FBL-3 Cells Research Articles

Abstract 733: Modulating T cell immunity in tumors by targeting PD-L1 and neoantigens using a live attenuated oral Salmonella platform

Abstract Significant progresses have been achieved recently in cancer vaccines that aim at engaging or reengaging tumor directed T cells, in particular in the rapidly growing field of personalized immunotherapy. Yet novel immunization solutions to produce effective tumor-associated (neo)antigens T cell responses while simultaneously overcoming the immunosuppressive tumor microenvironment are urgently needed. We are developing a unique and versatile oral T-cell vaccination platform based on the FDA-approved live-attenuated Salmonella Typhi strain Ty21a vaccine Vivotif®, capable of delivering tumor-associated antigens encoded in DNA expression construct to the gut-associated lymphoid tissue, breaking immune tolerance and inducing anti-tumor immunity. This study summarizes the immunogenicity and antileukemia efficacy of VXM10 vaccines based on the live-attenuated Salmonella Typhimurium strain SL7207, transformed with a eukaryotic expression plasmid encoding the murine programmed death-ligand 1 (PD-L1) protein. It also describes for the first time the systemic immunogenicity of Salmonella based polyepitope oral vaccines, supporting the design of Salmonella based neoantigen vaccines. The antileukemia activity of VXM10 was evaluated in the FBL-3 disseminated model of leukemia, in which the tumor cells express high levels of PD-L1. Oral administration of VXM10 produced a strong anti-tumor effect in the FBL-3 leukemia model, with 100% of surviving animals 80 days after leukemia challenge in the highest dose groups. In contrast, administration of the empty vector control did not show any anti-cancer effect. Moreover, 100% of long-term surviving mice resisted re-challenge with FBL-3 cells, demonstrating that vaccination with VXM10 generated a potent memory T cell response against the leukemia. Importantly, full leukemia control was achieved in both prophylactic and therapeutic settings. The anti-tumor effect observed in these experiments was as strong as in previous experiments using a Salmonella Typhimurium transformed with a plasmid encoding Wilm's tumor 1 (WT1). The anti-tumor efficacy was accompanied by an increased systemic antibody response, and the activation of T cells directed against PD-L1 epitopes. Finally, different polyepitope vaccines encoding model epitopes from VEGFR2, Mesothelin, WT1, CEA, and Ovalbumin, induced a significant systemic immunogenicity for up to 6 out of 9 epitopes, 10 days after vaccination of healthy C57BL/6 mice via the oral route, as measured in the spleen by flow cytometry using peptide-MHC class I pentamers. These studies demonstrate that the Salmonella Ty21a platform can be used to generate anti PD-L1 antibody and T cell responses, as well as CD8-positive T cell responses against a majority of MHC class I epitopes of a polyepitope construct. We are now designing a clinical neo-antigen based vaccine that employs the concepts presented. Citation Format: Sébastien Wieckowski, Lilli Podola, Heiko Smetak, Anne-Lucie Nugues, Philippe Slos, Amine Adda Berkane, Ming Wei, Klaus Breiner, Albrecht Meichle, Philipp Beckhove, Marc Mansour, Heinz Lubenau. Modulating T cell immunity in tumors by targeting PD-L1 and neoantigens using a live attenuated oral Salmonella platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 733.

Live attenuated oral Salmonella platform for effective T- and B-cell targeting of PD-L1.

