Protective immunity to progressive tumors can be induced by antigen presented on regressor tumors

Immunization of animals with 1591-RE tumor cells, a highly immunogenic UV-induced epithelia cell tumor from C3H/HeN mice, that were haptenated with trinitrophenol (TNP) leads to protective immunity against a challenge of TNP-haptenated 3152-PRO tumor cells, a progressive highly malignant. MCA-induced fibrosarcoma from syngeneic mice. Animals that rejected TNP-1591-RE and subsequently TNP-3152-PRO tumor cells showed increased tumor-specific resistance to another challenge of 3152-PRO tumor cells, even when these fibrosarcoma cells had not been haptenated with TNP. Induction of protection required the presence of TNP-hapten groups on both 1591-RE and 3152-PRO during the initial immunization, and could be induced by immunization with other haptenated syngeneic highly immunogenic regressor tumor lines. In addition, TNP-haptenated progressor variants of the 1591-RE were ineffective in generating protection, suggesting that the immunogenicity of the haptenated tumor used for the initial immunization was a determining factor in whether or not protective immunity against the highly malignant tumor was later generated. Protection required at least two T cell types: a Lyt-1-2+ T cells, and a Lyt-1+2- T cell that also expressed I-J determinants and was Vicia villosa lectin adherent, suggesting it was not a classical helper T cell. These results suggest that presentation of a hapten by highly immunogenic tumor cells can lead to enhanced protective immunity to poorly immunogenic noncross-reactive tumors that co-express the same hapten, and rejection of these haptenated poorly immunogenic tumors leads to enhanced protection against a subsequent challenge of the same, but not noncross-reactive progressor tumors.     

Stepwise immunologic selection of antigenic variants during tumor growth

Using tumor-specific effector cells as probes, we have studied the immunologic changes that occur in tumor cells during continuous growth in a host. As a model, we used a highly immunogenic ultraviolet light (UV)-induced tumor that is rejected regularly by normal mice but grows progressively when transplanted into UV-irradiated mice. The immunogenic tumor growing continuously in these partially immunocompromised mice gave rise to genetically stable progressor variants that were poorly immunogenic. A sequence of changes in susceptibility to activated macrophages and tumor-specific cytolytic T cells was observed when serial reisolates from the continuously growing tumors were analyzed. First, the tumor cells developed resistance to the cytocidal effects of activated macrophages. This was followed by the loss of one and then a second tumor-specific antigen defined by syngeneic cytolytic T cells. The phenotypes of the developing antigen loss variants and their sequence of appearance were the same in several independent experiments, and the process was apparently determined by a hierarchy of the host's immune response to multiple independent tumor-specific antigens expressed by a single malignant cell. Our ability to generate the predicted variants in vitro before they actually appear in vivo suggests a possible approach to preventing the outgrowth of such immunoselected variants from a tumor.     

Rescue of the tumor-specific immune response of aged mice in vitro

In contrast to young mice, old mice fail to reject a transplanted challenge of the highly immunogenic, ultraviolet light-induced tumor 1591-RE. Old mice also fail to mount a cytolytic tumor-specific immune response in vivo, and spleen cells of old mice are defective in their ability to generate tumor-specific T cells in vitro. In the present study we report the results of cell culture mixing experiments that show that this deficiency is due to a decreased responsiveness of the Lyt-2+ tumor-specific cytolytic T cell precursors of the old animals. We also demonstrate with limiting dilution analysis that the defective responsiveness is not due to a clonal exhaustion of the precursors. In fact, the responsiveness could be restored in vitro by culturing the spleen cells of old animals at high density or by the addition of excess Lyt-1-/Lyt-2-/2000-rad-resistant spleen cells from young or old mice. Our results suggest that the rescue of tumor immunity in old individuals may be possible, perhaps by educating effector cells in vitro for adoptive immunotherapy.     

Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. VII. Dominant expression of variant antigens in somatic cell hybrids

After mutagenesis of mouse mastocytoma P815, it is possible to obtain at high frequency stable tumor cell variants (tum-) that are rejected by syngeneic DBA/2 mice. Most of the variants express one or more new individual antigens specific for each variant, that are detectable in vitro by cytolytic T cells (CTL). Somatic hybrids were prepared either between tum- variants and the original P815 clone, or between different variants. Antigen expression of the hybrids was assessed by using long-term CTL clones that recognize specifically the new antigen present on the variants. Expression of tum- variant antigens behaved as a dominant trait in the hybrids. By submitting the somatic hybrids to selection with CTL clones, it was possible to obtain antigen-loss hybrid variants. The analyses of these antigen-loss variants showed that two variant-specific antigenic determinants associated with one of the variant fusion partners could be lost independently. Like the parental tum- variants, both the (tum+ X tum-) and (tum- X tum-) hybrids failed to form tumors in normal mice but formed tumors in irradiated mice.     

Defining the heterogeneity of anti-tumor antibody responses.

Studies were undertaken to analyze the humoral in vivo and in vitro antibody response of BALB/c mice to a syngeneic MSV-induced tumor cell line. With a sensitive radioimmunoassay, sera obtained from individual progressor and regressor mice were shown to vary greatly in total tumor-specific antibody concentration as well as immunoglobulin class distribution of the antibody, but no significant difference existed between the groups of progressor and regressor mice as a whole. In addition, serum antibodies from all animals were shown to have extensive cross-reactivity against a variety of cell lines chosen to share one or more antigens with the cell line used for immunization. Conversely, when in vitro fragment cultures of splenic tissue from progressor and regressor mice were stimulated with tumor-related antigen, differences in responsiveness among normal, progressor, and regressor mice were observed. In addition, antibodies derived from fragment cultures displayed several different cross-reactivity patterns all of which were more restricted in specificity than serum antibodies.     

Different phenotypic variants of the mouse B cell tumor A20/2J are selected by antigen- and mitogen-triggered cytotoxicity of L3T4-positive, I-A-restricted T cell clones

The L3T4+, Lyt-2-, cloned BALB/c T cell lines 5.9.24 and 5.8.6 are cytotoxic for the BALB/c B cell tumor line A20/2J. The T cell cytotoxicity against A20/2J cells could be triggered either by the specific antigen ovalbumin (OVA), which is recognized by the T cell clones in association with I-Ad determinants, or by the T cell mitogens Con A and rabbit anti-mouse brain (RaMBr) antiserum. Repeated exposure of A20/2J cells to 5.9.24 and 5.8.6 T cell cytotoxicity selected variant cell lines that had developed resistance to cytotoxicity. The variant lines could be classified into four different variant phenotypes of which three were stably maintained in vitro. The type of variant obtained appeared to be related to the nature of the ligand used to trigger T cell cytotoxicity during selection. Cytotoxicity triggered by the antigen OVA generated type 1 variants that expressed abnormally low levels of I-Ad determinants at the cell surface. Type 1 variants were resistant to OVA-triggered 5.9.24 T cell cytotoxicity, but were fully susceptible to cytotoxicity triggered by Con A or RaMBr antiserum. RaMBr-triggered cytotoxicity generated two unique types of variant cell lines: type 3 variants that were deficient in cell surface Fc receptors and resistant to 5.9.24 cytotoxicity only when triggered by RaMBr antiserum, and type 4 variants that were resistant to cytotoxicity triggered by all three ligands. One type 4 variant, the IC-1 cell line, appeared to be resistant to soluble cytotoxic factors released by 5.9.24 T cells after activation by antigen. All of these variant lines retained sensitivity to cytotoxicity by classic Lyt-2+ cytotoxic T lymphocytes (CTL), a finding that indicates that L3T4a+ T cells and Lyt-2+ CTL use different molecules to attack their target cells. The variant phenotypes were inherited by clones derived from the original cell lines. Because the variants were generated without mutagenesis, they are thought to have been derived by the immunoselection of pre-existing variant cells that arose spontaneously in the parental A20/2J cell line. It is postulated that inheritable variation of A20/2J cells may represent changes that normally occur during B cell differentiation in response to T cell signals. The variant A20/2J cell lines described here provide material for the investigation of B cell surface structures that may regulate T-B cell interactions.     

