Generation of therapeutic T lymphocytes from tumor-bearing mice by in vitro sensitization. Culture requirements and characterization of immunologic specificity

We have previously established an in vitro sensitization (IVS) procedure with which lymphocytes from tumor-bearing mice could be expanded and sensitized to acquire antitumor reactivity capable of mediating the regression of established pulmonary metastases from the weakly immunogenic MCA 105 murine sarcoma. Culture conditions required for the optimal generation of therapeutic effector cells were evaluated in the current study. Generation of effector cells by IVS required stimulation by intact tumor cells. Tumor cells killed by heat or disrupted by sonication were ineffective, but the antigenicity of tumor cells was not affected by gamma-irradiation. Long term established tumor cell lines could also serve as antigenic stimulator cells albeit with lower efficiency than fresh tumor cells. IL-2 was essential for cellular proliferation during IVS. The concentration of 1000 U/ml of IL-2 also induced nonspecific lymphokine-activated killer (LAK) activity. However, cytotoxic cells were generated during IVS in response to a broad range of IL-2 concentrations. At low IL-2 concentrations (2 to 10 U/ml), IVS cells were generated which displayed little or no LAK activity, had a greater therapeutic efficacy than those generated with high concentrations of IL-2 (100 to 1000 U/ml). Despite having high LAK activity, IVS cells, from cultures where IL-2 was added 3 or more days after initiation, had no therapeutic effect. Thus, the generation of therapeutic cells occurred independently of LAK cell production. Adoptive immunotherapy with IVS cells from MCA 105 tumor-bearing mice demonstrated cross-reactivity with the immunologically distinct MCA 106 but not the nonimmunogenic MCA 102 tumor. In contrast, IVS cells from MCA 106 tumor-bearing mice exhibited specific in vivo reactivity. In vitro cytotoxicity analyses revealed that IVS cells from MCA 105 and MCA 106 tumor-bearing mice were able to lyse both MCA 105 and MCA 106 target cells, but the reactivity toward inoculating tumors was highest. Considering previous findings that the MCA 105 and MCA 106 sarcomas possessed distinct tumor-specific transplantation Ag, the cross-reactivity observed in this study suggests that the immune response during progressive tumor growth may be different from that elicited in response to active immunization.     

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.     

Adoptive immunotherapy of a newly induced sarcoma: immunologic characteristics of effector cells

A newly induced syngeneic transplantable sarcoma, MCA 105, was used for studies of the biologic characteristics of fresh noncultured and secondarily in vitro sensitized (IVS) cells with antitumor reactivity. Fresh spleen cells harvested from mice immunized to the MCA 105 tumor by a mixture of viable tumor cells and Corynebacterium parvum exhibited no detectable cytotoxic activity to MCA 105 tumor targets in a 4-hr chromium-release assay, and adoptive transfer of these cells mediated the specific regression of established MCA 105 tumors. Phenotypic analysis of fresh, noncultured immune cells revealed that the therapeutically effective cells expressed both the Lyt-1 and the Lyt-2 T cell differentiation antigens. The therapeutic efficacy of fresh noncultured immune cells was not augmented by the concomitant administration of exogeneous interleukin 2 (IL 2). Secondary IVS of fresh immune cells with irradiated MCA 105 tumor stimulator cells resulted in the generation of tumor-specific cytotoxic effector cells. The generation of cytotoxic effector cells required Lyt-1+, 2+ cytotoxic precursor cells. Effective adoptive immunotherapy with these IVS immune cells, unlike fresh noncultured immune cells, depended on the concomitant administration of IL 2. Furthermore, the generation of therapeutically effective cells did not require the specific stimulation by MCA 105 tumor cells, because cultures of MCA 105 immune spleen cells with FBL-3 lymphoma cells in vitro also contained in vivo functional immune effector cells. These cells, however, possessed no detectable MCA 105 cytotoxic activity in vitro. Although this observation suggests that a noncytotoxic cell population is sufficient to initiate tumor regression in vivo, it does not exclude the possibility that cytolytic cells are generated in vivo after adoptive transfer of these cells. As a whole, our results indicate that secondary IVS functional immune effector cells are characteristically distinct from freshly harvested immune cells.     

