Immune Response following FLASH and Conventional Radiation in Diffuse Midline Glioma.

PURPOSE: Diffuse midline glioma (DMG) is a fatal tumor traditionally treated with radiation therapy (RT) and previously characterized as having a noninflammatory tumor immune microenvironment (TIME). FLASH is a novel RT technique using ultra-high dose rate that is associated with decreased toxicity and effective tumor control. However, the effect of FLASH and conventional (CONV) RT on the DMG TIME has not yet been explored. METHODS AND MATERIALS: Here, we performed single-cell RNA sequencing (scRNA-seq) and flow cytometry on immune cells isolated from an orthotopic syngeneic murine model of brainstem DMG after the use of FLASH (90 Gy/sec) or CONV (2 Gy/min) dose-rate RT and compared to unirradiated tumor (SHAM). RESULTS: At day 4 post-RT, FLASH exerted similar effects as CONV in the predominant microglial (MG) population, including the presence of two activated subtypes. However, at day 10 post-RT, we observed a significant increase in the type 1 interferon α/ß receptor (IFNAR+) in MG in CONV and SHAM compared to FLASH. In the non-resident myeloid clusters of macrophages (MACs) and dendritic cells (DCs), we found increased type 1 interferon (IFN1) pathway enrichment for CONV compared to FLASH and SHAM by scRNA-seq. We observed this trend by flow cytometry at day 4 post-RT in IFNAR+ MACs and DCs, which equalized by day 10 post-RT. DMG control and murine survival were equivalent between RT dose rates. CONCLUSIONS: Our work is the first to map CONV and FLASH immune alterations of the DMG TIME with single-cell resolution. Although DMG tumor control and survival were similar between CONV and FLASH, we found that changes in immune compartments differed over time. Importantly, although both RT modalities increased IFN1, we found that the timing of this response was cell-type and dose-rate dependent. These temporal differences, particularly in the context of tumor control, warrant further study.

Ultraviolet radiation shapes dendritic cell leukaemia transformation in the skin.

Tumours most often arise from progression of precursor clones within a single anatomical niche. In the bone marrow, clonal progenitors can undergo malignant transformation to acute leukaemia, or differentiate into immune cells that contribute to disease pathology in peripheral tissues1-4. Outside the marrow, these clones are potentially exposed to a variety of tissue-specific mutational processes, although the consequences of this are unclear. Here we investigate the development of blastic plasmacytoid dendritic cell neoplasm (BPDCN)-an unusual form of acute leukaemia that often presents with malignant cells isolated to the skin5. Using tumour phylogenomics and single-cell transcriptomics with genotyping, we find that BPDCN arises from clonal (premalignant) haematopoietic precursors in the bone marrow. We observe that BPDCN skin tumours first develop at sun-exposed anatomical sites and are distinguished by clonally expanded mutations induced by ultraviolet (UV) radiation. A reconstruction of tumour phylogenies reveals that UV damage can precede the acquisition of alterations associated with malignant transformation, implicating sun exposure of plasmacytoid dendritic cells or committed precursors during BPDCN pathogenesis. Functionally, we find that loss-of-function mutations in Tet2, the most common premalignant alteration in BPDCN, confer resistance to UV-induced cell death in plasmacytoid, but not conventional, dendritic cells, suggesting a context-dependent tumour-suppressive role for TET2. These findings demonstrate how tissue-specific environmental exposures at distant anatomical sites can shape the evolution of premalignant clones to disseminated cancer.

Moderate Low UVB Irradiation Modulates Tumor-associated Macrophages and Dendritic Cells and Promotes Antitumor Immunity in Tumor-bearing Mice.

