Regulatory T cells are not a strong predictor of survival for patients with glioblastoma.

BACKGROUND: Regulatory T cells (Tregs) are potentially prognostic indicators in patients with glioblastoma. If differences in frequency of Tregs in tumor or blood account for substantial variation in patient survival, then reliably measuring Tregs may enhance treatment selection and improve outcomes. METHODS: We measured Tregs and CD3+ T cells in tumors and blood from 25 patients with newly diagnosed glioblastoma. Tumor-infiltrating Tregs and CD3+ T cells, measured by quantitative DNA demethylation analysis (epigenetic qPCR) and by immunohistochemistry, and peripheral blood Treg proportions measured by flow cytometry were correlated with patient survival. Additionally, we analyzed data from The Cancer Genome Atlas (TCGA) to correlate the expression of Treg markers with patient survival and glioblastoma subtypes. RESULTS: Tregs, as measured in tumor tissue and peripheral blood, did not correlate with patient survival. Although there was a correlation between tumor-infiltrating Tregs expression by epigenetic qPCR and immunohistochemistry, epigenetic qPCR was more sensitive and specific. Using data from TCGA, mRNA expression of Forkhead box protein 3 (FoxP3) and Helios and FoxP3 methylation level did not predict survival. While the classical glioblastoma subtype corresponded to lower expression of Treg markers, these markers did not predict survival in any of the glioblastoma subtypes. CONCLUSIONS: Although immunosuppression is a hallmark of glioblastoma, Tregs as measured in tissue by gene expression, immunohistochemistry, or demethylation and Tregs in peripheral blood measured by flow cytometry do not predict survival of patients. Quantitative DNA demethylation analysis provides an objective, sensitive, and specific way of identifying Tregs and CD3+ T cells in glioblastoma.

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.

High immune response rates and decreased frequencies of regulatory T cells in metastatic renal cell carcinoma patients after tumor cell vaccination.

Our previously reported phase I clinical trial with the allogeneic gene-modified tumor cell line RCC-26/CD80/IL-2 showed that vaccination was well tolerated and feasible in metastatic renal cell carcinoma (RCC) patients. Substantial disease stabilization was observed in most patients despite a high tumor burden at study entry. To investigate alterations in immune responses that might contribute to this effect, we performed an extended immune monitoring that included analysis of reactivity against multiple antigens, cytokine/chemokine changes in serum and determination of the frequencies of immune suppressor cell populations, including natural regulatory T cells (nTregs) and myeloid-derived suppressor cell subsets (MDSCs). An overall immune response capacity to virus-derived control peptides was present in 100% of patients before vaccination. Vaccine-induced immune responses to tumor-associated antigens occurred in 75% of patients, demonstrating the potent immune stimulatory capacity of this generic vaccine. Furthermore, some patients reacted to peptide epitopes of antigens not expressed by the vaccine, showing that epitope-spreading occurred in vivo. Frequencies of nTregs and MDSCs were comparable to healthy donors at the beginning of study. A significant decrease of nTregs was detected after vaccination (p = 0.012). High immune response rates, decreased frequencies of nTregs and a mixed T helper 1/T helper 2 (T(H)1/T(H)2)-like cytokine pattern support the applicability of this RCC generic vaccine for use in combination therapies.

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.