Immunity to Plasmodium yoelii: kinetics of the generation of T and B lymphocytes that passively transfer protective immunity against virulent challenge

Adoptive immunization of A/Tru mice with splenic B cells or T cells from syngeneic donors with a primary, nonvirulent, Plasmodium yoelii (17X) infection confers on these recipients the capacity to resist a challenge infection with a virulent strain (YM) of P. yoelii. Unfractionated spleen cells as well as spleen cells enriched for T or B cells capable of transferring protective immunity were detected as early as Day 7 of the primary nonvirulent infection, and reached peak levels on Day 14. Spleen cells that were harvested from donor animals after resolution of the immunizing infection [on Days 21 or 28] were incapable of transferring protective immunity. The capacity of 7-day immune spleen cells to transfer immunity could be abolished by pretreatment with mitomycin C. In addition, it was found that immunocompetent recipient mice were required for successful adoptive immunization, since thymectomized, irradiated, bone marrow reconstituted mice infused with immune spleen cells failed to survive lethal challenge infections.     

Studies on the transfer of protective immunity with lymphoid cells from mice immune to malaria sporozoites.

In an effort to understand the mechanisms involved in the protective immunity to malarial sporozoites, an A/J mouse/Plasmodium berghei model was studied. Protective immunity could consistently be adoptively transferred only by using sublethal irradiation of recipients (500 R); a spleen equivalent (100 X 10(6))of donor cells from immune syngeneic mice; and a small booster immunization (1 X 10(4)) of recipients with irradiation-attenuated sporozoites. Recipient animals treated in this manner were protected from lethal challenge with 1 X 10(4) nonattenuated sporozoites. Immune and nonimmune serum and spleen cells from nonimmune animals did not protect recipient mice. Fewer immune spleen cells (50 X 10(6)) protected some recipients. In vitro treatment of immune spleen cells with anti-theta sera and complement abolished their ability to transfer protection. This preliminary study suggests that protective sporozoite immunity can be transferred with cells, and that it is T cell dependent.     

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.     

Transfer Agent of Immunity

An immune ribonucleic acid (RNA) was extractable from the spleen cells of mice hyperimmunized with live vaccine of Salmonella enteritidis. The RNA was capable of inducing cellular immunity and developing cellular antibody in the peritoneal macrophages of mice injected with this agent. It was found that cellular immunity was detectable even 90 days after injection in the peritoneal macrophages of mice which had received an intraperitoneal injection with this agent. Results of serial passive transfers of cellular immunity through immune RNA led us to the conclusion that this agent does not contain antigen or fragment thereof and may replicate actively in the recipient cells, although the mechanism still remains to be elucidated. The development of cellular immunity by immune RNA was inhibited by puromycin but not by actinomycin D. However, serial passive transfers of cellular immunity through immune RNA was inhibited by treatment of recipient mouse with actinomycin D, implying the role of DNA-dependent RNA polymerase in the processing of immune RNA in recipient cells. Using these results, the role of immune RNA and the possible mechanisms of immune RNA replication are discussed.     

Mechanism of loss of natural killer activity in P815 ascites tumor bearing DBA/2 mice

P815 tumor cells (10(7] were administered intraperitoneally to DBA/2 mice. As the ascites tumor grew in the syngeneic host, a decline leading to a total loss of host spleen natural killer (NK) activity could be demonstrated. Removal of T and B cells or macrophages from the tumor-bearing (TB) mouse spleen cells did not raise the level of NK activity. Spleen cells from TB mice did not inhibit the NK activity of normal spleen cells. Comparable target (YAC cells) binding capacity could be demonstrated in spleen cells derived from normal or TB mice, but interferon failed to significantly stimulate the NK activity of TB mouse spleen cells. In adoptive transfer experiments, transfer of spleen or bone marrow cells from TB mice resulted in the development of significant levels of spleen NK activity in lethally X-irradiated recipient DBA/2 mice. These results indicate that the impairment of NK cell differentiation pathway rather than active suppression at the level of effector cells may be the mechanism of loss of NK activity in P815 TB DBA/2 mice.     

