Induction Of Cell-mediated Immune Responses Research Articles

Comparative study of the effects of Montanide™ ISA 763A VG and ISA 763B VG adjuvants on the immune response against Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus)

Streptococcus agalactiae is regarded as a major bacterial pathogen of farmed fish, with outbreaks in Nile tilapia causing significant losses. Vaccination is considered the most suitable method for disease control in aquaculture, with the potential to prevent such outbreaks if highly efficacious vaccines are available for use. Several vaccines have been produced to protect against S. agalactiae infection in tilapia, including inactivated vaccines, live attenuated vaccines, and subunit vaccines, with variable levels of protection seen. Two commercial adjuvants, Montanide™ ISA 763A VG and ISA 763B VG, have been developed recently and designed to improve the safety and efficacy of oil-based emulsions delivered by intraperitoneal injection. In particular, their mode of action may help identify and stimulate particular immunological pathways linked to the intended protective response, which is an important tool for future vaccine development. Therefore, this study aimed to characterize the potential of two adjuvanted-bacterial vaccines against S. agalactiae (SAIV) comparatively, to determine their usefulness for improving protection and to analyse the immune mechanisms involved. Nile tilapia were divided into four groups: 1) fish injected with PBS as a control, 2) fish injected with the SAIV alone, 3) fish injected with the SAIV + Montanide™ ISA 763A VG, and 4) fish injected with the SAIV + Montanide™ ISA 763B VG. Following immunization selected innate immune parameters were analysed, including serum lysozyme, myeloperoxidase, and bactericidal activity, with significantly increased levels seen after immunization. Cytokines associated with innate and adaptive immunity were also studied, with expression levels of several genes showing significant up-regulation, indicating good induction of cell-mediated immune responses. Additionally, the specific IgM antibody response against S. agalactiae was determined and found to be significantly induced post-vaccination, with higher levels seen in the presence of the adjuvants. In comparison to the protection seen with the unadjuvanted vaccine (61.29% RPS), both Montanide™ ISA 763A VG and Montanide™ ISA 763B VG improved the RPS, to 77.42% and 74.19% respectively. In conclusion, Montanide™ ISA 763A VG and Montanide™ ISA 763B VG have shown potential for use as adjuvants for fish vaccines against streptococcosis, as evidenced by the enhanced immunoprotection seen when given in combination with the SAIV vaccine employed in this study.

Lumpfish (Cyclopterus lumpus) Is Susceptible to Renibacterium salmoninarum Infection and Induces Cell-Mediated Immunity in the Chronic Stage.

Renibacterium salmoninarum is a Gram-positive, intracellular pathogen that causes Bacterial Kidney Disease (BKD) in several fish species in freshwater and seawater. Lumpfish (Cyclopterus lumpus) is utilized as a cleaner fish to biocontrol sea lice infestation in Atlantic salmon (Salmo salar) farms. Atlantic salmon is susceptible to R. salmoninarum, and it can transfer the infection to other fish species. Although BKD outbreaks have not been reported in lumpfish, its susceptibility and immune response to R. salmoninarum is unknown. In this study, we evaluated the susceptibility and immune response of lumpfish to R. salmoninarum infection. Groups of lumpfish were intraperitoneally (i.p.) injected with either R. salmoninarum (1×107, 1×108, or 1×109 cells dose-1) or PBS (control). R. salmoninarum infection kinetics and mortality were followed for 98 days post-infection (dpi). Transcript expression levels of 33 immune-relevant genes were measured in head kidney (n = 6) of fish infected with 1×109 cells/dose and compared to the control at 28 and 98 dpi. Infected lumpfish displayed characteristic clinical signs of BKD. Lumpfish infected with high, medium, and low doses had a survival rate of 65%, 93%, and 95%, respectively. Mortality in the high-dose infected group stabilized after 50 dpi, but R. salmoninarum persisted in the fish tissues until 98 dpi. Cytokines (il1β, il8a, il8b), pattern recognition receptors (tlr5a), interferon-induced effectors (rsad2, mxa, mxb, mxc), and iron regulation (hamp) and acute phase reactant (saa5) related genes were up-regulated at 28 dpi. In contrast, cell-mediated adaptive immunity-related genes (cd4a, cd4b, ly6g6f, cd8a, cd74) were down-regulated at 28 dpi, revealing the immune suppressive nature of R. salmoninarum. However, significant upregulation of cd74 at 98 dpi suggests induction of cell-mediated immune response. This study showed that R. salmoninarum infected lumpfish in a similar fashion to salmonid fish species and caused a chronic infection, enhancing cell-mediated adaptive immune response.

