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Cancer patients with low or absent pre-existing anti-tumour immunity ("cold" tumours) respond poorly to treatment with immune checkpoint inhibitors (ICPI). In order to render these patients susceptible to ICPI, initiation of de novo tumour-targeted immune responses is required. This involves triggering of inflammatory signalling, innate immune activation including recruitment and stimulation of dendritic cells (DCs), and ultimately priming of tumour-specific T cells. The ability of tumour localised therapies to trigger these pathways and act as in situ tumour vaccines is being increasingly explored, with the aspiration of developing combination strategies with ICPI that could generate long-lasting responses. In this effort, it is crucial to consider how therapy-induced changes in the tumour microenvironment (TME) act both as immune stimulants but also, in some cases, exacerbate immune resistance mechanisms. Increasingly refined immune monitoring in pre-clinical studies and analysis of on-treatment biopsies from clinical trials have provided insight into therapy-induced biomarkers of response, as well as actionable targets for optimal synergy between localised therapies and ICB. Here, we review studies on the immunomodulatory effects of novel and experimental localised therapies, as well as the re-evaluation of established therapies, such as radiotherapy, as immune adjuvants with a focus on ICPI combinations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Appleton, Hassan, Chan Wah Hak, Sivamanoharan, Wilkins, Samson, Ono, Harrington, Melcher and Wennerberg.)

CD19 chimeric antigen receptors (CARs) have demonstrated great efficacy against a range of B cell malignancies. However, antigen escape and, more generally, heterogeneous antigen expression pose a challenge to applying CAR therapy to a wide range of cancers. We find that low-dose radiation sensitizes tumor cells to immune rejection by locally activated CAR T cells. In a model of pancreatic adenocarcinoma heterogeneously expressing sialyl Lewis-A (sLeA), we show that not only sLeA + but also sLeA - tumor cells exposed to low-dose radiation become susceptible to CAR therapy, reducing antigen-negative tumor relapse. RNA sequencing analysis of low-dose radiation-exposed tumors reveals the transcriptional signature of cells highly sensitive to TRAIL-mediated death. We find that sLeA-targeted CAR T cells produce TRAIL upon engaging sLeA + tumor cells, and eliminate sLeA - tumor cells previously exposed to systemic or local low-dose radiation in a TRAIL-dependent manner. These findings enhance the prospects for successfully applying CAR therapy to heterogeneous solid tumors. Local radiation is integral to many tumors' standard of care and can be easily implemented as a CAR conditioning regimen., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Radiofrequency ablation (RFA) can not only effectively kill hepatocellular carcinoma (HCC) tumour cells but also release tumour antigens that can provoke an immune response. However, there is no consensus regarding which antigens could constitutively be generated after RFA and could potentiate the immune response. The aim of this study was to identify these immune-potentiating antigens., Methods: We performed two-dimensional electrophoresis (2-DE) and MALDI-TOF-MS/MS analyses on serum obtained before and after RFA from 5 HCC patients. Further validation for selected proteins was performed utilizing ELISA analysis on another 52 HCC patients. Disease-free survival (DFS) analysis according to the differential expression of the interested protein before and after RFA was performed., Results: Twelve decreased and 6 increased proteins after RFA were identified by MS. Three proteins, including clusterin, Ficolin-3, and serum retinol binding protein-4, were further verified by ELISA on the 52 HCC patients. Only Ficolin-3 proved to be significantly changed after RFA. The 52 patients were divided into two groups according to the expression of Ficolin-3 before and after RFA. The 1-, 2- and 3-year DFS rates were 59.1%, 31.8%, and 22.7%, respectively, for patients in the low Ficolin-3 group (22 patients) and 73.3%, 60.0%, and 50.0%, respectively, for patients in the high Ficolin-3 group (30 patients) (P = 0.038)., Conclusions: In conclusion, Ficolin-3 was overexpressed in the serum of most HCC patients after RFA. Ficolin-3 might be a biomarker for RFA treatment efficacy and a potential target for HCC immunotherapy.

Background: We previously demonstrated that tumor irradiation potentiates cancer vaccines using genetic modification of tumor cells in murine tumor models. To investigate whether tumor irradiation augments the immune response to MUC1 tumor antigen, we have tested the efficacy of tumor irradiation combined with an MVA-MUC1-IL2 cancer vaccine (Transgene TG4010) for murine renal adenocarcinoma (Renca) cells transfected with MUC1., Methods: Established subcutaneous Renca-MUC1 tumors were treated with 8 Gy radiation on day 11 and peritumoral injections of MVA-MUC1-IL2 vector on day 12 and 17, or using a reverse sequence of vaccine followed by radiation. Growth delays were monitored by tumor measurements and histological responses were evaluated by immunohistochemistry. Specific immunity was assessed by challenge with Renca-MUC1 cells. Generation of tumor-specific T cells was detected by IFN-γ production from splenocytes stimulated in vitro with tumor lysates using ELISPOT assays., Results: Tumor growth delays observed by tumor irradiation combined with MVA-MUC1-IL-2 vaccine were significantly more prolonged than those observed by vaccine, radiation, or radiation with MVA empty vector. The sequence of cancer vaccine followed by radiation two days later resulted in 55-58% complete responders and 60% mouse long-term survival. This sequence was more effective than that of radiation followed by vaccine leading to 24-30% complete responders and 30% mouse survival. Responding mice were immune to challenge with Renca-MUC1 cells, indicating the induction of specific tumor immunity. Histology studies of regressing tumors at 1 week after therapy, revealed extensive tumor destruction and a heavy infiltration of CD45 + leukocytes including F4/80 + macrophages, CD8 + cytotoxic T cells and CD4 + helper T cells. The generation of tumor-specific T cells by combined therapy was confirmed by IFN-γ secretion in tumor-stimulated splenocytes. An abscopal effect was measured by rejection of an untreated tumor on the contralateral flank to the tumor treated with radiation and vaccine., Conclusions: These findings suggest that cancer vaccine given prior to local tumor irradiation augments an immune response targeted at tumor antigens that results in specific anti-tumor immunity. These findings support further exploration of the combination of radiotherapy with cancer vaccines for the treatment of cancer.

