Establishment of a tumor-specific immunotherapy model utilizing TNP-reactive helper T cell activity and its application to the autochthonous tumor system

Preinduction of potent hapten-reactive helper T cell activity and subsequent immunization with hapten-coupled syngeneic tumor cells result in enhanced induction of tumor-specific immunity through T-T cell collaboration between anti-hapten helper T cells and tumor-specific effector T cells. On the basis of this augmenting mechanism, a tumor-specific immunotherapy protocol was established in which a growing tumor regresses by utilizing a potent trinitrophenyl (TNP)-helper T cell activity. C3H/He mice were allowed to generate the amplified (more potent) TNP-helper T cell activity by skin painting with trinitrochlorobenzene (TNCB) after pretreatment with cyclophosphamide. Five weeks later, the mice were inoculated intradermally with syngeneic transplantable X5563 tumor cells. When TNCB was injected into X5563 tumor mass, an appreciable number of growing tumors, in the only group of C3H/He mice in which the amplified TNP-helper T cell activity had been generated were observed to regress (regressor mice). These regressor mice were shown to have acquired tumor-specific T cell-mediated immunity. Such immunity was more potent than that acquired in mice whose tumor was simply removed by surgical resection. These results indicate that in situ TNP haptenation of the tumor cells in TNP-primed mice can induce the enhanced tumor-specific immunity leading to the regression of a growing tumor. Most importantly, the present study further investigates the applicability of this TNP immunotherapy protocol to an autochthonous tumor system. The results demonstrate that an appreciable percent of growing methylcholanthrene-induced autochthonous tumors regressed by the above TNP immunotherapy protocol. Thus, the present model provides an effective maneuver for tumor-specific immunotherapy in syngeneic transplantable as well as autochthonous tumor systems.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary Preinduction of potent haptenic muramyl dipeptide (MDP)-reactive helper T cell activity and subsequent immunization with MDP hapten-coupled syngeneic tumor cells resulted in enhanced induction of tumor-specific immunity through T-T cell collaboration between anti-MDP hapten helper T cells and tumor-specific effector T cells. The present study establishes two types of tumor-specific immunotherapy protocols utilizing helper T cells against MDP hapten cross-reactive with Bacillus Calmette Guérin (BCG). In the first model, naive normal C3H/He mice or mice in which MDP hapten-reactive helper T cells had been generated by BCG-sensitization were inoculated i.d. with syngeneic X5563 tumor cells. When both groups of mice were allowed to generate MDP hapten-modified tumor cells in the tumor mass in situ by intratumoral injection of MDP hapten, an appreciable number of growing tumors in the BCG-presensitized but not in the unsensitized group were observed to regress. In the second model, a growing X5563 tumor mass was removed by the surgical resection 9 days after the tumor implantation. Approximately 90% of C3H/He mice receiving such treatment died from tumor metastasis by about 30 days after the tumor resection. However, immunization of mice with MDP hapten-coupled X5563 tumor cells subsequent to the tumor resection resulted in an increased survival rate. Such protection from the tumor metastasis was appreciably stronger when compared to the protection obtained by immunization with MDP hapten-uncoupled tumor cells. The mice surviving in both models were also demonstrated to retain X5563 tumor-specific immunity. These results indicate that the presentation of MDP hapten-modified tumor cells to BCG-sensitized recipients results in potent tumor-specific immunity which contributes to the regression of the primary tumor or inhibition of metastatic tumor growth.This work was supported by a Grant-in-Aid for the Special Project Cancer Bioscience from the Ministry of Education, Science and Culture, Japan     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A previous paper has demonstrated that enhanced tumor-specific immunity could be induced by priming mice with Bacillus Calmette Guerin (BCG) and subsequently immunizing them with syngeneic tumor cells modified with BCG-cross-reactive muramyl dipeptide (MDP) hapten [15]. The present study establishes a tumorspecific immunotherapy protocol for a murine chronic leukemia based on the above T-T cell collaboration between antitumor effector T cells and anti-MDP hapten helper T cells induced by BCG priming. BALB/c mice which had been primed to BCG were injected intravenously (i.v.) with viable, syngeneic BCL1 leukemia cells. One week later, these mice were immunized intraperitoneally (i.p.) with unmodified or MDP hapten-modified, 10,000 R X-irradiated BCL1 cells, followed by 4 booster immunizations at 5-day intervals. The administration of unmodified BCL1 tumor cells into BCG-primed mice failed to prevent them from tumor death due to the persistent growth of preinjected BCL1 cells. In contrast, the immunization of BCG-primed, BCL1 leukemia-cell-bearing mice with MDP-modified BCL1 cells resulted in a high growth inhibition of leukemia cells and protection of these mice from death by leukemia. It was also revealed that potent tumorspecific, T-cell-mediated immunity was generated in mice which survived in this immunotherapy model. Thus, these results indicate that administration of MDP hapten-modified, syngeneic leukemia cells into leukemia-bearing mice which have been primed with BCG results in potent tumor-specific, T-cell-mediated immunity attributable to preventing the growth of disseminated leukemic cells.This work was supported by a Grant-in-Aid for the Special Project Cancer-Bioscience from the Ministry of Education, Science, and Culture, Japan Abbreviations used: TATA, tumor-associated transplantation antigens; MDP, muramyl dipeptide; MTP, muramyl tripeptide; BCG, Bacillus Calmette Guerin     

