Alpha 1-interferon gene transfer into metastatic Friend leukemia cells abrogated tumorigenicity in immunocompetent mice: antitumor therapy by means of interferon-producing cells.

Highly metastatic alpha/beta-interferon (IFN-alpha/beta)-resistant Friend leukemia cells (FLC) were transfected with a retroviral vector (pLTneoL-5) containing the mouse IFN-alpha 1 gene. Transfected clones were isolated and tested for their capacity to secrete IFN-alpha 1 and their tumorigenicity when injected s.c. into immunocompetent syngeneic DBA/2 mice. Almost all FLC clones producing IFN in the range of 16-512 units/ml failed to grow when injected s.c. or i.p. into normal mice, whereas control FLC (transfected with a vector without the IFN gene) exhibited the highly malignant phenotype of the original FLC. High levels of IFN were detected in peritoneal fluid, tumor extracts, and sera of mice given injections of IFN-producing cells. Injection of mice with antibodies to IFN-alpha/beta resulted in the development of tumor ascites in mice transplanted i.p. with IFN-producing FLC. In contrast to the tumor rejection observed in immunocompetent mice, IFN-producing FLC were highly tumorigenic when transplanted into immunosuppressed nude mice. Mice given injections of IFN-producing FLC developed a long-lasting tumor-specific immune resistance to subsequent injection with highly metastatic FLC. Simultaneous s.c. injection of both metastatic FLC (approximately 10(3) 50% lethal doses) and IFN-producing cells resulted in potent inhibition of the tumor growth, with a survival rate of approximately 50% for injected mice. Contralateral injection (s.c.) of IFN-producing FLC into mice with established metastatic tumors produced a marked inhibition of tumor growth, with a survival rate of 10% for injected mice. These results indicate that: (a) the genetic modification of highly metastatic FLC by means of transfer of the IFN-alpha 1 gene results in potent tumor cell rejection, which is mediated by an IFN-induced host immune response; (b) injections of IFN-producing tumor cells are effective in inhibiting tumor growth in mice with established metastatic tumors. These data suggest that tumor cells transfected with the IFN-alpha gene might be used as an effective therapy for the treatment of certain human metastatic tumors, provided that suitable strategies are defined to prevent growth of the cytokine-producing cells.

The induction of in vivo proliferation of long-lived CD44hi CD8+ T cells after the injection of tumor cells expressing IFN-alpha1 into syngeneic mice.

The tumorigenicity of transplantable tumor cells in mice is reduced by transduction with cytokine genes, including IFN-alpha and interleukin (IL) 12. Although T cells are considered important in tumor rejection, the mechanism by which genetically modified tumor cells stimulate the immune system has not been examined. In this study, the in vivo proliferation of T-cell subsets in mice transplanted with cytokine-producing syngeneic tumor cells was assessed by administering the DNA precursor bromodeoxyuridine. The injection of viable cells producing IFN-alpha or IL-12 caused a marked proliferation of CD8+ T lymphocytes in both the spleen and lymph nodes. Proliferation was most prominent among memory-phenotype CD44hi CD8+ T cells. In contrast, proliferation of CD8+ T cells did not occur in mice injected with control cells or with cells expressing IL-4, granulocyte colony-stimulating factor, or IFN-gamma. Pulse-chase studies in mice injected with IFN-alpha-producing cells showed that a proportion of proliferating CD8+ T cells survived for at least 70 days, suggesting that long-lived memory cells are induced using such an approach. In summary, these results, together with previous studies on the host immune reactivity triggered by the injection of tumor cells expressing IFN-alpha, represent a strong rationale for considering IFN-alpha as a powerful T-cell adjuvant for the generation of more effective cancer vaccines.

Cure of mice with established metastatic friend leukemia cell tumors by a combined therapy with tumor cells expressing both interferon-alpha 1 and herpes simplex thymidine kinase followed by ganciclovir.

