Functional analysis of mononuclear cells infiltrating into tumors. II. Differential ability of mononuclear cells obtained from various tissues to produce helper factors that are involved in the generation of cytotoxic cells

The requirement of CGF in the generation of cytotoxic cells against syngeneic tumor cells (T-9) and in the rejection of transplanted T-9 cells has been investigated. Spleen cells obtained from sensitized rats showed strong cytotoxicity against 51Cr-labeled T-9 cells upon incubation with CGF for 48 hr. Human recombinant IL 2 and rat IFN failed to generate cytotoxic cells from spleen cells of sensitized rats. CGF are produced by spleen cells upon inoculation of T-9 cells into sensitized rats as a host in vivo immune response. Production of CGF preceded the appearance of cytotoxic cells in regional lymph node and tumor tissues. In those rats, inoculated tumor cells were eventually rejected. In contrast, spleen cells failed to produce CGF upon inoculation of T-9 cells in unsensitized rats. Cytotoxic cells were not detected in unsensitized rats, and inoculated tumor grew in those rats. Thus, CGF is likely to be involved in the generation of cytotoxic cells and in the rejection of inoculated syngeneic tumor cells. A Mono Q anion-exchange column with an FPLC system allowed the chromatographic separation of CGF from IL 1, IL 2, IL 3, and CSF.     

Functional analysis of mononuclear cells infiltrating into tumors. III. Soluble factors involved in the regulation of T lymphocyte infiltration into tumors

We have analyzed the mechanisms controlling the accumulation of T lymphocytes in tumor tissues. Spleen cells, left or right popliteal lymph node cells, and tumor-infiltrating cells were obtained from tumor-inoculated rats and were cultured for 24 h. Culture supernatants were obtained and assessed for lymphocyte migration factor (LMF) activity with the use of a modified Boyden chamber. We found that tumor-infiltrating cells derived from T-9-sensitized rats produced LMF. Two waves of LMF production were observed. The first wave of LMF production was detected between 6 and 12 h (LMF-a) and the second wave of LMF production was detected between 4 and 6 days (LMF-4d and -6d) after tumor inoculation. The tumor-infiltrating cells consisted of heterogenous cell populations. We found that only tumor-infiltrating neutrophils of T-9-sensitized rats produced LMF-a. Five peaks of LMF (A through E) were detected upon fractionation of LMF-a using Mono Q anion exchange column chromatography. Peak D exhibited the strongest activity. The action of peak D was chemotactic, but not chemokinetic. The m.w. of peak D was 33,000 and 70,000. Only W3/25 (+) (helper/inducer) T cells were found to be sensitive to peak D. The production of LMF-a by purified tumor-infiltrating neutrophils in vitro is in agreement with the histologic observation that the infiltration of neutrophils precedes the appearance of W3/25 (+) T cells in tumor tissues of T-9-sensitized rats. It is thus likely that peak D of LMF-a is responsible for the infiltration of T lymphocytes into tumor tissues.     

Functional analysis of mononuclear cells infiltrating into tumors. V. A. soluble factor involved in the regulation of cytotoxic/suppressor T cell infiltration into tumors

We have analyzed the mechanisms controlling the accumulation of cytotoxic/suppressor T lymphocytes in tumor tissues. We found that tumor-infiltrating helper/inducer T cells isolated from T-9 gliosarcoma-sensitized rats between 4 and 6 days after T-9 gliosarcoma inoculation produced a lymphocyte migration factor (LMF) during in vitro culture. Four peaks of LMF activity (A through D) were detected upon fractionation of LMF by using a Mono Q anion exchange column chromatography. Peak C exhibited the strongest activity among the four peaks of LMF. The action of peak C was chemotactic, but not chemokinetic. Peak C had an isoelectric point of 8.0 and a Mr of 26,000 Da. Only cytotoxic/suppressor T cells were found to be sensitive to peak C in vitro as well as in vivo. It is thus likely that peak C is responsible for the infiltration of cytotoxic/suppressor T cells into tumor tissues. The infiltration of lymphocytes into tumor tissues might also be regulated by the expression of lymphocyte sensitivity for LMF. The target molecule for LMF at 4 days may involve an asparagine-linked oligosaccharide.     

