Lymphocytes generated by in vivo priming and in vitro sensitization demonstrate therapeutic efficacy against a murine tumor that lacks apparent immunogenicity

The adoptive transfer of sensitized lymphocytes is an effective means to mediate the regression of established tumors. However, successful therapy can only be demonstrated in animal models where tumors are intrinsically immunogenic, capable of eliciting systemic immunity. To explore the potential of this therapeutic approach to tumors of less immunogenicity, we have selected and used a murine tumor, MCA 102, for the current study because all attempts to immunize syngeneic mice failed. We report here that inoculation of mice with a mixture of tumor cells and a bacterial adjuvant, Corynebacterium parvum led to the production of sensitized, but not fully functional, lymphocytes in the draining lymph nodes (LN). These cells, termed pre-effector cells, could nevertheless further differentiate to acquire full immunologic function by an established in vitro sensitization culture method. In adoptive immunotherapy experiments, transfer of as few as 1.5 X 10(7) in vitro sensitized cells not only reduced established pulmonary MCA 102 metastases but also prolonged survival and cured tumors in a majority of the treated animals. In order to elicit pre-effector cells in vivo, inoculation with both tumor cells and C. parvum was essential. Although a broad range of numbers of MCA 102 tumor cells appeared to be effective, generation of pre-effector cells was dependent on the dose of C. parvum. We have found that a C. parvum dose of 25 micrograms was optimal, whereas higher doses of the adjuvant had suppressive effects. Analysis of the kinetics of their appearance revealed that the generation of pre-effector cells was transient. They were detectable 7 days after in vivo priming followed by a rapid decline. Furthermore, pre-effector cells were detected only in the regional draining LN. No reactivity was demonstrable in the spleen, mesenteric LN, PBL, or bone marrow. Taken together, these results expand the scope of immunotherapy by demonstrating the feasibility of manipulating a limited and obscure immune response to the MCA 102 tumor for therapeutic efficacy.     

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.     

Recombinant tumor necrosis factor: species specificity for a variety of human and murine transformed cell lines

Tumor necrosis factor (TNF) exhibits cytotoxic or cytostatic activity on a wide range of animal and human transformed cell lines. Using pure, recombinant human and mouse TNF, we examined the degree of species specificity of the in vitro TNF activity on a variety of human and murine transformed cell lines. This species specificity was studied for the TNF activity alone or in synergism with IFN-gamma. Recombinant human and mouse TNF behave remarkably similarly regarding the in vitro cytolytic/cytostatic activity. However, a certain degree of species-specific preference could be revealed as human cell lines needed a higher concentration of recombinant mouse TNF than of recombinant human TNF to attain a similar effect, while on mouse cells the reverse was true. Also, synergism with IFN-gamma seemed more effective when the target cell was treated with homologous TNF.     

The role of interferon-alpha in a successful murine tumor therapy

Combination therapy using reovirus type 3 and the chemo-therapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is sufficient to cure approximately 80% of EL-4 lymphoma tumor-bearing BD2F1 male mice. Cured animals can be challenged with the EL-4 tumor, in the absence of the therapy, to yield 100% survival, whereas those challenged with heterologous tumor produce 0% survival. These results strongly suggest that a host-immune response is responsible for the observed therapeutic effect. Reovirus, a double-stranded RNA virus, is an efficient inducer of type I interferon. In an effort to determine the role of virus in this therapy, we substituted interferon-alpha (IFN-alpha) for reovirus in the therapy. Doses of IFN-alpha from 1000-10,000 U were capable of replacing reovirus to produce cure rates similar to reovirus. Spleen cells isolated from therapy-treated animals demonstrated high levels of cytotoxicity against the natural killer cell-sensitive cell line YAC-1, but not against EL-4 tumor. In vitro stimulation of isolated spleen cells by IFN-alpha resulted in a high level of natural killer cell activity, but no cytotoxicity against the EL-4 tumor. A significant antiproliferative effect against the EL-4 tumor in cell culture was demonstrated by IFN-alpha. Finally, therapy-treated, tumor-bearing mice that were injected with anti-IFN-alpha + -beta antibodies had similar survival levels as control mice, indicating that other cytokines might also play a role in promoting tumor killing. These investigations suggest that IFN-alpha may be a mediator of antitumor activity in the reovirus therapy system.     

In vitro and in vivo evaluation of intrinsic immunogenicity of reporter and insulin gene therapy plasmids

