Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors.

The 4-1BB glycoprotein is a member of the tumor necrosis factor receptor superfamily and binds to a high-affinity ligand (4-1BBL) expressed on several antigen-presenting cells such as macrophages and activated B cells. Expression of 4-1BB is restricted to primed CD4+ and CD8+ T cells, and 4-1BB signaling either by binding to 4-1BBL or by antibody ligation delivers a dual mitogenic signal for T-cell activation and growth. These observations suggest an important role for 4-1BB in the amplification of T cell-mediated immune responses. We now show that administration of anti-4-1BB monoclonal antibodies can eradicate established large tumors in mice, including the poorly immunogenic Ag104A sarcoma and the highly tumorigenic P815 masto cytoma. The immune response induced by anti-4- 1BB monoclonal antibodies is mediated by both CD8+ and CD4+ T cells and is accompanied by a marked augmentation of tumor-selective cytolytic T-cell activity. Our data suggest that a similar approach may be efficacious for immunotherapy of human cancer.

Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes.

Dendritic cells (DCs) are professional antigen presenting cells with the unique capacity to induce primary and secondary immune responses in vivo. Here, we show that DCs secrete antigen presenting vesicles, called exosomes, which express functional Major Histocompatibility Complex class I and class II, and T-cell costimulatory molecules. Tumor peptide-pulsed DC-derived exosomes prime specific cytotoxic T lymphocytes in vivo and eradicate or suppress growth of established murine tumors in a T cell-dependent manner. Exosome-based cell-free vaccines represent an alternative to DC adoptive therapy for suppressing tumor growth.

Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. I. Rejection by syngeneic mice.

We have reported that it is possible to obtain variants that are incapable of forming progressive tumour in syngeneic mice (tum-) by mutagenesis and cloning of a teratocarcinoma and a Lewis lung carcinoma cell line. These observations were extended to the ascitic P815 mastocytoma of mouse strain DB/2. After a treatment with the mutagen N-methyl-N"-nitro-N-nitrosoguanidine, we obtained a high frequency (14%) of tum- clones. A colony assay indicated that after a period of rapid multiplication extending to approximately 10 d after injection, the P815 tum- cells were rejected by a process that was usually completed by day 15. No rejection was observed in sublethally irradiated animals. The immunological nature of the rejection of the P815 variants was further inferred because, upon rejection, the mice acquired a radioresistant specific protection that could be transferred adoptively with spleen cells. Cross-immunization patterns demonstrated the presence of singular antigenic specificities on three of the five variants that were examined. In addition, a common antigen was found on all the tum- variants and the original cells that were capable of forming progressive tumors in syngeneic mice (tum+). Mice injected with tum- cells were significantly protected against a tum+ challenge, even though no significant protection was generated by irradiated tum+ cells. A study of the T lymphocyte-mediated cytolysis against the P815 variants described here is presented in the accompanying report (9).

Cellular immunoabsorbents in transplantation immunity. Specific in vitro deletion and recovery of mouse lymphoid cells sensitized against allogeneic tumors.

Mouse lymphoid cells, sensitized against tumor allografts, can be deprived of the immunoreactive cells by in vitro absorption with specific fibroblast monolayers. Populations of lymphocytes so depleted are less effective in retarding tumor growth in vivo and in lysing tumor cells in vitro. Moreover, the adsorbed immunoreactive cells can be recovered specifically and are subsequently efficient in inhibiting tumor growth in vivo and in killing tumor cells in vitro. Further evidence is presented for the suggestion that the destruction of target cells in vitro by sensitized lymphoid cells is truly representative of the mode of destruction of grafted cells in vivo.

Induction of anterior chamber-associated immune deviation requires an intact, functional spleen.