Radiation-inducible neo-antigens are proteins expressed on cancer cell surface after exposure to ionizing radiation (IR). These neo-antigens provide opportunities to specifically target cancers while sparing normal tissues. Tax interacting protein-1 (TIP-1) is induced by irradiation and is translocated to the surface of cancer cells. We have developed a monoclonal antibody, 2C6F3, against TIP-1.Epitope mapping revealed that 2C6F3 binds to the QPVTAVVQRV epitope of the TIP-1 protein. 2C6F3 binds to the surface of lung cancer (A549, LLC) and glioma (D54, GL261) cell lines. 2C6F3 binds specifically to TIP-1 and ELISA analysis showed that unconjugated 2C6F3 efficiently blocked binding of radiolabeled 2C6F3 to purified TIP-1 protein. To study in vivo tumor binding, we injected near infrared (NIR) fluorochrome-conjugated 2C6F3 via tail vein in mice bearing subcutaneous LLC and GL261 heterotopic tumors. The NIR images indicated that 2C6F3 bound specifically to irradiated LLC and GL261 tumors, with little or no binding in un-irradiated tumors.We also determined the specificity of 2C6F3 to bind tumors in vivo using SPECT/CT imaging. 2C6F3 was conjugated with diethylene triamine penta acetic acid (DTPA) chelator and radiolabeled with 111Indium (111In). SPECT/CT imaging revealed that 111In-2C6F3 bound more to the irradiated LLC tumors compared to un-irradiated tumors. Furthermore, injection of DTPA-2C6F3 labeled with the therapeutic radioisotope, 90Y, (90Y-DTPA-2C6F3) significantly delayed LLC tumor growth. 2C6F3 mediated antibody dependent cell-mediated cytotoxicity (ADCC) and antibody dependent cell-mediated phagocytosis (ADCP) in vitro.In conclusion, the monoclonal antibody 2C6F3 binds specifically to TIP-1 on cancer and radio-immunoconjugated 2C6F3 improves tumor control., Competing Interests: Dennis Hallahan licensed 2C6F3 antibody to Medical Guidance Systems for the development of the humanized antibody for planned clinical trials.

Background and Objective: The mRNA levels of 59 genes, detected by cDNA microarray, were up-regulated in the radioresistant human esophageal cacinoma cell line TE13R120 as compared with its parental cell line TE13 before and after radiation, and the expression of NRAGE gene showed a gradually up-regulating tendency. This study aimed to further detect the differences of NRAGE gene and protein expression and apoptosis between TE13R120 and TE13 cells, and to investigate the relationship between the NRAGE and the radioresistance of TE13R120 cells and its mechanism., Methods: The two cell lines were irradiated by ⁶⁰Co γ-ray at different conditions. Reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry were used to detect the expression of NRAGE. Flow cytometry (FCM) was used to detect the cell apoptosis before and after irradiation., Results: The mRNA level of NRAGE was higher in TE13R120 cells than in TE13 cells before and after irradiation (before radiation: 0.25 ± 0.03 vs. 0.49 ± 0.03; 4 Gy 4 h: 0.31 ± 0.03 vs. 0.53 ± 0.02; 4 Gy 16 h: 0.32 ± 0.04 vs. 0.59 ± 0.04; 4 Gy 24 h: 0.36 ± 0.05 vs. 0.72 ± 0.04; 2 Gy 12 h: 0.32 ± 0.02 vs. 0.64 ± 0.04; 6 Gy 12 h: 0.36 ± 0.02 vs. 0.79 ± 0.05; 10 Gy 12 h: 0.46 ± 0.04 vs. 0.85 ± 0.01; P < 0.01), and the mRNA level of NRAGE was increased gradually with the increase of radiation dose and time in the two cell lines (P < 0.05 and P < 0.01). Western blot results showed no difference of NRAGE protein level in cytoplasm between TE13R120 cells and TE13 cells before and after irradiation, but its level in nuclei was higher in TE13R120 cells than in TE13 cells at different radiation time and dosages. Immunocytochemistry showed similar results as Western blot. FCM showed no significant difference in apoptosis rate between TE13R120 and TE13 cells before and after radiation., Conclusion: NRAGE may play an important role in the radiation responses of the two cell lines, and may participate in the formation of radioresistance of TE13R120 cells by changing its subcellular localization, but its relationship with cell apoptosis has not been confirmed.