74 Background: Significant progresses have been recently achieved in cancer vaccines, yet novel immunization solutions to deliver efficiently tumor-associated antigens to professional antigen-presenting cells, and to overcome the peripheral tolerance and the immunosuppressive tumor microenvironment that prevent the eradication of cancer in most of patients, are urgently needed. VAXIMM is developing a unique and versatile oral T-cell vaccination platform based on the FDA-approved live-attenuated Salmonella Typhi strain Ty21a vaccine Vivotif, capable of delivering tumor-associated antigens encoded in DNA expression construct to the gut-associated lymphoid tissue, breaking immune tolerance and inducing effective anti-tumor immunity. Methods: This study summarizes the immunogenicity and antileukemia efficacy of VXM10 vaccines based on the live-attenuated Salmonella Typhimurium strain SL7207, transformed with a eukaryotic expression plasmid encoding different variants of the murine PD-L1 protein. Results: The antileukemia activity of VXM10 was evaluated in the FBL-3 disseminated model of leukemia, in which the tumor cells strongly express PD-L1. Multiple oral administrations of VXM10 vaccines produced a strong anti-tumor effect, with 100% of surviving animals 80 days after challenge with FBL3 leukemia in the highest dose groups. Moreover, 100% of long-term surviving mice resisted re-challenge with FBL-3 cells, demonstrating that vaccination with VXM10 generated a potent memory T cell response against the leukemia. Importantly, full leukemia control was achieved in both prophylactic and therapeutic settings. Upon immunization with VXM10 vaccines, T cell response was raised against PD-L1 epitopes after in vitro restimulation of the splenocytes, and anti-PD-L1 antibodies were detected in the serum. The precise mechanism of action of VXM10 vaccines is currently being investigated. Conclusions: This study provides further evidence that VAXIMM’s oral T-cell vaccination platform can be employed to stimulate anti-tumor immunity against antigens of the immune checkpoint regulatory protein PD-L1. These data paved the way for advancing the development of VXM10 into clinical development.

Abstract B196: Live attenuated oral Salmonella platform for effective targeting of multiple tumor-associated epitopes and PD-L1

Abstract Significant progress has been achieved recently in cancer vaccines that aim at engaging or reengaging the tumor immunity, in particular in the rapidly growing field of personalized immunotherapy. Yet novel immunization solutions to deliver efficiently tumor-associated (neo)antigens to professional antigen-presenting cells, and to overcome the peripheral tolerance and the immunosuppressive tumor microenvironment that prevent the eradication of cancer in most patients, are urgently needed. VAXIMM is developing a unique and versatile oral T-cell vaccination platform based on the FDA-approved live-attenuated Salmonella Typhi strain Ty21a vaccine Vivotif®, capable of delivering tumor-associated antigens encoded in DNA expression construct to the gut-associated lymphoid tissue, breaking immune tolerance and inducing antitumor immunity. This study summarizes the immunogenicity and antileukemia efficacy of VXM10 vaccines based on the live-attenuated Salmonella Typhimurium strain SL7207, transformed with a eukaryotic expression plasmid encoding the murine programmed death-ligand 1 (PD-L1) protein. It also describes for the first time the systemic immunogenicity of Salmonella Typhimurium-based polyepitope oral vaccines in healthy animals. The antileukemia activity of VXM10 was evaluated in the FBL-3 disseminated model of leukemia, in which the tumor cells express high levels of PD-L1. Multiple oral administrations of VXM10 used at 1010 colony-forming units (CFU) were generally well tolerated, and neither sign of toxicity nor body weight loss were observed throughout the study. Oral administration of VXM10 produced a strong antitumor effect in the FBL-3 leukemia model, with 100% of surviving animals 80 days after leukemia challenge in the highest-dose groups. In contrast, administration of the empty vector control did not show any anticancer effect. Moreover, 100% of long-term surviving mice resisted rechallenge with FBL-3 cells, demonstrating that vaccination with VXM10 generated a potent memory T-cell response against the leukemia. Importantly, full leukemia control was achieved in both prophylactic and therapeutic settings. Immunogenicity and antibody response towards PD-L1 are being evaluated to dissect the exact mechanism of action of these novel vaccines. Finally, different polyepitope vaccines encoding dominant epitopes from VEGFR2, Mesothelin, WT1, CEA, and Ovalbumin induced a significant systemic immunogenicity for up to 6 out of 9 epitopes, 10 days after vaccination of healthy C57BL/6J mice via the oral route, with doses up to 1010 CFU, as measured in the spleen by flow cytometry using peptide-MHC class I pentamers. This study provides further evidence that VAXIMM’s oral T-cell vaccination platform can be employed to stimulate antitumor immunity against antigens of the immune checkpoint regulatory protein PD-L1, and also against T-cell antigens encoded by polyepitope constructs. These data pave the way for advancing the development of VXM10 and neoantigen-based vaccines into clinical development. Citation Format: Sébastien Wieckowski, Lilli Podola, Marco Springer, Iris Kobl, Heiko Smetak, Anne-Lucie Nugues, Philippe Slos, Amine Adda Berkane, Ming Wei, Klaus Breiner, Albrecht Meichle, Beckhove Philipp, Marc Mansour, Matthias Schroff, Heinz Lubenau. Live attenuated oral Salmonella platform for effective targeting of multiple tumor-associated epitopes and PD-L1 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B196.

