Modification of tumor cells by a low dose of Newcastle disease virus. III. Potentiation of tumor-specific cytolytic T cell activity via induction of interferon-alpha/beta

To investigate possibilities of augmenting tumor-specific immune responses against the highly metastatic murine lymphoma ESb, we tested the effects of the interferon inducer newcastle disease virus (NDV) or of interferon-alpha/beta as costimulator in mixed lymphocyte-tumor cell cultures (MLTC) on the tumor-specific cytolytic T cell (CTL) response. Both approaches, namely stimulation of ESb immune spleen cells with NDV-modified stimulator cells or with ESb stimulator cells and exogenous IFN-alpha/beta, led to a selective potentiation of tumor-specific CTL activity. The potent activation of tumor-specific CTL precursor (CTLP) required the simultaneous presence of the specific ESb tumor antigen--possibly to mediate a signal via the corresponding T cell receptor--and costimulators--possibly to mediate second activation signals. Increased CTL activity required only very low amounts of NDV or IFN-alpha/beta. The generation of CTL activity in the MLTC cultures could be blocked by antisera to IFN-alpha/beta, not, however by control sera. Similar effects were observed in vivo, suggesting that IFN-alpha/beta not only caused an increase in CTL activity, but was essential for the generation of CTL activity. The reduction of the generation of CTL by antiserum to IFN-alpha/beta could be overcome by excess interferon, especially when using ESb-NDV as stimulator cells.     

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     

Prevention of lymph node metastases by adoptive transfer of CD4+ T lymphocytes admixed with irradiated tumor cells

CD4⁺8^– T lymphocytes with potent antitumor activity in vivo were obtained in peritoneal exudate cells by immunizing mice with irradiated MM48 tumor cells admixed with OK-432. These immune CD4⁺ T cells were used in adoptive immunotherapy for prevention of lymph node metastases after removal of the primary tumor. Complete cure of metastases was obtained by adoptive transfer of CD4⁺ T cells admixed with irradiated MM48 tumor cells, but not by CD4⁺ T cells alone. To analyze the curative effect of admixing tumor cells on the prevention of metastases, a model of 1-day tumor inoculated with macrophages was used. Administration of immune CD4⁺ T cells alone resulted in the regression of local tumor in more than half of the mice, although all of them eventually died of lymph node metastases. On the other hand, adoptive transfer of immune CD4⁺ T cells plus irradiated tumor cells resulted in the complete regression of local tumors in all the mice, which survived without any sign of metastasis. The curative effect of the immune CD4⁺ T cells obtained by admixing irradiated tumor cells was tumor-specific. Macrophages induced by OK-432 (tumoricidal), implanted together with tumor, assisted tumor regression more than did macrophages elicited by proteose peptone (nontumoricidal) in the same adoptive transfer system. Administration of recombinant interleukin-2 instead of stimulant tumor cells did not enhance, but rather eliminated the constitutive antitumor activity of CD4⁺ T cells. On the other hand, exogenous recombinant interleukin-1 was more effective in the enhancement of antitumor activity of the CD4⁺ T cells as compared with stimulant tumor cell administration. In this case, the activating states of macrophages at the implanted tumor site had no influence on the therapeutic efficacy. A possible role of macrophages for induction of tumor-specific cytotoxic T cells that were mediated by tumor-specific CD4⁺ T cells is discussed.     

Tumor-specific CD4+ T cells develop cytotoxic activity and eliminate virus-induced tumor cells in the absence of regulatory T cells

The important role of tumor-specific cytotoxic CD8⁺ T cells is well defined in the immune control of the tumors, but the role of effector CD4⁺ T cells is poorly understood. In the current research, we have used a murine retrovirus-induced tumor cell line of C57BL/6 mouse origin, namely FBL-3 cells, as a model to study basic mechanisms of immunological control and escape during tumor formation. This study shows that tumor-specific CD4⁺ T cells are able to protect against virus-induced tumor cells. We show here that there is an expansion of tumor-specific CD4⁺ T cells producing cytokines and cytotoxic molecule granzyme B (GzmB) in the early phase of tumor growth. Importantly, we demonstrate that in vivo depletion of regulatory T cells (Tregs) and CD8⁺ T cells in FBL-3-bearing DEREG transgenic mice augments IL-2 and GzmB production by CD4⁺ T cells and increases FV-specific CD4⁺ T-cell effector and cytotoxic responses leading to the complete tumor regression. Therefore, the capacity to reject tumor acquired by tumor-reactive CD4⁺ T cells largely depends on the direct suppressive activity of Tregs. We suggest that a cytotoxic CD4⁺ T-cell immune response may be induced to enhance resistance against oncovirus-associated tumors.     

