Modification of anti-tumor immunity by tolerogenic dendritic cells

Immunosuppressive functions of glucocorticoids (GC) can be mediated via various mechanisms, including the modulation of dendritic cells (DC). Our study investigates the effects of tolerogenic GC-treated DCs on NK and T cell anti-tumor responses in OT-1/Rag^?/? mice, expressing a transgenic TCR in CD8⁺ T cells. The effects caused by GC-treated DCs were compared to the responses to immunogenic, CpG-activated DCs. The effects of DCs on anti-tumor immune responses were analyzed using the EG7 tumor model, where the tumor cells express the peptide epitope recognized by OT-1 T cells. We observed that immunization with CpG and peptide-treated DCs protected against tumor growth by activation of NK cell response. Also, immunogenic DCs induced the expansion of cytotoxic CD8⁺OT-1 cells, expressing activation markers CD44 and CD69 and producing IFNγ. In contrast, the peptide and GC-treated DCs in OT-1 mice increased the numbers of immature Mac-1⁺CD27^? NK cells as well as Foxp3⁺ and IL-10 secreting CD8⁺OT-1 cells with suppressive properties. We conclude that the generation of tolerogenic DCs is one of many immunosuppressive mechanisms that can be induced by GC. Our study demonstrated that tolerogenic DCs modify anti-tumor immune response by suppressing NK cell activity and stimulating the formation of IL-10-secreting CD8⁺ Tregs.     

用糖基化磷脂酰肌醇B7-1锚定肿瘤细胞膜进行免疫治疗

目的 研制抗肿瘤免疫治疗的新疫苗.方法 用蛋白转化法将B7-1锚定在肿瘤细胞膜上,免疫C57BL-6小鼠后,一组小鼠取脾细胞进行T细胞扩增和细胞毒T淋巴细胞(CTL)功能检测,另一组小鼠进行肿瘤细胞接种试验.结果 用GPI-B7-1修饰的肿瘤细胞膜免疫小鼠后诱发了肿瘤特异性T细胞扩增和CTL.且该疫苗有一定的保护小鼠免受肿瘤细胞侵袭的作用.结论 GPI蛋白转化法为人类抗肿瘤免疫治疗提供了新的、有效的修饰肿瘤细胞膜的方法.     

肿瘤抗原联合CD40L致敏树突状细胞诱导CTLs杀伤K562细胞的实验研究

目的：以健康人外周血单核细胞为前体细胞，体外诱导为树突状细胞（DCs），负载K562细胞冻融抗原，并联合CD40L诱导产生特异性细胞毒性T淋巴细胞（CTLs）对K562细胞的杀伤作用。方法:密度梯度离心法、贴壁法分离健康人外周血单核细胞，应用rhGM-CSF、rhIL-4、rhTNF-α等细胞因子诱导扩增，培养DCs，用K562细胞冻融抗原联合rhsCD40L致敏DCs。实验分4组：K562细胞冻融抗原致敏DCs为实验组A，联合CD40L致敏DCs为实验组B，未致敏DCs为对照组A，单核细胞+异体淋巴细胞组为对照组B，观察CTLs对K562细胞的杀伤效应。结果:培养出具有典型特征的DCs，表达CD40最高达96%、CD86达97%、CD80为77%、CD1a为 69%，体外能诱导强烈的同种异体混合淋巴细胞增殖反应。在效靶比为 20∶1 时，实验组A对K562细胞的杀伤率为71.3%，实验组B为86.9%，对照组A为37.6%，对照组B为21.1%。实验组均显示高水平杀伤率，与对照组比较差异显著（P＜0.05），实验组B加CD40L杀伤率高于实验组A（P＜0.05） 。结论：K562细胞冻融抗原冲击致敏DCs能有效诱导T细胞特异性抗白血病作用，联合CD40L能增强其CTL的杀伤作用。     

The interaction between HMGB1 and TLR4 dictates the outcome of anticancer chemotherapy and radiotherapy

Summary: For the last four decades, the treatment of cancer has relied on four treatment modalities, namely surgery, radiotherapy, cytotoxic chemotherapy, and hormonotherapy. Most of these therapies are believed to directly attack and eradicate tumor cells. The emerging concept that cancer is not just a disease of a tissue or an organ but also a host disease relies on evidence of tumor-induced immunosuppression and polymorphisms in genes involved in host protection against tumors. This theory is now gaining new impetus, based on our recent data showing that optimal therapeutic effects require the immunoadjuvant effect of tumor cell death induced by cytotoxic anticancer agents. Here, we show that the release of the high mobility group box 1 protein (HMGB1) by dying tumor cells is mandatory to license host dendritic cells (DCs) to process and present tumor antigens. HMGB1 interacts with Toll-like receptor 4 (TLR4) on DCs, which are selectively involved in the cross-priming of anti-tumor T lymphocytes in vivo . A TLR4 polymorphism that affects the binding of HMGB1 to TLR4 predicts early relapse after anthracycline-based chemotherapy in breast cancer patients. This knowledge may be clinically exploited to predict the immunogenicity and hence the efficacy of chemotherapeutic regimens.     

