Lotta von Boehmer

γ-Radiation Promotes Immunological Recognition of Cancer Cells through Increased Expression of Cancer-Testis Antigens In Vitro and In Vivo

Background γ-radiation is an effective treatment for cancer. There is evidence that radiotherapy supports tumor-specific immunity. It was described that irradiation induces de novo protein synthesis and enhances antigen presentation, we therefore investigated whether γ-radiation results in increased expression of cancer-testis (CT) antigens and MHC-I, thus allowing efficient immunological control. This is relevant because the expression of CT-antigens and MHC-I on tumor cells is often heterogeneous. We found that the changes induced by γ-radiation promote the immunological recognition of the tumor, which is illustrated by the increased infiltration by lymphocytes after radiotherapy. Methods/Findings We compared the expression of CT-antigens and MHC-I in various cancer cell lines and fresh biopsies before and after in vitro irradiation (20 Gy). Furthermore, we compared paired biopsies that were taken before and after radiotherapy from sarcoma patients. To investigate whether the changed expression of CT-antigens and MHC-I is specific for γ-radiation or is part of a generalized stress response, we analyzed the effect of hypoxia, hyperthermia and genotoxic stress on the expression of CT-antigens and MHC-I. In vitro irradiation of cancer cell lines and of fresh tumor biopsies induced a higher or de novo expression of different CT-antigens and a higher expression of MHC-I in a time- and dose-dependent fashion. Importantly, we show that irradiation of cancer cells enhances their recognition by tumor-specific CD8+ T cells. The analysis of paired biopsies taken from a cohort of sarcoma patients before and after radiotherapy confirmed our findings and, in addition showed that irradiation resulted in higher infiltration by lymphocytes. Other forms of stress did not have an impact on the expression of CT-antigens or MHC-I. Conclusions Our findings suggest that γ-radiation promotes the immunological recognition of the tumor. We therefore propose that combining radiotherapy with treatments that support tumor specific immunity may result in increased therapeutic efficacy.

Quantitative computed tomography liver perfusion imaging using dynamic spiral scanning with variable pitch: feasibility and initial results in patients with cancer metastases.

Purpose:To assess the feasibility and image quality of computed tomography (CT) liver perfusion imaging using an adaptive 4D spiral-mode, developed to extend the z-axis coverage, and to report initial qualitative and quantitative results in patients with cancer metastases. Materials and Methods:A total of 21 patients with liver metastases of various origins underwent CT perfusion imaging (100 kV and 150 mAs/rot) using a 4D spiral-mode with single-source 64-slice CT (n = 7) with a scan range of 6.7cm (protocol A: 16 cycles, 46.5 seconds examination time), or dual-source 128-slice CT with a scan range of 14.8 cm (protocol B: 16 cycles, 46.5 seconds examination time, n = 7; protocol C: 12 cycles, 51.0 seconds examination time, n = 7). Ability to suspend respiration during perfusion imaging was monitored. Two independent readers assessed image quality on a 4-point scale, both before and after motion correction, and performed a qualitative (ie, arterial enhancement pattern and enhancement change over time) and quantitative perfusion (ie, arterial liver perfusion [ALP]; portal-venous perfusion [PVP]; hepatic perfusion index [HPI]) analysis. Results:Of 21 patients, 7 (33%) could suspend respiration throughout the perfusion study and 14 (67%) resumed shallow breathing during the perfusion scan. The 21 patients had a total of 88 metastases. The scan range of protocol A covered at least 1 metastasis in all patients (total 20/34 [58.8%] metastases). The scan range of protocol B and C covered 53 of 54 (98.1%) metastases, whereas one metastasis in segment VIII was only partially imaged. Image quality was diagnostic both before and after motion correction, whereas being significantly better after motion correction (P < 0.001). Qualitative perfusion analysis of 67 metastases revealed diffuse arterial enhancement in 3 (4.5%), sparse enhancement in 11 (16.4%), peripheral-nodular enhancement in 9 (13.4%), rim-like enhancement in 15 (22.4%), and none in 29 (43.3%) metastases. Enhancement over time of 67 metastases showed a centripetal progression in 6 (8.9%), sustained portal phase in 16 (23.9%), wash-out in 16 (23.9%), and none in 29 (43.3%) metastases. Quantitative perfusion analysis revealed significantly higher arterial liver perfusion and HPI in metastases and metastasis borders than in adjacent normal liver tissue (P < 0.001 each). Portal-venous perfusion was significantly lower in metastases and metastasis borders than in normal liver tissue (P < 0.001). There were no significant differences in image quality and qualitative perfusion analysis between the 3 protocols (P = n.s.). Calculated effective radiation doses were 13.4 mSv for protocol A, 30.7 mSv for protocol B, and 23.0 mSv for protocol C. Conclusion:CT perfusion imaging of the liver using the 4D spiral-mode is feasible with diagnostic image quality, and enables the reliable qualitative and quantitative analysis of the normal and metastatic liver parenchyma. Radiation dose issues must be considered when determining the scan range, number of cycles, and scan duration of the perfusion CT protocol.