Chemical and Immunological Characteristics of Aluminum-Based, Oil-Water Emulsion, and Bacterial-Origin Adjuvants.

Adjuvants are a diverse family of substances whose main objective is to increase the strength, quality, and duration of the immune response caused by vaccines. The most commonly used adjuvants are aluminum-based, oil-water emulsion, and bacterial-origin adjuvants. In this paper, we will discuss how the election of adjuvants is important for the adjuvant-mediated induction of immunity for different types of vaccines. Aluminum-based adjuvants are the most commonly used, the safest, and have the best efficacy, due to the triggering of a strong humoral response, albeit generating a weak induction of cell-mediated immune response. Freund's adjuvant is the most widely used oil-water emulsion adjuvant in animal trials; it stimulates inflammation and causes aggregation and precipitation of soluble protein antigens that facilitate the uptake by antigen-presenting cells (APCs). Adjuvants of bacterial origin, such as flagellin, E. coli membranes, and monophosphoryl lipid A (MLA), are known to potentiate immune responses, but their safety and risks are the main concern of their clinical use. This minireview summarizes the mechanisms that classic and novel adjuvants produce to stimulate immune responses.

Micellar nano-carriers for the delivery of STAT3 dimerization inhibitors to melanoma.

The objective of this research was to develop polymeric micellar formulations of inhibitors of signal transducer and activator of transcription 3 (STAT3) dimerization, i.e., S3I-1757 and S3I-201, and evaluate the activity of successful formulations in B16-F10 melanoma, a STAT3 hyperactive cancer model, in vitro and in vivo. STAT3 inhibitory agents were encapsulated in methoxy poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO114-b-PCL22) and methoxy poly(ethylene oxide)-b-poly(α-benzyl carboxylate-ε-caprolactone) (PEO114-b-PBCL20) micelles using co-solvent evaporation. Polymeric micelles of S3I-1757 showed high encapsulation efficiency (>88%), slow release profile (<32% release in 24h) under physiological conditions, and a desirable average diameter for tumor targeting (33-54 nm). The same formulations showed low encapsulation efficiencies and rapid drug release for S3I-201. Further studies evidenced the delivery of functional S3I-1757 by polymeric micelles to B16-F10 melanoma cells, leading to a dose-dependent inhibition of cell growth and vascular endothelial growth factor (VEGF) production comparable with that of free drug. Encapsulation of S3I-1757 in polymeric micelles significantly reduced its cytotoxicity in normal bone marrow-derived dendritic cells (DCs). Micelles of S3I-1757 were able to significantly improve the function of B16-F10 tumor-exposed immunosuppressed DCs in the production of IL-12, an indication for functionality in the induction of cell-mediated immune response. In a B16-F10 melanoma mouse model, S3I-1757 micelles inhibited tumor growth and enhanced the survival of tumor-bearing mice more than free S3I-1757. Our findings show that both PCL- and PBCL-based polymeric micelles have potential for the solubilization and delivery of S3I-1757, a potent STAT3 inhibitory agent.