Transduction of the murine interferon-alpha (IFN-alpha) gene into various malignant mouse tumor cells has resulted in the loss of tumorigenicity and an acquired capacity to induce long-lasting antitumor immunity following their injection into immunocompetent syngeneic mice. In the present study, we investigated the effectiveness of IFN-alpha-producing tumor cells in the therapy of mice with established mouse tumors. In DBA/2 mice bearing subcutaneous (s.c.) Friend erythroleukemia cell (FLC) tumors, we found that to achieve some antitumor response (i) it was necessary to inject high numbers of IFN-alpha-producing FLC, which occasionally lead to the formation of slowly growing tumors; and, that (ii) repeated injections of irradiated IFN-alpha-FLC did not result in any antitumor effect. The therapeutic potential of IFN-alpha-producing FLC rendered sensitive to ganciclovir (GCV), by transfer of the herpes simplex virus thymidine kinase (tk) gene, was investigated. Complete tumor rejection and cure was observed in > or = 70% of the animals after injection of high numbers (10(7)) of IFN-alpha-producing tk-expressing tumor cells followed 4 days later by repeated GCV treatments, whereas only a slight increase in survival time was obtained after administration of control tk-expressing tumor cells (not producing IFN) and GCV. Tumor rejection was associated with a dramatic destruction of tumor tissue and with the subsequent development of a potent and long-lasting antitumor immunity. No therapeutic effect was observed in immunosuppressed nude mice. These data indicate that this approach may represent an effective and safe therapeutic strategy for antitumor cytokine gene therapy.

Type I consensus IFN (IFN-con1) gene transfer into KSHV/HHV-8-infected BCBL-1 cells causes inhibition of viral lytic cycle activation via induction of apoptosis and abrogates tumorigenicity in sCID mice.

In this study, we investigated the effects of human type I consensus interferon (IFN-con1) (Amgen) gene transfer into body cavity-based lymphomas (BCBL)-1 cells, which are latently infected with Kaposi's sarcoma-associated herpesvirus (KSHV) human herpesvirus-8 (HHV-8). Both the basal and 12-O-tetradecanoyl phorbol-13-acetate (TPA)-stimulated production of KSHV/HHV-8 mature virions was strongly inhibited in genetically modified IFN-producing BCBL-1 cells as compared with parental or control transduced counterparts. A similar inhibition was obtained on treatment of parental BCBL-1 cells with exogenous IFN-con1. The reduction in KSHV/HHV-8 production was associated with a decrease in the basal and TPA-stimulated intracellular amount of the linear form of the viral genome. Interestingly, 25%40% of the IFN-producing BCBL-1 cell population underwent spontaneous apoptosis in vitro. TPA treatment, which did not significantly affect the viability of the parental and control BCBL-1 cells, resulted in the apoptotic death of up to 70% of the IFN-producing cell population. Addition of exogenous IFN-con1 to parental BCBL-1 cells produced similar effects, although less intense. Injection of either parental or control-transduced BCBL-1 cells into SCID mice resulted in progressively growing tumors characterized by an unusually high level of tumor angiogenesis. In contrast, complete tumor regression was observed in all the mice injected either subcutaneously (s.c.) or intraperitoneally (i.p.) with the IFN-producing BCBL-1 cells. These results represent the first evidence that type I IFN can counteract the activation of a productive herpesvirus infection in latently infected tumor cells by the induction of apoptosis, providing an interesting link between the antiviral and antitumor activities of this cytokine. These data suggest the possible advantages of strategies of type I IFN gene transfer (with respect to the use of the exogenous cytokine) for the treatment of patients with some HHV-8-induced malignancies.

Murine interferon-alpha1 gene-transduced ESb tumor cells are rejected by host-mediated mechanisms despite resistance of the parental tumor to interferon-alpha/beta therapy.

The highly metastatic ESb tumor is totally resistant to murine interferon-alpha/beta (IFN-alpha/beta) therapy, regardless of the number of cells injected or the route of inoculation. In contrast, as we show herein, mouse IFN-alpha1-transduced ESb tumor cells were inhibited markedly when injected subcutaneously into immunocompetent mice. IFN-producing ESb tumor rejection was mediated by the immune system, because these tumor cells grew normally in immunosuppressed mice. Tumor regression was accompanied by extensive necrosis and cellular infiltrates in the tumor area. These results further support the use of IFN-alpha in cytokine gene therapy of cancer and suggest the advantage of using gene transfer rather than cytokine administration to enhance an antitumor immune response.

Antitumor activity of recombinant adenoviral vectors expressing murine IFN-alpha in mice injected with metastatic IFN-resistant tumor cells.

