Porcine-Stimulated Human Tr1 Cells Showed Enhanced Suppression in Xenoantigen Stimulation Response.

Type 1 regulatory T (Tr1) cells play a fundamental role in maintaining and inducing immune tolerance. Our preliminary study demonstrated that an interleukin- (IL-) 10-mediated pathway is a possible regulatory mechanism underlying the xenoantigen-specific human Treg enhanced suppressive capacity. Here, we developed a feasible protocol for expanding IL-10-induced xenoantigen-specific human Tr1 cells in vitro which would be more efficient in transplantation immunotherapy efficiency. In this study, xenoantigen-specific Tr1 cells are generated from human naive CD4+ T cells expanded for two subsequent xenoantigen-stimulation cycles with recombinant human IL-10. The phenotype and suppressive capacity of xenoantigen-stimulated Tr1 cells are assessed, and the mechanism of their suppression is studied. Tr1 cells can be induced by porcine xenoantigen stimulation combined with IL-10, IL-2, and IL-15, displaying an increased expression of CD49b, CTLA-4, and LAG-3 without expressing Foxp3 which also showed an effector memory Treg phenotype and expressed high levels of CD39. After xenoantigen stimulation, the IL-10 and IL-5 gene expression in Tr1 cells increased, secreting more IL-10, and xenoantigen-stimulated Tr1 cells changed their T cell receptor (TCR) Vß repertoire, increasing the expression of TCR Vß2, TCR Vß9, and TCR Vß13. In a pig to human mixed lymphocyte reaction (MLR), xenoantigen-stimulated Tr1 cells displayed enhanced suppressive capacity via CD39 in a dose-dependent manner. Moreover, IL-5 could affect the proliferation of xenoantigen-specific Tr1 cells, but not their phenotypes' expression. This study provides a theory and feasible method for immune tolerance induction in clinical xenotransplantation.

Construction and Application of Newcastle Disease Virus-Based Vector Vaccines.

Paramyxoviruses are able to stably express a wide-variety of heterologous antigens at relatively high levels in various species and are consequently considered as potent gene delivery vehicles. A single vaccination is frequently sufficient for the induction of robust humoral and cellular immune responses. Here we provide detailed methods for the construction and application of Newcastle disease virus (NDV)-based vector vaccines. The in silico design and in vitro rescue as well as the in vivo evaluation of NDV based vectors are described in this chapter.

Development of a Recombinant Xenogeneic Tumor Necrosis Factor Alpha Protein Vaccine To Protect Mice from Experimental Colitis.

Previous studies have highlighted the efficacy of tumor necrosis factor alpha (TNF-α) inhibitors, including monoclonal antibodies and soluble receptors, in the treatment and management of intestinal bowel disease (IBD). However, because of the immunogenicity of xenogeneic TNF-α inhibitors, antidrug antibodies (ADAs) can be triggered after repeated administration. An alternative way to target TNF-α is active immunization to elicit the production of high titers of neutralizing antibodies. In this study, we prepared a xenogeneic TNF-α protein vaccine and studied the protective effects in experimental colitis models. The xenogeneic TNF-α protein vaccine could overcome self-tolerance and induce TNF-α-specific neutralizing antibody. Moreover, the xenogeneic TNF-α protein vaccine could protect mice from acute and chronic colitis induced by dextran sodium sulfate (DSS). One possible explanation for this protective effect is the production of TNF-α-specific neutralizing antibody, which absorbed the biological activity of mouse TNF-α (mTNF-α) and failed to induce T lymphocyte apoptosis. In summary, use of the xenogeneic TNF-α protein vaccine may be a potent therapeutic strategy for IBD.

Recombinant lactic acid bacteria as delivery vectors of heterologous antigens: the future of vaccination?

UNLABELLED: Lactic acid bacteria (LABs) are good candidates for the development of new oral vaccines and are attractive alternatives to attenuated pathogens. This review focuses on the use of wild-type and recombinant lactococci and lactobacilli with emphasis on their molecular design, immunomodulation and treatment of bacterial infections. The majority of studies related to recombinant LABs have focused on Lactococcus lactis, however, molecular tools have been successfully used for Lactobacillus spp. RESEARCH: Recombinant lactobacilli and lactococci have several health benefits, such as immunomodulation, restoration of the microbiota, synthesis of antimicrobial substances and inhibition of virulence factors. In addition, protective immune responses that are well tolerated are induced by the expression of heterologous antigens from recombinant probiotics.

