Targeting antigen to MHC Class I and Class II antigen presentation pathways for malaria DNA vaccines

An effective malaria vaccine which protects against all stages of Plasmodium infection may need to elicit robust CD8(+) and CD4(+) T cell and antibody responses. To achieve this, we have investigated strategies designed to improve the immunogenicity of DNA vaccines encoding the Plasmodium yoelii pre-erythrocytic stage antigens PyCSP and PyHEP17, by targeting the encoded proteins to the MHC Classes I and II processing and presentation pathways. For enhancement of CD8(+) T cell responses, we targeted the antigens for degradation by the ubiquitin (Ub)/proteosome pathway following the N-terminal rule. We constructed plasmids containing PyCSP or PyHEP17 genes fused to the Ub gene: plasmids where the N-terminal antigen residues were mutated from the stabilizing amino acid methionine to destabilizing arginine, plasmids where the C-terminal residues of Ub were mutated from glycine to alanine, and plasmids in which the potential hydrophobic leader sequences of the antigens were deleted. For enhancement of CD4(+) T cell and antibody responses, we targeted the antigens for degradation by the endosomal/lysosomal pathway by linking the antigen to the lysosome-associated membrane protein (LAMP). We found that immunization with DNA vaccine encoding PyHEP17 fused to Ub and bearing arginine induced higher IFN-gamma, cytotoxic and proliferative T cell responses than unmodified vaccines. However, no effect was seen for PyCSP using the same targeting strategies. Regarding Class II antigen targeting, fusion to LAMP did not enhance antibody responses to either PyHEP17 or PyCSP, and resulted in a marginal increase in lymphoproliferative CD4(+) T cell responses. Our data highlight the antigen dependence of immune enhancement strategies that target antigen to the MHC Class I and II pathways for vaccine development.     

Immunogenicity and protection of a recombinant human adenovirus serotype 35-based malaria vaccine against Plasmodium yoelii in mice

Given the promise of recombinant adenovirus type 5 (rAd5) as a malaria vaccine carrier in preclinical models, we evaluated the potency of rAd35 coding for Plasmodium yoelii circumsporozoite protein (rAd35PyCS). We chose rAd35 since a survey with serum samples from African subjects demonstrated that human Ad35 has a much lower seroprevalence of 20% and a much lower geometric mean neutralizing antibody titer (GMT) of 48 compared to Ad5 (seroprevalence, 85%; GMT, 1,261) in countries with a high malaria incidence. We also demonstrated that immunization with rAd35PyCS induced a dose-dependent and potent, CS-specific CD8(+) cellular and humoral immune response and conferred significant inhibition (92 to 94%) of liver infection upon high-dose sporozoite challenge. Furthermore, we showed that in mice carrying neutralizing antibody activity against Ad5, mimicking a human situation, CS-specific T- and B-cell responses were significantly dampened after rAd5PyCS vaccination, resulting in loss of inhibition of liver infection upon sporozoite challenge. In contrast, rAd35 vaccine was as potent in naive mice as in Ad5-preimmunized mice. Finally, we showed that heterologous rAd35-rAd5 prime-boost regimens were more potent than rAd35-rAd35 because of induction of anti-Ad35 antibodies after rAd35 priming. The latter data provide a further rationale for developing rAd prime-boost regimens but indicate that priming and boosting Ad vectors must be immunologically distinct and also should be distinct from Ad5. Collectively, the data presented warrant further development of rAd35-based vaccines against human malaria.     

CD4+ T-Cell- and Gamma Interferon-Dependent Protection against Murine Malaria by Immunization with Linear Synthetic Peptides from a Plasmodium yoelii 17-Kilodalton Hepatocyte Erythrocyte Protein

