Detection of human MCP-4/CCL13 isoforms by SELDI immunoaffinity capture

Monocyte Chemoattractant Proteins 4 (MCP-4/CCL13) is a member of a distinct, structurally-related subclass of CC chemokines mainly involved in recruitment of eosinphils to inflammatory sites. Recent evidence demonstrates that serum level of this protein strongly increases following high dose IL-2 immunotherapy. The physiological form of human MCP-4/CCL13 has yet to be purified. Therefore, the primary structure of the biologically relevant (mature) form has not been established. By using SELDI immunoaffinity capture technology we describe two mature isoforms both present in serum before and after high-dose IL-2 immunotherapy

Polarized monocyte response to cytokine stimulation

BACKGROUND: Mononuclear phagocytes (MPs) stand at the crossroads between the induction of acute inflammation to recruit and activate immune effector cells and the downmodulation of the inflammatory process to contain collateral damage. This decision is extensively modulated by the cytokine microenvironment, which includes a broad array of cytokines whose direct effect on MPs remains largely unexplored. Therefore, we tested whether polarized responses of MPs to pathogens are related to the influence of selected cytokines or represent a mandatory molecular switch through which most cytokines operate. RESULTS: Circulating CD14+ MPs were exposed to bacterial lipopolysaccharide (LPS) followed by exposure to an array of cytokines, chemokines and soluble factors involved in the immune response. Gene expression was studied by global transcript analysis. Two main classes of cytokines were identified that induced a classical or an alternative pathway of MP activation. Expression of genes affected by NFkappaB activation was most predictive of the two main classes, suggesting that this pathway is a fundamental target of cytokine regulation. As LPS itself induces a classical type of activation, the most dramatic modulation was observed toward the alternative pathway, suggesting that a broad array of cytokines may counteract the pro-inflammatory effects of bacterial components. CONCLUSIONS: This analysis is directly informative of the primary effect of individual cytokines on the early stages of LPS stimulation and, therefore, may be most informative of the way MP maturation may be polarized at the early stages of the immune response

In support of descriptive studies; relevance to translational research

The contemporary scientific establishment equates hypothesis testing to good science. This stance bypasses the preliminary need to identify a worthwhile hypothesis through rigorous observation of natural processes. If alleviation of human suffering is claimed as the goal of a scientific undertaking, it would be unfair to test a hypothesis whose relevance to human disease has not been satisfactorily proven. Here, we argue that descriptive investigations based on direct human observation should be highly valued and regarded essential for the selection of worthwhile hypotheses while the pursuit of costly scientific investigations without such evidence is a desecration of the cause upon which biomedical research is grounded

Potency analysis of cellular therapies: the emerging role of molecular assays

Potency testing is an important part of the evaluation of cellular therapy products. Potency assays are quantitative measures of a product-specific biological activity that is linked to a relevant biological property and, ideally, a product's in vivo mechanism of action. Both in vivo and in vitro assays can be used for potency testing. Since there is often a limited period of time between the completion of production and the release from the laboratory for administration to the patient, in vitro assays such are flow cytometry, ELISA, and cytotoxicity are typically used. Better potency assays are needed to assess the complex and multiple functions of cellular therapy products, some of which are not well understood. Gene expression profiling using microarray technology has been widely and effectively used to assess changes of cells in response to stimuli and to classify cancers. Preliminary studies have shown that the expression of noncoding microRNA which play an important role in cellular development, differentiation, metabolism and signal transduction can distinguish different types of stem cells and leukocytes. Both gene and microRNA expression profiling have the potential to be important tools for testing the potency of cellular therapies. Potency testing, the complexities associated with potency testing of cellular therapies, and the potential role of gene and microRNA expression microarrays in potency testing of cellular therapies is discussed

Tumor-infiltrating macrophages and dendritic cells in human colorectal cancer: relation to local regulatory T cells, systemic T-cell response against tumor-associated antigens and survival

<p>Abstract</p> <p>Introduction</p> <p>Although systemic T-cell responses against tumor antigens and tumor infiltration by T cells have been investigated in colorectal cancer (CRC), the initiation of spontaneous immune responses <it>in situ </it>is not well understood. Macrophages and dendritic cells (DC) play an important role as a link between innate and adaptive immune response. The aim of the present study was to analyze macrophage and DC infiltration in CRC and to investigate whether there is a correlation to systemic T-cell response, regulatory T cell (Treg) infiltration, and survival.</p> <p>Methods</p> <p>Immunohistological staining was performed with nine markers for macrophages and DC (CD68, CD163, S100, CD11c, CD208, CD209, CD123, CD1a, Langerin) in 40 colorectal cancer samples from patients, in whom the state of systemic T-cell responses against tumor-associated antigens (TAA) and Treg infiltration had previously been determined.</p> <p>Results</p> <p>All specimens contained cells positive for CD68, CD163, S100 and CD1a in epithelial tumor tissue and tumor stroma. Only a very few (less than median 3/HPF) CD123+, CD1a+, CD11c+, CD 208+, CD209+, or Langerin+ cells were detected in the specimens. Overall, we found a trend towards increased infiltration by S100-positive DC and a significantly increased number of stromal S100-positive DC in patients without T-cell response. There was an increase of stromal S100 DC and CD163 macrophages in limited disease (S100: 11.1/HPF vs. 7.3/HPF, p = 0.046; CD163: 11.0/HPF vs. 8.1/HPF, p = 0.06). We found a significant, positive correlation between S100-positive DC and FOXP3-positive Tregs. Survival in patients with high DC infiltration was significantly better than that in those with low DC infiltration (p < 0.05). Furthermore, we found a trend towards better survival for increased infiltration with CD163-positive macrophages (p = 0.07).</p> <p>Conclusion</p> <p>The present <it>in situ </it>study adds new data to the discussion on the interaction between the innate and adoptive immune system. Our data strongly support the hypothesis that tumor-infiltrating DC are a key factor at the interface between innate and adaptive immune response in malignant disease. Tumor infiltrating S100-positive DC show an inverse relationship with the systemic antigen-specific T-cell response, a positive correlation with regulatory T cells, and a positive association with survival in CRC. These data put tumor-infiltrating DC at the center of the relevant immune response in CRC.</p