Selective localization of radiolabeled immune lymphocytes into syngeneic tumors.

Newly formed long-lived small lymphocytes (LLSL) generated during the immunization of mice to tumor specific transplantation antigens (TSTA) of syngeneic MCA-induced sarcomas were labeled. We then studied the localization of these labeled cells into tumor implants. "Crisis-cross" experiments were performed in which MCA sarcomas with individually distinct TSTA were studied in parallel. A selective localization of LLSL into tumors to which the lymphocytes were immune was found when small tumor pieces were implanted into immune mice whose LLSL had been labeled. Selective localization was also detected upon adoptive transfer of immune LLSL to tumor-bearing mice, but only when these mice had, before transfer, received a sublethal dose of whole body irradiation.     

Generation from tumor-bearing mice of lymphocytes with in vivo therapeutic efficacy

Systemic transfer of sensitized lymphocytes can effectively mediate the regression of established tumors. However, virtually all prior experimental applications of this approach have utilized lymphocytes from animals that have been immunized to reject tumor challenge. A similar source of cells is not available in the human. With the use of a weakly immunogenic murine tumor, MCA 105, we demonstrate here that following in vitro sensitization (IVS) with viable tumor cells and interleukin 2, the nontherapeutic lymphoid cells from mice bearing a progressively growing tumor acquired antitumor reactivity capable of mediating the regression of established pulmonary metastases. Although the IVS system induced nonspecific lymphokine-activated killer-like cytotoxic activity from lymphoid cells of normal as well as tumor-bearing mice, therapeutically active cells could only be generated from cultures initiated with lymphoid cells from tumor-bearing animals, indicating that the IVS was a secondary in vitro immune response. Without other treatment, the IVS cells could mediate antitumor effects. However, low doses of exogenous interleukin 2 administration could enhance their therapeutic efficacy. By in vivo T cell subset depletion with monoclonal antibodies, the primary effector cells were identified as belonging to cytotoxic/suppressor T cell lineage expressing the Lyt-2 phenotype. In addition, these therapeutic effector cells could be further expanded in numbers in vitro with continuous stimulation by tumor cells in the presence of interleukin 2. Compared to the number of cells initiating the culture, as many as 126 times the number of cells were obtained after 9 days of IVS followed by in vitro expansion for an additional 5 days. Studies on the kinetics of the occurrence of the pre-effector lymphocytes during tumor growth revealed that they were readily obtained from draining lymph nodes of mice with a broad range of tumor burdens as well as durations of tumor growth. The ability to generate and expand, in vitro, therapeutically active lymphocytes from tumor-bearing hosts has important implications for cellular therapy of human cancers.     

In vitro sensitization and expansion with viable tumor cells and interleukin 2 in the generation of specific therapeutic effector cells

We have investigated the efficacy and immunologic characteristics of immune effector cells generated from cultures containing large numbers of viable tumor cells and interleukin 2 (IL 2) in the adoptive immunotherapy of experimentally induced pulmonary metastases from the newly developed, weakly immunogenic MCA 105 sarcoma in mice. The current culture conditions allowed increases of either normal or MCA 105 immune spleen cells up to 94-fold in 15 days. The in vitro expanded normal and MCA 105 immune cells displayed nonspecific in vitro cytotoxicity against several syngeneic tumor targets. However, therapeutically effective cells could only be obtained from cultures initiated with MCA 105 immune spleen cells. Immunotherapy with expanded immune effector cells could lead to the reduction of established 3 day pulmonary metastases, prolongation of survival, and cure of tumor in the majority of animals. The generation and proliferation of therapeutic effector cells in vitro depended on the presence in cultures of specific tumor stimulator cells as well as the presence of IL 2. Although immunotherapy with either fresh noncultured or secondarily in vitro-sensitized (IVS) MCA 105 immune spleen cells was immunologically specific, the efficacy of the adoptive cellular therapy with cultured but not fresh immune cells could be improved by the administration to tumor-bearing hosts of exogenous IL 2. In addition to numerical expansion, the IVS immune cells, on a per cell basis, afforded an eightfold to 10-fold increase in therapeutic efficacy when compared with fresh noncultured MCA 105 immune cells. Our results indicate that the current culture procedure induced in vitro antigenic stimulation and expansion of tumor-specific immune effector cells that was otherwise not possible by conventional mixed lymphocyte-tumor cultures.     