Most work on protective immunity against the pre-erythrocytic stages of malaria has focused on induction of antibodies that prevent sporozoite invasion of hepatocytes, and CD8(+) T-cell responses that eliminate infected hepatocytes. We recently reported that immunization of A/J mice with an 18-amino-acid synthetic linear peptide from Plasmodium yoelii sporozoite surface protein 2 (SSP2) in TiterMax adjuvant induces sterile protection that is dependent on CD4(+) T cells and gamma interferon (IFN-gamma). We now report that immunization of inbred A/J mice and outbred CD1 mice with each of two linear synthetic peptides from the 17-kDa P. yoelii hepatocyte erythrocyte protein (HEP17) in the same adjuvant also induces protection against sporozoite challenge that is dependent on CD4(+) T cells and IFN-gamma. The SSP2 peptide and the two HEP17 peptides are recognized by B cells as well as T cells, and the protection induced by these peptides appears to be directed against the infected hepatocytes. In contrast to the peptide-induced protection, immunization of eight different strains of mice with radiation-attenuated sporozoites induces protection that is absolutely dependent on CD8(+) T cells. Data represented here demonstrate that CD4(+) T-cell-dependent protection can be induced by immunization with linear synthetic peptides. These studies therefore provide the foundation for an approach to pre-erythrocytic-stage malaria vaccine development, based on the induction of protective CD4(+) T-cell responses, which will complement efforts to induce protective antibody and CD8(+) T-cell responses.     

pHSP65疫苗增强结核杆菌特异性T细胞产生IFN-γ

为提高BCG的免疫效果,以BCG进行初次免疫,比较pHSP65基因疫苗和BCG疫苗加强免疫的效果,寻找新型结核疫苗的初免-加强免疫策略。于第0周皮下接种BCG,第6、8周给予pHSP65滴鼻免疫或BCG加强免疫,末次免疫后2周检测全身及肺脏局部IFN-γ的产生。与BCG初免/BCG加强免疫相比,经pHSP65基因疫苗滴鼻加强免疫后,ELISPOT结果显示脾脏和肺局部分泌IFN-γ的淋巴细胞数量显著增加,流式检测显示IFN-γ+CD4+T细胞数量显著增加,ELISA结果证实淋巴细胞IFN-γ的分泌显著增高,提示经BCG初次免疫后,以pHSP65 DNA滴鼻加强免疫可显著增强全身和肺局部T淋巴细胞IFN-γ的产生,具有良好的抗结核感染潜能。     

Protection of rhesus macaques against lethal Plasmodium knowlesi malaria by a heterologous DNA priming and poxvirus boosting immunization regimen

We tested a cytokine-enhanced, multiantigen, DNA priming and poxvirus boosting vaccine regimen for prevention of malaria in the Plasmodium knowlesi-rhesus macaque model system. Animals were primed with a mixture of DNA plasmids encoding two preerythrocytic-stage proteins and two erythrocytic-stage proteins from P. knowlesi and combinations of the cytokines granulocyte-macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor alpha and were boosted with a mixture of four recombinant, attenuated vaccinia virus strains encoding the four P. knowlesi antigens. Two weeks after boosting, the geometric mean immunofluorescence titers in the immunized groups against sporozoites and infected erythrocytes ranged from 160 to 8,096 and from 1,810 to 5,120, respectively. The geometric mean anti-P. knowlesi circumsporozoite protein (PkCSP) titers ranged from 1,761 to 24,242. Peripheral blood mononuclear cells (PBMC) from the immunized monkeys produced gamma interferon (IFN-gamma) in response to incubation with pooled peptides from the PkCSP at frequencies of 10 to 571 spot-forming cells/10(6) PBMC. Following challenge with 100 infectious P. knowlesi sporozoites, 2 of 11 immunized monkeys were sterilely protected, and 7 of the 9 infected monkeys resolved their parasitemias spontaneously. In contrast, all four controls became infected and required treatment for overwhelming parasitemia. Early protection was strongly associated with IFN-gamma responses against a pool of peptides from the preerythrocytic-stage antigen, PkCSP. These findings demonstrate that a multistage, multiantigen, DNA priming and poxvirus boosting vaccine regimen can protect nonhuman primates from an otherwise lethal malaria sporozoite challenge.     

