Bacillus thuringiensis protein production, signal transduction, and insect control in chemically inducible PR-1a/cry1Ab broccoli plants

In an effort to develop a chemically inducible system for insect management, we studied production of Cry1Ab Bacillus thuringiensis (Bt) protein and control of the diamondback moth (DBM), Plutella xylostella L., in inducer-treated and untreated tissues of a broccoli line transformed with a PR-1a/cry1Ab expression cassette. Spraying leaves of these plants with the inducer acibenzolar-S-methyl (= 1,2,3 benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester) (ASM) triggered expression of the cry1Ab gene and produced a high level of Cry1Ab protein within 2–3 days. Cry1Ab protein persisted in leaves for at least 8 weeks, providing prolonged protection from P. xylostella attack. Signals generated in inducer-treated leaves were transferred to untreated newly emerged leaves or heads, as seen by production of Cry1Ab protein and/or protection from insect damage in these plant parts. Signal transduction proceeded in an attenuated manner up to the sixth newly emerged leaf. No Cry1Ab protein was detectable by ELISA in uninduced young leaves, but small amounts of the protein were present in uninduced leaves older than 3 weeks and caused some insect mortality. Such basal expression of Bt genes without induction may favor the evolution of resistant insect populations and therefore limits the application of the PR-1a/cry1Ab system for insect management. However, the rapid production and steady maintenance of a high level of transgenic protein upon induction, the signal transduction observed, and the fact that the chemical inducer can be used in field conditions make the PR-1a promoter attractive for chemical regulation of other agriculturally or pharmaceutically important genes for which low expression in the absence of induction is not a concern.     

Characterization of antibodies to idiotypic determinants of monoclonal anti-sperm antibodies

Rabbit polyclonal antibodies to the idiotype of murine monoclonal anti-sperm antibodies were developed and characterized. M29.6 and M42.15 are monoclonal antibodies (mAbs) that inhibit fertilization in vivo and in vitro. Sera from rabbits inoculated with purified mAbs (Ab1) were absorbed with normal mouse and isotype-specific immunoglobulin (Ig); the anti-idiotype Ig fraction (Ab2) was isolated by protein A-chromatography or by chromatography on the corresponding idiotype column. Binding specificity of Ab2 was confirmed by measuring the reactivity of Ab2 with homologous and heterologous mAbs. Ab2 competitively inhibited 125I-labeled Ab1 binding to mouse sperm, suggesting that the Ab2 preparation possessed subpopulations directed against idiotopes similar or adjacent to the antigen-binding site of the mAb. Anti-idiotype antibodies reactive with the antigen-combining site of the anti-sperm mAb may contain subpopulations that mimic the mouse sperm epitope recognized by Ab1. Immunization with Ab2 induced anti-(anti-idiotype) antibodies (Ab3), which competitively inhibited binding of 125I-labeled Ab1 to immobilized Ab2. These results are consistent with the hypothesis that immunization of mice with antibodies to the idiotype of sperm-specific mAbs can induce antibodies that share structural similarities with the anti-sperm mAb used for their induction. Immunization with anti-idiotype antibodies that mimic sperm antigen structure represents a possible strategy for induction of immunity to sperm.     

Parasporin-2Ab,a Newly Isolated Cytotoxic Crystal Protein from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A novel crystal protein that exhibited potent cytotoxicity against human leukemic T-cells was cloned from the Bacillus thuringiensis TK-E6 strain. The protein, designated as parasporin-2Ab (PS2Ab), was a polypeptide of 304 amino acid residues with a predicted molecular weight of 33,017. The deduced amino acid sequence of PS2Ab showed significant homology (84% identitiy) to parasporin-2Aa (PS2Aa) from the B. thuringiensis A1547 strain. Upon processing of PS2Ab with proteinase K, the active form of 29 kDa was produced. The activated PS2Ab showed potent cytotoxicity against MOLT-4 and Jurkat cells and the EC₅₀ values were estimated as 0.545 and 0.745 ng/mL, respectively. The cytotoxicity of PS2Ab was significantly higher than that of PS2Aa reported elsewhere. Although both cytotoxins were structurally related, it was thought that the minor differences found were responsible for the different cytotoxicities of PS2Ab and PS2Aa.     

