminum hydroxide gel (alum)-precipitated ovalbumin (OVA) and challenged with aerosolized OVA. The serum immunoglobulin (Ig) E and IgG1 levels in C57BL/6 mice were higher than those in DBA/2 mice. In contrast, IgG2a levels in C57BL/6 mice were lower than that in DBA/2 mice. C57BL/6 mice were also much more susceptible than DBA/2 mice to OVA-induced pulmonary eosinophilia. Furthermore, patterns of cytokine generation in lung tissue were different between C57BL/6 and DBA/2 mice after OVA challenge. Th2-type cytokine interleukin (IL-) 4 and IL-5 generation in C57BL/6 mice was higher than that in DBA/2 mice, while Thl-type cytokine interferon-gamma (IFN-gamma) generation in C57BL/6 mice was lower than that in DBA/2 mice. Similar patterns of IL-4 and IL-5, and IFN-gamma production in splenocytes from both strains after OVA stimulation in vitro were also observed. The participation of IL-4 and IL-5, and IFN-gamma in the regulation of eosinophil infiltration into the lung was confirmed by injection of anti-IL-5, -IL-4 and -IFN-gamma monoclonal antibodies. These results indicate that C57BL/6 mice preferentially induce IL-4 and IL-5-mediated Th2-type response, while DBA/2 mice induce IFN-gamma-mediated Thl-type response. Thus, the genotype of laboratory strains partially determines whether Th1- or Th2-type immune responses are elicited.     

A STAT4-dependent Th1 response is required for resistance to the helminth parasite Taenia crassiceps

To determine the role of STAT4-dependent Th1 responses in the regulation of immunity to the helminth parasite Taenia crassiceps, we monitored infections with this parasite in resistant mice lacking the STAT4 gene. While T. crassiceps-infected STAT4(+/+) mice rapidly resolved the infection, STAT4(-/-) mice were highly susceptible to infection and displayed large parasite loads. Moreover, the inability of STAT4(-/-) mice to control the infection was associated with the induction of an antigen-specific Th2-type response characterized by significantly higher levels of Th2-associated immunoglobulin G1 (IgG1) and total IgE as well as interleukin-4 (IL-4), IL-10, and IL-13 than those in STAT4(+/+) mice, who produced significantly more gamma interferon. Furthermore, early after infection, macrophages from STAT4(-/-) mice produced lower levels of the pro-inflammatory cytokines IL-12, tumor necrosis factor alpha, IL-1 beta, and nitric oxide (NO) than those from STAT4(+/+) mice, suggesting a pivotal role for macrophages in mediating protection against cysticercosis. These findings demonstrate a critical role for the STAT4 signaling pathway in the development of a Th1-type immune response that is essential for mediating protection against the larval stage of T. crassiceps infection.     

TSH receptor-adenovirus-induced Graves' hyperthyroidism is attenuated in both interferon-gamma and interleukin-4 knockout mice; implications for the Th1/Th2 paradigm

The role of the Th1/Th2 balance in the pathogenesis of murine Graves' hyperthyroidism is controversial. In BALB/c mice injected with adenovirus expressing TSH receptor (TSHR-adeno model), we found that suppression of TSHR-specific Th1 immune responses by exogenous interleukin-4 (IL-4), alpha-galactosylceramide or helminth (Schistosoma mansoni) infection was associated with inhibition of hyperthyroidism, indicating the critical role for Th1 cytokines. In contrast, BALB/c IL-4 knockout (KO), but not interferon-gamma (IFN-gamma) KO mice failed to develop Graves' hyperthyroidism when injected with TSHR-expressing M12 B lymphoma cells (TSHR-M12 model), suggesting the importance of Th2 cytokine IL-4. To reconcile differences in these two models, we used IL-4 KO and IFN-gamma KO BALB/c mice in the TSHR-adeno model. Unlike wild-type (wt) BALB/c mice in which 60% developed hyperthyroidism, only 13 and 7% of IL-4 KO and IFN-gamma KO mice, respectively, became hyperthyroid. Thyroid stimulating antibodies were positive in most hyperthyroid mice. TSHR antibody titres determined by TSH binding inhibition and ELISA were comparable in all three groups. IgG1 and IgG2a TSHR antibody titres were similar in IFN-gamma KO and wt mice, whereas IgG1 TSHR antibody titres and TSHR-specific splenocyte IFN-gamma secretion were lower in IL-4 KO than in IFN-gamma KO and wt mice, respectively. Our results clearly implicate both IFN-gamma and IL-4 in development of hyperthyroidism in the TSHR-adeno model. These data, together with the previous report, also indicate different cytokine requirements in these two Graves' models, with IFN-gamma being more important in the TSHR-adeno than the TSHR-M12 model. Moreover, our previous and present observations indicate a difference in the role of exogenous versus endogenous IL-4 in TSHR-adenovirus induced Graves' hyperthyroidism.     