Recent studies have shown that gene therapy with type I interferon (IFN) in an adenovirus vector is a powerful tool to suppress the growth of human tumors transplanted in immune-deficient mice. However, in these studies the host immune-mediated effects, which may be important in mediating the long-term control of tumor growth by these cytokines, was not studied. In this paper, we evaluate the antitumor efficacy of different adenoviral vectors containing mouse IFN-alpha genes (i.e., a first-generation replication-defective vector containing IFN-alpha1 and two different second-generation vectors containing IFN-alpha2) in immunocompetent DBA/2 mice transplanted with highly metastatic Friend leukemic cells resistant in vitro to type I IFN. We found that injection of all the different adenovirus vectors containing mouse IFN-alpha( genes resulted in a marked antitumor response in mice transplanted either subcutaneously or intravenously with IFN-resistant Friend leukemic cells compared to tumor-bearing animals inoculated with a control vector. Tumor growth inhibition after injection of IFN-adenovirus vectors was associated with a prolonged presence of high IFN levels in the sera of the injected mice. Suppression of metastatic tumor growth was also observed after a single injection of the IFN--adenovirus recombinant vectors, whereas a comparable antitumor response generally required several injections of high doses of IFN. Altogether, these results demonstrate that IFN--adenoviral vectors can efficiently inhibit metastatic tumor growth by host-mediated mechanisms and suggest that adenovirus-mediated IFN-alpha gene therapy may represent an attractive alternative to the conventional clinical use of this cytokine, which generally requires multiple injections of high IFN doses for a prolonged period of time.

Wheat germ agglutinin-binding protein changes in highly malignant Friend leukemia cells metastasizing to the liver.

We have used binding of radioactive lectins (i.e. Concanavalin A (ConA), Wheat Germ Agglutinin (WGA) and Ricinus communis agglutinin I (RCAI)) to membrane glycoproteins separated in SDS gel electrophoresis, to detect specific carbohydrate changes in plasma membrane proteins of in vivo passaged Friend erythroleukemia cells (FLC). These cells are highly metastatic to the liver, whereas the original in vitro passaged tumor cells do not metastasize. Marked qualitative differences in the high molecular weight region of the gels (100-200 kD) were observed between the WGA binding glycoproteins of metastatic in vivo passaged FLC and nonmetastatic in vitro passaged FLC. Furthermore, the binding of WGA to plasma membrane proteins of in vivo passaged FLC was much greater than the binding of WGA to plasma membrane proteins of in vitro passaged FLC. Lectin binding experiments after sialic acid removal by in situ mild acid hydrolysis of FLC glycoproteins indicated that an increased sialylation of the 120 and 145 kD glycoproteins was responsible for the increased WGA reactivity of in vivo passaged FLC plasma membranes. Besides the increased sialylation, other changes in glycosylation of the 100-200 kD glycoproteins of in vivo passaged FLC were observed: (1) qualitative differences between the WGA binding patterns of the two cell types were restored after treatment of the gels with mild acid and subsequent Smith degradation; (2) after chemical removal of sialic acid residues from the gels, qualitative differences in the RCA binding patterns to the glycoproteins of the two cell types were apparent.

Studies on the expression of H-2 antigens in non-metastatic and highly metastatic Friend erythroleukemia cells: correlation with the in vivo behaviour of tumor cells.

The levels of expression of histocompatibility antigens on the cell membrane and their gene expression in non-metastatic and in highly metastatic Friend leukemia cells (FLC) were measured and the levels of expression of these antigens were correlated with the different in vivo behaviour of the tumor cells. Highly metastatic in vivo passaged FLC (either interferon-sensitive 745 or interferon alpha/beta-resistant 3Cl-8 cells) expressed higher levels of class I H-2K and H-2D antigens on their cell membrane with respect to the non-metastatic in vitro passaged counterparts. The increased expression of H-2 class I antigens was associated with an increased transcription of H-2K and H-2D genes. As both in vitro and in vivo passaged FLC have been shown to be resistant in vitro to the natural killer (NK) cell activity, we tried to correlate the levels of expression of histocompatibility antigens with the in vivo clearance of [125I]UDR-labeled FLC. However, no correlation was found between the levels of expression of H-2 antigens and the in vivo clearance of tumor cells. In fact, in vivo passaged FLC (tested either after 1 or after 15 in vitro passages) expressed virtually identical levels of H-2 antigens; however, the freshly explanted in vivo passaged FLC exhibited markedly lower levels of clearance from the lung, spleen and liver (when injected i.v. in DBA/2 mice) with respect to the corresponding FLC cultivated for several passages in vitro. Pretreatment of in vitro passaged 745 FLC with either interferon alpha/beta or interferon gamma resulted in the acquisition of some metastatic potential of FLC to the liver when interferon-treated FLC were subsequently injected i.v. in DBA/2 mice; such in vitro treatments resulted in a 2-3-fold increase in the expression of H-2K antigens versus the control untreated FLC. We suggest that such increases could represent some advantages for the homing properties of tumor cells and/or for the tumor progression, by mechanisms different from the resistance to the NK cells.

