Intermolecular electron transfer in radical SAM enzymes as a new paradigm for reductive activation.

Radical S-adenosyl-L-methionine (rSAM) enzymes bind one or more Fe-S clusters and catalyze transformations that produce complex and structurally diverse natural products. One of the clusters, a 4Fe-4S cluster, binds and reductively cleaves SAM to generate the 5'-deoxyadenosyl radical, which initiates the catalytic cycle by H-atom transfer from the substrate. The role(s) of the additional auxiliary Fe-S clusters (ACs) remains largely enigmatic. The rSAM enzyme PapB catalyzes the formation of thioether cross-links between the ß-carbon of an Asp and a Cys thiolate found in the PapA peptide. One of the two ACs in the protein binds to the substrate thiol where, upon formation of a thioether bond, one reducing equivalent is returned to the protein. However, for the next catalytic cycle to occur, the protein must undergo an electronic state isomerization, returning the electron to the SAM-binding cluster. Using a series of iron-sulfur cluster deletion mutants, our data support a model whereby the isomerization is an obligatorily intermolecular electron transfer event that can be mediated by redox active proteins or small molecules, likely via the second AC in PapB. Surprisingly, a mixture of FMN and NADPH is sufficient to support both the reductive and the isomerization steps. These findings lead to a new paradigm involving intermolecular electron transfer steps in the activation of rSAM enzymes that require multiple iron-sulfur clusters for turnover. The implications of these results for the biological activation of rSAM enzymes are discussed.

Capturing the Binuclear Copper State of Peptidylglycine Monooxygenase Using a Peptidyl-Homocysteine Lure.

Peptidylglycine monooxygenase is a copper-dependent enzyme that catalyzes C-alpha hydroxylation of glycine extended pro-peptides, a critical post-translational step in peptide hormone processing. The canonical mechanism posits that dioxygen binds at the mononuclear M-center to generate a Cu(II)-superoxo species capable of H atom abstraction from the peptidyl substrate, followed by long-range electron tunneling from the CuH center. Recent crystallographic and biochemical data have challenged this mechanism, suggesting instead that an "open-to-closed" transition brings the copper centers closer, allowing reactivity within a binuclear intermediate. Here we present the first direct observation of an enzyme-bound binuclear copper species, captured by the use of an Ala-Ala-Phe-hCys inhibitor complex. This molecule reacts with the fully reduced enzyme to form a thiolate-bridged binuclear species characterized by EXAFS of the WT and its M314H variant and with the oxidized enzyme to form a novel mixed valence entity characterized by UV/vis and EPR. Mechanistic implications are discussed.

Peptide Selenocysteine Substitutions Reveal Direct Substrate-Enzyme Interactions at Auxiliary Clusters in Radical S-Adenosyl-l-methionine Maturases.

Radical S-adenosyl-l-methionine (SAM) enzymes leverage the properties of one or more iron- and sulfide-containing metallocenters to catalyze complex and radical-mediated transformations. By far the most populous superfamily of radical SAM enzymes are those that, in addition to a 4Fe-4S cluster that binds and activates the SAM cofactor, also bind one or more additional auxiliary clusters (ACs) of largely unknown catalytic significance. In this report we examine the role of ACs in two RS enzymes, PapB and Tte1186, that catalyze formation of thioether cross-links in ribosomally synthesized and post-translationally modified peptides (RiPPs). Both enzymes catalyze a sulfur-to-carbon cross-link in a reaction that entails H atom transfer from an unactivated C-H to initiate catalysis, followed by formation of a C-S bond to yield the thioether. We show that both enzymes tolerate substitution of SeCys instead of Cys at the cross-linking site, allowing the systems to be subjected to Se K-edge X-ray spectroscopy. The EXAFS data show a direct interaction with the Fe of one of the ACs in the Michaelis complex, which is replaced with a Se-C interaction under reducing conditions that lead to the product complex. Site-directed deletion of the clusters in Tte1186 provide evidence for the identity of the AC. The implications of these observations in the context of the mechanism of these thioether cross-linking enzymes are discussed.