Development of systemic immunity to glioblastoma multiforme using tumor cells genetically engineered to express the membrane-associated isoform of macrophage colony-stimulating factor.

We investigated the ability of Fischer rat T9 glioblastoma cells transduced with cDNA genes for the secreted (s) or membrane-associated (m) isoform of M-CSF to elicit an antitumor response when implanted into syngeneic animals. Intracranial (i.c.) implantation of 1 x 10(5) T9 cells expressing mM-CSF (T9/mM-CSF) resulted in 80% tumor rejection. Electron microscopy of the T9/mM-CSF tumor site, 2-4 days postimplantation, showed marked infiltration by macrophages, many of which were in physical contact with the T9/mM-CSF cells. Animals that rejected T9/mM-CSF cells were resistant to i.c. rechallenge with T9 cells, but not syngeneic MadB106 breast adenocarcinoma cells, suggesting that T9-specific immunity can be generated within the brain via the endogenous APCs. Intracranial injection of parental T9, vector control (T9/LXSN), or T9 cells secreting M-CSF (T9/sM-CSF) was 100% fatal. Subcutaneous injection of 1 x 10(7) T9/sM-CSF, T9/LXSN, or parental T9 cells resulted in progressive tumors. In contrast, T9/mM-CSF cells injected s.c. were destroyed in 7-10 days and animals developed systemic immunity to parental T9 cells. Passive transfer of CD3+ T cells from the spleens of immune rats into naive recipients transferred T9 glioma-specific immunity. In vitro, splenocytes from T9/mM-CSF-immunized rats specifically proliferated in response to various syngeneic glioma stimulator cells. However, only marginal T cell-mediated cytotoxicity was observed by these splenocytes in a CTL assay against T9 target cells, regardless of restimulation with T9 cells. Subcutaneous immunization with viable T9/mM-CSF cells was effective in eradicating i.c. T9 tumors.     

Host immune responses to tumor cells augmented by bleomycin and their therapeutic effects on rat fibrosarcoma

The administration--timing-dependent therapeutic effects of bleomycin (BLM) were observed on a fibrosarcoma implanted SC in WKA rats. Five consecutive IP administrations of BLM (5 mg/kg/d) were found to be more effective when BLM was given from Day 8 than when it was given from Day 1 for tumors implanted on Day 0. The therapeutic effects correlated well with antitumor immune responses, which were examined on Day 13 when the tumor had not yet regressed even in surviving rats. The tumor-neutralizing activity of spleen cells was augmented in rats treated with BLM from Day 8 to Day 12, and the suppressor cell activity detected in the spleen cells of tumor-bearing rats was eliminated by the BLM treatment. The tumoricidal activity of peritoneal exudate cells (PEC) was detected in rats treated from Day 8 but not in rats untreated or treated from Day 1. The in vitro treatment of KMT-17 cells with BLM (20 micrograms/ml) for two hours enhanced the sensitivity of the tumor cells to the activity of tumoricidal PEC. This suggests that the direct action of BLM on tumor cells also plays an immunologic role in BLM treatment. The findings reveal that the therapeutic effect of BLM is elicited by its ability to augment the host immune responses to tumor cells.     

Characterization of bovine mononuclear cell populations with natural cytolytic activity against bovine herpesvirus 1-infected cells

Freshly isolated or overnight cultured peripheral blood mononuclear cells from immune or nonimmune animals had natural cytolytic activity against bovine herpesvirus 1 (BHV-1)-infected tumor target cells. No lysis was demonstrated against tumor target cells alone. This natural cytolytic activity was present in mononuclear cells from the spleen, lymph node, and peripheral blood but little or no cytolytic activity was detected in bone marrow or thymus cells. When monoclonal antibodies and complement to deplete bovine mononuclear cell subpopulations from the nonadherent cells were used, results indicated the effector cell was not a T cell, B cell, or activated monocyte. From nonadherent populations separated on density gradients, it was determined that the effector cells were large, low density mononuclear cells. These results indicate the nonadherent effector cells mediating lysis of BHV-1-infected xenogeneic adherent target cells were large null lymphocytes and/or immature monocytes.     