Particle size and activation threshold: a new dimension of danger signaling

Previous studies have shown that single-stranded RNA (ssRNA) mixed with protamine forms particles and activates immune cells through Toll-like receptors (TLRs). We have found that the size of protamine-RNA particles generated depends on the electrolyte content when mixing the 2 components. Moreover, we have evidenced that (1) nanometric particles induce production of interferon-alpha, whereas (2) micrometric particles mainly induce production of tumor necrosis factor-alpha (TNF-alpha) in human immune cells. We found that the mechanisms underlying these observations are (1) nanoparticles but not microparticles are selectively phagocytosed by plasmacytoid dendritic cells (pDCs), which produce interferon-alpha and (2) monocytes that produce TNF-alpha have a higher activation threshold than that of pDCs. Thus, at the same time as sensing pathogen-associated molecular patterns such as ssRNA, the immune system distinguishes the size of the associated structure in such a way as to trigger the adapted antivirus (nanometric) or antibacterial/antifungal (micrometric) immune response. Our results introduce a new dimension in danger signaling--how size qualitatively affects innate response.

Tumor-associated macrophages subvert T-cell function and correlate with reduced survival in clear cell renal cell carcinoma

Although malignant cells can be recognized and controlled by the immune system, in patients with clinically apparent cancer immunosurveillance has failed. To better understand local immunoregulatory processes that impact on cancer progression, we correlated intratumoral immunological profiles with the survival of patients affected by primary clear cell renal cell carcinoma (ccRCC). A retrospective analysis of 54 primary ccRCC samples for 31 different immune response-related transcripts, revealed a negative correlation of CD68 (a marker of tumor-associated macrophages, TAMs) and FOXP3 (a marker of regulatory T cells, Tregs) with survival. The subsequent analysis of 12 TAM-related transcripts revealed an association between the genes coding for CD163, interferon regulatory factor 4 (IRF4) and fibronectin 1 (FN1), all of which have been linked to the M2 TAM phenotype, with reduced survival and increased tumor stage, whereas the opposite was the case for the M1-associated gene coding for inducible nitric oxide synthetase (iNOS). The M2 signature of (CD68+) TAMs was found to correlate with CD163 expression, as determined in prospectively collected fresh ccRCC tissue samples. Upon co-culture with autologous tumor cells, CD11b+ cells isolated from paired blood samples expressed CD163 and other M2-associated proteins, suggesting that the malignant cells promote the accumulation of M2 TAMs. Furthermore, the tumor-associated milieu as well as isolated TAMs induced the skewing of autologous, blood-derived CD4+ T cells toward a more immunosuppressive phenotype, as shown by decreased production of effector cytokines, increased production of interleukin-10 (IL-10) and enhanced expression of the co-inhibitory molecules programmed death 1 (PD-1) and T-cell immunoglobulin mucin 3 (TIM-3). Taken together, our data suggest that ccRCC progressively attracts macrophages and induces their skewing into M2 TAMs, in turn subverting tumor-infiltrating T cells such that immunoregulatory functions are increased at the expense of effector functions.