Induction of cell-mediated immune responses after vaccination with inactivated oil-emulsion Nocardia seriolae in amberjack Seriola dumerili

Induction of cell-mediated immune responses after vaccination with inactivated oil-emulsion Nocardia seriolae in amberjack Seriola dumerili

Vaginal memory T cells induced by intranasal vaccination are critical for protective T cell recruitment and prevention of genital HSV-2 disease.

Protective immunity against genital pathogens causing chronic infections, such as herpes simplex virus 2 (HSV-2) or human immunodeficiency virus, requires the induction of cell-mediated immune responses locally in the genital tract. Intranasal immunization with a thymidine kinase-deficient (TK(-)) mutant of HSV-2 effectively induces HSV-2-specific gamma interferon (IFN-γ)-secreting memory T cell production and protective immunity against intravaginal challenge with wild-type HSV-2. However, the precise mechanism by which intranasal immunization induces protective immunity in the distant genital mucosa more effectively than does systemic immunization is unknown. Here, we showed that intranasal immunization with live HSV-2 TK(-) induced the production of effector T cells and their migration to, and retention in, the vaginal mucosa, whereas systemic vaccination barely established a local effector T cell pool, even when it induced the production of circulating memory T cells in the systemic compartment. The long-lasting HSV-2-specific local effector T cells induced by intranasal vaccination provided superior protection against intravaginal wild-type HSV-2 challenge by starting viral clearance at the entry site earlier than with intraperitoneal immunization. Intranasal immunization is an effective strategy for eliciting high levels of cell-mediated protection of the genital tract by providing long-lasting antigen (Ag)-specific local effector T cells without introducing topical infection or inflammation. Intranasal (i.n.) vaccines against sexually transmitted diseases that are caused by viruses such as herpes simplex virus 2 (HSV-2) have long been in development, but no vaccine candidate is currently available. Understanding the cellular mechanisms of immune responses in a distant vaginal mucosa induced by i.n. immunization with HSV-2 will contribute to designing such a vaccine. Our study demonstrated that i.n. immunization with an attenuated strain of HSV-2 generated long-lasting IFN-γ-secreting T cells in vaginal mucosa more effectively than systemic immunization. We found that these vaginal effector memory T cells are critical for the early stage of viral clearance at natural infection sites and prevent severe vaginal inflammation and herpes encephalitis.

Induction of cell-mediated immune responses in mice by DNA vaccines that express hepatitis C virus NS3 mutants lacking serine protease and NTPase/RNA helicase activities.

Effective therapeutic vaccines against virus infection must induce sufficient levels of cell-mediated immune responses against the target viral epitopes and also must avoid concomitant risk factors, such as potential carcinogenic properties. The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) carries a variety of CD4+ and CD8+ T cell epitopes, and induces strong HCV-specific T cell responses, which are correlated with viral clearance and resolution of acute HCV infection. On the other hand, NS3 possesses serine protease and nucleoside triphosphatase (NTPase)/RNA helicase activities, which not only play important roles in viral life cycle but also concomitantly interfere with host defense mechanisms by deregulating normal cellular functions. In this study, we constructed a series of DNA vaccines that express NS3 of HCV. To avoid the potential harm of NS3, we introduced mutations to the catalytic triad of the serine protease (H57A, D81A and S139A) and the NTPase/RNA helicase domain (K210N, F444A, R461Q and W501A) to eliminate the enzymatic activities. Immunization of BALB/c mice with each of the DNA vaccine candidates (pNS3[S139A/K210N], pNS3[S139A/F444A], pNS3[S139A/R461Q] and pNS3[S139A/W501A]) that expresses an NS3 mutant lacking both serine protease and NTPase/helicase activities induced T cell immune responses to the degree comparable to that induced by the wild type NS3 and the NS3/4A complex, as demonstrated by interferon-γ production and cytotoxic T lymphocytes activities against NS3. The present study has demonstrated that plasmids expressing NS3 mutants, NS3(S139A/K210N), NS3(S139A/F444A), NS3(S139A/R461Q) and NS3(S139A/W501A), which lack both serine protease and NTPase/RNA helicase activities, would be good candidates for safe and efficient therapeutic DNA vaccines against HCV infection.

