T cells as key players for bone destruction in gouty arthritis?

The deposition of monosodium urate (MSU) crystals in synovial fluid and tissue leads to gouty arthritis frequently associated with synovial inflammation and bone erosions. The cellular mechanism that links MSU crystals to an increased number of osteoclasts has not yet been fully understood. In a recent issue of Arthritis Research & Therapy Lee and colleagues proposed that bone destruction in chronic gouty arthritis is at least in part dependent on expression by T cells of receptor activator of NF-κB ligand (RANKL). The authors showed that pro-resorptive cytokines such as IL-1β, IL-6, and TNFα are expressed within tophi and stromal infiltrates. In vitro stimulation with MSU crystals revealed monocytes as a source for these cytokines, whereas T cells produce RANKL, the major trigger of osteoclastogenesis

Recruitment kinetics of DNA repair proteins Mdc1 and Rad52 but not 53BP1 depend on damage complexity.

The recruitment kinetics of double-strand break (DSB) signaling and repair proteins Mdc1, 53BP1 and Rad52 into radiation-induced foci was studied by live-cell fluorescence microscopy after ion microirradiation. To investigate the influence of damage density and complexity on recruitment kinetics, which cannot be done by UV laser irradiation used in former studies, we utilized 43 MeV carbon ions with high linear energy transfer per ion (LET = 370 keV/µm) to create a large fraction of clustered DSBs, thus forming complex DNA damage, and 20 MeV protons with low LET (LET = 2.6 keV/µm) to create mainly isolated DSBs. Kinetics for all three proteins was characterized by a time lag period T(0) after irradiation, during which no foci are formed. Subsequently, the proteins accumulate into foci with characteristic mean recruitment times τ(1). Mdc1 accumulates faster (T(0) = 17 ± 2 s, τ(1) = 98 ± 11 s) than 53BP1 (T(0) = 77 ± 7 s, τ(1) = 310 ± 60 s) after high LET irradiation. However, recruitment of Mdc1 slows down (T(0) = 73 ± 16 s, τ(1) = 1050 ± 270 s) after low LET irradiation. The recruitment kinetics of Rad52 is slower than that of Mdc1, but exhibits the same dependence on LET. In contrast, the mean recruitment time τ(1) of 53BP1 remains almost constant when varying LET. Comparison to literature data on Mdc1 recruitment after UV laser irradiation shows that this rather resembles recruitment after high than low LET ionizing radiation. So this work shows that damage quality has a large influence on repair processes and has to be considered when comparing different studies

Fractionated radiotherapy is the main stimulus for the induction of cell death and of Hsp70 release of p53 mutated glioblastoma cell lines

BACKGROUND: Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Despite a multimodal therapy consisting of resection followed by fractionated radiotherapy (RT) combined with the chemotherapeutic agent (CT) temozolomide (TMZ), its recurrence is almost inevitable. Since the immune system is capable of eliminating small tumor masses, a therapy should also aim to stimulate anti-tumor immune responses by induction of immunogenic cell death forms. The histone deacetylase inhibitor valproic acid (VPA) might foster this. METHODS: Reflecting therapy standards, we applied in our in vitro model fractionated RT with a single dose of 2Gy and clinically relevant concentrations of CT. Not only the impact of RT and/or CT with TMZ and/or VPA on the clonogenic potential and cell cycle of the glioblastoma cell lines T98G, U251MG, and U87MG was analyzed, but also the resulting cell death forms and release of danger signals such as heat-shock protein70 (Hsp70) and high-mobility group protein B1 (HMGB1). RESULTS: The clonogenic assays revealed that T98G and U251MG, having mutated tumor suppressor protein p53, are more resistant to RT and CT than U87MG with wild type (WT) p53. In all glioblastoma cells lines, fractionated RT induced a G2 cell cycle arrest, but only in the case of U87MG, TMZ and/or VPA alone resulted in this cell cycle block. Further, fractionated RT significantly increased the number of apoptotic and necrotic tumor cells in all three cell lines. However, only in U87MG, the treatment with TMZ and/or VPA alone, or in combination with fractionated RT, induced significantly more cell death compared to untreated or irradiated controls. While necrotic glioblastoma cells were present after VPA, TMZ especially led to significantly increased amounts of U87MG cells in the radiosensitive G2 cell cycle phase. While CT did not impact on the release of Hsp70, fractionated RT resulted in significantly increased extracellular concentrations of Hsp70 in p53 mutated and WT glioblastoma cells. CONCLUSIONS: Our results indicate that fractionated RT is the main stimulus for induction of glioblastoma cell death forms with immunogenic potential. The generated tumor cell microenvironment might be beneficial to include immune therapies for GBM in the future