A subdominant CD8(+) cytotoxic T lymphocyte (CTL) epitope from the Plasmodium yoelii circumsporozoite protein induces CTLs that eliminate infected hepatocytes from culture

Previous studies indicated that the Plasmodium yoelii circumsporozoite protein (PyCSP) 57-70 region elicits T cells capable of eliminating infected hepatocytes in vitro. Herein, we report that the PyCSP58-67 sequence contains an H-2(d) binding motif, which binds purified K(d) molecules in vitro with low affinity (3, 267 nM) and encodes an H-2(d)-restricted cytotoxic T lymphocyte (CTL) epitope. Immunization of BALB/c mice with three doses of a multiple antigen peptide (MAP) construct containing four branches of amino acids 57 to 70 linked to a lysine-glycine core [MAP4(PyCSP57-70)] and Lipofectin as the adjuvant induced both T-cell proliferation and a peptide-specific CTL response that was PyCSP59-67 specific, H-2(d) restricted, and CD8(+) T cell dependent. Immunization with either DNA encoding the PyCSP or irradiated sporozoites demonstrated that this CTL epitope is subdominant since it is not recognized in the context of whole CSP immunization. The biological relevance of this CTL response was underlined by the demonstration that it could mediate genetically restricted, CD8(+)- and nitric-oxide-dependent elimination of infected hepatocytes in vitro, as well as partial protection of BALB/c mice against sporozoite challenge. These findings indicate that subdominant epitopes with low major histocompatibility complex affinity can be used to engineer epitope-based vaccines and have implications for the selection of epitopes for subunit-based vaccines.     

Intradermal NKT cell activation during DNA priming in heterologous prime-boost vaccination enhances T cell responses and protection against Leishmania

Heterologous prime-boost vaccination employing DNA-vaccinia virus (VACV) modality using the Leishmania homologue of receptors for activated C kinase (LACK) (p36) antigen has been shown to elicit protective immunity against both murine cutaneous and visceral leishmaniasis. However, DNA priming is known to have limited efficacy; therefore in the current study the effect of NKT cell activation using alpha-galactosyl-ceramide (alphaGalCer) during intradermal DNAp36 priming was examined. Vaccinated mice receiving alphaGalCer + DNAp36 followed by a boost with VVp36 appeared to be resolving their lesions and had at ten- to 20-fold higher reductions in parasite burdens. NKT cell activation during alphaGalCer + DNAp36 priming resulted in higher numbers of antigen-reactive effector CD4(+) and CD8(+) T cells producing granzyme and IFN-gamma, with lower levels of IL-10. Although immunodepletion studies indicate that both CD4 and CD8 T cells provide protection in the vaccinated mice, the contribution of CD4(+) T cells was significantly increased in mice primed with DNAp36 together with alphaGalCer. Notably 5 months after boosting, mice vaccinated with DNAp36 + alphaGalCer continued to show sustained and heightened T cell immune responses. Thus, heterologous prime-boost vaccination using alphaGalCer during priming is highly protective against murine cutaneous leishmaniasis, resulting in the heightened activation and development of CD4 and CD8 T cells (effector and memory T cells).     

In vivo activation of dendritic cells and T cells during Salmonella enterica serovar Typhimurium infection

The present study was initiated to gain insight into the interaction between splenic dendritic cells (DC) and Salmonella enterica serovar Typhimurium in vivo. Splenic phagocytic cell populations associated with green fluorescent protein (GFP)-expressing bacteria and the bacterium-specific T-cell response were evaluated in mice given S. enterica serovar Typhimurium expressing GFP and ovalbumin. Flow cytometry analysis revealed that GFP-positive splenic DC (CD11c+ major histocompatibility complex class II-positive [MHC-II+] cells) were present following bacterial administration, and confocal microscopy showed that GFP-expressing bacteria were contained within CD11c+ MHC-II+ splenocytes. Furthermore, splenic DC and T cells were activated following Salmonella infection. This was shown by increased surface expression of CD86 and CD40 on CD11c+ MHC-II+ cells and increased CD44 and CD69 expression on CD4+ and CD8+ T cells. Salmonella-specific gamma interferon (IFN-gamma)-producing cells in both of these T-cell subsets, as well as cytolytic effector cells, were also generated in mice given live bacteria. The frequency of Salmonella-specific CD4+ T cells producing IFN-gamma was greater than that of specific CD8+ T cells producing IFN-gamma in the same infected animal. This supports the argument that the predominant source of IFN-gamma production by cells of the specific immune response is CD4+ T cells. Finally, DC that phagocytosed live or heat-killed Salmonella in vitro primed bacterium-specific IFN-gamma-producing CD4+ and CD8+ T cells as well as cytolytic effector cells following administration into na?ve mice. Together these data suggest that DC are involved in priming na?ve T cells to Salmonella in vivo.     