Immunoglobulin isotype regulation by antigen-presenting cells in vivo

The isotype and magnitude of the B cell response clearly depends on the in vivo activation of T helper (Th) cells which secrete different lymphokines. Since Th are activated by the presentation of the antigen on specialized cells, we wished to test whether the nature of the antigen-presenting cells (APC) influences the isotypic profile of the humoral response. Data are presented showing that antigen-pulsed dendritic cells (DC) and peritoneal macrophages induce the synthesis of specific antibodies when injected in syngeneic animals. By contrast, a single injection of antigen-pulsed resting B cells does not prime the mice in vivo. Moreover, the injection of antigen-pulsed DC induces the synthesis of specific IgG2a and IgG1 antibodies, whereas peritoneal macrophages favor the production of IgG1 and IgE antibodies specific for the antigen. These data show that the isotype and the amplitude of the B cell response can be regulated by the nature of the APC, and indirectly suggest that Th cell differentiation is controlled at the level of antigen presentation.     

Simultaneous blocking of human Toll-like receptors 2 and 4 suppresses myeloid dendritic cell activation induced by Mycobacterium bovis bacillus Calmette-Guérin peptidoglycan

The Mycobacterium bovis bacillus Calmette-Guérin (BCG) cell wall skeleton (CWS) consists of mycolic acids, arabinogalactan, and peptidoglycan (PGN) and activates Toll-like receptor 2 (TLR2) and TLR4. Here we investigated the ability of the essential portion of highly purified BCG CWS to support the TLR agonist function by using the following criteria: myeloid dendritic cell (DC) maturation, i.e., tumor necrosis factor alpha (TNF-alpha) production and CD83/CD86 up-regulation. The purified PGN region was sufficient to activate TLR2 and TLR4 in mouse DCs and macrophages; in TLR2 and TLR4 double-knockout cells the BCG PGN-mediated TNF-alpha production ability was completely impaired. Likewise, stimulation with BCG CWS of HEK293 cells expressing either human TLR2 or TLR4, MD-2, and CD14 resulted in NF-kappa B activation as determined by a reporter assay. Notably, specific blockers of extracellular human TLR2 (an original cocktail of monoclonal antibodies TLR2.45 and TH2.1) and TLR4 (E5531) inhibited BCG CWS-mediated NF-kappa B activation by 80%. Using this human TLR blocking system, we tested whether human myeloid DC maturation was TLR2 and TLR4 dependent. BCG PGN-mediated DC maturation was blocked by 70% by suppression of both TLR2 and TLR4 and by 30 to 40% by suppression of either of these TLRs. Similar but less profound suppression of BCG CWS-mediated DC maturation was observed. Hence, the presence of BCG PGN is a minimal requirement for activation of both TLR2 and TLR4 in human DCs, unlike the presence of PGNs of gram-positive bacteria, which activate only TLR2. Unexpectedly, however, BCG PGN, unlike BCG CWS, barely activated NF-kappa B in HEK293 cells coexpressing TLR2 plus TLR1, TLR2 plus TLR4, TLR2 plus TLR6, or TLR2 plus TLR10, suggesting that PGN receptors other than TLR2 and TLR4 present on human DCs but not on HEK293 cells are involved in TLR signaling for DC activation.     