Xenogeneic therapeutic cancer vaccines as breakers of immune tolerance for clinical application: to use or not to use?

Accumulation of firm evidence that clinically apparent cancer develops only when malignant cells manage to escape immunosurveillance led to the introduction of tumor immunotherapy strategies aiming to reprogramm the cancer-dysbalanced antitumor immunity and restore its capacity to control tumor growth. There are several immunotherapeutical strategies, among which specific active immunotherapy or therapeutic cancer vaccination is one of the most promising. It targets dendritic cells (DCs) which have a unique ability of inducing naive and central memory T cell-mediated immune response in the most efficient manner. DCs can be therapeutically targeted either in vivo/in situ or by ex vivo manipulations followed by their re-injection back into the same patient. The majority of current DC targeting strategies are based on autologous or allogeneic tumor-associated antigens (TAAs) which possess various degrees of inherent tolerogenic potential. Therefore still limited efficacy of various tumor immunotherapy approaches may be attributed, among various other mechanisms, to the insufficient immunogenicity of self-protein-derived TAAs. Based on such an idea, the use of homologous xenogeneic antigens, derived from different species was suggested to overcome the natural immune tolerance to self TAAs. Xenoantigens are supposed to differ sufficiently from self antigens to a degree that renders them immunogenic, but at the same time preserves an optimal homology range with self proteins still allowing xenoantigens to induce cross-reactive T cells. Here we discuss the concept of xenogeneic vaccination, describe the cons and pros of autologous/allogeneic versus xenogeneic therapeutic cancer vaccines, present the results of various pre-clinical and several clinical studies and highlight the future perspectives of integrating xenovaccination into rapidly developing tumor immunotherapy regimens.

[Therapeutic intervention alternatives in cancer, using attenuated live bacterial vectors: Salmonella enterica as a carrier of heterologous molecules]. / Alternativas de intervención terapéutica en cáncer usando vectores bacterianos vivos atenuados: Salmonella enterica como acarreador de moléculas heterólogas.

Salmonella enterica is a facultative anaerobic bacteria, whose ability to colonize antigen-presenting cells (APCs) such as dendritic cells and macrophages, has allowed its successful use as an alive, attenuated bacterial vector for vaccination. Salmonella enterica elicits efficient cellular, humoral and mucosal immune responses, against heterologous antigens including viruses, parasites, other bacterial species and tumor-associated antigens, since it is capable of delivering these antigens to cells of the immune system. The extracellular expression of heterologous antigens on the surface of Salmonella enterica via its type I, III and V secretion systems, and their delivery into infected cells is essential for its stimulation of immune responses against these antigens. Moreover, Salmonella enterica is a promising therapeutic agent against cancer, as demonstrated by reports of pre-clinical and clinical studies indicating that, after systemic administration, Salmonella enterica preferentially localizes in solid tumors and metastases as compared to normal tissues. In this review, we focus on novel prophylactic and therapeutic anti-cancer approaches using Salmonella enterica as a delivery system of heterologous molecules with the aim of inhibiting tumor growth.

[The condition of lipid peroxidation in mice and the effect of fullerene C60 during immune response].

The aim of this study was to assess the influence of fullerene C60 on lipid peroxidation (POL) and antioxidant protection during the induction of the immune response to heteroantigen. Balb/c mice were immunized intraperitoneal (i.p.) with sheep erythrocytes for the primary immunization. Water dispersion of fullerene C60 was injected i.p. once at the dose 50 ng to mice on first, third and sixth days after immunization. During immune response, the increment ofmalonic dialdehide (MDA) was enhanced in liver, kidneys and heart tissues. Fullerene C60 induced POL during the latent phase of immune response, but inhibited this process during progression of immune response. Activities of superoxide dismutase (SOD) and catalase in liver and spleen tissues were induced after injection of fullerene C60 to intact mice. After immunization, high level of activity of antioxidant enzymes and low level of organs mass factor were determined. Injection of fullerene C60 reduced the activities of SOD and catalase in spleen tissues. The results of our study indicate that fullerene C60 can display positive effect on POL processes and antioxidant enzymes activity which is probably due to membrane's stabilization action or the ability of fullerene C60 to bind free radicals independently.