Effect of arctigenin on apoptosis of mouse erythroleukemia FBL-3 cells and its mechanism

Objective To investigate the effect of arctigenin on the apoptosis of FBL-3 cells and its mechanisms. Methods The mouse erythroleukemia FBL-3 cells were taken as subjects. The untreated FBL-3 parental cells were taken as the control group, and the FBL-3 cells treated with 20 mg/L arctigenin for 24 h were taken as the experimental group. The effects of arctigenin on the apoptosis of the mouse erythroleukemia FBL-3 cells were determined by agarose gel electrophoresis and flow cytometry assay. The changes of apoptosis-related genes were analyzed by reverse transcriptase-polymerase chain reaction(RT-PCR). Results The agarose gel electrophoresis results revealed that the DNA ladder was displayed in the experimental group compared to the control group. The flow cytometry data demonstrated that the apoptosis number of FBL-3 cells in the experimental group was markedly increased compared to that in the control group (t=60.681, P=0.000). The RT-PCR assay results suggested that the expressions of Bcl-2 and IAP-1 gene were down-regulated in the experimental group compared to the control group (t=14.732, 29.702, all P=0.000), while the expressions of Bax and Smac gene were up-regulated(t=6.721, 8.499; P=0.003, 0.001), but the expression of Bcl-XL did not change(t=0.209, P=0.844). Conclusions Arctigenin can induce the apoptosis of the mouse erythrolenkemia FBL-3 cells. Apoptosis of the mouse erythrolenkemia FBL-3 cells induced by arctigenin may be correlated to the down-regulation of Bcl-2 and IAP-1 and up-regulation of Bax as well as Smac. Key words: Arctigenin; Leukemia; Apoptosis; Anti-apoptosis gene; Pro-apoptosis gene

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-012-1329-y) contains supplementary material, which is available to authorized users.

Effects of ICOSLG expressed in mouse hematological neoplasm cell lines in the GVL reaction

As a member of the B7 family, inducible co-stimulator ligand (ICOSLG) expressed on tumor cell has been reported to have an important role in tumor immunity. In this study, we sought to determine whether the expression of ICOSLG in mouse hematological malignancy cells influences GVL reaction after mouse allogeneic BMT. In our study, we analyzed the expression of ICOSLG in six mice hematological malignancy cell lines for the first time, and found that FBL3, A20 and P388 cells expressed high levels of ICOSLG. Then, we chose A20 cells as targets to construct a GVL model and study the effects on the GVL reaction by silencing the ICOSLG gene. The survival was analyzed. We found that in GVL model, mortality of interference groups was significantly delayed compared with the control group (P=0.0005). Our results indicate that knockdown of ICOSLG of mouse leukemic cells may significantly enhance GVL effect after allogeneic BMT.