Effective induction of acquired resistance to Listeria monocytogenes by immunizing mice with in vivo-infected dendritic cells

Splenic dendritic cells (DCs) obtained from mice at 48 h after Listeria monocytogenes infection exhibited up-regulation of CD80 and produced higher titers of gamma interferon (IFN-gamma) and interleukin-12 (IL-12) than did DCs obtained from uninfected mice. Mice immunized with DCs obtained from mice that had been infected with L. monocytogenes 48 h before acquired host resistance to lethal infection with L. monocytogenes at 4 and 8 weeks. Immunization with DCs from heat-killed L. monocytogenes failed to induce resistance. Acquired antilisterial resistance is specific, since the immunized mice could not be protected from Salmonella enterica serovar Typhimurium infection. Infected DCs stimulated proliferation of naive CD4(+) and CD8(+) cells in vitro, suggesting that in vivo-infected DCs activate CD8(+) T cells, which are critical in acquired antilisterial resistance, as well as CD4(+) T cells. When wild-type mice were immunized with DCs from IFN-gamma-deficient mice, they were protected against a lethal L. monocytogenes challenge. In contrast, when mice were immunized with DCs from anti-IL-12 p40 monoclonal antibody-injected mice, they failed to gain acquired antilisterial resistance. These results suggest that DC-derived IL-12, but not IFN-gamma, may play a critical role in induction of acquired antilisterial resistance. Our present results suggest that splenic DCs obtained from mice infected with L. monocytogenes in vivo may be an effective immunogen with which to induce antigen-specific immunity.     

Mutating the anchor residues associated with MHC binding inhibits and deviates CD8+ T cell mediated protective immunity against malaria

We investigated whether immune responses induced by immunization with plasmid DNA are restricted predominantly to immunodominant CD8+ T cell epitopes, or are raised against a breadth of epitopes including subdominant CD8+ and CD4+ T cell epitopes. Site-directed mutagenesis was used to change one or more primary anchor residues of the immunodominant CD8+ T cell epitope on the Plasmodium yoelii circumsporozoite protein, and in vivo protective efficacy and immune responses against defined PyCSP CD8+ and/or CD4+ epitopes were determined. Mutation of the P2 but not P9 or P10 anchor residues decreased protection and completely abrogated the antigen-specific CD8+ CTL activity and CD8+ dependent IFN-gamma responses to the immunodominant CD8+ epitope and overlapping CD8+/CD4+ epitope. Moreover, mutation deviated the immune response towards a CD4+ T cell IFN-gamma dependent profile, with enhanced lymphoproliferative responses to the immunodominant and subdominant CD4+ epitopes and enhanced antibody responses. Responses to the subdominant CD8+ epitope were not induced. Our data demonstrate that protective immunity induced by PyCSP DNA vaccination is directed predominantly against the single immunodominant CD8+ epitope, and that although responses can be induced against other epitopes, these are mediated by CD4+ T cells and are not capable of conferring optimal protection against challenge.     

GP96NTD-CSP重组DNA疟疾疫苗诱导小鼠产生保护性免疫的研究

目的探讨pFLAG CMV8 gp96NTD-CSP重组DNA疟疾疫苗免疫能否诱导小鼠产生保护性免疫及其效应机制。方法以pFLAG CMV8质粒为载体,构建免疫用重组质粒,按照DNA疫苗免疫方法免疫小鼠;野生子孢子进行攻击后,采用Real-time PCR和吉氏染色观察被攻击小鼠的肝脏虫荷和原虫血症,即免疫小鼠抵御野生子孢子攻击的能力;并通过ELISA和ELISPOT方法探讨免疫小鼠保护性免疫的可能机制。结果核酸疫苗pFLAG CMV8 gp96NTD-CSP免疫小鼠能显著抵御野生子孢子的攻击,并且能诱导小鼠产生较高的抗体水平和较高的CSP特异的CD8+T细胞频率。结论 pFLAG CMV8 gp96NTD-CSP重组DNA疫苗可能通过诱导小鼠CSP特异抗体和CSP特异的CD8+T细胞的产生,一定程度上抵御野生子孢子的攻击。     

Isolation and characterization of protective cytolytic T cells in a rodent malaria model system.

