Membrane-bound and soluble Fas ligands have opposite functions in photoreceptor cell death following separation from the retinal pigment epithelium.

Fas ligand (FasL) triggers apoptosis of Fas-positive cells, and previous reports described FasL-induced cell death of Fas-positive photoreceptors following a retinal detachment. However, as FasL exists in membrane-bound (mFasL) and soluble (sFasL) forms, and is expressed on resident microglia and infiltrating monocyte/macrophages, the current study examined the relative contribution of mFasL and sFasL to photoreceptor cell death after induction of experimental retinal detachment in wild-type, knockout (FasL-/-), and mFasL-only knock-in (ΔCS) mice. Retinal detachment in FasL-/- mice resulted in a significant reduction of photoreceptor cell death. In contrast, ΔCS mice displayed significantly more apoptotic photoreceptor cell death. Photoreceptor loss in ΔCS mice was inhibited by a subretinal injection of recombinant sFasL. Thus, Fas/FasL-triggered cell death accounts for a significant amount of photoreceptor cell loss following the retinal detachment. The function of FasL was dependent upon the form of FasL expressed: mFasL triggered photoreceptor cell death, whereas sFasL protected the retina, indicating that enzyme-mediated cleavage of FasL determines, in part, the extent of vision loss following the retinal detachment. Moreover, it also indicates that treatment with sFasL could significantly reduce photoreceptor cell loss in patients with retinal detachment.

Cytotoxic T cells play no essential role in acute rejection of orthotopic corneal allografts in mice.

PURPOSE: To determine whether cytotoxic T cells of the direct alloreactive type are activated and responsible for early, acute failure of orthotopic corneal allografts observed in eyes of C57BL/6 but not of BALB/c mice. METHODS: Corneas from BALB/c and BALB.B mice were placed orthotopically in eyes of C57BL/6 and beta-2 microglobulin knockout mice (deficient in CD8(+) cytotoxic T cells). Graft fates were assessed clinically, and the T lymphocytes of recipients were assayed for the capacity to lyse target cells bearing donor major (MHC) and/or minor histocompatibility (minor H) antigens (direct and indirect pathways, respectively). RESULTS. Similar to BALB/c recipients, C57BL/6 mice with rejected cornea allografts acquired donor minor H-specific T cells. Unlike BALB/c recipients, C57BL/6 mice-both rejectors and acceptors-acquired donor MHC-specific T cells. beta-2 Microglobulin knockout mice showed rejection of corneal allografts in a manner indistinguishable from C57BL/6 mice, including early, acute rejection, yet T cells from beta-2 microglobulin knockout recipients of corneal allografts displayed no cytotoxic T cells specific for either donor MHC or minor H alloantigens. CONCLUSIONS: Although C57BL/6 mice acquired donor MHC-specific cytotoxic T cells (direct alloreactive cells), neither these cells nor donor minor H-specific cytotoxic T cells (indirect alloreactive cells) play any essential role in corneal allograft rejection, including the early acute failure uniquely observed in C57BL/6 eyes.

Characterization of complex chromosomal abnormalities in uveal melanoma by fluorescence in situ hybridization, spectral karyotyping, and comparative genomic hybridization.

Several nonrandom recurrent chromosomal changes are observed in uveal melanoma. Some of these abnormalities, e.g., loss of chromosome 3, gain of the q arm of chromosome 8, and chromosome 6 abnormalities, are of prognostic value. Cytogenetic analysis and/or fluorescence in situ hybridization (FISH) are used to detect these changes. In some cases, however, detailed cytogenetic analysis is not possible due to the presence of complex abnormalities. To define more accurately these cytogenetic changes, we have applied comparative genomic hybridization (CGH) and/or spectral karyotyping (SKY) to two uveal melanoma cell lines and five primary uveal melanomas, with partially defined and/or complex abnormalities. SKY provided additional information on 34/39 partially defined aberrant chromosomes and revealed a new abnormality, a der(17)t(7;17)(?;q?), that had not been recognized by conventional cytogenetics. Additionally, using SKY, abnormalities involving chromosome 6 or 8 were found to be twice as common as observed with cytogenetic analysis. CGH was especially useful in assigning the abnormalities identified by SKY to specific chromosomal regions and, in addition, resulted in the detection of a small deletion of chromosome region 3q13 approximately 21. We conclude that SKY and CGH, as methods complementary to cytogenetic and FISH analysis, provide more complete information on the chromosomal abnormalities occurring in uveal melanoma.