CAF01 potentiates immune responses and efficacy of an inactivated influenza vaccine in ferrets.

Trivalent inactivated vaccines (TIV) against influenza are given to 350 million people every year. Most of these are non-adjuvanted vaccines whose immunogenicity and protective efficacy are considered suboptimal. Commercially available non-adjuvanted TIV are known to elicit mainly a humoral immune response, whereas the induction of cell-mediated immune responses is negligible. Recently, a cationic liposomal adjuvant (dimethyldioctadecylammonium/trehalose 6,6′-dibehenate, CAF01) was developed. CAF01 has proven to enhance both humoral and cell-mediated immune responses to a number of different experimental vaccine candidates. In this study, we compared the immune responses in ferrets to a commercially available TIV with the responses to the same vaccine mixed with the CAF01 adjuvant. Two recently circulating H1N1 viruses were used as challenge to test the vaccine efficacy. CAF01 improved the immunogenicity of the vaccine, with increased influenza-specific IgA and IgG levels. Additionally, CAF01 promoted cellular-mediated immunity as indicated by interferon-gamma expressing lymphocytes, measured by flow cytometry. CAF01 also enhanced the protection conferred by the vaccine by reducing the viral load measured in nasal washes by RT-PCR. Finally, CAF01 allowed for dose-reduction and led to higher levels of protection compared to TIV adjuvanted with a squalene emulsion. The data obtained in this human-relevant challenge model supports the potential of CAF01 in future influenza vaccines.

Differentiation and maturation of human dendritic cells modulated by an exopolysaccharide from an anamorph of Cordyceps sinensis

Dendritic cells are central players in initiating and regulating immune responses. Effective induction of cell-mediated immune responses strongly depends on stimulating T cells by dendritic cells through antigen-presenting complex and co-stimulatory molecules. In this study, dendritic cells were differentiated from human peripheral blood mononuclear cells by using rhGM-CSF and rhIL-4. Then the effects of exopolysaccharide (EPS) from an anamorph of Cordyceps sinensis on human DCs were investigated. The results of flow cytometry showed that surface HLA-DR and co-stimulatory molecules of DCs were increased and DC's phagocytosis of FITC-dextran were significantly decreased after EPS treatment. EPS-treated DCs also displayed enhanced T cell stimulatory capacity in mixed lymphocyte reaction (MLR). ELISA and RT-PCR assay showed EPS increased IL-12 level of DCs. RT-PCR assay also showed EPS decreased VEGF mRNA level of DCs. Furthermore, Western blotting demonstrated that phosphorylated STAT3 were decreased after EPS treatment, suggesting that EPS stimulated the maturation of DCs probably by inhibiting STAT3 activation.

A DNA Vaccine-Encoded Nucleoprotein of Influenza Virus Fails To Induce Cellular Immune Responses in a Diabetic Mouse Model

Influenza virus infections cause yearly epidemics and are a major cause of lower respiratory tract illnesses in humans worldwide. Influenza virus has long been recognized to be associated with higher morbidity and mortality in diabetic patients. Vaccination is an effective tool to prevent influenza virus infection in this group of patients. Vaccines employing recombinant-DNA technologies are an alternative to inactivated virus and live attenuated virus vaccines. Internal highly conserved viral nucleoprotein (NP) can be delivered as a DNA vaccine to provide heterosubtypic immunity, offering resistance against various influenza virus strains. In this study, we investigated the efficacy of an NP DNA vaccine for induction of cell-mediated immune responses and protection against influenza virus infection in a mouse model of diabetes. Healthy and diabetic BALB/c mice were immunized on days 0, 14, and 28 by injection of NP DNA vaccine. Two weeks after the last immunization, the cellular immune response was evaluated by gamma interferon (IFN-gamma), lymphocyte proliferation, and cytotoxicity assays. The mice were challenged with influenza virus, and the viral titers in the lungs were measured on day 4. Diabetic mice showed significantly smaller amounts of IFN-gamma production, lymphocyte proliferation, and cytotoxicity responses than nondiabetic mice. Furthermore, higher titers of the influenza virus were detected after challenge in the lungs of the diabetic mice. The present data suggest that the NP DNA vaccine with the protocol of immunization described here is not able to induce efficient cellular immune responses against influenza virus infection in diabetic mice.