Anterior chamber-associated immune deviation (ACAID) expresses itself in BALB/c mice inoculated intracamerally with P815 cells in three ways: progressive growth of the tumor within the eye, transient growth of P815 cells injected subcutaneously, and prolonged acceptance of DBA/2 skin allografts. The spleen was found to play a crucial role in the development of ACAID. Splenectomized animals bearing intracameral P815 tumors reject DBA/2 skin grafts in an accelerated manner. A functioning spleen was required during the first 10 d after intracameral inoculation of P815 cells, but not thereafter. Reconstitution experiments revealed that the spleen's ability to support the induction of ACAID depends partly upon its constituent lymphoid cells, but also upon either a stromal component or a unique architectural arrangement that can only be restored with splenic fragments. The data hold promise that therapeutic protocols using appropriately timed splenectomy and specific immunization can be devised to induce hosts bearing intraocular tumors to mount an immune response sufficiently vigorous to destroy the tumor within the eye, and sufficiently precise to preserve the functional and anatomic integrity of the eye.

Immunologically mediated regression of a murine lymphoma after treatment with anti-L3T4 antibody. A consequence of removing L3T4+ suppressor T cells from a host generating predominantly Lyt-2+ T cell-mediated immunity.

This study shows that intravenous injection of 1 mg of anti-L3T4 mAb (GK1.5) into thymectomized mice bearing the syngeneic L5178Y lymphoma results, after a delay of 2-3 d, in complete regression of this tumor and in long-term host survival. A flow cytofluorometric examination of the spleen cells of mAb-treated mice revealed that antibody treatment resulted in the elimination of greater than 98% of L3T4+ T cells, but had no effect on the Lyt-2+ T cells subset. Tumor regression was immunologically mediated, because L5178Y lymphoma cells were shown to be L3T4-, and regression of the tumor failed to occur in mice that had been lethally irradiated before anti-L3T4 mAb was given. Tumor regression was mediated by tumor-sensitized Lyt2+ T cells, as evidenced by the finding that treatment of tumor-bearing mice with anti-Lyt-2 mAb alone, or in combination with anti-L3T4 mAb, resulted in enhancement of tumor growth and a significant decrease in host survival time. Moreover, the spleens of mice whose tumors were undergoing regression in response to anti-L3T4 mAb treatment contained Lyt-2+ T cells capable, on passive transfer, of causing regression of a tumor in recipient mice. These results can be interpreted as showing that removal of tumor-induced L3T4+ suppressor T cells results in the release of Lyt-2+ effector T cells from suppression, and consequently in the generation of enough Lyt-2+ T cell-mediated immunity to cause tumor regression. This can only be achieved, however, if immunity to the tumor is mediated exclusively by Lyt-2+ T cells, as is the case for the L5178Y lymphoma. In the case of the P815 mastocytoma, treatment with anti-L3T4 mAb was without a therapeutic effect, and this was in keeping with the finding that immunity to this tumor is mediated by L3T4+, as well by Lyt-2+ T cells.

Systemic immune unresponsiveness induced in adult mice by anterior chamber presentation of minor histocompatibility antigens.

The ability to introduce carefully controlled numbers of viable cells into the anterior chamber of mouse eyes made it possible to examine the interrelationship between presentation of antigens into the anteior chamber and into conventional body sites and their synergistic/antagonistic effects on the immune system. P815 mastocytoma (DBA/2; H-2d) cells are syngeneic with BALB/c hosts at the major histocompatibility locus, but differ at multiple minor histocompatibility loci. When P815 cells were injected subcutaneously, they were rejected by BALB/c recipients who became specifically immune. By contrast, when P815 cells were injected intracamerally, they grew progressively into massive intraocular tumors; moreover, these BALB/c hosts proved subsequently unable to reject subcutaneously injected P815 cells, and, more impressively, failed to reject DBA/2 skin allografts placed orthotopically. Minor histocompatibility antigens, presented first through the anterior chamber of mouse eyes, elicit a suppressive rather than an aggressive host immune response that protects cells that bear these antigens from a destructive alloimmune reaction at both intracameral and systemic sites.

Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. II. T lymphocyte-mediated cytolysis.