Augmented induction of tumor-specific resistance by priming with Mycobacterium tuberculosis (TBC) and subsequent immunization with PPD-coupled syngeneic tumor cells

The present study investigates the augmenting effect of tuberculin- (PPD) reactive amplifier T cells on the induction of syngeneic tumor immunity. PPD-reactive helper (amplifier) T cell activity was generated in C3H/HeJ mice by appropriate immunization with heat-killed Mycobacterium (Tbc). Immunization of these Tbc-primed mice with PPD-coupled syngeneic X5563 tumor cells led to augmented generation of in vivo tumor-neutralizing activity contingent on the presence of PPD-reactive amplifier T cell activity. Splenic T cells from these mice exhibited potent tumor-neutralizing activity using Winn's assay, whereas spleen cells from mice not primed with Tbc before PPD-X5563 immunization failed to neutralize viable X5563 tumor cells. After establishing that the neutralizing activity was tumor specific and mediated by T cells, the applicability of this augmentation of tumor-specific immunity to an immunotherapy model was explored. Immunization with PPD-X5563 in the early stages of the tumor-bearing state induced potent anti-tumor activity sufficient to reject the growing tumor. Pretreatment of mice with cyclophosphamide or light x-irradiation (250 R), procedures that eliminate suppressor cell activity nonspecifically, before priming with Tbc further potentiated the anti-tumor activity under these conditions. Thus, the present study elucidates the augmenting effect of PPD-reactive amplifier T cells in the induction of tumor-specific immunity and provides an effective method of immunotherapy in tumor-bearing animals.     

The generation of tumor-specific in vivo protective immunity in the tumor mass from mice rendered tolerant to tumor antigens

Summary C3H/He mice were inoculated i.v. with 10⁶ heavily X-irradiated syngeneic X5563 plasmacytoma cells 3 times at 4 day intervals. When these mice received an appropriate immunization procedure consisting of i. d. inoculation of viable tumor cells plus the surgical resection of the tumor which enables i.v. nonpresensitized mice to produce anti-X5563 immunity, they failed to develop tumor-specific immunity. This was demonstrated by the abrogation in potential of spleen and lymph node cells to generate in vivo protective immunity. In contrast, the tumor mass from X5563 tumor-bearing mice which had received the i.v. presensitization contained comparable anti-X5563 tumor neutralizing activity to that obtained from the tumor mass from nonpresensitized, X5563 tumor-bearing mice. Such an in vivo protective immunity was revealed to be mediated by tumor-specific T cells. These results demonstrate the differential generation and antitumor capability of tumor infiltrating T cells and T cells in lymphoid organs from mice which are in the tumor-specific tolerant state. The results are discussed in the context of potential utilization of tumor infiltrating in vivo protective T cells to enhance the local tumor-specific immunity in tumor-specific tolerant mice.This work was supported by Special Project Research-Cancer Bioscience from the Ministry of Education, Science and Culture     