Two-step maturation of immature DCs with proinflammatory cytokine cocktail and poly(I:C) enhances migratory and T cell stimulatory capacity

Effective induction of cell-mediated immune responses strongly depends on the ability of dendritic cells (DCs) to produce Th1-polarizing cytokines, migrate to lymph nodes and stimulate T cells through antigen-presenting complex and costimulatory molecules. While various protocols for optimizing DC maturation with single or multiple stimuli mimicking infections or inflammatory milieu have been proposed for the generation of DCs with features desired for clinical application, stepwise maturation of DCs by these multiple stimuli has not been systemically assessed. Among the combinations of several immune-modulating factors with known effects on DC maturation, we found that stepwise DC maturation with cytokine cocktail (TNF-α + IL-6 + IL-1β + PGE 2) followed by poly(I:C) stimulation enhanced the production of IL-12 with strong allostimulatory capacity. While there were no significant differences between DC matured by simultaneous or sequential activation by cytokine cocktail and poly(I:C) in expression of markers and costimulatory molecules of mature DCs, the delivery of inflammatory signal prior to poly(I:C) results in sustained interleukin-12 expression with reduced IL-10 than DC matured by simultaneous stimulation. This sequential stimulation significantly increased migratory capacity in response to CCL21 and CXCL12 compared to DC matured with cytokine cocktail. Furthermore, these DCs retained their responsiveness to CD40L stimulation in secondary IL-12 production and efficiently generated autologous antigen-specific effector T cells as evidenced by ELISPOT assay. Thus, we propose a novel DC maturation protocol in which stimulation of DCs with cytokine cocktail and subsequently with poly(I:C) generates DCs with a high migratory capacity with a preferential Th1 inducing capacity.

Induction of Cell-Mediated Immune Responses to Peptide Antigens of P. vivax in Microparticles Using Intranasal Immunization

T-cells play a critical role in resistance to malaria, not only because they function as helper cells for an antibody response, but also because they serve as effector cells. Such cellular immunity is directly implicated in protection from sporozoites as well as from blood stage parasites. The aim of this study was to induce cell mediated immune responses to peptide antigens of Plasmodium vivax co-encapsulated with CpG oligodeoxynucleotide (ODN) in microparticles. In the present study, we have investigated the immunomodulatory effects of two CpG adjuvants, CpG 1826 and CpG 2006 to the five peptide antigens of Plasmodium vivax derived from circumsporozoite protein, merozoite surface protein-1, apical membrane antigen-1 and gametocyte surface antigen (Pvs24) in microparticle delivery. The T-cell proliferation response study of the cells collected from spleen, lamina propria and peyer's patches showed significantly high (p<0.001) stimulation index when primed with peptide antigens in microparticles co-encapsulating CpG ODN adjuvant as compared to peptide alone primed mice. The cytokine measurement profile of IFN-γ, TNF-α, IL-2, IL-4 and IL-10 in culture supernatants of cells primed with peptide antigens in microparticles co-encapsulating CpG ODN showed higher levels of IFN- γ followed by TNF-α and IL-2, with relatively low levels of IL-4 and IL-10.

