The cellular ratio of immune tolerance (immunoCRIT) is a definite marker for aggressiveness of solid tumors and may explain tumor dissemination patterns.

The adaptive immune system is involved in tumor establishment and aggressiveness. Tumors of the ovaries, an immune-privileged organ, spread via transceolomic routes and rarely to distant organs. This is contrary to tumors of non-immune privileged organs, which often disseminate hematogenously to distant organs. Epigenetics-based immune cell quantification allows direct comparison of the immune status in benign and malignant tissues and in blood. Here, we introduce the "cellular ratio of immune tolerance" (immunoCRIT) as defined by the ratio of regulatory T cells to total T lymphocytes. The immunoCRIT was analyzed on 273 benign tissue samples of colorectal, bronchial, renal and ovarian origin as well as in 808 samples from primary colorectal, bronchial, mammary and ovarian cancers. ImmunoCRIT is strongly increased in all cancerous tissues and gradually augmented strictly dependent on tumor aggressiveness. In peripheral blood of ovarian cancer patients, immunoCRIT incrementally increases from primary diagnosis to disease recurrence, at which distant metastases frequently occur. We postulate that non-pathological immunoCRIT values observed in peripheral blood of immune privileged ovarian tumor patients are sufficient to prevent hematogenous spread at primary diagnosis. Contrarily, non-immune privileged tumors establish high immunoCRIT in an immunological environment equivalent to the bloodstream and thus spread hematogenously to distant organs. In summary, our data suggest that the immunoCRIT is a powerful marker for tumor aggressiveness and disease dissemination.

Plasticity of human regulatory T cells in healthy subjects and patients with type 1 diabetes.

Regulatory T cells (Tregs) constitute an attractive therapeutic target given their essential role in controlling autoimmunity. However, recent animal studies provide evidence for functional heterogeneity and lineage plasticity within the Treg compartment. To understand better the plasticity of human Tregs in the context of type 1 diabetes, we characterized an IFN-γ-competent subset of human CD4(+)CD127(lo/-)CD25(+) Tregs. We measured the frequency of Tregs in the peripheral blood of patients with type 1 diabetes by epigenetic analysis of the Treg-specific demethylated region (TSDR) and the frequency of the IFN-γ(+) subset by flow cytometry. Purified IFN-γ(+) Tregs were assessed for suppressive function, degree of TSDR demethylation, and expression of Treg lineage markers FOXP3 and Helios. The frequency of Tregs in peripheral blood was comparable but the FOXP3(+)IFN-γ(+) fraction was significantly increased in patients with type 1 diabetes compared to healthy controls. Purified IFN-γ(+) Tregs expressed FOXP3 and possessed suppressive activity but lacked Helios expression and were predominately methylated at the TSDR, characteristics of an adaptive Treg. Naive Tregs were capable of upregulating expression of Th1-associated T-bet, CXCR3, and IFN-γ in response to IL-12. Notably, naive, thymic-derived natural Tregs also demonstrated the capacity for Th1 differentiation without concomitant loss of Helios expression or TSDR demethylation.

Epigenetic quantification of tumor-infiltrating T-lymphocytes.

The immune system plays a pivotal role in tumor establishment. However, the role of T-lymphocytes within the tumor microenvironment as major cellular component of the adaptive effector immune response and their counterpart, regulatory T-cells (Treg), responsible for suppressive immune modulation, is not completely understood. This is partly due to the lack of reliable technical solutions for specific cell quantification in solid tissues. Previous reports indicated that epigenetic marks of immune cells, such as the Treg specifically demethylated region (TSDR) within the FOXP3 gene, may be exploited as robust analytical tool for Treg-quantification. Here, we expand the concept of epigenetic immunophenotyping to overall T-lymphocytes (oTL). This tool allows cell quantification with at least equivalent precision to FACS and is adoptable for analysis of blood and solid tissues. Based on this method, we analyse the frequency of Treg, oTL and their ratio in independent cohorts of healthy and tumorous ovarian, colorectal and bronchial tissues with 616 partly donor-matched samples. We find a shift of the median ratio of Treg-to-oTL from 3-8% in healthy tissue to 18-25% in all tumor entities. Epigenetically determined oTL frequencies correlate with the outcome of colorectal and ovarian cancers. Together, our data show that the composition of immune cells in tumor microenvironments can be quantitatively assessed by epigenetic measurements. This composition is disturbed in solid tumors, indicating a fundamental mechanism of tumor immune evasion. Epigenetic quantification of T-lymphocytes serves as independent clinical parameter for outcome prognosis.