Highly immunogenic regressor tumor cells can prevent development of postsurgical tumor immunity

Highly immunogenic malignant cells form small tumors that spontaneously regress after initial growth because the tumor induces specific immunity. However, variants may arise during the initial tumor growth that lose antigens, grow progressively, often become the predominant tumor population, and eventually kill the host. These progressively growing variants usually have not lost all tumor antigens and remain susceptible to rejection by T cells specific for antigens present on the parental tumor and retained by the progressively growing variants. Thus, it would seem logical for therapy to actively immunize with the parental highly immunogenic tumor (or sublines made similarly immunogenic by tumor heterogenization) after maximal surgical removal of the growing tumor. However, the present findings suggest that such a strategy may be ineffective and have adverse effects: the parental highly immunogenic tumor cells, either remaining or reintroduced, may perpetuate unresponsiveness to both the parental and the variant tumor. These findings suggest that unless tumor-induced suppression is first abrogated, immunization with highly immunogenic tumor cells may be counterproductive because this maneuver may maintain preexisting immune suppression and prevent development of postsurgical tumor immunity.     

Expression and thermal stability of simian virus 40 tumor-specific transplantation antigen and tumor antigen in wild type- and tsA mutant-transformed cells.

We have explored aspects of a suggested relationship between the expression of simian virus 40 tumor-specific transplantation antigen (TSTA) and tumor antigen (TA). A unique rat embryo cell line transformed by a temperature-sensitive A mutant that loses TA during exposure to the nonpermissive temperature (A28-RE) was found to lose TSTA. On the other hand, a typical control tsA-transformed cell line (A239-MB) expressed both TA and TSTA at the non-permissive temperature. TA in lysates obtained from A239-MB cells was found to be three to four times more thermolabile by covwt-mb) when incubated at either 33 or 40 degrees C. These data complement previous reports using TA from lytic infection and are consistent with the suggestion that TA is virus encoded. In contrast to TA, which even in wild-type-transformed cells was completely destroyed in less than 10 min at 50 degrees C, TSTA, assayed in vivo by tumor rejection, and tumor-specific surface antigen(s) TSSA) defined by an in vitro cytolytic assay, were thermostabile. Even after 24 h of incubation of extracts of 50 degrees C, high levels of TSTA and TSSA activity were present. Since these surface antigens when obtained from cells transformed by tsA mutants were also thermostabile, they could not be distinguished from the wild-type antigens. These results (i) indicate a coordinate expression of TA and TSTA in transformed cells; (ii) confirm that TA is virus encoded; and (iii) confirm that tha antigenic and immunogenic determinants that characterize TA and TSTA activities are distinct. However, the possibility that TSTA, like TA, is of viral rather than cellular origin is not excluded.     

Distinct proliferative T cell clonotypes are generated in response to a murine retrovirus-induced syngeneic T cell leukemia: viral gp70 antigen-specific MT4+ clones and Lyt-2+ cytolytic clones which recognize a tumor-specific cell surface antigen

After immunization of B6 mice with the syngeneic retrovirus-induced T cell leukemia/lymphoma FBL-3, two major tumor-specific proliferative T cell clonotypes were derived. T cell clones derived from long-term lines propagated by in vitro culture with irradiated tumor cells and syngeneic spleen cells were exclusively of the Lyt-2+ phenotype. Such clones were cytolytic, retained their proliferative phenotype indefinitely when expanded by repeated cycles of reactivation and rest, and recognized a tumor-specific cell surface antigen in association with class I MHC molecules. This tumor cell antigen was not present on nontransformed virus-infected cells. Class II MHC-restricted MT4+ clones specific for the viral antigen gp70 were derived from lymph node T cells of FBL-3 tumor-immune mice only by in vitro culture with purified Friend virus in the presence of syngeneic splenic APC. Once derived, however, such clones could be stimulated in the presence of FBL-3 tumor cells and syngeneic spleen cells, demonstrating the reprocessing of tumor-derived gp70 antigen by APC in the spleen cell population. In contrast, no reprocessing of the tumor cell surface antigen by splenic APC for presentation to the class I MHC-restricted T cell clones could be demonstrated. Evidence is presented that FBL-3 T leukemia/lymphoma cells function as APC for Lyt-2+ class I MHC-restricted clones, and that no concomitant recognition of Ia molecules is required to activate these clones. Both Lyt-2+ and MT4+ clones were induced to proliferate in the presence of exogenous IL2 alone, but this stimulus failed to result in significant release of immune interferon. In contrast, antigen stimulation of both clones resulted in proliferation as well as significant immune interferon release. Immune interferon production is not required for the generation of MHC-restricted cell-mediated cytolytic function.     