Ex-vivo expanded baboon CD4+ CD25 Hi Treg cells suppress baboon anti-pig T and B cell immune response.

BACKGROUND: CD4(+) CD25(+) FoxP3(+) regulatory T (Treg) cells play an important role in regulating immune responses. A very small number of Treg cells are present in peripheral blood and lymphoid organs, but due to their ability to suppress the immune response, they have a high potential for immunotherapy in clinics. Successful ex-vivo expansion of naturally occurring CD4(+) CD25(+) T cells has been achieved after TCR stimulation in the presence of T cell growth factors. In this study, we evaluated the role of these Treg cells in suppressing proliferative response of baboon T and B cells to pig xenoantigens. METHODS: Naturally occurring baboon CD4(+) CD25(+) regulatory T cells (nTreg) were sorted from peripheral blood and expanded in the presence of either anti-CD3/CD28 beads or irradiated pig peripheral blood mononuclear cells with IL-2. Treg cells were also enriched directly from CD4(+) T cells cultured in the presence of rapamycin (0.1-10 nm). Mixed lymphocyte culture and polyclonal B cell stimulation with ex-vivo Treg cells were performed to assess the function of ex-vivo expanded Treg cells. RESULTS: The nTreg cells were expanded to more than 200-fold in 4 weeks and retained all the nTreg cell phenotypic characteristics, including high levels of FoxP3 expression. 2-fold increase in enrichment of CD4(+) CD25(+) FoxP3(+) Treg cells from CD4(+) cells was observed with rapamycin compared to cultures without rapamycin. The ex-vivo expanded Treg cells obtained from both methods were able to suppress the baboon anti-porcine xenogeneic T and B cell immune response in-vitro efficiently (more than 90% suppression at 1:1 ratio of T regulatory cells: T effector cells), and their suppression potential was retained even at 1:256 ratio. However, freshly isolated nTreg cells had only 70% suppression at 1:1 ratio, and their suppressive ability was reduced to ≤ 50% at 1:16 ratio. Furthermore, we have found that ex-vivo expanded Treg can also suppress the proliferation of B cells after polyclonal stimulation. Forty to 50 percent reduction in B cell proliferation was observed when ex-vivo expanded Treg cells were added to the culture at a 1:1 ratio. The addition of CD4(+) CD25(Neg) cells however induced vigorous proliferation. CONCLUSION: Ex-vivo expanded CD4(+) CD25(+) FoxP3(+) Treg cells can be used to efficiently suppress xenogeneic immune responses by inhibiting T and B cell proliferation. These ex-vivo expanded Treg cells may also be used with other immunosuppressive agents to overcome xenograft rejection in preclinical xenotransplantation models.

Heterologous prime-boost-boost immunisation of Chinese cynomolgus macaques using DNA and recombinant poxvirus vectors expressing HIV-1 virus-like particles.

BACKGROUND: There is renewed interest in the development of poxvirus vector-based HIV vaccines due to the protective effect observed with repeated recombinant canarypox priming with gp120 boosting in the recent Thai placebo-controlled trial. This study sought to investigate whether a heterologous prime-boost-boost vaccine regimen in Chinese cynomolgus macaques with a DNA vaccine and recombinant poxviral vectors expressing HIV virus-like particles bearing envelopes derived from the most prevalent clades circulating in sub-Saharan Africa, focused the antibody response to shared neutralising epitopes. METHODS: Three Chinese cynomolgus macaques were immunised via intramuscular injections using a regimen composed of a prime with two DNA vaccines expressing clade A Env/clade B Gag followed by boosting with recombinant fowlpox virus expressing HIV-1 clade D Gag, Env and cholera toxin B subunit followed by the final boost with recombinant modified vaccinia virus Ankara expressing HIV-1 clade C Env, Gag and human complement protein C3d. We measured the macaque serum antibody responses by ELISA, enumerated T cell responses by IFN-γ ELISpot and assessed seroneutralisation of HIV-1 using the TZM-bl ß-galactosidase assay with primary isolates of HIV-1. RESULTS: This study shows that large and complex synthetic DNA sequences can be successfully cloned in a single step into two poxvirus vectors: MVA and FPV and the recombinant poxviruses could be grown to high titres. The vaccine candidates showed appropriate expression of recombinant proteins with the formation of authentic HIV virus-like particles seen on transmission electron microscopy. In addition the b12 epitope was shown to be held in common by the vaccine candidates using confocal immunofluorescent microscopy. The vaccine candidates were safely administered to Chinese cynomolgus macaques which elicited modest T cell responses at the end of the study but only one out of the three macaques elicited an HIV-specific antibody response. However, the antibodies did not neutralise primary isolates of HIV-1 or the V3-sensitive isolate SF162 using the TZM-bl ß-galactosidase assay. CONCLUSIONS: MVA and FP9 are ideal replication-deficient viral vectors for HIV-1 vaccines due to their excellent safety profile for use in humans. This study shows this novel prime-boost-boost regimen was poorly immunogenic in Chinese cynomolgus macaques.