Cutting edge: antilisterial activity of CD8+ T cells derived from TNF-deficient and TNF/perforin double-deficient mice

The mechanisms by which CD8+ T cells mediate immunity against bacterial pathogens remain largely unknown. Perforin-dependent cytolysis plays a role, but is not required for CD8+ T cell-mediated immunity against Listeria monocytogenes. TNF is essential for CD8+ T cell immunity to L. monocytogenes, but the cellular source of TNF is undefined. TNF-deficient and TNF/perforin double-deficient mice were used to generate CD8+ T cells specific for an L. monocytogenes-derived Ag. Wild-type and TNF-deficient CD8+ T cells mediated antilisterial immunity in wild-type but not TNF-deficient host mice, revealing that CD8+ T cell-derived TNF is not required for CD8+ T cell-mediated antilisterial immunity, but demonstrating a role for TNF derived from other cell types. TNF/perforin double-deficient CD8+ T cells mediated antilisterial immunity in the liver, but not in the spleen, of wild-type recipient mice, suggesting that perforin-independent immunity in the spleen requires CD8+ T cell-derived TNF.     

Intracamerally induced concomitant immunity: mice harboring progressively growing intraocular tumors are immune to spontaneous metastases and secondary tumor challenge

Intracameral inoculation of allogeneic P815 mastocytoma cells (DBA/2) into BALB/c mice resulted in progressively growing intraocular tumors. Intraocular tumor cells disseminated rapidly to the spleen and cervical lymph nodes, yet extraocular nests of tumor cells never developed into fulminant tumors. Further experiments showed that tumor cells were continuously seeded from the primary intraocular tumor and were rapidly cleared from extraocular sites. Hosts harboring intraocular P815 mastocytomas rejected tumorigenic doses of P815 cells inoculated subcutaneously or even into the contralateral anterior chamber. This systemic tumor immunity was found to be radiosensitive and T cell dependent. Spleen cells from animals with progressively growing intraocular tumors protected recipient mice challenged with intracamerally inoculated tumor cells and thus suggests that a cell-mediated mechanism is the underlying basis for this form of tumor immunity. The data indicate that mice harboring progressively growing intraocular tumors develop a potent state of "concomitant immunity," that prevents the development of metastases, yet is ineffective in controlling the primary tumor.     

Prophylactic immunization against experimental leishmaniasis. VI. Comparison of protective and disease-promoting T cells

In previous studies, we reported that mice immunized i.v. with lethally irradiated Leishmania major promastigotes developed substantial resistance to a subsequent L. major infection. However, such protection could be totally suppressed by prior s.c. injection with the same antigens. Both the protective immunity and the inhibition of its induction could be adoptively transferred with specific Lyt-2- T cells. Here, we present evidence showing that protection and disease promotion resulting from i.v. or s.c. immunization, respectively, are mediated by functionally distinct subsets of T cells. In a series of titration experiments, it was found that freshly isolated T cells derived from prophylactically i.v. immunized BALB/c mice were either protective (greater than 10(7) cells/recipient) or ineffective (less than 10(7) cells/recipient). No exacerbation of disease was observed at any dose. Conversely, T cells from mice immunized s.c. either accelerated disease development and inhibited protective immunization (greater than 10(7) cells/recipient) or had no effect (less than 10(7) cells/recipient). No protection was observed at any dose tested. In mixed transfer experiments, increasing numbers of T cells from s.c. immunized donors progressively inhibited the protective effect of T cells from i.v. immunized donors. Supernatant of T cell cultures from protectively immunized donors contained substantial macrophage-activating factor whereas such activity was not detectable in the supernatant of T cell culture from s.c. immunized donors. Analysis by flow cytometry showed that the spleen and lymph nodes of normal, i.v., or s.c. immunized BALB/c mice contained similar ratios of L3T4+ cells and Lyt-2+ cells.     

Studies of delayed hypersensitivity to L. Monocytogenes in mice: nature of cells involved in passive transfers

C3Hf/Umc mice were immunized by an intravenous injection of a sublethal dose of live Listeria monocytogenes. The animals developed delayed-type hypersensitivity (DH) concomitant with infectious immunity to this organism. Delayed hypersensitivity could be transferred to normal lethally irradiated mice with spleen cells from immune animals. The immune cells cells responsible for transfer of adoptive immunity were susceptible to in vitro cytolytic action of anti-theta iso-antibody and complement, since such treatment rendered these cells incapable of further passive transfer of specific immunity to Listeria. The acquired DH to Listeria persisted in mice after 900 R lethal irradiation, provided normal syngeneic bone marrow cells were also administered, thus indicating the persistance of a cell population in the immune irradiated mice, resistant to effects of radiation. The radio resistant nature of this immune cell population was further demonstrated by passive transfer with spleen cells, derived from preimmunized lethally irradiated mice to normal syngeneic mice or to lethally irradiated nonimmunized hosts reconstituted with normal bone marrow which then responded to antigenic challenge with DH.Treatment of the immune radio resistant spleen cells in vitro with anti-theta and complement eliminated passive transfers of DH by these cells; however, this effect was less obvious than similar treatment of the immune, nonirradiated, spleen cells.     