Protective immunity against malaria is induced by immunization with irradiation-attenuated sporozoites. Here we report the isolation of cytolytic T-cell (CTL) clones from BALB/c (H-2d) mice immunized with either Plasmodium berghei or Plasmodium yoelii sporozoites. The epitopes recognized by these CTL can be mimicked by synthetic peptides corresponding to a homologous region in the CS proteins of both rodent malaria species. Both peptides are recognized by the CTL in the context of the same MHC class I molecule, H-2 Kd. In vivo adoptive transfer of the CTL clones into non-immune syngeneic mice protected them from a lethal challenge of infectious sporozoites.     

Complete protection against Plasmodium yoelii by adoptive transfer of a CD8+ cytotoxic T-cell clone recognizing sporozoite surface protein 2.

BALB/c mice immunized with irradiated Plasmodium yoelii sporozoites produce antibodies and cytotoxic T lymphocytes against the circumsporozoite protein and against a 140-kDa protein, sporozoite surface protein 2 (PySSP2). Approximately 50% of mice immunized with P815 cells transfected with the gene encoding PySSP2 are protected against malaria, and this protection is reversed by in vivo depletion of CD8+ T cells. To determine if CD8+ T cells against PySSP2 are adequate to protect against malaria in the absence of other malaria-specific immune responses, we produced three CD8+ T-cell clones by stimulating spleen cells from mice immunized with irradiated P. yoelii sporozoites with a mitomycin-treated P815 cell clone transfected with the PySSP2 gene. Adoptive transfer of clone TSLB7 protected 100% of mice against P. yoelii. The second clone protected 58% of mice, and the third clone provided no protection. Clone TSLB7 protected even when administered 3 h after sporozoite inoculation at a time when sporozoites had entered hepatocytes, suggesting that it is recognizing and eliminating infected hepatocytes. These studies demonstrate that cytotoxic T lymphocytes against PySSP2 can protect against P. yoelii sporozoite challenge in the absence of other parasite-specific immune responses.     

Heterologous immunity in the absence of variant-specific antibodies after exposure to subpatent infection with blood-stage malaria

We examined immunity induced by subpatent blood-stage malaria (undetectable by microscopy) using the rodent malaria parasite, Plasmodium chabaudi chabaudi, postulating that limited infection may allow expansion of antigen-specific T cells that are normally deleted by apoptosis. After three infections drug cured at 48 h, mice were protected against high-dose challenge with homologous or heterologous parasites (different strain or variant). Immunity differed from that generated by three untreated, patent infections. Subpatently infected mice lacked immunoglobulin G (IgG) to variant surface antigens, despite producing similar titers of total malaria-specific IgG to those produced by patently infected mice, including antibodies specific for merozoite surface antigens conserved between heterologous strains. Antigen-specific proliferation of splenocytes harvested prechallenge was significantly higher in subpatently infected mice than in patently infected or naive mice. In subpatently infected mice, lymphoproliferation was similar in response to homologous and heterologous parasites, suggesting that antigenic targets of cell-mediated immunity were conserved. A Th1 cytokine response was evident during challenge. Apoptosis of CD4+ and CD8+ splenic lymphocytes occurred during patent but not subpatent infection, suggesting a reason for the relative prominence of cell-mediated immunity after subpatent infection. In conclusion, subpatent infection with blood stage malaria parasites induced protective immunity, which differed from that induced by patent infection and targeted conserved antigens. These findings suggest that alternative vaccine strategies based on delivery of multiple parasite antigens at low dose may induce effective immunity targeting conserved determinants.     