Induction of tumor-reactive T helper responses by a posttranslational modified epitope from tumor protein p53

Posttranslational modifications regulate the function and stability of proteins, and the immune system is able to recognize some of these modifications. Therefore, the presence of posttranslational modifications increases the diversity of potential immune responses to a determinant antigen. The stimulation of tumor-specific CD4⁺ helper T lymphocytes (HTLs) is considered important for the production of anti-tumor antibodies by B cells and for the generation and persistence of CD8⁺ cytotoxic T lymphocytes, and in some instances, HTLs can directly reduce tumor cell growth. Identification of MHC class II-restricted peptide epitopes from tumor-associated antigens including those generated from posttranslational protein modifications should enable the improvement of peptide-based cancer immunotherapy. We describe here an MHC class II binding peptide from the tumor protein p53, which possesses an acetylated lysine at position 120 (p53_{110-124/AcK120}) that is effective in eliciting CD4⁺ T cell responses specific for the acetylated peptide. Most importantly, the acetylated peptide-reactive CD4 HTLs recognized the corresponding naturally processed posttranslational modified epitope presented by either dendritic cells loaded with tumor cell lysates or directly on tumors expressing p53 and the restricting MHC class II molecules. Treatment of tumor cells with a histone deacetylase inhibitor augmented their recognition by the p53_{110-124/AcK120}-reactive CD4⁺ T cells. These findings prove that the epitope p53_{110-124/AcK120} is immunogenic for anti-tumor responses and is likely to be useful for cancer immunotherapy.     

Immunotherapy in multiple myeloma: Id-specific strategies suggested by studies in animal models

Multiple myeloma (MM) cells produce monoclonal immunoglobulin (Ig) which serves as a truly tumor-specific antigen. The tumor-specific antigenic determinants are localized in the variable (V)-regions of the monoclonal Ig and are called idiotopes (Id). We review here the evidence obtained in a T-cell receptor (TCR) transgenic mouse model that Id-specific, MHC class II–restricted CD4⁺ T cells play a pivotal role in immunosurveillance and eradication of MHC class II-negative MM cells. In brief, monoclonal Ig secreted by MM cells is endocytosed and processed by antigen-presenting cells (APCs) in the tumor. Such tumor-resident dendritic cell APCs in turn present Id peptide on their class II molecules to Id-specific CD4⁺ T cells which become activated and indirectly kill the MHC class II-negative myeloma cells. However, if the Id-specific CD4⁺ cells fail to eliminate the MM cells during their initial encounter, the increasing number of tumor cells secretes so much monoclonal Ig that T-cell tolerance to Id is induced. Extending these findings to MM patients, Id-specific immunotherapy should be applied at a time of minimal residual disease and when new Id-specific T cells have been educated in the thymus, like after high-dose chemotherapy and autologous stem cell transplantation.Abbreviations APC antigen-presenting cell - ASCT autologous stem cell transplantation - CDR complementarity-determining region - CFA complete Freunds adjuvant - DC dendritic cell - GM-CSF granulocyte-macrophage colony-stimulating factor - H heavy - Id idiotope or idiotype - Ig immunoglobulin - IL interleukin - L light - M-component monoclonal component - MGUS monoclonal gammopathy of undetermined significance - MHC major histocompatibility complex - MM multiple myeloma - MOPC mineral oil–induced plasmacytoma - TCR T-cell antigen receptor - V variableA. Corthay and B. Bogen are joint corresponding authors for this article.     