Inhibition of lung colonisation of a mouse mammary carcinoma by therapeutic vaccination with interferon-alpha gene-transduced tumor cells.

A spontaneously metastatic murine mammary adenocarcinoma, TSA, has been transduced with the gene for interferon alpha1 (IFN-alpha). Transfectants were used for the immunotherapy of mice bearing lung colonies induced by the intravenous inoculation of non-transduced parental cells. A significant reduction in the number of tumor colonies was obtained when repeated subcutaneous administrations of mitomycin C-blocked transfectant cells were given, commencing 3 days after an intravenous challenge with TSA cells. Intraperitoneal vaccination induced a stronger anti-tumor response than subcutaneous vaccination, and the proportion of tumor-free mice reached 50%. The potency of IFN-alpha transfectants was similar to that of IFN-gamma transfectants previously obtained from TSA. Admixture of IFN-alpha and IFN-gamma transfectant cells in the same vaccine did not increase the curative effect over that of single vaccines. In nude mice vaccination with IFN-alpha or IFN-gamma transfectants did not lead to a reduction in the number of lung colonies, indicating that an intact T cell response was required for the therapeutic effect observed in immunocompetent mice.

Gene therapy of cancer with interferon: lessons from tumor models and perspectives for clinical applications.

Cytokine gene transfer is a current approach in studies of gene therapy of cancer IFNs represent valuable cytokines for these studies, since they exert multiple biological effects, including anti-tumor activities. Early studies have been focused on IFN-gamma. Recently, several reports have shown that the transfer of type I IFN (especially IFN-alpha) genes represents a powerful approach for inducing tumor suppression. Recent studies have underscored new IFN-induced activities on immune cells. This knowledge adds a further rationale for the use of IFN-alpha in strategies of gene therapy of cancer and can be exploited for the design of more selective and effective anticancer treatments.

Role of endogenous alpha/beta interferon in the selection of virus nonproducer Friend leukemia cells after serial intraperitoneal passages in syngeneic mice.

Serial intraperitoneal passage of interferon (IFN)-sensitive Friend leukemia cells (FLCs) and L1210-S and RBL-5 tumor cells in syngeneic mice resulted in the selection of tumor cells exhibiting a marked decrease in the capacity to release reverse transcriptase (RT) activity. The virus nonproducer phenotype was a stable characteristic of clones derived from in vivo-passaged IFN-sensitive 745 FLCs. In contrast, in vivo passages of IFN-resistant 3Cl-8 FLCs and L1210-R cells did not result in any significant decrease in the capacity of these tumor cells to release in vitro RT activity. Although in vitro treatment of IFN-sensitive FLCs with mouse alpha/beta IFN (IFN-alpha/beta) for 1 or 10 passages resulted in a marked inhibition in the release of RT activity, these effects were completely reversible after removal of IFN from the culture medium. In addition, in vitro treatment of 745 FLCs with IFN resulted in a marked increase in the expression of H-2 (class I) and gp70 Friend virus antigens on the cell membrane. These effects were not observed in IFN-resistant 3Cl-8 cells. To investigate the possible role of endogenous IFN in the in vivo selection of virus nonproducer tumor cells, IFN-sensitive virus producer FLCs were serially passaged intraperitoneally in mice treated with antibodies to IFN-alpha/beta and in control mice, and the recovered tumor cells were cloned in vitro. Most (83 to 91%) of the clones derived from 745 cells recovered from control mice did not produce any detectable RT activity in the culture supernatants. In contrast, 96% of the clones (26 of 27) derived from 745 cells recovered from mice serially treated with antibodies to IFN-alpha/beta were still capable of releasing high levels of RT activity in the culture medium, indicating that endogenous IFN-alpha/beta was indeed an important host component for the in vivo selection of virus nonproducer tumor cell variants. The results reported in this article indicate that both direct effects of IFN on tumor cells and host-mediated effects are involved in this phenomenon.