Alteration of GABAergic synapses and gephyrin clusters in the thalamic reticular nucleus of GABAA receptor α3 subunit‐null mice

Multiple GABAA‐receptor subtypes are assembled from α, β and γ subunit variants. GABAA receptors containing the α3 subunit represent a minor population with a restricted distribution in the CNS. In addition, they predominate in monoaminergic neurons and in the nucleus reticularis thalami (nRT), suggesting a role in the regulation of cortical function and sleep. Mice with a targeted deletion of the α3 subunit gene (α30/0) are viable and exhibit a subtle behavioural phenotype possibly related to dopaminergic hyperfunction. Here, we investigated immunohistochemically the consequences of the loss of α3 subunit for maturation of GABAA receptors and formation of GABAergic synapses in the nRT. Throughout postnatal development, the regional distribution of the α1, α2, or α5 subunit was unaltered in α30/0 mice and the prominent α3 subunit staining of nRT neurons in wildtype mice was not replaced. Subcellularly, as seen by double immunofluorescence, the α3 and γ2 subunit were clustered at postsynaptic sites in the nRT of adult wildtype mice along with the scaffolding protein gephyrin. In α30/0 mice, γ2 subunit clustering was disrupted and gephyrin formed large aggregates localized at the cell surface, but unrelated to postsynaptic sites, indicating that nRT neurons lack postsynaptic GABAA receptors in mutant mice. Furthermore, GABAergic terminals were enlarged and reduced in number, suggesting a partial deficit of GABAergic synapses. Therefore, GABAA receptors are required for gephyrin clustering and long‐term synapse maintenance. The absence of GABAA‐mediated transmission in the nRT may have a significant impact on the function of the thalamo‐cortical loop of α30/0 mice.

Efficient in-vivo priming by vaccination with recombinant NY-ESO-1 protein and CpG in antigen naïve prostate cancer patients

Purpose: NY-ESO-1, one of the most immunogenic tumor antigens, is expressed in 15% to 25% of metastatic prostate cancers. The immunological and clinical effects of vaccination with recombinant NY-ESO-1 protein combined with CpG as adjuvant were evaluated. Experimental Design: In a phase I clinical study, patients with advanced prostate cancer were vaccinated with recombinant NY-ESO-1 protein (100 μg) mixed with CpG 7909 (2.5 mg) every 3 weeks intradermally for 4 doses. Objectives of the study were the safety of the vaccine and changes of specific humoral and cellular immunological responses to NY-ESO-1 in relation to detectable NY-ESO-1 expression in the individual tumor. Results: All 12 baseline sero-negative patients developed high-titer NY-ESO-1 antibody responses. B-cell epitope mapping identified NY-ESO-1 p91–110 to be recognized most frequently by vaccine-induced antibodies. Two patients developed significant antibody titers against the adjuvant CpG. NY-ESO-1-specific CD4+ and/or CD8+ T-cell responses were induced in 9 patients (69%). Five of these 9 patients did not express NY-ESO-1 in the autologous tumor. Postvaccine CD8+ T-cell clones recognized and lyzed HLA-matched tumor cell lines in an antigen-specific manner. Conclusion: Our data provide clear evidence for the capacity of NY-ESO-1 protein/CpG vaccine to induce integrated antigen-specific immune responses in vivo and to efficiently prime CD8+ T-cell responses in NY-ESO-1 antigen-negative patients. Our results may also support further clinical vaccination protocols with NY-ESO-1 protein not only focused on the treatment of existing cancer, but also to prevent further development of NY-ESO-1 positive cancers in vivo. Clin Cancer Res; 17(4); 1–10. ©2010 AACR.

MAGE-C2/CT10 Protein Expression Is an Independent Predictor of Recurrence in Prostate Cancer

