ESb Tumor Cells Research Articles

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.

AUGMENTATION OF PROTECTIVE IMMUNOTHERAPY AGAINST METASTATIC ESB LYMPHOMA-CELLS BY COMBINING IMMUNE CELL TRANSFER WITH HOST IRRADIATION, ANTIGEN RESTIMULATION AND/OR ANTI-TNF-ALPHA ANTIBODY TREATMENT

To develop effective immunotherapies against disseminated metastatic tumor cells we have tried to improve the overall protective immunity effect by combining adoptive immune cell therapy (ADI) with other treatment modalities. In contrast to other groups who use in vitro cultured effector cells we use for ADI only in situ activated anti-tumor immune cells. The immunotherapy studies were performed with the lymphoma variant ESb which metastasizes to multiple visceral organs and can develop immune escape variants. In the absence of additional exogenous cytokines, peritoneal effector cells (PEC), generated from intra-pinna tumor immunized and i.p. re-stimulated mice, were able to transfer protective immunity and anti-tumor DTH reactivity into syngeneic mice with disseminated ESb tumor cells. We describe the protective immune capacity of PEC in comparison to immune cells from lymph nodes or spleens of immunized mice and test for possible synergistic interactions of the latter with PEC. The adoptive immunotherapy effect seen with immune PEC could be augmented by host irradiation and by combination with active specific immunization (ASI) using previously established virus modified tumor vaccines. In an attempt to study the possible role of cytokines in this system, we tested the effect of neutralizing antibodies to TNF-alpha and IFN-gamma on the therapeutic effect. Anti-TNF-alpha but not anti IFN-gamma antibody treatment augmented the immunotherapeutic effectiveness of immune PEC mediated ADI.

ADOPTIVE TRANSFER OF PROTECTIVE IMMUNITY AGAINST A HIGH METASTATIC TUMOR-CELL VARIANT BY SMALL NUMBERS OF TUMOR-SPECIFIC IN-SITU ACTIVATED PERITONEAL EFFECTOR T-CELLS

We describe the generation and immunotherapeutic properties of syngeneic immune T cells activated in situ against the lymphoma variant ESb which metastasizes to multiple organs (liver, lung, spleen, bone) and can develop sophisticated immune escape mechanisms. Peritoneal effector cells (PEC) generated from intra-pinna tumor immunized and i.p. restimulated mice were able to transfer protective immunity in the absence of exogenous cytokines into normal or 5Gy irradiated or SCID mice which had been injected either s.c. or i.v. with 5x10(3) to 10(5) metastatic ESb tumor cells. Protective immunity was specific for ESb cells and could be followed by growth retardation of s.c. tumors as well as by prevention or retardation of death from metastases. Expression of protective immunity was independent of host T cells since it was similarly expressed in SCID-mice and sublethally irradiated mice. Preincubation in vitro for 24 h of the PEC before adoptive transfer led to pronounced decrease of protective immunity while the tumor specific cytotoxic T lymphocyte (CTL) activity was maintained or even enhanced. These results demonstrate i) that protective immunity in vivo requires more than tumor specific cytotoxic activity and ii) that in vitro culture of immune cells can change their in vivo functional properties.

GAMMA-IRRADIATION SUPPRESSES T-CELL MEDIATED PROTECTIVE IMMUNITY AGAINST A METASTATIC TUMOR IN THE AFFERENT PHASE OF THE IMMUNE-RESPONSE BUT ENHANCES IT IN THE EFFERENT PHASE WHEN GIVEN BEFORE IMMUNE CELL TRANSFER