Induction of T-cell-mediated immunity against MethA fibrosarcoma by intratumoral injections of a bacillus Calmette-Guérin nucleic acid fraction

Summary MY-1, which consists of DNA and RNA extracted and purified from bacillus Calmette-Guérin (BCG), has been shown to have strong antitumor activity against various experimental tumors. To examine the role of T cells in the antitumor mechanism of MY-1, the effect of MY-1 injection on the development of tumor-specific immunity against MethA fibrosarcoma was investigated. MY-1 injections inhibited tumor growth less effectively in T-cell-deficient nude mice than in normal BALB/c mice. MethA tumor growth was suppressed after inoculation with L3T4-positive lymphocytes from tumor-bearing mice treated with MY-1. MethA-specific delayed-type hypersensitivity was also detected in tumor-bearing mice treated with MY-1. Immunohistochemical analyses showed that many L3T4-positive and a few Lyt2-positive cells infiltrated the regressing tumors. These results indicate that intratumoral MY-1 injections induce a MethA-specific, L3T4-positive cell-mediated, delayed-type hypersensitivity, which is necessary for the tumor regression.     

Studies on the recovery from tolerance to tumor antigens

C3H/He mice were injected i.v. with heavily X-irradiated syngeneic X5563 tumor cells three times at 4-day intervals. This regimen resulted in the abrogation of the potential to generate X5563 tumor-specific T cell-mediated immunity as induced by i.d. inoculation of viable X5563 tumor cells followed by surgical resection of the tumor, representing the tolerance induction. Although such a tumor-specific tolerant state was long-lasting, the recovery of anti-X5563 effector T cell responses was observed when the above ordinary immunization procedure was performed 6 months after the tolerance induction. The present study investigated whether the recovery from the tolerance can be accelerated by applying a helper-effector T-T cell interaction model in which enhanced anti-X5563 immunity is obtained by priming mice with BCG and by immunizing X5563 tumor cells modified with BCG cross-reactive MDP hapten (designated as L4-MDP) in the presence of anti-L4-MDP helper T cells preinduced with BCG. The results demonstrated that BCG-primed mice which received the tolerance regimen failed to generate anti-X5563 immunity when the ordinary immunization was performed 2 or 3 months after the tolerance induction. In contrast, the immunization of BCG-primed and X5563-tolerant mice with L4-MDP-coupled X5563 tumor cells at comparable timing to that of the ordinary immunization were capable of generating potent X5563-specific in vivo protective T cell-mediated immunity. As control groups, BCG-primed or unprimed tolerant mice did not develop anti-X5563 immunity when immunized with L4-MDP-uncoupled or L4-MDP-coupled tumor cells, respectively. These results indicate that immunization of BCG-primed, tumor-tolerant mice with L4-MDP-modified tumor cells results in accelerated recovery from the tumor tolerance.     

Oncolytic Adenovirus Expressing IL-23 and p35 Elicits IFN-γ- and TNF-α-Co-Producing T Cell-Mediated Antitumor Immunity