Excessive, high doses of ultraviolet B (UVB) UVB irradiation are known to cause skin cancer, aging and immunosuppression. On the contrary, moderate low doses of UVB irradiation are shown to be essential and beneficial to human health, including a tumor-suppressive effect. However, the mechanism by which low levels of UVB suppress tumorigenesis remains unclear. Here, using tumor-bearing mouse models, we show that moderate low repetitive UVB irradiation increases the percentage of activated CD4+ and CD8+ T cells, and CD103+ conventional type 1 dendritic cells (cDC1s), while it decreases the number of immunosuppressive, M2-like macrophages in the tumors. Finally, in mice, deletion of Batf3, a transcription factor critical for the development of conventional dendritic cells, including the CD103+ cDC1s, showed increased tumor growth in both sham- and UVB-irradiated mice. Our findings demonstrate that moderate low UVB irradiation inhibits M2-like tumor-associated macrophages, increases CD103+ cDC1s and promotes antitumor immunity in mice with an established tumor.

Theranostic near-infrared-IIb emitting nanoprobes for promoting immunogenic radiotherapy and abscopal effects against cancer metastasis.

Radiotherapy is an important therapeutic strategy for cancer treatment through direct damage to cancer cells and augmentation of antitumor immune responses. However, the efficacy of radiotherapy is limited by hypoxia-mediated radioresistance and immunosuppression in tumor microenvironment. Here, we construct a stabilized theranostic nanoprobe based on quantum dots emitting in the near-infrared IIb (NIR-IIb, 1,500-1,700 nm) window modified by catalase, arginine-glycine-aspartate peptides and poly(ethylene glycol). We demonstrate that the nanoprobes effectively aggregate in the tumor site to locate the tumor region, thereby realizing precision radiotherapy with few side-effects. In addition, nanoprobes relieve intratumoral hypoxia and reduce the tumor infiltration of immunosuppressive cells. Moreover, the nanoprobes promote the immunogenic cell death of cancer cells to trigger the activation of dendritic cells and enhance T cell-mediated antitumor immunity to inhibit tumor metastasis. Collectively, the nanoprobe-mediated immunogenic radiotherapy can boost the abscopal effect to inhibit tumor metastasis and prolong survival.

Vitamin D-independent benefits of safe sunlight exposure.

This review examines the beneficial effects of ultraviolet radiation on systemic autoimmune diseases, including multiple sclerosis and type I diabetes, where the epidemiological evidence for the vitamin D-independent effects of sunlight is most apparent. Ultraviolet radiation, in addition to its role in the synthesis of vitamin D, stimulates anti-inflammatory pathways, alters the composition of dendritic cells, T cells, and T regulatory cells, and induces nitric oxide synthase and heme oxygenase metabolic pathways, which may directly or indirectly mitigate disease progression and susceptibility. Recent work has also explored how the immune-modulating functions of ultraviolet radiation affect type II diabetes, cancer, and the current global pandemic caused by SARS-CoV-2. These diseases are particularly important amidst global changes in lifestyle that result in unhealthy eating, increased sedentary habits, and alcohol and tobacco consumption. Compelling epidemiological data shows increased ultraviolet radiation associated with reduced rates of certain cancers, such as colorectal cancer, breast cancer, non-Hodgkins lymphoma, and ultraviolet radiation exposure correlated with susceptibility and mortality rates of COVID-19. Therefore, understanding the effects of ultraviolet radiation on both vitamin D-dependent and -independent pathways is necessary to understand how they influence the course of many human diseases.

More Than Effects in Skin: Ultraviolet Radiation-Induced Changes in Immune Cells in Human Blood.

Cells of the skin and circulation are in constant two-way communication. Following exposure of humans to sunlight or to phototherapy, there are alterations in the number, phenotype and function of circulating blood cells. In this review, only data obtained from human studies are considered, with changes induced by UV radiation (UVR) exposure described for phagocytic leukocytes and peripheral blood mononuclear cells plus their component T and B cells, natural killer cells and dendritic cells. These immune modulations illustrate the potential of UVR to have therapeutic effects beyond the skin, and that sunlight exposure is an important environmental influence on human health.

The abscopal effect of radiation therapy.