Using a metastasizing animal tumor as a model we describe experimental conditions for obtaining either synergistic or antagonistic effects between host irradiation and T cell mediated antitumor immunity. The results were obtained in the well defined murine lymphoma ESb which is a spontaneous highly metastatic tumor variant expressing a tumor associated antigen that can induce protective immunity and tumor specific cytotoxic T lymphocyte (CTL) activity. Sublethal irradiation of mice during the afferent (induction) phase but not during the efferent (effector) phase of the antitumor immune response had a strong suppressive effect on protective immunity. The radiation mediated defect could be reconstituted by transfer of immune spleen cells. The immune system of syngeneic (DBA/2) and allogeneic (B10.D2) mice changed within 24 h after first contact with the ESb tumor cells from radio-sensitivity to radio-resistance possibly reflecting an active cellular response associated with the change from virgin to an antigen sensitized (primed) state of T lymphocyte differentiation. T cell depletion experiments revealed that the afferent phase was dependent on both CD4 and CD8 host T lymphocytes while the efferent phase was mainly CD8 T cell dependent. A synergistic effect between gamma-irradiation and antitumor immunity was observed in adoptive immunotherapy experiments. When tumor-bearing hosts were irradiated before intravenous transfer of either syngeneic or allogeneic antitumor immune T cells the expression of anti-metastatic protective immunity was greatly enhanced in comparison to identically treated non-irradiated hosts.

Expression of increased immunogenicity by thiol-releasing tumor variants

Even moderate variations of the extracellular cysteine concentration were previously shown to affect T cell functions in vitro despite high concentrations of cystine. We therefore analyzed the membrane transport activities of T cells for cysteine and cystine, and the role of low molecular weight thiol in T cell-mediated host responses against a T cell tumor in vivo. A series of T cell clones and tumors including the highly malignant lymphoma L5178Y ESb and its strongly immunogenic variant ESb-D was found to express extremely weak transport activity for cystine but strong transport activity for cysteine. However, not all cells showed the expected requirement for cysteine (or 2-mercaptoethanol (2-ME)) in the culture medium. One group of clones and tumors including the malignant ESb-lymphoma did not respond to changes of extracellular cystine concentrations and was strongly thiol dependent. This group released only little acid soluble thiol (cysteine) if grown in cystine-containing cultures. The other T cell lines, in contrast, were able to maintain high intracellular GSH levels and DNA synthesis activity in cystine-containing culture medium without cysteine or 2-ME and released substantial amounts of thiol. This group included the immunogenic ESb-D line. Additional thiol-releasing ESb variants were obtained by culturing large numbers of L5178Y ESb tumor cells in cultures without cysteine or 2-ME. All of these ESb variants showed a significantly decreased tumcrigenicity and some of them induced cytotoxic and protective host responses even against the malignant ESb parent tumor. Taken together, our experiments suggest that the host response against a tumor may be limited in certain cases by the failure of the stimulator (i.e., the tumor) cell to deliver sufficient amounts of cysteine to the responding T cells.

Transfer of long-lasting tumor immunity by immune T cells from MHC congenic mice: Migration, survival and tumor-protectivity of cytotoxic donor cells

Immunocompetent B10.D2 (H-2d) mice are able to reject the highly malignant lymphoma ESb of DBA/2 (H-2d) origin very effectively. Seven days after intravenous injection of the ESb tumor cells, B10.D2 mice developed a strong tumor-rejection response which was associated with the generation of anti-tumor T cells in their spleens with direct cytotoxic activity. Most of the cytotoxic potential was directed against the minor histocompatibility differences as demonstrated by the lysis of unrelated DBA/2 derived Eb tumor cells and normal DBA/2 but no B10.D2 derived ConA lymphoblasts. A previously performed clonal analysis, however, revealed a minority population of CTL clones which specifically recognized the ESb specific transplantation antigen (ESb-TATA). When transferred systemically into DBA/2 mice, the B10.D2 anti-ESb immune spleen cells could delay the outgrowth of s.c. transplanted ESb tumor cells. When the ESb tumor cells were experimentally distributed in a s.c. implanted sponge-matrix, the i.v. injected B10.D2 immune cells could confer complete protective immunity against the metastatic tumor, provided the recipients were pre-treated with 5 Gy to allow a better take of the allogeneic cells. The distribution of intravenously injected B10.D2 donor spleen cells was assessed in the recipients up to 50 days by cytotoxicity testing and assaying for the expression of the beta 2 microglobulin allelic form b (beta 2mb). These tests revealed a high propensity of donor cells to populate the spleen and lymph nodes of the DBA/2 recipients. Again this was particularly marked in sublethally irradiated mice where a long-lasting lymphoid chimerism was established.

Host humoral and cellular immune mechanisms in the continued suppression of Friend erythroleukemia metastases after interferon alpha/beta treatment in mice.