Irradiated tumor cell vaccine for treatment of an established glioma. I. Successful treatment with combined radiotherapy and cellular vaccination

Rats bearing a 5-day intracranial (i.c.) syngeneic glioma were treated with a subcutaneous (s.c.) vaccination consisting of irradiated glioma cells or a multimodality approach composed of radiotherapy plus s.c. vaccination. Vaccination of rats harboring a T9 glioma with 5 x 10(6) irradiated T9.F glioma cells (a clone derived from the T9 glioblastoma cell line) resulted in a marked enhancement of i.c. glioma growth and a significant decrease in survival. Histopathology of the tumors from vaccinated rats revealed a massive glioma composed of healthy tumor tissue lacking any marked inflammation, edema or hemorrhage. Analysis of the tumor-infiltrating mononuclear cells indicated that gliomas from vaccinated rats contained a 10-fold greater lymphoid infiltrate per milligram of tumor as compared to tumors from non-vaccinated rats, suggesting that the vaccination had induced immune cells to localize to the i.c. glioma. Combined treatment consisting of 15 Gy of whole head irradiation of the 5-day glioma followed by vaccination with T9.F cells resulted in a significant increase in survival compared to that of non-treated rats, 45% of which remained tumor-free. Microscopic evaluation in survivors of the tumor implantation site revealed the presence of hemosiderin-laden macrophages and other mononuclear cells, with the absence of tumor cells within the residual lesion. When survivors were challenged s.c. with viable T9.F glioma cells, a delayed-type hypersensitivity (DTH) reaction appeared at the challenge site. T cells purified from these rats proliferated and secreted Th(1)-associated cytokines when stimulated with irradiated T9.F glioma cells, and lysed T9.F target cells. In contrast, when these rats were challenged s.c. with the unrelated MadB106 adenocarcinoma, tumor formation was observed. These findings indicate that the treatment of an established i.c. glioma with a cellular vaccination alone may induce enhanced tumor growth; however, when the vaccination is combined with radiation therapy, the results are beneficial in terms of increased survival time or complete remission that is accompanied by the development of tumor-specific cellular immunity.     

Increase in tumor size following intratumoral injection of immunostimulatory CpG-containing oligonucleotides in a rat glioma model

The immunosuppressive environment of malignant gliomas is likely to suppress the anti-tumor activity of infiltrating microglial cells and lymphocytes. Macrophages and microglial cells may be activated by oligonucleotides containing unmethylated CpG-motifs, although their value in cancer immunotherapy has remained controversial. Following injection of CpG-containing oligonucleotides (ODN) into normal rat brain, we observed a local inflammatory response with CD8+ T cell infiltration, upregulation of MHC 2, and ED1 expression proving the immunogenic capacity of the CpG-ODN used. This was not observed with a control ODN mutated in the immunostimulatory sequence (m-CpG). To study their effect in a syngeneic tumor model, we implanted rat 9L gliosarcoma cells into the striatum of Fisher 344 rats. After 3 days, immunostimulatory CpG-ODN, control m-CpG-ODN, or saline was injected stereotactically into the tumors (day 3 group). In another group of animals (day 0 group), CpG-ODN were mixed with 9L cells prior to implantation without further treatment on day 3. After 3 weeks, the animals were killed and the brains and spleens were removed. Rather unexpectedly, the tumors in several of the animals treated with CpG-ODN (both day 0 and day 3 group) were larger than in saline or m-CpG-ODN treated control animals. The tumor size in CpG-ODN-treated animals was more variable than in both control groups. This was associated with inflammatory responses and necrosis which was observed in most tumors following CpG treatment. This, however, did not prevent excessive growth of solid tumor masses in the CpG-treated animals similar to the control-treated animals. Dense infiltration with microglial cells resembling ramified microglia was observed within the solid tumor masses of control- and CpG-treated animals. In necrotic areas (phagocytic), activation of microglial cells was suggested by ED1 expression and a more macrophage-like morphology. Dense lymphocytic infiltrates consisting predominantly of CD8+ T cells and fewer NK cells were detected in all tumors including the control-treated animals. Expression of perforin serving as a marker for T cell or NK cell activation was detected only on isolated cells in all treatment groups. Tumors of all treatment groups revealed CD25 expression indicating T cells presumed to maintain peripheral tolerance to self-antigens. Cytotoxic T cell assays with in vitro restimulated lymphocytes (⁵¹chromium release assay) as well as interferon-gamma production by fresh splenocytes (Elispot assay) revealed specific responses to 9L cells but not another syngeneic cell line (MADB 106 adenocarcinoma). Surprisingly, the lysis rates with lymphocytes from CpG-ODN-treated animals were lower compared to control-treated animals. The tumor size of individual animals did not correlate with the response in both immune assays. Taken together, our data support the immunostimulatory capacity of CpG-ODN in normal brain. However, intratumoral application proved ineffective in a rat glioma model. CpG-ODN treatment may not yield beneficial effects in glioma patients.     