Radiation therapy (RT) in some cases results in a systemic anticancer response known as the abscopal effect. Multiple hypotheses support the role of immune activation initiated by RT-induced DNA damage. Optimal radiation dose is necessary to promote the cGAS-STING pathway in response to radiation and initiate an IFN-1 signaling cascade that promotes the maturation and migration of dendritic cells to facilitate antigen presentation and stimulation of cytotoxic T cells. T cells then exert a targeted response throughout the body at areas not subjected to RT. These effects are further augmented through the use of immunotherapeutic drugs resulting in increased T-cell activity. Tumor-infiltrating lymphocyte presence and TREX1, KPNA2 and p53 signal expression are being explored as prognostic biomarkers.

Enlightening the Immune Mechanism of the Abscopal Effect in a Murine HCC Model and Overcoming the Late Resistance With Anti-PD-L1.

PURPOSE: The establishment of a preclinical model of the abscopal effect on hepatocellular carcinoma (HCC) and evaluation of whether the hypofractionated radiation therapy (RT) multitumor Hepa1-6 mouse HCC model could be used to suppress nonradiated tumor mass was performed in this study. METHODS AND MATERIALS: Hepa1-6 mouse liver cancer cell lines were used to form tumors. Immunogenicity was analyzed using ELISpot and immune cell labeled antibody. Interferon (IFN) ß expression was confirmed through polymerase chain reaction. RESULTS: After investigation, the intratumoral transcription of type Ⅰ IFN increased by 2-fold. The antitumor immune response to Hepa 1-6 cells induced by radiation was increased. Moreover, the influx of activated CD8+ T cells was increased in nonirradiated tumors. The number of dendritic cells and activation status were evaluated by flow cytometry on the second day after irradiation. Flow cytometry revealed a significantly increased dendritic cell population expressing the CD11c molecule in tumor-draining lymph nodes. Furthermore, because irradiation leads to adaptation of immune resistance of tumor cells against RT, we sought to elucidate a potent tool to overcome the resistance and confirm the ability of PD-L1 antibody to survive late RT resistance. CONCLUSIONS: The immunologic mechanism of the abscopal effect was revealed and the application of PD-L1 inhibitor successfully performed as a breakthrough in late RT resistance in the Hepa1-6 tumor model.

Use of Ultraviolet Blood Irradiation Against Viral Infections.

Ultraviolet blood irradiation (UBI) was used with success in the 1930s and 1940s for a variety of diseases. Despite the success, the lack of understanding of the detailed mechanisms of actions, and the achievements of antibiotics, phased off the use of UBI from the 1950s. The emergence of novel viral infections, from HIV/AIDS to Ebola, from SARS and MERS, and SARS-CoV-2, bring back the attention to this therapeutical opportunity. UBI has a complex virucidal activity, mostly acting on the immune system response. It has effects on lymphocytes (T-cells and B-cells), macrophages, monocytes, dendritic cells, low-density lipoprotein (LDL), and lipids. The Knott technique was applied for bacterial infections such as tuberculosis to viral infections such as hepatitis or influenza. The more complex extracorporeal photopheresis (ECP) is also being applied to hematological cancers such as T-cell lymphomas. Further studies of UBI may help to create a useful device that may find applications for novel viruses that are resistant to known antivirals or vaccines, or also bacteria that are resistant to known antibiotics.

Single High-Dose Radiation Enhances Dendritic Cell Homing and T Cell Priming by Promoting Reactive Oxygen Species-Induced Cytoskeletal Reorganization.