Analysis of primed donor-specific T cells in recipient mice bearing orthotopic corneal allografts.

BACKGROUND: Orthotopic corneal allografts placed in normal eyes of mice are often not rejected, whereas grafts placed in high-risk (neovascularized) eyes are routinely destroyed. Because rejection of solid tissue allografts is usually mediated by donor-specific T cells, we wished to determine the extent to which donor-specific T cells become primed in mice bearing orthotopic corneal allografts in normal and "high-risk" eyes. METHODS AND RESULTS: Our data indicate corneal allografts placed in neovascularized eyes were rejected within 2 weeks, and lymph nodes draining these grafts contained primed donor-specific T cells that proliferated in vitro and displayed cytotoxic activity. By contrast, only 50% of corneal allografts placed in normal eyes experienced rejection. Lymphoid cells from all of these mice displayed donor-specific proliferative activity, irrespective of whether the graft was accepted or rejected. At no time were donor-specific cytotoxic T cells detected. Failure to detect primed cytotoxic T cells was not the result of anergy or deletion of unprimed donor-specific precursors of CTL. CONCLUSIONS: We conclude that primed donor-specific proliferative and cytotoxic T cells directed at MHC alloantigens correlate well with rejection of orthotopic corneal allografts in neovascularized high-risk eyes. However, rejection of cornea allografts in normal eyes does not correlate well with proliferative T cells, nor are donor MHC-specific cytotoxic T cells detected. The possibility is discussed that graft rejection in normal eyes is not mediated by T cells that recognize MHC alloantigens via the direct pathway, but via T cells that recognize donor alloantigens presented by recipient MHC molecules (indirect pathway).

Langerhans cells, orthotopic corneal allografts, and direct and indirect pathways of T-cell allorecognition.

PURPOSE: To determine after orthotopic corneal allografting the role of Langerhans cells in activation of T cells via the direct and indirect pathways of allorecognition and the relationship between these pathways and the rapidity of graft rejection. METHODS: Corneas from eyes of normal mice and from eyes after superficial cauterization were grafted to eyes of major histocompatibility complex (MHC) and/or minor histocompatibility (H)-disparate recipient mice. The grafts were analyzed through time for content of class 1 MHC- bearing Langerhans cells and for rejection or acceptance. Graft recipients were evaluated for acquisition of delayed hypersensitivity (DH) and cytotoxic T cells (Tc) directed at donor MHC and minor H alloantigens. RESULTS: Langerhans cells migrated more rapidly into epithelium of cauterized grafts than normal grafts. Unlike normal grafts, the vast majority of cauterized allografts were rejected within 2 weeks. Normal grafts induced neither DH nor Tc directed at donor MHC antigens, whereas cauterized grafts induced both DH and Tc specific for donor MHC. All grafts induced DH directed at donor minor H antigens, but only rejected grafts correlated with acquisition of Tc directed at donor minor H antigens. CONCLUSIONS. The rapidity of orthotopic corneal allograft rejection correlated with density of Langerhans cells within epithelium and with acquisition of donor-specific DH and Tc. Although recipient-derived Langerhans cells promoted minor H-specific, self-MHC-restricted T cells (indirect pathway) and subacute graft rejection, donor-derived Langerhans cells promoted early, acute rejection in conjunction with allogeneic MHC-specific Tc (direct pathway).

Anterior chamber-associated immune deviation-inducing cells activate T cells, and rescue them from antigen-induced apoptosis.