Expression of recombinant enterotoxigenic Escherichia coli colonization factor antigen I by Salmonella typhimurium elicits a biphasic T helper cell response

Protective immunity to enterotoxigenic Escherichia coli (ETEC) is antibody (Ab) dependent; however, oral immunization with purified ETEC fimbriae fails to elicit protective immunity as a consequence of antigenic alteration by the gastrointestinal (GI) tract. Unless unaltered ETEC fimbriae can reach the inductive lymphoid tissues of the GI tract, immunity to ETEC cannot be induced. To produce immunity, live vectors, such as Salmonella typhimurium, can effectively target passenger antigens to the inductive lymphoid tissues of the GI tract. By convention, oral immunizations with Salmonella vectors induce CD4(+) T helper (Th) cell responses by gamma interferon (IFN-gamma)-dominated pathways both to the vector and passenger antigen, resulting in serum immunoglobulin G2a (IgG2a) and modest mucosal IgA Ab responses. In the present study, mice orally immunized with a Salmonella vector engineered to stably express ETEC colonization factor antigen I (CFA/I) showed initially elevated serum IgG1 and mucosal IgA anti-CFA/I Ab responses. As expected, mice orally immunized with an E. coli-CFA/I construct elicited poor anti-CFA/I Ab responses. In fact, the addition of cholera toxin during oral E. coli-CFA/I immunization failed to greatly enhance mucosal IgA Ab responses. Seven days after immunization with the Salmonella-CFA/I construct, cytokine-specific ELISPOT showed induction of predominant Th2-type responses in both mucosal and systemic immune compartments supporting the early IgG1 and IgA anti-CFA/I Abs. By 4 weeks, the Th cell response became Th1 cell dominant from the earlier Th2-type responses, as evidenced by increased mucosal and systemic IFN-gamma-producing T cells and a concomitant elevation of serum IgG2a Ab responses. This biphasic response offers an alternative strategy for directing Salmonella vector-induced host immunity along a Th2 cell-dependent pathway, allowing for early promotion of mucosal and systemic Abs.     

Antigen dose defines T helper 1 and T helper 2 responses in the lungs of C57BL/6 and BALB/c mice independently of splenic responses

To investigate the effect of antigen dose on immune response, C57BL/6 and BALB/c mice were sensitized with aluminum hydroxide gel (alum)-precipitated ovalbumin (OVA) then challenged with aerosolized OVA. Low-dose sensitization (less than 8 microg of OVA) elicited T helper 2 (Th2)-type immunoglobulins (Igs) secretion from C57BL/6 mice, including high levels of serum IgE, IgG1 and low levels of IgG2a, while BALB/c mice secreted T helper 1 (Th1)-type Igs, including low levels of IgE, IgG1 and high levels of IgG2a. In contrast, high-dose sensitization (more than 50 microgram) elicited Th1-type Igs secretion in C57BL/6mice, while BALB/c mice exhibited Th2-type Igs secretion. Furthermore, the number of eosinophils infiltrating into the lungs of low-dose OVA-sensitized C57BL/6 mice was significantly greater than in BALB/c mice sensitized with the same amount of OVA. Only a very high dose of OVA (1 mg) could induce greater eosinophil infiltration into the lungs of BALB/c mice compared with C57BL/6 mice. Additionally, low-dose sensitization generated Th2-type cytokines, including high levels of interleukin (IL) -4, IL-5 and a low level of interferon-gamma (IFN-gamma) in the lungs of C57BL/6 mice, while BALB/c mice generated Th1-type cytokines in their lungs, including low levels of IL-4, IL-5 and a high level of IFN-gamma. In contrast, high-dose sensitization elicited Th1-type cytokines production in the lungs of C57BL/6 mice, while BALB/c mice generated Th2-type cytokines in their lungs. Interestingly, splenocyte cultures from C57BL/6 mice produced Th1-type cytokines, while cultures from BALB/c mice produced Th2-type cytokines regardless of OVA sensitization dose (100 ng-1 mg). These results indicate that C57BL/6 and BALB/c mice have different susceptibilities to OVA-sensitization and OVA-induced pulmonary eosinophilia regulated by Th1- and Th2-type cytokines, independent of splenic Th1- and Th2-type cytokines production.     