Tumor cell variants that were rejected by syngeneic mice (tum-) were obtained from mastocytoma P815 by mutagenesis (as described in the accompanying report (13). A considerable T lymphocyte-mediated lysis was observed upon incubation of these tum- variants with peritoneal exudate cells collected a few days after an intraperitoneal challenge of immune animals. Spleen cells from these animals were cytolytic after stimulation in vitro with the immunizing variant. New antigens, absent from the original P815 tum+ cells, were detected on 15 of the 21 tum- variants that were tested. All these antigens appeared to be different. No new antigen was detected on any of 10 mutagenized P815 clones that had retained their ability to form tumors. We compared the evidence obtained in vivo and in vitro for the presence of specific antigens on five tum- variants. Three variants were shown both in vivo and in vitro to carry an individual antigen. One showed no specificity either in vivo or in vitro. However, for one variant, no specificity was observed in vivo, although cytolysis tests demonstrated the existence of a singular antigenic specificity.

Inhibition of T-lymphocyte-mediated tumor-specific lysis by alloantisera directed against the H-2 serological specificities of the tumor.

After appropriate in vivo or in vitro immunization, cytotoxic T lymphocytes (CTL) are generated which efficiently kill cells bearing particular membrane antigens in common with the immunizing cell (reviewed in reference 1). Such CTL have been most thoroughly studied in mice, employing alloimmunization with cells differing at the major histocompatibility locus, H-2. in such cases, the predominant cell surface antigens recognized by the CTL appear to be the molecules carrying the serologically defined H-2 specificities, coded for by the K and D regions of the H-2 complex (2). In other syngeneic models of cell-mediated specific cytolysis, involving lymphocyte chariomeningitis (LCM) virus- or ectromelia virus-infected cells or TNP-modified lymphoid cells, thymus-derived cells also constitute the main effector cell type. The CTL generated in these latter systems function most efficiently when virus-infected or TNP-modified target cells share identitites at the H-2K or H-2D loci with the effector CTL and stimulator cells (3-5). Another set of experimental systems in which CTL are generated and play a significant biological role is that of immunity to tumor-associated antigens (TAA) (6). The nature of the TAA which the CTL recognize is only beginning to be understood. Several recent reports indicated the existence of physiochemical and/or antigenic relationships between TAA and H-2 antigens (7,8). These relationships, together with the genetic restrictions cited above in the generation of CTL involving products of the H-2K or H-2D loci suggested the possibility that in certain tumor systems, the TAA which are able to most effectively stimulate CTL responses might be structurally similar to, or linked with, the H-2K or H- 2D molecules on the tumor surface. It has been previously demonstrated in allogenic models that antisera specific for the appropriate H-2K or H-2D products present on a target cell could specifically block CTL-mediated lysis (1,9). This report demonstrates that certain anti-H-2 alloantisera specific for the target tumor cells can block lysis of those target cells mediated by syngeneic tumor-specific CTL effector cells.

Binding of bacillus Calmette-Guérin-activated macrophages to tumor targets. Selective inhibition by membrane preparations from homologous and heterologous neoplastic cells.

The binding of tumor cells by activated macrophages is an initial and necessary event in the cytolysis of these targets. The data here indicate that membrane preparations from RL sigma 1 leukemia targets, EL-4 lymphoma targets, and P815 mastocytoma targets each inhibited binding of its homologous target to bacillus Calmette-Guérin (BCG)-activated murine macrophages in a dose-dependent fashion. Similar amounts of membrane from lymphocytes did not alter binding of the three neoplastic target to BCG-macrophages. Membranes of the three targets also inhibited binding of the heterologous neoplastic targets. Inhibitory activity of membrane preparations from P815, EL-4, and RL sigma 1 targets could be adsorbed by incubation of limiting concentrations of the membrane preparations with BCG-activated macrophages but not with thioglycollate broth-elicited macrophages. Exposure of BCG macrophages to membrane preparations from RL sigma 1, FL-4, or P815 targets inhibited subsequent cytolysis of the three targets. Inhibitory activity was increased in preparations enriched for plasma membrane. The data suggest that binding of three murine, nonadherent neoplastic targets to BCG-activated murine macrophages is mediated, in part, by recognition structures present within the plasma membranes of the three targets.

Separation of stages in the development of the "T" cells involved in cell-mediated immunity.