Expression of ICOSLG on Mouse Hematologic Neoplasm Cell Lines and Their Influence on Cytotoxicity in Allogeneic Mixed Lymphocyte Reactions

In this study, we sought to determine whether the expression of inducible co-stimulator ligand (ICOSLG) on mouse hematologic neoplasm cells influences cytotoxicity. Using flow cytometry, we examined the expression of ICOSLG in mouse hematologic neoplasm cell lines for the first time and used FBL3/A20 as targets to study. Effectors and targets were incubated at effector:target (E:T) ratios ranging from 5:1 to 50:1 for 10 h. Enzyme-linked immunosorbent assay was used to assess the expression of cytokines associated with type 2 T helper cells. We found that FBL3, A20 and P388 cells expressed ICOSLG. At E:T ratios ranging from 20:1 to 30:1, the cytotoxic activity of alloreactive T cells was significantly increased when ICOSLG was blocked (p < 0.05). We also found that ICOSLG blockade was associated with decreased expression of interleukin 4 and interleukin 10. Our results indicate that ICOSLG blockade may enhance cytotoxity in allogeneic mixed lymphocyte–hematologic neoplasm cell reactions.

IFN-γ对白血病细胞株FBL-3细胞生物学行为的影响

Objective To investigate the effects of interferon-γ(IFN-γ) on proliferation,cell cycle,apoptosis and invasion of mouse leukemia cell line FBL-3.Methods The leukemia cellular proliferation capacity was determined with MTT assay,cell cycle distribution and apoptotic rate were measured with flow cytometry and the invasion capacity was assayed in a Transwell cell culture chamber.Result(1)Low dose of IFN-γ(5 ng/ml) had no significant effects on proliferation of FBL-3(P0.05).The survival rate of FBL-3 stayed up to about 80% after treated by IFN-γ(50~200 ng/ml) for 48 h while higher dose of IFN-γ(500 ng/ml) had significant suppressive effects(less than 50%)(P0.05);(2)The FBL-3 cell number at G0/G1 phase was increased and cell number at G2/M phase was relatively reduced after treatment,indicating that cells were arrested at G0/G1 phase by IFN-γ;(3)IFN-γ had promotive effects on apoptosis and suppressive effects on cell invasion capacity of FBL-3 cells in a dose-dependent manner(P0.05).Conclusion IFN-γ had definite effects against leukemia through suppressing proliferation,promoting apoptosis and decreasing cell invasion directly in a dose-dependent manner.

Study of the mechanism of dendritic cells induce FBL-3 cells to differentiate into the monocytes in vitro

Objective To investigate mechanism involved in the differentiation of FBL-3 cells(eryth-roleukemia cells)induced by dendritic cells(DC). Methods To cultural the FBL-3 cells together with the DC supematant of different concentration for 72 hours,then we used the Wright's staining methed to record the ma-ture monocyte cell population, the transmission electron microscope to observe the ultramicrostructure, the flow eytoraetry to detect the expression rate of the surface molecular CD14. Results The Wright's staining methed, the transmission electron microscope and the flow cytometry all presented that after being induced by the super-natant of the DC ,the FBL-3 cells can partly differentiate into monocytes,inversion monocyte consistent with the personal characteristics. And the intensity of the DC supernatant was connected with interleukin-12. Conclusion The DC supernatant can induce the FBL-3 cells into monocytoid cell, the differentiated cells correspond to monocyte in macro-appearance,uhramicrostructure and phenotype. The competence of the DC supematant are partly concerned with interleukin-12. Key words: Dendritic cells; Leukemia; Interleukin-12; Manocytes

Retroviral immunotoxin gene therapy of leukemia in mice using leukemia-specific T cells transduced with an interleukin-3/Bax fusion protein gene.