Th1 adjuvant N-acetyl-D-glucosamine polymer up-regulates Th1 immunity but down-regulates Th2 immunity against a mycobacterial protein (MPB-59) in interleukin-10-knockout and wild-type mice.

Treatment of mice with heat-killed (HK) Mycobacterium bovis BCG or 1- to 10-microm chitin particles (nonantigenic N-acetyl-D-glucosamine polymers) is known to induce innate immune responses, including gamma interferon (IFN-gamma) production, which plays a Th1 adjuvant role. However, HK BCG further induces prostaglandin E2-releasing spleen macrophages (Mphi) (PGE2-Mphi), which potentially inhibit Th1 adjuvant activities. We found that chitin particles did not induce PGE2-Mphi formation. To further assess whether chitin has Th1 adjuvant effects, interleukin-10 (IL-10)-knockout (KO) mice and their wild-type (WT, C57BL/6) controls were immunized with a 30-kDa MPB-59 mycobacterial protein mixed with chitin. Immunization with MPB-59 alone induced Th2 responses, characterized by increases in total serum immunoglobulin E (IgE) and specific serum IgG1 levels and spleen Th2 cells producing IL-4, IL-5, and IL-10. No IFN-gamma-producing spleen Th1 cells, specific serum IgG2a, or delayed-type hypersensitivity (DTH) footpad reactions were detected. On the other hand, chitin-MPB-59 immunization significantly increased spleen Th1 responses, DTH reaction, and serum IgG2a levels along with decreases of Th2 responses. The magnitude of these Th1 adjuvant effects was greater in IL-10-KO mice than in WT mice. In contrast, immunization with HK BCG-MPB-59 showed little or no Th1 adjuvant effect. These data indicate that chitin has a unique Th1 adjuvant effect on the development of Th1 immunity against a mycobacterial antigen. IL-10 down-regulates the adjuvant effect of chitin.     

The in vitro production of cytokines by mucosal lymphocytes immunized by oral administration of keyhole limpet hemocyanin using cholera toxin as an adjuvant.

The in vitro production of a variety of cytokines by lymphocytes isolated from spleen mesenteric lymph node (MLN), Peyer's patches (PP) and lamina propria (LP) was measured, after oral immunization with keyhole limpet hemocyanin using cholera toxin as an adjuvant. LP responses were characterized by very high levels of interleukin (IL) 4, IL 5 and IL 6 with lower levels of IL 2 and interferon-gamma (IFN-gamma). The PP had lower levels of IL 4, IL 5 and IL 6 than LP but higher levels of IL 2, IFN-gamma was only present at very low levels in this organ. The MLN had a pattern of cytokine production similar to the PP but did produce IFN-gamma. The spleen produced all cytokines measured except IL 5. Antibody production was characterized by IgA in the LP and PP but IgG was the dominant isotype in the spleen, the MLN was a poor source of antibody-producing cells. We interpret the results to show that (a) the LP response to cholera toxin/keyhole limpet hemocyanin is dominated by Th2-type cytokines compared to a lower production of Th1 type and (b) that the PP has responses typical of an organ with a high proportion of resting lymphocytes which develop mainly into Th2-types cells. The spleen is less dominated by Th2-type cytokines than the mucosal sites and this difference is paralleled by IgA antibody production at the mucosal sites and IgG antibody dominance in the spleen.     

