Dagmar Quandt

Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors.

Activation of the NLRP3 inflammasome enables monocytes and macrophages to release high levels of interleukin-1β during inflammatory responses. Concentrations of extracellular calcium can increase at sites of infection, inflammation or cell activation. Here we show that increased extracellular calcium activates the NLRP3 inflammasome via stimulation of G protein-coupled calcium sensing receptors. Activation is mediated by signalling through the calcium-sensing receptor and GPRC6A via the phosphatidyl inositol/Ca2+ pathway. The resulting increase in the intracellular calcium concentration triggers inflammasome assembly and Caspase-1 activation. We identified necrotic cells as one source for excess extracellular calcium triggering this activation. In vivo, increased calcium concentrations can amplify the inflammatory response in the mouse model of carrageenan-induced footpad swelling, and this effect was inhibited in GPRC6A−/− mice. Our results demonstrate that G-protein-coupled receptors can activate the inflammasome, and indicate that increased extracellular calcium has a role as a danger signal and amplifier of inflammation.

The expression, function, and clinical relevance of B7 family members in cancer

The modulation and suppression of anti-tumor immune responses is a characteristic feature of tumor cells to escape immune surveillance. Members of the B7 family are involved in this process, since the level of activation of the anti-tumor immune response depends on the balance between co-stimulatory and co-inhibitory signals. Some molecules are often overexpressed in tumors, which has been associated with the pathogenesis and progression of malignancies as well as their immunological and non-immunological functions. The B7 homologs play a key role in the maintenance of self-tolerance and the regulation of both innate and adaptive immunity in tumor-bearing hosts. Furthermore, the blockade of negative signals mediated by the interaction of co-inhibitory ligands and counter-receptors of the B7 family is currently being studied as a potential immunotherapeutic strategy for the treatment of cancer in humans.

Association of anticytomegalovirus seropositivity with more severe joint destruction and more frequent joint surgery in rheumatoid arthritis.

OBJECTIVE Expansion of autoreactive CD4+CD28(null) T cells is associated with extraarticular disease manifestations, including rheumatoid vasculitis, and it has recently been demonstrated that expansion of these T cells is associated with anticytomegalovirus (anti-CMV) seropositivity. This study was undertaken to investigate a possible link between latent CMV infection and rheumatoid arthritis (RA). METHODS In a retrospective analysis, anti-CMV antibodies and clinical, serologic, and radiologic parameters of joint destruction were examined in 202 RA patients and 272 healthy controls. In addition, frequencies of CD4+CD28(null) T cells; concentrations of the cytokines monocyte chemotactic protein 1 (MCP-1), interferon-α (IFNα), and IFN-inducible protein 10; and anti-CMV-specific T cell responses were analyzed in RA patients. RESULTS Overall, no significant difference in the frequency of anti-CMV seropositivity between RA patients and healthy controls was observed. Among individuals older than age 55 years, however, anti-CMV IgG antibodies were significantly more frequent in RA patients than controls (65.3% and 54.7%, respectively; P = 0.05). Anti-CMV seropositivity in RA patients was associated with an increased frequency of CD4+CD28(null) T cells and increased serum concentrations of MCP-1. The frequency of anti-CMV-specific CD4+ T cells producing IFNγ was increased in RA patients compared to controls. Most importantly, anti-CMV-seropositive RA patients showed radiographic evidence of more advanced joint destruction and had increased frequencies of joint-related surgical procedures, indicating more severe joint disease. CONCLUSION Our findings indicate that latent CMV infection aggravates the clinical course of RA and is associated with increased frequencies of CD4+CD28(null) T cells and of CMV-specific IFNγ-secreting CD4+ T cells.