Induction of anti-idiotypic humoral and cellular immune responses by a murine monoclonal antibody recognizing the ovarian carcinoma antigen CA125 encapsulated in biodegradable microspheres

The use of biodegradable poly(dl-lactic-co-glycolic acid) microspheres as a cancer vaccine delivery system for induction of anti-idiotypic responses was investigated using a murine monoclonal antibody B43.13 that recognizes the human ovarian cancer antigen CA125. Immunization of mice with mAb B43.13 encapsulated in poly(dl-lactic-co-glycolic acid) microspheres resulted in enhanced humoral and cellular immune responses compared with mAb B43.13 alone or mAb B43.13 mixed with microspheres. The antibody responses could be further enhanced by the co-encapsulation of mAb B43.13 with monophosphoryl lipid A, a non-toxic adjuvant, in microspheres. Anti-idiotypic humoral responses were shown to result in Ab2 antibodies mimicking the nominal antigen CA125 and Ab3 antibodies recognizing CA125. Further, microsphere delivery of mAb B43.13 also resulted in induction of T cell responses involving T2 cells reactive with mAb B43.13 epitopes and T3 cells recognizing CA125. These results indicate that microsphere delivery of Ab1 can induce both humoral and cellular anti-idiotypic responses relevant to cancer antigens. This raises the possibility of the use of such formulations for anti-idiotypic induction immunotherapy for cancer. Received: 27 August 1997 / Accepted: 24 April 1998     

Possibly similar genetic basis of resistance to Bacillus thuringiensis Cry1Ab protein in 3 resistant colonies of the sugarcane borer collected from Louisiana,USA

The sugarcane borer, Diatraea saccharalis (F.), is a major maize borer pest and a target of transgenic maize expressing Bacillus thuringiensis (Bt) proteins in South America and the mid‐southern region of the United States. Evolution of resistance in target pest populations is a great threat to the long‐term efficacy of Bt crops. In this study, we compared the genetic basis of resistance to Cry1Ab protein in 3 resistant colonies of sugarcane borer established from field populations in Louisiana, USA. Responses of larvae to the Cry1Ab protein for the parental and 10 other cross colonies were assayed in a diet‐incorporated bioassay. All 3 resistant colonies were highly resistant to the Cry1Ab protein with a resistance ratio of >555.6 fold. No maternal effect or sex linkage was evident for the resistance in the 3 colonies; and the resistance was functionally nonrecessive at the Cry1Ab concentrations of ≤ 3.16 μg/g, but it became recessive at ≥10 μg/g. In an interstrain complementation test for allelism, the F₁ progeny from crosses between any 2 of the 3 resistant colonies exhibited the similar resistance levels as their parental colonies, indicating that the 3 colonies most likely shared a locus of Cry1Ab resistance. Results generated from this study should provide useful information in developing effective strategies for managing Bt resistance in the insect.     

Cry1Ab protein levels in phytophagous insects feeding on transgenic corn: implications for secondary exposure risk assessment

Enzyme-linked immunosorbent assays (ELISA) and bioassays were used to estimate levels of Cry1Ab protein in four species of phytophagous insects after feeding on transgenic Bt-corn plants expressing Cry1Ab protein or artificial diets containing Cry1Ab protein. The level of Cry1Ab in insects feeding on sources containing the Cry1Ab protein was uniformly low but varied with insect species as well as food source. For the corn leaf aphid, Rhopalosiphum maidis (Fitch), feeding on diet solutions containing Cry1Ab protein, the level of the protein in the aphid was 250–500 times less than the original levels in the diet, whereas no Cry1Ab was detected by ELISA in aphids feeding on transgenic Bt-Corn plants. For the lepidopteran insects, Ostrinia nubilalis (Hübner), Helicoverpa zea (Boddie), and Agrotis ipsilon (Hufnagel), levels of Cry1Ab in larvae varied significantly with feeding treatment. When feeding for 24 h on artificial diets containing 20 and 100 ppm of Cry1Ab, the level of Cry1Ab in the larvae was about 57 and 142 times lower, respectively, than the original protein level in the diet for O. nubilalis, 20 and 34 times lower for H. zea, and 10 to 14 times lower for A. ipsilon. Diet incorporation bioassays with a susceptible insect (first instar O. nubilalis) showed significant Cry1Ab bioactivity present within whole body tissues of R. maidis and O. nubilalis that had fed on diet containing a minimum of 20 ppm or higher concentrations (100 or 200 ppm) of Cry1Ab, but no significant bioactivity within the tissues of these insects after feeding on transgenic Bt-corn plants. The relevance of these findings to secondary exposure risk assessment for transgenic Bt crops is discussed.     