Cytokine immunogene therapy is a promising strategy for cancer treatment. Interleukin (IL)-12 boosts potent antitumor immunity by inducing T helper 1 cell differentiation and stimulating cytotoxic T lymphocyte and natural killer cell cytotoxicity. IL-23 has been proposed to have similar but not overlapping functions with IL-12 in inducing Th1 cell differentiation and antitumor immunity. However, the therapeutic effects of intratumoral co-expression of IL-12 and IL-23 in a cancer model have yet to be investigated. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral inoculation of oncolytic adenovirus co-expressing IL-23 and p35, RdB/IL23/p35. Intratumoral administration of RdB/IL23/p35 elicited strong antitumor effects and increased survival in a murine B16-F10 syngeneic tumor model. The levels of IL-12, IL-23, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were elevated in RdB/IL23/p35-treated tumors. Moreover, the proportion of regulatory T cells was markedly decreased in mice treated with RdB/IL23/p35. Consistent with these data, mice injected with RdB/IL23/p35 showed massive infiltration of CD4⁺ and CD8⁺ T cells into the tumor as well as enhanced induction of tumor-specific immunity. Importantly, therapeutic mechanism of antitumor immunity mediated by RdB/IL23/p35 is associated with the generation and recruitment of IFN-γ- and TNF-α-co-producing T cells in tumor microenvironment. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-23 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.     

Tumor regression induced by intratumor therapy with a disabled infectious single cycle (DISC) herpes simplex virus (HSV) vector, DISC/HSV/murine granulocyte-macrophage colony-stimulating factor, correlates with antigen-specific adaptive immunity

Direct intratumor injection of a disabled infectious single cycle HSV-2 virus encoding the murine GM-CSF gene (DISC/mGM-CSF) into established murine colon carcinoma CT26 tumors induced a significant delay in tumor growth and complete tumor regression in up to 70% of animals. Pre-existing immunity to HSV did not reduce the therapeutic efficacy of DISC/mGM-CSF, and, when administered in combination with syngeneic dendritic cells, further decreased tumor growth and increased the incidence of complete tumor regression. Direct intratumor injection of DISC/mGM-CSF also inhibited the growth of CT26 tumor cells implanted on the contralateral flank or seeded into the lungs following i.v. injection of tumor cells (experimental lung metastasis). Proliferation of splenocytes in response to Con A was impaired in progressor and tumor-bearer, but not regressor, mice. A potent tumor-specific CTL response was generated from splenocytes of all mice with regressing, but not progressing tumors following in vitro peptide stimulation; this response was specific for the gp70 AH-1 peptide SPSYVYHQF and correlated with IFN-gamma, but not IL-4 cytokine production. Depletion of CD8(+) T cells from regressor splenocytes before in vitro stimulation with the relevant peptide abolished their cytolytic activity, while depletion of CD4(+) T cells only partially inhibited CTL generation. Tumor regression induced by DISC/mGM-CSF virus immunotherapy provides a unique model for evaluating the immune mechanism(s) involved in tumor rejection, upon which tumor immunotherapy regimes may be based.     

Intratumoral interferon-α gene transfer enhances tumor immunity after allogeneic hematopoietic stem cell transplantation

One of the major challenges in the treatment of solid cancers by allogenic hematopoietic stem cell transfer (alloHSCT) is the specific enhancement of antitumor immunity. Interferon (IFN) is a cytokine with pleiotropic biological functions including an immunomoduration, and our preclinical studies have shown that an intratumoral IFN-α gene transfer induced strong local tumor control and systemic tumor-specific immunity. In the present study, we examined whether the IFN-α gene transfer could enhance recognition of tumor-associated antigens by donor T cells and augment the antitumor activity of alloHSCT. First, when a mouse IFN-α adenovirus vector (Ad-mIFN) was injected into subcutaneous xenografts of syngeneic renal and colon cancer cells, tumor growth was significantly suppressed in a dose-dependent manner. A significant tumor cell death and infiltration of immune cells was recognized in the Ad-mIFN-injected tumors, and the dendrtic cells isolated from the tumors showed a strong Th1-oriented response. The antitumor effect of Ad-mIFN was then examined in a murine model of minor histocompatibility antigen-mismatched alloHSCT. The intratumoral IFN-α gene transfer caused significant tumor suppression in the alloHSCT recipients, and this suppression was evident not only in the gene-transduced tumors but also in simultaneously inoculated distant tumors which did not receive the vector injection. A cytotoxicity assay showed specific tumor cell lysis by donor T cells responding to IFN-α. Graft-versus-host disease was not exacerbated serologically or clinically in the mice treated with IFN-α. This combination strategy deserves evaluation in future clinical trials for human solid cancers.     