Early detection of immunization: a study based on an animal model using 1H nuclear magnetic resonance spectroscopy.

Vaccines require a period of at least three months for clinical trials, hence a method that can identify elicitation of immune response a few days after the first dose is a necessity. Evolutionary variable selections are modeling approaches for proper manipulation of available data which were used to set up an animal model for classification of time dependent 1HNMR metabolomic profiles and pattern recognition of fluctuations of metabolites in two groups of male rabbits. One group of rabbits was immunized with human red blood cells and the other used as control. Blood was obtained every 48 h from each rabbit for a period of six weeks and the serum monitored for antibodies and metabolites by 1HNMR spectra. Evaluation of data was carried out using orthogonal signal correction followed by principal component analysis and partial least square. A neural network was also set up to predict immunization profiles. A distinct separation in patterns of significant metabolites was obtained between the two groups, just a few days after the first and the second dose. These metabolites were used as targets of neural networks where each sample was used as test, validation and training and their quantitative influence predicted by regression. This model could be used for prediction of immunization in rabbits a few days after the first dose with 96% accuracy. Similar animals and human vaccine trials would assist greatly in reaching early conclusions in advance of the usual two month immunization schedule; resulting in an appreciable saving of cost and time.

Immune suppression produced by intrathymic inoculation with xenogeneic antigen and whole-body γ-irradiation in a pig-to-monkey heart transplantation model.

OBJECTIVE: To validate the use of pig-to-monkey transplantation as an experimental animal model for study of the induction of immune suppression of cardiac xenografts via intrathymic inoculation with xenogeneic spleen cells and whole-body γ-irradiation. MATERIALS AND METHODS: Donors (Meishan pigs) and recipients (Rhesus monkeys) were randomized into 3 groups. Group 2 was the control group; group 2 received whole-body irradiation, and group 3 received whole-body irradiation and intrathymic inoculation. In each group, survival time of heterotopic heart xenografts in monkeys were observed. A mixed lymphocyte reaction assay with (3)H thymidine was used to examine changes in lymphocyte function in all groups on the day of transplantation. In each group, recipient serum was harvested for analysis of IL-2 and IL-10 concentrations. RESULTS: Mean (SD) survival time of donor hearts in group 3 was significantly longer (91.1 [22.8] hours) than in group 1 (36.6 [5.8] hours) (P < .01). The results of mixed lymphocyte reaction assay demonstrated a significant reduction in the stimulation index in group 3 compared with groups 1 and 2 (P < .01) when recipient splenocytes responded to the donor stimulator. The IL-2 concentration in recipients in all groups was much higher during rejection of xenografts than before transplantation (P < .01). CONCLUSION: These results suggest that pretreatment with intrathymic inoculation or whole-body irradiation induces T-cell immunosuppression. The IL-2 concentration is closely correlated with xenograft rejection.

Immunologic response to xenogeneic gp100 DNA in melanoma patients: comparison of particle-mediated epidermal delivery with intramuscular injection.