Background: Human epidermal growth factor receptor-2 (HER-2) and tumor-associated glycoprotein 72 (TAG-72) have proven to be excellent molecular targets for cancer imaging and therapy. Trastuzumab, which binds to HER-2, is effective in the treatment of disseminated intraperitoneal disease when labeled with (213)Bi or (212)Pb. (213)Bi-humanized CC49 monoclonal antibody (HuCC49DeltaCH2), which binds to TAG-72, inhibits the growth of subcutaneous xenografts. A next logical step to improve therapeutic benefit would be to target tumors with both molecules simultaneously., Methods: Athymic mice bearing intraperitoneal human colon carcinoma xenografts were treated with a combination of trastuzumab and HuCC49DeltaCH2 labeled with (213)Bi administered through an intraperitoneal route. The sequence of administration also was examined., Results: Before combining the 2 monoclonal antibodies, the effective doses of (213)Bi-CC49DeltaCH2 and (213)Bi-trastuzumab for the treatment of peritoneal disease were determined to be 500 muCi for each labeled antibody. Treatment with (213)Bi-HuCC49DeltaCH2 resulted in a median survival of 45 days and was comparable to the median survival achieved with (213)Bi-trastuzumab. Each combination provided greater therapeutic efficacy than either of the agents given alone. However, the greatest therapeutic benefit was achieved when (213)Bi-HuCC49DeltaCH2 and (213)Bi-trastuzumab were coinjected, and a median survival of 147 days was obtained., Conclusions: Dual targeting of 2 distinct molecules in tumors such as TAG-72 and HER-2 with alpha-particle radiation resulted in an enhanced, additive, therapeutic benefit. The authors also observed that this radioimmunotherapeutic strategy was well tolerated., (Published 2010 by the American Cancer Society.)

The immune response to cancer is critically determined by the way in which tumor cells die. As necrotic, stress-associated death can be associated with activation of antitumor immunity, whole tumor cell antigen loading strategies for dendritic cell (DC)-based vaccination have commonly used freeze-thaw "necrotic" lysates as an immunogenic source of tumor-associated antigens. In this study, the effect of such lysates on the ability of DCs to mature in response to well-established maturation stimuli was examined, and methods to enhance lysate-induced DC activation explored. Freeze-thaw lysates were prepared from murine tumor cell lines and their effects on bone marrow-derived DC maturation and function examined. Unmodified freeze-thaw tumor cell lysates inhibited the toll-like receptor-induced maturation and function of bone marrow-derived DCs, preventing up-regulation of CD40, CD86, and major histocompatibility complex class II, and reducing secretion of inflammatory cytokines [interleukin (IL)-12 p70, tumor necrosis factor-alpha, and IL-6]. Although IL-10 secretion was increased by lysate-pulsed DCs, this was not responsible for the observed suppression of IL-12. Although activation of the nuclear factor-kappaB pathway remained intact, the kinase activity of phosphorylated p38 mitogen-activated protein kinase was inhibited in lysate-pulsed DCs. Lysate-induced DC suppression was partially reversed in vitro by induction of tumor cell stress before lysis, and only DCs loaded with stressed lysates afforded protection against tumor challenge in vivo. These data suggest that ex vivo freeze-thaw of tumor cells does not effectively mimic in vivo immunogenic necrosis, and advocates careful characterization and optimization of tumor cell-derived vaccine sources for cancer immunotherapy.

The unique immunoglobulin (Ig) idiotype on the surface of each B-cell lymphoma represents an ideal tumor-specific antigen for use as a therapeutic vaccine. We have used an Escherichia coli-based, cell-free protein-expression system to produce a vaccine within hours of cloning the Ig genes from a B-cell tumor. We demonstrated that a fusion protein consisting of an idiotypic single chain Fv antibody fragment (scFv) linked to a cytokine (GM-CSF) or to an immunostimulatory peptide was an effective lymphoma vaccine. These vaccines elicited humoral immune responses against the native Ig protein displayed on the surface of a tumor and protected mice against tumor challenge with efficacy equal to that of the conventional Ig produced in a mammalian cell and chemically coupled to keyhole limpet hemocyanin. The cell-free E coli system offers a platform for rapidly generating individualized vaccines, thereby allowing much more efficient application in the clinic.

Immunotherapy of cancer is attractive because of its potential for specificity and limited side effects. The efficacy of this approach may be improved by providing adjuvant signals and an inflammatory environment for immune cell activation. We evaluated antitumor immune responses in mice after treatment of OVA-expressing B16-F0 tumors with single (15 Gy) or fractionated (5 x 3 Gy) doses of localized ionizing radiation. Irradiated mice had cells with greater capability to present tumor Ags and specific T cells that secreted IFN-gamma upon peptide stimulation within tumor-draining lymph nodes than nonirradiated mice. Immune activation in tumor-draining lymph nodes correlated with an increase in the number of CD45(+) cells infiltrating single dose irradiated tumors compared with nonirradiated mice. Similarly, irradiated mice had increased numbers of tumor-infiltrating lymphocytes that secreted IFN-gamma and lysed tumor cell targets. Peptide-specific IFN-gamma responses were directed against both the class I and class II MHC-restricted OVA peptides OVA(257-264) and OVA(323-339), respectively, as well as the endogenous class I MHC-restricted B16 tumor peptide tyrosinase-related protein 2(180-188). Adoptive transfer studies indicated that the increased numbers of tumor Ag-specific immune cells within irradiated tumors were most likely due to enhanced trafficking of these cells to the tumor site. Together these results suggest that localized radiation can increase both the generation of antitumor immune effector cells and their trafficking to the tumor site.