Cellular basis of immunologic interactions in adoptive T cell therapy of established metastases from a syngeneic murine sarcoma

The adoptive transfer of specifically sensitized T lymphocytes can effectively mediate the regression of established local and metastatic tumors. Previous experiments using the weakly immunogenic MCA 105 sarcoma indicated that cellular interactions between transferred L3T4+ helper and Lyt-2+ cytotoxic immune T cells were necessary for mediating tumor regression. In this study, the kinetics of T-T cell interactions were analyzed by in vivo depletion of T cell subsets with mAb. The anti-tumor efficacy of transferred immune cells was abrogated by in vivo administration of either L3T4 or Lyt-2 mAb on the day of cellular therapy. However, if mAb were given 3 days after the transfer of immune cells, depletion of Lyt-2+ but not L3T4+ cells abrogated anti-tumor efficacy. T cell depletion on day 6 after transfer of immune cells had no adverse effect on tumor regression, indicating the period required for T cell reactivity in vivo. Furthermore, depletion of Ia+ cells by in vivo mAb treatment abrogated the anti-tumor efficacy of immune cells. It is thus hypothesized that there are two distinct but sequential phases of in vivo T cell interactions leading to the regression of established tumors after adoptive immunotherapy. An initial "helper/inducer" phase apparently requires the interaction of L3T4+ immune cells and the tumor Ag involving Ia+ cells. The inducement of L3T4+ cell activation is to provide helper function via the secretion of IL-2. The second phase designated as an "effector phase" involves differentiation of immune Lyt-2+ cells under the influence of IL-2 secreted during the helper/inducer phase for generation of mature Lyt-2+ effector cells. To further support the hypothesis of a two-phase process we have examined the phenotype and kinetics of tumor regression mediated by effector cells generated by secondary in vitro sensitization (IVS). Although the IVS cells were generated from fresh MCA 105 immune spleen cells, their anti-tumor efficacy was mediated solely by Lyt-2+ lymphocytes. Kinetic studies revealed that the in vivo requirement of IVS Lyt-2+ effector cells to mediate tumor regression was less than 3 days, and the anti-tumor reactivity of these cells was not affected by in vivo depletion of Ia+ cells. Thus, the IVS reaction is likely representative of the in vivo counterpart of the helper/inducer phase leading to the generation of mature Lyt-2+ immune effector cells. Tumor regression after transfer of Lyt-2+ cells generated in IVS therefore required a relatively shorter period of time than that required after the transfer of fresh noncultured MCA 105 immune spleen cells.     

Induction of cell-mediated immunity against B16-BL6 melanoma in mice vaccinated with cells modified by hydrostatic pressure and chemical crosslinking