Peripheral CD4CD8 Double Positive T Cells with a Distinct Helper Cytokine Profile Are Increased in Rheumatoid Arthritis

Peripheral CD4CD8 double positive (DP) T cells have been reported to play a role in several autoimmune diseases, virus infections and cancer. In rheumatoid arthritis (RA), both CD4 and CD8 single positive (SP) T cells are known to be involved in the pathogenesis, but the role of peripheral CD4CD8 DP T cells has not been investigated in detail. Anti cyclic citrullinated antibodies (ACPA) positive and ACPA negative RA patients, patients with systemic lupus erythematodes (SLE) and age matched healthy donors (HD) were enrolled in the analysis. The frequencies and phenotype of DP T cells in PBMC were investigated. In addition, DP T cells were quantified in biopsies from rheumatoid synovium. After in vitro restimulation, the cytokine production of DP T cells was investigated in cultures of PBMC. CMV specific cytokine secretion as well as proliferation was analyzed following antigen specific restimulation after an appropriate culture duration. DP T cells were found more frequently in RA patients than in healthy controls or patients with SLE. These DP T cells express αβ TCRs, are of a memory phenotype and share features of both CD4 as well as CD8 SP T cells. Importantly, DP T cells were found to also be present in the rheumatoid synovium. Further characterization of DP T cells from RA patients revealed increased production of IL-21 and IL-4, implying a possible role as T helper cells. In addition, DP T cells in RA seem to contribute to the inflammatory process, because they produce significantly more IFNγ than counterparts from HD and are increased in CMV+ RA patients. Given their capacity to produce a variety of cytokines (IL4, IL21 and IFNγ), their association with ACPA positive RA and their presence in the synovium, we suggest an important role of double positive T cells in the pathogenesis of rheumatoid arthritis.

Implementing liquid biopsies into clinical decision making for cancer immunotherapy

During the last decade, novel immunotherapeutic strategies, in particular antibodies directed against immune checkpoint inhibitors, have revolutionized the treatment of different malignancies leading to an improved survival of patients. Identification of immune-related biomarkers for diagnosis, prognosis, monitoring of immune responses and selection of patients for specific cancer immunotherapies is urgently required and therefore areas of intensive research. Easily accessible samples in particular liquid biopsies (body fluids), such as blood, saliva or urine, are preferred for serial tumor biopsies. Although monitoring of immune and tumor responses prior, during and post immunotherapy has led to significant advances of patients’ outcome, valid and stable prognostic biomarkers are still missing. This might be due to the limited capacity of the technologies employed, reproducibility of results as well as assay stability and validation of results. Therefore solid approaches to assess immune regulation and modulation as well as to follow up the nature of the tumor in liquid biopsies are urgently required to discover valuable and relevant biomarkers including sample preparation, timing of the collection and the type of liquid samples. This article summarizes our knowledge of the well-known liquid material in a new context as liquid biopsy and focuses on collection and assay requirements for the analysis and the technical developments that allow the implementation of different high-throughput assays to detect alterations at the genetic and immunologic level, which could be used for monitoring treatment efficiency, acquired therapy resistance mechanisms and the prognostic value of the liquid biopsies.

Synergistic effects of IL-4 and TNFα on the induction of B7-H1 in renal cell carcinoma cells inhibiting allogeneic T cell proliferation

BackgroundThe importance of B7-H molecules for the T cell/tumor communication and its impact on renal cell carcinoma (RCC) progression and prognosis has been recently described. Cytokine treatment of RCC has earlier been shown to be beneficial in preclinical settings, but its clinical implementation has not proven to be as effective. This might be partially explained by the yet incomplete picture of cellular alterations in tumor cells upon cytokine treatment investigated in detail in this study.MethodsRCC tumor cell lines were treated with different cytokines alone or in combination. The constitutive and/or cytokine-induced expression of cytokine receptors signaling components and B7-H molecules in RCC cells were analysed by qPCR and flow cytometry. A mcherry reporter gene construct containing B7-H1 promoter was cloned and its activity was determined upon transfection in cytokine-stimulated cells. Cytokine pretreated tumor cells were co-cultured with allogeneic CD8+ T cells from healthy donors and T cell proliferation as well as cytokine secretion was determined.ResultsA heterogeneous, but constitutive B7-H1,-H2,-H3 and H4 expression was found on human RCC cell lines. IL-4 and TNFα treatment led to strong synergistic induction of B7-H1 in RCC cells, whereas B7-H2 was only increased by TNFα. In contrast, B7-H3 and B7-H4 expression were not altered by these cytokines. Treatment of RCC cells with TNFα and IL-4 was accompanied by an activation of signaling molecules like NF-κB, IκB and STAT6. The cytokine-mediated up-regulation of B7-H1 was due to transcriptional control as determined by an increased B7-H1 promoter activity in the presence of IL-4 and TNFα. Despite HLA class I and LFA-1 were also increased, the cytokine-mediated up-regulation of B7-H1 was more pronounced and caused an inhibition of allospecifc CD8+ T cell proliferation.ConclusionThus, IL-4 and TNFα, which could be released by immune cells of the tumor microenvironment, are able to control the B7-H1 expression in RCC thereby altering T cell responses. These data are of importance for understanding the complex interplay of tumor cells with immune cells orchestrated by a number of different soluble and membrane bound mediators and for the implementation of check point antibodies directed against B7-H1.