Bacteriophages support anti-tumor response initiated by DC-based vaccine against murine transplantable colon carcinoma

Bacteriophages in eukaryotic hosts may behave as particulate antigens able to activate the innate immune system and generate adaptive immunity. Dendritic cells (DCs) play a key role in the initiation of the immune response, mainly by priming T cell-mediated immunity. For this reason, they are increasingly applied as an adjuvant for effective anti-tumor therapies in animal models as well as in a few clinical trials. The presented study focused on the application of mouse DCs which were activated with T4 bacteriophages (T4 phages, T4) and further loaded with tumor antigens (TAg) in inducing an anti-tumor response. The activation of bone marrow-derived DCs with T4 phages and TAg resulted in augmentation of their differentiation marker expression accompanied by an enhanced ability to prime T cells for IFN-gamma production. These activated DCs (BM-DC/T4+TAg) were used in experimental immunotherapy of C57BL/6 mice bearing advanced MC38 colon carcinoma tumors. As a result of their triple application, a significant tumor growth delay, up to 19 days, was observed compared with the controls - treated with BM-DCs activated only with T4 phages, TAg, or lipopolysaccharide solution ["solvent"], where the tumor growth delay did not exceed 7 days. The percentage of tumor growth inhibition estimated 10 days after the third cell injection ranged from 32% (for animals treated with BM-DC/TAg cells) to 76% (for animals treated with BM-DC/T4+TAg cells) over the tumor-bearing untreated control mice. The obtained data indicate that in vitro interactions between T4 phages and BM-DCs followed by TAg activation caused augmentation of the anti-tumor effect when DCs were used as a vaccine for tumor-bearing mice treatment. Therefore, pretreatment of DCs with the phages may be considered as a beneficial element of a novel strategy in anti-tumor immunotherapy.     

Lymphocyte activation gene-3 induces tumor regression and antitumor immune responses

The lymphocyte activation gene-3 (LAG-3) product is an MHC class II ligand related to CD4. We investigated whether LAG-3 could be used in vivo to stimulate MHC class II(+) antigen-presenting cells (APC), such as resident macrophages or dendritic cells known to play a crucial role in processing and presenting of antigens to the immune system. We first introduced human (h) LAG-3 or mouse LAG-3 into three types of tumor cells (MCA 205, TS / A and RENCA) to evaluate its capacity to stimulate a tumor-specific immune response in vivo. In contrast to the progressive growth of wild-type cells in syngeneic mice, LAG-3-transfected tumors completely regressed or their growth was markedly reduced. Mice were significantly to completely protected against a rechallenge with parental tumor cells. Protection induced by hLAG-3(+) tumor cells involved recruitment of a CD8(+) T cell response since nu / nu mice and CD8-depleted mice did not reject tumors, and a systemic tumor-specific CTL activity was induced. Co-administration of soluble LAG-3 with wild-type tumor cells also markedly reduced primary tumor growth. Interestingly, immunization with LAG-3(+) tumor cells or co-administration of soluble LAG-3 with irradiated wild-type tumor cells reduced the growth of pre-established tumors. We therefore suggest that LAG-3 could be used as a vaccine adjuvant for its ability to trigger APC via MHC class II molecules.     

Optimizing dendritic cell vaccine for immunotherapy in multiple myeloma: tumour lysates are more potent tumour antigens than idiotype protein to promote anti-tumour immunity

Dendritic cells (DCs) are the most potent antigen-presenting cells and are the mediators of T cell immunity. Many investigators have explored the potential of using DCs as a vaccine for tumour-derived antigens in immunotherapy of B cell malignancies, and the results have been disappointing. To search for better tumour antigens to improve the efficacy of DC-based immunotherapy in myeloma, we evaluated and compared the efficacy of the vaccination of DCs pulsed with idiotype (Id) or tumour lysate in the 5TGM1 myeloma mouse model. Our results showed that Id- or tumour lysate-pulsed DC vaccines protected mice efficiently against developing myeloma, retarded tumour growth, induced tumour regression against established tumour and protected surviving mice from tumour rechallenge. The therapeutic responses were associated with an induction of strong humoral immune responses, including anti-Id or anti-lysate antibodies, and cellular immune responses including myeloma-specific CD8⁺ cytotoxic T lymphocytes, CD4⁺ type 1 T helper cells and memory T cells in mice receiving Id- or tumour lysate-pulsed DC vaccines. In addition, our studies showed that tumour lysate-pulsed DCs were more potent vaccines than the Id-pulsed DC vaccines to promote anti-tumour immunity in the model. This information will be important for improving the strategies of DC-based immunotherapy for patients with myeloma and other B cell tumours.     