MHC-class I-restricted CD4 T cells: a nanomolar affinity TCR has improved anti-tumor efficacy in vivo compared to the micromolar wild-type TCR

Clinical studies with immunotherapies for cancer, including adoptive cell transfers of T cells, have shown promising results. It is now widely believed that recruitment of CD4⁺ helper T cells to the tumor would be favorable, as CD4⁺ cells play a pivotal role in cytokine secretion as well as promoting the survival, proliferation, and effector functions of tumor-specific CD8⁺ cytotoxic T lymphocytes. Genetically engineered high-affinity T-cell receptors (TCRs) can be introduced into CD4⁺ helper T cells to redirect them to recognize MHC-class I-restricted antigens, but it is not clear what affinity of the TCR will be optimal in this approach. Here, we show that CD4⁺ T cells expressing a high-affinity TCR (nanomolar K _d value) against a class I tumor antigen mediated more effective tumor treatment than the wild-type affinity TCR (micromolar K _d value). High-affinity TCRs in CD4⁺ cells resulted in enhanced survival and long-term persistence of effector memory T cells in a melanoma tumor model. The results suggest that TCRs with nanomolar affinity could be advantageous for tumor targeting when expressed in CD4⁺ T cells.     

Recruitment and activation of tumor-specific immune T cells in situ. CD8+ cells predominate the secondary response in sponge matrices and exert both delayed-type hypersensitivity-like and cytotoxic T lymphocyte activity

This study analyzes the involvement of CD4+ and CD8+ T cells in a secondary cellular immune response to the highly metastatic murine lymphoma ESb in situ. This tumor line expresses tumor-associated transplantation Ag which can induce protective immunity in vivo and specific CTL in vitro. In tumor-immune mice the injection of a tumor vaccine (x-irradiated ESb tumor cells) into s.c. implanted vascularized sponges resulted in the generation of a specific secondary immune response characterized by massive leukocyte recruitment and generation of strong CTL activity at the restimulation site. During the antitumor immune response the CD4+:CD8+ T cell ratio decreased significantly and specifically in the restimulated sponges. Depletion of CD8+ but not CD4+ T cells from the tumor immune mice before restimulation significantly reduced the delayed-type hypersensitivity-like response and totally blocked the generation of tumor-specific CTL activity in situ. Only a minority of the CD8+ immune T cells which predominated the secondary response in situ expressed IL-2R and lymph node homing receptors as detected by the mAb MEL-14.     

Polyclonal activation of human lymphocytes and induction of cytotoxic lymphocytes by streptococcal preparations

Polyclonal activation of human peripheral blood lymphocytes (PBLs)in vitro by preparations ofStreptococcus pyogenes Su strain (OK-432) and other heat-killed strains was investigated. The streptococcal preparations tested induce a proliferative response of PBLs via interleukin-2 (IL-2)-independent pathways. The proliferative response is accompanied by the generation of lymphoblastic cells (LBCs), which consist of heterologous lymphocyte populations: CD4⁺ helper type of T cells, and CD4^–CD8^– double-negative (DN) lymphocytes, including both CD3⁺ TcR ⁺ T cells and CD2⁺CD3^– immature type of T or non-T cell type of lymphocytes. Almost all the LBCs express Leu19, TfR (transferrin receptor), LFA-1 and CD38 (OKT10) antigens, which are expressed on activated T cells, NK cells and some other lymphocytes. The proliferative response of human PBLs is also accompanied by the generation of potent cytotoxic activity against NK-sensitive and -resistant targets. C-dependent cytolysis and cell sorting experiments of OK-432-activated LBCs revealed that both CD3⁺ and CD3^– types of CD4^–CD8^– DN lymphocytes, but not CD4⁺ helper T cells, may be major populations responsible for the cytotoxicity induced. On the other hand, CD4^–CD8 T cells may be required for the proliferation of PBLs and generation of cytotoxic effector cells. These results suggest that the OK-432 and other streptococcal preparations stimulate the human PBLsin vitro to induce the proliferation/activation of CD4⁺ T cells, mediating the following generation of DN cytotoxic effector lymphocytes.     