DBA/2 mice were injected intravenously with 2 x 10(6) 3C18 Friend erythroleukemia cells (FLC), a cell line resistant to interferon alpha/beta (IFN-alpha/beta). Although daily administration of mouse IFN-alpha/beta markedly increased the mean survival time, most IFN-treated mice continued to harbor FLC in different organs. To investigate the mechanisms responsible for this persistent suppression of FLC growth in IFN-treated mice, we undertook a series of adoptive transfer experiments with sera and spleen cells. Sera from FLC-injected, IFN-treated mice were very effective in conferring protection on DBA/2 mice even when injected systemically (intravenously) 18-24 h before intravenous challenge with FLC. These sera also exhibited antitumor activity when injected subcutaneously or intraperitoneally together with FLC. The protective factor in serum was shown to be an immunoglobulin. FLC-injected, IFN-treated mice developed antibodies to FLC demonstrable by radioimmunoassay and complement-dependent cytotoxicity. Sera from these mice recognized a specific 65-kD FLC membrane antigen(s) not detectable on membrane extracts from RBL-5 or ESb tumor cells, or on normal spleen cells. FLC-injected, IFN-treated mice also developed a specific cellular response demonstrable by transfer of protection with spleen cells injected intravenously or subcutaneously. Analysis of the responsible spleen cell populations indicated that the effector cells were neither T nor B cells. These results demonstrating the importance of host humoral and cellular immune mechanisms in the persistent suppression of FLC in IFN-treated mice may be relevant to the use of IFN-alpha/beta in patients in whom tumors may regress and tumor cells may then remain latent for extended periods of time.

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.

Recruitment and activation of tumor-specific immune T cells in situ: functional studies using a sponge matrix model.

The activation of tumor-specific precursor cytotoxic T lymphocytes (CTLP) into cytotoxic T cells (CTL) was demonstrated in situ using the well-defined, highly metastatic ESb tumor as murine model system. Ten days after optimal immunization of syngeneic mice with a sublethal dose of live ESb tumor cells in the pinna, tumor-sensitized non-cytotoxic CTLP were recovered from the spleen and lymph nodes. These cells mature into tumor-specific CTL upon restimulation in vitro. Using a confined sponge matrix compartment, in combination with a specific tumor vaccine (autologous inactivated tumor cells), we induced a CD8+ (Lyt 2+) T-cell-mediated, highly cytotoxic anti-tumor immune response in situ in immunized mice. It was not possible to activate a similar response directly in lymphoid organs such as the spleen. The cytotoxic CD8+ T cells, recovered by simple mechanical pressing of the sponge, were active against the specific tumor cells in a 51Cr-release assay in vitro and also in a Winn neutralization assay in vivo. CTL activity was increased and remained in the non-adherent fraction when the cell mixture, squeezed out of the sponges, was passed over nylon wool. In a cell recruitment assay, the delayed-type hypersensitivity (DTH) potential of the activated sponge-infiltrating T cells was demonstrated by their capacity to recruit circulating host lymphocytes to sites of tumor-cell location in situ.

Modification of tumor cells by a low dose of Newcastle disease virus. Augmentation of the tumor-specific T cell response in the absence of an anti-viral response.

The present study elucidates the mechanism whereby viral xenogenization of highly metastatic ESb lymphoid tumor cells increases tumor immunogenicity and syngeneic tumor-specific T cell responses in comparison to nonmodified tumor cells. It was found that the frequency of cytotoxic T lymphocytes specific for the Esb tumor-associated transplantation antigen (TATA) and the cytotoxic anti-tumor activity in bulk cultures of immune spleen cells were significantly increased (by factor 3 and 25, respectively) when using virus-modified tumor cells. An amplified response was observed both in vivo and in vitro which might explain the demonstrated effectiveness of this approach for postoperative immunotherapy of ESb metastases. For the stimulation of tumor-specific cytolytic T lymphocytes (CTL) the ESb tumor cells which are highly metastatic were infected with an avirulent strain of the paramyxovirus Newcastle Disease Virus (NDV). Infection of ESb cells with low amounts of NDV was sufficient to lead to an increase in cytolytic activity of tumor-specific CTL after sensitization in vivo and restimulation in vitro. In a sensitive limiting dilution mixed leukocyte-tumor cell microculture system the direct effect of viral modification on the frequency and specificity of CTL was investigated. The number of ESb-specific CTL per spleen could be raised from about 3300 (without modification) to 9100 by both in vivo and in vitro application of ESb-NDV. One application of ESb-NDV (in vivo or in vitro) increased the number of CTL to 4900 and 4600, respectively. In split-type experiments it could be shown at the clonal level that viral modification did not alter the specificity of ESb-specific CTL.(ABSTRACT TRUNCATED AT 250 WORDS)