Studies on the recovery from tolerance to tumor antigens

Summary The present study investigates the potential of bone marrow cells from mice tolerant to tumor antigens to repopulate tumor-specific effector T cells. C3H/He mice were inoculated i.v. with 10⁶ 10000 R X-irradiated syngeneic X5563 plasmacytoma tumor cells three times at 4-day intervals. This regimen abrogated the ability of spleen cells from these mice to develop anti-X5563 cytotoxic and in vivo protective (tumor-neutralizing) T cell-mediated immunity as induced by i.d. inoculation of viable X5563 cells followed by surgical resection of the tumor. Since such suppression was induced in a tumor-specific way, this represented a state of antitumor tolerance. When bone marrow cells from normal or X5563-tolerant mice were transferred i.v. into 950 R X-irradiated syngeneic C3H/He mice, both groups of recipient mice generated anti-X5563 tumor immunity over a similar time course and to almost the same degree. Anti-X5563 tumor immunity induced in (C3H/He×C57BL/6) F1 mice which had been transferred with bone marrow cells from normal or X5563-tolerant C3H/He mice were mediated by T cells expressing the Ly phenotype of C3H/He, but not of C57BL/6, excluding the possibility that the antitumor effector cells were derived from recipient mice. It was also demonstrated that C3H/He mice which had been reconstituted with normal marrow were rendered tolerant when the tolerance regimen was started 7 weeks, but not 1 week after the bone marrow reconstitution. These results indicate that bone marrow cells from antitumor tolerant mice are not rendered tolerant to the tumor but can provide the potential to repopulate antitumor CTL and in vivo protective effector T cells.This work was supported by the Special Project Cancer-Bioscience from the Ministry of Education, Science and Culture, Japan Abbreviations used: MHC, major histocompatibility complex; CTL, cytotoxic T lymphocytes; TNP, trinitrophenyl; C, complement; TNBS; trinitrobenzene sulfonate; MMC, mitomycin C     

Recombinant cholera toxin B subunit activates dendritic cells and enhances antitumor immunity

Activation of dendritic cells (DC) is crucial for priming of cytotoxic T lymphocytes (CTL), which have a critical role in tumor immunity, and it is considered that adjuvants are necessary for activation of DC and for enhancement of cellular immunity. In this study, we examined an adjuvant capacity of recombinant cholera toxin B subunit (rCTB), which is non-toxic subunit of cholera toxin, on maturation of murine splenic DC. After the in vitro incubation of DC with rCTB, the expression of MHC class II and B7-2 on DC was upregulated and the secretion of IL-12 from DC was enhanced. In addition, larger DC with longer dendrites were observed. These data suggest that rCTB induced DC maturation. Subsequently, we examined the induction of tumor immunity by rCTB-treated DC by employing Meth A tumor cells in mice. Pretreatment with subcutaneous injection of rCTB-treated DC pulsed with Meth A tumor lysate inhibited the growth of the tumor cells depending on the number of DC. Moreover, intratumoral injection of rCTB-treated DC pulsed with tumor lysate had therapeutic effect against established Meth A tumor. Immunization with DC activated by rCTB and the tumor lysate increased number of CTL precursor recognizing Meth A tumor. The antitumor immune response was significantly inhibited in CD8+ T cell-depleted mice, although substantial antitumor effect was observed in CD4+ T cell-depleted mice. These results indicated that rCTB acts as an adjuvant to enhance antitumor immunity through DC maturation and that CD8+ T cells play a dominant role in the tumor immunity. Being considered to be safe, rCTB may be useful as an effective adjuvant to raise immunity for a tumor in clinical application.     