Previously, we reported that 100 Gy X-ray irradiation followed by 24 hr incubation up-regulates CD80 expression in murine B lymphoma cells, A20-2J. In the present study, we analysed the underlying mechanisms of such up-regulation using A20-HL cells derived from A20-2J cells. Irradiation of A20-HL cells with 100 Gy enhanced CD80 expression. Incubation of untreated A20-HL cells with those 100 Gy irradiated induced up-regulation of CD80 expression. Irradiation of A20-HL cells also up-regulated the expression of tumour necrosis factor-alpha (TNF-alpha) and CD40 ligand (CD40L), and the amount of immunoprecipitable TNF-alpha and CD40L in cell lysates. The addition of anti-TNF-alpha or anti-CD40L monoclonal antibody (mAb) to the incubation of irradiated A20-HL cells partially inhibited up-regulation of CD80 expression, and the addition of both antibodies together almost completely inhibited the up-regulation, suggesting that irradiation up-regulated the CD80 expression through the induction of TNF-alpha and CD40L expression. Irradiation also increased the accumulation of CD80, TNF-alpha and CD40L mRNA. n-tosyl-l-phenylalanine chloromethyl ketone (TPCK), a nuclear factor (NF)-kappaB inhibitor, markedly decreased irradiation-induced accumulation of CD80 mRNA and CD80 expression. FK506, a calcineurin inhibitor, and nifedipine, a calcium channel inhibitor, inhibited not only the expression of TNF-alpha and CD40L, but also the up-regulation of CD80 on irradiated A20-HL cells. These results strongly suggested that irradiation induced TNF-alpha and CD40L expression, which then up-regulated CD80 mRNA and CD80 expression through activation of NF-kappaB transcription factor in A20-HL cells.

Multiple clinically applicable methods have been used to induce dendritic cells (DCs) to express whole cell tumor antigens, including pulsing DCs with tumor lysate, and mixing DCs with apoptotic or live tumor cells. Herein we demonstrate, using two different tumor systems, that these methods are equipotent inducers of systemic antitumor immunity. Furthermore, tumor lysate pulsed DC vaccines generate more potent antitumor immunity than immunization with irradiated tumor cells plus the classic adjuvant, Corynebacterium parvum.

Object: Glioblastoma multiforme (GBM) is a malignant tumor of the central nervous system that directly suppresses immunological defenses in vitro and in vivo. The authors used the peripheral delivery of continuously infused granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence of irradiated tumor antigens as a tumor-specific stimulant to dendritic cells to initiate an immune response to GBM in rats., Methods: The 9L gliosarcoma tumors were established in the flanks of syngeneic Fischer 344 rats. Osmotic minipumps implanted in the animals' contralateral flanks continuously delivered recombinant GM-CSF (0, 0.1, 1, or 10 ng/day) for 28 days. Irradiated gliosarcoma cells were intermittently injected at the site of the GM-CSF infusion. Animals in the saline control group (0 ng/day GM-CSF) died on Day 59 with average tumor volumes greater than 30,000 mm3. This control group was significantly different from the GM-CSF-treated animals, which all survived with average tumor volumes that peaked on Day 23 and later regressed completely. Tumor growth as well as peak tumor volumes (5833+/-2284 mm3, 3294+/-1632 mm3, and 1979+/-1142 mm3 for 0.1, 1, and 10 ng/day GM-CSF, respectively) in the different treatment groups reflected a significant dose-response relationship with the GM-CSF concentrations. All animals treated with GM-CSF and irradiated cells were resistant to additional challenges of peripheral and intracerebral gliosarcoma, even when they were inoculated 8 months after initial immunotherapy. The colocalization of GM-CSF and inactivated tumor antigens was required to stimulate immunoprotection. To test the efficacy of a peripherally administered immunological therapy on intracerebral brain tumors the authors transplanted 10(6) gliosarcoma cells into the striatum of treated and control animals. Subcutaneous pumps that released GM-CSF (10 ng/day) and irradiated gliosarcoma cells were placed in the treated animals. The control animals all died within 31 days after intracerebral tumor implantation. In contrast, 40% of the animals receiving GM-CSF-irradiated cell vaccinations survived beyond 300 days. These long-term survivors showed no evidence of gliosarcoma at the injection site on evaluation by magnetic resonance imaging., Conclusions: These results suggest that the continuous localized delivery of subcutaneous GM-CSF in conjunction with inactivated tumor antigens can initiate a systemic response that leads to the regression of distant peripheral and intracerebral tumors. The success of this treatment illustrates the feasibility of tumor-specific peripheral immunological stimulation after tumor resection to prevent the recurrence of malignant brain tumors.

Purpose: Studies were designed to analyse the effects of high doses of gamma-irradiation on the expression of a tumour rejection antigen (heat shock protein gp96) in human cervical carcinoma cell lines., Materials and Methods: The expression of heat shock protein gp96 was evaluated at the transcriptional (Northern blot) and post-transcriptional levels (Western blot) in two human cervical carcinoma cell lines following exposure to high doses of gamma-irradiation., Results: Doses of gamma-irradiation ranging from 25 to 100 Gy significantly and consistently increased the expression of heat shock protein gp96 on CaSki and HT-3 cervical cancer cells. The increase in the amount of protein was due to transcriptional up-regulation of this gene. Radiation doses unable to inhibit completely cell replication in the totality of tumour cells (i.e. 25 Gy), as well as higher (fully lethal) doses of irradiation (i.e. 50 to 100 Gy), were shown to up-regulate significantly the expression of heat shock protein gp96 mRNA in a dose-dependent manner., Conclusions: Recently, gp96 molecules have been implicated in the presentation of endogenous and viral antigens. A number of key elements in this pathway, including major histocompatibility complex (MHC) class I molecules as well as adhesion/co-stimulation molecules such as ICAM-1, are known to be sensitive to irradiation effects. The results show that radiation can also increase the expression of other immunologically important cell molecules such as a tumour rejection antigen (heat shock protein gp96) in human cervical cancer. Such findings may partially explain the increased immunogenicity of tumour cells following irradiation and further support a role for local radiation therapy as a powerful biologic response modifier.