Combination treatment with IL-2 and anti-IL-2 mAbs reduces tumor metastasis via NK cell activation

Combination treatment consisting of IL-2 together with anti-IL-2 mAbs results in markedly larger increases in the numbers of CD8(+) T cells, dendritic cells (DCs) and NK cells in vivo compared with the results observed with injections of IL-2 or the antibodies alone. We previously showed that this combination treatment overcomes the problems associated with the short half-life of IL-2 in vivo. Importantly, the combination treatment but not IL-2 or the anti-IL-2 mAbs alone protected the mice against tumor metastases in the lungs. Here we have investigated which cell types are responsible for this protective immunity against tumors. We analyzed tumor metastases in mice that were depleted of DCs, CD8(+) T cells or NK cells. DC-deficient, diphtheria toxin receptor-expressing mice injected with diphtheria toxin as well as B cell- and T cell-deficient RAG-2-knockout mice were protected against tumors after they were administered the combination treatment. On the other hand, mice that were depleted of NK cells using anti-asialo-GM1 antibodies did not exhibit the anti-tumor activity after treatment with IL-2 combined with anti-IL-2 mAbs. Thus, these data demonstrate that NK cells, but not DCs, or CD8(+) T cells mediate the anti-tumor effect induced by this combination treatment. Therefore, combining neutralizing anti-IL-2 mAbs with IL-2 may be clinically useful to effectively enhance IL-2-mediated NK cell activities.     

The role of T helper 17 and regulatory T cells in tumor microenvironment

T helper 17 (Th17) cells were first described as a novel T helper cell lineage independent from Th1 and Th2 subsets. Th17 cells play vital roles in inflammation and tumor immunity. It causes the dissipation of antitumor immunity and contribution to the survival of tumor cells, worsening tumor growth and metastasis. Tumor-infiltrating Th17 cells were seen innumerous cancers in mice and humans. There has been an association between intratumoral Th17 cell infiltration and both good and bad prognoses. Besides the protumoral roles defined for IL-17 andTh17 cells, several reports have shown that Th17 cells also drive antitumoral immunity. Various mechanisms by which Th17 cells control tumor growth are as following: recruitment of several immune cells including DCs, CD4⁺ T cells, and CD8⁺ T cells within tumors, activation of CD8⁺ T cells, and probably plasticity toward Th1 phenotype, related to IFN-γ and TNF-α production. Regulatory T cells (Tregs) have been exhibited to infiltrate human tumors and are believed to restrict antitumor immunity. The effect of Treg cells has been more controversial. Whereas some studies have proposed that a high density of Treg cells within the tumor associated with a poor clinical prognosis, other studies have presented a positive clinical prognosis, underlining the importance of elucidating the clinical significance of Treg cells further. Treg and Th17 cells play both positive and negative roles in regulating antitumor immune responses. In spite of the presence of these cells, yet some tumors develop and grow. These T cells by themselves are not adequate to efficiently mount antitumor immune responses.     

Differential mediator production by dendritic cells upon toll-like receptor stimulation does not impact T cell cytokine expression

Dendritic cells are key components of successful immunological responses bridging innate and adaptive defenses. In this study we wanted to know whether ligation of toll-like receptors (TLR) expressed by dendritic cells would induce differential proinflammatory mediator expression and whether these dendritic cells would differentially impact T cell function. For this purpose bone marrow-derived dendritic cells from OTII mice were used. The dendritic cells showed detectable levels of TLR1, 2, 4, 6, 7, 8 and 9, with TLR2 and TLR4 expressed at the highest levels. To determine whether TLR ligation differentially influenced proinflammatory mediator expression the dendritic cells were stimulated with peptidoglycan (PGN) or lipopolysaccharide (LPS) for TLR2 or TLR4, respectively. Comparisons were made to dendritic cells exposed to TNF-alpha or saline as controls. Whereas, both LPS and PGN were equally effective at inducing CXCL1 and TNF-alpha expression from the dendritic cells, LPS was unique at inducing CCL2 expression, and PGN was unique at inducing IL-1beta expression. Despite these differences, LPS and PGN treated dendritic cells were equally effective at eliciting IFN-gamma expression from T cells in an antigen-specific manner. These data indicate that ligation of TLR by components of Gram+ and Gram- bacteria differentially influence dendritic cell proinflammatory mediator expression, and that differential mediator production by dendritic cells upon TLR stimulation does not impact T cell cytokine production.     

Murine dendritic cells pulsed in vitro with tumor antigen induce tumor resistance in vivo

The aim of this work is to induce tumor resistance to a B cell lymphoma in BALB/c mice using elements of the immune system. It has indeed been shown by us and by others that antigen-presenting cells (APC) like dendritic cells can induce efficient immune responses and can even substitute for Freund's adjuvant. Here we show that mice immunized with syngeneic dendritic cells pulsed in vitro with tumor antigen (BCL1 idiotype expressed by lymphoma cells) are protected against a subsequent tumor inoculation. The in vivo resistance can be correlated with the induction of a humoral response specific for the idiotype expressed by the tumor. No such protection can be achieved when B cells are used as APC. These data show that effector cells in tumor-bearing animals can be recruited and activated using dendritic cells, providing long-lasting immune surveillance.     