Relative fitness of susceptible and Cry1A.105/Cry2Ab2-single-/dual-protein-resistant Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) on non-Bt diet and a diet containing a low concentration of two proteins

Helicoverpa zea (Boddie) is a destructive agricultural pest species that is targeted by both Bacillus thuringiensis (Bt) maize and cotton in the United States. Cry1A.105 and Cry2Ab2 are two Bt proteins expressed in a widely planted maize event MON 89034. In this study, two tests (Test-I and Test-II) were conducted to evaluate the relative fitness of Bt-susceptible and -resistant H. zea on non-Bt diet (Test-I and Test-II) and a diet containing a mix of Cry1A.105 and Cry2Ab2 at a low concentration (Test-II only). Insect populations evaluated in Test-I were two Bt-susceptible strains and three Bt-resistant strains (a single-protein Cry1A.105-, a single-protein Cry2Ab2-, and a dual-protein Cry1A.105/Cry2Ab2-resistant strains). Test-II analyzed the same two susceptible strains, three backcrossed-and-reselected Cry1A.105/Cry2Ab2-single-/dual-protein-resistant strains, and three F₁ heterozygous strains. Measurements of life table parameters showed that neither the single- nor dual-protein Cry1A.105/Cry2Ab2 resistance in H. zea was associated with fitness costs under the test conditions. The single Cry protein resistances at a concentration of a mix of Cry1A.105 and Cry2Ab2 that resulted in a zero net reproductive rate for the two susceptible strains were functionally incomplete recessive or codominant, and the dual-protein resistance was completely dominant. The lack of fitness costs could be a factor contributing to the rapid revolution of resistance to the Cry proteins in this species. Data generated from this study should aid our understanding of Cry protein resistance evolution and help in refining IRM programs for H. zea.     

Ingestion and excretion of two transgenic Bt corn varieties by slugs

The release of transgenic Bacillus thuringiensis (Bt) corn expressing various Cry endotoxins has raised concern that these endotoxins are disseminated in the food web and may adversely affect non-target beneficial organisms, such as predators and organisms of the decomposer food web. We therefore investigated in a laboratory study, whether the Cry1Ab and Cry3Bb1 protein from Bt corn could potentially be transferred to such organisms by measuring the Cry protein content in the two common agricultural slug pests Arion lusitanicus and Deroceras reticulatum and their feces. We measured Cry1Ab and Cry3Bb1 protein concentration in leaves, intestines, and feces of corn leaf-fed slugs using ELISA and determined how much of the ingested protein is excreted by the slugs. Cry3Bb1 concentration in leaves of DKC5143Bt corn was significantly higher than Cry1Ab concentration in leaves of N4640Bt corn. While slugs were feeding on corn leaves, the Cry3Bb1 and Cry1Ab proteins were found in intestines and feces of both slug species. Bt protein concentrations in intestines of Cry3Bb1 corn-fed slugs were in both slug species higher than in Cry1Ab corn fed slugs, whereas no differences between Cry3Bb1 and Cry1Ab protein in feces were found. After slugs had ceased feeding on Bt corn, Cry1Ab was detectable in fresh slug feces for a significantly longer time and often in higher amounts than the Cry3Bb1. Our results indicate that both Cry proteins are likely to be transferred to higher trophic levels and to the decomposer food web. Since different Bt proteins seem to vary in their degradation, they have different transfer probabilities. This should be considered in risk assessments for non-target arthropods.     