BACKGROUND: Plasmid DNA vectors offer the potential of safe gene therapy avoiding viral vector-mediated toxicity and immunogenicity. As plasmid DNA is bacterial in origin, presence of bacterial lipopolysaccharide (LPS) or unmethylated CpG dinucleotides may stimulate host innate immunity. METHODS: Primary cultures of mouse and rat dendritic cells were established and incubated with bacterial lipopolysaccharide; immunostimulatory CpG oligodeoxynucleotide; control GpC oligodeoxynucleotide; and a range of (pVR1012) plasmids encoding transgenes with increasing CpG content (wild-type and mutant human preproinsulin; non-mammalian eukaryotic eGFP reporter gene; and bacterial beta-galactosidase reporter gene). IL-12 secretion was assayed to determine in vitro plasmid immunogenicity. Local inflammatory response following intramuscular injection of these plasmids, with or without a non-ionic carrier SP1017, was characterised in vivo. RESULTS: Dose-responsive LPS and CpG stimulation of IL-12 secretion from dendritic cells was demonstrated. All plasmids induced significant IL-12 secretion in comparison to control unstimulated cells. The beta-galactosidase plasmid had highest CpG content and induced significantly higher IL-12 secretion than constructs containing a eukaryotic transgene. Injection of rat muscle with the beta-galactosidase construct induced greater inflammatory response than human preproinsulin constructs. This was further enhanced by SP1017. At 2 days post-injection, monocyte/macrophage injection site infiltration predominated with CD8-positive lymphocytes predominating at 7 days. There was no evidence of transgene expression in infiltrating immune cells. CONCLUSIONS: Dendritic cell immunostimulation may be employed as an in vitro bioassay of innate immune response to plasmid DNA vectors during evaluation for clinical gene therapy.     

Identification of a new murine tumor necrosis factor receptor locus that contains two novel murine receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

Tumor necrosis factor (TNF) ligand and receptor superfamily members play critical roles in diverse developmental and pathological settings. In search for novel TNF superfamily members, we identified a murine chromosomal locus that contains three new TNF receptor-related genes. Sequence alignments suggest that the ligand binding regions of these murine TNF receptor homologues, mTNFRH1, -2 and -3, are most homologous to those of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors. By using a number of in vitro ligand-receptor binding assays, we demonstrate that mTNFRH1 and -2, but not mTNFRH3, bind murine TRAIL, suggesting that they are indeed TRAIL receptors. This notion is further supported by our demonstration that both mTNFRH1:Fc and mTNFRH2:Fc fusion proteins inhibited mTRAIL-induced apoptosis of Jurkat cells. Unlike the only other known murine TRAIL receptor mTRAILR2, however, neither mTNFRH2 nor mTNFRH3 has a cytoplasmic region containing the well characterized death domain motif. Coupled with our observation that overexpression of mTNFRH1 and -2 in 293T cells neither induces apoptosis nor triggers NFkappaB activation, we propose that the mTnfrh1 and mTnfrh2 genes encode the first described murine decoy receptors for TRAIL, and we renamed them mDcTrailr1 and -r2, respectively. Interestingly, the overall sequence structures of mDcTRAILR1 and -R2 are quite distinct from those of the known human decoy TRAIL receptors, suggesting that the presence of TRAIL decoy receptors represents a more recent evolutionary event.     

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.     

Modulation of hematologic and immunologic effects of high dose chemotherapy by interleukin-2 in a murine tumor model.

To determine if intensive chemotherapy consisting of cyclophosphamide (C), etoposide (E), and cisplatin (P) (CEP) may be usefully combined with recombinant human interleukin-2 (rhIL-2), we examined a murine tumor model designed to approximate a common clinical situation: macroscopic, drug-resistant cancer. Using C57BL/6 mice with extensive tumor burden 10 days after intravenous B16 melanoma cell injection, we observed (1) C, E, and P synergize to enhance survival but do not cure mice at the highest tolerable dose (C = 200 mg/kg, E = 60 mg/kg, and P = 3 mg/kg); (2) rhIL-2 at 3 x 10(5) U (subcutaneously) daily for 4 days administered 10-18 days after B16 injection significantly improves survival; (3) CEP plus rhIL-2 is more effective than CEP alone only when rhIL-2 is administered before CEP; (4) CEP suppresses IL-2-induced lymphokine-activated killer cell activity in the spleen; and (5) rhIL-2 protects mice incompletely from the immunologic and hematologic suppression of CEP. Our results suggest that intensive chemotherapy combined with rhIL-2 may be beneficial. The success of any such combination may be schedule dependent.     

Species specificity of human and murine tumor necrosis factor. A comparative study of tumor necrosis factor receptors

Recombinant murine and human tumor necrosis factor (mTNF and hTNF, respectively) were radioiodinated to high specific activity using a solid-phase lactoperoxidase method. A single class of high affinity receptors for 125I-TNF was identified on TNF-sensitive murine L cells and human HeLa S2 cells. Competitive radioligand binding assays were used to study the species specificity of TNF preparations. Unlabeled hTNF competed 30-fold less effectively than mTNF for binding to L cell receptors, whereas mTNF competed to approximately the same extent as hTNF for binding to HeLa cell receptors. A similar species specificity was observed in cytotoxicity assays; hTNF was more cytotoxic for HeLa cells than mTNF. Conversely, mTNF was more growth inhibitory and cytotoxic for L cells than hTNF. mTNF. and hTNF.receptor complexes were compared by gel filtration chromatography and polyacrylamide gel electrophoresis before and after cross-linking with bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES). These complexes eluted in gel filtration at a position corresponding to a globular protein of 350,000 Mr. Gel autoradiographs of the fractions containing cross-linked complexes showed bands of 95,000 and 75,000 Mr as well as small amounts of higher Mr bands. mTNF and hTNF treated with BSOCOES formed cross-linked dimers and trimers. Therefore, we were unable to determine whether the 95,000 and 75,000 Mr bands represented two distinct subunits of receptors or one subunit to which either a dimer or a monomer of TNF was cross-linked. These results demonstrate species specificity in the TNF-receptor interaction. In addition, the affinity labeling studies in two species give an identical pattern for the TNF X receptor complexes, suggesting that the receptors have similar subunit composition.     

