Adaptive immune defects and delayed rejection of allogeneic tumor cells in beige mice

The effect of the beige (bg) mutation on adaptive allogeneic tumor rejection was examined by monitoring tumor cell survival in vivo using [131I]iododeoxyuridine-prelabeled cells. Accelerated elimination of allogeneic tumor cells normally begins 8 days after ip injection and is due to active immune responses. Two independent mutations to beige on two different inbred backgrounds (C57BL/6J bgJ and DBA/2JCo bg8J) were tested, and bg/bg mice showed a 1-day delay in immune elimination of allogeneic cells. This delayed rejection was not due to a defect in clearing label from dead cells, nor to an inability to effect antibody-induced killing in vivo. Both humoral and cell-mediated responses against the allogeneic tumor cells were significantly lower in bg/bg than in +/bg mice.     

Modulation of tissue-specific immune response to cardiac myosin can prolong survival of allogeneic heart transplants

The role of immune response to tissue-specific Ags in transplant rejection is poorly defined. We have previously reported that transplantation of cardiac allografts triggers a CD4(+) Th1 cell response to cardiac myosin (CM), a major contractile protein of the heart, and that pretransplant activation of proinflammatory CM-specific T cells accelerates rejection. In this study, we show that administration of CM together with IFA (CM/IFA) can prevent acute rejection of an allogeneic heart transplant. Prolongation of cardiac graft survival is associated with activation of CM- and allo-specific T cells secreting type 2 cytokines (IL-4, IL-5) and reduction of the frequency of proinflammatory IFN-gamma-secreting (type 1) alloreactive T cells. Blocking of IL-4 cytokine with Abs abrogates the prolongation. CM/IFA treatment prevents acute rejection of MHC class I-mismatched, but not fully mismatched grafts. However, if donor heart is devoid of MHC class II expression, CM-IFA administration delays rejection of fully allogeneic cardiac transplants. This finding suggests that the effect of CM modulation depends on the type (direct vs indirect) and strength of recipient's CD4(+) T cell alloresponse. Our results underscore the important role of host immunity to tissue-specific Ags in the rejection of an allograft. This study demonstrates that modulation of the immune response to a tissue-specific Ag can significantly prolong cardiac allograft survival, an observation that may have important implications for the development of novel selective immune therapies in transplantation.     

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.     

Therapeutic immune response induced by electrofusion of dendritic and tumor cells

To elicit a therapeutic antitumor immune response, dendritic cells (DCs) have been employed as a cellular adjuvant. Among various DC-based approaches, fusion of DCs and tumor cells potentially confers not only DC functionality, but also a continuous source of unaltered tumor antigens. We have recently demonstrated successful generation of fusion hybrids by a large-scale electrofusion technique. The immunogenicity and therapeutic potential of fusion hybrids were further analyzed in a model system of a murine melanoma cell line expressing beta-galactosidase (beta-gal) as a surrogate tumor antigen. A single vaccination with fusion hybrids plus IL-12 induced a therapeutic immune response against 3-day established pulmonary metastases. This immunotherapy was beta-gal specific and involved both CD4 and CD8 T cells. In vitro, fusion hybrids stimulated specific IFN-gamma secretion from both CD4 and CD8 immune T cells. They also nonspecifically induced IL-10 secretion from CD4 but not CD8 T cells. Compared to other DC loadings, our results demonstrate the superior immunogenicity of fusion. The current technique of electrofusion is adequately developed for clinical use in cancer immunotherapy.     

Immune responses to weakly immunogenic virally induced tumors. III. Genetically unrestricted cytolysis of allogeneic tumor target cells.

Splenocytes from A mice injected with YAC-1 or RBL5 could generate, after in vitro culture with or without stimulation, a genetically nonrestricted cytotoxic response against the allogenic tumor RBL5. YAC-1 tumor is an in vitro carried tumor induced in A mice (H-2a) by Moloney virus. RBL5 tumor is a Rauscher virus-induced tumor of C57BL/6 mice (H-2b). These tumors cross-react serologically. The effector cells that were generated after the in vitro cultivation recognized tumor-associated antigens on the target cells. H-2 alloantigens were not recognized by the effector cells. The effector cells that killed RBL5 tumor in a genetically nonrestricted manner were identified as T cells. The in vivo carried tumor YAC, in contrast to the in vitro carried tumor YAC-1, could not induce anti-RBL5 reactive cells in A mice. Instead, YAC tumor induced suppressor cells in A mice, which could abrogate the anti-RBL5 cytotoxic response of RBL5-primed splenocytes, but not that of YAC-1 primed splenocytes.     