MHC class II and CD80 tumor cell–based vaccines are potent activators of type 1 CD4<Superscript>+</Superscript> T lymphocytes provided they do not coexpress invariant chain

We are developing vaccines that activate tumor-specific CD4⁺ T cells. The cell-based vaccines consist of MHC class I⁺ tumor cells that are genetically modified to express syngeneic MHC class II and costimulatory molecules. Previous studies demonstrated that treatment of mice with established tumors with these vaccines resulted in regression of solid tumors, reduction of metastatic disease, and increased survival time. Optimal vaccines will prime naïve T cells and activate T cells to tumor peptides derived from diverse subcellular compartments, since potential tumor antigens may reside in unique cellular locales. To determine if the MHC class II / costimulatory molecule vaccines fulfill these conditions, the vaccines have been tested for their ability to activate antigen-specific, naïve, transgenic CD4⁺ T lymphocytes. MHC class II⁺CD80⁺ vaccine cells were transfected with hen eggwhite lysozyme targeted to the cytosol, nuclei, mitochondria, or endoplasmic reticulum, and used as antigen-presenting cells to activate I-A^k–restricted, lysozyme-specific CD4⁺ 3A9 transgenic T cells. Regardless of the cellular location of lysozyme, the vaccines stimulated release of high levels of IFN- and IL-2. If the vaccines coexpressed the MHC class II accessory molecule invariant chain, then IFN- and IL-2 release was significantly reduced. These studies demonstrate that in the absence of invariant chain the MHC class II and CD80 tumor cell vaccines (1) function as antigen-presenting cells to activate naïve, tumor-specific CD4⁺ cells to endogenously synthesized tumor antigens; (2) polarize the activated CD4⁺ T cells toward a type 1 response; and (3) present epitopes derived from varied subcellular locales.Abbreviations APC antigen-presenting cells - CIITA MHC class II transactivator - CytoHEL HEL targeted to cytoplasm - ER endoplasmic reticulum - ErHEL HEL targeted to ER - HEL hen eggwhite lysozyme - 3A9 HEL_46–61–specific, I-A^k–restricted TCR - Hph hygromycin - Ii invariant chain - MAb monoclonal antibody - MitoHEL HEL targeted to mitochondria - NucHEL HEL targeted to nucleus - Puro puromycin - TG transgenic - Zeo Zeocin     

Effects of CD4+ and CD8+ T cells in tumor-bearing mice on antibody production

The function of T cell subsets in tumor-bearing mice was examined using an in vitro culture system of anti-(sheep red blood cell) antibody production, which is known to be dependent on T cells. The helper function of T cells of fibrosarcoma-MethA-bearing mice in antibody production decreased with the tumor stage of the mice. T cells were separated into CD4⁺ and CD8⁺ cells for further analysis of T cell subsets by the panning method using monoclonal antibodies. The helper function of CD4⁺ T cells in antibody production began to decrease significantly in tumor-bearing mice 1 week after the tumor transplantation. On the other hand, the suppressive function of CD8⁺ T cells was retained and had not decreased in the mice even 3 weeks after the transplantation. The same changes in function of CD4⁺ and CD8⁺ T cells were also observed in Methl-bearing mice. These results suggested that this tumor-associated immunosuppression in antibody production is attributable to the decrease in helper activity of CD4⁺ T cells and the maintenance of the suppressive activity of CD8⁺ T cells.     

Galectins and their ligands: negative regulators of anti-tumor immunity

Cytotoxic CD8⁺ T cells are major players of anti-tumor immune responses, as their functional activity can limit tumor growth and progression. Data show that cytotoxic T cells efficiently control the proliferation of tumor cells through major histocompatibility complex class I-mediated mechanisms; nevertheless, the presence of tumor-infiltrating CD8⁺ T cells in lesional tissue does not always correlate with better prognosis and increased survival of cancer patients. Similarly, adoptive transfer of tumor-specific cytotoxic T cells has only shown marginal improvement in life spans of patients with metastatic disease. In this report, we discuss experimental evidence showing that expression of tumor-derived galectins, galectin (Gal)-1, Gal-3 and Gal-9, and concomitant presence of their ligands on the surface of anti-tumor immunocytes directly compromise anti-tumor CD8⁺ T cell immune responses and, perhaps, undermine the promise of adoptive CD8⁺ T cell immunotherapy. Furthermore, we describe novel strategies designed to counteract Gal-1-, Gal-3- and Gal-9-mediated effects and highlight their targeting potential for creating more effective anti-tumor immune responses. We believe that Gal and their ligands represent an efficacious targeted molecular paradigm that warrants clinical evaluation.     