Identification and cytotoxic reactivity of inflammatory cells recovered from progressing or regressing syngeneic UV-induced murine tumors.

Most tumors induced in C3H mice by ultraviolet (UV) light are immunologically rejected by normal syngeneic recipients, but will grow progressively in immunosuppressed mice and in mice treated with UV light. In this study we compared the composition and cytotoxic activity of the inflammatory cell infiltrate from tumors transplanted into syngeneic UV-irradiated or unirradiated mice. Tumor fragments were implanted in either normal (regressor) or UV-treated (progressor) mice, and removed on various days after implantation and mechanically dissociated. The cells were examined by immunofluorescence for theta and immunoglobulin markers, stained for morphologic examination, and tested for cytotoxicity against the tumor. No significant differences were noted in numbers of macrophages, granulocytes, or B cells recovered from progressing or regressing tumors on day 6, the time of greatest activity. However, the numbers of T cells recovered from tumor fragments implanted in normal mice was approximately 3-fold that recovered from tumor fragments implanted in UV-treated mice. Lymphocytes recovered from regressing tumor fragments were specifically cytotoxic for that tumor in a microcytotoxicity test; those from progressing tumor fragments were not cytotoxic.     

Engagement of the OX-40 receptor in vivo enhances antitumor immunity

The OX-40 receptor (OX-40R), a member of the TNFR family, is primarily expressed on activated CD4+ T lymphocytes. Engagement of the OX-40R, with either OX-40 ligand (OX-40L) or an Ab agonist, delivers a strong costimulatory signal to effector T cells. OX-40R+ T cells isolated from inflammatory lesions in the CNS of animals with experimental autoimmune encephalomyelitis are the cells that respond to autoantigen (myelin basic protein) in vivo. We identified OX-40R+ T cells within primary tumors and tumor-invaded lymph nodes of patients with cancer and hypothesized that they are the tumor-Ag-specific T cells. Therefore, we investigated whether engagement of the OX-40R in vivo during tumor priming would enhance a tumor-specific T cell response. Injection of OX-40L:Ig or anti-OX-40R in vivo during tumor priming resulted in a significant improvement in the percentage of tumor-free survivors (20-55%) in four different murine tumors derived from four separate tissues. This anti-OX-40R effect was dose dependent and accentuated tumor-specific T cell memory. The data suggest that engagement of the OX-40R in vivo augments tumor-specific priming by stimulating/expanding the natural repertoire of the host's tumor-specific CD4+ T cells. The identification of OX-40R+ T cells clustered around human tumor cells in vivo suggests that engagement of the OX-40R may be a practical approach for expanding tumor-reactive T cells and thereby a method to improve tumor immunotherapy in patients with cancer.     

Specific inhibition of cytotoxic memory cells produced against UV-induced tumors in UV-irradiated mice.

Cytotoxic responses of UV-irradiated mice against syngeneic UV-induced tumors were measured by using a 51Cr-release assay to determine if UV treatment induced a specific reduction of cytotoxic activity. The in vivo and in vitro primary responses against syngeneic tumors and allogeneic cells were unaffected, as was the "memory" response (in vivo stimulation, in vitro restimulation) against alloantigens. In contrast, the memory response of UV-treated mice against syngeneic, UV-induced tumors was consistently and significantly depressed. The cytotoxicity generated by tumor cell stimulation in vivo or in vitro was tumor-specific and T cell-dependent. Since the primary response against syngeneic UV-induced tumors produces apparently normal amounts of tumor-specific cytotoxic activity, UV-treated mice may not reject transplanted syngeneic tumors because of too few T effector memory cells. These results imply that, at least in this system, tumor rejection depends mostly on the secondary responses against tumor antigens and that at least one carcinogen can, indirectly, specifically regulate immune responses.     

