Tumor-Treating Fields Induce RAW264.7 Macrophage Activation Via NK-κB/MAPK Signaling Pathways.

OBJECTIVE: Tumor-treating fields are currently used to successfully treat various cancers; however, the specific pathways associated with its efficacy remain unknown in the immune responses. Here, we evaluated tumor-treating fields-mediated initiation of the macrophage-specific immune response. MATERIALS AND METHODS: We subjected RAW 264.7 mouse macrophages to clinically relevant levels of tumor-treating fields (0.9 V/cm, 150 kHz) and evaluated alterations in cytokine expression and release, as well as cell viability. Additionally, we investigated the status of immunomodulatory pathways to determine their roles in tumor-treating fields-mediated immune activation. RESULTS AND DISCUSSION: Our results indicated that tumor-treating fields treatment at 0.9 V/cm decreased cell viability and increased cytokine messenger RNA/protein levels, as well as levels of nitric oxide and reactive oxygen species, relative to controls. The levels of tumor necrosis factor α, interleukin 1ß, and interleukin 6 were markedly increased in tumor-treating fields-treated RAW 264.7 cells cocultured with 4T1 murine mammary carcinoma cells compared with those in 4T1 or RAW 264.7 cells with or without tumor-treating fields treatment. Moreover, the viability of 4T1 cells treated with the conditioned medium of tumor-treating fields-stimulated RAW 264.7 cells decreased, indicating that macrophage activation by tumor-treating fields effectively killed the tumor cells. Moreover, tumor-treating fields treatment activated the nuclear factor κB and mitogen-activated protein kinase pathways involved in immunomodulatory signaling. CONCLUSION: These results provide critical insights into the mechanisms through which tumor-treating fields affect macrophage-specific immune responses and the efficacy of this method for cancer treatment.

Ultraviolet B-induced loss of HLA class II antigen expression on lymphocytes is dose, time, and locus dependent.

Ultraviolet B (UVB) irradiation interferes with the afferent and efferent loops of the immune response. One mechanism that has been suggested is the decline of class II histocompatibility antigen expression on the cell surface. However, data in the literature are controversial. In the present study, we examined the effect of UVB light (peak emission at 302 nm), at doses of 0.05-300 mJ/cm2, on class II antigen expression on normal peripheral blood lymphocytes enriched for non-T cells. Monoclonal antibodies directed at nonpolymorphic determinants on HLA-DR, -DQ, and -DP were used to label cells before and at time intervals up to 72 h after irradiation. Immediately following UVB exposure, fluorescence intensity for all three antigens was equal to or slightly increased above control values. Subsequently, there was a decline in antigen expression that was UVB dose dependent and varied for HLA-DR, -DP and -DQ. Although there was only a very moderate loss of surface labeling for HLA-DR up to 72 h, there was a steep decline for HLA-DQ and -DP. There was no significant decline in class II antigen expression on cells exposed to 2000 or 4000 cGy of gamma irradiation. Also, there was no effect of either gamma or UVB irradiation on class I antigen expression. These data explain some of the discrepancies in previous reports on the effects of UVB on class II antigens; they show not only a dose effect but also an effect of time after exposure and, most importantly, the class II antigen under study.

Experimental photoimmunology: immunologic ramifications of UV-induced carcinogenesis.

The use of animal model systems to investigate the sequence of events which lead to the induction and progression of skin tumors following chronic ultraviolet light (UVL) exposure has clearly shown that the direct mutagenic effects of UVL is only one of the components involved in this process. In spite of the fact that overt carcinogenesis is only one of the many effects produced by UV light, most hypotheses as to the mechanism by which UVL can cause the mutations necessary to achieve the transformed phenotype have focused on the direct effects of UVL on DNA and the generation of carcinogenic compounds. Investigations during the last 5 yr, however, have clearly demonstrated that immunologic factors are also critically important in the pathogenesis of UV-induced skin cancers. A complete understanding of UV-carcinogenesis must therefore consider the mechanisms which allow the transformed cell to evade immunologic rejection by the host in addition to those aspects which deal with conversion of a normal cell to a cancer cell. It is the object of this review to provide both a historical account of the work which established the immunologic consequences of chronic UVL exposure and the results of recent experiments designed to investigate the kinetics and mechanisms by which UVL affects the immunologic apparatus. In addition, a hypothetical model is presented to explain the sequence of events which ultimately lead to the emergence of the suppressor T-cells which regulate antitumor immune responses.