Reducing CO2 Emissions of a Coal-Fired Power Plant via Accelerated Weathering of Limestone: Carbon Capture Efficiency and Environmental Safety.

Reducing CO2 emissions is a key task of modern society to attenuate climate change and its environmental effects. Accelerated weathering of limestone (AWL) has been proposed as a tool to capture CO2 from effluent gas streams and store it primarily as bicarbonate in the marine environment. We evaluated the performance of the biggest AWL-reactor to date that was installed at a coal-fired power plant in Germany. Depending on the gas flow rate, approximately 55% of the CO2 could be removed from the flue gas. The generated product water was characterized by an up to 5-fold increase in alkalinity, which indicates the successful weathering of limestone and the long-term storage of the captured CO2. A rise of potentially harmful substances in the product water (NO2-, NOx-, NH4+, SO42-, and heavy metals) or in unreacted limestone particles (heavy metals) to levels of environmental concern could not be observed, most likely as a result of a desulfurization of the flue gas before it entered the AWL reactor. At locations where limestone and water availability is high, AWL could be used for a safe and long-term storage of CO2.

Experiences with EPAs, potential benefits and pitfalls.

Reforms in postgraduate medical education (PGME) exposed a gap between educational theory and clinical practice. Entrustable Professional Activities (EPAs) were introduced to assist clinicians in bridging this gap and to create better consonance between the intended and the enacted curriculum. In this viewpoint paper, we discuss the potential and the pitfalls of using EPAs in PGME. EPAs promise an effective way of teaching abstract competencies in a curriculum based on real-life professional activities that are suitable for clinical assessment. Summative judgement is used to entrust a resident step by step in a certain EPA, resulting in an increase of independent practice. However, we argue that the success of EPAs depends on (1) a balance: brief focussed descriptions against the requirements for detail and (2) a precondition: a mature and flexible workplace for learning.

Cultural diversity: blind spot in medical curriculum documents, a document analysis.

BACKGROUND: Cultural diversity among patients presents specific challenges to physicians. Therefore, cultural diversity training is needed in medical education. In cases where strategic curriculum documents form the basis of medical training it is expected that the topic of cultural diversity is included in these documents, especially if these have been recently updated. The aim of this study was to assess the current formal status of cultural diversity training in the Netherlands, which is a multi-ethnic country with recently updated medical curriculum documents. METHODS: In February and March 2013, a document analysis was performed of strategic curriculum documents for undergraduate and postgraduate medical education in the Netherlands. All text phrases that referred to cultural diversity were extracted from these documents. Subsequently, these phrases were sorted into objectives, training methods or evaluation tools to assess how they contributed to adequate curriculum design. RESULTS: Of a total of 52 documents, 33 documents contained phrases with information about cultural diversity training. Cultural diversity aspects were more prominently described in the curriculum documents for undergraduate education than in those for postgraduate education. The most specific information about cultural diversity was found in the blueprint for undergraduate medical education. In the postgraduate curriculum documents, attention to cultural diversity differed among specialties and was mainly superficial. CONCLUSIONS: Cultural diversity is an underrepresented topic in the Dutch documents that form the basis for actual medical training, although the documents have been updated recently. Attention to the topic is thus unwarranted. This situation does not fit the demand of a multi-ethnic society for doctors with cultural diversity competences. Multi-ethnic countries should be critical on the content of the bases for their medical educational curricula.

Association and Mutation Analyses of the IRF6 Gene in Families With Nonsyndromic and Syndromic Cleft Lip and/or Cleft Palate.