Enhanced immunoglobulin A response and protection against Salmonella enterica serovar typhimurium in the absence of the substance P receptor

The development of the neurokinin-1 receptor-deficient (NK1R(-/-)) mouse permitted inquiry into the regulation of secretory immunoglobulin A (S-IgA) responses by substance P (SP) after oral immunization with a Salmonella enterica serovar Typhimurium vector expressing colonization factor antigen I (CFA/I) from enterotoxigenic Escherichia coli. In NK1R(-/-) mice, mucosal and serum IgA anti-CFA/I fimbrial responses were augmented, while secreted IgG anti-CFA/I fimbrial responses remained unaffected compared to those of BALB/c (NK1R(+/+)) mice. Supportive antibody-forming cells were present in the small intestinal lamina propria and spleen. To gain insight as to why the augmented S-IgA responses occurred, minimally, the responses were not attributed to differences in vaccine colonization of Peyer's patch (PP) and spleen or in their respective tissue weights. However, these S-IgA responses were supported by increased numbers of PP CD4(+) T helper (Th) cells secreting interleukin-5 (IL-5) and IL-6 and splenic CD4(+) Th cells secreting IL-6 compared to NK1R(+/+) mice. Challenge of naive NK1R(-/-) mice with wild-type Salmonella showed improved median survival compared to naive NK1R(+/+) mice. Data from peritoneal macrophage infection studies suggest that this survival is in part contributed by increased IL-10 production. Oral vaccination with Salmonella CFA/I or Salmonella vector showed no significant differences in conferred protection against wild-type challenge for either NK1R(-/-) or NK1R(+/+) mice. Thus, these studies suggest that SP mediation contributes to proinflammatory responses to Salmonella infections.     

Interleukin 7 can enhance antigen-specific cytotoxic-T-lymphocyte and/or Th2-type immune responses in vivo

Interleukin 7 (IL-7) protein has been reported to be important in the development of cytotoxic-T-lymphocyte (CTL) responses. However, other studies also support a partial Th2 phenotype for this cytokine. In an effort to clarify this unusual conflict, we compared IL-7 along with IL-12 (Th1 control) and IL-10 (Th2 control) for its ability to induce antigen (Ag)-specific CTL and Th1- versus Th2-type immune responses using a well established DNA vaccine model. In particular, IL-7 codelivery showed a significant increase in immunoglobulin G1 (IgG1) levels compared to IgG2a levels. IL-7 coinjection also decreased production of Th1-type cytokine IL-2, gamma interferon, and the chemokine RANTES but increased production of the Th2-type cytokine IL-10 and the similarly biased chemokine MCP-1. In herpes simplex virus (HSV) challenge studies, IL-7 coinjection decreased the survival rate after lethal HSV type 2 (HSV-2) challenge compared with gD plasmid vaccine alone in a manner similar to IL-10 coinjection, whereas IL-12 coinjection enhanced the protection, further supporting that IL-7 drives immune responses to the Th2 type, resulting in reduced protection against HSV-2 challenge. Moreover, coinjection with human immunodeficiency virus type 1 env and gag/pol genes plus IL-12 or IL-7 cDNA enhanced Ag-specific CTLs, while coinjection with IL-10 cDNA failed to influence CTL induction. Thus, IL-7 could drive Ag-specific Th2-type cellular responses and/or CTL responses. These results support that CTLs could be induced by IL-7 in a Th2-type cytokine and chemokine environment in vivo. This property of IL-7 allows for an alternative pathway for CTL development which has important implications for host-pathogen responses.     

Immunity to vaginal HSV-2 infection in immunoglobulin A knockout mice.