Suppression of in vivo tumorigenicity of rat hepatoma cell line KDH-8 cells by soluble TGF-beta receptor type II

We have previously reported that transforming growth factor beta (TGF-beta) produced by rat hepatoma cell line KDH-8 cells suppressed the interleukin-2 (IL-2) production of T cells and the tumoricidal activity of macrophages in KDH-8 tumor-bearing rats and that the inhibition of TGF-beta production by low-dose bleomycin restored these activities significantly. In this study, we established three transfectant clones with stable expression of soluble TGF-beta receptor type II (sTRII), namely KT1, KT2 and KT3, and one with an empty vector used as control vector (KV), and then investigated the effects of sTRII on the tumorigenicity of KDH-8 cells and immune responses in syngeneic Wistar King Aptekman/Hok (WKAH) rats. We found that sTRII expressed in sTRII transfectants could abolish growth inhibition of Mv1Lu cells by TGF-beta1 produced by the cells themselves, and that tumor growth of KT2 and KT3 clones in vivo was suppressed significantly compared with that of parent, KV and KT1 clones. Furthermore, we demonstrated that IL-2 production of splenocytes and IL12p40 mRNA expression in tumor tissues were restored in rats inoculated with KT2 and KT3 clones, whereas such restoration was not observed in rats inoculated with parent, KV and KT1 clones. Combined with a low expression of sTRII in KT1 tumor tissues, these results suggest that sTRII may to some extent be able to abolish the tumor-promoting activity of TGF-beta, and imply that sTRII might have a therapeutic effect on TGF-beta-producing tumors.     

Identification of tumor-infiltrating macrophages as the killers of tumor cells after immunization in a rat model system.

Immunization can prevent tumor growth, but the effector cells directly responsible for tumor cell killing in immunized hosts remain undetermined. The present study compares tumor grafts that progress in naive syngeneic rats with the same grafts that completely regress in hosts preimmunized with an immunogenic cell variant. The progressive tumors contain only a few macrophages that remain at the periphery of the tumor without direct contact with the cancer cells. These macrophages do not kill tumor cells in vitro. In contrast, tumors grafted in immunized hosts and examined at the beginning of tumor regression show a dramatic infiltration with mature macrophages, many of them in direct contact with the cancer cells. These macrophages are strongly cytotoxic for the tumor cells in vitro. In contrast to macrophages, tumor-associated lymphocytes are not directly cytotoxic to the tumor cells, even when obtained from tumor-immune rats. However, CD4(+) and CD8(+) T cells prepared from the regressing tumors induce tumoricidal activity in splenic macrophages from normal or tumor-bearing rats and in macrophages that infiltrate progressive tumors. These results strongly suggest that the main tumoricidal effector cells in preimmunized rats are macrophages that have been activated by adjacent tumor-immune lymphocytes.     

Functional analysis of mononuclear cells infiltrating into tumors. IV. Purification and functional characterization of cytotoxic cell-generating factor

Rat cytotoxic cell-generating factor (CGF) was purified from cell-free supernatants of a T cell hybridoma (6B2-B8) that constitutively produces CGF. CGF activity was assessed by its ability to generate cytotoxic cells against 51Cr-labeled T-9 cells from spleen cells of T-9-immunized rats. The purification scheme consisted of ammonium sulfate precipitation, AcA 54 gel permeation, Mono Q anion exchange chromatography, Superose 12HR 10/30 gel permeation, SDS-PAGE with subsequent electroelution, and ProRPC HR5/10 reverse phase column chromatography. Overall, CGF was purified approximately 13,000-fold, with a maximum 2.5% recovery of activity, and the sp. act. of the purified CGF was approximately 19,000 U/mg. The purified CGF is distinct from the other lymphokines such as IL-1, IL-2, IL-3, IL-4, T cell-replacing factor/IL-5, IL-6, and IFN-gamma. It is capable of promoting the generation of cytotoxic T cells from R1-10B5 (+) spleen cells of T-9-immunized rats and also stimulates a W3/25 (+) T cell hybridoma to express the IL-2R. The CGF has an apparent m.w. of 28,000 under non-reducing and 14,000 and 16,000 under reducing conditions. 125I-labeled CGF binds to normal thymocytes as well as splenic T cells. The highest level of binding of CGF was detected on splenic T cells derived from T-9-immunized rats that were previously shown to contain CTL precursors. The binding analysis with 125I-labeled CGF demonstrated that CGF binds to a specific cell surface molecule with an approximate m.w. of 60,000 to 70,000.     