OBJECTIVES: (1) To detect interferon regulatory factor 6 gene (IRF6) mutations in newly recruited Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) families. (2) To test for association, in nonsyndromic cleft lip and/or cleft palate (NSCL/P) and in VWS/PPS families, the single nucleotide polymorphism (SNP) rs642961, from the IRF6 enhancer AP-2α region, alone or as haplotype with rs2235371, a coding SNP (Val274Ile). DESIGN: IRF6 mutation screening was performed by direct sequencing and genotyping of rs642961 and rs2235371 by TaqMan technology. PATIENTS: Seventy-one Swedish NSCL/P families, 24 Finnish cleft palate (CP) families, and 24 VWS/PPS families (seven newly recruited) were studied. RESULTS: Allelic and genotypic frequencies in each phenotype were compared to those of the controls, and no significant difference could be observed. IRF6 gene mutation was detected in six of the seven new VWS/PPS families. Association analysis of the entire VWS/PPS sample set revealed the A allele from rs642961 to be a risk allele. Significant association was detected in the Swedish CP subset of our NSCL/P collection where the G-C haplotype for rs642961-rs2235371 were at risk (P = .013). CONCLUSIONS: Our results do not support the previously reported association between the A allele of rs642961 and the NSCL phenotype. However, in the VWS/PPS families, the A allele was a risk allele and was, in a large majority (>80%), transmitted on the same chromosome as the IRF6 mutation.

Laser light activation of a second-generation photosensitiser and its use as a potential photomodulatory agent in skin rejuvenation.

Photodynamic rejuvenation therapy (PDRT) is a growing field in cosmetic dermatology. In this study, different sources of light (a yellow laser, a red laser and ultraviolet A (UVA) lamps) were used to activate a second-generation photosensitiser, hypericin. Uptake of hypericin was monitored over 24 h and efficacy of PDRT was assessed using cell viability and reactive oxygen species (ROS) quantification assays. In addition, we show for the first time, a quantifiable assay for ROS production in human dermal fibroblasts incubated with hypericin and exposed to yellow laser light or UVA lamps. Furthermore, we optimised a protocol with regard to hypericin concentration and irradiation parameters using the XTT cell viability kit. This study showed that this photosensitiser, hypericin, was taken up by the cells in a concentration-dependent manner over 24 h with cell saturation occurring after approximately 16 h. The uptake seemed to be localised to the cell cytoplasm with no hypericin appearing in the nucleus. The levels of ROS increased in the cell when irradiated with the yellow laser (561 nm) however, it did not increase further with the addition of hypericin. Hypericin and UVA showed a significant increase in the amount of ROS produced. The results also show that cell viability is not affected by low power light (2 mW) from the yellow laser irrespective of the dose used. However, an increase to 10 mW power with 5 J/cm(2) light dose, resulted in a significant drop (p < 0.05) in cell viability at both 0.5 (77.53 ± 9.67 %) and 1 µM (48.51 ± 13.27 %) hypericin concentrations. In contrast, a 20 % increase in cell viability was seen with 1 J/cm(2) and 20 mW and 0.25 µM hypericin. Overall, this study highlights an optimised protocol for hypericin-induced photorejuvenative therapy using laser light and proposes that parameters of 0.25 µM hypericin as a photosensitiser activated via a dosage of 1 J/cm(2) yellow laser light produces an effective in vitro outcome to be considered as an important contribution towards optimising PDRT.

Diffuse reflectance spectroscopy as a tool to measure the absorption coefficient in skin: system calibration.

An individualised laser skin treatment may enhance the treatment and reduces risks and side-effects. The optical properties (absorption and scattering coefficients) are important parameters in the propagation of laser light in skin tissue. The differences in the melanin content of different skin phototypes influence the absorption of the light. The absorption coefficient at the treatment wavelength for an individual can be determined by diffuse reflectance spectroscopy, using a probe containing seven fibres. Six of the fibres deliver the light to the measurement site and the central fibre collects the diffused reflected light. This is an in vivo technique, offering benefits for near-real-time results. Such a probe, with an effective wavelength band from 450 to 800 nm, was used to calibrate skin-simulating phantoms consisting of intralipid and ink. The calibration constants were used to calculate the absorption coefficients from the diffuse reflectance measurements of three volunteers (skin phototypes, II, IV and V) for sun-exposed and non-exposed areas on the arm.