Regression mechanisms of mouse fibrosarcoma cells after in vitro exposure to quercetin: Diminution of tumorigenicity with a corresponding decrease in the production of prostaglandin E2

Summary We have previously reported that both regressor (QR) and progressor (metastatic, QP) clones were obtained after the in vitro exposure of a mouse fibrosarcoma BMT-11 cl-9 to quercetin [17]. In this study, we investigated possible mechanisms of spontaneous regression of QR clones as compared with tumorigenic QP and BMT-11 cl-9 tumor clones. We observed that BMT-11 cl-9 cells produced relatively high amounts of prostaglandin E₂ (PGE₂) during in vitro culture. The average production by 11 subclones of BMT-11 cl-9 cells was 9236±2829 pg/ml whereas that by 9 QR clones was 3411±2213 pg/ml (P <0.02). Indomethacin not only inhibited in vitro PGE₂ synthesis by QP clones (high-PGE₂ producers) but also the s.c. growth of QP clones in mice. Chronological changes in host immune responses to tumor-associated antigen were measured by cytotoxic T lymphocyte (CTL) activity examined after mixed lymphocyte/tumor cell culture of spleen cells obtained from tumor-bearing mice. The CTL activity disappeared abruptly in the spleen of QP-clonebearing mice 21 days after the inoculation of tumors, whereas the spleen cells of QR-clone-inoculated mice retained their CTL activity. We determined that the mechanism responsible for the regression of these regressor clones is not due to any qualitative or quantitative increase in pre-existing membrane antigens, nor the emergence of new antigen(s) on the cell surface of the QR clones; nor was it due to enhanced susceptibility of QR clones to natural killer cells, lymphokine-activated killer cells and macrophages. These finding suggest that the regression mechanism of QR clones may be the diminished inhibition of host response to tumor-associated antigen caused by the reduced production of PGE₂ by QR clones.     

Functional activity in vivo of effector T cell populations. II. Anti-tumor activity exhibited by syngeneic anti-MoMULV-specific cytolytic T cell clones

The functional activity in vivo of murine tumor-specific cytolytic T lymphocyte populations and clones was studied. Tumor cell destruction induced after the i.v. injection of cytolytic effector cells was quantitated by monitoring the elimination of 131IUdR-labeled tumor cells in the peritoneal cavity by using whole-body counting techniques. Mixed leukocyte-tumor cell cultures were established by using spleen cells from C57BL/6 regressor mice that had rejected an intramuscular tumor induced by the injection of MSV-MoMuLV virus. This effector cell population was observed to eliminate syngeneic MoMuLV-induced tumor cells in a dose-dependent manner. Treatment of the effector cell population with monoclonal anti-Lyt-2 antibodies plus complement totally abrogated their ability to induce tumor cell destruction in the peritoneal cavity. MSV-MoMuLV-specific Lyt-2+ cytolytic T cell clones derived by micro-manipulation of T lymphocyte-tumor cell conjugates were also tested for functional activity in vivo. Several clones induced a rapid, specific elimination of 131I-labeled MBL-2 tumor cells from the peritoneal cavity after i.v. injection, whereas others were inactive. Both active and inactive clones were highly cytolytic and secreted MAF/IFN-gamma lymphokines. In contrast to previous results obtained in a tumor allograft model, the MSV-MoMuLV-specific cytolytic T cell clones that were active in vivo did not proliferate in vitro in response to stimulation with irradiated tumor cells plus filler spleen cells in the absence of an added source of interleukin 2.     