PURPOSE: Radiation therapy (RT) affects tumor-infiltrating immune cells, cooperatively driving tumor growth inhibition. However, there is still no absolute consensus on whether the homing ability of dendritic cells (DCs) is affected by direct x-ray irradiation. Most importantly, the underlying mechanisms are poorly understood. METHODS AND MATERIALS: Using noninvasive imaging, we systematically examined the dose effect of RT on the in vivo homing and distribution of bone marrow-derived DCs and elucidated the detailed mechanisms underlying these events. After exposure to 2, 5, 10, 15, and 20 Gy, DCs were analyzed for maturation, in vivo homing ability, and T cell priming. RESULTS: At ranges of 2 to 20 Gy, irradiation did not cause direct cellular apoptosis or necrosis, but it induced mitochondrial damage in DCs independent of dose. In addition, upregulation of CD40, CD80, CD86, CXCR4, and CCR7 were detected on irradiated DCs. Secretion of IL-1ß and IL-12p70 remained unchanged, whereas decreased secretion of IL-6 and promotion of tumor necrosis factor α secretion were observed. In particular, the homing ability of both the local residual and blood circulating DCs to lymphoid tissues was significantly higher in groups that received ≥5 Gy radiation than in the group that received 2 Gy. Furthermore, improved homing ability was associated with rearrangement of the cytoskeleton, which was regulated by reactive oxygen species accumulation through the RhoA/ROCK1 signaling pathway. Finally, more robust T cell activation was observed in mice inoculated with 20 Gy-treated DCs than in those inoculated with 2 Gy-irradiated DCs, and T cell activation also correlated with reactive oxygen species production. CONCLUSIONS: An RT dose ≥5 Gy has distinct advantages over 2 Gy in facilitating DC homing to lymph nodes and cross-priming T cells.

RIG-I-Like Receptor LGP2 Is Required for Tumor Control by Radiotherapy.

Dendritic cells (DC) play an essential role in innate immunity and radiation-elicited immune responses. LGP2 is a RIG-I-like receptor involved in cytoplasmic RNA recognition and antiviral responses. Although LGP2 has also been linked to cell survival of both tumor cells and T cells, the role of LGP2 in mediating DC function and antitumor immunity elicited by radiotherapy remains unclear. Here, we report that tumor DCs are linked to the clinical outcome of patients with breast cancer who received radiotherapy, and the presence of DC correlates with gene expression of LGP2 in the tumor microenvironment. In preclinical models, host LGP2 was essential for optimal antitumor control by ionizing radiation (IR). The absence of LGP2 in DC dampened type I IFN production and the priming capacity of DC. In the absence of LGP2, MDA5-mediated activation of type I IFN signaling was abrogated. The MDA5/LGP2 agonist high molecular weight poly I:C improved the antitumor effect of IR. This study reveals a previously undefined role of LGP2 in host immunity and provides a new strategy to improve the efficacy of radiotherapy. SIGNIFICANCE: These findings reveal an essential role of LGP2 in promoting antitumor immunity after radiotherapy and provide a new strategy to enhance radiotherapy.

Bendamustine Conditioning Skews Murine Host DCs Toward Pre-cDC1s and Reduces GvHD Independently of Batf3.

Graft-versus-host disease (GvHD) remains the second leading cause of death in allogeneic hematopoietic stem cell transplantation recipients, highlighting the need for improved preventative strategies. Our laboratory has previously demonstrated in an experimental bone marrow transplantation (BMT) model that bendamustine combined with total body irradiation (BEN+TBI) is a safer alternative to cyclophosphamide with TBI (CY+TBI). The biological mechanisms of action of BEN have not been fully elucidated and likely involve multiple cell populations. Host dendritic cells (DCs) can prime naïve donor T-cells immediately following transplantation, making host DCs critical for the initiation phase of GvHD. We hypothesized that BEN+TBI conditioning favorably alters host DC composition to reduce GvHD. We demonstrate that host DCs treated with BEN+TBI induce less allogeneic T-cell proliferation than those conditioned with CY+TBI. We further show that BEN+TBI conditioning results in greater total numbers of all host DC subsets but with a more favorable composition compared to CY+TBI with significantly larger proportions of type 1 conventional DCs (cDC1), a highly regulatory DC subset capable of suppressing GvHD. Our studies using recipient Batf3 KO mice indicate that CD8α+ cDC1s are largely dispensable for the reduced GvHD following BEN+TBI conditioning. We found a higher frequency of host pre-cDC1s with BEN+TBI conditioning in both wild-type (WT) and Batf3 KO mice, which was inversely associated with GvHD. Additionally, we observed that BEN treatment results in greater expression of Flt3 receptor (CD135) on host DCs compared to CY, potentially contributing to the skewing of host DCs toward cDC1s. Further, BEN+TBI conditioning results in host cDCs with greater expression of PIR-B, an inhibitory receptor capable of preventing lethal GvHD. We conclude that BEN+TBI is a safer alternative to CY+TBI, resulting in a greater frequency of host pre-cDC1s and limiting GvHD.