In the preceding paper we have demonstrated an increase in presentation of both major histocompatibility complex antigens (MHC) and a tumor-associated antigen of the weakly immunogenic B16 melanoma by a straight-forward technique. The method consists in modulating the tumor cell membrane by hydrostatic pressure and simultaneous chemical crosslinking of the cell-surface proteins. In B16-BL6 melanoma, the induced antigenic modulation was found to persist for over 48 h, which permitted the evaluation of the ability of modified B16-BL6 cells to induce immunity against unmodified B16-BL6 cells. In the present study, we have shown that a significant systemic immunity was induced only in mice that were immunized with modified B16-BL6 melanoma cells, whereas immunization with unmodified B16-BL6 cells had only a marginal effect when compared to the results in control sham-immunized mice. The induced immunity was specific since a single immunization affected the growth of B16-BL6 tumors but had no effect on MCA 106, an antigenically unrelated tumor. The addition of interleukin-2 to the immunization regimen had no effect on the antitumor responses induced by the modified B16-BL6 cells. The cell-mediated immunity conferred by immunization with treated B16-BL6 cells was confirmed in experiments in vitro where splenocytes from immunized mice could be sensitized to proliferate by the presence of B16-BL6 cells. In addition, the altered antigenicity of these melanoma cells appeared to correlate with their increased susceptibility to specific effectors. Thus,⁵¹Cr-labeled B16-BL6 target cells, modified by pressure and crosslinking, in comparison to control labeled target cells, were lysed in much greater numbers by effectors such as lymphokine-activated killer cells and allogeneic cytotoxic lymphocytes (anti-H-2^b), while such cells remained resistant to lysis by natural killer cells. Our findings indicate that the physical and chemical modifications of the tumor cells that are described here may be considered as a simple yet effective method for the preparation of tumor vaccines, which could be applied in tumor-bearing hosts.     

Cellular interactions and the role of interleukin 2 in the expression and induction of immunity against a syngeneic murine sarcoma

We have previously demonstrated that following the adoptive transfer of immune cells, the regression of established pulmonary metastases from a weakly immunogenic sarcoma, MCA 105, required the collaboration of two T cell subsets. In this study, we found that the critical role played by L3T4+ immune cells was to provide a helper function since tumor regression proceeded in the absence of L3T4+ immune cells if exogenous interleukin 2 (IL-2) was administered. To extend these observations, we analyzed the events leading to the induction and generation of L3T4+ and Lyt-2+ immune T cells after immunization of mice with viable tumor cells admixed with Corynebacterium parvum. The basic protocol involved immunization, surgical excision of the immunization site on day 7, and challenge with viable tumor cells on day 21. The ability of mice to reject tumor challenge provided a means to evaluate the occurrence of a systemic antitumor immunity. With the use of this experimental protocol, we have found that depletion of T cell subsets in vivo with either L3T4 or Lyt-2 monoclonal antibodies after active immunization abrogated the development of antitumor immunity. Mice immunized and depleted of L3T4+ but not Lyt-2+ T cells were able to reject tumor challenge if exogenous IL-2 was given for 7 days. However, the rejection of tumor challenge required 3 days of additional exogenous IL-2 administration. These results indicate that the induction of Lyt-2+ immune T cells depended on the helper function of L3T4+ T cells via the secretion of IL-2. In the absence of L3T4+ immune lymphocytes, the expression of antitumor immunity by Lyt-2+ immune cells could be facilitated by in vivo administration of exogenous IL-2. The induction of L3T4+ immune T cells, on the other hand, occurred independently of the Lyt-2+ T cell response because the transfer of spleen cells from Lyt-2+ cell-depleted, immunized animals was able to restore antitumor reactivity in L3T4+ cell-depleted, immunized mice. These results demonstrate the intricate cellular interactions leading to the induction as well as the expression of antitumor immunity.     

Successful adoptive immunotherapy of murine poorly immunogenic tumor with specific effector cells generated from gene-modified tumor-primed lymph node cells