The onset of immune protection in acute experimental chagas' disease in C3H(HE) mice

The onset of protective immunity against Trypanosoma cruzi in mice was determined by adoptively immunizing newly infected recipients with spleen cells from normal or infected donor mice. It was found that spleen cells from animals with 3 day and 6 day infections did not provide protection but that spleen cells from infections of 9, 12, 15 and 18 days significantly increased longevity in infected recipient animals. The protective capacity per spleen cell was found to increase in proportion to the duration of infection of donor mice. It was further noted that immune protection, as reflected in increased longevity, did not result in decreased development of parasitemia. Immunized mice which demonstrated the greatest longevity developed parasitemias over twice that observed in contrrol groups.     

Ribonucleic acid in the immune response

Summary In the studies of experimental salmonellosis, immunization of mice with a live vaccine SER of S. enteritidis was found to be effective against further infection with virulent S. enteritidis 116-54. Macrophages obtained from the peritoneal cavity, subcutaneous tissue or liver of immunized mice inhibited intracellular growth of bacteria and resisted cell degeneration caused by engulfment of virulent 116-54 bacteria. This immunity was called cellular immunity.We discovered by chance in 1961 a transfer agent of immunity (TA) from the culture fluid of immunized macrophages. This agent is RNA in nature and can be extracted from the spleen, peritoneal exudate cells or the lymph node of immunized animals and is called immune (i) RNA. We could demonstrate antibody activity in macrophages treated in vitro or in vivo with iRNA by the immune adherence hemagglutination technique.Cellular immunity against tumor cells could be transferred in vitro or in vivo to lymphocytes through iRNA prepared from the spleen cells of syngeneic, allogeneic and xenogeneic animals immunized with the tumor cells.We prepared iRNA against antigens capable of inducing humoral antibody production in animals, i.e., RBCs, bacterial toxin, bacterial flagella and hapten-protein conjugates. Serum antibody was not demonstrated in recipient animals of iRNAs by single or repeated injections of these agents. However, in these animals an increase in the number of specific antibody-carrying cells was found as rosette-formers. It was found further that prior injection of iRNA could induce immunologic memory and produced a high titer of humoral antibody after a boosting stimulation with a small dose of the corresponding antigen. The required interval between the first iRNA and the second antigenic stimulation, and the minimal effective doses of iRNA and antigen are described.We studied the interaction of iRNA with either T- or B-cells and with both cells using adoptive transfer system, athymic nude mice and neonatally thymectomized (NT) mice. Immune RNAs against T-dependent and T-independent antigens could not induce the proliferation of antibody-carrying cells in cyclophosphamide-treated (B-cell depleted) mice. But these agents could induce the proliferation of rosette-formers, implying that iRNAs can replace some role of T-cells even against T-dependent antigens. B-cells can be directly activated by treatment with iRNA against both T-dependent and T-independent antigens, and they differentiated into rosette-formers.Passive transfers of iRNA were successful in establishing immunity against infection with S. enteritidis, or immunity to Salmonella flagella, RBCs and hapten-protein conjugates. The ability of iRNA to confer a secondary response of antibody formation is serially and passively transmissible in recipient animals. These facts suggest the presence of some mechanism that is responsible for the amplification of antigenic stimulation in the immune response. The RNA-dependent RNA polymerase and RNA-dependent DNA polymerase are presented and their role in the immune response is discussed.     

Interdependence of CD4+ T cells and malarial spleen in immunity to Plasmodium vinckei vinckei. Relevance to vaccine development

We studied immunity to the blood stage of the rodent malaria, Plasmodium vinckei vinckei, which is uniformly lethal to mice. BALB/c mice develop solid immunity after two infections and drug cure. The following experiments define the basis of this immunity. Transfer of pooled serum from such immune mice renders very limited protection to BALB/c mice and no protection to athymic nu/nu mice. Moreover, B cell-deficient C3H/HeN mice develop immunity to P. vinckei reinfection in the same manner as immunologically intact mice, an observation made earlier. In vivo depletion of CD4+ T cells in immune mice abrogates their immunity. This loss of immunity could be reversed through reconstitution of in vivo CD4-depleted mice with fractionated B-, CD8-, CD4+ immune spleen cells; however, adoptive transfer of fractionated CD4+ T cells from immune spleen into naive BALB/c or histocompatible BALB/c nude mice does not render recipients immune. In vivo depletion of CD8+ T cells did not influence the parasitemia in nonimmune or immune mice. Splenectomy of immune mice completely reverses their immunity. Repletion of splenectomized mice with their own spleen cells does not reconstitute their immunity. We conclude that some feature of the malaria-modified spleen acts in concert with the effector/inducer function of CD4+ T cells to provide protection from P. vinckei. To be consistent with this finding, a malaria vaccine may require a combination of malaria Ag to induce immune CD4+ T cells and an adjuvant or other vaccine vehicle to alter the spleen.     