A Promiscuous rSAM Enzyme Enables Diverse Peptide Cross-linking.

Ribosomally produced and post-translationally modified polypeptides (RiPPs) are a diverse group of natural products that are processed by a variety of enzymes to their biologically relevant forms. PapB is a member of the radical S-adenosyl-l-methionine (rSAM) superfamily that introduces thioether cross-links between Cys and Asp residues in the PapA RiPP. We report that PapB has high tolerance for variations in the peptide substrate. Our results demonstrate that branched side chains in the thiol- and carboxylate-containing residues are processed and that lengthening of these groups to homocysteine and homoglutamate does not impair the ability of PapB to form thioether cross-links. Remarkably, the enzyme can even cross-link a peptide substrate where the native Asp carboxylate moiety is replaced with a tetrazole. We show that variations to residues embedded between the thiol- and carboxylate-containing residues are tolerated by PapB, as peptides containing both bulky (e.g., Phe) and charged (e.g., Lys) side chains in both natural L- and unnatural D-forms are efficiently cross-linked. Diastereomeric peptides bearing (2S,3R)- and (2S,3S)-methylaspartate are processed by PapB to form cyclic thioethers with markedly different rates, suggesting the enzymatic hydrogen atom abstraction event for the native Asp-containing substrate is diastereospecific. Finally, we synthesized two diastereomeric peptide substrates bearing E- and Z-configured γ,δ-dehydrohomoglutamate and show that PapB promotes addition of the deoxyadenosyl radical (dAdo•) instead of hydrogen atom abstraction. In the Z-configured γ,δ-dehydrohomoglutamate substrate, a fraction of the dAdo-adduct peptide is thioether cross-linked. In both cases, there is evidence for product inhibition of PapB, as the dAdo-adducts likely mimic the native transition state where dAdo• is poised to abstract a substrate hydrogen atom. Collectively, these findings provide critical insights into the arrangement of reacting species in the active site of the PapB, reveal unusual promiscuity, and highlight the potential of PapB as a tool in the development peptide therapeutics.

Invariant NKT cells as novel targets for immunotherapy in solid tumors.

Natural killer T (NKT) cells are a small population of lymphocytes that possess characteristics of both innate and adaptive immune cells. They are uniquely poised to respond rapidly to infection and inflammation and produce cytokines that critically shape the ensuing adaptive cellular response. Therefore, they represent promising therapeutic targets. In cancer, NKT cells are attributed a role in immunosurveillance. NKT cells also act as potent activators of antitumor immunity when stimulated with a synthetic agonist in experimental models. However, in some settings, NKT cells seem to act as suppressors and regulators of antitumor immunity. Here we briefly review current data supporting these paradoxical roles of NKT cells and their regulation. Increased understanding of the signals that determine the function of NKT cells in cancer will be essential to improve current strategies for NKT-cell-based immunotherapeutic approaches.

Leveraging Substrate Promiscuity of a Radical S-Adenosyl-L-methionine RiPP Maturase toward Intramolecular Peptide Cross-Linking Applications.

Radical S-adenosyl-l-methionine (RS) enzymes operate on a variety of substrates and catalyze a wide range of complex radical-mediated transformations. Radical non-α-carbon thioether peptides (ranthipeptides) are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs). The RS enzyme PapB catalyzes the formation of thioether cross-links between Cys/Asp (or Cys/Glu) residues located in six Cys-X3-Asp/Glu motifs. In this report, using a minimal substrate that contains a single cross-link motif, we explore the substrate scope of the PapB and show that the enzyme is highly promiscuous and will accept a variety of Cys-X n -Asp sequences where n = 0-6. Moreover, we show that the enzyme will introduce in-line and nested thioether cross-links independently in peptide sequences that contain two motifs derived from the wild-type sequence. Additionally, the enzyme accepts peptides that contain d-amino acids at either the Cys or the Asp position. These observations are leveraged to produce a thioether cyclized analogue of the FDA-approved therapeutic agent octreotide, with a Cys-Glu cross-link replacing the disulfide that is found in the drug. These findings highlight the remarkable substrate tolerance of PapB and show the utility of RS RiPP maturases in biotechnological applications.