Structure of the gene of tum- transplantation antigen P91A: the mutated exon encodes a peptide recognized with Ld by cytolytic T cells

Mutagen treatment of mouse P815 tumor cells produces immunogenic mutants that express new transplantation antigens (tum- antigens) recognized by cytolytic T cells. We found that the gene conferring expression of tum- antigen P91A contains 12 exons, encoding a 60 kd protein lacking a typical N-terminal signal sequence. The sequence shows no significant similarity with sequences in current data bases. A mutation that causes expression of the antigen is located in exon 4; it is the only apparent difference between the normal and the antigenic alleles. A short synthetic peptide corresponding to a region of exon 4 located around this mutation makes P815 cells sensitive to lysis by anti-P91A cytolytic T cells. The mutation creates a strong aggretope enabling the peptide to bind the H-2 Ld molecule. Several secondary tumor cell variants that no longer express tum- antigen P91A were found to carry deletions in the gene.     

Adoptive tumor therapy with T lymphocytes enriched through an IFN-gamma capture assay

Successful adoptive T-cell therapy has been demonstrated in viral disease and selected forms of cancer. However, it is limited by the difficulty to efficiently isolate and amplify autologous tumor-reactive T-cell clones. Tetramers of major histocompatibility complex (MHC) class I and peptide have facilitated the characterization of CD8+ T cells specific for tumor-associated antigens. However, for adoptive T-cell therapy, MHC-tetramers have limitations: they require knowledge of tumor antigens, which is often not available; they select T cells with a single specificity, thereby posing risk for selection of tumor escape variants; they do not select for function, so that T cells may be anergic when isolated from cancer patients; and they do not allow the isolation of CD4+ T cells that can be essential for tumor rejection. Because interferon (IFN)-gamma is essential for tumor rejection, we isolated live T cells based on their IFN-gamma production. IFN-gamma secreted by previously activated T cells is retained on the cell surface, allowing their specific isolation and expansion. We show here that IFN-gamma+ but not IFN-gamma- T cells from tumor-immunized mice are cytolytic and mediate tumor rejection upon adoptive transfer. Importantly, tumor-specific T cells can be enriched from lymphocytes infiltrating human renal cell carcinoma by the IFN-gamma capture assay.     

Bystander elimination of antigen loss variants in established tumors

Cancers express antigens that are targets for specific cytotoxic T lymphocytes (CTLs). However, cancer cells are genetically unstable. Consequently, sub-populations of cancer cells that no longer express the target antigen may escape destruction by CTLs and grow progressively. We show that cytotoxic T cells indirectly eliminate these antigen loss variants (ALVs) in a model system when the parental cancer cells express sufficient antigen to be effectively cross-presented by the tumor stroma. When the parental tumor expressed lower levels of antigen, cytotoxic T cells eradicated the antigen-positive parental cancer cells, but the ALVs escaped, grew and killed the host. By contrast, when the parental tumor expressed higher levels of antigen, cytotoxic T cells eradicated not only the parental cancer cells but also the ALVs. This 'bystander' elimination of ALVs required stromal cells expressing major histocompatibility complex (MHC) molecules capable of presenting the antigen, and occurred in tumors showing evidence of stromal destruction. ALVs were apparently eliminated indirectly when tumor-specific CTLs killed stromal cells that were cross-presenting antigen produced by and released from antigen-positive cancer cells. These results highlight the general importance of targeting the tumor stroma to prevent the escape of variant cancer cells.     