Radio-Immunotherapy-Induced Immunogenic Cancer Cells as Basis for Induction of Systemic Anti-Tumor Immune Responses – Pre-Clinical Evidence and Ongoing Clinical Applications

Radiotherapy (RT) primarily aims to locally destroy the tumor via the induction of DNA damage in the tumor cells. However, the so-called abscopal, namely systemic and immune–mediated, effects of RT move over more and more in the focus of scientists and clinicians since combinations of local irradiation with immune therapy have been demonstrated to induce anti-tumor immunity. We here summarize changes of the phenotype and microenvironment of tumor cells after exposure to irradiation, chemotherapeutic agents, and immune modulating agents rendering the tumor more immunogenic. The impact of therapy-modified tumor cells and damage-associated molecular patterns on local and systemic control of the primary tumor, recurrent tumors, and metastases will be outlined. Finally, clinical studies affirming the bench-side findings of interactions and synergies of radiation therapy and immunotherapy will be discussed. Focus is set on combination of radio(chemo)therapy (RCT) with immune checkpoint inhibitors, growth factor inhibitors, and chimeric antigen receptor T-cell therapy. Well-deliberated combination of RCT with selected immune therapies and growth factor inhibitors bear the great potential to further improve anti-cancer therapies

Modern Radiotherapy Concepts and the Impact of Radiation on Immune Activation

Even though there is extensive research carried out in radiation oncology, most of the clinical studies focus on the effects of radiation on the local tumor tissue and deal with normal tissue side effects. The influence of dose fractionation and timing particularly with regard to immune activation is not satisfactorily investigated so far. This review, therefore, summarizes current knowledge on concepts of modern radiotherapy (RT) and evaluates the potential of RT for immune activation. Focus is set on radiation-induced forms of tumor cell death and consecutively the immunogenicity of the tumor cells. The so-called non-targeted, abscopal effects can contribute to anti-tumor responses in a specific and systemic manner and possess the ability to target relapsing tumor cells as well as metastases. The impact of distinct RT concepts on immune activation is outlined and pre-clinical evidence and clinical observations on RT-induced immunity will be discussed. Knowledge on the radiosensitivity of immune cells as well as clinical evidence for enhanced immunity after RT will be considered. While stereotactic ablative body radiotherapy seem to have a beneficial outcome over classical RT fractionation in pre-clinical animal models, in vitro model systems suggest an advantage for classical fractionated RT for immune activation. Furthermore, the optimal approach may differ based on the tumor site and/or genetic signature. These facts highlight that clinical trials are urgently needed to identify whether high-dose RT is superior to induce anti-tumor immune responses compared to classical fractionated RT and in particular how the outcome is when RT is combined with immunotherapy in selected tumor entities. The primary goal of radiotherapy (RT) in cancer therapy is to eliminate the disease by restricting the reproductive potential of tumor cells. This is achieved by the induction of tumor cell death as well as the inhibition of the proliferating capacity of the cells (1). RT is a valuable therapy that is able to control tumor growth, eliminate the tumor, reduce the risk of cancer recurrence, and ultimately to improve survival (2). Radiation predominantly induces DNA damage in the cells (3) and some of its most prominent consequences are apoptosis, necrosis, mitotic catastrophe (MC), autophagy, cell cycle arrest, and/or senescence (Figure 1). About 60% of patients with solid tumors receive RT [15% RT monotherapy, 45% radiochemotherapy (RCT)], making it the most common treatment option for cancer (4, 5). Recent advances in radiation technologies have opened the field for new and promising radiation strategies, such as the stereotactic ablative body radiotherapy (SABR). However, while it has become generally accepted that RT is capable of inducing anti-tumor immunity (6), little is known about the effects of particular high-dose RT on the immune system. In this review, we will, therefore, deal with radiation-induced cell responses, current state-of-the-art radiation protocols, as well as the direct or indirect impact of fractionation schemes and radiation doses on the cells of the immune system, including limitations and draw-backs of today’s radiation research

Chemoradiation Increases PD-L1 Expression in Certain Melanoma and Glioblastoma Cells