Radiation Therapy and the In Situ Vaccination Approach.

During the past century, from the advent of preclinical modeling to the establishment of clinical trials, the hypothesis that host defenses regulate tumor growth (posited and refined by leaders in the field of cancer immunity) has become accepted as a scientific pillar in oncology. Since the turn of the millennium, a search has been under way for the best therapeutic approach to reprogram the immune system to recognize tumor cells that have undergone "immune escape." This quest has led some to question conventional scientific views of tumor cell kill, including the role of host immunity in patients treated with radiation therapy. In the last two decades, evidence has accumulated that radiation therapy can effectively convert a potentially lethal cancer into an in situ personalized vaccine. Herein, we review the underlying mechanisms and maneuvers responsible for in situ vaccine production.

Wee1 kinase inhibitor AZD1775 potentiates CD8+ T cell-dependent antitumour activity via dendritic cell activation following a single high dose of irradiation.

As standard treatments for cancer, DNA-damaging chemotherapeutic agents and irradiation therapy improve survival in patients with various cancers. Wee1, a kinase associated with the cell cycle, causes G2/M cell cycle arrest to allow repair of injured DNA in cancer cells, and a Wee1 inhibitor has been confirmed to lead to apoptosis in cancer cells. Recently, there has been renewed interest in exploring the immune environment which plays a significant role in tumour suppression. A Wee1 inhibitor combined with radiotherapy has been tested in lung, pancreatic, and prostate cancer and melanoma in vivo or in vitro. There is still no research evaluating the immunoregulatory effects of AZD1775 plus high-dose irradiation (IR) in vivo. T cell killing and CD8+ T cell depletion assays demonstrated that the combination of AZD1775 and IR delayed tumour growth in breast cancer mouse models. Additionally, combination treatment also suppressed the expression of PD-L1, a co-inhibitor, through the STAT3-IRF1 axis. The importance and originality of this study are that it explores the internal and external mechanisms of AZD1775 combined with a single high dose of IR and provides a rationale for applying the combination therapy described above in a clinical trial.

Radiation with STAT3 Blockade Triggers Dendritic Cell-T cell Interactions in the Glioma Microenvironment and Therapeutic Efficacy.

PURPOSE: Patients with central nervous system (CNS) tumors are typically treated with radiotherapy, but this is not curative and results in the upregulation of phosphorylated STAT3 (p-STAT3), which drives invasion, angiogenesis, and immune suppression. Therefore, we investigated the combined effect of an inhibitor of STAT3 and whole-brain radiotherapy (WBRT) in a murine model of glioma. EXPERIMENTAL DESIGN: C57BL/6 mice underwent intracerebral implantation of GL261 glioma cells, WBRT, and treatment with WP1066, a blood-brain barrier-penetrant inhibitor of the STAT3 pathway, or the two in combination. The role of the immune system was evaluated using tumor rechallenge strategies, immune-incompetent backgrounds, immunofluorescence, immune phenotyping of tumor-infiltrating immune cells (via flow cytometry), and NanoString gene expression analysis of 770 immune-related genes from immune cells, including those directly isolated from the tumor microenvironment. RESULTS: The combination of WP1066 and WBRT resulted in long-term survivors and enhanced median survival time relative to monotherapy in the GL261 glioma model (combination vs. control P < 0.0001). Immunologic memory appeared to be induced, because mice were protected during subsequent tumor rechallenge. The therapeutic effect of the combination was completely lost in immune-incompetent animals. NanoString analysis and immunofluorescence revealed immunologic reprograming in the CNS tumor microenvironment specifically affecting dendritic cell antigen presentation and T-cell effector functions. CONCLUSIONS: This study indicates that the combination of STAT3 inhibition and WBRT enhances the therapeutic effect against gliomas in the CNS by inducing dendritic cell and T-cell interactions in the CNS tumor.