Dendritic cells efficiently acquire and present antigen derived from lung cancer cells and induce antigen-specific T-cell responses

Active immunotherapy of cancer requires the availability of a source of tumor antigens. To date, no such antigen associated with lung cancer has been identified. We have therefore investigated the ability of dendritic cells (DC) to capture whole irradiated human lung tumor cells and to present a defined surrogate antigen derived from the ingested tumor cells. We also describe an in vitro system using a modified human adenocarcinoma cell line (A549-M1) that expresses the well-characterized, immunogenic influenza M1 matrix protein as a surrogate tumor antigen. Peripheral blood monocyte-derived DC, when co-cultured with sub-lethally irradiated A549 cells or primary lung tumor cells derived from surgical resection of non-small cell carcinoma (NSCLC), efficiently ingested the tumor cells as determined by flow cytometry analysis and confocal microscopic examination. More importantly, DC loaded with irradiated A549-M1 cells efficiently processed and presented tumor cell-derived M1 antigen to T cells and elicited antigen-specific immune responses that included IFNgamma release from an M1-specific T-cell line, expansion of M1 peptide-specific Vbeta17+ and CD8+ peripheral T cells and generation of M1-specific cytotoxic T lymphocytes (CTL). We also compared DC loaded with irradiated tumor cells to those loaded with tumor cell lysate or killed tumor cells and found that irradiated lung tumor cells as a source of tumor antigen for DC loading is superior to tumor cell lysate or killed tumor cells in efficient induction of antigen-specific T-cell responses. Our results demonstrate the feasibility of using lung tumor cell-loaded DC to induce immune responses against lung cancer-associated antigens and support ongoing efforts to develop a DC-based lung cancer vaccine.     

Transfection and expression of a gene coding for a human melanoma antigen recognized by autologous cytolytic T lymphocytes

Human melanoma line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocytes (CTL). As a first step towards the cloning of the gene coding for one of these antigens, we tried to obtain transfectants expressing the antigen. The DNA recipient cell was a variant of MZ2-MEL which had been selected with a CTL clone for the loss of antigen E. It was cotransfected with genomic DNA of the original melanoma line and with selective plasmid pSVtkneo. Geneticin-resistant transfectants were obtained at a frequency of 2 × 10^–4. These transfectants were then screened for their ability to stimulate the production of tumor necrosis factor by the anti-E CTL clone. One transfectant expressing antigen E was identified among 70 000 drug-resistant transfectants. Its sensitivity to lysis by the anti-E CTL was equal to that of the original melanoma cell line. When this transfectant was submitted to immunoselection with the anti-E CTL clone, the resulting antigen-loss variants were found to have lost several of the transfected pSVtkneo sequences. This indicated that the gene coding for the antigen had been integrated in the vicinity of pSVtkneo sequences, as expected for cotransfected DNA. Address correspondence and offprint requests to: T. Boon.     

T cell responses to select Ia determinants using the I-A mutant mouse strain B6.C-H-2bm12

The T cell repertoire of B6.C-H-2bm12 mice (an I-A mutant mouse strain) to wild-type Iab antigens was investigated using both secondary proliferative cultures and cloned T cell lines. Because bm12 mice have a gain-loss mutation of their gene encoding the Ia beta-chain polypeptide, bm12 anti-B6 T cell responses are specific for the select component of Iab specificities that was lost as a result of the mutation. Although stimulator cells bearing Iab antigens elicited the strongest responses, Iaq, d, and s antigens also resulted in reproducible stimulations of these bm12 anti-B6-primed T cells. Cloned T cell lines isolated from bm12 anti-b6 cultures revealed similar findings, with most clones recognizing determinants unique for Iab antigens; however, clones showing cross-reactions with Iad and/or q were also selected. Using F1 hybrid responder T cells (mutant x cross-reactive strain), we further dissected this cross-reactivity into several distinct cross-reactive determinants. Because bm12 mice lack the serologically defined Ia differentiation antigen W39, T cell recognition of this determinant was investigated by using bm12 anti-B6-primed cells. Stimulation by Ia.W39+ cells was appreciably better than by Ia.W39- (Xid-defective) cells, suggesting that bm12 T cells recognize an Xid-regulated, W39-like Ia differentiation antigen.     