Latent CMV infection in rheumatoid arthritis increases frequencies of cytolytic LIR‐1+ CD8+ T cells

Leukocyte immunoglobulin‐like receptor 1 (LIR‐1) is up‐regulated by cytomegalovirus (CMV), which in turn, has been associated with premature aging and more severe joint disease in patients with rheumatoid arthritis (RA). The aim of this study was to investigate the expression and functional significance of LIR‐1 in CMV‐positive RA patients.

Latent Cytomegalovirus Infection in Rheumatoid Arthritis and Increased Frequencies of Cytolytic LIR-1+CD8+ T Cells.

Leukocyte immunoglobulin‐like receptor 1 (LIR‐1) is up‐regulated by cytomegalovirus (CMV), which in turn, has been associated with premature aging and more severe joint disease in patients with rheumatoid arthritis (RA). The aim of this study was to investigate the expression and functional significance of LIR‐1 in CMV‐positive RA patients.

GPRC6A mediates Alum-induced Nlrp3 inflammasome activation but limits Th2 type antibody responses.

Alum adjuvanticity is still an unknown mechanism despite the frequent use as vaccine adjuvant in humans. Here we show that Alum-induced inflammasome activation in vitro and in vivo is mediated by the G protein-coupled receptor GPRC6A. The Alum-induced humoral response in vivo was independent of the inflammasome because Nlrp3−/− and ASC−/− mice responded normally to Alum and blockade of IL-1 had no effect on antibody production. In contrast, Alum adjuvanticity was increased in GPRC6A−/− mice resulting in increased antibody responses and increased Th2 cytokine concentrations compared to wildtype mice. In vitro activation of GPRC6A−/− splenic B cells also induced increased IgG1 concentrations compared to wildtype B cells. For the first time, we show GPRC6A expression in B cells, contributing to the direct effects of Alum on those cells. B cell produced immunostimulatory IL-10 is elevated in GPRC6A−/− B cells in vitro and in vivo. Our results demonstrate a dual role of GPRC6A in Alum adjuvanticity. GPCR6A activation by Alum leads to the initiation of innate inflammatory responses whereas it is an important signal for the limitation of adaptive immune responses induced by Alum, partially explained by B cell IL-10.

HLA-E expression and its clinical relevance in human renal cell carcinoma

The non-classical human leukocyte antigen E (HLA-E) expression is frequently overexpressed in tumor diseases, transplants and virus-infected cells and represents an immunomodulatory molecule by binding to the receptors CD94/NKG2A, -B and –C on NK and T cells. Due to its immune suppressive features HLA-E expression might represent an important mechanism of tumors to escape immune surveillance. While an aberrant expression of the non-classical HLA-G antigen in human renal cell carcinoma (RCC) has been demonstrated to be associated with a worse outcome of patients and reduced sensitivity to immune effector cell-mediated cytotoxicity, the expression and function of HLA-E has not yet been analyzed in this tumor entity. Higher levels of HLA-E transcripts were detected in all RCC cell lines and tumor lesions, which were tested in comparison to normal kidney epithelium. Immunohistochemical staining of a tissue microarray (TMA) using the HLA-E-specific monoclonal antibody TFL-033 recognizing the cytoplasmic HLA-E α-chain as monomer revealed a heterogeneous HLA-E expression in RCC lesions with the highest frequency in chromophobe RCC when compared to other RCC subtypes. HLA-E expression did not correlate with the frequency of CD3+, CD4+, CD8+ and FoxP3+ immune cell infiltrations, but showed an inverse correlation with infiltrating CD56+ cells. In contrast to HLA-G, HLA-E expression in RCCs was not statistically significant associated with a decreased disease specific survival. These data suggest that HLA-E overexpression frequently occurs in RCC and correlates with reduced immunogenicity.