Improving Graduate Medical Education Through Faculty Empowerment Instead of Detailed Guidelines.

Calls for improvement and reform in graduate medical education (GME) have led to more detail in educational and curricular guidelines. The current level of detail in curriculum guidelines for GME training programs is high, encompassing, for example, competency frameworks, entrustable professional activities, and milestones. In addition, faculty must employ an increasing number of assessment tools and elaborate portfolio systems for their residents. It is questionable whether any further increase in curriculum detail and assessment formats leads to better GME programs. Focusing on this type of system development may even lead to less engaged faculty if faculty are not encouraged to use their own professional judgment and creativity for teaching residents. Therefore, faculty members must be empowered to engage in curricular innovation, since system development alone will not result in better training programs. Raising faculty members' awareness of their virtues and value as teachers and involving them in the debate about how GME can be enhanced might increase their engagement in resident training.

Invariant natural killer T cells regulate breast cancer response to radiation and CTLA-4 blockade.

PURPOSE: Immunoregulatory and suppressive mechanisms represent major obstacles to the success of immunotherapy in cancer patients. We have shown that the combination of radiotherapy to the primary tumor and CTL-associated protein 4 (CTLA-4) blockade induces antitumor immunity, inhibiting metastases and extending the survival of mice bearing the poorly immunogenic and highly metastatic 4T1 mammary carcinoma. Similarly to patients with metastatic cancer, however, mice were seldom cured. Here we tested the hypothesis that invariant natural killer T (iNKT) cells, a subset with unique regulatory functions, can regulate the response to radiotherapy and CTLA-4 blockade. EXPERIMENTAL DESIGN: The growth of 4T1 primary tumors and lung metastases was compared in wild-type and iNKT cell-deficient (iNKT-/-) mice. Treatment was started on day 13 when the primary tumors were palpable. Mice received radiotherapy to the primary tumor in two doses of 12 Gy in combination or not with 9H10 monoclonal antibody against CTLA-4. Response to treatment was assessed by measuring primary tumor growth delay/regression, survival, and number of lung metastases. RESULTS: The response to radiotherapy plus 9H10 was markedly enhanced in the absence of iNKT cells, with 50% of iNKT-/- versus 0% of wild-type mice showing complete tumor regression, long-term survival, and resistance to a challenge with 4T1 cells. Administration of the iNKT cell activator alpha-galactosylceramide did not enhance the response of wild-type mice to radiotherapy plus 9H10. Tumor-infiltrating iNKT cells were markedly reduced in wild-type mice treated with radiotherapy plus 9H10. CONCLUSIONS: iNKT cells play a major role in regulating the response to treatment with local radiotherapy and CTLA-4 blockade.

IL15 synergizes with radiotherapy to reprogram the tumor immune contexture through a dendritic cell connection.

IL15 is a key cytokine for the activation and survival of anti-tumor effectors CD8+ T and NK cells. Recently published preclinical studies demonstrate that the therapeutic activity of IL15 requires conventional dendritic cells type 1 (cDC1). Radiotherapy cooperates with IL15 by enhancing cDC1 tumor infiltration via interferon type 1 activation.

Radiotherapy Cooperates with IL15 to Induce Antitumor Immune Responses.