Purpose: To investigate the changes in antigenic expression of intercellular adhesion molecule-1 (ICAM-1) caused by ionizing radiation of cultured human adenocarcinoma cells., Methods and Materials: Human colonic BM314 and gastric MKN45 adenocarcinoma cells were irradiated to investigate the expression of ICAM-1 on the cell membrane and in the supernatant. In addition, the ICAM-1 gene expression (mRNA) was analyzed using a ICAM-1 cDNA as a probe., Results: The expression of ICAM-1 on the membrane was found to increase by irradiation. This effect was also observed in the supernatant. In addition, the irradiated cell population showed slight, but clear increases in ICAM-1 mRNA expression., Conclusions: These results show that the enhancement of expression of ICAM-1 by radiation takes place at the ICAM-1 gene expression (mRNA) level. The results suggest that the low dose radiation may be useful for accumulating LFA-1 positive cytotoxic T lymphocytes (CTL) at the local tumor tissue, by which tumor cells may be attacked.

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.

A causative role of UV light in the development of melanocytic neoplasms has often been suggested. In order to investigate the short-term effects of UV light on melanocytic nevi, the morphological and immunohistochemical changes in nevi after a single UV irradiation are studied in 12 nevi from 10 patients and compared with the nonirradiated part of the same nevus. After irradiation more melanocytes above the dermal-epidermal junction are observed in seven nevi, simulating a melanoma in situ in three nevi. Moreover, a marked increase in the expression of HMB-45 is found after irradiation in all investigated nevi, indicating an activation of the melanocytes and active melanosome formation. The metabolic activity correlates with the ultrastructural findings, which show a large cytoplasm, hypertrophic Golgi apparatus, abundant mitochondria, and an increased number of melanosomes of different stages. One week after irradiation, no increase in the proliferative activity of the melanocytes is found. The morphological and immunohistochemical changes after one low dose of UV irradiation should be considered in the differential diagnosis of pigmented skin lesions. The UV-irradiated nevus should be added to the list of so-called simulators of malignant melanoma.

Objectives: To determine if tissue expression of prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), and a prostate-associated monoclonal antibody (TURP-27) is retained after irradiation therapy and to compare these results with serum levels., Methods: Immunohistochemical tests were performed on prostatic tissue obtained by needle biopsy or transurethral resection prior to and following definitive irradiation therapy for clinically localized prostatic carcinoma. PSA, PAP, and TURP-27 were studied. Results were compared with serum PSA and PAP values., Results: All 20 preirradiation specimens stained positively for PSA and PAP; 19 of 20 stained for TURP-27. All 5 of the initial post-treatment biopsy specimens that showed recurrent tumor stained for all 3 markers. In 2 cases, staining for the 3 markers was greatly diminished. Only 8 of 15 post-treatment biopsy-negative specimens stained for all 3 markers. Six of 15 demonstrated loss of tissue expression for all 3 antigens. One specimen stained for PAP and TURP-27 but failed to stain for PSA. Serum PSA levels paralleled tissue expression in recurrent tumor specimens. However, 3 of the post-treatment biopsy-positive cases with PAP expressing tissue had normal serum PAP levels., Conclusions: No cases of recurrent tumor with marker-negative tissue were identified. However, benign epithelial prostate cells appear to sustain sufficient damage from irradiation to lose the capacity to produce certain proteins. Diminished contribution of benign glands to circulating PSA, in addition to decreased expression in malignant tissues, may explain the lower than anticipated serum PSA levels in patients who progress after irradiation therapy.

Bladder washings can be used for simultaneous karyometry and proliferation analysis using MiB-1 as a proliferation marker. We analyzed 42 problem cases with a discordance between cytologic and karyometric classification (QUANTICYT), in which the karyometric classification was based on a combination of a nuclear shape parameter and DNA (2cDI). Moreover, 25 concordant cases were analyzed: 5 normal samples, 6 low-grade tumors and 7 high-grade tumors. All normal samples and all low-grade tumors had labelling indices below 10%, and all high-grade tumors over 10%. For the discordant low-grade tumors, the QUANTICYT classification correlated better with the MiB-1 labelling than the cytologic diagnosis. There was a clear correlation between 2cDI and MiB-1 labelling index. Slightly elevated MiB-1 labelling indices might have some prognostic value.

In diagnostic tumor pathology, immunohistochemical detection of proliferating cell populations is increasingly used. With the event of microwave-antigen retrieval it has become possible to detect proliferating cells in cervical smears staining positive for MiB-1. We report that with the PAPNET system, using neural network computing, it is possible to collect from the smears the images with epithelial fragments containing positive nuclei. We used this system for quantification of staining results. Cases with carcinoma in situ contained many epithelial fragments having a large number of positive-staining nuclei and with labelling indices of 60 +/- 16. Dysplasias were often completely devoid of cells with positive nuclei. In addition, we could not detect differences between progressive and regressive dysplasias. Automatic prescreening of immunostained smears using PAPNET is useful when it is desired to quantify staining results.

For 105 patients with locoregional carcinoma of the prostate, prostate specific antigen (PSA) levels were evaluated before, during and after external beam radiotherapy. The median follow-up is 17 months. In 51 patients (48.5%) initial PSA levels exceeded the maximum normal value of 20 ng/ml. Nine patients kept non-declining high levels just after radiotherapy. Only one of these is free of disease. Assuming PSA levels decrease exponentially during radiotherapy, a mean half-life of 62 days (median 54, SD 26 days) was calculated. Three out of five patients with a PSA half-life of more than 88 days relapsed as compared to a 8% (3/37) relapse rate in patients with a "normal" half-life. Prolonged PSA half-life suggests residual disease. PSA levels are expected to further decrease after radiation. Six months after irradiation persistent high PSA levels were found in 14/51 (27.5%) patients. Only four of them had no evidence of manifest disease. Important negative prognostic factors for disease control in our series were non-declining high levels of PSA, a PSA serum half-life exceeding 88 days and persistence of elevated PSA values longer than six months after treatment. In our opinion, PSA is a valuable marker in the follow-up of prostate cancer patients during and after radiotherapy.