TLR2 engagement on CD8 T cells lowers the threshold for optimal antigen-induced T cell activation

TLR have a crucial role in the detection of microbial infection in mammals. Until recently, most investigations on TLR have focused on cells of the innate immune system and on the role of TLR in the initiation of antigen-specific responses following recognition of microbial products by APC. Here, we report that murine T cells express TLR1, TLR2, TLR6, TLR7 and TLR9 mRNA. Using CD8 T cells from F5 TCR-transgenic mice, we demonstrate that the lipopeptide Pam(3)CysSK(4) (Pam), a synthetic analog of bacterial and mycoplasmal lipoproteins that recognizes TLR1/2 complex, costimulates antigen-activated T cells. Costimulation with Pam permits an increased cell proliferation and survival associated with a sustained CD25 expression and an enhanced expression of Bcl-xL anti-apoptotic protein. In addition, we show that costimulation with Pam up-regulates IFN-gamma production but also granzyme B secretion and cytotoxic activity of antigen-activated T cells, indicating that TLR2 engagement enhances the major effector functions of CD8 T cells. Finally, we demonstrate that TLR2 engagement on T cells lowers the activation threshold for costimulatory signals delivered by APC.     

A comparative study of the JAM test and 51Cr-release assay to assess the cytotoxicity of dendritic cells on hematopoietic tumor cells

Dendritic cells (DCs) are potent antigen presenting cells and possess a direct anti-tumor cytotoxic ability. Nevertheless, the mechanism of anti-tumor cytotoxicity by DCs and the methods for its evaluation are not fully elucidated. In order to clarify this mechanism of cytotoxicity, we examined the ability of DCs 1) to suppress [³H] thymidine (³H-TdR) uptake by tumor cells; 2) to induce cytolysis on ⁵¹Cr-labeled tumor cells; 3) and to induce DNA fragmentation on ³H-TdR labeled tumor cells (JAM test). Cytolysis and DNA fragmentation are markers of necrotic and apoptotic mechanisms of cytotoxicity in vitro, respectively. DCs inhibited approximately 38.6% to 54.8% of the growth of B4D6, NB4, U937, and Daudi cells as evaluated by the uptake of ³H-TdR. However no cytolysis was verified by ⁵¹Cr-release assay. On the other hand, cytotoxicity rates found using the JAM test ranged from 3 to 81% depending on the cell line and the effector to target cell ratio. The discrepancy of cytotoxicity between ⁵¹Cr-release assay and the JAM test may be due to the phagocytosis of apoptotic tumor cells or the absorption of released ⁵¹Cr by DCs surrounding the target cells. In conclusion, the JAM test was more sensitive than the 4-h and the 10-h ⁵¹Cr-release assay to investigate cytotoxicity mediated by DCs toward hematopoietic tumor cell lines in vitro.     

Engagement of Toll-like receptor 2 enhances interleukin (IL)-17+ autoreactive T cell responses via p38 mitogen-activated protein kinase signalling in dendritic cells

Functional analysis of single Toll-like receptors (TLRs) in vivo is necessary to understand how they shape the ocular inflammation involved in uveitis. In this study we explored the role and mechanisms of TLR-2 agonists on the autoreactive T helper type 17 (Th17) response in experimental autoimmune uveitis (EAU). Treatment by peptidoglycan (PGN), a specific TLR-2 agonist, remarkably increased mRNA levels of Th17-lineage genes interleukin (IL)-17A, IL-21 and RAR-related orphan receptor (ROR)γt and promoted antigen-specific Th17 response in EAU mice. A mixture of PGN and interphotoreceptor retinoid-binding protein peptide (IRBP_161–180) could effectively induce EAU in the absence of complete Freund''s adjuvant (CFA). PGN treatment also enhanced the pathogenic activities of activated antigen-specific Th17 cells in vivo. PGN significantly increased the production of IL-1β, IL-6 and IL-23 of dendritic cells (DCs) and enhanced their ability to promote IL-17⁺ uveitogenic T cells. Enhanced immunostimulatory activities of PGN-DCs depend upon p38 activation. Inhibition of p38 mitogen-activated protein kinase (MAPK) activity dramatically decreased IL-17 gene expression and antigen-specific Th17 responses stimulated by PGN-DCs. Our findings suggest that PGN treatment dramatically promotes the IL-17⁺ uveitogenic T cell responses via enhancing the immunostimulatory activities of DCs. This effect may be mediated, at least in part, by activation of the p38 signalling pathway in DCs.     