Focal radiotherapy can promote cross-presentation of tumor antigens to T cells, but by itself, it is insufficient to induce therapeutically effective T-cell responses. The common gamma-chain cytokine IL15 promotes and sustains the proliferation and effector function of CD8+ T cells but has limited activity against poorly immunogenic tumors that do not elicit significant spontaneous T-cell responses. Here, we show that radiotherapy and subcutaneous IL15 had complementary effects and induced CD8+ T-cell-mediated tumor regression and long-term protective memory responses in two mouse carcinoma models unresponsive to IL15 alone. Mechanistically, radiotherapy-induced IFN type I production and Batf3-dependent conventional dendritic cells type 1 (cDC1) were required for priming of tumor-specific CD8+ T cells and for the therapeutic effect of the combination. IL15 cooperated with radiotherapy to activate and recruit cDC1s to the tumor. IL15 alone and in complex with a hybrid molecule containing the IL15α receptor have been tested in early-phase clinical trials in patients with cancer and demonstrated good tolerability, especially when given subcutaneously. Expansion of natural killer (NK) cells and CD8+ T cells was noted, without clear clinical activity, suggesting further testing of IL15 as a component of a combinatorial treatment with other agents. Our results provide the rationale for testing combinations of IL15 with radiotherapy in the clinic.

Converging focal radiation and immunotherapy in a preclinical model of triple negative breast cancer: contribution of VISTA blockade.

Antibodies targeting the co-inhibitory receptor programmed cell death 1 (PDCD1, best known as PD-1) or its main ligand CD274 (best known as PD-L1) have shown some activity in patients with metastatic triple-negative breast cancer (TNBC), especially in a recent Phase III clinical trial combining PD-L1 blockade with taxane-based chemotherapy. Despite these encouraging findings, however, most patients with TNBC fail to derive significant benefits from PD-L1 blockade, calling for the identification of novel therapeutic approaches. Here, we used the 4T1 murine mammary cancer model of metastatic and immune-resistant TNBC to test whether focal radiation therapy (RT), a powerful inducer of immunogenic cell death, in combination with various immunotherapeutic strategies can overcome resistance to immune checkpoint blockade. Our results suggest that focal RT enhances the therapeutic effects of PD-1 blockade against primary 4T1 tumors and their metastases. Similarly, the efficacy of an antibody specific for V-set immunoregulatory receptor (VSIR, another co-inhibitory receptor best known as VISTA) was enhanced by focal RT. Administration of cyclophosphamide plus RT and dual PD-1/VISTA blockade had superior therapeutic effects, which were associated with activation of tumor-infiltrating CD8+ T cells and depletion of intratumoral granulocytic myeloid-derived suppressor cells (MDSCs). Overall, these results demonstrate that RT can sensitize immunorefractory tumors to VISTA or PD-1 blockade, that this effect is enhanced by the addition of cyclophosphamide and suggest that a multipronged immunotherapeutic approach may also be required to increase the incidence of durable responses in patients with TNBC.

Formal Versus Informal Judgments: Faculty Experiences With Entrustment in Graduate Medical Education.

BACKGROUND: Entrustment of residents has been formalized in many competency-based graduate medical education programs, but its relationship with informal decisions to entrust residents with clinical tasks is unclear. In addition, the effects of formal entrustment on training practice are still unknown. OBJECTIVE: Our objective was to learn from faculty members in training programs with extensive experience in formal entrustment how formal entrustment relates to informal entrustment decisions. METHODS: A questionnaire was e-mailed to all Dutch obstetrics and gynecology program directors to gather information on how faculty entrusts residents with clinical independence. We also interviewed faculty members to explore the relationship between formal entrustment and informal entrustment. Interviews were analyzed with conventional content analysis. RESULTS: Of 92 programs, 54 program directors completed the questionnaire (59% response rate). Results showed that formal entrustment was seen as valuable for generating formative feedback and giving insight into residents' progress in technical competencies. Interviewed faculty members (n = 12) used both formal and informal entrustment to determine the level of resident independence. Faculty reported they tended to favor informal entrustment because it can be reconsidered. In contrast, formal entrustment was reported to feel like a fixed state. CONCLUSIONS: In a graduate medical education program where formal entrustment has been used for more than a decade, faculty used a combination of formal and informal entrustment. Informal entrustment is key in deciding if a resident can work independently. Faculty members reported being unsure how to optimally use formal entrustment in practice next to their informal decisions.