Influence of ultraviolet radiation treatment on the survival of heterotopic skin grafts in the mouse.

Isolated mouse tail skin was UV-irradiated in vitro at a dose of 40 mJ/cm2 from both sides to remove the Ia immunogenicity. Immediately after irradiation the skin was transplanted onto the flank of allogeneic mice. When there was a total H-2 difference between donor and recipient, the UV-irradiated skin did not show a prolonged survival compared to control grafts. In the case of an I-region difference only, i.e., B10.AQR grafts onto B10.T (6R) recipients, a significant prolongation of the survival time was observed, whereas 50% of the UV-treated grafts were not rejected at all.

Effect of glucocorticoids and gamma radiation on epidermal Langerhans cells.

The effect of 750 rads of gamma radiation on the rate of return of epidermal Langerhans cells (LC) following suppressive doses of topical glucorticoids was studied in guinea pigs. Gamma radiation alone had no effect on the LC as assessed by staining for cell membrane ATPase activity and Ia antigen. It did, however, delay the expected return of Ia but not ATPase surface markers on the LC after perturbation with glucocorticoids. The delayed return of surface Ia antigen is possibly related to a radiation-induced defect in the production of a required lymphokine and/or in intracellular Ia transport. Although our data do not rule out a cytolytic effect of steroids on the LC, they do strongly suggest that, at least in part, glucocorticoids act on the LC by altering cell surface characteristics.

Effects of irradiation on the expression of major histocompatibility complex class I antigen and adhesion costimulation molecules ICAM-1 in human cervical cancer.

PURPOSE: We initiated studies to analyze the effects of high doses of gamma irradiation on the surface antigen expression of MHC Class I, Class II, and ICAM-1 on human cervical carcinoma cell lines. METHODS AND MATERIALS: The expression of surface antigens (MHC Class I, Class II, and ICAM-1) was evaluated by FACS analysis on two cervical cell lines at different time points, following their exposure to high doses of gamma irradiation (i.e., 25.00, 50.00, and 100.00 Gy). RESULTS: The CaSki and SiHa cervical cancer cells we analyzed in this study expressed variable levels of MHC Class I and ICAM-1 antigens, while Class II surface antigens were not detectable. Whereas irradiation doses of 25.00 Gy were not sufficient to totally block cell replication in both cell lines, exposure to 50.00 or 100.00 Gy was able to completely inhibit cell replication. Range doses from 25.00 to 100.00 Gy significantly and consistently increased the expression of all surface antigens present on the cells prior to irradiation but were unable to induce neoexpression of antigens previously not expressed by these cells (i.e., MHC Class II). Importantly, such upregulation was shown to be dose dependent, with higher radiation doses associated with increased antigen expression. Moreover, when the kinetic of this upregulation was studied after 2 and 6 days after irradiation, it was shown to be persistent and lasted until all the cells died. CONCLUSIONS: These findings may partially explain the increased immunogenicity of tumor cells following irradiation and may suggest enhanced immune recognition in tumor tissue in patients receiving radiation therapy.

Fetal pancreas transplantation in miniature swine. V. The functional and immunodulatory effects of ultraviolet light on fetal pig islets.