An immunoglobulin A (IgA) knockout (KO) mouse was used to study the role of IgA in protective immunity against vaginal infection with herpes simplex virus-type 2 (HSV-2). Intact and KO mice were immunized intravaginally (IVAG) with attenuated HSV-2, challenged IVAG with wild-type virus 6 weeks later and evaluated for vaginal infection and neurological disease. Non-immunized/challenged intact and KO mice showed vaginal infection and succumbed to neurological disease, while immunized/challenged mice exhibited reduced or no vaginal infection and no neurological disease. Log 2.5 enzyme-linked immunoassay (ELISA) titres of viral IgA, immunoglobulin G (IgG) and immunoglobulin M (IgM) in vaginal secretions collected from intact immune mice before challenge were 0.6+/-0.3, 6.4+/-0.32 and 0.0, while those in KO immune mice were 0.0, 6.7+/-0.19 and 3.0+/-0.29, respectively. Twenty-four hours after challenge, the percentage of vaginal epithelium that was infected in non-immune intact and KO mice was 2.0+/-0.6 and 2.4+/-0.6, which was reduced to 0.2+/-0.1 and 0.1+/-0.06 in immune intact and KO mice, respectively. No shed virus protein was detected in vaginal secretions 3 days after challenge in any immune mouse, whereas titres were 1400 and 1700 in the two groups of non-immune mice. Thus, immune protection against vaginal HSV-2 infection was similar in both KO and intact mice, indicating that this mucosal immunity does not depend mainly on IgA.     

C57BL/6 mice are more susceptible to antigen-induced pulmonary eosinophilia than BALB/c mice, irrespective of systemic T helper 1/T helper 2 responses

Inflammatory response differences between C57BL/6 and BALB/c mice following ovalbumin (OVA) sensitization and a single challenge were investigated. Serum immunoglobulin (Ig)E and IgG1 levels were higher in C57BL/6 mice than in BALB/c mice. In contrast, IgG2a levels in C57BL/6 mice were lower than in BALB/c mice. Furthermore, the number of eosinophils infiltrating into lungs in C57BL/6 mice was significantly higher than in BALB/c mice after OVA challenge. The levels of the T helper 2 (Th2)-type cytokines interleukin (IL)-4 and IL-5, generated in challenged C57BL/6 lung tissue, were also higher than in BALB/c lung tissue. The participation of IL-4 and IL-5 in the induction of eosinophil infiltration into the lungs was confirmed in both strains of mice by injection of anti-IL-4 and anti-IL-5 monoclonal antibodies (mAbs). However, following OVA stimulation, in vitro IL-4 and IL-5 production in splenocyte cultures from C57BL/6 mice was lower than in splenocyte cultures from BALB/c mice. These results indicate that C57BL/6 mice induce Th2-type responses in the lungs, while BALB/c mice induce T helper 1 (Th1)-type responses in the lungs, despite considerable production of IL-4 and IL-5 from splenocytes. Therefore, local immune responses are more important in the induction of allergic inflammation in the lungs and are different from systemic immune responses, which are thought to depend on genetic background.     

Protection against rotavirus shedding after intranasal immunization of mice with a chimeric VP6 protein does not require intestinal IgA

Intranasal immunization of mice with chimeric VP6 and the adjuvant LT(R192G) consistently elicits >95% reductions in fecal rotavirus shedding following challenge. To determine the association between mucosal antibody and protection, we immunized BALB/c wt and J chain knockout (Jch-/-) mice with VP6 and either LT(R192G) or cholera toxin (CT). Both strains developed nearly equal levels of serum rotavirus IgG, but Jch-/- mice, which cannot transport dimeric IgA across epithelial cell surfaces, developed >4-fold higher levels of serum rotavirus IgA. Stool rotavirus IgA was present in wt but undetectable in Jch-/- mice. When challenged with rotavirus strain EDIM, reductions in rotavirus shedding were nearly identical in VP6-immunized wt and Jch-/- mice (i.e., 97% and 92%, respectively; P > 0.01). Th1 CD4 T cell responses were also detected in VP6-immunized animals based on high levels of IFN-gamma and IL-2 found after in vitro VP6 stimulation of spleen cells. Therefore, protection induced by intranasal immunization of mice with VP6 and adjuvant does not depend on intestinal rotavirus IgA antibody but appears to be associated with CD4 T cells.     