Expression of the full-length and 3'-spliced cry1Ab gene in the 135-kDa crystal protein minus derivative of Bacillus thuringiensis subsp. kyushuensis

Bacillus thuringiensis produces a 130–135-kDa insecticidal protein in the form of bipyramidal crystal which is toxic to lepidopteran larvae. Part of the C-terminal region of the native Cry1Ab was replaced by a heterologous sequence of Cry11Aa C-terminus to get a 3′-spliced cry1Ab gene. The full-length cry1Ab and 3′-spliced cry1Ab, which were both cloned into the E. coli–B. thuringiensis shuttle expression vector pHZB1, were expressed in a 135-kDa crystal protein minus derivative of B. thuringiensis subsp. kyushuensis (4U1-Cry⁻¹³⁵). The crystal shape of Cry1Ab proteins from both recombinants was regularly bipyramidal, while the crystal size of the intact Cry1Ab was approximately fivefold larger than the 3′-spliced Cry1Ab. In addition, these two kinds of Cry1Ab proteins had similar toxicity against Argyrogramma agnata larvae. Received: 19 October 2001 / Accepted: 7 December 2001     

Idiotype vaccine for tumor by anti-idiotypic antibody prepared against anti-(bacillus Calmette Guèrin)BCG monoclonal antibody

Summary The anti-idiotypic antibody (Ab2) prepared against the anti-BCG monoclonal antibody (mAb) (Ab1) exhibited potential vaccine activity against Meth A fibrosarcoma that shared a common antigen(s) withMycobacterium bovis strain bacillus Calmette Guèrin (BCG). Mice vaccinated with the anti-idiotypic antibody (Ab2) were protected significantly against growth of the transplanted Meth A tumor (66%), and the presence of anti-(anti-idiotypic antibody) (Ab3) was proved in the Ab2-vaccinated mice by enzyme-linked immunosorbent assay and indirect immunofluorescence analyses using unabsorbed or absorbed sera against the BCG antigen(s) and Meth A tumor cells. This indicated that the anti-idiotypic antibody (Ab2) mimicked the structures of the BCG antigen(s) and behaved as the BCG antigen(s) to induce the Abl-like antibody (Ab3) in vivo. Presumably the Ab2-induced Ab3 plays a significant role in preventing growth of the transplanted tumor in animals. By contrast, the control mice treated with normal mouse serum failed to inhibit the tumor growth. These results suggest the possible development of a tumor vaccine from the anti-idiotypic antibody (Ab2) prepared against the anti-BCG monoclonal antibody, for tumors sharing a common antigen(s) withMycobacterium bovis strain BCG.

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Idiotype vaccine for tumor by anti-idiotypic antibody prepared against anti-(bacillus Calmette Guèrin)BCG monoclonal antibody

     

Susceptibility of northern corn rootworm Diabrotica barberi (Coleoptera: Chrysomelidae) to mCry3A and eCry3.1Ab Bacillus thuringiensis proteins

The susceptibility of the northern corn rootworm Diabrotica barberi (Smith & Lawrence) to mCry3A and eCry3.1Ab proteins derived from Bacillus thuringiensis (Bt) was determined using a diet bioassay. Northern corn rootworm neonates were exposed to different concentrations of mCry3A and eCry3.1Ab, incorporated into artificial diet. Larval mortality was evaluated after 7 d. The mCry3A and eCry3.1Ab proteins were found to be toxic to the northern corn rootworm larvae. The LC₅₀ and LC₉₉ values for mCry3A were 5.13 and 2482.31 μg/mL, respectively. For eCry3.1Ab, the LC₅₀ and LC₉₉ values were 0.49 and 213.01 μg/mL. Based on the estimated lethal concentrations, eCry3.1Ab protein was more efficacious to northern corn rootworm larvae than mCry3A. These lethal concentration values will be used as diagnostic doses for routine annual monitoring for change in susceptibility of field collected northern corn rootworm to mCry3A, and eCry3.1Ab toxins.     

Cry2Ab蛋白对龟纹瓢虫的安全性研究

【目的】转基因作物对非靶标昆虫的影响是转基因作物环境安全评价的重要内容,研究Cry2Ab蛋白对龟纹瓢虫的影响,对转基因作物的环境安全评价具有重要意义。【方法】采用实验动物学、分子生物学等方法,研究Cry2Ab蛋白对龟纹瓢虫发育历期、成虫体重、雌雄比例及体内氨基酸种类和含量的影响。【结果】与蔗糖对照组相比,Cry2Ab蛋白对龟纹瓢虫不同龄期的发育历期、成虫体重和雌雄比例均无明显差异,对体内氨基酸种类和含量也没有显著差异。【结论】Cry2Ab蛋白对龟纹瓢虫的生长发育及代谢无显著影响。     