New antigens presented on tumor cells can cause immune rejection without influencing the frequency of tumor-specific cytolytic T cells

The specific immune response against syngeneic tumors by T cells is dependent on the existence of tumor-associated transplantation antigens (TATA). In the case of the chemically induced DBA/2-derived lymphoma Eb and its highly metastatic variant ESb the immunogenicity of these antigens is not sufficient to prevent tumor growth. Therefore we tested in two systems the influence of additional antigens as possible helper determinants for the generation of tumor-specific immune responses. In the Eb tumor system additional antigens were induced by mutagenization. The frequency of cytotoxic T lymphocytes (CTL) in response to mutagenized Eb cells was higher than that in response to untreated Eb cells. Fine specificity analysis revealed there there was no increase in the CTL response against the original TATA, but an activation of additional CTL clones responding to mutagen-induced antigens. In the ESb tumor system we tested the effect of additional recognition of minor histocompatibility antigens on the frequency of TATA-specific CTL. Transplantation of ESb tumor cells into B10.D2 mice, which are H-2-identical but differ in minor antigens, results in strong tumor rejection responses. In a limiting dilution mixed-leukocyte-tumor microculture system it was found that the minor antigens are recognized at the clonal level as independent antigens. The overall frequency of anti-tumor CTL in ESb-immunized B10.D2 mice was about 1 3000 . Among these, the frequency of TATA-specific CTL was 1 16,709 and thus not significantly different from that of syngeneic DBA/2 mice. Thus neither minor antigens nor mutagen-induced antigens acted in the Eb/ESb tumor system as helper determinants and did not increase the frequency of tumor-specific CTLs.

Prolongation of survival of mice bearing the Eb and ESb lymphoma by treatment with interferon inducers alone or in combination with Corynebacterium parvum.

The objective of this study was to evaluate if pretreatment with Corynebacterium parvum (C. parvum) augments the effects of interferon (IFN) inducers on survival of DBA/2 mice transplanted with two syngeneic lymphoma variants, the low metastatic Eb and the high metastatic ESb tumor. The involvement of IFN in the treatment effects was investigated. As inducers of IFN-alpha/beta Newcastle disease virus (NDV), polyinosinic-polycytidylic acid (polyI:polyC), and 10-carboxymethyl-9-acridanone (CMA) were injected i.p. at the site of tumor transplantation. The Eb tumor was found to be sensitive to the antiproliferative action of IFN-alpha/beta in vitro. In vivo single injections of each of the inducers retarded growth of the Eb tumor. In C. parvum-pretreated mice the effects of the inducers on survival were markedly increased. There was a correlation between prolonged survival and local IFN levels in response to polyI:polyC or CMA but not upon NDV. Injections of each of the inducers increased cytotoxicity of peritoneal exudate cells against the Eb tumor cells in vitro especially when mice were pretreated with C. parvum. Although other mechanisms cannot be excluded IFN-mediated activation of host defence and also direct antiproliferative effects of endogenously produced IFN seem to be involved in the antitumor effects by these IFN inducers in the Eb model. In the ESb tumor model irrespective of additional pretreatment with C. parvum survival was only slightly prolonged by the treatments and endogenous IFN induction did not result in any real benefit for the animals. When compared with Eb cells the ESb cells were less sensitive to the antiproliferative action of IFN-alpha/beta in vitro and less sensitive to in vitro cytotoxicity by the host cells. Although other mechanisms may additionally be active in vivo the different susceptibility of the Eb and ESb tumor cells to the direct and indirect actions of IFN seems to contribute to the different responsiveness of these tumor cell lines to the treatments with IFN inducers.