Density distribution analysis in continuous gradients of albumin has been used to study the development of cytotoxic lymphocytes (CL), to separate and characterize the progenitors of CL, and to determine their relationship to subpopulations of T cells. CL progenitors in the thymus were a homogeneous, medium-density population, distinct from the typical, dense, thymus small-lymphocyte. Activity seemed to be associated with one minor subpopulation of cells with surface antigenic properties characteristic of peripheral T cells (high levels of H-2 antigen, low levels of theta-antigen). CL progenitors in the spleen differed from those in the thymus and normally had the high buoyant density of typical small T lymphocytes. In states of antigenic stimulation, some lighter-density CL progenitors were found in the spleen. The buoyant density of the CL population developing in the spleens of immunized animals showed progressive changes with time. Early, "immature" CL had the light-density characteristics of large, activated lymphocytes. As the response developed, the density of the CL population increased, and finally approached that of CL progenitors and of typical small lymphocytes. The data suggest that density subpopulations of T cells represent stages in the development of immunocompetent cells. Possible differentiation pathways of T lymphocytes in the thymus and in the spleen are discussed.

Generation of cytotoxic T lymphocytes in vitro. III. Velocity sedimentation studies of the differentiation and fate of effector cells in long-term mixed leukocyte cultures.

Separation of cells by velocity sedimentation at unit gravity was utilized to investigate the physical properties of cytotoxic thymus-derived lymphocytes (CTL) generated in long-term mixed leukocyte cultures (MLC). In kinetic studies, CTL were found almost exclusively in the large cell fractions at the peak of the response on day 4, whereas the majority of CTL in day 14 MLC had the sedimentation properties of small lymphocytes. Reculture until day 14 of cells fractionated on the basis of size on day 4 indicated that the small CTL were derived exclusively from cells which had been large on day 4. Re-exposure of day 14 MLC cells to the original stimulating alloantigens resulted in significant cell proliferation and rapid regeneration of CTL activity. Cell fractionation experiments demonstrated that the cells in the day 14 MLC population which responded to the secondary allogeneic stimulus were small T lymphocytes, and that these cells rapidly developed into large, highly cytotoxic CTL following stimulation. Moreover, by restimulating on day 14 fractions which were selected on the basis of size on day 4, it was found that the responding small lymphocytes were themselves the progeny of cells which were large at the peak of the response. Since CTL and CTL progenitors showed concomitant changes in physical properties with time, the possibility exists that they belong to the same cell lineage, and hence that CTL can differentiate into cells which are no longer cytotoxic, but capable of mounting an anamnestic response.

The gene coding for a major tumor rejection antigen of tumor P815 is identical to the normal gene of syngeneic DBA/2 mice.

We showed previously that mouse mastocytoma P815 expresses several distinct antigens that are recognized by cytolytic T lymphocytes (CTL) of syngeneic DBA/2 mice. Antigens P815A and P815B are usually lost jointly and are targets for immune rejection responses in vivo. We used a cosmid library and a CTL stimulation assay to obtain transfectants expressing tumor rejection antigen P815A. From these transfectants we retrieved gene P1A which transferred the expression of both P815A and B. This gene is unrelated to three previously isolated genes coding for tum-antigens. It encodes a putative protein of 224 amino acids which contains two highly acidic domains showing homology with similar regions of nuclear proteins. The P1A gene expressed by tumor P815 is completely identical to the gene present in normal DBA/2 cells. Expression of the gene was tested by Northern blots. Cells from liver, spleen, and a number of mast cell lines were negative, but mast cell line L138.8A produced a high level of P1A message and was lysed by CTL directed against antigens P815A and B. We conclude that major tumor rejection antigens of P815 are encoded by a gene showing little or no expression in most normal cells of adult mice.

Association of immunity and tolerance to host H-2 determinants in irradiated F1 hybrid mice reconstituted with bone marrow cells from one parental strain.