PURPOSE: Prior studies show that i.m. injection of xenogeneic orthologues of melanosomal antigens (tyrosinase, gp100) induces CD8(+) T-cell responses to the syngeneic protein. To further define the optimal vaccination strategy, we conducted a pilot clinical trial comparing i.m. injection with particle-mediated epidermal delivery (PMED). EXPERIMENTAL DESIGN: Human leukocyte antigen (HLA)-A*0201(+) disease-free melanoma patients were randomized to the PMED or i.m. arm, receiving eight vaccinations over 4 months. Patients received 4 microg or 2,000 microg per injection, respectively, of mouse gp100 DNA. Peripheral blood mononuclear cells were collected, cultured with gp100 peptides, and analyzed by tetramer and intracellular cytokine staining for responses to HLA-A*0201-restricted gp100 epitopes [gp100(209-217) (ITDQVPFSV) and gp100(280-288) (YLEPGPVTA)]. RESULTS: Twenty-seven patients with stage IIB-IV melanoma were analyzable for immune response. The only common toxicity was grade 1 injection site reaction in nine patients with no intergroup difference, and one dose-limiting toxicity of acute hypersensitivity occurred in a PMED patient with undiagnosed gold allergy. Four of 27 patients produced gp100 tetramer(+)CD8(+) T cells, all carrying the CCR7(lo)CD45RA(lo) effector-memory phenotype. Five of 27 patients generated IFN-gamma(+)CD8(+) T cells, one who was also tetramer-positive. Overall, vaccination induced a response in 30% of patients, which was not significantly associated with study arm or clinical outcome. However, the PMED group showed a trend toward increased IFN-gamma(+)CD8(+) T-cell generation (P = 0.07). CONCLUSION: A comparable efficacy and safety profile was shown between the i.m. and PMED arms, despite a significantly decreased dose of DNA used for PMED injection.

Steering orally induced deviation of xenogeneic immunity with exogenous cytokines in rats fed porcine proteins.

Immune response to xenoantigens is a barrier to xenotransplantation. The objective of this study was to determine if rat cell-mediated immunity (CMI) and antibody (Ab) to discordant porcine xenoantigens could be suppressed by oral administration of porcine protein with adjunct systemic cytokine therapy. Based on principles of oral tolerance, it was hypothesized that: a. Feeding proteins from porcine blood mononuclear cells (PBMC) would induce a type 2 response, inhibiting CMI and type 1 Ab (associated with xenograft rejection) but increasing the amount of type 2 Ab. b. IL-4, a type 2 cytokine would exaggerate type 2 bias, enhancing immune deviation. c. IFN-gamma, a type 1 cytokine was expected to down-regulate overall Ab production, but increase CMI and type 1 Ab. DA rats fed porcine proteins with or without subcutaneous and intraperitoneal injections of IFN-gamma or IL-4 received PBMC by subcutaneous injection. Dermal delayed-type hypersensitivity to PBMC was the indicator of CMI. The IgG Ab to porcine proteins was quantified and type 2 (IgG(1)+IgG(2a)) to type 1 (IgG(2b)) Ab ratios indicated IR bias. Unfed, PBMC-challenged controls had a type 1 response bias but feeding PBMC induced a type 2 bias that suppressed CMI and type 1 Ab and increased type 2 Ab but not total IgG Ab. Contrary to the T(H)1/T(H)2 paradigm, IL-4 decreased oral-induced type 2 IR deviation and did not provide significant benefits relative to feeding alone. Treatment with IFN-gamma prevented a switch to type 2 IR but feeding-induced suppression of CMI was not significantly compromised. The IFN-gamma potently suppressed Ab, including, surprisingly, type 1 IgG isotypes. Contrary to the hypothesis tested, feeding Ag in combination with systemic delivery of recombinant IFN-gamma apparently created an immunoregulatory environment most favorable to xenograft survival.

Xenogeneic immunization with human tyrosine hydroxylase DNA vaccines suppresses growth of established neuroblastoma.

Neuroblastoma (NB) is a challenging malignancy of the sympathetic nervous tissue characterized by a very poor prognosis. One important marker for NB is the expression of tyrosine hydroxylase (TH), the first-step enzyme of catecholamine biosynthesis. We could show stable and high TH gene expression in 67 NB samples independent of the clinical stage. Based on this observation, we addressed the question of whether xenogeneic TH DNA vaccination is effective in inducing an anti-NB immune response. For this purpose, we generated three DNA vaccines based on pCMV-F3Ub and pBUD-CE4.1 plasmids encoding for human (h)THcDNA (A), hTH minigene (B), and hTHcDNA in combination with the proinflammatory cytokine interleukin 12 (C), and tested prophylactic and therapeutic efficacy to suppress primary tumor growth and spontaneous metastasis. Here we report that xenogeneic TH DNA vaccination was effective in eradicating established primary tumors and inhibiting metastasis. Interestingly, this effect could not be enhanced by adding the Th1 cytokine interleukin 12. However, increased IFN-gamma production and NB cytotoxicity of effector cells harvested from vaccinated mice suggested the participation of tumor-specific CTLs in the immune response. The depletion of CD8(+)T cells completely abrogated the hTH vaccine-mediated anti-NB immune response. Furthermore, rechallenging of surviving mice resulted in reduced primary tumor growth, indicating the induction of a memory immune response. In conclusion, xenogeneic immunization with TH-derived DNA vaccines is effective against NB, and may open a new venue for a novel and effective immunotherapeutic strategy against this challenging childhood tumor.