Intracamerally induced concomitant immunity: mice harboring progressively growing intraocular tumors are immune to spontaneous metastases and secondary tumor challenge

Intracameral inoculation of allogeneic P815 mastocytoma cells (DBA/2) into BALB/c mice resulted in progressively growing intraocular tumors. Intraocular tumor cells disseminated rapidly to the spleen and cervical lymph nodes, yet extraocular nests of tumor cells never developed into fulminant tumors. Further experiments showed that tumor cells were continuously seeded from the primary intraocular tumor and were rapidly cleared from extraocular sites. Hosts harboring intraocular P815 mastocytomas rejected tumorigenic doses of P815 cells inoculated subcutaneously or even into the contralateral anterior chamber. This systemic tumor immunity was found to be radiosensitive and T cell dependent. Spleen cells from animals with progressively growing intraocular tumors protected recipient mice challenged with intracamerally inoculated tumor cells and thus suggests that a cell-mediated mechanism is the underlying basis for this form of tumor immunity. The data indicate that mice harboring progressively growing intraocular tumors develop a potent state of "concomitant immunity," that prevents the development of metastases, yet is ineffective in controlling the primary tumor.     

Early antigen elimination by cytotoxic T cells results in diminished cellular immune responses to allogeneic tumor

Previous reports have suggested that repeated alloantigenic challenge increases humoral responses to alloantigens, but may cause decreasing cellular responses. We stimulated BALB/c (H-2^d) mice with intraperitoneal EL-4 tumor (H-2^b) and serially assessed cytotoxic responses in spleen and peritoneal lymphocytes using ⁵¹Cr-labeled EL-4 target cells. We observed that cytotoxicity generated in the spleen of nonimmune BALB/c mice was much greater than that in immunized mice; similar peak responses were generated in peritoneal lymphocytes in normal and immunized hosts. Complement-mediated cytotoxicity was not necessary for diminished splenic responses in hyperimmune hosts, for the same phenomenon was seen in the Hzl anti-BL/6 system which is free of humoral responses. Irradiation (2000 rad) of the EL-4 tumor challenge prevented tumor cell proliferation and markedly reduced splenic responses in nonimmune mice. We suggest that cytotoxic cells suppressed further generation of cyto-toxicity; by effecting an early elimination of tumor inoculum, tumor proliferation was abrogated and dose of cellular antigen was, consequently, markedly reduced.     

Autoimmunity and tumor immunity induced by immune responses to mutations in self

Little is known about the consequences of immune recognition of mutated gene products, despite their potential relevance to autoimmunity and tumor immunity. To identify mutations that induce immunity, here we have developed a systematic approach in which combinatorial DNA libraries encoding large numbers of random mutations in two syngeneic tyrosinase-related proteins are used to immunize black mice. We show that the libraries of mutated DNA induce autoimmune hypopigmentation and tumor immunity through cross-recognition of nonmutated gene products. Truncations are present in all immunogenic clones and are sufficient to elicit immunity to self, triggering recognition of normally silent epitopes. Immunity is further enhanced by specific amino acid substitutions that promote T helper cell responses. Thus, presentation of a vast repertoire of antigen variants to the immune system can enhance the generation of adaptive immune responses to self.     

Macrophage tumor necrosis factor-alpha release is induced by contact with some tumors

The purpose of this study was to determine whether macrophages were directly stimulated by tumor cells to release TNF-alpha. We found that several murine and human tumor cell lines and crude cell membrane vesicles prepared from these tumor cells stimulated pyran copolymer-elicited murine peritoneal macrophages (PEM) to release as much as 362 +/- 69 (mean +/- SE) units of TNF activity per 10(6) PEM in vitro. By contrast, several nontransformed cells, including Con A-stimulated splenic leukocytes and CTLL cloned T lymphocytes, failed to stimulate PEM to release TNF. Antibody and complement-mediated depletion of macrophages abrogated the release of TNF; whereas depletion of NK cells and T lymphocytes did not affect tumor-stimulated TNF release, suggesting that tumor cells directly stimulated PEM to release TNF. Tumor-stimulated TNF release was rapid, peaking in 2 to 3 h with subsequent loss of TNF activity from the medium. In the absence of tumor, PEM contained detectable levels of TNF mRNA, but did not release functionally active TNF. The addition of P815 tumor cell membrane vesicles increased both TNF mRNA levels, peaking at 1 to 2 h, and release of high levels of TNF activity. Confounding effects of endotoxin were excluded by the resistance of tumor-stimulated TNF release to neutralization by polymixin B, and by the equivalent responsiveness of PEM from endotoxin-resistant (C3H/HeJ) and endotoxin-sensitive (C3H/HeN) mice to stimulation by tumor cells. Factors which stimulated PEM to release TNF could be extracted from tumor cell membrane, with 77% of the macrophage-stimulating activity recoverable in aqueous phase. In conclusion, we have demonstrated that some tumor cell lines express specific characteristics which can be recognized by macrophages and which stimulate macrophages to release TNF.     