We previously reported that cytokine gene transfer into weakly immunogenic tumor cells could enhance the generation of precursor cells of tumor-reactive T cells and subsequently augment antitumor efficacy of adoptive immunotherapy. We investigated whether such potent antitumor effector T cells could be generated from mice bearing poorly immunogenic tumors. In contrast to similarly modified weakly immunogenic tumors, MCA102 cells, which are chemically induced poorly immunogenic fibrosarcoma cells transfected with cDNA for IL-2, IL-4, IL-6, IFN-gamma, failed to augment the host immune reaction. Because priming of antitumor effector T cells in vivo requires two important signals provided by tumor-associated Ags and costimulatory molecules, these tumor cells were cotransfected with a B7-1 cDNA. Transfection of both IFN-gamma and B7-1 (MCA102/B7-1/IFN-gamma) resulted in regression of s.c. tumors, while tumor transfected with other combinations of cytokine and B7-1 showed progressive growth. Cotransfection of IFN-gamma and B7-1 into other poorly immunogenic tumor B16 and LLC cells also resulted in the regression of s.c. tumors. Cells derived from lymph nodes draining MCA102/B7-1/IFN-gamma tumors showed potent antitumor efficacy, eradicating established pulmonary metastases, but this effect was not seen with parental tumors. This mechanism of enhanced antitumor efficacy was further investigated, and T cells with down-regulated L-selectin expression, which constituted all the in vivo antitumor reactivity, were significantly increased in lymph nodes draining MCA102/B7-1/IFN-gamma tumors. These T cells developed into potent antitumor effector cells after in vitro activation with anti-CD3/IL-2. The strategy presented here may provide a basis for developing potent immunotherapy for human cancers.     

T Lymphocytes with Modified Specificity in the Therapy of Malignant Diseases

Immunotherapy is one of the most rapidly progressing and promising fields in antitumor therapy. It is based on the idea of using immune cells of patient or healthy donors for elimination of malignant cells. T lymphocytes play a key role in cell-mediated immunity including the response to tumors. Recently developed approaches of altering antigen specificity of T cells consist of their genetic modification (introduction of additional T cell receptor or chimeric antigen receptor), as well as the use of bispecific molecules that crosslink target and effector cells. These approaches are used to retarget T lymphocytes with arbitrary specificity against tumor antigens in the context of antitumor immunotherapy. The high potential of T cell immunotherapy was demonstrated in a number of clinical trials. In the future, it is possible to develop approaches to the therapy of a wide spectrum of tumors. The selection of the optimal antigen is the main challenge in successful T cell immunotherapy, as it largely determines the effectiveness of the treatment, as well as the risk of side effects. In this review we discuss potential methods of modification of T cell specificity and targets for immunotherapy.     

Soluble OX40L favors tumor rejection in CT26 colon carcinoma model

OX40 receptor-expressing regulatory T cells (Tregs) populate tumors and suppress a variety of immune cells, posing a major obstacle for cancer immunotherapy. Different ways to functionally inactivate Tregs by triggering OX40 receptor have been suggested, including anti-OX40 antibodies and Fc:OX40L fusion proteins. To investigate whether the soluble extracellular domain of OX40L (OX40Lexo) is sufficient to enhance antitumor immune response, we generated an OX40Lexo-expressing CT26 colon carcinoma cell line and studied its tumorigenicity in immunocompetent BALB/c and T cell deficient nu/nu mice.We found that soluble OX40L expressed in CT26 colon carcinoma favors the induction of an antitumor response which is not limited just to cells co-expressing EGFP as an antigenic determinant, but also eliminates CT26 cells expressing another fluorescent protein, KillerRed. Tumor rejection required the presence of T lymphocytes, as indicated by the unhampered tumor growth in nu/nu mice. Subsequent re-challenge of tumor-free BALB/c mice with CT26 EGFP cells resulted in no tumor growth, which is indicative of the formation of immunological memory. Adoptive transfer of splenocytes from mice that successfully rejected CT26 OX40Lexo EGFP tumors to naïve mice conferred 100% resistance to subsequent challenge with the CT26 EGFP tumor.     