In past studies, we showed that T cells transduced with retroviral diphtheria immunotoxin (IT) target genes could serve as vehicles for delivering IT to tumors in vivo. We took advantage of the observation that antigen-specific T cells are able to penetrate tumors to design an approach delivering combined cellular and humoral therapy directly to the tumor site. To improve tumor specificity, we selected interleukin (IL)-3 as a ligand because its receptor is selectively overexpressed on myeloid leukemia progenitors. Because Bcl-2 family proteins show structural similarity to diphtheria toxin (DT), we constructed a unique retroviral IT using Bax, a proapoptotic member of the Bcl-2 family, in place of DT. Bax was chosen because several studies showed that its transduction induces lethal apoptosis in different cancers. The retroviral construct for gene therapy included IL-3 positioned downstream of its 80 amino acid leader, and permitted cotranslational protein synthesis of hybrid IL-3/human Bax fusion protein. Other vectors were constructed with IL-3 fused to DT or Pseudomonas exotoxin. Retroviral vectors were used to transiently transduce C8, a CD4(+) T cell clone that specifically recognized FBL-3, a lethal myeloid leukemia. Supernatants collected from transduced cells showed proapoptotic activity and selectively inhibited FBL-3 cells in vitro. Intraperitoneal injection of transduced but not nontransduced C8 into mice with subcutaneous tumors or systemic cancer significantly inhibited tumor growth. These results indicate that retroviral IT made with IL-3 and various toxic proteins may be useful in patients with acute myelogenous leukemia (AML). Furthermore, the Bax construct may be particularly useful as a nonimmunogenic substitute for bacterial toxins in retIT.

Granulocyte-macrophage colony-stimulating factor induces the differentiation of murine erythroleukaemia cells into dendritic cells.

Dendritic cells (DC) are professional antigen-presenting cells (APC) within the immune system and antigen-pulsed DC can be used as an effective vaccine for active immunotherapy of cancer. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the generation of DC. We previously showed that GM-CSF can induce murine erythroleukaemia cells (FBL-3) to differentiate into monocyte-like cells. To develop a new vaccinating method to stimulate the host immune response to leukaemia, we further investigate whether FBL-3 cells induced by GM-CSF can differentiate into DC in the present study. After being treated with GM-CSF, FBL-3 cells expressed high levels of 33D1 and NLDC-145, which are the specific markers of DC. The expression of MHC-II, B7-1, B7-2 and vascular cell adhesion molecule-1 (VCAM-1) was up-regulated markedly; the typical morphology of DC were also observed by electron microscopy. Functionally, the GM-CSF-induced FBL-3 cells could apparently stimulate the proliferation of naive allogeneic and autologous T lymphocytes and induce the generation of specific CTL more efficiently than the wild-type FBL-3 cells. Mice immunized with GM-CSF-induced FBL-3 cells could resist the subsequent challenge with the wild-type FBL-3 cells. Collectively, these data indicate that GM-CSF differentiates murine erythroleukaemia cells into DC phenotypically, morphologically and functionally. FBL-3-derived DC can be used as a new type of vaccine. Our results may have important implications for the immunotherapy of leukaemia.

Immune activation of erythroleukemia cells induced by interleukin 12

To investigate the antitumor activity of IL-12, the induction of differentiation of IL-12 was observed using erythroleukemia cells (FBL-3) as model. After incubation with 200 U/mL IL-12 for 48 h, DNA synthesis of FBL-3 cells in S phase decreased significantly; the expression of CD14 which is the specific marker of monocyte increased, the rate of NBT(+) cells was apparently higher than that of the untreated FBL-3 cells. After treating FBL-3 cells with IL-12 for 72 h, the expression of 33D1 and NLDC145 which are the specific markers of dendritic cells increased markedly, the surface molecules such as MHC-11, B7-1, B7-2, and VCAM-1 were up-regulated; morphological observation showed two kinds of cells: some cells had a ruffled surface and plentiful lysosome; the others had many dendritic projections on the surface, and contained numerous mitochondria. Functionally, the IL-12-treated FBL-3 cells could apparently stimulate the proliferation of allogeneic and autologous T lymphocytes, and improve the specific cytotoxic activity of CTL on FBL-3 cells. These results indicated that erythroleukemia cells were induced by IL12 to differentiate into the monocytes and dendritic cells, then exhibited the antigen-presenting function. The data outline a new mechanism for IL-12 to treat leukemia.