Immune responses to antigens injected into the anterior chamber of the eye are devoid of T helper 1 (Th1)-type responses of the delayed hypersensitivity type, which has been termed anterior chamber-associated immune deviation (ACAID). Recently, it has been found that peritoneal exudate cells (PEC) from normal mice can be made to acquire the capacity to induce ACAID in vivo when the cells are pulsed with antigen in vitro in the presence of transforming growth factor-beta2 (TGF-beta2), a major cytokine in the ocular microenvironment. We now report that when ovalbumin (OVA)-specific T cells from DO11.10 transgenic mice, or from OVA-primed normal mice, were activated in vitro by normal (untreated) PEC pulsed with OVA, the responding T cells were induced to undergo apoptosis. However, when PEC were first treated with TGF-beta2 and then used to stimulate DO11.10 T cells in the presence of OVA, T-cell proliferation occurred without evidence of increased apoptosis. The ability of TGF-beta2 to rescue responding T cells from apoptosis rested with the capacity of this cytokine to inhibit interleukin-12 (IL-12) production by PEC. Untreated PEC produced large amounts of IL-12 upon interaction with responding T cells. Under these conditions, tumour necrosis factor-alpha (TNF-alpha) production was up-regulated, and this cytokine, in turn, triggered apoptosis among T cells stimulated with OVA-pulsed PEC. From these results, we conclude that TGF-beta2-treated APC promote ACAID by rescuing antigen-activated T cells from apoptosis, and by conferring upon these cells the capacity to down-regulate delayed hypersensitivity.

Immune privilege is extended, then withdrawn, from allogeneic tumor cell grafts placed in the subretinal space.

PURPOSE: To determine whether the subretinal space can extend immune privilege to allogeneic tumor cell grafts that do not possess their own inherent immune privilege. METHODS: P815 tumor cells were injected into the anterior chamber (AC), the subretinal (SR) space, or subconjunctivally in eyes of BALB/c (allogeneic), SCID (immune incompetent), normal DBA/2 (syngeneic), or DBA/2 mice presensitized with P815 cells transfected with interleukin-12 and B7.1. Tumor growth was observed clinically and histologically for up to 50 days. BALB/c recipients were tested for suppression of DBA/2-specific delayed hypersensitivity and concomitant immunity. The SR space of tumor-containing eyes was assessed for its capacity to support ovalbumin (OVA)-specific anterior chamber associated immune deviation (ACAID). RESULTS: P815 cells injected into the SR space of presensitized and normal DBA/2 and SCID mice grew progressively, resulting eventually in recipient death. Tumor cells injected into the SR space of eyes of BALB/c mice grew progressively until day 14, followed by tumor regression resulting in phthisis bulbi (14/35) or tumor elimination (19/35) with preserved ocular anatomy by day 35. Despite elimination of tumors from the SR space, BALB/c recipients exhibited DBA/2-specific ACAID and concomitant immunity. In addition, OVA injected into the SR space of eyes from which tumor has been eliminated induced ACAID. CONCLUSIONS: Various parameters of immune privilege, originally described for the AC, are characteristic of immune privilege within the SR space. However, because P815 cells placed in the AC prove lethal for BALB/c recipients, but P815 cells placed in the SR space resolve without jeopardizing the host's life, immune privilege in the SR space can be distinguished from immune privilege in the AC, and this may have implications for grafts of retinal tissue placed within the SR space.

Detection of minor alloantigen-specific cytotoxic T cells after rejection of murine orthotopic corneal allografts: evidence that graft antigens are recognized exclusively via the "indirect pathway".

BACKGROUND: The immune mechanisms by which corneal allografts are rejected in normal ocular graft beds have not been identified. Both acceptors and rejectors of these types of grafts display donor-specific delayed hypersensitivity and in vitro proliferating primed T cells, yet neither develop conventional, donor-specific cytotoxic T cells. We wished to determine whether unconventional donor-specific cytotoxic T cells are generated in rejector mice that recognize donor minor alloantigens presented by recipient major histocompatibility complex (MHC) molecules. METHODS: BALB/c mice received orthotopic corneal allografts from C57BL/10 donors in normal eyes. At 4 weeks (when 50% of grafts can be designated as rejected), primed cytotoxic T lymphocyte (CTL) activity in draining lymph nodes and spleen was assayed on targets selected to present donor-type minor H antigens on recipient MHC molecules. Control mice received heterotopic corneal allografts and were similarly examined. RESULTS: Lymphoid organs of recipients that rejected orthotopic or heterotopic corneal allografts contained CTL that lysed targets expressing donor-type minor H antigens presented by recipient MHC molecules. By contrast, no CTL activity was detected from lymphoid cells of recipients that accepted orthotopic corneal allografts. Rejection of orthotopic corneal allografts placed into normal mouse eyes correlates directly with the generation of donor-specific CTL that recognize minor H antigens in the context of recipients MHC molecules. CONCLUSIONS: These results indicate the indirect pathway of alloantigen presentation is the only pathway operative in the process by which orthotopic corneal allografts are rejected. The roles of emigrant Langerhans cells and corneal lymphatics in the indirect pathway are discussed.