A method is described which leads to the production of large amounts of ascites containing antitumor antibody in small numbers of mice. The antibody was then used to identify and characterize tumor-associated antigens on an ultraviolet light-induced murine skin fibrosarcoma. The antibody showed specific complement-dependent cytotoxicity to the homologous tumor and to an allogeneic tumor line which displayed a glycoprotein viral determinant with a molecular weight of 70,000 on its surface. Absorption of the immune ascites with other tumor cell lines removed the cytotoxicity in relation to the presence of the glycoprotein. Isolation of the tumor cell surface components binding antibody revealed two components with molecular weights of approximately 70,000 and 60,000. The Mr 70,000 component was identified as viral gp70 by peptide mapping.

Female rats of the inbred strains BN/BiRij and WAG/Rij were irradiated with 30 kV X-rays, or 15 MeV or 0.5 MeV fast neutrons. Sera were collected several months after irradiation and found to be negative for antibodies reacting with the murine mammary tumour virus as tested by a solid-phase radioimmunoassay and an immunofluorescence absorption test. We found, however, that in an immunofluorescence assay several sera from irradiated rats reacted with a cytoplasmic antigen in rat mammary, ureter and skin carcinoma cell lines as well as a mouse mammary tumour and a transformed BALB/3T3 mouse fibroblast line. No reaction was found with normal fibroblast cell lines of rat or murine origin. Endpoint titres of the sera on tumour cells ranged from 1:20 to 1:160. Of 48 sera from unirradiated rats 18 also stained tumour cells, but usually at the low dilution of 1:10. Irradiation seems to enhance antibody activity to a ubiquitous tumour-associated antigen.

Tumor-specific immunity mediated by thymus-derived lymphocytes (T cells) was established in C3H/He mice against syngeneic X5563 plasmacytoma. Splenic T cells from mice, which exhibited resistance to tumor challenge, revealed a potent in vivo tumor-neutralizing activity as well as in vitro cytotoxicity. In contrast, spleen cells from mice 7 days after tumor cell inoculation (7-day tumor-bearing mice) exhibited no protective activity when assayed by in vivo tumor-neutralization test, whereas these cells exhibited almost comparable in vitro cytotoxic activity to that from the tumor-resistant mice. Any suppressor cell activity was not detected in 7-day tumor-bearing animals. While a slight in vivo protective activity was observed in the spleen cells from 14-day tumor-bearing mice, this activity was still significantly weaker than that of spleen cells from mice similarly inoculated with tumor 14 days before but whose tumor had been removed 7 days before the assay. The development of in vivo protective immunity in tumor-resected mice was suppressed by intravenous inoculation with 7000 R X-irradiated tumor cells. These results indicate that in vitro reactivity of immune T lymphocyte population is not always correlated with in vivo protective immunity, but there is a substantial decrease in in vivo immune capability of T cells from tumor-bearing animals, and that this suppression may be ascribed to the presence of a large amount of tumor-associated transplantation antigens rather than to suppressor cell activity.

Adult or newborn C57BL/6J mice were immunized with isogenic Moloney strain MuLV-induced leukemia cells irradiated with 10,000 rads or treated with low concentrations of formalin. Groups of immunized and control mice were challenged with a range of doses of viable leukemia cells, and tumor deaths were recorded for 90 days after challenge. Then, the doses of challenge cells which produced 50% tumor deaths were calculated for immunized and control mice. The logarithm of their ratio quantified the degree of protection provided by immunization. For adult C57BL/6J mice, a single immunization with MuLV-induced leukemia cells was not effective; either cells plus Bacillus Calmette-Guérin or Corynebacterium parvum, or else two immunizations with irradiated leukemia cells were needed to produce statistically significant increases in the values of the doses of challenge cells which produced 50% tumor deaths. Cross-protection was obtained by immunization with other isogenic MuLV-induced leukemias, but not by immunization with isogenic carcinogen-induced tumors or with an isogenic spontaneous leukemia. For newborn mice, a single injection of irradiated leukemia cells provided 1.3 to 1.5 logs of protection, and admixture of B. Calmette-Guérin or C. parvum increased this protection to 2.4 to 2.7 logs. Since irradiated and frozen-thawed MuLV-induced leukemia cells contained viable MuLV, leukemia cells treated with 0.5 or 1.0% formalin were tested as an alternative. A single injection of formalin-treated isogenic leukemia cells admixed with C. parvum provided between 1.7 and 2.8 logs of protection. These results demonstrate that a single vaccination of newborn animals against a highly antigenic virally induced leukemia produces strong protection against a subsequent challenge with viable leukemia cells.