The immunological mouse mutants nude (nu) and rhino (hr rh ) generate cytotoxic effector cells following adoptive immunotherapy but fail to reject a transplanted tumor

Summary Adoptive immunotherapy, consisting of cyclophosphamide injection and the i. v. transfer of tumor-sensitized T cells, resulted in rejection of the immunogenic fibrosarcoma, MCA/76-9, by syngeneic C57BL/6J (B6) mice. The same treatment of tumor-bearing congenic immunodeficient mice, homozygous for the deleterious mutations nude (nu) and rhino (hr ^rh ), did not result in tumor rejection. Paradoxically, the intratumor and intrasplenic changes taking place in each of the three strains after therapy were indistinguishable. There was an increase in Thy-1⁺, Lyt-2⁺, or L3T4⁺ cells at the tumor site 8 days after adoptive immunotherapy and a similar increase in Thy-1⁺ cells in the spleen. Moreover, the T cells isolated from the tumors or spleens from each genotype were shown to be specifically cytotoxic in vitro as well as in an in vivo Winn assay. Further evidence that immune amplification had occurred in the immunological mutant mice was provided by experiments showing (a) the ability of spleen cells from tumor-bearers and those tested after therapy to produce IL-2 in response to Con A stimulation and (b) an increase in class II-MHC antigen expression by tumor-associated macrophages. The data suggest that, although amplification of antitumor immune responses occurred in the immunological mutants, the absence of a critical host factor limited the potency of the antitumor response. Abbreviations used: CY, cyclophosphamide; TAM, tumor-associated macrophages; TAL, tumor-associated lymphocytes; TAC, tumor-associated cells; B6, C57BL/6J; class II MHC antigens: Ia; con A, concanavalin A; IL-1, IL-2, interleukin 1 and 2     

Vaccination with mRNAs encoding tumor-associated antigens and granulocyte-macrophage colony-stimulating factor efficiently primes CTL responses, but is insufficient to overcome tolerance to a model tumor/self antigen

Immunization of mice with dendritic cells transfected ex vivo with tumor-associated antigen (TAA)-encoding mRNA primes cytotoxic T lymphocytes (CTL) that mediate tumor rejection. Here we investigated whether direct injection of TAA mRNA, encapsulated in cationic liposomes, could function similarly as cancer immunotherapy. Intradermal and intravenous injection of ovalbumin (OVA) mRNA generated specific CTL activity and inhibited the growth of OVA-expressing tumors. Vaccination studies with DNA have demonstrated that co-administration of antigen (Ag)- and cytokine-encoding plasmids potentiate the T cell response; in analogous fashion, the inclusion of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA enhanced OVA-specific cytotoxicity. The ability of this GM-CSF-augmented mRNA vaccine to treat an established spontaneous tumor was evaluated in the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mouse, using the SV40 large T Ag (TAg) as a model tumor/self Ag. Repeated vaccination elicited vigorous TAg-specific CTL activity in nontransgenic mice, but tumor-bearing TRAMP mice remained tolerant. Adoptive transfer of naïve splenocytes into TRAMP mice prior to the first vaccination restored TAg reactivity, and slowed tumor progression. The data from this study suggests that vaccination with TAA mRNA is a simple and effective means of priming antitumor CTL, and that immunogenicity of the vaccine can be augmented by co-delivery of GM-CSF mRNA. Nonetheless, limitations of such vaccines in overcoming tolerance to tumor/self Ag may mandate prior or simultaneous reconstitution of the autoreactive T cell repertoire for this form of immunization to be effective.     

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.     

Spontaneous elicitation of potent antitumor immunity and eradication of established tumors by administration of DNA encoding soluble transforming growth factor-β II receptor without active antigen-sensitization

Since immunity is generally suppressed by immunoregulatory factors, such as transforming growth factor-beta (TGF-β), interleukin (IL)-10, and vascular endothelial growth factor (VEGF), produced by tumor cells or stromal cells surrounding tumor cells, various kinds of cancer immunotherapy mostly fail to elicit potent antitumor immunity. Herein, we tested whether neutralization of TGF-β can elicit strong antitumor immune responses and tumor regression in tumor-bearing mice. A plasmid DNA, pcDNA-sTGFβR/huIg, encoding a fusion protein consisting of the extracellular domain of TGF-β type II receptor (TGFβRII) and the Fc portion of human IgG heavy chain, was injected through different routes into B6 mice carrying established tumors of E.G7 cells, which consist of the poorly immunogenic tumor cells EL4, transfected with the ovalbumin (OVA) gene. The frequency of OVA-specific cytotoxic T lymphocytes (CTL), in the treated mice. increased resulting in the tumor eradication and relapse-free survival in around 70% of the E.G7-bearing mice. In contrast, administration of mock DNA into E.G7-bearing mice did not elicit tumor-specific immune responses. Therefore, administration of DNA encoding TGFβRII allowed tumor-bearing hosts to elicit sufficiently potent antitumor immune responses without requirement of further active antigen-immunization. This strategy seems to be applicable to clinical therapy against cancer, because it is low-cost, safe, and easy to manipulate.     