A Synergistic Antitumor Effect of Interleukin-2 Addition with CD80 Immunogene Therapy for Peritoneal Metastasis of Gastric Carcinoma

The co-stimulatory molecule CD80 is a ligand of CD28, which plays a key role in the induction of cell-mediated immune responses. Many tumors, including gastric cancer, decrease the expression of CD80, which results in the failure of immune recognition. We evaluated the effect of interleukin-2 addition combined with CD80 infection on the peritoneal metastasis in gastric cancer. CD80 infection combined with interleukin-2 addition significantly increased the activated cytotoxicity of mononuclear cells compared to CD80 gene infection and compared to the lacZ control group. In vivo, the survival of animals with intraperitoneal tumor was longest in those given CD80 infection with interleukin-2 addition (median survival, 46 days), followed by those given interleukin-2 (39 days), those given CD80 infection (37 days), and those given lacZ (29 days). These results suggest that interleukin-2 addition might contribute to improving the observed outcome of CD80 immunogene therapy in peritoneal metastasis of gastric carcinoma.

A CD46-binding Chimpanzee Adenovirus Vector as a Vaccine Carrier

A replication-defective chimeric vector based on the chimpanzee adenovirus serotype C1 was developed and tested as a vaccine carrier in mice. The AdC1 virus is closely related to human adenoviruses of subgroup B2 and uses CD46 for cell attachment. To overcome poor growth of E1-deleted AdC1 vectors on cell lines that provide the E1 of adenovirus of the human serotype 5 (AdHu5) virus in trans, the inverted terminal repeats and some of the early genes of AdC1 were replaced with those from AdC5, a chimpanzee origin adenovirus of subfamily E. The chimeric AdC1/C5 vector efficiently transduces CD46-expressing mouse dendritic cells (DCs) in vitro and initiates their maturation. Transduction of DCs in vivo is inefficient in CD46 transgenic mice. The AdC1/C5 vector induces transgene product-specific B- and CD8(+) T-cell responses in mice. Responses are slightly higher in wild-type mice than in CD46 transgenic mice. Transgene product-specific T-cell responses elicited by the AdC1/C5 vector can be increased by priming or boosting with a heterologous adenovirus vector. Pre-existing immunity to adenovirus of the common human serotype 5 does not affect induction of cell-mediated immune responses by the AdC1/C5 vector. This vector provides an additional tool in a repertoire of adenovirus-based vaccine vectors.

Immunoglobulin A and CD8+ T‐Cell Mucosal Immune Defenses Protect Against Intranasal Infection with Chlamydia pneumoniae

Chlamydia pneumoniae initiates infection in humans via the mucosal epithelia of the respiratory tract; therefore, immunity at this mucosal site is believed to be important to control infection with this pathogen. We compared the protective capacity of immunization in mice with two C. pneumoniae antigens, namely the major outer membrane protein (MOMP) and the heat shock protein 60 (HSP-60), against intranasal (i.n.) infection with the bacteria when given as protein or DNA and when administered by i.n. or intraperitoneal (i.p.) routes. Our data showed that i.n. immunizations with both antigens delivered as DNA were protective against C. pneumoniae infection, probably due to induction of cell-mediated immune responses. Our study also revealed that i.n. immunizations with MOMP, but not with HSP-60, given as protein induced protective local immune responses in the respiratory tract against C. pneumoniae infection. Moreover, no protection was induced by either antigen when the i.p. route of immunization was used. We further investigated in immunoglobulin (Ig)A-deficient mice whether the reduction in the bacterial loads observed when MOMP was administered intranasally was related to the strong local IgA responses induced by this route of immunization. Our data showed that IgA-deficient mice were more susceptible to infection than wild-type mice, suggesting that the induction of local IgA responses may play a role in the protection of the respiratory tract against C. pneumoniae infections.

Leukemia Specific Complement Dependent Cytotoxicity Contributes to Anti-Leukemia Immunity Induced by Leukemia Cell-Derived HSP70 Immunization in Mice.