The induction of lymphokine synthesis and cell growth in IL 3-dependent cell lines using antigen-antibody complexes

Several IL 3-dependent murine bone marrow-derived cell lines can be stimulated to grow with antigen-antibody (Ag.Ab) complexes. The Ag.Ab complexes induced lymphokine gene expression and the synthesis of IL 2, GM-CSF, IL 3, and BSF-1 (IL 4). The lymphokines produced by these IL 3-dependent cells appeared to stimulate their own growth, as both IL 3 and BSF-1 (IL 4) stimulated the growth of IL 3-dependent cells. Ag.Ab complexes also stimulate the growth of primary cultures of bone marrow cells that have been previously activated with IL 3. Normal bone marrow, IL 2-, and GM-CSF-dependent bone marrow cell lines could bind Ag.Ab complexes, but binding did not result in the induction of lymphokine synthesis or cell growth. Hyperimmune serum from mice also stimulated lymphokine synthesis and cell growth in IL 3-dependent cells, and the stimulatory activity was removed by treatment with Staphylococcus aureus protein A, suggesting the presence of Ag.Ab complexes.     

Contribution of Bacillus thuringiensis Spores to Toxicity of Purified Cry Proteins Towards Indianmeal Moth Larvae

The influence of Bacillus thuringiensis subsp. kurstaki HD-1 spores upon the toxicity of purified Cry1Ab and Cry1C crystal proteins toward susceptible and BT-resistant Indianmeal moth (IMM, Plodia interpunctella) larvae was investigated. With susceptible larvae, HD-1 spores were toxic in the absence of crystal protein and highly synergistic (approximately 35- to 50-fold) with either Cry1Ab or Cry1C protein. With BT-resistant IMM larvae, HD-1 spores were synergistic with Cry1Ab and Cry1C protein in all three resistant strains examined. Synergism was highest (approximately 25- to 44-fold) in insects with primary resistance toward Cry1C (IMM larvae with resistance to B. thuringiensis subsp. aizawai or entomocidus). However, HD-1 spores also synergized either Cry1Ab or Cry1C toxicity toward larvae resistant to B. thuringiensis subsp. kurstaki at a lower level (approximately five- to sixfold). With susceptible larvae, the presence of spores reduced the time of death when combined with each of the purified Cry proteins. Without spores, the speed of intoxication and eventual death for larvae treated with Cry1C and Cry1Ab proteins was much slower than for the HD-1 preparation containing both spores and crystals together. Neither spores nor toxin dose affected the mean time of death of resistant larvae treated with either Cry1Ab or Cry1C toxins. Both Cry1Ab and Cry1C toxins appeared to reduce feeding and consequently toxin consumption. Received: 1 December 1995 / Accepted: 3 January 1996     

The expression of the 14D9 catalytic antibody in suspended cells of Nicotiana tabacum cultures increased by the addition of protein stabilizers and by transference from erlenmeyer flasks to a 2‐L bioreactor

The effect of two protein stabilizers (polyvinylpyrrolidone [PVP] and gelatine) on growth and 14D9 yield of Nicotiana tabacum cell suspension cultures (Ab‐KDEL and sec‐Ab) was analyzed. The addition of PVP at a concentration of 1.0 g L^?1 produced the highest total 14D9 yield (biomass + culture medium) in the Ab‐KDEL line (4.82% total soluble protein [TSP]). With the addition of gelatine, the highest total 14D9 yield (2.48% TSP) was attained in the Ab‐KDEL line at 5.0 g L^?1 gelatine. When the Ab‐KDEL suspended cells were cultured in a 2‐L bioreactor, the highest 14D9 yield was 8.1% TSP at a 5% w/v inoculum size, which was the best 14D9 yield so far obtained in the platforms tested (E. coli, N. tabacum leaves and seeds, N. tabacum hairy roots, and cell suspension cultures). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1185–1189, 2014     

Phosphorylation of the zebrafish M6Ab at serine 263 contributes to filopodium formation in PC12 cells and neurite outgrowth in zebrafish embryos

Background

Mammalian M6A, a member of the proteolipid protein (PLP/DM20) family expressed in neurons, was first isolated by expression cloning with a monoclonal antibody. Overexpression of M6A was shown to induce filopodium formation in neuronal cells; however, the underlying mechanism of is largely unknown. Possibly due to gene duplication, there are two M6A paralogs, M6Aa and M6Ab, in the zebrafish genome. In the present study, we used the zebrafish as a model system to investigate the role of zebrafish M6Ab in filopodium formation in PC12 cells and neurite outgrowth in zebrafish embryos.