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     

Enhanced TNP-reactive helper T cell activity and its utilization in the induction of amplified tumor immunity that results in tumor regression

The present study was designed to investigate the generation of trinitrophenyl (TNP)-reactive helper T cell activity potent enough to induce the regression of a syngeneic tumor; this occurs by augmenting antitumor-specific immunity through T-T cell interaction. Mice whose skin was painted with trinitrochlorobenzene (TNCB) exhibited a variety of anti-TNP T cell responses, including delayed-type hypersensitivity (DTH) and cytotoxic T cell responses, as well as helper T cell activity. Pretreatment of C3H/He mice with TNP-conjugated copolymer of D-glutamic acid and lysine (TNP-D-GL) or cyclophosphamide, which have been shown, respectively, to inactivate TNP-specific suppressor T cells or suppressor T cells in general, exhibited a slight or marginal augmentation of DTH and cytotoxic potentials when tested 5 wk after TNCB painting. In contrast, the same pretreatment regimens induced an appreciably amplified generation of anti-TNP helper T cell activity. This amplified TNP-helper T cell activity was demonstrated to enhance cytotoxic responses to antigens other than TNP in an antigen-nonspecific way. In fact, such helper T cells enhanced antitumor CTL responses when co-cultured with spleen cells from syngeneic X5563 plasmacytoma-bearing mice in the presence of TNBS-modified X5563 tumor cells. This amplified TNP-helper cell system was utilized for its immunotherapeutic potential. When TNCB was injected into X5563 tumor mass of syngeneic C3H/He mice in which the amplified TNP-helper T cell activity had been generated, an appreciable number of growing tumors was observed to regress. This contrasted with the low incidence of tumor regression observed in mice in which TNP-helper activity had been induced by TNCB painting without inactivation of suppressors. Thus, the present model provides an effective immunotherapeutic manipulation for eliciting enhanced in vivo tumor regression, and emphasizes a role of helper T cells in augmentation of syngeneic tumor immunity.     

Effector phenotype and immunologic specificity of T-cell-mediated adoptive therapy for a murine tumor that lacks intrinsic immunogenicity

The MCA 102 sarcoma has been defined by a variety of immunologic studies as a tumor lacking intrinsic immunogenicity. Nevertheless, we have recently demonstrated the feasibility of generating therapeutically effective lymphocytes for adoptive immunotherapy of this tumor. Procedures to achieve this required in vivo priming of syngeneic mice to elicit preeffector cells followed by in vitro sensitization (IVS) with tumor cells in the presence of IL-2. By selective depletion of T cell subsets in vivo, we identified the involvement of both CD4+ (L3T4+) and CD8+ (Lyt-2+) T cells in mediating tumor regression. The CD4+ cells exerted their helper function via the secretion of IL-2 because antitumor effects abrogated by depletion of CD4+ cells could be reconstituted by exogenous IL-2. In order to elicit preeffector cells with reactivity against the MCA 102 tumor, we found that in vivo sensitization could be accomplished with either the MCA 102 or MCA 106 tumor but not with the MCA 101 or MCA 105 tumor. Analysis of specificity of tumor stimulation during IVS of MCA 102 tumor-primed preeffector cells demonstrated cross-reactivity between not only the MCA 102 and MCA 106 tumors but also the MCA 105 tumor whereas the MCA 101 tumor was ineffective. In adoptive immunotherapy, transfer of IVS cells generated from MCA 102 tumor-primed and stimulated lymph node cells was able to mediate reductions of pulmonary metastases established from the MCA 102, MCA 105, and MCA 106 tumors but not from the MCA 101 tumor. We conclude that regression of the MCA 102 tumor is probably mediated through T cell recognition of a set of common tumor-associated Ag shared by several other syngeneic tumors. Immunologically, the tumor-associated Ag are characteristically different from classical tumor-specific transplantation Ag (TSTA) because immunity to TSTA on the MCA 105 or MCA 106 tumor does not cross-react with the MCA 102 tumor. Thus, this study demonstrates that Ag other than TSTA on chemically induced tumors can serve as target molecules for T cell-mediated adoptive immunotherapy.     