Immunotherapy approaches currently make their way into the clinics to improve the outcome of standard radiochemotherapy (RCT). The programed cell death receptor ligand 1 (PD-L1) is one possible target that, upon blockade, allows T cell-dependent antitumor immune responses to be executed. To date, it is unclear which RCT protocol and which fractionation scheme leads to increased PD-L1 expression and thereby renders blockade of this immune suppressive pathway reasonable. We therefore investigated the impact of radiotherapy (RT), chemotherapy (CT), and RCT on PD-L1 surface expression on tumor cells of tumor entities with differing somatic mutation prevalence. Murine melanoma (B16-F10), glioblastoma (GL261-luc2), and colorectal (CT26) tumor cells were treated with dacarbazine, temozolomide, and a combination of irinotecan, oxaliplatin, and fluorouracil, respectively. Additionally, they were irradiated with a single dose [10 Gray (Gy)] or hypo-fractionated (2 × 5 Gy), respectively, norm-fractionated (5 × 2 Gy) radiation protocols were used. PD-L1 surface and intracellular interferon (IFN)-gamma expression was measured by flow cytometry, and IL-6 release was determined by ELISA. Furthermore, tumor cell death was monitored by AnnexinV-FITC/7-AAD staining. For first in vivo analyses, the B16-F10 mouse melanoma model was chosen. In B16-F10 and GL261-luc2 cells, particularly norm-fractionated and hypo-fractionated radiation led to a significant increase of surface PD-L1, which could not be observed in CT26 cells. Furthermore, PD-L1 expression is more pronounced on vital tumor cells and goes along with increased levels of IFN-gamma in the tumor cells. In melanoma cells CT was the main trigger for IL-6 release, while in glioblastoma cells it was norm-fractionated RT. In vivo, fractionated RT only in combination with dacarbazine induced PD-L1 expression on melanoma cells. Our results suggest a tumor cell-mediated upregulation of PD-L1 expression following in particular chemoradiation that is not only dependent on the somatic mutation prevalence of the tumor entity

The novel cytokine interleukin-36α is expressed in psoriatic and rheumatoid arthritis synovium

Background: Interleukin (IL)-36α is a recently described member of the IL-1 cytokine family with pro-inflammatory and clearly pathogenic properties in psoriasis. Objective: To determine the IL-36α expression in psoriatic arthritis (PsA) compared to rheumatoid arthritis (RA) and osteoarthritis (OA). Methods: Synovial tissues obtained from arthritis patients were stained for IL-36α, IL-36 receptor (IL-36R) and IL-36R antagonist (IL-36Ra) by immunohistochemistry and immunofluorescence. Lysates were examined for IL-36α by western blot analysis. Synovial fibroblasts (FLS) cultured in the presence of IL-36α were assayed for cytokine expression by quantitative real time PCR and multiplex assay. IL-36α-induced signal transduction in FLS was analysed by immunoblotting. Results: Expression of IL-36R and its ligands IL-36α and IL-36Ra was detected in the synovial lining layer and cellular infiltrates of patients with inflammatory arthritis. IL-36α was expressed significantly higher in PsA and RA than in OA synovium. CD138-positive plasma cells were identified as the main cellular source of IL-36α. No differences were observed for the expression of IL-36R and IL-36Ra between PsA, RA and OA. Functionally, IL-36α induced the expression of IL-6 and IL-8 in FLS through p38/NFkB activation. Conclusions: IL-36α is up-regulated in PsA and RA synovium, expressed by tissue plasma cells and leads to IL-6 and IL-8 production by synovial fibroblasts. Hence, IL-36α links plasma cells to inflammatory cytokine production by FLS and may represent a key link between autoimmunity and the induction of synovitis

Deletion of the receptor tyrosine kinase Tyro3 inhibits synovial hyperplasia and bone damage in arthritis

Objective: To test whether the tyrosine kinase Tyro3 affects arthritis. Tyro3, the ligand of growth arrest–specific protein 6 (GAS6) is a receptor tyrosine kinase involved in cell survival. Tyro3 and GAS6 are expressed in the arthritic synovium, and in vitro studies have shown their role in osteoclast differentiation. Methods: Bone was assessed by micro CT and histomorphometry in Tyro3-deficient (Tyro3−/−) and wild-type mice. Arthritis was induced in both genotypes, and Gas6 level was measured by ELISA. Synovitis, synovial hyperplasia, bone erosion, osteoclast activation and osteoclast gene expression were assessed by histomorphometry and reverse transcriptase–PCR, respectively. In vitro osteoclast differentiation assays were performed in Tyro3−/− and wild-type mice. Furthermore, effects of Tyro3 and GAS6 on human synovial fibroblast proliferation and osteoclastogenesis were assessed in human cells. Results: Tyro3−/− mice had significantly higher bone mass than wild-type littermates. Induction of arthritis increased GAS6 serum levels. Arthritic Tyro3−/− mice showed less synovial hyperplasia, osteoclast numbers and bone damage compared with controls. In vivo expression of osteoclast-associated receptor and receptor activator of nuclear factor-κB and in vitro osteoclastogenesis were impaired in Tyro3−/− mice. GAS6 also induced synovial fibroblast proliferation and osteoclast differentiation in human cells in Tyro3-dependent manner. Conclusions: These findings indicate that Tyro3 is a critical signal for synovial hyperplasia, osteoclast differentiation and bone erosion during arthritis. GAS6 and Tyro3 therefore constitute therapeutic targets to inhibit synovial hyperplasia and associated bone erosion