Analysis of protective and cytotoxic immune responses in vivo against metabolically inactivated and untreated cells of a mutagenized tumor line (requirements for tumor immunogenicity)

The immunogenicity of a mutagenized subline (ESb-D) of the weakly immunogenic T-cell lymphoma L 5178 Y ESb has been characterized. The injection of 10 6 ESb-D cells ip did not establish lethal tumors in untreated DBA/2 mice but established tumors in sublethally irradiated mice. Injection of ESb-D cells into otherwise untreated DBA/2 mice established also a state of protective immunity against the subsequent injection of otherwise lethal doses of ESb tumor cells. Protection was only obtained after injection of intact but not UV-irradiated or mitomycin-C-treated ESb-D cells. A direct T-cell-mediated cytotoxic activity was also demonstrable in the spleen cells of DBA/2 mice after injection of ESb-D cells but not ESb cells. The cytotoxic activity was variant specific for ESb-D target cells, and it was induced only with intact but not UV-irradiated or mitomycin C-treated ESb-D cells. This suggested that the induction of protective and cytotoxic immunity may require the persistence of the antigen or unusually high antigen doses. The in vivo priming for a secondary in vitro cytotoxic response, in contrast, was achieved with intact and also with mitomycin C-treated ESb-D cells but again not with UV-irradiated ESb-D cells. This indicated that the metabolic activity was a minimal requirement for the in vivo immunogenicity of the ESb-D tumor line. The secondary cytotoxic activity was demonstrable on ESb-D and ESb target cells and could be restimulated in vitro about equally well with ESb-D and ESb cells. But the in vivo priming was again only obtained with ESb-D cells and not with ESb cells. These experiments thus demonstrated that the requirements for immunogenicity are more stringent in vivo than in vitro, and more stringent for the induction of direct cytotoxic and protective immunity in vivo than for the in vivo priming for secondary in vitro responses.

Characterization of an extracellular matrix-degrading protease derived from a highly metastatic tumor cell line

A proteolytic activity associated with the microsomal fraction of L-5178Y/Esb tumor cells has been characterized. The enzyme has a molecular weight of 80–90 kD as determined by affinity-labelling with [ 3 H]DFP and SDS-gel electrophoresis. It cleaves ester substrates at the carboxyl position of lysine and arginine and can activate the proenzyme plasminogen. The enzyme is found to be associated with the plasma membranes of high and low metastatic tumor cell lines and is shed in high-molecular-weight form mainly by the high metastatic variant. The pH optimum for esterase and protease activities was 7.5–8.5. Although similar to trypsin in substrate specificity, the enzyme was not inhibited by lima-bean trypsin inhibitor but was inhibited by DFP, PMSF, aprotinin and leupeptin. Partially purified preparations of the protease can alone degrade 125 I-labelled endothelial cell extracellular matrix, pointing at the putative role of this enzyme in tumor invasion.

Interaction of high or low metastatic related tumor lines with normal or lymphokine-activated syngeneic peritoneal macrophages: in vitro analysis of tumor cell binding and cytostasis.

Peritoneal macrophages from normal DBA/2 mice were found to bind significantly more cells of a syngeneic low metastatic lymphoma line (Eb) than cells of a high metastatic variant (ESb) derived therefrom. These differences were observed in three different assays, at 4 degrees C and at 37 degrees C, and at various ratios of macrophages to tumor cells. Upon co-culture with normal macrophages, a tumor cytostatic effect was consistently observed with Eb but not with ESb tumor cells. Further experiments indicated that macrophages exerted their growth inhibitory effect via direct tumor cell contact. Pre-treatment of tumor cells with neuraminidase or pre-treatment of macrophages with lens culinaris lectin increased the numbers of macrophages binding Eb and ESb tumor cells. Addition of D-galactose or D-mannose at 50 mM concentration led to an increase of tumor cell binding and tumor cytostatic activity. Taken together, these results suggest (i) that carbohydrates play a role in tumor cell recognition by macrophages and (ii) that the differences observed between Eb and ESb tumor cells may be due to differences in the expression of carbohydrates. Pre-activation of the macrophages by lymphokine(s) led to a short increase in their tumor cell binding capacity. Lymphokine activation resulted in a strong but also short-lived increase of tumor cytostatic potential. This was effective against both the low and the high metastatic tumor line.