Phenotypic T Cell Exhaustion in a Murine Model of Bacterial Infection in the Setting of Pre-Existing Malignancy

While much of cancer immunology research has focused on anti-tumor immunity both systemically and within the tumor microenvironment, little is known about the impact of pre-existing malignancy on pathogen-specific immune responses. Here, we sought to characterize the antigen-specific CD8⁺ T cell response following a bacterial infection in the setting of pre-existing pancreatic adenocarcinoma. Mice with established subcutaneous pancreatic adenocarcinomas were infected with Listeria monocytogenes, and antigen-specific CD8⁺ T cell responses were compared to those in control mice without cancer. While the kinetics and magnitude of antigen-specific CD8⁺ T cell expansion and accumulation was comparable between the cancer and non-cancer groups, bacterial antigen-specific CD8⁺ T cells and total CD4⁺ and CD8⁺ T cells in cancer mice exhibited increased expression of the coinhibitory receptors BTLA, PD-1, and 2B4. Furthermore, increased inhibitory receptor expression was associated with reduced IFN-γ and increased IL-2 production by bacterial antigen-specific CD8⁺ T cells in the cancer group. Taken together, these data suggest that cancer''s immune suppressive effects are not limited to the tumor microenvironment, but that pre-existing malignancy induces phenotypic exhaustion in T cells by increasing expression of coinhibitory receptors and may impair pathogen-specific CD8⁺ T cell functionality and differentiation.     

The role of tumor-specific Lyt-1+2− T cells in eradicating tumor cells in vivo

Summary The present study investigates some of mechanisms for tumor-specific Lyt-1⁺2^– T cell-mediated tumor cell eradication in vivo through analyses of tumor specificity in the afferent tumor recognition and efferent rejection phases. When C3H/He mice which had acquired immunity against syngeneic MH134 hepatoma were challenged with other syngeneic X5563 plasmacytoma cells, these mice failed to exhibit any inhibitory effect on the growth of X5563 tumor cells. However, the inoculation of X5563 tumor cells into the MH134-immune C3H/He mice together with the MH134 tumor cells resulted in appreciable growth inhibition of antigenically distinct (bystander) X5563 tumor cells. Although the growth of X5563 cells was inhibited in an antigen-nonspecific way in mice immunized to antigenically unrelated tumor cells (bystander effect), the activation of Lyt-1⁺2^– T cells leading to this effect was strictly antigen-specific. Such a bystander growth inhibition also required the admixed inoculation of the bystander (X5563) and specific target (MH134) tumor cells into a single site in mice immunized against the relevant MH134 tumor cells. Furthermore, the results demonstrated that Lyt-1⁺2^– T cells specific to MH134 tumor cells were responsible for mediating the growth inhibition of antigenically irrelevant (bystander) and relevant tumor cells. These results are discussed in the context of cellular and molecular mechanisms involved in the Lyt-1⁺2^– T cell-initiated bystander phenomenon.This work was supported by Special Project Research-Cancer Bioscience from the Ministry of Education, Science and Culture     

Mast cells impair the development of protective anti-tumor immunity

Mast cells have emerged as critical intermediaries in the regulation of peripheral tolerance. Their presence in many precancerous lesions and tumors is associated with a poor prognosis, suggesting mast cells may promote an immunosuppressive tumor microenvironment and impede the development of protective anti-tumor immunity. The studies presented herein investigate how mast cells influence tumor-specific T cell responses. Male MB49 tumor cells, expressing HY antigens, induce anti-tumor IFN-??⁺ T cell responses in female mice. However, normal female mice cannot control progressive MB49 tumor growth. In contrast, mast cell-deficient c-Kit^Wsh (W^sh) female mice controlled tumor growth and exhibited enhanced survival. The role of mast cells in curtailing the development of protective immunity was shown by increased mortality in mast cell-reconstituted W^sh mice with tumors. Confirmation of enhanced immunity in female W^sh mice was provided by (1) higher frequency of tumor-specific IFN-??⁺ CD8⁺ T cells in tumor-draining lymph nodes compared with WT females and (2) significantly increased ratios of intratumoral CD4⁺ and CD8⁺ T effector cells relative to tumor cells in W^sh mice compared to WT. These studies are the first to reveal that mast cells impair both regional adaptive immune responses and responses within the tumor microenvironment to diminish protective anti-tumor immunity.     