Induction of anterior chamber-associated immune deviation (ACAID) by allogeneic intraocular tumors does not require splenic metastases

Anterior chamber-associated immune deviation (ACAID), induced by intracameral injection of allogeneic tumor cells, is expressed in three distinct ways: 1) progressive growth of intraocular tumors, 2) specific suppression of systemic allograft immunity, and 3) transient growth of allogeneic tumors injected subcutaneously. Induction of ACAID requires that alloantigen presentation occur via the anterior chamber; injection by other routes failed to elicit this phenomenon. Antigenic material must remain in the anatomically intact eye for at least 10 days; removal of the injected dye before this time prevented the establishment of ACAID. The similar temporal requirement for an anatomically intact spleen confirms the validity of the concept of a camero-splenic axis for processing of intracamerally injected alloantigens. Deployment of an alternate model of ACAID, using LP/J mice injected intracamerally with B16F10 melanoma, showed the antigen-specific inductive signal for ACAID (transmitted via the camero-splenic axis) was not in the form of viable alloantigen-bearing tumor cells that metastasize to the spleen. B16F10 melanoma cells were never found in the spleens or any other extraocular sites after intracameral injection, despite the fact these mice manifested ACAID and harbored enormous ocular tumors. The data emphasize that intraocular processing of antigens is a unique and dynamic phenomenon with significant, systemic immunologic consequences.     

Studies on the inhibition of rejection of Trichostrongylus colubriformis larvae from immune sheep

A total of 8 drugs were examined for their ability to suppress the rejection of Trichostrongylus colubriformis infective larvae (L₃) from immune sheep. Specific antagonists of leukotrienes (plroxicam), prostaglandin (indomethacin) and the immunosuppressant cyclosporin A were given orally, while injectable preparations of dexamethasone, chlorpheniramine, BN 52051, WEB 2086 (anti-platelet-activating factor) and theophylline were administered as directed. The drugs were given for 5 days prior to and 4 days after challenge with 20,000 L₃, when worm counts were done. Corticosteroids inhibited rejection by around 70% in two experiments, and none of the remaining compounds were effective. In the third study, six of the drugs were given to susceptible sheep and did not affect the establishment of a primary infection with T. colubriformis.     

Killer cells induced by stimulation with allogeneic tumor cells and subsequent culture with recombinant interleukin-2

Summary Peripheral blood lymphocytes were cultured for 5 days with allogeneic tumor cells (allogeneic mixed lymphocyte/tumor cell culture), and subsequently cultured with recombinant interleukin-2 for 12 days. These cultured cells were found to be cytotoxic to autologous tumor cells. Results of two-color analysis using monoclonal antibodies to cell markers showed that more than 80% of their cultured cells were CD3⁺ cells, and CD4⁺ cells showed a higher distribution than CD8⁺ cells. However, CD8⁺ cells had a much higher killing activity with autologous tumor than did CD4⁺ cells, when estimated by an elimination study using monoclonal antibodies to T cell phenotypes and complement. The cold-target inhibition test showed that the cytotoxicity of these cells for autologous tumor cells was inhibited by unlabeled autologous tumor cells but not by unlabeled stimulator cells. Furthermore, about 40% of the cytotoxicity was suppressed by blocking of HLA class I antigen with a monoclonal antibody on autologous tumor cells. Thus, cytotoxic activity of lymphocytes to autologous tumor restricted by target cell HLA class I antigen is possibly induced by allogeneic tumor-stimulation.     

HLA-DR restriction in suppression of hematopoiesis by T cells from allogeneic bone marrow transplants

Three allogeneic bone marrow transplantation patients who exhibited a suppressive subset of T cells for in vitro hematopoiesis have been investigated to determine whether this T cell suppressive effect was genetically restricted. In the three cases, T cells separated by sheep red cell rosetting inhibited blood colony-forming units granulocyte-monocyte (CFU-GM) and burst-forming unit erythroid (BFU-E) growth from the patients and from the bone marrow donors who were HLA identical, but not from randomly chosen unrelated subjects. In one case, cocultures were performed between the patient T cells and the T-depleted cells from eight siblings and from the mother. A marked inhibition (30 to 60%) of CFU-GM and BFU-E growth was found in the relatives who shared a haplo-identical HLA-DR 5. The same degree of suppression was found with respect to whether the siblings were homozygous or heterozygous for the HLA-DR 5 antigen, and whether or not they shared common class I antigens. This inhibition was totally abolished when a monoclonal antibody against HLA-DR was added, whereas a monoclonal antibody against class I histocompatibility antigen had no effect. To additionally demonstrate that this inhibition was mediated by a single HLA-DR haplotype, T cells from the patient were co-cultured with cells from three normal unrelated individuals, one with a phenotypically identical DR and two with only one haploidentical DR. Inhibition was similarly found in the subject exhibiting complete DR identity, and the subject with only the DR 5 haploidentical phenotype. These results demonstrate that a unique subset of T cells present in allogeneic bone marrow transplants specifically suppress differentiation of hemopoietic progenitors that bear one phenotypically haplo-identical HLA-DR antigen.     