Hypofractionated Irradiation Has Immune Stimulatory Potential and Induces a Timely Restricted Infiltration of Immune Cells in Colon Cancer Tumors

In addition to locally controlling the tumor, hypofractionated radiotherapy (RT) particularly aims to activate immune cells in the RT-modified microenvironment. Therefore, we examined whether hypofractionated RT can activate dendritic cells (DCs), induce immune cell infiltration in tumors, and how the chronology of immune cell migration into tumors occurs to gain knowledge for future definition of radiation breaks and inclusion of immunotherapy. Colorectal cancer treatments offer only limited survival benefit, and immunobiological principles for additional therapies need to be explored with preclinical models. The impact of hypofractionated RT on CT26 colon cancer tumor cell death, migration of DCs toward supernatants (SN) of tumor cells, and activation of DCs by SN were analyzed. The subcutaneous tumor of a BALB/c-CT26 mouse model was locally irradiated with 2 × 5 Gy, the tumor volume was monitored, and the infiltration of immune cells in the tumor was determined by flow cytometry daily. Hypofractionated RT induced a mixture of apoptotic and necrotic CT26 cells, which is known to be in particular immunogenic. DCs that migrated toward SN of CT26 cells particularly upregulated the activation markers CD80 and CD86 when in contact with SN of irradiated tumor cells. After hypofractionated RT, the tumor outgrowth was significantly retarded and in the irradiated tumors an increased infiltration of macrophages (CD11bhigh/F4-80+) and DCs (MHC-II+), but only between day 5 and 10 after the first irradiation, takes place. While CD4+ T cells migrated into non-irradiated and irradiated tumors, CD8+ T cells were only found in tumors that had been irradiated and they were highly increased at day 8 after the first irradiation. Myeloid-derived suppressor cells and regulatory T cells show regular turnover in irradiated and non-irradiated tumors. Tumor cell-specific anti-IgM antibodies were enhanced in the serum of animals with irradiated tumors. We conclude that hypofractionated RT suffices to activate DCs and to induce infiltration of innate and adaptive immune cells into solid colorectal tumors. However, the presence of immune cells in the tumor which are beneficial for antitumor immune responses is timely restricted. These findings should be considered when innovative multimodal tumor treatment protocols of distinct RT with immune therapies are designed and clinically implemented

Hypofractionated Radiotherapy Upregulates Several Immune Checkpoint Molecules in Head and Neck Squamous Cell Carcinoma Cells Independently of the HPV Status While ICOS-L Is Upregulated Only on HPV-Positive Cells

While the treatment of squamous cell carcinoma of the head and neck (HNSCC) with radiotherapy (RT) is complemented more and more by immunotherapy in clinical trials, little is known about the impact of the human papillomavirus (HPV) status or the applied RT scheme on the immune phenotype of the tumor cells. Therefore, we aimed to examine the impact of the HPV status of four human HNSCC cell lines on cell death and the expression of immune checkpoint molecules (ICMs) after RT with either hypofractionation irradiation (5x3.0Gy) or a high single dose (1x19.3Gy) via multicolor flow cytometry and quantitative PCR at an early time point after therapy. In our study, 5x3.0Gy RT induced high numbers of early and late apoptotic cells independent of the HPV status, but necrosis was only increased in the HPV-positive UM-Scc-47 cells. Generally, the immune stimulatory ICMs (CD70, CD137-L, ICOS-L) were less affected by RT compared to the immune suppressive ones (PD-L1, PD-L2, and the herpesvirus entry mediator (HVEM)). A significant higher surface expression of the analyzed ICMs was found after hypofractionated RT compared to a single high dose; however, regardless of the HPV status, with the exception of ICOS-L. Here, HPV-positive HNSCC tumor cells showed a stronger response to 5x3.0Gy than HPV-negative ones. On the RNA level, only minor alterations of ICMs were observed following RT, with the exception of the HPV negative cell line CAL33 treated with 5x3.0Gy, where PD-L2, HVEM and CD70 were significantly increased. We conclude that the HPV status may not distinctly predict immunological responses following RT, and thus cannot be used as a single predictive marker for therapy responses in HNSCC. In contrast, the patient-specific individual expression of ICMs following RT is preferable for the targeted patient selection for immune therapy directed against distinct ICM