Background: Tumor-derived heat shock proteins (HSPs) can be used for the vaccination against cancer because tumor-derived HSPs bind the tumor specific antigenic peptides and these peptides are carried onto MHC molecules by HSPs. We previously reported that immunotherapy using leukemia cell-derived HSPs against minimal residual leukemia after syngeneic bone marrow transplantation in mice prolonged survival by inducing leukemia-specific CD8 + cytotoxic T lymphocyte (Sato et al. Blood, 2001). It is known that induction of cell-mediated immune response plays a main role of tumor rejection in the HSP-based immunotherapy. However we also showed that CD4+ as well as CD8+ T cell is necessary for survival prolongation of HSP-immunized mice, suggesting that humoral immune response though CD4+ T cell contributes to the eradication of leukemia cells in our mouse model. To investigate the contribution of humoral immune responses in anti-leukemia immunity induced by HSP-based immunotherapy, we tried to detect the anti-leukemia cell antibody and analyzed cytotoxicities by the antibody in the leukemia cell-derived HSP70 immunization mice model.Methods: Balb/c mice and syngeneic A20 B-cell leukemia cell line were used in this study. HSP70 was purified from A20 cells or liver tissue from healthy mice. Three weeks after subcutaneous administration of A20-derived HSP70 (A20-HSP70), liver-derived HSP70 (liver-HSP70) or PBS to the healthy mice, the sera were harvested to perform following experiments. To detect anti-A20 antibodies, mean fluorescence intensity (MFI) of A20 cells with immunized mice sera and FITC-conjugated anti-mouse-IgG was analyzed by flowcytometry. Additionally, the sera were subjected to ELISA using HRP labeled anti-mouse-IgG to detect specific antibody against A20-HSP70. Complement dependent cytotoxicity (CDC) activities were determined by trypan blue uptake of mouse target cells (A20, YAC1: lymphoma, or T27A: myeloid leukemia) after incubation with mice sera and rabbit complement.Results: MIF of A20 with the sera from A20-HSP70 immunized mice was significantly higher than that from PBS or liver-HSP70 immunized mice. In addition, anti-A20-HSP70 IgG level by ELISA was significantly increased in the A20-HSP70 immunized mice. However, anti-A20-HSP70 IgG level in liver-HSP70 mice was not elevated, suggesting that A20-HSP70 immunization induced the specific IgG against not HSP70 itself but peptides bound for A20-HSP70. The sera of mice immunized with A20-HSP70 showed no cytotoxic activity without complement but showed significantly high CDC activity against A20 in vitro. This CDC activity was not observed against YAC-1 or T27A.Conclusions: Leukemia specific CDC by the antibodies against the peptides bound for leukemia cell-derived HSP70 is suggested to be one of the mechanisms of anti-leukemia immunity induced by immunization with leukemia cell-derived HSP70 in mice. This would be an important finding to eradicate leukemia cells effectively in HSP-based immunotherapy for leukemia patients.

Enhancement of Innate and Cell-Mediated Immunity by Antimycobacterial Antibodies

We investigated the ability of human antibodies induced by Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination to protect against mycobacterial infections. Serum samples containing mycobacterium-specific antibodies were obtained from volunteers who had received two intradermal BCG vaccinations 6 months apart. Significant increases in lipoarabinomannan (LAM)-specific immunoglobulin G (IgG) were detected after both the primary and booster vaccinations. Effects of mycobacterium-specific antibodies on surface binding and internalization of BCG by neutrophils and monocytes/macrophages were studied, using green fluorescent protein (gfp)-expressing BCG. Surface-bound gfp-expressing BCG were distinguished from intracellular BCG by surface labeling with LAM-specific monoclonal antibody. Internalization of BCG by phagocytic cells was shown to be significantly enhanced in postvaccination serum samples. Furthermore, the inhibitory effects of neutrophils and monocytes/macrophages on mycobacterial growth were significantly enhanced by BCG-induced antibodies. The growth-inhibiting effects of postvaccination sera were reversed by preabsorption of IgG with Protein G. Finally, the helper effects of antimycobacterial antibodies for the induction of cell-mediated immune responses were investigated. BCG-induced antibodies significantly enhanced proliferation and gamma interferon production in mycobacterium-specific CD4(+) and CD8(+) T cells, as well as the proportion of proliferating and degranulating CD8(+) T cells. We conclude that mycobacterium-specific antibodies are capable of enhancing both innate and cell-mediated immune responses to mycobacteria.