Photodepletion with 2-Se-Cl prevents lethal graft-versus-host disease while preserving antitumor immunity.

Acute graft-versus-host-disease (GVHD), limits the use of hematopoietic cell transplant (HCT) to treat a variety of malignancies. Any new therapeutic approach must satisfy three requirements: 1) Prevent GVHD, 2) Maintain anti-pathogen immunity, and 3) Maintain anti-tumor immunity. In prior studies we have shown that the selective photosensitizer 2-Se-Cl eliminates highly alloreactive lymphocytes from the graft prior to HCT preventing GVHD and that antiviral immune responses were preserved following incubation with 2-Se-Cl. In this report, we investigated whether 2-Se-Cl treatment preserves antitumor immunity, and then used high dimensional flow cytometry to identify the determinants of successful immune reconstitution. Donor C57BL/6 splenocytes were cocultured for 4 days with irradiated BALB/c splenocytes and then exposed to 2-Se-Cl. Photodepletion (PD)-treated splenocytes were then infused into lethally irradiated BALB/c mice inoculated with A20 leukemia/lymphoma cells. Recipient mice that received PD-treated splenocytes survived > 100 days without evidence of GVHD or leukemia. In contrast, mice that did not receive PD-treated cells at time of HCT died of leukemia progression. Multiparameter flow cytometry of cytokines and surface markers on peripheral blood samples 15 days after HCT demonstrated unique patterns of immune reconstitution. We found that before clinical disease onset GVHD was marked by functionally exhausted T cells, while tumor clearance and long-term survival were associated with an expansion of polyfunctional T cells, monocytes, and DCs early after transplantation. Taken together these results demonstrate that 2-Se-Cl photodepletion is a new treatment that can facilitate HCT by preventing GVHD while preserving antiviral and anti-tumor immunity.

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.

Radiation-induced augmentation in dendritic cell function is mediated by apoptotic bodies/STAT5/Zbtb46 signaling.

Purpose: To evaluate the effect of ionizing radiation (IR) exposure on differentiation and maturation of dendritic cells (DC).Materials and methods: Bone marrow progenitor cells irradiated in vitro or isolated from mice exposed to whole body or localized tumor irradiation were differentiated into DC. Phenotypic maturation of DC was characterized by labeling with specific antibodies and flow cytometry analysis. Cytokines were estimated by ELISA.Results: Splenic and bone marrow-derived DC (BMDC) from tumor-bearing mice exposed to localized irradiation showed abrogation of tumor-induced immunosuppression. This was not due to the effect of radiation on tumor cells as DC derived from normal mice exposed to whole-body irradiation (WBI) also showed increase in immune-activating potential of DC. This was observed in terms of increased phenotypic and functional activation of DCs. This phenomenon was also recapitulated if DC were differentiated from in vitro irradiated progenitor cells and was found to be due to STAT5/Zbtb46 signaling mediated by the irradiation-induced apoptotic bodies (ABs). When these ABs were depleted using annexin-beads, these effects were reversed confirming the involvement of this pathway. The role of ABs was further validated in DC derived from mice exposed to WBI in adaptive response experiments with 0.1 Gy priming dose prior to 2 Gy challenge dose. A corresponding reduction in DC maturation markers was observed with decrease in apoptosis in vivo. Further, these DCs derived from irradiated progenitors (IP) could resist the suppressive effects of tumor conditioned medium (TCM) and had increased immune-activating potential as seen in the tumor-bearing mice.Conclusions: Though radiation is the most commonly used therapeutic modality for cancer, its effects on dendritic cell differentiation is not completely understood. We demonstrate here for the first time that exposure to select doses of IR can increase immune-activating potential of DC through ABs. This can have implications in selection of appropriate doses of IR during radiotherapy of cancer patients.

pH-Sensitive Carbon Dots for Enhancing Photomediated Antitumor Immunity.