Radiotherapy and CTLA-4 Blockade Shape the TCR Repertoire of Tumor-Infiltrating T Cells.

Immune checkpoint inhibitors activate T cells to reject tumors. Unique tumor mutations are key T-cell targets, but a comprehensive understanding of the nature of a successful antitumor T-cell response is lacking. To investigate the T-cell receptor (TCR) repertoire associated with treatment success versus failure, we used a well-characterized mouse carcinoma that is rejected by CD8 T cells in mice treated with radiotherapy (RT) and anti-CTLA-4 in combination, but not as monotherapy, and comprehensively analyzed tumor-infiltrating lymphocytes (TILs) by high-throughput sequencing of the TCRΒ CDR3 region. The combined treatment increased TIL density and CD8/CD4 ratio. Assessment of the frequency of T-cell clones indicated that anti-CTLA-4 resulted in fewer clones and a more oligoclonal repertoire compared with untreated tumors. In contrast, RT increased the CD8/CD4 ratio and broadened the TCR repertoire, and when used in combination with anti-CTLA-4, these selected T-cell clones proliferated. Hierarchical clustering of CDR3 sequences showed a treatment-specific clustering of TCRs that were shared by different mice. Abundant clonotypes were commonly shared between animals and yet treatment-specific. Analysis of amino-acid sequence similarities revealed a significant increase in the number and richness of dominant CDR3 motifs in tumors treated with RT + anti-CTLA-4 compared with control. The repertoire of TCRs reactive with a single tumor antigen recognized by CD8+ T cells was heterogeneous but highly clonal, irrespective of treatment. Overall, data support a model whereby a diverse TCR repertoire is required to achieve tumor rejection and may underlie the synergy between RT and CTLA-4 blockade. Cancer Immunol Res; 6(2); 139-50. ©2017 AACR.

Exosomes Shuttle TREX1-Sensitive IFN-Stimulatory dsDNA from Irradiated Cancer Cells to DCs.

Radiotherapy (RT) used at immunogenic doses leads to accumulation of cytosolic double-stranded DNA (dsDNA) in cancer cells, which activates type I IFN (IFN-I) via the cGAS/STING pathway. Cancer cell-derived IFN-I is required to recruit BATF3-dependent dendritic cells (DC) to poorly immunogenic tumors and trigger antitumor T-cell responses in combination with immune checkpoint blockade. We have previously demonstrated that the exonuclease TREX1 regulates radiation immunogenicity by degrading cytosolic dsDNA. Tumor-derived DNA can also activate cGAS/STING-mediated production of IFN-I by DCs infiltrating immunogenic tumors. However, how DNA from cancer cells is transferred to the cytoplasm of DCs remains unclear. Here, we showed that tumor-derived exosomes (TEX) produced by irradiated mouse breast cancer cells (RT-TEX) transfer dsDNA to DCs and stimulate DC upregulation of costimulatory molecules and STING-dependent activation of IFN-I. In vivo, RT-TEX elicited tumor-specific CD8+ T-cell responses and protected mice from tumor development significantly better than TEX from untreated cancer cells in a prophylactic vaccination experiment. We demonstrated that the IFN-stimulatory dsDNA cargo of RT-TEX is regulated by TREX1 expression in the parent cells. Overall, these results identify RT-TEX as a mechanism whereby IFN-stimulatory dsDNA is transferred from irradiated cancer cells to DCs. We have previously shown that the expression of TREX1 is dependent on the RT dose size. Thus, these data have important implications for the use of RT with immunotherapy. Cancer Immunol Res; 6(8); 910-20. ©2018 AACR.