703. Combination Therapy with Bleomycin and IL-12 Gene Induces Regression of Breast Cancer

Electrochemotherapy has been used extensively in clinical studies for treating melanoma and squamous cell carcinoma, demonstrating that a low dose of drug such as bleomycin (BLM) has anti-tumor activity when it is delivered by electroporation. However, many tumors re-grow from the escaped tumor cells causing metastasis. Electrochemo-gene immunotherapy combines the local cytotoxic effect of BLM with the induction of cell-mediated immune response by cytokines. In this study, we used the IL-12 gene and BLM combination therapy delivered intratumorally by electroporation for treating breast cancer in Balb/c mice.

CD80 gene therapy for lymph node involvement by gastric carcinoma

The co-stimulatory molecule, CD80 (B7-1), is a ligand of CD28 and plays a key role in the induction of cell-mediated immune responses. Many tumors, including gastric cancer, have decreased expression of CD80 which leads to a failure of immune recognition. Lymph node spread is a factor of poor prognosis in gastric cancer. In this study, we transfected the CD80 gene by an adenovirus vector into a human gastric cancer cell line, OCUM-2MLN, and analyzed the effect on lymph node disease in vitro and in vivo. After transfection of CD80 in vitro, the adhesive ability of cancer cells for peripheral blood mononuclear cells and their cytotoxicity showed significant regression (p<0.01). Intratumoral injection of AdCD80 caused significantly growth of subcutaneous tumors. In vivo lymph node spread was suppressed by injection of AdCD80 into gastric tumors. Histopathologic findings revealed CD80-positive cells around the tumor. These results suggest that CD80 gene transfer into cancer cells using an adenovirus vector might be a promising approach for in vivo cancer therapy.

The role of antigenic peptide in CD4+ T helper phenotype development in a T cell receptor transgenic model.

CD4+ Th1 cells play a critical role in the induction of cell-mediated immune responses that are important for the eradication of intracellular pathogens. Peptide-25 is the major Th1 epitope for Ag85B of Mycobacterium tuberculosis and is immunogenic in I-Ab mice. To elucidate the role of the TCR and IFN-gamma/IL-12 signals in Th1 induction, we generated TCR transgenic mice (P25 TCR-Tg) expressing TCR alpha- and beta-chains of Peptide-25-reactive cloned T cells and analyzed Th1 development of CD4+ T cells from P25 TCR-Tg. Naive CD4+ T cells from P25 TCR-Tg differentiate into both Th1 and Th2 cells upon stimulation with anti-CD3. Naive CD4+ T cells from P25 TCR-Tg preferentially develop Th1 cells upon Peptide-25 stimulation in the presence of I-Ab splenic antigen-presenting cells under neutral conditions. In contrast, a mutant of Peptide-25 can induce solely Th2 differentiation. Peptide-25-induced Th1 differentiation is observed even in the presence of anti-IFN-gamma and anti-IL-12. Furthermore, naive CD4+ T cells from STAT1 deficient P25 TCR-Tg also differentiate into Th1 cells upon Peptide-25 stimulation. Moreover, Peptide-25-loaded I-Ab-transfected Chinese hamster ovary cells induce Th1 differentiation of naive CD4+ T cells from P25 TCR-Tg in the absence of IFN-gamma or IL-12. These results imply that interaction between Peptide-25/I-Ab and TCR may primarily influence determination of the fate of naive CD4+ T cells in their differentiation towards the Th1 subset.