致敏树突状细胞活化细胞毒性T细胞抗肿瘤作用的研究

目的：研究树突状细胞（DCs）激活的细胞毒性T细胞的抗肿瘤及预防肿瘤发生的作用。 方法： 细胞因子诱生人PBMC未成熟DCs，加入肿瘤细胞抗原提取物致敏DCs产生成熟DCs；通过细胞形态、表面标记鉴定成熟DCs，MTT法测成熟DCs活化的细胞毒性T细胞(CTL)的体外杀伤活性；裸鼠体内注射活化CTL观察其抑制移植瘤生长及发生的作用。 结果： 经过7 d培养，获得大量形态典型、具有强烈刺激增殖能力、高表达CD80(63.5%)、CD83（67.6%）和CD3/ HLA-DR(83.2%)的DCs。其活化的CTL在20∶1效靶比时对抗原来源细胞株自身的杀伤率达75%以上，对同系细胞株的杀伤活性为35%-45%，对其它种系肿瘤细胞仅有微弱杀伤力（P<0.01）。CTL对裸鼠结肠癌HT-29移植瘤有特异性的生长抑制和预防生成作用（P<0.05）。CTL治疗组肿瘤组织中PCNA表达水平显著低于对照组（P<0.05）。 结论： 肿瘤细胞抗原活化的DC诱导CTL对肿瘤有特异性的杀伤作用，体内应用可特异性抑制移植结肠癌的生长或预防小鼠结肠癌移植瘤的发生。     

Dendritic cells in the tumor microenvironment: prognostic and theranostic impact

Among all immune cells, dendritic cells (DC) are the most potent APCs in the immune system and are central players of the adaptive immune response. There are phenotypically and functionally distinct DC populations derived from blood and lymphoid organ including plasmacytoid DC (pDC), conventional DC (cDC1 and cDC2) and monocyte-derived DC (moDC). The interaction between these different DCs and tumors is a dynamic process where DC-mediated cross-priming of tumor specific T cells is critical in initiating and sustaining anti-tumor immunity. Their presence within the tumor tends to induce T cell responses and to reduce cancer progression and is associated with improved patient survival. This review will focus on the distinct tumor-associated DCs (TADC) subsets in the tumor microenvironment (TME), their roles in tumor immunology and their prognostic and/or predictive impact in human cancers. The development of therapeutic immunity strategies targeting TADC is promising to enhance their immune-stimulatory capacity in cancers and improve the efficacy of current immunotherapies including immune checkpoint inhibitor (ICI) blockade and DC-based therapies.     

Augmentation of anti-tumor activity by immunization with Mycobacterium tuberculosis (Tbc) and tuberculin-coupled tumor cells

The anti-tumor effect of immunization with heat-killed Mycobacterium tuberculosis (Tbc) and Tuberculin (PPD)-coupled syngeneic tumor cells was examined in vivo. Three tumor cell lines were employed. Immunization of Tbc-primed BALB/c mice with PPD-coupled syngeneic Meth-A tumor cells displayed a potent anti-tumor effect on viable Meth-A cells inoculated subcutaneously. Neither PPD-coupled LLC (Lewis Lung Carcinoma) cells nor sonicated PPD-coupled Meth-A cells were capable of immunizing these mice. PPD-coupled syngeneic whole tumor cells were indispensable for induction of this tumor-specific resistance. Immunization of Tbc-primed C3H/He mice with PPD-coupled syngeneic MH134 tumor cells did not elicit anti-tumor activity against MH134, but additional pretreatment of mice with cyclophosphamide brought on an anti-tumor effect. Antimetastatic reactivity was investigated in C57BL/6 mice bearing LLC, with a reduction in metastases noted. This antimetastatic effect was observed even when the mice were immunized with PPD-coupled LLC cells three days after removal of the initial tumor. Immunization with Tbc and PPD-coupled Meth-A cells together with intraperitoneal administration of murine or rat interleukin 2 (IL 2) further augmented anti-Meth-A resistance. Murine IL 2 further inhibited tumor growth during the early stage, while rat IL 2 showed an anti-tumor effect throughout the course of tumor growth.