Effect of tumor immunity on the distribution of labeled lymphoid cells in tumor-challenged mice

The distribution of ⁵¹Cr-labeled lymphoid cells from normal mice and mice immunized against a tumor were compared after intravenous inoculation of the labeled cells into normal syngeneic recipients. Spleen cell preparations from immune donors contained increased percentages of spleen and bone marrow-seeking cells, thus suggesting expansion of these cell populations when immunity to a tumor exists. Homing of labeled normal cells in tumor cell-injected normal animals was somewhat different from that seen in tumor cell-inoculated mice that were immunized against the tumor. In the latter case, accumulations of lymph node and spleen cells in recipient lymph nodes and bone marrow were consistently lower. In contrast, lymphoid cells from animals immunized against the tumor were found to accumulate in virtually the same percentages in lymphoid organs of normal and immune recipients. The behavior of lymphoid cell populations from thymus or bone marrow that consist mainly of precursor cells was unaffected by presence of malignancy and/or tumor immunity.     

γ-Irradiation facilitates the expression of adoptive immunity against established tumors by eliminating suppressor T cells

It was found that sublethal (550 rad) whole-body gamma-irradiation of mice bearing established immunogenic tumors enabled tumor-sensitized spleen cells infused intravenously 1 h later to cause complete tumor regression in all mice. In contrast, gamma-irradiation alone caused only a temporary halt in tumor growth, and immune cells gave practically no therapeutic effect at all. This result was obtained with the SA1 sarcoma, Meth A fibrosarcoma, P815 mastocytoma, and P388 lymphoma. Additional experiments with the Meth A fibrosarcoma revealed that the spleen cells from tumor-immune donors that caused tumor regression in gamma-irradiated recipients were T cells, as evidenced by their functional elimination by treatment with anti-Thy-1.2 antibody and complement. It was shown next that adoptive T-cell-mediated regression of tumors in gamma-irradiated recipients was inhibited by an intravenous infusion of spleen cells from donors with established tumors, but not by spleen cells from normal donors. The spleen cells that suppressed the expression of adoptive immunity were functionally eliminated by treatment with anti-Thy-1.2 antibody and complement. Moreover, they were destroyed by exposing the tumor-bearing donors to 500 rad of gamma-radiation 24 h before harvesting their spleen cells. The results are consistent with the interpretation that gamma-radiation facilitates the expression of adoptive T-cell-mediated immunity against established tumors by eliminating a population of tumor-induced suppressor T cells from the tumor-bearing recipient.     

Enterically induced immunologic tolerance. I. Induction of suppressor T lymphoyctes by intragastric administration of soluble proteins.

Specific immune unresponsiveness was induced in inbred mice (BDF1) by the administration of soluble ovalbumin (OVA) by gastric intubation. Anti-hapten (DNP) responses likewise were specifically diminished when animals were fed autologous carrier (OVA or keyhole limpet hemocyanin). Adoptive transfer of spleen cells demonstrated that the tolerant state could be maintained in irradiated recipient mice, and specific anergy could be transferred to normal recipient animals. Adoptive suppression was mediated by T lymphocytes, as demonstrated by nylon wool fractionation and susceptibility of the cells to anti-Thy 1.2 and complement. Transferred B cells had neither suppressive nor augmentative effects. Enteric administration of OVA also specifically diminished antigen-induced DNA synthesis of primed lymph node T cells, although suppressor cells were not identified in the lymph nodes per se.     

Factors responsible for immune resistance to L1210 murine leukemia in hyperimmune mice

Summary The serum of mice hyperimmune to L1210 leukemia was cytotoxic to L1210 cells and, to a much lesser extent, to P388 cells in the presence of complement. However, it did not suppress in vitro growth of L1210 cells, nor did it endow a recipient mouse with immunity to inoculated L1210 cells. This indicates that the serum did not play a significant role, if any, in immune protection of hyperimmune mice.Spleen and peritoneal exudate cells of hyperimmune mice suppressed the in vitro growth of L1210 but not of P388 cells. This is consistent with the fact that hyperimmune mice did not survive the inoculation of P388 cells. The immunocytes failed to suppress the in vitro growth of L1210 cells when preincubated with anti-Thy-1.2 antisera and complement. This, together with the finding that cell populations not adherent to a plastic dish suppressed in vitro growth of L1210 cells, indicates that T cells of immune spleen and peritoneal exudate cell populations were the effectors that suppressed in vitro growth of L1210 cells. Hyperimmune mice lost their immune protection in vivo following the administration of anti-thymocyte antisera, but not with carrageenan or silica, which resulted in the lethal growth of the inoculated L1210 cells. This indicates that T cells were in vivo effectors in immune protection.Hyperimmune spleen T cells endowed a recipient with immunity to L1210 leukemia when transferred in vivo. This confirmed the above results and suggests the applicability of immune cells in an adoptive immunotherapy approach.     