We have used the pig as a large animal model for studies of fetal pancreas transplantation. Fetal pig pancreas (FPP) has also been proposed as a potential source of endocrine cells for the treatment of diabetes mellitus. Among the approaches to prevent rejection, the irradiation of donor islets with ultraviolet B light has been used for its immunomodulating properties. Our goal was to study in vitro the effects of UV-B irradiation of FPP on the function and immunogenicity of the tissue. FPP were collagenase-digested and cultured for 1-29 days prior to UV-B irradiation. Static incubation tests were used to measure glucose-theophylline stimulated insulin release. Data obtained at 300 J/m2 revealed no impairment of insulin release (78% to 129% of controls, P = ns). At 500 J/m2, a significant reduction of glucose-theophylline stimulated insulin release was observed with 50-60-day-old FPP (35% to 66% of controls, P < 0.05), but not with 80-day-old FPP (93% of controls, P = ns). At both doses, prolonged observation in culture did not show any alteration of the growth and proliferation of islet cell clusters. UV-irradiated (300 J/m2) adult and fetal pig islet allografts released C-peptide and survived > 200 days. The immunogenicity of irradiated tissues was determined in vitro with allogeneic mixed islet-lymphocyte cultures (MILC). Proliferative responses of allogeneic lymphocytes to UV-irradiated FPP were very significantly decreased by 52-91% at both 300 and 500 J/m2 doses. This effect was observed from 1 to 10 days following UV irradiation and was not modulated by exposure of the tissues to gamma-interferon. We conclude that UVB-irradiation of FPP at a dose of 300 J/m2 does not alter its endocrine function and growth and is effective in reducing tissue immunogenicity. This treatment may be a useful approach for fetal islet transplantation in large animal models.

CD4+ and CD8+ T cells each can utilize a perforin-dependent pathway to mediate lethal graft-versus-host disease in major histocompatibility complex-disparate recipients.

Perforin-deficient (-/-) mice were used as T-cell donors for infusion into irradiated major histocompatibility complex (MHC)-disparate recipients to investigate the requirement for perforin-mediated cytolysis during graft-versus-host disease (GVHD) generation. Administration of 5x10(6) C57BL/6 (H2b) perforin -/- splenocytes was significantly less effective in inducing GVHD lethality when given to MHC class I + II disparate B10.BR (H2k) recipients, as compared with wild-type (+/+) controls. Perforin expression by donor T cells was not required for GVHD induction because recipients given fivefold higher numbers of perforin -/- donor splenocytes uniformly succumbed to lethal GVHD. Because both CD4+ and CD8+ donor T cells are required for optimal GVHD lethality in this strain combination, to discern the relative contribution of perforin-mediated cytolysis by CD4+ and CD8+ T cells, additional studies were performed. For these latter studies, we used a sensitive assay involving the infusion of highly purified CD4+ or CD8+ T cells into sublethally irradiated MHC class II or I disparate recipients, respectively. As compared with recipients of perforin +/+ T cells, recipients of either CD4+ or CD8+ perforin -/- T-cell subsets had a significant reduction in GVHD-mediated lethality at T-cell doses that were uniformly lethal. T-cell dose titration studies established that GVHD lethality in recipients of perforin -/- CD4+ or CD8+ T cells was reduced by approximately threefold. These data are the first to indicate that approaches to limit perforin-mediated cytolysis should be similarly effective in situations in which CD4+ or CD8+ T cells dominate the GVHD response.

Comparison of altered expression of histocompatibility antigens with altered immune function in murine spleen cells treated with ultraviolet radiation and/or TPA.

Previous studies in our laboratory demonstrated that several treatments that inhibited the ability of cells to stimulate the mixed lymphocyte reaction (MLR) also blocked the shedding of histocompatibility antigens and Ia antigens from murine spleen cells. In the present studies, one of these treatments, ultraviolet radiation (UV), was shown to cause an initial loss in the density of H-2K, IA, and IE antigens prior to the block in shedding observed after culture of these cells. Further analysis revealed that the UV-induced loss of antigens could be prevented by the presence of colchicine during irradiation. Biosynthetic analyses revealed the IA antigen synthesis was also inhibited in the UV-irradiated cells. Examination of the effects of a second agent, 12-0-tetradecanoylphorbol-13-acetate (TPA) on the turnover of histocompatibility antigens revealed that the biosynthesis and shedding of these antigens were accelerated by this agent. However, addition of TPA to UV-irradiated cells did not result in a reversal of the UV-induced block in biosynthesis of IA antigens. Results of immune function assays correlated with the biochemical studies: UV-irradiation inhibited the generation of the MLR, but TPA enhanced this reaction, and addition of TPA to mixed lymphocyte cultures with UV-irradiated stimulators did not reverse the UV-induced inhibition. These results suggest that, although the turnover of histocompatibility antigens may be affected by TPA and UV in an antagonistic fashion, additional factors other than the expression of histocompatibility antigens are operating in the inhibition of stimulation of an MLR by UV radiation or its enhancement by TPA.