Immunotherapy of melanoma: a dichotomy in the requirement for IFN-gamma in vaccine-induced antitumor immunity versus adoptive immunotherapy

The mechanism by which tumors are rejected following the adoptive transfer of tumor-specific T cells is not well characterized. Recent work has challenged the requirement for cytotoxicity mediated by either the perforin/granzyme or Fas/Fas ligand pathway in T cell-mediated tumor regression. Many reports, including ours, suggest that tumor-specific production of IFN-gamma is critical for T cell-mediated tumor regression. However, in most of these studies the evidence to support the role for IFN-gamma is only indirect. We have directly examined the requirement for IFN-gamma using IFN-gamma knockout (GKO) mice. The results show an interesting dichotomy in the requirement for IFN-gamma: Antitumor immunity induced by active-specific immunotherapy (vaccination) required IFN-gamma, whereas adoptive immunotherapy did not. In GKO mice vaccination with the GM-CSF gene-modified B16BL6-D5 tumor (D5-G6) failed to induce protective immunity against parental D5 tumor. However, adoptive transfer of effector T cells from GKO mice cured 100% of GKO mice with established pulmonary metastases and induced long term antitumor immunity and depigmentation of skin. Furthermore, in vivo neutralization of IFN-gamma by mAb treatment or adoptive transfer into IFN-gamma receptor knockout mice failed to block the therapeutic efficacy of effector T cells generated from wild-type or perforin knockout mice. Analysis of regressing metastases revealed similar infiltrates of macrophages and granulocytes in both wild-type and GKO mice. These results indicate that in this adoptive immunotherapy model, neither a direct effect on the tumor nor an indirect effect of IFN-gamma through activation of myeloid or lymphoid cells is critical for therapeutic efficacy.     

Distinct immunologic specificity of tumor regression mediated by effector cells isolated from immunized and tumor-bearing mice

Sensitized T lymphocytes can mediate potent antitumor effects when transferred to tumor-bearing animals. Employing the MCA 105 and MCA 106 sarcomas, we were able to generate antitumor effector cells by immunization of syngeneic mice with tumor cells admixed with Corynebacterium parvum. These immune splenocytes could be further sensitized and expanded in culture by the in vitro sensitization (IVS) method utilizing tumor stimulator cells and IL-2. Adoptive immunotherapy of pulmonary metastases mediated by noncultured splenocytes from immunized mice or immune IVS cells showed exquisite specificity between the two sarcomas. These results demonstrate the presence of tumor-specific antigens on MCA 105 and MCA 106 tumor cells which can serve as target molecules for immunotherapy. Recently, we have generated therapeutic T lymphocytes from mice bearing progressively growing tumors by the IVS method. However, IVS cells from tumor-bearing mice showed cross-reactivity between the MCA 105 and 106 sarcomas in adoptive immunotherapy experiments. Since these IVS cells did not affect other control tumors, the limited cross-reactivity suggests the presence of common tumor-associated antigens on MCA 105 and MCA 106 tumor cells which can also serve as the target for tumor rejection. Therefore, immune responses to progressive tumor growth and to immunization are distinct with respect to antigen recognition by T lymphocytes.     

Antitumor effect of intratumoral administration of dendritic cell combination with vincristine chemotherapy in a murine fibrosarcoma model