Effect of a neutrophilia-inducing tumor on hemopoietic stem cells in mice

The influence of neutrophilic stimulation on hemopoietic stem cells was studied in mice with tumor-induced neutrophilia. Transfusions of marrow cells from normal and neutrophilic tumor-bearing mice into lethally irradiated normal and tumor-bearing mice were performed. The number and the erythroid:granuloid (E:G) ratio of day 7 colonies in the recipient spleens and bones as well as the size of spleen colonies of recipient animals were determined. The E:G ratio of spleen and bone marrow colonies between normal and tumor-bearing mouse recipients and the number of spleen colonies did not differ significantly in either experiment. However, spleen colonies which developed in tumor-bearing irradiated mice were significantly larger than those which developed in normal recipients in both experiments. These studies indicated that while the line of differentiation taken by hemopoietic stem cells was not affected by the neutrophilic influence of the tumor, the tumor-bearing host environment appeared to enhance proliferation of transfused stem cells and/or their descendants. The stimulators of granulocytopoiesis in this model of neutrophilia appear to act on a population of progenitor cells more mature than the stem cells capable of forming 7-day colonies in the spleen and bone marrow of irradiated recipient mice.     

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.     

益气补肾方药对化疗荷瘤小鼠免疫功能的影响

目的　探讨益气补肾方药 (由金匮肾气方加味人参、黄芪组成 ,以下均称方药 ,TS)对环磷酰胺 (CY)处理的荷H2 2 瘤小鼠非特异免疫功能的影响。方法　用CY处理荷H2 2 瘤小鼠 ,建立免疫力低下动物模型。用乳酸脱氢酶 (LDH)释放法分别检测NK细胞、巨噬细胞 (M)的活性 ,用RT PCR法检测白细胞介素 12 (IL 12 )mRNA在脾脏细胞中的表达。结果　TS CY组小鼠吸光度A值为 1 332± 0 5 10 ,明显高于CY组小鼠 (P <0 0 1)。TS CY组小鼠A值为 1 12 9± 0 2 80 ,明显高于CY组小鼠 (P <0 0 1)。此方药能明显提高CY所致免疫力低下小鼠NK细胞、M的活性 ,增加IL 12在脾细胞中的表达。结论　该方药可以明显提高环磷酰胺所致免疫力低下荷瘤小鼠的非特异免疫功能。     

The augmentation of tumor-specific immunity by virus help

Summary The role of vaccinia virus-reactive helper T cells (Th) in augmenting in vivo generation of antitumor protective immunity and the Ly phenotype mediating the enhanced in vivo tumor immunity were investigated. C3H/HeN mice were inoculated i.p. with viable vaccinia virus to generate vaccinia virus-reactive Th activity. The mice were subsequently immunized i.p. with virus-infected syngeneic X5563 and MH134 tumor cells, and spleen cells from these mice were tested for in vivo tumor neutralizing activity. Immunization of virus-primed mice with virus-uninfected tumor cells and of virus-unprimed mice with virus-infected tumor cells failed to result in in vivo protective immunity. In contrast, spleen cells from mice immunized with virus-infected tumor cells subsequent to virus-priming exhibited potent tumor-specific neutralizing activities. Such an augmented generation of in vivo protective immunity was accompanied by enhanced induction of tumor-specific cytotoxic T lymphocyte (CTL) and antibody activities in X5563 and MH134 tumor systems, respectively. However, analysis of the effector cell type responsible for in vivo tumor neutralization revealed that enhanced in vivo immunity was mediated by Lyt-1⁺2^– T cells in both tumor systems. Moreover, the Lyt-1⁺2^– T cells exerted their function in vivo under conditions in which anti-X5563 tumor-specific CTL or anti-MH134 tumor-specific antibody activity was not detected in recipient mice. These results indicate that augmenting the generation of a tumor-specific Lyt-1⁺2^– T cell population is essential for enhanced tumor-specific immunity in vivo.This work was supported by Special Project Research-Cancer Bioscience from the Ministry of Education, Science and Culture