Resistance of primary CD8+ cytotoxic T lymphocytes to lysis by cytotoxic granules from cloned T cell lines

Recent evidence has shown that cloned, murine CTL cell lines are resistant to the cytotoxic components of the toxic granules they release upon specific interaction with their target cells. Inasmuch as the resistance might be due to selection in culture over many months by repeated exposure to these cytolytic components (which are released repeatedly as a result of the cultured CTL being periodically stimulated by target cells), we asked whether primary CTL are also resistant. The primary CTL were elicited in vivo by i.p. injection of allogeneic tumor cells or in vitro by 5- to 6-day MLC or by 48-h exposure to the lectin Con A. The responding cells were separated into purified CD8+ (i.e., CD4-, CD8+) and purified CD4+ (i.e., CD4+, CD8-) T cell populations that were analyzed for cytolytic activity and for resistance to lysis by toxic secretory granules derived from cloned CTL cell lines. The CD8+ T cells were highly cytolytic and relatively resistant; they retained their cytolytic activity and were lysed to a minimal extent (0 to 10%) by quantities of isolated granules that lysed 80 to 90% of the P815 tumor cell line (tested as a representative standard cell line). The CD4+ T cells, in contrast, had only minimal cytolytic activity and were far more susceptible to granule-mediated lysis. Although the resistance of primary CD8+ T cells is impressive, it is not as pronounced as the resistance of the cloned CTL cell lines, indicating that during long-term culture there is some selection for increased resistance to granule-mediated lysis. In contrast to T cells (especially CD8+ T cells), Ia+ macrophages, isolated from primary immune peritoneal exudates, were highly susceptible to granule-mediated lysis.     

Clonal analysis of expression of tumor-associated transplantation antigens and of metastatic capacity

Summary ESb, a spontaneous high metastatic variant of the chemically induced T lymphoma Eb, was found previously to express a tumor-associated transplantation antigen (TATA) that was different from that of the parental line. Syngeneic tumor-specific cytolytic T lymphocytes (CTL) were able to recognize the different TATAs of Eb and ESb in vitro and could therefore be used for routine typing. The object of this study was to investigate tumor antigen expression on a clonal level and to compare the in vitro data with the in vivo behavior of the same cell lines.Our CTL typing analysis of cloned tumor lines revealed that the two populations, Eb and ESb, are distinct and relatively homogeneous with regard to their TATA expression. Furthermore, all ESb clones formed rosettes with antibody-coated erythrocytes, while none of the parental type Eb clones showed this characteristic. The sensitivity to tumor-specific CTL lysis varied with time of tumor cell culture in vitro in a clone-dependent manner.Variability was also noted in vivo in tumor growth and metastatic spread. Of over 50 ESb clones tested, the majority were highly metastatic while a minority were significantly lower in metastatic capacity. High and low metastatic ESb clones could not be distinguished by their expression of TATAs and of Fc receptors. There was also a considerable individual variability in the hosts, although they were genetically identical. This variability was most probably due to differences in the immune status of the animals.     

Induction of antitumor immunity after cure of pulmonary metastases, using staphylococcal enterotoxin B and bispecific antibody

The combination of staphylococcal enterotoxin B (SEB) and anti-p97 x anti-CD3 bispecific antibody (bsAb) cures 60%-80% of mice with established pulmonary metastases of the syngeneic p97+ murine melanoma, CL62. We investigated the ability of cured mice to generate protective antitumor immunity. In tumor rechallenge experiments, CL62-cured mice developed protective immunity against rechallenge with CL62. The majority of mice also rejected the p97-negative parental cell line, K1735, indicating an immune response to tumor antigens common to both cell lines that were not bsAb-targeted. A significant humoral response developed against p97 antigen, but not against other antigens common to both CL62 and K1735. That the majority of cured mice nevertheless rejected K1735 suggests that tumor immunity is not antibody-dependent. Evidence of cellular immunity was obtained from the results of delayed-type hypersensitivity, proliferation and cytotoxicity assays, which revealed the presence of tumor-specific memory in bsAb-treated, CL62-cured mice. CD8+ T cells from cured, but not control mice were able to lyse tumor; however, memory CD4 cells had no cytolytic function. In vivo, however, both CD4 and CD8 T cells were required for effective protective immunity. These studies demonstrate that treatment with SEB and bsAb not only confers passive immune effects of tumor eradication, but also actively promotes the generation of a host antitumor immune response.