CpG-Oligodeoxynucleotides activate tyrosinase-related protein 2–specific T lymphocytes but do not lead to a protective tumor-specific memory response

Purpose: Peritumoral CpG-oligodeoxynucleotide (ODN) treatment has been successful in tumor mouse models expressing strong antigens to induce activation of tumor-specific CD8⁺ T lymphocytes which contribute to the control of tumor growth. To get near to clinical reality, the tumor-specific CD8⁺ response was investigated in mice bearing the weakly immunogenic B16 melanoma tumor and using the melanocyte differentiation tyrosinase-related protein 2 (TRP-2) as a tracking antigen. Methods: The expansion and activation of TRP-2–specific T lymphocytes by CpG-ODNs was analyzed by tetramer staining and IFN- production assays, while the activity of these cells in both memory and primary response was evaluated in vivo. Results: After CpG-ODN treatment, the number of TRP-2 tetramer-stained CD8⁺ T lymphocytes was not significantly modified, but these cells produced higher levels of interferon (IFN-) in response to the antigen than those from untreated mice. Mice possessing these activated T lymphocytes, when evaluated for their antitumor memory response, showed marginal protection against intravenous (i.v.) and subcutaneous (s.c.) tumor rechallenge. These cells were not crucial for the control of primary tumor growth since strong reduction of subcutaneous tumor was observed after CpG-ODN treatment in both CD8⁺ T cell depleted or nondepleted mice. On the contrary, NK cell depletion markedly reduced CpG-ODN-induced tumor growth inhibition. Conclusions: Altogether, these data indicate the CpG treatment activates tumor-reactive effector CD8⁺ T lymphocytes, but, paralleling recent clinical observations, our model indicates that the mere activation of antitumor T cells is insufficient to result in a clinical response.Abbreviations CpG unmethylated CpG dinucleotides - ODNs oligodeoxynucleotides - TLR9 toll-like receptor 9 - TRP-2 tyrosinase-related protein 2     

Tumor antigen presentation: changing the rules

 Cell-based tumor vaccines have been developed on the basis of the hypothesis that tumor cells can be genetically modified to present antigen to T lymphocytes directly. Contrary to expectations, cross-priming is the predominant pathway for activation of tumor-specific CD8⁺ T cells, while direct presentation of antigen dominates activation of tumor-specific CD4⁺ T cells. These results pose interesting paradoxes for the generation of immune responses, and have definite implications for the development of anti-cancer vaccines. Received: 10 October 1997 / Accepted 10 January 1998     

Activation and expansion of cytotoxic T lymphocytes from tumor-draining lymph nodes

Summary Large numbers of cytotoxic T lymphocytes (CTL) could be generated from tumor-draining lymph nodes (DLN) from mice bearing PHS-5 tumor by culturing at low density with autologous tumor cell stimulators and 20 U/ml recombinant interleukin-2 (IL-2). Outgrowth of metastatic tumor cells in culture was prevented by use of this hypoxanthine/aminopterin/thymidine-sensitive mutant of P815, PHS-5. After 9 days in culture, lymphoid cells demonstrated specific cytotoxicity against autologous tumor target cells. Lymph node cells could be expanded continuously in culture with repeated tumor stimulation with up to 7500-fold increase in cell number by 6 weeks; although CTL could be activated from tumor-bearing host spleen cells in short-term culture, they showed no significant growth in long-term cultures. Phenotypically, DLN cells were a mixture of CD8⁺ and CD4⁺ cells immediately after harvest but after 2 weeks in culture they were predominantly CD8⁺ CD4^–. CTL could be generated from tumor-bearing mice 10–14 days after i.d. tumor inoculation into the abdominal wall, but the immune response declined both in spleen and DLN by 21 days. Much greater CTL activity could be generated from axillary DLN that contained metastases than from non-draining popliteal nodes that were free of metastatic tumor cells. Some CTL activity could be generated from DLN with the addition of IL-2 alone but was further increased by the addition of more tumor cells as stimulators. When adoptively transferred to a host with 3-day P815 liver metastases, lymphocytes from DLN activated in vitro were able to reduce or eliminate metastases with very little or no IL-2 administered concomitantly. As few as 10⁶ cells were therapeutically effective, and in vivo efficacy was tumor-specific, since L5178Y liver metastases were not affected.This work was supported in part by grants CA42443, CA48075 and T32-CA09210 from the National Cancer Institute, Department of Health and Human ServicesRecipient of the Canadian Cancer Society McEachern Fellowship.     