Semiallogenetic radiation chimeras were prepared by injecting heavily irradiated F1 hybrid mice with bone marrow cells from one parental strain; the bone marrow cells were treated with anti-theta serum and complement to remove T cells and injected in large numbers (2 times 10-7 cells). The mice survived in excellent health until sacrifice 6 mo later. Thoracic duct cannulation at this stage showed that the mice possessed normal numbers of recirculating lymphocytes. Close to 100% of thoracic duct lymphocytes and lymph node cells were shown to be of donor strain origin. The capacity of lymphocytes from the chimeras to respond to host-type determinants was tested in mixed leukocyte culture and in an assay for cell-mediated lympholysis (CML). Mixed leukocyte reactions (MLR) were measured both in vitro and in vivo; tumor cells and phytohemmaglutinin-stimulated blast cells were used as target cells for measuring CML. While responding normally to third party determinants, cells from the chimeras gave a definite, though reduced MLR when exposed to host-type determinants. However, this proliferative response to host-type determinants, unlike that to third party determinants, was not associated with differentiation into cytotoxic lymphocytes. No evidence could be found that unresponsiveness in this situation was due to blocking serum factors or suppressor T cells. It is argued that the results support the concept that lymphocytes responsive in mixed leukocyte culture have a different specificity to those exerting cell-mediated lympholysis.

Monoclonal antibody to murine gamma interferon inhibits lymphokine-induced antiviral and macrophage tumoricidal activities.

Fusion of rat immune spleen cells with mouse myeloma cells resulted in the formation of a stable hybridoma that secretes monoclonal antibody (MAb) directed against murine gamma interferon ( MuIFN -gamma). This MAb specifically neutralized the antiviral activity of a variety of MuIFN -gamma preparations, including a sample produced by recombinant DNA technologies. In contrast, the antiviral activities of a mixture of MuIFN -alpha plus MuIFN -beta, as well as those of rat or human IFN-gamma, were not neutralized by this antibody. The ability of the MAb to inhibit lymphokine-induced macrophage activation was also tested. It was found that in relation to the quantity of antibody needed to completely neutralize antiviral activity, much higher concentrations of MAb were required to abolish the capacity of lymphokine preparations to induce macrophage tumoricidal activity in vitro. The MAb was also coupled to cyanogen bromide-activated Sepharose beads and used as an immunoadsorbent. By reacting lymphokines with MAb coupled to an insoluble matrix, it was possible to show that this immobilized antibody completely and specifically removed from the lymphokine preparations the ability both to invoke macrophage tumoricidal activity and to mediate antiviral activity.

Expression of passively transferred immunity against an established tumor depends on generation of cytolytic T cells in recipient. Inhibition by suppressor T cells.

The results of this study with the P815 mastocytoma confirm the results of previous studies that showed that the passive transfer of tumor-sensitized T cells from immunized donors can cause the regression of tumors growing in T cell-deficient (TXB) recipients, but not in normal recipients. The key additional finding was that the expression of adoptive immunity against tumors growing in TXB recipients is immediately preceded by a substantial production of cytolytic T cells in the recipients' draining lymph node. On the other hand, failure of adoptive immunity to be expressed against tumors growing in normal recipients was associated with a cytolytic T cell response of much lower magnitude, and a similar low magnitude response was generated in TXB recipients infused with normal spleen cells and in tumor-bearing control mice. Because the passively transferred sensitized T cells possessed no cytolytic activity of their own, the results indicate that the 6-8-d delay before adoptive immunity is expressed represents the time needed for passively transferred helper or memory T cells to give rise to a cytolytic T cell response of sufficient magnitude to destroy the recipient's tumor. In support of this interpretation was the additional finding that inhibition of the expression of adoptive immunity by the passive transfer of suppressor T cells from tumor-bearing donors was associated with a substantially reduced cytolytic T cell response in the recipient's draining lymph node. The results serve to illustrate that interpretation of the results of adoptive immunization experiments requires a knowledge of the events that take place in the adoptively immunized recipient. They support the interpretation that suppressor T cells function in this model to "down-regulate" the production of cytolytic effector T cells.

Stimulus specificity of the generation of leukotrienes by dog mastocytoma cells.