Role of tumor cell apoptosis in tumor antigen migration to the draining lymph nodes

Establishment of an immune response against cancer may depend on the capacity of dendritic cells to transfer tumor Ags into T cell-rich areas. To check this possibility, we used a colon cancer cell variant that yields tumors undergoing complete T cell-dependent rejection when injected into syngeneic rats. We previously demonstrated that immunogenicity of these tumors depended on the early apoptosis of a part of these tumor cells. In this paper we show that fluorescent tumor cell proteins are released from FITC-labeled tumor cells and undergo engulfment by tumor-infiltrating monocytes without a phenotype of mature dendritic cells or macrophages. Fluorescence-labeled mononuclear cells with a phenotype of MHC class II+ dendritic cells are also found in the T cell areas of the draining lymph nodes. Interestingly, no fluorescent cell can be found in lymph nodes after a s.c. injection of Bcl2-transfected apoptosis-resistant tumor cells that yielded progressive tumors. Proliferation of tumor-immune T lymphocytes was induced by dendritic cells isolated from the draining lymph nodes recovered after a s.c. injection of apoptosis-sensitive, but not apoptosis-resistant, tumor cells. These results show that tumor cell apoptosis releases proteins that are engulfed by inflammatory cells in the tumor, then transported to lymph node T cell areas where they can induce a specific immune response leading to tumor rejection.     

The rationale and practice of CTL therapy against cancer in the mouse and human

It is essential to investigate and elucidate the immune response especially T cell response to either syngeneic or autologous tumor for establishing a rational immunotherapy of cancer. We reported that major immune effector cells capable of inducing tumor regression are cytotoxic T lymphocytes (CTL). We found that there are at least two distinct CTL subsets directed to syngeneic tumor. One CTL subset which is selectively induced by syngeneic solid tumor is independent from CD4 positive helper T cells but requires a soluble factor (s) released from macrophage-like accessory cells designated killer T cell activating factor (KAF) in its induction and generation directed to the homologous tumor. The other CTL subset which is usually induced by syngeneic tumor of hematocytic origin is dependent on CD4 positive helper T cells in its induction. On the basis of our findings regarding the induction and activation mechanism of CTL to syngeneic tumors in the mouse, we have investigated the mechanisms of human CTL generation to autochthonous tumor in peripheral blood mononuclear cells of cancer patients. It was found that the nature of human CTL and its generation to autochthonous tumor are similar to those of murine CTL to syngeneic solid tumor. We are now establishing a rational cancer specific immunotherapy utilizing intravenous passive cell transfer of in vitro activated CTL to autochthonous tumor into an original cancer patient.     

The generation of anti-tumoral cells using dentritic cells from the peripheral bloood of patients with malignant brain tumors

 Dendritic cells (DCs) can be the principal initiators of antigen-specific immune responses. We analyzed the in vitro-responses against brain tumor cells using DCs from the peripheral blood of patients with brain tumors. Peripheral blood mononuclear cells (PBMC) were obtained from 19 patients with malignant brain tumors: 12 metastatic brain tumors of lung adenocarcinoma, 7 high-grade astrocytomas. PBMC were cultured with 100 ng/ml of GM-CSF and 10 ng/ml of IL-4 for 5–7 days in order to produce mature DCs. The autologous tumor lysate (5 mg/ml, containing 1 × 10⁶ cells) was then added to the cultured DCs. Using the DCs generated by these treatments, we assessed the changes that occurred in their immune responses against brain tumor via ⁵¹Cr-release and lymphocyte proliferation assays. We found that the matured DCs displayed the typical surface phenotype of CD3⁺, CD45⁺, CD80⁺ and CD86⁺. After the pulsation treatment with tumor lysate, DCs were found to have strong cytotoxic T lymphocyte activity, showing 42.5 ± 12.7% killing of autologous tumor cells. We also found an enhancement of allogeneic T cell proliferation after pulsing the DC with tumor lysate. These data support the efficacy of DC-based immunotherapy for patients with malignant brain tumors. Received: 2 October 2000 / Accepted: 26 April 2001     