Heterologous priming-boosting immunization of cattle with Mycobacterium tuberculosis 85A induces antigen-specific T-cell responses

Heterologous priming-boosting vaccination regimens involving priming with plasmid DNA antigen constructs and inoculating (boosting) with the same recombinant antigen expressed in replication-attenuated poxviruses have recently been demonstrated to induce immunity, based on CD4(+)- and CD8(+)-T-cell responses, against several diseases in both rodents and primates. We show that similar priming-boosting vaccination strategies using the 85A antigen of Mycobacterium tuberculosis are effective in inducing antigen-specific gamma interferon-secreting CD4(+) and CD8(+) T cells, detected by a bovine enzyme-linked immunospot assay, in Bos indicus cattle. T-cell responses induced by priming with either plasmid DNA or fowlpox virus 85A constructs were enhanced by boosting with modified vaccinia virus Ankara expressing the same antigen administered intradermally. On the basis of the data, it appears that intradermal priming was more effective than intramuscular delivery of the priming dose for boosting with the modified vaccinia virus Ankara strain in cattle. Using either fowlpox virus or DNA priming, there was a significant bias toward induction of CD4(+)- rather than CD8(+)-T-cell responses. These data illustrate the general applicability of priming-boosting vaccination strategies for induction of antigen-specific T-cell responses and suggest that the method may be useful for development of veterinary vaccines.     

Dendritic Cells Can Initiate Protective Immune Responses against Malaria

An understanding of the antigen presentation mechanisms that mediate induction of protective immune responses against malaria is essential for the development of successful immunization approaches. Here we show that dendritic cells presenting Plasmodium yoelii sporozoite antigens are able to activate specific CD4(+) and CD8(+) T cells and initiate protective immune responses against malaria in mice.     

CD8(+) T-cell priming against a nonsecreted Listeria monocytogenes antigen is independent of the antimicrobial activities of gamma interferon

Sublethal infection of mice with recombinant Listeria monocytogenes expressing a model epitope in either secreted or nonsecreted form results in similar CD8(+) T-cell priming. Since nonsecreted bacterial proteins have no obvious access to the endogenous major histocompatibility complex (MHC) class I presentation pathway, presentation of these antigens requires destruction of the bacterium to reveal the nonsecreted molecules to an exogenous MHC class I presentation pathway. Gamma interferon (IFN-gamma), a cytokine made by multiple cell types in response to L. monocytogenes infection, could be required for exogenous presentation of nonsecreted bacterial antigens via its capacity to upregulate the expression of molecules involved in antigen presentation, its capacity to activate macrophages to kill bacteria to expose nonsecreted molecules or both. IFN-gamma knockout (KO) mice were used to address the requirement for IFN-gamma in CD8(+) T-cell priming against (i) a model exogenous antigen and (ii) secreted and nonsecreted L. monocytogenes antigens. We demonstrate that IFN-gamma KO mice are capable of cross-presenting the model exogenous antigen ovalbumin to prime CD8(+) T-cell responses that are only slightly weaker than that in wild-type (WT) mice. Despite their extreme susceptibility to primary L. monocytogenes infection, previously immunized and naive IFN-gamma KO mice were able to generate CD8(+) T-cell responses against both secreted and nonsecreted L. monocytogenes antigens which were similar to responses of WT mice. Interestingly, IFN-gamma KO mice were as capable as WT mice in mediating the characteristic drop in bacterial load in the liver at 4 h postinfection, although the IFN-gamma KO mice have exacerbated bacterial loads as early as 24 h postinfection. These results demonstrate that the regulatory functions of IFN-gamma are not required for priming of CD8(+) T cells by cross-presentation of a model exogenous antigen or in response to a nonsecreted L. monocytogenes antigen. In addition, the capacity of IFN-gamma to activate the microbicidal activities of macrophages is not required for the very early innate immune response to L. monocytogenes or priming of CD8(+) T cells against a nonsecreted bacterial antigen.     