Isolated dog mastocytoma cells sensitized with dog anti-ragweed IgE and challenged with ragweed antigen or incubated with ionophore A23187 or the carboxy-terminal dodecapeptide of platelet factor 4, PF4(59-70), release histamine and concurrently generate leukotrienes B4, C4, and D4. In contrast, the exposure of mastocytoma cells to 0.1-3 micrograms/ml of 15-hydroxyeicosatetraenoic acid (15-HETE) stimulates selectively the generation of leukotrienes, in the absence of histamine release, while 0.1-1 micrograms/ml of compound 48/80 releases histamine without enhancing the generation of leukotrienes. That natural stimuli are capable of selectively activating one synthetic or secretory compartment of mast cells suggests that separate subsets of receptors as well as different biochemical events may serve to mobilize each class of mediators.

Escape of mouse mastocytoma P815 after nearly complete rejection is due to antigen-loss variants rather than immunosuppression.

Even though mastocytoma P815 often undergoes a nearly complete rejection in syngeneic mice, the tumor cells almost always escape to form progressive tumors. We found that this was not due to the establishment of an immunosuppressed state because genetically marked P815 cells, that were injected in mice where tumor escape was occurring, were readily rejected. An analysis of escaping tumor cell populations with anti-P815 cytolytic T lymphocyte (CTL) clones showed the presence of stable resistant variants. Using antigen-loss variants found in escaping populations or selected in vitro with CTL clones, we were able to define four different tumor-associated antigenic specificities, each recognized by a specific CTL clone. One of these specificities was absent from all escaping tumor cells and another had been lost by some of them.

Cells mediating specific in vitro cytotoxicity. I. Detection of receptor-bearing lymphocytes.

Spleen cells from mice immunized with allogeneic tumor cells are incubated on different fibroblast monolayers. The nonadsorbed cells are tested for cytotoxicity against (51)Cr-labeled target cells. The cytotoxicity of nonadsorbed cells is much lower after incubation on fibroblasts syngeneic to the immunizing tumor cells than after incubation on fibroblasts syngeneic to the immune cells. This specific decrease of cytotoxic activity depends on the duration and temperature of incubation on monolayers. After incubation the monolayers are trypsinized and pure populations of adsorbed lymphocytes isolated by density gradient fractionation. The cytotoxicity of such trypsin-eluted, gradient-purified lymphocytes is much higher when these lymphocytes are isolated from fibroblasts syngeneic to the immunizing tumor cells than when they are isolated from fibroblasts syngeneic to the immune cells. These experiments demonstrate specific adsorption of immune cells onto fibroblasts carrying the immunizing antigens, and thus prove the existence of specific receptors at the surface of these immune cells. Spleen cells from mice immunized with two types of allogeneic tumor cells bearing different H-2 antigen alleles are incubated on different fibroblast monolayers. The results of such experiments show a differential specific adsorption pattern, suggesting independent adsorption of two populations of immune cells bearing receptors directed against either one or the other immunizing H-2 antigen. The existence of at least a majority of cells, each of which is homogeneous as to the specificity of its receptors, makes it likely that specific receptors are synthetized by the cells that bear them. The role of specific receptor-bearing cells in the killing process is discussed.

Nature of lymphocyte-tumor interaction. A general method for cellular immunoabsorption.

The binding of sensitized lymphocytes to tumor cells that leads to tumor cell lysis in vitro has been investigated using poly-L-lysine-fixed tumor cell monolayers and lymphocytes obtained from the anatomical site of tumor allograft rejection. The results show that magnesium is an important prerequisite for this interaction and that the extent of lymphocyte-tumor cell binding depends upon temperature as well as pH. Binding can occur in the absence of serum, although serum factors are necessary for the completion of the cytolytic process. The poly-L-lysine technique is applicable to the formation of confluent monolayers with virtually any normal or neoplastic cell type, including those that are otherwise nonadherent to surfaces. Cells immobilized by this technique can be used for the specific immunoabsorption and subsequent recovery of effector lymphocytes sensitized against transplantation or tumor cell antigens.