Normal cerebrospinal fluid suppresses the in vitro development of cytotoxic T cells: role of the brain microenvironment in CNS immune regulation.

The regulatory role of cerebrospinal fluid (CSF) in brain physiology is well established, while our understanding of its role in brain immunity is undefined. We demonstrate that normal rat CSF suppresses the in vitro development of mastocytoma-specific CTL activity in restimulated splenocytes from Balb/c mice, a strain unable to reject this tumor from the brain. Suppression is dependent on TGF-beta, revealed by reversal of suppression with specific neutralizing antibody. In contrast, mice which can reject this tumor from the brain, such as Balb/c mice with immunological memory to the tumor or CD-1 mice with major histo-incompatibility with the tumor, have populations of precursor CTL which are resistant to CSF-induced suppression, in the in vitro restimulation protocol. We propose that the susceptibility to CSF-induced suppression of peripherally generated immune cells that traffic to the brain plays an important role in determining whether growing tumor cells survive in the brain.

Uveal melanomas contain antigenically specific and non-specific infiltrating lymphocytes.

PURPOSE: Tumor-infiltrating lymphocytes (TIL) were recovered from a series of human choroidal melanomas and expanded in cultures containing interleukin-2 (IL-2) to determine whether TIL contained cytotoxic cells that could be activated in vitro. METHODS: TIL were recovered from six ocular melanoma patients and expanded in vitro with IL-2. Cytotoxic activity was tested in a standard 4-hr 51Cr release assay. The HLA class I phenotype of patients was determined, using peripheral blood lymphocytes and the Amos modified-cytotoxicity test. HLA class I expression on tumor cells was determined by flow cytometry. RESULTS: TIL from four patients lysed autologous ocular melanoma cells. Two of these patients possessed TIL that displayed specific cytotoxic activity and failed to lyse tumor cells from other patients (HLA-mismatched, or -matched). TIL from the remaining two patients possessed non-specific cytotoxic cells that lysed ocular melanoma cells from a variety of other patients (HLA-mismatched). TIL from patients that failed to lyse autologous tumor cells possessed cytotoxic activity for ocular melanoma cells from other HLA-mismatched patients. CONCLUSIONS: Ocular melanomas accumulate lymphocytes with the potential to kill tumor cells. Our results imply that elimination of tumor cells may be possible by activation of cytotoxic cells present within progressively growing ocular tumors.

Melanomas that develop within the eye inhibit lymphocyte proliferation.

Experiments were performed to compare the ability of ocular and skin melanoma cells to stimulate T cells. Primary melanoma cell lines were obtained from a series of patients with either eye or skin melanoma. The ability of tumor cells to stimulate T cells in the absence of exogenous growth factors was assessed in mixed-lymphocyte tumor cell cultures in which allogeneic lymphocytes were stimulated with irradiated ocular or skin melanoma cells. Expression of HLA class I and class II on tumor cells, in the presence or absence of IFN-gamma, was determined by flow cytometry. The ability of tumor cells to inhibit T-cell proliferation was determined by adding various concentrations of irradiated tumor cells to standard mixed-lymphocyte cultures. Our results indicate that primary skin melanoma cells induce vigorous proliferation of allo-antigen-specific T cells. By contrast, ocular melanoma cells failed to induce significant T-cell proliferation. The failure of ocular melanoma cells to stimulate lymphocyte proliferation was not due to low levels of either class I or class II on tumor cells since tumor cells treated with IFN-gamma expressed high levels of class I and class II but still failed to induce lymphocyte proliferation. Ocular melanoma cells inhibited lymphocyte proliferation, as shown by experiments in which a small number of tumor cells prevented proliferation of T cells in mixed-lymphocyte cultures. Inhibition of lymphocyte proliferation required cell-to-cell contact, and supernatants from tumor cell cultures did not prevent lymphocyte proliferation. Moreover, the ability of ocular melanoma cells to inhibit T-cell proliferation was lost when tumor cells migrated from the eye and formed hepatic metastases. We conclude that there is a fundamental difference in the immunogenicity of ocular and skin melanoma cells. Ocular melanomas, but not primary skin melanomas, are poorly immunogenic tumors that inhibit T-cell proliferation. Our results imply that the immunogenicity of melanoma cells is altered when they develop within the unique ocular micro-environment.