Quantitative DNA methylation analysis of FOXP3 as a new method for counting regulatory T cells in peripheral blood and solid tissue.

Regulatory T-cells (Treg) have been the focus of immunologic research due to their role in establishing tolerance for harmless antigens versus allowing immune responses against foes. Increased Treg frequencies measured by mRNA expression or protein synthesis of the Treg marker FOXP3 were found in various cancers, indicating that dysregulation of Treg levels contributes to tumor establishment. Furthermore, they constitute a key target of immunomodulatory therapies in cancer as well as transplantation settings. One core obstacle for understanding the role of Treg, thus far, is the inability of FOXP3 mRNA or protein detection methods to differentiate between Treg and activated T cells. These difficulties are aggravated by the technical demands of sample logistics and processing. Based on Treg-specific DNA demethylation within the FOXP3 locus, we present a novel method for monitoring Treg in human peripheral blood and solid tissues. We found that Treg numbers are significantly increased in the peripheral blood of patients with interleukin 2-treated melanoma and in formalin-fixed tissue from patients with lung and colon carcinomas. Conversely, we show that immunosuppressive therapy including therapeutic antibodies leads to a significant reduction of Treg from the peripheral blood of transplantation patients. In addition, Treg numbers are predictively elevated in the peripheral blood of patients with various solid tumors. Although our data generally correspond to data obtained with gene expression and protein-based methods, the results are less fluctuating and more specific to Treg. The assay presented here measures Treg robustly in blood and solid tissues regardless of conservation levels, promising fast screening of Treg in various clinical settings.

DNA methylation analysis as novel tool for quality control in regenerative medicine.

Cell-based regenerative medicine, including tissue engineering, is a novel approach to reconstituting tissues that do not spontaneously heal, such as damaged cartilage, and to curing diseases caused by malfunctioning cells. Typically, manufacturing processes to generate cartilage for replacement therapies involve isolation and expansion of cells from cartilage biopsies. A challenge in the field is potential contamination by other cell types (e.g., fibroblast-like cells), which can overgrow the desired cells during culturing and may ultimately compromise clinical efficacy. No standard analytical system has been absolutely effective in ensuring the identity of these cell-based products. Therefore, we tested deoxyribonucleic acid methylation analysis as a quality assessment tool, applying it to Genzyme's Carticel product, a chondrocyte implant that the Food and Drug Administration has approved. We identified 7 potent discriminators by assaying candidate genomic regions derived from methylation discovery approaches and literature searches regarding a functional role of genes in chondrocyte biology. Using a support vector machine, we trained an optimal cell type classifier that was absolutely effective in discriminating chondrocytes from synovial membrane derived cells, the major potential contaminant of chondrocyte cultures. The abundant marker availability and high quality of this assay format also suggest it as a potential quality control test for other cell types grown or manipulated in vitro.

DNA methylation analysis as a tool for cell typing.

Cell therapeutic approaches currently lack definitive quality control measures which guarantee safety in clinical applications and create consistent standards for regulatory approval. These approaches rely on isolation, purification and possibly ex vivo manipulation of donor cells. Since such cells are exposed to artificial environments, there is potential for deviations from natural growth processes. The resulting heterogeneity of cell cultures is an inherent problem. Therefore, verification of cell identity and quantification of subpopulations is mandatory. Focusing on cultured human primary cells, we tested whether DNA methylation patterns serve as distinctive cell type markers. We identified panels of cell type specific differentially methylated gene regions (CDMs) which produce unambiguous profiles for these cell types. Applying methylation sensitive single nucleotide primer extension generated binary cell type descriptors ("barcodes") which allow quantification of cell mixtures. Thus, methylation based analytics suggest themselves as promising tools for the characterization and quality control of ex vivo manipulated cells.

Interaction of a PDZ Protein Domain with a Synthetic Library of All Human Protein C Termini.

New interaction partners of a PDZ protein domain were identified through use of a library made up of all known human protein C-terminal peptides ("P" in the schematic representation). The library was displayed on a cellulose membrane by positional resolution of inverted peptides.

In Vitro Evolution and Selection of Proteins: Ribosome Display for Larger Libraries.

The cell-free production of large protein libraries can now be achieved with the ribosome display technique. The use of intact ribosomes (R) in the translation of a mRNA library enables the rapid selection of the generated proteins (P) according to their affinity to a given immobilized binding partner (B).