A GRA1 DNA vaccine primes cytolytic CD8(+) T cells to control acute Toxoplasma gondii infection

Protective immunity against Toxoplasma gondii is known to be mediated mainly by T lymphocytes and gamma interferon (IFN-gamma). The contribution of CD4(+) and CD8(+) T-lymphocyte subsets to protective immune responses against T. gondii infection, triggered by a GRA1 (p24) DNA vaccine, was assessed in this study. In vitro T-cell depletion experiments indicated that both CD4(+) and CD8(+) T-cell subsets produced IFN-gamma upon restimulation with a T. gondii lysate. In addition, the GRA1 DNA vaccine elicited CD8(+) T cells that were shown to have cytolytic activity against parasite-infected target cells and a GRA1-transfected cell line. C3H mice immunized with the GRA1 DNA vaccine showed 75 to 100% protection, while 0 to 25% of the mice immunized with the empty control vector survived challenge with T. gondii cysts. In vivo T-cell depletion experiments indicated that CD8(+) T cells were essential for the survival of GRA1-vaccinated C3H mice during the acute phase of T. gondii infection, while depletion of CD4(+) T cells led to an increase in brain cyst burden during the chronic phase of infection.     

The suppression of delayed-type hypersensitivity by CD8+ regulatory T cells requires interferon-gamma

CD8(+) regulatory (suppressor) T cells are induced by complex cellular pathways in the spleens of mice that have received an injection of antigen into the anterior chamber (AC) of an eye, an immune-privileged site. Although these CD8(+) regulatory T cells perform an antigen-specific regulatory function for an immune response to self and non-self antigens, the mechanisms of the activation or function of these regulatory cells are not clear. Here, we describe a novel mechanism for the activation of splenic CD8(+) regulatory T cells induced by injection of antigen into the AC. Immunization of mice with trinitrophenyl and bovine serum albumin (TNP-BSA) amplified AC-induced splenic CD8(+) regulatory T cells that suppressed the initiation of contact sensitivity when transferred to immunized, challenged mice. These CD8(+) regulatory T cells were produced independently of perforin, indicating that they are not canonical cytotoxic T cells. Fas ligand (FasL)-deficient CD8(+) regulatory T-cell function was rescued by inclusion of exogenous interferon-gamma (IFN-gamma), demonstrating that the expression of FasL by CD8(+) regulatory T cells was dispensable, but IFN-gamma was not. Ultimately, we demonstrated that the generation of these CD8(+) regulatory T cells occurred independently of IFN-gamma, but their suppressor function required IFN-gamma receptor stimulation.     

Prenatal exposure to bisphenol A up-regulates immune responses, including T helper 1 and T helper 2 responses, in mice

The effect of prenatal exposure to bisphenol A (BPA) on the immune system in mice was investigated. Virgin female mice were fed varying doses of BPA, on a daily basis, over a period of 18 days commencing on the day of pairing with stud males (day 0). On day 77, their male offspring of 8 weeks were immunized with hen egg lysozyme (HEL). Three weeks later, anti-HEL immunoglobulin G (IgG) in sera, and proliferative responses of spleen cells to the antigen, were measured. Anti-HEL IgG2a and interferon-gamma (IFN-gamma), secreted from splenic lymphocytes, were measured as indicators of T helper 1 (Th1) immune responses, while anti-HEL IgG1 and interleukin-4 (IL-4) were measured as indicators of Th2 responses. The results showed that fetal exposure to BPA was followed by significant increases in anti-HEL IgG as well as antigen-specific cell proliferation. Both Th1 responses (including anti-HEL IgG2a and IFN-gamma production) and Th2 responses (including anti-HEL IgG1 and IL-4 production) were augmented by prenatal exposure to BPA, although the augmentation of Th1 responses appeared to be greater than that of Th2 responses. Two-colour flow cytometric analysis showed that mice exposed prenatally to BPA had 29% and 100% more splenic CD3(+) CD4(+) and CD3(+) CD8(+) cells, respectively, than control animals. Similar results were obtained from females whose mothers had consumed BPA during pregnancy. These results suggest that prenatal exposure to BPA may result in the up-regulation of immune responses, especially Th1 responses, in adulthood.