Friend murine leukemia virus and spleen focus-forming virus expression in highly malignant interferon-sensitive and interferon-resistant Friend leukemia cells.

Analysis of expression of the Friend murine leukemia virus (F-MuLV) and of the spleen focus forming virus (SFFV) has been undertaken in highly malignant interferon (IFN)-sensitive (745) and IFN-resistant (3Cl-8) Friend leukemia cells (FLC), serially passaged intraperitoneally in DBA/2 mice. In vivo passaged 745 cells, as well as the clones derived thereof, did not release Friend virus (FV). Western blot analysis of the plasma membrane fractions of the virus nonproducer 745 cells revealed the lack of gp69/70 glycoprotein expression. At least 10 intraperitoneal passages of virus producer in vitro passaged of virus producer in vitro passaged 745 cells were necessary to obtain the selection of the virus nonproducer phenotype. In contrast in vivo passaged 3Cl-8 cells continued to produce FV even after 100 in vivo passages and expressed gp69/70 antigens to a similar extent as the original in vitro passaged FLC. The expression of F-MuLV and SFFV RNAs in virus producer and virus nonproducer FLC clones has been investigated by means of Northern blot technique using probes specific for either F-MuLV or SFFV. No F-MuLV specific RNA sequences were detected in virus nonproducer 745 clones. SFFV specific RNA transcripts and gp52/55 glycoprotein production could be revealed in all the FLC tested. Southern blot analysis showed the presence of F-MuLV specific sequences in the cellular DNA of virus nonproducer 745 clones. As both in vivo passaged F-MuLV producer 3Cl-8 and F-MuLV nonproducer 745 cells were equally barely immunogenic and highly malignant when injected into syngeneic DBA/2 mice, these results indicate that F-MuLV expression does not result per se in a high immunogenic potential of tumor cells. For the time being, as a specific property of 3Cl-8 versus 745 cells is the interferon-resistant phenotype, it is tempting to speculate that the selection of virus nonproducer cell variants after in vivo passages of interferon-sensitive 745 cells could depend on the presence of low levels of endogenous interferon in normal young mice.

IFN-alpha 1 gene transfection completely abolishes the tumorigenicity of murine B16 melanoma cells in allogeneic DBA/2 mice and decreases their tumorigenicity in syngeneic C57BL/6 mice.

The murine B16 melanoma (H-2b) was transfected with a retroviral vector containing the mouse IFN-alpha 1 gene. IFN-alpha 1-transfected cells produced IFN-alpha in vitro and exhibited an altered phenotype characterized by a decreased rate of multiplication, enhanced expression of H-2 antigens, an antiviral state to VSV, and decreased pigmentation. Control and IFN-alpha 1-transfected cells were tested for their ability to grow in syngeneic (H-2b) C57Bl/6 and allogeneic (H-2d) DBA/2 mice. IFN-alpha 1-producing B16 clones were less tumorigenic after s.c., i.p., and i.v. routes of injection than IFN-non-producer B16 clones in syngeneic C57Bl/6 mice. IFN-alpha 1-producing B16 cells were, however, totally rejected by allogeneic DBA/2 mice regardless of the routes and inocula tested, while control B16 cells grew in and killed DBA/2 mice. The total rejection of IFN-alpha 1-transfected B16 cells in allogeneic mice appeared to be dependent on T cells as these cells grew in DBA/2 nude mice. Incubation of IFN-alpha-producing clones with anti-mouse IFN-alpha/beta prior to injection into C57Bl/6 mice did not enhance their tumorigenicity. Likewise, injection of C57Bl/6 and DBA/2 mice with antibody to IFN-alpha/beta did not enhance the tumorigenicity of IFN-alpha 1-transfected cells. C57Bl/6 mice immunized with irradiated IFN-alpha 1 cells were only slightly protected against a subsequent challenge with parental B16 cells. In contrast, DBA/2 mice immunized with irradiated IFN-alpha 1 cells exhibited tumor-specific, long-lasting immunity to subsequent challenge with parental B16 cells.

Persistent infection of normal mice with human immunodeficiency virus.