Methodology/Principal Findings

We demonstrated that zebrafish M6Ab promoted extensive filopodium formation in NGF-treated PC12 cells, which is similar to the function of mammalian M6A. Phosphorylation at serine 263 of zebrafish M6Ab contributed to this induction. Transfection of the S263A mutant protein greatly reduced filopodium formation in PC12 cells. In zebrafish embryos, only S263D could induce neurite outgrowth.

Conclusions/Significance

Our results reveal that the phosphorylation status of zebrafish M6Ab at serine 263 is critical for its role in regulating filopodium formation and neurite outgrowth.     

The insecticidal crystal protein Cry2Ab10 from Bacillus thuringiensis: cloning, expression, and structure simulation

The cry2Ab-type gene was cloned from Bacillus thuringiensis and designated as cry2Ab10. The recombinant Cry2Ab10 protein expressed in E. coli cells shows high toxicity against Plutella xylostella. The protein structure was constructed by homology modeling, and the receptor-binding sites were predicted by a molecular docking method.     

Resistance of sugarcane borer to Bacillus thuringiensis Cry1Ab toxin

The sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), strain (F52‐3‐R) was developed from F₃ survivors of a single‐pair mating on commercial Cry1Ab Bacillus thuringiensis (Bt) corn plants in the greenhouse. The susceptibility of a Bt‐susceptible and the F52‐3‐R strain of D. saccharalis to trypsin‐activated Cry1Ab toxin was determined in a laboratory bioassay. Neonate‐stage larvae were fed a meridic diet incorporating Cry1Ab toxin at a concentration range of 0.0625 to 32 µg g^?1. Larval mortality, larval weight, and number of surviving larvae that did not gain significant weight (<0.1 mg per larva) were recorded on the 7th day after inoculation. The F52‐3‐R strain demonstrated a significant level of resistance to the activated Cry1Ab toxin. Larval mortality of the Bt‐susceptible strain increased in response to higher concentrations of Cry1Ab toxin, exceeding 75% at 32 µg g^?1, whereas mortality of the F52‐3‐R strain was below 8% across all Cry1Ab concentrations. Using a measure of practical mortality (larvae either died or gained no weight), the median lethal concentration (LC₅₀) of the F52‐3‐R strain was 102‐fold greater than that of the Bt‐susceptible insects. Larval growth of both Bt‐susceptible and F52‐3‐R strains was inhibited on Cry1Ab‐treated diet, but the inhibition of the F52‐3‐R strain was significantly less than that of the Bt‐susceptible insects. These results confirm that the survival of the F52‐3‐R strain on commercial Bt corn plants was related to Cry1Ab protein resistance and suggest that this strain may have considerable value in studying resistance management strategies for Bt corn.     

Induction of cellular immunity by anti-idiotypic antibodies mimicking GD2 ganglioside

Gangliosides are potentially useful targets for tumor destruction by antibodies. However, the role of gangliosides in T cell-mediated immunity to tumors is unclear. We produced three murine monoclonal anti-idiotypic antibodies (Ab2) against a monoclonal antibody (Ab1) that binds strongly to melanoma-associated GD2 ganglioside and weakly to GD3 ganglioside. All three Ab2 induced anti-anti-idiotypic antibodies (Ab3) with Ab1-like binding specificity to tumor cells and antigen in rabbits. The Ab3 specifically bound to GD2(+) tumor cells and isolated GD2, and shared idiotopes with the Ab1. Two of the three Ab2 induced GD2-specific delayed-type hypersensitivity responses in BALB/c and C57BL/6 mice, but not in C57BL/6/CD4(-/-) mice. Peripheral blood mononuclear cells (PBMC) from a melanoma patient proliferated specifically in response to in vitro stimulation with Ab2. Proliferation was accompanied by Th1-type cytokine production. Our studies demonstrate the induction of ganglioside-specific T cell-dependent immunity by Ab2 in mice. These T cells showed specific reactivity to ganglioside expressed by tumor cells.