Molecular signatures of maturing dendritic cells: implications for testing the quality of dendritic cell therapies

<p>Abstract</p> <p>Background</p> <p>Dendritic cells (DCs) are often produced by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) stimulation of monocytes. To improve the effectiveness of DC adoptive immune cancer therapy, many different agents have been used to mature DCs. We analyzed the kinetics of DC maturation by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) induction in order to characterize the usefulness of mature DCs (mDCs) for immune therapy and to identify biomarkers for assessing the quality of mDCs.</p> <p>Methods</p> <p>Peripheral blood mononuclear cells were collected from 6 healthy subjects by apheresis, monocytes were isolated by elutriation, and immature DCs (iDCs) were produced by 3 days of culture with GM-CSF and IL-4. The iDCs were sampled after 4, 8 and 24 hours in culture with LPS and IFN-γ and were then assessed by flow cytometry, ELISA, and global gene and microRNA (miRNA) expression analysis.</p> <p>Results</p> <p>After 24 hours of LPS and IFN-γ stimulation, DC surface expression of CD80, CD83, CD86, and HLA Class II antigens were up-regulated. Th1 attractant genes such as CXCL9, CXCL10, CXCL11 and CCL5 were up-regulated during maturation but not Treg attractants such as CCL22 and CXCL12. The expression of classical mDC biomarker genes CD83, CCR7, CCL5, CCL8, SOD2, MT2A, OASL, GBP1 and HES4 were up-regulated throughout maturation while MTIB, MTIE, MTIG, MTIH, GADD45A and LAMP3 were only up-regulated late in maturation. The expression of miR-155 was up-regulated 8-fold in mDCs.</p> <p>Conclusion</p> <p>DCs, matured with LPS and IFN-γ, were characterized by increased levels of Th1 attractants as opposed to Treg attractants and may be particularly effective for adoptive immune cancer therapy.</p

A systematic approach to biomarker discovery; Preamble to "the iSBTc-FDA taskforce on immunotherapy biomarkers"

The International Society for the Biological Therapy of Cancer (iSBTc) has initiated in collaboration with the United States Food and Drug Administration (FDA) a programmatic look at innovative avenues for the identification of relevant parameters to assist clinical and basic scientists who study the natural course of host/tumor interactions or their response to immune manipulation. The task force has two primary goals: 1) identify best practices of standardized and validated immune monitoring procedures and assays to promote inter-trial comparisons and 2) develop strategies for the identification of novel biomarkers that may enhance our understating of principles governing human cancer immune biology and, consequently, implement their clinical application. Two working groups were created that will report the developed best practices at an NCI/FDA/iSBTc sponsored workshop tied to the annual meeting of the iSBTc to be held in Washington DC in the Fall of 2009. This foreword provides an overview of the task force and invites feedback from readers that might be incorporated in the discussions and in the final document

Emerging concepts in biomarker discovery; The US-Japan workshop on immunological molecular markers in oncology

Supported by the Office of International Affairs, National Cancer Institute (NCI), the "US-Japan Workshop on Immunological Biomarkers in Oncology" was held in March 2009. The workshop was related to a task force launched by the International Society for the Biological Therapy of Cancer (iSBTc) and the United States Food and Drug Administration (FDA) to identify strategies for biomarker discovery and validation in the field of biotherapy. The effort will culminate on October 28th 2009 in the "iSBTc-FDA-NCI Workshop on Prognostic and Predictive Immunologic Biomarkers in Cancer", which will be held in Washington DC in association with the Annual Meeting. The purposes of the US-Japan workshop were a) to discuss novel approaches to enhance the discovery of predictive and/or prognostic markers in cancer immunotherapy; b) to define the state of the science in biomarker discovery and validation. The participation of Japanese and US scientists provided the opportunity to identify shared or discordant themes across the distinct immune genetic background and the diverse prevalence of disease between the two Nations

‘1-8 interferon inducible gene family': putative colon carcinoma-associated antigens

Db−/−xβ2 microglobulin (β2m) null mice transgenic for a chimeric HLA-A2.1/Db-β2m single chain (HHD mice) are an effective biological tool to evaluate the antitumour cytotoxic T-lymphocyte response of known major histocompatibility-restricted peptide tumour-associated antigens, and to screen for putative unknown novel peptides. We utilised HHD lymphocytes to identify immunodominant epitopes of colon carcinoma overexpressed genes. We screened with HHD-derived lymphocytes over 500 HLA-A2.1-restricted peptides derived from colon carcinoma overexpressed genes. This procedure culminated in the identification of seven immunogenic peptides, three of these were derived from the ‘human 1-8D gene from interferon inducible gene' (1-8D). The 1-8D gene was shown to be overexpressed in fresh tumour samples. The three 1-8D peptides were both antigenic and immunogenic in the HHD mice. The peptides induce cytotoxic T lymphocytes that were able to kill a colon carcinoma cell line HCT/HHD, in vitro and retard its growth in vivo. One of the peptides shared by all the 1-8 gene family primed efficiently normal human cytotoxic T lymphocyte precursors. These results highlight the 1-8D gene and its homologues as putative immunodominant tumour-associated antigens of colon carcinoma