Tumor-specific granulocyte/macrophage colony-stimulating factor and interferon γ secretion is associated with in vivo therapeutic efficacy of activated tumor-draining lymph node cells

In this study, cytokine release by tumor-draining lymph node cells sensitized in vitro (IVS-TDLN) was examined and correlated with therapeutic efficacy in adoptive immunotherapy. Mice bearing immunologically distinct MCA 207 and MCA 205 sarcoma tumors were utilized in criss-cross experiments. IVS-TDLN obtained from mice bearing 10-day subcutaneous (s. c.) tumors mediated immunologically specific regression of established 3-day pulmonary metastases, but demonstrated non-specific cytolytic reactivity against both tumors in a 4-h⁵¹Cr-release assay. By contrast, these IVS-TDLN cells were found specifically to secrete granulocyte/macrophage colony-stimulating factor (GM-CSF) and interferon (IFN) when restimulated in vitro with irradiated tumor cells. To determine the predictive value of tumor-specific cytokine release with in vivo therapeutic efficacy, a kinetic analysis of antitumor activities of TDLN obtained from animals bearing MCA 207 tumors for increasing lengths of time was performed. IVS-TDLN cells from mice bearing day-7, -10 and-14 s. c. tumors manifested tumor-specific release of GM-CSF and IFN, and mediated significant antitumor reactivity in vivo. In contrast IVS-LN cells from day-0 and day-21 tumor-bearing animals did not release significant amounts of GM-CSF and IFN, and were not therapeutically efficacious in vivo. Day-4 IVS-TDLN released high levels of GM-CSF and IFN non-specifically, and were not therapeutic in adoptive immunotherapy at doses effective for day-7 and day-14 IVS-TDLN cells. In other experiments, IVS cells generated from different lymph node groups in animals bearing 10-day established s. c. tumors were examined and found to have unique profiles of cytokine release. In these studies, the ability of IVS cells to release specifically both cytokines as opposed to one was associated with greater therapeutic efficacy on a per cell basis. Our findings suggest that the tumor-specific releases of GM-CSF and IFN are useful parameters to assess the in vivo therapeutic efficacy of immune lymphocytes.     

Specific adoptive immunotherapy mediated by tumor-draining lymph node cells sequentially activated with anti-CD3 and IL-2.