Homogenates of L1210 cells infected in vitro with vesicular stomatitis virus (VSV) were immunogenic agains a tumor graft of 100 times the LD50 dose of L1210 cells" whereas those of uninfected cells were not. The immunogenicity of intact X-irradiated L1210 cells was distinguishable from that of VSV-infected cell homogenates on the basis of the susceptibility of immunogenicity to experimental procedures used in preparation of the immunogenic homogenates: Homogenization of intact X-irradiated cells or their infection with VSV prior to irradiation led to loss of immunogenicity. In addition, uninfected cell homogenates were not made immunogenic nor was the immunogenicity of VSV-infected cell homogenates eliminated by X-irradiation. At the time of tumor challenge, sera from mice that were effectively immunized with VSV-infected cell homogenate showed a high VSV-neutralizing titer but no complement-dependent cytotoxicity for L1210 cells. Quantitative absorption studies demonstrated that VSV infection led to a marked reduction in L1210 surface antigens recognized by cytotoxic alloantibody; spatial association between these antigens and VSV antigens was not demonstrable on VSV-infected cells. Antigens recognized by heterologous antiserum to L1210 cells were also reduced following VSV infection.

Tumor resistance could be induced against the transplantation of cell lines derived from spontaneous lymphomas that occurred in the third of three lymphoma epizootics in a hamster colony. Immunization of normal hamsters with irradiated lymphoma cells promoted resistance to homologous lymphoma challenge and prevented the development of spontaneous lymphomas when immunized hamsters were exposed to the contaminated colony. This immunity could be transferred in an adoptive transfer assay. Resistance to direct challenge was not extended to simian virus 40(SV40)-induced sarcomas carrying SV40 tumor-specific transplantation antigen nor to herpesvirus-induced carcinoma cells, indicating specificity. The nature of the antigen(s) involved was discussed.

In previous studies, intradermal inoculation of small doses of L2C leukemic cell suspensions into strain 2 guinea pigs induced immunity against challenging reinoculation with leukemic cells; however, 50% of the guinea pigs developed leukemia in the course of immunization. We have now attempted to induce immunity by inoculation of L2C leukemic cells inactivated by in vitro gamma irradiation ranging from 750 to 8000 rads (1 rad = 0.01 gray). In preliminary experiments, irradiation with 750 to 2750 rads had no significant effect on leukemogenic potency of leukemic cells; however, doses exceeding 3000 rads inactivated the leukemogenic potency of L2C cells. Eighty-nine guinea pigs that survived intradermal, subcutaneous, or intraperitoneal inoculations with irradiated (1000-8000 rads) L2C cells were subsequently challenged by reinoculation with nonirradiated leukemic cells, and 83 of them (93%) developed leukemia. L2C leukemic cells contain spherical particles, about 103 nm in diameter; it is reasonable to assume that these particles represent the causative virus responsible for the development of L2C leukemia. Inactivation of leukemogenic potency of L2C leukemic cells by gamma irradiation in vitro does not necessarily imply that the virus particles consistently present in these cells were also inactivated. In previous experiments carried out on mouse leukemia, doses exceeding 1,000,000 rads were needed in order to inactivate the mouse leukemia virus in vitro.

In vitro exposure of nonimmunogenic murine tumor cells to UV radiation (UVR) generates highly antigenic variants that are immunologically rejected by normal, syngeneic mice. The purpose of this study was to determine whether these antigenic variants cross-react immunologically with the parental tumor and whether the UVR-associated antigen unique to UVR-induced tumors is also present on the variants. Antigenic (regressor) variants and nonimmunogenic (progressor) clones derived from UV-irradiated cultures of the C3H K1735 melanoma and SF19 spontaneous fibrosarcoma cell lines were used to address these questions. In an in vivo immunization and challenge assay, the antigenic variants did not induce cross-protection among themselves, but each induced immunity against the immunizing variant, the parent tumor cells, and nonimmunogenic clones derived from UV-irradiated parent cultures. Therefore, the variants can be used to induce in mice a protective immunity that prevents the growth of the parent tumor and nonimmunogenic clones, but not other antigenic variants. In contrast, immunization with cells of the parental tumor or the nonimmunogenic clones induced no protective immunity against challenge with any of the cell lines. Utilizing the K1735 melanoma-derived cell lines in vitro, T-helper (Th) cells isolated from tumor-immunized mice were tested for cross-reactivity by their ability to collaborate with trinitrophenyl-primed B-cells in the presence of trinitrophenyl-conjugated tumor cells. Also, the cross-reactivity of cytotoxic T-lymphocytes from tumor-immunized mice was assessed by a 4-h 51Cr-release assay. Antigenic variants induced cytotoxic T-lymphocytes and Th activity that was higher than that induced by the parent tumor and nonimmunogenic clones from the UVR-exposed parent tumor and cross-reacted with the parental tumor cells and nonimmunogenic clones, but not with other antigenic variants. Furthermore, upon transplantation, the UVR-induced antigenic variants grew in UV-irradiated and immunosuppressed mice, but not in untreated mice indicating that the variants expressed the determinant recognized by suppressor T-cells present in UV-irradiated mice. These results demonstrate that highly antigenic cells generated by the in vitro exposure of two different murine tumors to UV radiation express a determinant shared with the parental tumor cells and nonimmunogenic clones, a unique variant-specific determinant and the suppressor cell-defined determinant present on UVR-induced tumors. Based on these results, two models are proposed to explain the make-up of the antigenic determinants present on the UVR-induced antigenic variants.