In vitro efficacy of carboplatin and hyperthermia in a murine retinoblastoma cell line.

PURPOSE: To determine the cell-killing activity of varying doses of carboplatin, graded hyperthermia, and the combination of carboplatin and hyperthermia in the treatment of a transgenic murine retinoblastoma cell line. METHODS: Replicate cell wells (more than six wells per dose point) from an established transgenic murine retinoblastoma cell line (Rb-6) were exposed to a single application of hyperthermia for 15, 30, 60, and 120 minutes at temperatures of 37 degrees C (control), 40 degrees C, and 43 degrees C. Carboplatin dose response treatment was studied at doses of 2000, 1000, 500, 400, 300, 200, 100, and 50 ng per well. Combined treatment studies used these carboplatin dosages with each of the graded hyperthermia exposure temperatures at each exposure time. At 24 hours, all wells were pulsed with 3H-thymidine for 24 hours, washed three times, harvested, and counted. Raw counts (3H-thymidine) were fitted to a linear regression model to calculate the lethal dose for 50% (LD50) of cells. RESULTS: The LD50 for carboplatin exposure at 37 degrees C occurred at 542 ng. The LD50 for hyperthermia at 40 degrees C occurred at 90 minutes and at 43 degrees C it occurred at 62 minutes. Combined hyperthermia and carboplatin exposure yielded a synergistic interaction with an LD50 of 327 ng at 43 degrees C for 30 minutes. Determination of a thermal enhancement ratio yielded an enhancement range of 1.1 to 25.8. CONCLUSIONS: The synergistic cytocidal interaction of heat and carboplatin in a transgenic murine retinoblastoma cell line has been established in this study. The increased thermal enhancement ratio documents the potential utility of combined treatment applications in reducing treatment levels of single-modality therapy, potentially allowing for a decrease in treatment-related morbidity.

Gene transfer of the CD80 costimulatory molecule into ocular melanoma cells using a novel episomal vector.

PURPOSE: The CD80 (B7.1) molecule, which is a necessary costimulatory signal for T-cell activation and proliferation, is a powerful inducer of antitumor immunity. In this study, primary human ocular melanoma cells were transfected with a novel vector (B45-Neo episomal vector) containing the complementary DNA (cDNA) for human CD80 to determine if this vector system is useful for stimulating CD8+ T cells. METHODS: Ocular melanoma cells were transfected with the B45-Neo episomal vector containing the cDNA for human CD80 and were positively selected in medium containing geneticin. The transcription of plasmid cDNA, plasmid copy number, and cell surface expression were determined on transfected tumor cell lines, and cloned tumor cells were obtained by limiting dilution techniques. The stability of CD80 expressed on tumor cells was determined after prolonged culture without geneticin and on irradiated cells. Autologous lymphocytes were restimulated with CD80+ tumor cells in the presence of recombinant interleukin-2 to determine whether CD8+ T cells were stimulated. RESULTS: CD80 was expressed on tumor cells transfected with the B45-Neo vector containing the cDNA for CD80. The level of CD80 expressed on different transfected tumor cell lines was heterogeneous and dependent on the plasmid copy number. High CD80 expression was observed on cloned tumor cells that possessed more than 520 plasmids per cell; intermediate levels were observed on tumor cells with approximately 240 to 520 plasmids. CD80+ ocular melanoma cells maintained a stable CD80 expression even after prolonged culture without geneticin, and on irradiated tumor cells. CD80 expressed on tumor cells was biologically functional and stimulated autologous CD8+ cells. CONCLUSIONS: The B45-Neo episomal vector induces stable expression of the CD80 costimulatory molecule on ocular melanoma cells. Our results indicate that this vector is suitable for experiments designed to genetically engineer ocular melanoma cells to stimulate CD8+ T cells.