Deficiency of CD26 results in a change of cytokine and immunoglobulin secretion after stimulation by pokeweed mitogen

To investigate the role of CD26 in the immune system, CD26 gene knockout mice with C57BL/6 background were used to study the immune response after stimulation with PWM. CD26(-/-) mice display an apparently normal phenotype. However, in their spleen lymphocyte population the percentage of CD4(+) T cells is lower, and that of NK cells is higher, than that in CD26(+/+) mice. In their peripheral blood, CD26(-/-) mice present a conspicuously decreased proportion of CD4(+) NKT lymphocytes. In vitro, the PWM-stimulated IL-4 production was decreased by 60-80% in the supernatants of spleen lymphocytes of CD26(-/-) mice compared to that of CD26(+/+) mice, whereas levels of IL-10 and IFN-gamma were increased. No significant differences were found in the production of IL-2, IL-5, IL-6 and IL-13 between knockout and wild-type mice. After immunization of mice with PWM in vivo, serum levels of total IgG, IgG1, IgG2a and IgE were markedly lower in CD26(-/-) mice than those in CD26(+/+) mice, while no difference was found in IgM production. Further analysis of cytokine levels in vivo revealed a reduced IL-4, IL-2 and delayed IFN-gamma production in sera of CD26(-/-) mice upon immunization with PWM. These results indicate that CD26 contributes to the regulation of development, maturation and migration of CD4(+) T, NK and NKT cells, cytokine secretion, T cell-dependent antibody production and immunoglobulin isotype switching of B cells.     

Interleukin 7 Can Enhance Antigen-Specific Cytotoxic-T-Lymphocyte and/or Th2-Type Immune Responses In Vivo

Interleukin 7 (IL-7) protein has been reported to be important in the development of cytotoxic-T-lymphocyte (CTL) responses. However, other studies also support a partial Th2 phenotype for this cytokine. In an effort to clarify this unusual conflict, we compared IL-7 along with IL-12 (Th1 control) and IL-10 (Th2 control) for its ability to induce antigen (Ag)-specific CTL and Th1- versus Th2-type immune responses using a well established DNA vaccine model. In particular, IL-7 codelivery showed a significant increase in immunoglobulin G1 (IgG1) levels compared to IgG2a levels. IL-7 coinjection also decreased production of Th1-type cytokine IL-2, gamma interferon, and the chemokine RANTES but increased production of the Th2-type cytokine IL-10 and the similarly biased chemokine MCP-1. In herpes simplex virus (HSV) challenge studies, IL-7 coinjection decreased the survival rate after lethal HSV type 2 (HSV-2) challenge compared with gD plasmid vaccine alone in a manner similar to IL-10 coinjection, whereas IL-12 coinjection enhanced the protection, further supporting that IL-7 drives immune responses to the Th2 type, resulting in reduced protection against HSV-2 challenge. Moreover, coinjection with human immunodeficiency virus type 1 env and gag/pol genes plus IL-12 or IL-7 cDNA enhanced Ag-specific CTLs, while coinjection with IL-10 cDNA failed to influence CTL induction. Thus, IL-7 could drive Ag-specific Th2-type cellular responses and/or CTL responses. These results support that CTLs could be induced by IL-7 in a Th2-type cytokine and chemokine environment in vivo. This property of IL-7 allows for an alternative pathway for CTL development which has important implications for host-pathogen responses.     

Mucosal immunization of CD4+ T cell-deficient mice with an inactivated virus induces IgG and IgA responses in serum and mucosal secretions

Immunoglobulin (Ig) class switching can occur in the absence of alphabeta+ or gammadelta+ T cells when mice are infected with certain live viruses, although CD4 T helper cells are believed to be essential for induction of a high-affinity antibody response and for efficient isotype switching from IgM to IgG and IgA production. However, little information is available about the immune responses after mucosal immunization of CD4+ T cell-deficient mice with inactivated virus. In this study, we show that intranasal immunization with formalin-inactivated influenza A/PR8/34 virus induces IgG and IgA responses in serum and IgA responses in mucosal secretions in CD4+ T cell-deficient mice. All four subclasses of IgG were produced. IgG1/IgG2a ratios were found to be from 1 to 1.75, indicating that both Th1 and Th2 immune responses are induced by the inactivated influenza virus. The sera and mucosal secretions were found to have neutralizing activity against influenza virus in vitro. In addition, the mucosally immunized CD4+ T cell-deficient mice were protected completely from challenge with a lethal dose of live, pathogenic influenza virus. To our knowledge, this is the first demonstration that mucosal immunization with an inactivated virus induces immune responses in serum and mucosal secretions in CD4+ T cell-deficient mice.