In this article, we report the establishment of persistent HIV type 1 infection of normal Swiss mice after a single intraperitoneal injection with high-producing HIV-infected U937 cells. Anti-HIV antibodies were found more than 500 days after the original injection, and p24 antigenemia was detected in approximately 50% of the mice. By polymerase chain reaction (PCR) techniques, HIV-specific gag and env sequences were detected in DNA samples from peripheral blood mononuclear cells (PBMC) and peritoneal cells of seropositive mice 300 to 500 days after inoculation with HIV-infected cells. These DNA samples did not contain human DNA sequences, as determined by PCR analysis using primers and the probe for the HLA-DQ alpha gene. Low levels of p24 and detectable human reverse transcriptase activity were found in cultures of PBMC and peritoneal macrophages. Cocultivation of PBMC, peritoneal cells, and spleen cells with human uninfected U937 or CEM (a T lymphoma cell line) cells resulted in HIV infection of the target cells, as determined by PCR analysis and/or p24 assays. The intravenous injection of untreated Swiss mice with the PBMC from PCR-positive mice resulted in the development of an increasing antibody response to HIV in the recipient animals. Together these results indicate that cells from seropositive Swiss mice were persistently infected with HIV and were capable of producing infectious virus. The development of persistent HIV infection in an immunocompetent mouse may represent the starting point for further studies aimed at defining the host mechanisms involved in the restriction of virus replication, defining the pathogenesis of HIV infection, and testing antiviral compounds and vaccines.

Biologic and biochemical differences between in vitro and in vivo passaged Friend erythroleukemia cells. II. Changes in cell surface glycoproteins associated with a highly malignant phenotype.

Friend erythroleukemia cells (FLC), serially passaged in vitro or by intraperitoneal injection in DBA/2 mice, exhibit markedly different tumorigenicity and capacity to metastasize. We have attempted to determine whether the differences in tumorigenicity between these two lines of FLC were correlated with any biochemical changes in their cell membranes. Although consistent modifications of FLC membrane gangliosides were detected after FLC multiplied in the peritoneum, the pattern of FLC gangliosides was not a stable characteristic and did not correlate with tumorigenicity. In contrast, analysis of FLC membrane glycoproteins by cell surface labelling techniques (i.e., galactose-oxidase-borohydride techniques and polyacrylamide gel electrophoresis-fluorography) or by metabolic labelling of glycoproteins with 3H-galactose, revealed consistent differences in the high MW region of the gels between parental in vitro passaged FLC (either 745 or 3Cl-8 cells) and clones derived from in vivo passaged cells. No significant differences in the membrane proteins were detected between in vitro and in vivo passaged FLC when lactoperoxidase-catalyzed iodination and polyacrylamide gel electrophoresis-autoradiography were used. It is seen that repeated in vivo passages of FLC resulted in the appearance of different patterns of membrane glycoproteins and that these changes appeared to be associated consistently with the capacity of these cells to grow as tumor ascites and to metastasize to the liver and spleen.

Biologic and biochemical differences between in vitro and in vivo passaged Friend erythroleukemia cells. I. Tumorigenicity and capacity to metastasize.

Cloned interferon-sensitive (745) and interferon-resistant (3Cl-8) Friend erythroleukemia cells (FLC) passaged in vitro, are not very tumorigenic when first injected intraperitoneally (i.p.) into syngeneic DBA/2 mice although they do form solid tumors when injected subcutaneously (s.c.). By serially passaging FLC (either 745 or 3Cl-8 cells) i.p. in DBA/2 mice, we obtained two different FLC lines capable of growing i.p. and inducing tumor ascites. The s.c. injection of DBA/2 mice with these in vivo passaged FLC resulted in tumor metastases in the liver and spleen, whereas metastases were not observed in mice inoculated s.c. with in vitro passaged FLC. The capacity of in vivo passaged FLC to metastasize was acquired after several i.p. passages. This highly malignant behavior was a stable characteristic of these cells. All the clones derived from in vivo passaged FLC and passaged more than 14 times in vitro induced hemorrhagic ascites when injected i.p., and metastasized to the liver and spleen when injected s.c. The phenotype of sensitivity or resistance to the inhibitory effect of alpha/beta mouse interferon on virus replication and cell multiplication was conserved during serial i.p. passages and maintained in the clones derived from in vivo passaged cells. These FLC showed a decreased capacity to differentiate in vitro upon treatment with dimethylsulfoxide (DMSO) and a reduced production of Friend leukemia virus with respect to the original clones passaged in vitro.

Local and systemic response of mice to interferon-alpha 1-transfected Friend leukemia cells.