Carbon monoxide generated by heme oxygenase-1 suppresses the rejection of mouse-to-rat cardiac transplants

Mouse-to-rat cardiac transplants survive long term after transient complement depletion by cobra venom factor and T cell immunosuppression by cyclosporin A. Expression of heme oxygenase-1 (HO-1) by the graft vasculature is critical to achieve graft survival. In the present study, we asked whether this protective effect was attributable to the generation of one of the catabolic products of HO-1, carbon monoxide (CO). Our present data suggests that this is the case. Under the same immunosuppressive regimen that allows mouse-to-rat cardiac transplants to survive long term (i.e., cobra venom factor plus cyclosporin A), inhibition of HO-1 activity by tin protoporphyrin, caused graft rejection in 3--7 days. Rejection was associated with widespread platelet sequestration, thrombosis of coronary arterioles, myocardial infarction, and apoptosis of endothelial cells as well as cardiac myocytes. Under inhibition of HO-1 activity by tin protoporphyrin, exogenous CO suppressed graft rejection and restored long-term graft survival. This effect of CO was associated with inhibition of platelet aggregation, thrombosis, myocardial infarction, and apoptosis. We also found that expression of HO-1 by endothelial cells in vitro inhibits platelet aggregation and protects endothelial cells from apoptosis. Both these actions of HO-1 are mediated through the generation of CO. These data suggests that HO-1 suppresses the rejection of mouse-to-rat cardiac transplants through a mechanism that involves the generation of CO. Presumably CO suppresses graft rejection by inhibiting platelet aggregation that facilitates vascular thrombosis and myocardial infarction. Additional mechanisms by which CO overcomes graft rejection may involve its ability to suppress endothelial cell apoptosis.     

Broadening of epitope recognition during immune rejection of ErbB-2-positive tumor prevents growth of ErbB-2-negative tumor

Tumor heterogeneity is a limiting factor in Ag-specific vaccination. Ag-negative variants may arise after tumor cells bearing the immunizing Ags are destroyed. In situ priming to tumor-associated epitopes distinct from and not cross-reactive with the immunizing Ags may be crucial to the ultimate success of cancer vaccination. Immunization of BALB/c mice with DNA encoding wild-type human ErbB-2 (Her-2/neu, E2) or cytoplasmic ErbB-2 (cytE2), activated primarily CD4 or CD8 T cells, respectively, and both vaccines protected against ErbB-2-positive D2F2/E2 tumors. In > or =50% of protected mice, a second challenge of ErbB-2-negative D2F2 tumor cells was rejected. Recognition of non-ErbB-2, tumor-associated Ags was demonstrated by immune cell proliferation upon stimulation with irradiated D2F2 cells. This broadening of epitope recognition was abolished if CD4 T cells were depleted before D2F2/E2 tumor challenge, demonstrating their critical role in Ag priming. Similarly, mice that rejected D2F2/cytE2 tumor cells, which express only MHC I epitopes of ErbB-2, were not protected from a second challenge with D2F2 cells. Depletion of CD8 T cells abolished protection against D2F2, indicating the activation of D2F2-specific CTL. Therefore, long term protection may be achieved by immunization with dominant Ag(s), followed by a general enhancement of CD4 T cell activity to promote priming to multiple tumor-associated Ags.     

Structural studies of migration inhibitory activity induced during murine tumor allograft rejection.

Lymphocytes from mice rejecting a tumor allograft produce a soluble activity that inhibits the migration of murine macrophages. The present studies show that this activity is stable with 56 °C heating for 30 min, is inactivated by trypsin, has a molecular size of about 45,000, and has an isoelectric point of 5.0–5.5. In addition, it does not inhibit the migration of guinea pig macrophages. These results indicate that this lymphokine is similar in its physicochemical properties to murine, guinea pig and human migration inhibitory factors (MIF) studied previously, but they also suggest that there may be structural differences in the functionally active portions of MIF from different species.