The cancer-testis (CT) family of antigens is expressed in a variety of malignant neoplasms. In most cases, no CT antigen is found in normal tissues, except in testis, making them ideal targets for cancer immunotherapy. A comprehensive analysis of CT antigen expression has not yet been reported in prostate cancer. MAGE-C2/CT-10 is a novel CT antigen. The objective of this study was to analyze extent and prognostic significance of MAGE-C2/CT10 protein expression in prostate cancer. 348 prostate carcinomas from consecutive radical prostatectomies, 29 castration-refractory prostate cancer, 46 metastases, and 45 benign hyperplasias were immunohistochemically analyzed for MAGE-C2/CT10 expression using tissue microarrays. Nuclear MAGE-C2/CT10 expression was identified in only 3.3% primary prostate carcinomas. MAGE-C2/CT10 protein expression was significantly more frequent in metastatic (16.3% positivity) and castration-resistant prostate cancer (17% positivity; p<0.001). Nuclear MAGE-C2/CT10 expression was identified as predictor of biochemical recurrence after radical prostatectomy (p = 0.015), which was independent of preoperative PSA, Gleason score, tumor stage, and surgical margin status in multivariate analysis (p<0.05). MAGE-C2/CT10 expression in prostate cancer correlates with the degree of malignancy and indicates a higher risk for biochemical recurrence after radical prostatectomy. Further, the results suggest MAGE-C2/CT10 as a potential target for adjuvant and palliative immunotherapy in patients with prostate cancer.

Radiotherapy of Human Sarcoma Promotes an Intratumoral Immune Effector Signature

Purpose: The tumor immune microenvironment plays a crucial role in the development and progression of cancer. Sarcomas are a group of heterogeneous soft tissue malignancies that are often treated with radiotherapy as a part of the treatment concept. There is increasing evidence that radiotherapy leads to alterations in the tumor microenvironment, particularly with respect to the immune infiltrate. This study has been carried out to develop a better understanding of such changes following radiotherapy. Experimental Design: We retrospectively analyzed the expression of 35 immune response-related genes by quantitative reverse transcription PCR analysis and immunohistochemistry on paired formalin-fixed paraffin-embedded tumor samples from 38 sarcoma patients before and after radiotherapy. Results: We observed that radiotherapy results in a significant upregulation of several immune effectors and cancer-testis antigens and a concomitant downregulation of immune suppressors, indicating that radiotherapy may support the immune defense in sarcomas. Conclusions: These novel findings may have implications for the design of therapeutic regimens which exploite the immune system in sarcoma patients by combining standard radiotherapy with immunotherapeutic strategies. Clin Cancer Res; 19(17); 4843–53. ©2013 AACR.

Radiotherapy supports protective tumor-specific immunity.

Radiotherapy is an important therapeutic option for the treatment of cancer. Growing evidence indicates that, besides inducing an irreversible DNA damage, radiotherapy promotes tumor-specific immune response, which significantly contribute to therapeutic efficacy. We postulate that radiotherapy activates tumor-associated dendritic cells, thus changing the tolerogenic tumor environment into an immunogenic one.

Fine analysis of spontaneous MAGE-C1/CT7–specific immunity in melanoma patients

Cancer/testis (CT) antigens represent prime candidates for immunotherapy in cancer patients, because their expression is restricted to cancer cells and germ cells of the testis. MAGE-C1/CT7 is a CT antigen that is highly expressed in several types of cancers. Spontaneous occurrence of CT7-specific antibodies was previously detected by SEREX screen in a melanoma patient. However, naturally occurring CT7-specific T-cell responses have thus far not been detected. Peripheral blood mononuclear cells (PBMCs) from 26 metastatic melanoma patients expressing CT7 in their tumor lesions (CT7+) were analyzed for CT7-specific T-cell responses using overlapping peptides. CT7-specific CD4+ T-cell responses were detected in three patients (11.5%). These CT7-specific CD4+ T-cell responses were detectable in melanoma patients’ PBMCs exclusively from preexisting CD45RA− memory CD4+ T-cell pool. Additional CT7-specific memory CD4+ T-cell responses were detected in CT7+ melanoma patients after depletion of CD4+CD25high Treg cells showing that Treg cells impact on CT7-specific CD4+ T cells in melanoma patients. CT7-specific CD4+ T-cell clones were generated and used to define minimal epitopes, restriction elements, and confirm the recognition of naturally processed antigen. Surprisingly, these clones were able to secrete perforin and exert cytotoxicity. This study shows that CT7 can induce specific cellular immunity in melanoma patients. Based on these findings, CT7 will be further explored as a potential vaccine for melanoma immunotherapy.