Blood-borne signals that induce anterior chamber-associated immune deviation after intracameral injection of antigen.

PURPOSE: Anterior chamber-associated immune deviation (ACAID) is elicited by an antigen-specific signal that escapes the antigen-containing eye and travels through the blood to the spleen. Two types of ACAID-inducing signals have been described: those associated with blood-borne monocytes, and a soluble factor found in serum. The authors sought to understand the basis for the existence of two distinct types of ACAID-inducing signals. METHODS: Different kinds of antigens (soluble, cell associated, particulate) were injected into the anterior chamber (AC) of normal, presensitized, and immunodeficient mice. In addition, peritoneal exudate cells were pulsed in vitro with different kinds of antigen in the presence of transforming growth factor beta and then evaluated for the ability to induce ACAID in naive (nonsensitized) as well as T- and B-cell-deficient recipients. RESULTS: Among antigens injected into the AC, inert particulate antigens could not induce ACAID, but soluble and cell-associated (minor histocompatibility) antigens generated cell-associated ACAID-inducing signals. In contrast, antigens injected into the AC of presensitized mice generated ACAID-inducing signals that were soluble and located in the plasma fraction of blood. All ACAID-inducing signals created in vitro with soluble, particulate, or cell-associated antigens induced ACAID in vivo. CONCLUSIONS: Cell-associated ACAID-inducing signals are generated in naive mice regardless of the kind of antigen, and these signals arise from mobile intraocular antigen-presenting cells. However, when antigen is injected into the AC of presensitized mice, a soluble signal emerges, perhaps derived from T cells that enter the antigen-containing eye. Together, these signals dictate that subsequent exposures to ocular antigen will not evoke immunogenic inflammation.

Expression of MAGE genes in ocular melanoma during progression from primary to metastatic disease.

Primary melanomas that form within the eye have a unique pattern of disease progression as compared with melanomas that form within the skin. A high percentage of patients (approximately 50%) develop metastatic tumors that occur predominately in the liver. An unusual characteristic of ocular melanomas is the prolonged disease-free interval that extends for many years between the development of primary and metastatic tumors. It is estimated that the shortest interval between dissemination of tumor cells from the eye and the appearance of clinically detectable metastases is 6 years. A recent report indicated that fresh uveal melanoma tissue and metastatic tumor biopsies failed to express melanoma antigen gene (MAGE)-1, MAGE-2, or MAGE-3. In the present study, we examined the expression of MAGE genes on fresh and cultured tumor cells obtained from an ocular melanoma patient during different stages of progressive disease. MAGE gene expression was determined by reverse transcription-polymerase chain reaction using MAGE-1, MAGE-2 and MAGE-3 specific primers. Our results demonstrate that primary ocular tumor tissue and cultured tumor cells both express significant levels of MAGE-1, 2, and 3 at the time of enucleation. A high percentage of tumor cells within the primary tumor appear to express MAGE as demonstrated by consistent MAGE expression in 16 tumor cell clones. Metastatic liver tumors that developed 3 years after enucleation and 18 years after the initial formation of the primary tumor also expressed high levels of MAGE-1, -2, and -3. MAGE was expressed on fresh tumor tissue from a single biopsy and cultured tumor cells obtained from three of four different metastatic tumor nodules. When the MAGE-negative metastatic tumor cells were treated with the demethylating agent 5-Aza-2-Deoxycytidine (5-Aza-dC), transcription of MAGE-1 was restored, indicating the MAGE genes were not deleted. Our results demonstrate that in some patients, MAGE genes are expressed on primary and metastatic ocular melanomas.