Lymph nodes (LN) draining progressively growing tumors contain tumor-sensitized but not fully functional preeffector lymphocytes. These cells could acquire therapeutic efficacy and be expanded upon sequential culture with anti-CD3 mAb for 2 days followed by incubation in IL-2 for 3 days. Using the weakly immunogenic MCA 106 and MCA 205 murine sarcomas, we have further defined conditions of this anti-CD3/IL-2 activation with which preeffector cells differentiated into immune effector cells. In vitro activation and expansion of effector cells required sequential but independent stimulation with anti-CD3 and IL-2 because the simultaneous presence of both anti-CD3 and IL-2 at either stage did not enhance the efficacy of activation. Generation of effector cells by this two-stage activation was critically dependent on the optimal concentrations of anti-CD3 (1.0 microgram/ml) and IL-2 (2-10 U/ml). However, these conditions were not optimal for inducing the greatest cellular proliferation. In adoptive immunotherapy experiments, although the transfer of anti-CD3/IL-2-activated cells alone could mediate the regression of established metastases, the concomitant administration of IL-2 enhanced the in vivo activity of these cells. More importantly, tumor regression mediated by the anti-CD3/IL-2-activated cells was found to be immunologically specific. The specificity was determined by the tumor that stimulated the preeffector cell response. In spite of their in vivo antitumor effects, the anti-CD3/IL-2-activated tumor-draining LN cells did not exhibit detectable in vitro cytotoxicity against the tumor target in the 4-h 51Cr-release assay. In mice bearing progressive tumor, draining LN contained most preeffector cells. Some preeffector cells were also detected in the spleen whereas mesenteric LN did not demonstrate any reactivity. In kinetics studies, sensitization of preeffector cells in the draining LN occurred between 4 to 6 days after tumor inoculation. As the tumor progressed, the presence of preeffector cells declined gradually suggesting a tumor-induced suppression. These results define the conditions whereby tumor-draining LN cells could be stimulated, in the absence of tumor Ag, to develop into specific therapeutic effector cells. Our findings also raise the possibility of using similar approaches for isolating immune effector cells from cancer patients for adoptive immunotherapy.     

Therapeutic efficacy of tumor-targeted IL2 in LTα−/− mice depends on conditioned T cells

An effective immunological eradication of tumors by the adaptive immune system depends on T cell priming, expansion of specific T cells and their effector function. It has been shown that either step may be impaired in the tumor-bearing host, and several strategies have been used to improve antitumor immune responses. In this regard, tumor-targeted IL2 therapy leads to the destruction of established melanoma metastases in fully immune competent mice as previously demonstrated. This effect has been attributed, but never directly confirmed, to the boost of antigen-experienced T cells. To this end, we demonstrate the absence of any antitumor effect of targeted IL2 in mice characterized by an impaired priming of T cell responses. Notably, in these animals tumor-targeted IL2 therapy induced tumor regression only after adoptive transfer of tumor-conditioned splenocytes. A detailed analysis revealed that T cells present within the transferred splenocytes were actively participating in the immune response as these were clonally expanded after targeted IL2 therapy. In summary, we demonstrate here that in LTα^−/− mice lacking sufficient numbers of tumor-specific T cells only the passive transfer of such cells prior to therapy restores the efficacy of tumor-targeted IL2 therapy. Thus, the antitumor effect of tumor-targeted IL2 is indeed based on the boost of pre-existing T cell responses.     

EBV-induced molecule 1 ligand chemokine (ELC/CCL19) promotes IFN-gamma-dependent antitumor responses in a lung cancer model

The antitumor efficacy of EBV-induced molecule 1 ligand CC chemokine (ELC/CCL19) was evaluated in a murine lung cancer model. The ability of ELC/CCL19 to chemoattract both dendritic cells and T lymphocytes formed the rationale for this study. Compared with diluent-treated tumor-bearing mice, intratumoral injection of recombinant ELC/CCL19 led to significant systemic reduction in tumor volumes (p < 0.01). ELC/CCL19-treated mice exhibited an increased influx of CD4 and CD8 T cell subsets as well as dendritic cells at the tumor sites. These cell infiltrates were accompanied by increases in IFN-gamma, MIG/CXCL9, IP-10/CXCL10, GM-CSF, and IL-12 but a concomitant decrease in the immunosuppressive molecules PGE(2) and TGFbeta. Transfer of T lymphocytes from ELC/CCL19 treated tumor-bearing mice conferred the antitumor therapeutic efficacy of ELC/CCL19 to naive mice. ELC/CCL19 treated tumor-bearing mice showed enhanced frequency of tumor specific T lymphocytes secreting IFN-gamma. In vivo depletion of IFN-gamma, MIG/CXCL9, or IP-10/CXCL10 significantly reduced the antitumor efficacy of ELC/CCL19. These findings provide a strong rationale for further evaluation of ELC/CCL19 in tumor immunity and its use in cancer immunotherapy.