A new strategy to induce effective antitumour response in vitro and in vivo.

To induce Her2-specific cell immune response, we used xenogeneic antigen rat neu L2-S2 domains as the vaccine antigen. The antigenic protein was engineered as a chimeric protein with human IgG1 Fc region (neu-Fc). Neu-Fc could stimulate the cell proliferation in mixed lymphocyte reaction effectively. Simultaneous neu-Fc and IFN-gamma stimulation dramatically elevated IL-12 secretion and reduced IL-10 production in PBMC. To further augment the activating effects on Th1-type response, Bacille Calmette-Guerin (BCG) was utilized as a non-specific stimulus. Neu-Fc, IFN-gamma and BCG costimulation exhibited the most conspicuous effects on the reversal of the Th1-type inhibitory effects by MCF-7 cell supernatant compared with neu-Fc alone or IFN-gamma and BCG costimulation. The lytic activity of effector cells to Her2 overexpressing cells was greatly promoted by neu-Fc, IFN-gamma and BCG stimulation simultaneously. Neu-Fc led to considerable retardation in EMT6/Her2 tumour growth in Balb/c mice. IFN-gamma and BCG efficiently enhanced the antitumour activity. A large amount of inflammatory cells were found to be accumulated in the tumour tissues or surrounded tumours in mice treated with neu-Fc, IFN-gamma and BCG but no inflammatory cell infiltration was observed in control tumours, indicating that the strategy is potent enough to support the initiation and propagation of tumour-specific immune response in an established tumour and generate a proinflammatory environment.

Deviation of xenogeneic immune response and bystander suppression in rats fed porcine blood mononuclear cells.

Reducing or deviating xenogeneic immune response prior to xenotransplantation may enhance the efficacy of conventional immunosuppressive therapies in prolonging xenograft survival. The potential to suppress or steer immune responses by oral administration of xenoantigens was evaluated. Based on knowledge of oral tolerance, hypotheses tested were that feeding xenoantigens would inhibit cell-mediated immune response (CMIR) and production of antibodies associated with graft rejection and induce bystander suppression. DA and LEW rats, high and low responders to xenoantigens, respectively, were fed dead porcine blood mononuclear cells (PBMC) and subsequently received live PBMC and hen egg-white lysozyme (HEWL, a third-party antigen) by subcutaneous injection. Delayed-type hypersensitivity (DTH) to PBMC was an indicator of CMIR. Quantification of T(H)1 (IgG(2b)) and T(H)2 (IgG(1))-associated antibodies and their ratio measured magnitude and bias of the antibody-mediated response to PBMC and HEWL. Feeding PBMC reduced IgG(2b) antibody production by 90% (DA) and 71% (LEW) and increased IgG(1) antibodies by 116% in DA but not LEW rats (p

Double-negative T cells, activated by xenoantigen, lyse autologous B and T cells using a perforin/granzyme-dependent, Fas-Fas ligand-independent pathway.