Induction of immune response by IL-6 gene-modified leukemia cells

Human IL-6 gene was transduced into FBL-3 murine erythroleukemia cellsin vitro by calcium phosphate co-participation. After selection in the presence of G418, limiting dilution and biological activity assay, G418 resistant clone that secreted the highest level of IL-6 (225.6 U/ml) was selected out of 24 IL-6-secreting clones. The FBL-3 cells secreting the highest level of IL-6 (FBL-3-IL-6) showed decreased growth potential and clonogenicityin vitro. Inhibition of cell growth and clone formation was found to be closely related to the level of IL-6 secretion. FBL-3-IL-6 cells grew more slowly than wild-type FBL-3 leukemia cells and FBL-3 cells secreting lower level of IL-6 (21.3 U/ml) when inoculated s.c. into C57BL/6 mice. The mice inoculated with FBL-3-IL-6 cells showed prolonged survival period than those inoculated with control leukemia cells. Increased cytotoxic activities of splenic NK and CTL were found in mice inoculated with FBL-3-IL-6 cells. The secretions of IL-2, TNF and GM-CSF from murine splenocytes were also found to be greatly elevated after the inoculation of FBL-3-IL-6 leukemia cells. These data suggested that transduction of IL-6 gene into FBL-3 cells magnificently decreased the tumorigenicity and increased the immunogenicity of the leukemia cells, could induce specific and nonspecific antitumor immune responses. IL-6 gene-modified leukemia cells might be of great interests to be used as vaccine for the treatment of leukemia.

Immunological characteristics of the leukemia cells transfected with oncostatin M gene by recombinant adenovieus

In the present study, FBL-3 murine erythroleukemia cells were transacted with human OSM(hOSM) gene by recombinant adenovirus, then the immunological properties of hOSM gene-transfected FBL-3 cells(FBL-3-OSM+) were investigated. 4 hours after transfection with hOSM gene, hOSM could be detected in the supernatant of FBL-3-OSM+ cells and hOSM secretion peaked at 24 h. The proliferation of FBL-3-OSM+ cells was inhibited markedly. The clonal formation of FBL-3-OSM+ cells was suppressed more obviously in comparison with wild-type FBL-3 cells when analysed in clonal argar culture. Flow cytometry analysis showed that FBL-3-OSM+ cells expressed higher levels of Fas protein, B7 and ICAM-1 molecuIes.FBL-3-OSM+ cells also expressed higher level of MHC class I molecules(H-2Kb) but remained unchanged in expression of MHC class II molecules (la). CD14, which is a specific marker of monocyte/macrophage and not expressed on the wild-type FBL-3 cells, was also detected on the surface of FBL-3-OSM+ cells. The results suggested that OSM gene transfer could increase the immunogenicity of FBL-3 cells and promote their differentiation into macrophage-like cells. The data outline a promising approach to OSM gene therapy of leukemia mediated by recombinant adenovirus.

Eradication of disseminated murine leukemia by treatment with high-dose interleukin 2.