A mouse hepatocyte carbohydrate-specific receptor and its interaction with liver-metastasizing tumor cells.

Spontaneous high-metastatic variants (ESb) of the DBA/2 mouse lymphoma L5178Y which show heavy liver involvement were found to form rosettes in vitro with isolated autologous hepatocytes, whilst low-metastatic sublines of the same tumor (Eb) did not. An analysis of the molecules involved in the hepatocyte:tumor cell interaction was performed by affinity adsorption and SDS-polyacrylamide gel electrophoresis of 125I-labelled membrane components from either the hepatocytes or the tumor cells. The hepatocytes were found to bind ESb tumor cells through lectin-like hepatic binding proteins (HBP) with molecular weights of 52, 56 and 110 Kd and specificity for D-galactosyl and N-acetyl-D-galactosaminyl residues. More than 10 different cell surface glycoproteins of ESb tumor cells and none of Eb-type tumor cells served as ligands in the hepatocyte interaction. The low-metastatic subline Eb formed hepatocyte rosettes only after neuraminidase pretreatment, indicating that lectin binding carbohydrate structures existed in a cryptic form masked on these cells by sialic acid. Although lectin-carbohydrate interactions have been found to play a crucial role in many intercellular recognition processes, this apparently is the first molecular description of such an interaction between organ-derived normal parenchymal cells and tumor cells. The possible relevance of such an interaction for cancer metastasis is suggested by the finding that spleen-selected ESb sublines differed from liver-selected ones in their organotropism as well as in their ability to form hepatocyte rosettes.

Tumor metastases and cell‐mediated immunity in a model system in DBA/2 mice. VI. Similar specificity patterns of protective anti‐tumor immunity in vivo and of cytolytic T cells in vitro

AbstractIn an attempt to analyze mechanisms of immunity against tumor metastases, protective anti‐tumor immunity in vivo was compared with cytotoxic T‐cell activity in vitro in a well‐defined syngeneic tumor model system. The system consists of a chemically induced parental tumor cell line (Eb) with little or no metastatic potential and a spontaneous variant thereof (ESb) with pronounced metastatic properties. Tumor protection experiments revealed the presence of tumor‐associated transplantation antigens (TATAs) on both Eb and ESb tumor cells. TATAs of Eb and ESb were found to be distinct and non‐cross‐reactive. One of several unrelated tumors, however, RL♂1, expressed TATAs which cross‐reacted with those of Eb. Protective immunity against the non‐metastasizing tumor was much stronger than that against the metastasizing variant. Furthermore, the optimal procedures for induction of immunity in vivo were strikingly different for each tumor. Tumor‐specific cytotoxic T lymphocytes (CTLs) were obtained after sensitization in vivo with viable tumor cells and restimulation in vitro for 4–5 days with mitomycin‐C‐treated autologous tumor cells. Both anti‐Eb and anti‐ESb CTLs showed high cytolytic activity in a 4‐h 51Cr release assay against the autologous tumor lines. The target antigens recognized by these cells were similar to the TATAs as defined in the protection experiments. (1) The target antigens of Eb and ESb were distinct and non‐cross‐reactive. (2) Only one of 14 unrelated syngeneic and allogeneic tumors expressed a target antigen which cross‐reacted with that of Eb. (3) This tumor was the radiation‐induced BALB/c lymphoma RL♂1 which also cross‐reacted at the level of the TATAs. The correlations between protective immunity obtained in vivo and cytolytic T cells induced in vitro suggest that cytolytic T cells can recognize TATAs and may thus play an important role in the establishment of protective immunity.