Recent cancer immunotherapy has attracted much attention due to high specificity and recurrence prevention of tumor. Nevertheless, its therapeutic effects are still challenging in solid cancer. To establish superior antitumor immunity, chlorin e6 (Ce6)-loaded pH sensitive carbon dots were investigated (Ce6@IDCDs). At tumoral pH 6.5, Ce6 was released four times compared with the release at physiological pH 7.4 due to an imbalance between hydrophilic and hydrophobic forces via protonation of imidazole groups in Ce6@IDCDs. This result led to the superior singlet oxygen generating activity of Ce6@IDCDs without Ce6 quenching. The maturation effects of dendritic cells after co-incubation with supernatant media obtained from Ce6@IDCDs with laser-treated cells at pH 6.5 were much higher than at physiological pH. Furthermore, Ce6@IDCDs following a laser at pH 6.5 significantly promoted calreticulin exposure and high-mobility group box 1 release, as major immunogenic cell death markers. In bilateral CT-26-bearing mice model, the Ce6@IDCDs elicited significant antitumoral effects at laser treated-primary tumor regions via therapeutic reactive oxygen species. Furthermore, Ce6@IDCDs upon laser irradiation induced a large amount of activated CD8+ T cells, natural killer cells, and mature dendritic cells recruitment into tumoral tissue and hampered tumor growth even at untreated sites approximately four-fold compared with those of others. Overall, this pH-sensitive immunoinducer can accomplish primary and distant tumor ablation via photomediated cancer immunotherapy.

Therapeutic potential of lipids obtained from γ-irradiated PBMCs in dendritic cell-mediated skin inflammation.

BACKGROUND: Since numerous pathological conditions are evoked by unwanted dendritic cell (DC) activity, therapeutic agents modulating DC functions are of great medical interest. In regenerative medicine, cellular secretomes have gained increasing attention and valuable immunomodulatory properties have been attributed to the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCs). Potential effects of the PBMC secretome (PBMCsec) on key DC functions have not been elucidated so far. METHODS: We used a hapten-mediated murine model of contact hypersensitivity (CH) to study the effects of PBMCsec on DCs in vivo. Effects of PBMCsec on human DCs were investigated in monocyte-derived DCs (MoDC) and ex vivo skin cultures. DCs were phenotypically characterised by transcriptomics analyses and flow cytometry. DC function was evaluated by cytokine secretion, antigen uptake, PBMC proliferation and T-cell priming. FINDINGS: PBMCsec significantly alleviated tissue inflammation and cellular infiltration in hapten-sensitized mice. We found that PBMCsec abrogated differentiation of MoDCs, indicated by lower expression of classical DC markers CD1a, CD11c and MHC class II molecules. Furthermore, PBMCsec reduced DC maturation, antigen uptake, lipopolysaccharides-induced cytokine secretion, and DC-mediated immune cell proliferation. Moreover, MoDCs differentiated with PBMCsec displayed diminished ability to prime naïve CD4+T-cells into TH1 and TH2 cells. Furthermore, PBMCsec modulated the phenotype of DCs present in the skin in situ. Mechanistically, we identified lipids as the main biomolecule accountable for the observed immunomodulatory effects. INTERPRETATION: Together, our data describe DC-modulatory actions of lipids secreted by stressed PBMCs and suggest PBMCsec as a therapeutic option for treatment of DC-mediated inflammatory skin conditions. FUNDING: This research project was supported by the Austrian Research Promotion Agency (Vienna, Austria; grant "APOSEC" 862068; 2015-2019) and the Vienna Business Agency (Vienna, Austria; grant "APOSEC to clinic" 2343727).