Destructive and nondestructive patterns of immune rejection of syngeneic intraocular tumors

Two immunogenic, syngeneic murine tumors were used to analyze the immunopathological processes associated with the immune rejection of primary intraocular tumors. Intracameral inoculation of P91 mastocytoma, an immunogenic variant of P815 mastocytoma, into DBA/2 mice resulted in progressive tumor growth for several weeks before immune rejection eradicated the intraocular neoplasm. The histopathologic characteristics of the tumor rejection included: a) destruction of the vascular endothelium of the microvasculature feeding the tumor; b) ischemic bulk necrosis; c) extensive innocent bystander damage to normal ocular structures; and d) absence of direct inflammatory cell-to-tumor cell contact. Thus, the immunopathological features resembled a delayed-type hypersensitivity (DTH) lesion. A second intraocular tumor model was similarly studied. UV5C25 fibrosarcoma grew slowly in the eyes of syngeneic BALB/c hosts. In sharp contrast to P91 tumors, a mononuclear cellular infiltrate was prominent within the tumor. After 5 wk, the intraocular tumors were completely rejected without detectable damage to normal ocular structures. The rejection of UV5C25 tumors did not produce scar tissue, damage to vascular endothelium, bulk necrosis, or atrophy of the globe. Although tumor-specific cytotoxic T lymphocytes (CTL) and DTH responses were readily detected, there was no histological evidence for DTH-mediated tumor rejection. Moreover, in situ immunoperoxidase staining indicated that the majority of the infiltrating lymphocytes were CTL, based on their characteristic phenotype: Thy-1+, Lyt-2+. Furthermore, the growth of UV5C25 fibrosarcoma in athymic, natural killer (NK) cell competent BALB/c nude mice demonstrated progressive tumor growth without infiltrating host cells. Collectively, the results indicate that immunogenic intraocular tumors can undergo strikingly different patterns of immune rejection with profoundly different pathological consequences. In one case (P91), tumor rejection occurs by a process that strongly resembles DTH and produces extensive nonspecific damage to normal tissues, resulting in irrevocable loss of vision. In contrast, the second intraocular tumor undergoes an immune rejection that is characterized by precision and a notable absence of damage to normal ocular tissues. The weight of evidence presented here strongly supports the hypothesis that the latter form of tumor rejection is mediated by CTL. Thus, the immunologic pathway invoked for tumor rejection in the eye has a profound effect on the fate of this delicate organ and the preservation of vision.     

Thy and Ly markers on lymphocytes initiating tumor rejection

Nonadherent spleen cells from mice specifically immune to the syngeneic methylcholanthrene-induced fibrosarcoma H-7 caused suppression of tumor growth in footpads of nonimmune mice. The cells were Thy-1 positive, largely Ly-1 positive, but Ly-2 and Ly-3 negative, and resembled T cells causing delayed-type hypersensitivity reactions rather than cytolytic T cells. It is suggested that some tumors fail to elicit the production of rapidly cytolytic T cells but can be rejected by a mechanism akin to that of delayed-type hypersensitivity, mediated by a different class of T cells working in collaboration with macrophages.     

T9 glioma cells expressing membrane-macrophage colony stimulating factor produce CD4+ T cell-associated protective immunity against T9 intracranial gliomas and systemic immunity against different syngeneic gliomas

Cloned T9 glioma cells (T9-C2) expressing the membrane form of macrophage colony stimulating factor (mM-CSF) inoculated subcutaneously into rats do not grow and glioma-specific immunity is stimulated. Immunotherapy experiments showed that intracranial T9 tumors present for one to four days could be successfully eradicated by peripheral vaccination with T9-C2 cells. CD4+ and CD8+ T splenocytes from immunized rats, when restimulated in vitro with T9 cells, produced interleukin-2 and -4. Protective immunity against intracranial T9 gliomas could only be adoptively transferred into naive rats by the CD4+ splenocytes obtained from T9-C2 immunized rats. Rats immunized by the T9-C2 tumor cells also resisted two different syngeneic gliomas (RT2 and F98) but allowed a syngeneic NUTU-19 ovarian cancer to grow. Such cross-protective immunity against unrelated gliomas suggests that mM-CSF transfected tumor cells have immunotherapeutic potential for use as an allogeneic tumor vaccine.