Reduced MHC class II antigen expression on rat heart and kidney cells after in vitro exposure to longwave ultraviolet irradiation.

The pretreatment of both kidney and heart donor with the photosensitizer 8-methoxy-psoralen followed by in vitro longwave ultraviolet graft irradiation (PUVA therapy) significantly prolonged graft survival times in rats even across a strong major histocompatibility complex (MHC) barrier. Immunohistological studies using monoclonal antibodies (Mab) to rat MHC class I and II antigens showed a significant reduction of class II antigen expression after PUVA therapy in both Sprague-Dawley rat kidneys and hearts. Using MHC class I Mab there were no differences in distribution pattern in untreated as well as in PUVA treated organs. Thus, our results represent the first in vivo evidence that photochemotherapy-induced graft survival time prolongation is closely connected with its ability to reduce MHC class II antigen expression.

Dissection of the D-region of the human major histocompatibility complex by means of induced mutations in a lymphoblastoid cell line.

This paper describes part of a mutagenic dissection of the human D-region. Twenty-six human lymphoblastoid cell mutants that had lost expressions of HLA-DR were created with a two-step procedure: (i) A mutant from which one entire haplotype had been physically deleted by gamma-rays was isolated by means of immunoselection against cells expressing a specific HLA-B antigen. (ii) This heterozygous deletion mutant was irradiated with gamma-rays or treated with ICR 191, a frameshift mutagen, and mutants that no longer expressed the remaining DR1 antigen were selected with a monoclonal antibody directed against a monomorphic DR determinant. Monoclonal antibody GENOX 3.53 was used to show that four of the gamma-ray induced DR-null mutants did not express the cis-linked MB1/MT1 locus. Since MB1/MT1 was still expressed in the other 16 gamm-ray induced and 6 ICR 191-induced DR-null mutants, the separate loss of expression of MB1/MT1 and DR1 is strong evidence that the DR1 and MB1/MT1 alloantigens are under separate genetic control in the cells we used. Since DR-null mutants bound SB2-specific monoclonal antibody ILR1, whether or not they expressed MB1/MT1, the results mean that gamma-rays resolved the genetic determinants for DR1, MB1/MT1, and SB2. Additional complexity of determinants encoded by D-region genes is indicated by the following results. The amount of MB1/MT1 antigen that was detected with ELISA tests for binding of GENOX 3.53 antibody to cells varied inversely with the number of expressed copies of DR or of a locus near DR. This could result from an increased amount of MB1/MT1 antigen or from increased binding accessibility of GENOZ 3.53-reactive antigen in DR-null mutants. Monoclonal antibodies CC 11.23 and CC 6.4 displayed patterns of binding to parental and diverse mutant cells that differed from that of GENOX 3.53, suggesting the existence of at least one additional D-region antigen that is neither SB, DR, nor MB/MT.

A deletion mutant defines DQ beta variants with DR4 positive DQw3 positive haplotypes.

We describe the production of an HLA deletion mutation by radiation mutagenesis of a DR4- and DQw3-homozygous, Dw4- and Dw14-heterozygous cell line designed to analyze polymorphisms associated with DR4 and DQw3. Southern blot analysis confirms a deletion of class I and class II genes on one haplotype. Variation in DQ beta alleles associated with DQw3 was previously described by characteristic RFLP patterns for a DQ beta bene. One pattern, which correlated precisely with A-10-83 monoclonal antibody reactivity (TA10), defined an allele which we call DQ"3.1". The mutant cell line has lost the polymorphic bands on Southern blots corresponding to the DQ"3.1" allele, while the intact Dw14 haplotype retains the alternate allele at DQ beta which is DQw-3 positive. TA10-negative. These data demonstrate the segregation of two DQw3 positive DQ beta allelic variants, both associated with DR4, which can be distinguished on the basis of both RFLP and monoclonal antibody reactivity.

The influence of ultraviolet irradiation on the blood transfusion effect.