Interleukin 2 (IL 2) in high concentration induces lymphocytes to become nonspecifically cytolytic to a wide variety of tumor targets. We evaluated the therapeutic potential of such lymphokine-activated killer (LAK) cells in vivo and high-dose II 2 in vivo against disseminated murine leukemia. To quantitate the potential anti-leukemia effect of LAK cells in vivo, B6 mice were injected i.p. with graded doses of FBL-3 leukemia cells followed by LAK cells. In this Winn-type assay, 1 X 10(7) LAK cells were able to prevent the outgrowth of 1 X 10(2) FBL-3 cells in only 50% of mice and did not prevent the outgrowth of 1 X 10(6) tumor cells. Thus LAK cells, highly cytolytic to FBL-3 in vitro, mediated only a limited anti-tumor effect when applied directly to leukemia cells in vivo. LAK cells used as an adjunct to chemotherapy induced a small but non-curative effect against FBL-3, however. In this circumstance, LAK cells were markedly less effective than were immune spleen cells from mice previously sensitized to FBL-3. To test the anti-leukemia effect of high-dose IL 2 in vivo, B6 mice were inoculated with 5 X 10(6) FBL-3 cells followed by repeated doses of IL 2 at dose levels shown to induce LAK in vivo. "LAK-inducing" IL 2 doses on days 5 to 9 after FBL-3 inoculation, when tumor was disseminated, cured 50% of the mice. Treatment on days 5 to 9 was far more effective than on days 0 to 4, implying that the evolution of a host-tumor interaction was essential for the therapeutic effect of IL 2. Mice cured of FBL-3 by high-dose IL 2 were found to be immune to FBL-3, suggesting that tumor eradication resulted from a collaboration between LAK activity and tumor-specific immunity.

Effect of interleukin 2 on cytotoxic effectors: I. Short-term culture of the cytotoxic effectors and the in vivo anti-tumor activity of the cultured effectors isolated from tumor site.

The effects of interleukin 2 (IL2) on the in vitro and in vivo activity of cytotoxic T cells have been studied. IL2 was produced by W/Fu rat spleen cells cultured with concanavalin A. The IL2 thus prepared gave an optimal T-cell growth-promoting effect at a concentration of 5-20% equivalents of the original preparation. In the primary syngeneic mixed lymphocyte/tumor cell cultures (MLTC) against FBL-3 tumor cells, the addition of IL2 failed to generate a cytotoxic response. However, the cytotoxic response could be generated in MLTC by addition of exogenous macrophages. On the other hand, IL2 could maintain the growth of performed cytotoxic T cells for 3 to 5 weeks. These cytotoxic T cells were generated either by in vitro sensitization (MLC or MLTC) or by in vivo sensitization of B6 mice against a syngeneic tumor FBL-3. In short-term cultures, augmentation of the cytotoxic activity was seen after 10 days' culturing with IL2. The antigenic specificity of the cytotoxic reaction was altered after 28-35 days in culture, and the effectors broadly reactive. When growing a nonadherent population of lymphocytes isolated from FBL-3 ascites tumor, supplementation with IL2 selectively promoted the growth of a T-cell population, resulting in the elimination of the contaminating tumor cells. These purified T cells were highly cytotoxic for FBL-3 cells in vitro and also possessed strong in vivo anti-tumor activity against FBL-3 cells in the adoptive transfer experiments. The present study demonstrates that short-term culture (2-3 weeks) in IL2 promotes the growth of T cells and augments their cytotoxic activity with the appropriate antigenic specificity. IL2 also promoted the selective growth of T cells isolated from tumor site and these T cells showed augmented in vitro and in vivo anti-tumor activity.

Regulation of the In Vitro Secondary Cell‐Mediated Cytotoxic Response against Syngeneic FBL‐3 Leukemia by Macrophages

Depletion of macrophages from immune spleen cells by treatment with carbonyl iron and magnet or by in vivo treatment with carrageenan enhanced the in vitro secondary cell-mediated cytotoxic response against a syngeneic Friend virus-induced leukemia, FBL-3 cells of C57BL/6 mice. However, further depletion of macrophages by passing the carbonyl iron-treated immune spleen cells through a nylon wool column abrogated the cytotoxic response. The addition of splenic macrophage-enriched preparations from either FBL-3-immune or normal mice suppressed the cytotoxic response of immune spleen cells treated with carbonyl iron and magnet. This suppressive effect of splenic macrophages presented a marked contrast with the enhancing effect of normal peritoneal macrophages on the same cell-mediated cytotoxic response, indicating regulation of the generation of killer T cells against a syngeneic tumor by functionally distinct macrophages. The suppressed cell-mediated cytotoxic response against FBL-3 cells by immune spleen cells was augmented by the addition of indomethacin to the culture medium, and this augmentation with indomethacin was greatly decreased by depletion of phagocytic cells from the immune spleen by treatment with carbonyl iron and magnet. The mechanisms of regulation of the cell-mediated cytotoxic response with soluble factors released from macrophages are discussed.