Selection of microRNA for providing tumor specificity of transgene expression in cancer gene therapy

The use of tumor-specific microRNA loss to inhibit transgene expression in normal cells is considered as a way to increase the specificity of gene-therapeutic antitumor drugs. This method assumes the introduction of recognition sites of suppressed in tumor cells microRNAs into transgene transcipt. In the presented work, the efficiency of the strategy for providing the tumor specificity of transgene expression depending on parameters of microRNA expression in normal and tumor cells was studied. It was established that microRNA suppression in tumor cells and the determination of absolute microRNA levels in tumor and normal cells are not sufficient for the adequate estimation of the possibility of specific microRNA usage in the scheme of cancer gene therapy, and particularly do not allow to exclude a significant decrease in the efficiency of the gene-therapeutic drug upon the introduction of microRNA recognition sites. These parameters are only suitable for the preliminary selection of microRNA. The effect of introduction of microRNA recognition sites on transgene expression level in target tumor cells should be validated experimentally. It is suggested that this should be done directly in the cancer gene therapy scheme with monitoring of the therapeutic transgene activity.     

Isolation of a Rhodobacter capsulatus mutant that lacks c-type cytochromes and excretes porphyrins.

A Rhodobacter capsulatus mutant lacking cytochrome oxidase activity was isolated by Tn5 mutagenesis. Difference spectroscopy of crude extracts and extracted c-type cytochromes demonstrated that this mutant completely lacked all c-type cytochromes. The strain did, however, synthesize normal amounts of b-type cytochromes and nonheme iron. This mutant also excreted large amounts of coproporphyrin and protoporphyrin and synthesized reduced amounts of bacteriochlorophyll, suggesting a link between the synthesis of c-type cytochromes and the expression of the tetrapyrrole biosynthetic pathway.     

Generation and characterization of hamster monoclonal antibodies that neutralize murine tumor necrosis factors

mAb to murine TNF (MuTNF) were produced after immunization of Armenian hamsters with purified, Escherichia coli-derived rMuTNF-alpha. Antibody produced from clone TN3-19.12, was purified and was found to inhibit 100% of the lytic activity of either recombinant or natural MuTNF-alpha at an antibody input of 25 ng/U. TN3-19.12 also inhibited all the lytic activity in culture supernatants from a variety of T cell sources, including activated T cell clones and T cell hybridomas (all of which expressed high levels of TNF-alpha and TNF-beta (lymphotoxin, LT) mRNA). Western blot analysis was used to document the physical form(s) of MuTNF recognized by TN3-19.12. Recombinant and macrophage-derived TNF displayed identical patterns of a single band with Mr 17 kDa. In contrast, T cell culture supernatants exhibited patterns consisting of two bands with Mr 17 and 24.7 kDa. The higher m.w. form was glycosylated based on its sensitivity to n-glycanase and displayed a m.w. consistent with that of TNF-beta (LT). These data suggest that TN3-19.12 recognizes both MuTNF-alpha and MuTNF-beta (LT). Monoclonal TN3-19.12 and polyvalent rabbit anti-rTNF were used to establish a MuTNF-specific ELISA capable of detecting picogram quantities of recombinant or natural TNF. This assay was used to detect TNF in the sera of mice challenged with a lethal dose of LPS. Peak TNF serum levels of 11 ng/ml were observed in these animals 90 min after i.p. LPS administration and then rapidly declined to near base line levels by 3 h. These values were confirmed by quantitating levels of TNF functional activity in the same samples. TN3-19.12 injected into mice subsequently treated with LPS prevented the detection of TNF in the circulation by either assay and protected mice from the lethal effects of endotoxin shock. Thus, TN3-19.12 effectively neutralizes endogenously produced TNF in vivo.     

Distinct phenotypic expression of immunogenicity and immunosensitivity in sublines of a tumor

Continuous in vitro or in vivo passage of a BALB/c leukemia has resulted in generation of 2 immunologically distinct sublines. The subline maintained by in vitro passage failed to stimulate an allogeneic response but was susceptible to lysis by alloreactive cytotoxic cells. Conversely, the subline maintained by in vivo passage induced an allogeneic response but was resistant to lysis by cytotoxic T lymphocytes (CTL) reactive to H-2d antigens. Resistance to lysis occurred despite expression of H-2d antigens in a form recognizable by differentiated alloreactive CTL, as determined by cold-target inhibition experiments. Moreover, resistance was immunologically specific, in that the subline was susceptible to immune lysis mediated through recognition of other determinants. The results imply that the display and/or orientation of antigen in the cell membrane of these sublines that is required for a lytic event is distinct from the antigen expression necessary for immunologic recognition.