DBA/2 mice were injected subcutaneously with an interferon (IFN)-alpha/beta-resistant line of Friend erythroleukemia cells (FLC) transfected with the mouse IFN-alpha 1 gene. These tumor cells produced IFN constitutively, and mice had persistently high levels of IFN in the circulation. We examined the IFN-induced host mechanisms responsible for the local inhibition of growth of these IFN-alpha-transfected FLC and some of the unusual systemic effects of constant interferonemia such as extramedullary hematopoiesis in the liver, an increase in myeloid cells in the spleen, and persistently elevated splenic natural killer (NK) cell activity. In addition, both DBA/2 +/bg and beige mice developed a rapid and specific resistance to intravenous challenge with parental FLC. In previous experiments DBA/2 beige mice could not be protected by exogenous IFN-alpha/beta. The differences in the response of mice to the constitutive production of IFN-alpha by IFN-alpha-transfected tumor cells and their response to exogenous IFN is discussed in terms of the effects of IFN on the host and of antitumor therapy.

IFN-alpha 1 gene expression into a metastatic murine adenocarcinoma (TS/A) results in CD8+ T cell-mediated tumor rejection and development of antitumor immunity. Comparative studies with IFN-gamma-producing TS/A cells.

Cells from a spontaneous, invasive, and metastasizing mouse mammary adenocarcinoma (TS/A-pc) were transfected with a retroviral vector containing the mouse IFN-alpha 1 gene. TS/A clones secreting varying amounts of IFN-alpha 1 were isolated and their tumorigenicity was evaluated after s.c. or i.v. injection into immunocompetent BALB/c mice. Almost all of the IFN-alpha-secreting TS/A clones failed to grow in a high percentage of mice or formed small tumors after a long latency time, whereas TS/A-pc or transfection control cells always grew into large s.c. tumors. Rejection was mainly mediated by CD8+ T lymphocytes and partially by polymorphonuclear cells, as demonstrated by selective immunosuppression experiments and histologic and ultrastructural data. After rejection, a significant portion of mice displayed an immune resistance to the subsequent challenge with TS/A-pc. When the metastatic ability of IFN-alpha-secreting clones was compared with that of previously characterized IFN-gamma-secreting TS/A clones, it was found that the expression of IFN-alpha into TS/A tumor cells resulted in a potent inhibition of metastases formation, whereas IFN-gamma expression either did not affect or even enhanced the metastatic behavior of TS/A cells. These results provide strong evidence for the usefulness of IFN-alpha-producing tumor cells for the development of gene therapy strategies and vaccines against metastatic tumors.

Local and systemic antitumor response after combined therapy of mouse metastatic tumors with tumor cells expressing IFN-alpha and HSVtk: perspectives for the generation of cancer vaccines.

In this study, we have evaluated the local versus systemic antitumor response in tumor-bearing mice subjected to a combined therapeutic regimen based on the injection of genetically modified Friend erythroleukemia cells (FLC) producing IFN-alpha and expressing the HSVtk (tk) gene, and we have investigated the host immune mechanisms involved in tumor rejection and development of antitumor immunity. Repeated subcutaneous (s.c.) injections of IFNtk-expressing tumor cells, followed by GCV administration, were effective in counteracting the growth of both contralateral parental tumors as well as visceral metastases, whereas similar treatments with control tk cells (i.e. nonproducing IFN) were ineffective. Morphologic analyses of the homolateral and contralateral tumor tissues and in vivo immunosuppression experiments with specific monoclonal antibodies revealed that both CD4+ and CD8+ T lymphocytes played essential roles in the generation of a definite antitumor response after the combined therapeutic regimen. We have also compared the effectiveness of irradiated versus viable tumor vaccines co-expressing the two genes in the FLC model and in the poorly immunogenic metastasizing TS/A adenocarcinoma tumor system. Repeated injections of high doses of irradiated IFN-alpha-tk-expressing tumor cells followed by GCV administration resulted in the cure of the majority of mice bearing established metastatic tumors, while repeated inoculations of the same number of viable tumor vaccines were much less effective. We conclude that; (1) IFN-alpha is an essential cofactor in the generation of a systemic antitumor immunity following the prodrug-induced tumor cell killing; (2) vaccines co-expressing an autotoxic gene and a cytokine gene may represent promising new tools for the treatment of some cancer patients.