The ability to control the response of B cells is of particular interest in xenotransplantation as Ab-mediated hyperacute and acute xenograft rejection are major obstacles in achieving long-term graft survival. Regulatory T cells have been proven to play a very important role in the regulation of immune responses to self or non-self Ags. Previous studies have shown that TCRalphabeta+CD3+CD4-CD8- (double-negative (DN)) T cells possess an immune regulatory function, capable of controlling antidonor T cell responses in allo- and xenotransplantation through Fas-Fas ligand interaction. In this study, we investigated the possibility that xenoreactive DNT cells suppress B cells. We found that DNT cells generated from wild-type C57BL/6 mice expressed B220 and CD25 after rat Ag stimulation. These xenoreactive B220+CD25+ DNT cells lysed activated, but not naive, B and T cells. This killing, which took place through cell-cell contact, required participation of adhesion molecules. Our results indicate that Fas ligand, TGF-beta, TNF-alpha, and TCR-MHC recognition was not involved in DNT cell-mediated syngenic cell killing, but instead this killing was mediated by perforin and granzymes. The xenoreactive DNT cells expressed high levels of granzymes in comparison to allo- or xenoreactive CD8+ T cells. Adoptive transfer of DNT cells in combination with early immune suppression by immunosuppressive analog of 15-deoxyspergualin, LF15-0195, significantly prolonged rat heart graft survival to 62.1 +/- 13.9 days in mice recipients. In conclusion, this study suggests that xenoreactive DNT cells can control B and T cell responses in perforin/granzyme-dependent mechanisms. DNT cells may be valuable in controlling B and T cell responses in xenotransplantation.

Protective immunization against "Candidatus Helicobacter suis" with heterologous antigens of H. pylori and H. felis.

"Helicobacter (H.) heilmannii" type 1 colonizes the human stomach. It has been shown to be identical to "Candidatus H. suis", a Helicobacter species colonizing the stomach of more than 60% of slaughter pigs. This bacterium is, until now, not isolated in vitro. The effect of vaccination on "Candidatus H. suis" infection was studied in a mouse model. Mice were vaccinated intranasally or subcutaneously with whole bacterial cell lysate of Helicobacter pylori or Helicobacter felis and subsequently challenge infected with "Candidatus H. suis". Intranasal and subcutaneous immunisation caused a decrease in faecal excretion of "Candidatus H. suis" DNA. Urease tests on stomach tissue samples at 16 weeks after challenge infection were negative in all H. felis intranasally immunized animals and in the majority of the animals of the other immunisation groups. Since PCR on stomach tissue samples at 16 weeks after challenge infection could still detect "Candidatus H. suis DNA" in all immunisation-challenge groups, complete clearance of challenge bacteria was not achieved.

Induction of immunological tolerance in chickens inoculated with xenogeneic antigens at an early stage of embryonic development.

To induce immunotolerance, laboratory chicken embryos were inoculated with casein via embryonic blood vessel microinjection or with bovine serum albumin (BSA) by either embryonic blood vessel or yolk sac injection. All hatched chickens were challenged with the same protein four times at 10-day intervals beginning at 3 weeks old. Serum anti-casein and anti-BSA antibodies were analyzed by enzyme-linked immunosorbent assay (ELISA). Significantly reduced serum specific antibody, demonstrating immunotolerance, was observed in 53.8% of chickens exposed to casein in embryo by microinjection at 65-70 h of incubation; and in 62.5% (inoculating at 65-67 h of incubation) and 33.33% (inoculating at 67-70 h of incubation) of chickens exposed to BSA. Tolerant chickens presented in those groups inoculated with BSA at 5-7 days of embryogenesis by in ovo injection. The results showed tolerance could be induced by injecting xenogeneic protein into early developing embryo by both inoculation methods.

Direct antigen presentation by a xenograft induces immunity independently of secondary lymphoid organs.

The location of immune activation is controversial during acute allograft rejection and unknown in xenotransplantation. To determine where immune activation to a xenograft occurs, we examined whether splenectomized alymphoplastic mice that possess no secondary lymphoid organs can reject porcine skin xenografts. Our results show that these mice rejected their xenografts, in a T cell-dependent fashion, at the same tempo as wild-type recipients, demonstrating that xenograft rejection is not critically dependent on secondary lymphoid organs. Furthermore, we provide evidence that immune activation in the bone marrow did not take place during xenograft rejection. Importantly, immunity to xenoantigens was only induced after xenotransplantation and not by immunization with porcine spleen cells, as xenografted mutant mice developed an effector response, whereas mutant mice immunized by porcine spleen cells via i.p. injection failed to do so. Moreover, we provide evidence that antixenograft immunity occurred via direct and indirect Ag presentation, as recipient T cells could be stimulated by either donor spleen cells or recipient APCs. Thus, our data provide evidence that direct and indirect Ag presentation by a xenograft induces immunity in the absence of secondary lymphoid organs. These results have important implications for developing relevant xenotransplantation protocols.