Reports of improved survival of allografts in recipients of donor-specific blood prompted an attempt to determine the relationship of the antigenic composition of the blood product transfused to the development of immunologic unresponsiveness in rats. Cardiac allografts were transplanted from Fischer, Brown-Norway (BN), and Lewis (L) X BN (LBN) f1 hybrids to recipients treated with three weekly transfusions of 1 ml of donor-specific whole blood, erythrocytes, or ultraviolet-irradiated whole blood. Despite moderate improvement in survival with whole blood alone in the LBN- greater than L group (11.6 +/- 1.0 days), it was only with the ultraviolet-irradiated whole blood that marked prolongation was seen in all three strain combinations: Fischer- greater than L: 25.5 +/- 5.2, LBN- greater than L: 17.3 +/- 1.2, and BN- greater than L: 11.1 +/- 0.4 days compared with respective controls: 10.3 +/- 1.2, 7.3 +/- 0.5, and 7.4 +/- 0.6 days. Unlike reports for renal allografts, erythrocyte suspensions provided minimal protection for the cardiac allografts (14.2 +/- 0.8, 9.0 +/- 1.1, and 11.0 +/- 0.4 days, respectively), and adjunctive treatment with antilymphocyte serum had a similar small effect (16.3 +/- 1.4, 13.4 +/- 1.9, and 8.3 +/- 0.8 days, respectively). The elimination or inactivation of functional class 2 major histocompatibility complex antigens from the blood used for donor-specific blood transfusion may be an effective means of prolonging allograft survivals over those seen with whole blood alone; however, the degree of resultant unresponsiveness is still clearly influenced by dosage schedule, the organ transplanted, histocompatibility barrier, and adjunctive immunosuppression.

The effect of ultraviolet radiation on humoral immune responses to T-dependent antigens.

Multiple exposures to broadband ultraviolet (uv) radiation resulted in alterations in the ability of BALB/c mice to produce antibodies to the T-dependent antigens ovalbumin and sheep erythrocytes following intraperitoneal, intradermal, and intravenous inoculation. A number of differences were observed in the influence of uv radiation on the specific cells producing antibody, as opposed to pure serological analyses. While both enhancement and reduction of the humoral response occurred, the experiments have suggested that the alterations in antibody responses following uv irradiation appear to be dependent on the nature of the antigen, the route of antigen administration, and the sometimes transient nature of the responses. The intradermal route of antigen inoculation was under the greatest influence of uv radiation.

Ultraviolet light-induced suppression of antigen presentation.

Ultraviolet (UV) light irradiation of animals results in the development of specific T suppressor cells that inhibit antitumor immune responses. It is thought that suppression may arise as a consequence of altered antigen presentation by UV-irradiated epidermal cells. This hypothesis is based on evidence demonstrating that specific lymphoid tissues from UV-irradiated hosts exhibit impaired antigen-presenting function and that animals cannot be contact sensitized when antigens are applied to a UV-irradiated skin site. Langerhans cells of the skin are likely candidates as targets of UV-induced defects in antigen presentation as they bear Fc and C3b receptors, express Ia antigens, are of bone marrow origin, and are capable of presenting antigen in vitro. We speculate on the possible clinical usefulness of UV-induced tolerance to specific antigens such as those encountered in monoclonal antibody therapy and tissue transplantation.

Dissimilar effects of ultraviolet light on HLA-D and HLA-DR antigens.

Since HLA-DR antigens are closely related to HLA-D antigens, we studied the effect of low-dose ultraviolet irradiation on the ability of human lymphocytes to induce a proliferative response in mixed lymphocyte culture (HLA-D) and on the serologic reactivity of B lymphocyte HLA-DR antigens. We found (1) no alteration in the serologic definition of DR antigens despite the abrogation of the induction of proliferative responses in mixed lymphocyte culture, (2) no evidence that ultraviolet light (UVL) stimulated suppressor cells contributed to the loss of allostimulation, and (3) no evidence that the abrogation of the induction of proliferative responses could be modified by increasing the number of UVL exposed lymphocytes in mixed lymphocyte culture. These findings suggest that HLA-D and HLA-DR antigens are different molecularly, but are also consistent with the concept that certain active metabolic processes are required of stimulator cells for allostimulation and that these processes are UVL sensitive.