Adult A/J mice inoculated with 1 x 10(6) syngeneic C1300 neuroblastoma cells had a palpable tumor after 1 week, and the tumor grew uniformly. The hypertonic KCl extract of the tumor induced blastogenic response of syngeneic spleen cells from tumor-bearing mice, and tumor antigens were considered to be solubilized by KCl from tumor cells. Although a higher blastogenic response to insoluble tumor antigens coupled to Sepharose 4B beads could have been expected as demonstrated in this mixed lymphocyte-tumor cell reaction (MLTR) assays, the blastogenic activity, which was approximately equal to that of soluble tumor antigens, was less than one-third of that in MLTR. The initial information of blastogenic response was found to be transmitted to the responder cells with out the entrance of tumor antigens into the cells by the use of insoluble tumor antigens. Blastogenic responses to soluble tumor antigens and to irradiated tumor cells (MLTR) in spleen cells from tumor-bearing mice were serially assayed after tumor inoculation. The response to soluble tumor antigens reached a peak 2 weeks after inoculation but a progressive depression of the responses was observed after a marked tumor growth. Although the blastogenic activity of soluble tumor antigens was small, changes in consecutive response to soluble tumor antigens in tumor-bearing mice were well correlated with those in MLTR. The blastogenic responses to soluble tumor antigens and MLTR were considered to be the manifestation of tumor-specific cell-mediated immunity. Furthermore, the serial blastogenic responses to concanavalin-A and lipopolysaccharide were also coincident with those of tumor-specific immunity.

The pre-thymic cell phenotypes arising in spleen and bone marrow (BM) of fractionated total lymphoid irradiation (TLI)-treated mice are characterized in this paper. Different strains of mice received 200 rad lymphoid irradiation daily for 8 to 10 days. At different time intervals after treatment, BM or spleen cells of treated mice were stained with a battery of antisera directed specifically to TL and Thy-1 antigens. Analysis of the stained cells on a fluorescence-activated cell sorter (FACS) apparatus revealed that 10 to 30% of spleen or BM cells of TLI-treated mice were TL+ cells whereas no TL+ cells could be found in the same organs in the normal untreated mice. The appearance of TL+ cells in the spleen and BM of TLI-treated mice was found to be thymic independent, as thymectomy of mice neither prevented nor altered the kinetics of TL+ cell appearance. The sequence of antigenic marker appearance reveals that TL markers precede Thy-1 markers and that TLaa markers precede TL4 antigen. The relevance of the TL4+ cells to the development of leukemias is discussed.

Tumor-specific immunoprophylaxis was achieved in C57BL/6J mice against EL 4 leukosis cell challenge by sensitization of the syngeneic host with multiple ip injections of irradiated EL 4 cells. A minimal radiation dose was used to replication-block EL 4 cells before inoculation, as defined by dose-response analysis of irradiated EL 4 cells. Multiple ip injections of irradiated EL 4 cells stimulated development of significant, yet relatively low, levels of cytotoxic lymphoid activity (CLA) in lymphoid cells of the peritoneal exudate as measured by in vitro 51Cr-release cytotoxicity assays. The specific temporal and frequency dependencies of the inoculation regimen for achieving immunoprophylaxis indicated that, in addition to CLA, other, short-lived, immune processes were important in the tumor rejection. These observations showed the capacity of the C57BL/6J host for tumor-specific immune recognition and rejection of the syngeneic EL 4 leukemia. The tumor rejection could be elicited solely by inoculations of irradiated EL 4 cells and did not require exogenous amplifiers, such as immunoadjuvants, chemical modifiers, and/or allogeneic immune information transfer.

The effect of the protease inhibitor antipain (1.25 micrograms/ml) on the radiation-induced expression of a tumor-associated antigen in human cell hybrids has been investigated. A variety of treatment protocols have been studied where antipain was present before, during and at various times post-irradiation. It was found that antipain suppressed the radiation-induced expression of the tumor-associated antigen in all treatment protocols. The most effective suppression was obtained in those protocols where the protease inhibitor was present for the first 4 h post-irradiation. A possible explanation for this observation is that antipain may inhibit an error-prone DNA repair process. However, it is clear that this is not the only mechanism whereby the inhibitor can exert its effect since suppression was obtained even when antipain was added 10 days post-irradiation, a time when any DNA repair processes would be expected to be over.

The treatment in vivo with anti-tumour drugs can induce an antigenic alteration of L1210 leukemia resulting in the rejection of an inoculum of 10 X 10(6) viable cells in syngeneic mice. As drug-induced antigens appeared in excess of any pre-existing tumour-associated transplantation antigens (TATA), viable altered cells have been used to sensitize syngeneic animals. Experiments showed that viable altered cells elicited stronger anti-TATA reaction than X-inactivated parental cells, as measured by host survival to a challenge of L1210 leukemia. TATA immunogenicity of parental cells has been preminent, in the strain of animals used, to determine the sensitizing effectiveness of 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) cells. Host protection to an inoculum of parental tumours has been more proficient with the DTIC subline derived from highly immunogenic L1210Ha cells than from poorly immunogenic L1210Cr cells. Immunoprophylactic inocula, proper chemotherapeutic treatments and adoptive transfer of immune lymphocytes used in combination exerted a synergic host protection.

Six tumors of varying immunorejective strengths were used to compare the response of their isogenic hosts to standardized regimens of immunoprophylaxis and immunotherapy. The tumors were generated spontaneously or induced chemically [with 9,10-dimethyl-1,2-benzanthracene (CAS: 57-97-6)], virally (with murine leukemia virus), or radiogenically (with strontium-90). The hosts were C57BL/6J or BALB/cByJ mice. Immunoprophylaxis and immunotherapy were performed with isogenic irradiated tumor cells, with Corynebacterium parvum, or with both. The results of challenge experiments were quantified as the doses of viable tumor cells that produced 50% tumor deaths for immunized and for control mice. The results for these quantitative "classical" immunoprophylaxis and immunotherapy experiments were consistent with two theses: that only immunorejective tumors give positive results with classical immunotherapy and that classical immunoprophylaxis is more effective than classical immunotherapy when identical materials are used for immunizations. These results have important consequences for the clinical use of classical immunotherapy.