A new antitumor therapeutic strategy utilizing the combined effect of chemotherapy and DC (dendritic cell)-based immunotherapy was designed, and the effect of intratumoral injections of unpulsed, immature DCs was evaluated after in vivo pretreatment of vincristine on tumor growth in a murine fibrosarcoma tumor model. Vincristine exerted a much more potent apoptosis/necrosis-inducing effect on MCA-102 tumor cells than on DCs both in vitro and in vivo. Moreover, CD11c, CD40, CD80 and CD86 molecules on DCs were not downregulated after treatment with vincristine either in vitro or in vivo. The growth of tumor significantly regressed in the group which received the combined vincristine chemotherapy with intratumoral administration of DCs in contrast to the untreated group, the group treated with DCs alone, and the group treated with vincristine alone. In particular, an upregulated expression of CD40, CD80 and CD86 molecules on DCs was found in the combination treatment group. Furthermore, the number of CD4+ and CD8+ T cells and the staining intensity of their CD4 and CD8 surface molecules also increased after the combination treatment. Therefore, our results indicate the feasibility of this combination therapy with vincristine chemotherapy and DC-based immunotherapy as an efficient antitumor strategy for the treatment of fibrosarcoma.     

Secondary lymphoid tissue chemokine mediates T cell-dependent antitumor responses in vivo

Secondary lymphoid tissue chemokine (SLC, also referred to as Exodus 2 or 6Ckine) is a recently identified high endothelial-derived CC chemokine. The ability of SLC to chemoattract both Th1 lymphocytes and dendritic cells formed the rationale to evaluate this chemokine in cancer immunotherapy. Intratumoral injection of recombinant SLC evidenced potent antitumor responses and led to complete tumor eradication in 40% of treated mice. SLC-mediated antitumor responses were lymphocyte dependent as evidenced by the fact that this therapy did not alter tumor growth in SCID mice. Studies performed in CD4 and CD8 knockout mice also revealed a requirement for both CD4 and CD8 lymphocyte subsets for SLC-mediated tumor regression. In immunocompetent mice, intratumoral SLC injection led to a significant increase in CD4 and CD8 T lymphocytes and dendritic cells, infiltrating both the tumor and the draining lymph nodes. These cell infiltrates were accompanied by the enhanced elaboration of Th1 cytokines and chemokines monokine induced by IFN-gamma and IFN-gamma-inducible protein 10 but a concomitant decrease in immunosuppressive cytokines at the tumor site. In response to irradiated autologous tumor, splenic and lymph node-derived cells from SLC-treated tumor-bearing mice secreted significantly more IFN-gamma, GM-CSF, and IL-12 and reduced levels of IL-10 than did diluent-treated tumor-bearing mice. After stimulation with irradiated autologous tumor, lymph node-derived lymphocytes from SLC-treated tumor-bearing mice demonstrated enhanced cytolytic capacity, suggesting the generation of systemic immune responses. These findings provide a strong rationale for further evaluation of SLC in tumor immunity and its use in cancer immunotherapy.     

Potent tumor-specific protection ignited by adoptively transferred CD4+ T cells

Administration of anti-CD25 mAb before an aggressive murine breast tumor inoculation provoked effective antitumor immunity. Compared with CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that did not reject tumor, CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that rejected tumor stimulated by dendritic cells (DCs) produced more IFN-gamma and IL-2, and less IL-17 in vitro, and ignited protective antitumor immunity in vivo in an adoptive transfer model. Tumor Ag-loaded DCs activated naive CD8(+) T cells in the presence of these CD4(+) T cells in vitro. Tumor Ag and adoptively transferred CD4(+) T cells were both required for inducing a long-term tumor-specific IFN-gamma-producing cellular response and potent protective antitumor activity. Although adoptively transferred CD4(+) T cells ignited effective tumor-specific antitumor immunity in wild-type mice, they failed to do so in endogenous NK cell-depleted, Gr-1(+) cell-depleted, CD40(-/-), CD11c(+) DC-depleted, B cell(-/-), CD8(+) T cell-depleted, or IFN-gamma(-/-) mice. Collectively, the data suggest that adoptively transferred CD4(+) T cells orchestrate both endogenous innate and adaptive immunity to generate effective tumor-specific long-term protective antitumor immunity. The data also demonstrate the pivotal role of endogenous DCs in the tumor-specific protection ignited by adoptively transferred CD4(+) T cells. Thus, these findings highlight the importance of adoptively transferred CD4(+) T cells, as well as host immune components, in generating effective tumor-specific long-term antitumor activity.