Simian virus 40 large-T-antigen-specific rejection of mKSA tumor cells in BALB/c mice is critically dependent on both strictly tumor-associated, tumor-specific CD8(+) cytotoxic T lymphocytes and CD4(+) T helper cells

Protective immunity of BALB/c mice immunized with simian virus 40 (SV40) large T antigen (TAg) against SV40-transformed, TAg-expressing mKSA tumor cells is critically dependent on both CD8(+) and CD4(+) T lymphocytes. By depleting mice of T-cell subsets at different times before and after tumor challenge, we found that at all times, CD4(+) and CD8(+) cells both were equally important in establishing and maintaining a protective immune response. CD4(+) cells do not contribute to tumor eradication by directly lysing mKSA cells. However, CD4(+) lymphocytes provide help to CD8(+) cells to proliferate and to mature into fully active cytotoxic T lymphocytes (CTL). Depletion of CD4(+) cells by a single injection of CD4-specific monoclonal antibody at any time from directly before injection of the vaccinating antigen to up to 7 days after tumor challenge inhibited the generation of cytolytic CD8(+) lymphocytes. T helper cells in this system secrete the typical Th-1 cytokines interleukin 2 (IL-2) and gamma interferon. Because in this system TAg-specific CD8(+) cells secrete only minute amounts of IL-2, it appears that T helper cells provide these cytokines for CD8(+) T cells. Moreover, this helper effect of CD4(+) T cells in mKSA tumor rejection in BALB/c mice does not simply improve the activity of TAg-specific CD8(+) CTL but actually enables them to mature into cytolytic effector cells. Beyond this activity, the presence of T helper cells is necessary even in the late phase of tumor cell rejection in order to maintain protective immunity. However, despite the support of CD4(+) T helper cells, the tumor-specific CTL response is so weak that only at the site of tumor cell inoculation and not in the spleen or in the regional lymph nodes can TAg-specific CTL be detected.     

Adjuvant IL-15 does not enhance the efficacy of tumor cell lysate-pulsed dendritic cell vaccines for active immunotherapy of T cell lymphoma

There has been a recent interest in using IL-15 to enhance antitumor activity in several models because of its ability to stimulate CD8⁺ T cell expansion, inhibit apoptosis and promote memory T cell survival and maintenance. Previously, we reported that C6VL tumor lysate-pulsed dendritic cell vaccines significantly enhanced the survival of tumor-bearing mice by stimulating a potent tumor-specific CD8⁺ T cell response. In this study, we determined whether IL-15 used as immunologic adjuvant would augment vaccine-primed CD8⁺ T cell immunity against C6VL and further improve the survival of tumor-bearing mice. We report that IL-15 given after C6VL lysate-pulsed dendritic cell vaccines stimulated local and systemic expansion of NK, NKT and CD8⁺ CD44^hi T cells. IL-15 did not, however, augment innate or cellular responses against the tumor. T cells from mice infused with IL-15 following vaccination did not secrete increased levels of tumor-specific TNF-α or IFN-γ or have enhanced C6VL-specific CTL activity compared to T cells from recipients of the vaccine alone. Lastly, IL-15 did not enhance the survival of tumor-bearing vaccinated mice. Thus, while activated- and memory-phenotype CD8⁺ T cells were dramatically expanded by IL-15 infusion, vaccine-primed CD8⁺ T cell specific for C6VL were not significantly expanded. This is the first account of using IL-15 as an adjuvant in a therapeutic model of active immunotherapy where there was not a preexisting pool of tumor-specific CD8⁺ T cells. Our results contrast the recent studies where IL-15 was successfully used to augment tumor-reactivity of adoptively transferred transgenic CD8⁺ T cells. This suggests that the adjuvant potential of IL-15 may be greatest in settings where it can augment the number and activity of preexisting tumor-specific CD8⁺ T cells.