Detection of Cellular Immunity to Murine Sarcoma Virus-Induced Tumors by Leukocyte Adherence Inhibition&lt;xref ref-type="fn" rid="FN1"&gt;2&lt;/xref&gt;

The leukocyte adherence inhibition (LAI) response of C57BL/6 mice inoculated with a regressing strain of Moloney murine sarcoma virus (M-MuSV) was measured in a microassay with 3 M KCI extracts of either the RBL-5 or FBL-3 leukemia cell line. The LAI reactivity of spleen and peritoneal cells of mice bearing M-MuSV-induced tumors was antigen-specific, reached its highest level when the tumors began to regress (12–14 days), and developed more rapidly in M-MuSV-immune mice following a secondary challenge with RBL-5 or FBL-3 cells. The kinetics of the LAI response was monophasic, and the development and magnitude of the reactivity correlated with the size of the MMuSV-induced tumor. Extensive cross-reactive LAI activity was observed with extracts from RBL-5 and FBL-3 cells. Extracts from normal syngeneic tissues, allogeneic tumor cells, and syngeneic tumor cells induced by nonviral agents did not elicit an LAI response with lymphoid cells from mice with M-MuSV-induced tumors. Indirect assays detected the production of a mediator(s) of LAI in response to both intact cells and extracts of RBL-5 and FBL-3. LAI reactivity was both lower in magnitude and rapidly lost in unsensitized C57BL/6 mice bearing progressively growing, as opposed to regressing, FBL-3 tumors. These results showed that LAI reactivity is a good indicator of host responsiveness to M-MuSV-induced tumors.

Murine lymphoma-induced immunosuppression: requirement for direct tumour cell contact.

The FBL-3 lymphoma cell line caused impaired antibody formation in vivo when injected into mice intraperitoneally, and in vitro when added to normal syngeneic spleen cells immunized in vitro with sheep erythrocytes. Immunosuppression occurred only when intact viable tumor cells were cocultivated with the normal spleen cells. As few as 10(5) FBL-3 cells, when added to 5 X 10(6) normal cells, impaired antibody formation. However, cell-free extracts of filtrates from even much larger numbers of tumor cells did not affect antibody formation, either in vitro or in vivo. Heating the tumor cells at 56 degrees C or irradiation with as little as 1000 rads completely abolished immunosuppressive activity, both in vitro and in vivo. Separation of viable tumor cells from target antibody-forming cells by cell-impermeable membranes prevented immunosuppression, showing that direct cell-to-cell contact is required for immunosuppression.

Solubilized TSTA and the major viral structural proteins, gp70 and p30, in the immune response to murine leukemias induced by Friend and Rauscher virus.

Antigens present in gp70 and p30 purified from Rauscher virus, were tested for immunogenicity in various assays measuring the anti-tumor immune response against lymphocytic leukemias of Friend (FBL-3) or Rauscher (RBL-5) virus origin. p30 had no effect on in vitro cytotoxicity against tumor cell targets mediated by either an anti RBL-5 serum or lymphocytes from animals immunized with FBL-3 cells. gp70 had had no effect on serum-mediated cytotoxicity but used at high concentrations it inhibited cell-mediated cytotoxicity. When used to immunize mice directly against subsequent challenge with the RBL-5 and FBL-3 leukemias, p30 had no discernible effect, while gp70 afforded partial protection against RBL-5 but only at high concentrations. Cell-free preparations of tumor membranes containing negligible amounts of gp70 were antigenically superior to gp70 in both the in vitro and in vivo assays. It is concluded that antigens on these purified proteins that are also expressed on tumor cells are not major targets of the anti-tumor immune response in this system.