Tumor metastases and cell‐mediated immunity in a model system in DBA/2 mice. V. Transfer of protective immunity with H‐2 identical immune T cells from B10.D2 mice

AbstractEvidence is presented for the existence of immune T lymphocytes which have protective activity in vivo against the lethal effects of a metastasizing tumor. ESb, the metastasizing tumor cell line of our model system, which is highly malignant in syngeneic DBA/2 mice, can be rejected when transplanted into B10.D2 mice. Since B10.D2 and DBA/2 mice are identical at the H‐2 complex, cells could be transferred from the tumor‐resistant into the susceptible strain to determine whether they could confer protective immunity in a syngeneic environment containing disseminated ESb tumor cells. Protective immunity against ESb could be transferred into DBA/2 with spleen cells but not with ascites fluid from B10.D2 mice preimmunized against the ESb tumor cells. B10.D2 spleen cells taken between 6 and 22 days after tumor transplantation had protective immunity, while cells taken from animals 3 days after tumor transplantation, or from normal animals, had no significant protective effect. The cells in B10.D2 immune spleens with protective activity were sensitive to treatment with anti‐theta serum and complement and did not adhere to nylon‐wool columns. Such nylon‐wool column‐passed cells which were enriched for T lymphocytes had a higher protective activity then the unfractionated cells. DBA/2 mice which received viable ESb tumor cells subcutaneously on day 0 could be protected by intravenous inoculation of B10.D2 immune spleen cells only when these were given after the tumor cells (day +1, +3, +5, +7), not when given before (day −3 or −1). The mechanisms of protective immunity against tumor metastasis and the significance of our findings for completely syngeneic tumor systems will be discussed.

Tumor metastases and cell-mediated immunity in a model system in DBA/2 mice. I. Tumor invasiveness in vitro and metastasis formation in vivo.

AbstractA syngeneic model system for the study of tumor metastases and cell‐mediated immunity is described. The system consists of two related, chemically induced murine lymphomas, the non‐metastasizing parental line Eb and its metastasizing variant ESb. An unrelated, chemically induced tumor (MDAY) is included for specificity controls. Serological typing revealed that both Eb and ESb were of T lymphoid origin and expressed the H‐2K and H‐2D molecules of the host strain DBA/2. By various electron microscope techniques, morphological differences were observed between the two cell lines. In comparison to Eb cells, ESb tumor cells had a more polymorphic nucleus with many in vagi‐nations of the nuclear envelope and a more prominent expression of microvilli on the cell surface. An in vitro organ culture test for tumor invasiveness, presented here for the first time in a syngeneic murine system, revealed that ESb but not Eb tumor cells had the ability to attach to and invade normal tissue. Accordingly, ESb tumor cells showed higher malignancy in vivo. This was apparent from their higher tumorigenicity and their ability to disseminate and metastasize and to kill recipient mice more quickly. Upon histological examination of the local primary tumors a striking difference was noticed with regard to the degree of infiltration by host‐derived mononuclear cells, mostly histiocytes. The non‐metastasizing tumor Eb was heavily infiltrated while tumor ESb contained only a few of these cells. The differences between the tumor lines ESb and Eb are considered in the light of their possible relevance for metastases in general. The etiology of the two tumors is discussed in particular with respect to their relatedness.

Tumor metastases and cell-mediated immunity in a model system in DBA/2 mice. IV. Antigenic differences between a metastasizing variant and the parental tumor line revealed by cytotoxic T lymphocytes.

The syngeneic cytotoxic T-cell response against a metastasizing murine lymphoma variant was investigated and compared with the response against the non-metastasizing parental tumor line Eb. Anti-tumor cytotoxicity was not detectable in a 4-h 51Cr release assay in spleens taken directly from tumor-bearing animals (primary CMC). After restimulation in vitro (secondary CMC) however, high anti-tumor cytotoxic activity was detected. This activity was mediated by immune T lymphocytes as shown by its sensitivity to treatment with anti-Thy 1.2 serum and complement. Ten cells of the metastasizing tumor ESb, inoculated subcutaneously, were sufficient to raise a local tumor and metastases and to induce cytotoxic T memory cells in the spleens. In contrast, about 104 cells were required to raise a local tumor and to induce splenic cytotoxic T memory cells, when the parental tumor Eb was tested. The specificity studies of the anti-tumor cytotoxic activity demonstrated that cytotoxic T cells could distinguish unrelated, chemically induced syngeneic tumors and also recognize antigenic differences between the parental tumor Eb and its variant ESb. Eb and ESb tumor cells were recognized as carrying distinct antigens at the responder cell level, the stimulator cell level and the target cell level. The in vivo significance of these findings is discussed.