Tregs In Cancer Research Articles

CD4+ Regulatory T Cells in Human Cancer: Subsets, Origin, and Molecular Regulation.

CD4+CD25hiFOXP3+ regulatory T cells (Treg) play major roles in the maintenance of immune tolerance, prevention of inflammation, and tissue homeostasis and repair. In contrast with these beneficial roles, Tregs are abundant in virtually all tumors and have been mechanistically linked to disease progression, metastases development, and therapy resistance. Tregs are thus recognized as a major target for cancer immunotherapy. Compared with other sites in the body, tumors harbor hyperactivated Treg subsets whose molecular characteristics are only beginning to be elucidated. Here, we describe current knowledge of intratumoral Tregs and discuss their potential cellular and tissue origin. Furthermore, we describe currently recognized molecular regulators that drive differentiation and maintenance of Tregs in cancer, with a special focus on those signals regulating their chronic immune activation, with relevant implications for cancer progression and therapy.

A tumor-associated heparan sulfate-related glycosaminoglycan promotes the generation of functional regulatory T cells.

Functional Tregs play a key role in tumor development and progression, representing a major barrier to anticancer immunity. The mechanisms by which Tregs are generated in cancer and the influence of the tumor microenvironment on these processes remain incompletely understood. Herein, by using NMR, chemoenzymatic structural assays and a plethora of in vitro and in vivo functional analyses, we demonstrate that the tumoral carbohydrate A10 (Ca10), a cell-surface carbohydrate derived from Ehrlich's tumor (ET) cells, is a heparan sulfate-related proteoglycan that enhances glycolysis and promotes the development of tolerogenic features in human DCs. Ca10-stimulated human DCs generate highly suppressive Tregs by mechanisms partially dependent on metabolic reprogramming, PD-L1, IL-10, and IDO. Ca10 also reprograms the differentiation of human monocytes into DCs with tolerogenic features. In solid ET-bearing mice, we found positive correlations between Ca10 serum levels, tumor size and splenic Treg numbers. Administration of isolated Ca10 also increases the proportion of splenic Tregs in tumor-free mice. Remarkably, we provide evidence supporting the presence of a circulating human Ca10 counterpart (Ca10H) and show, for the first time, that serum levels of Ca10H are increased in patients suffering from different cancer types compared to healthy individuals. Of note, these levels are higher in prostate cancer patients with bone metastases than in prostate cancer patients without metastases. Collectively, we reveal novel molecular mechanisms by which heparan sulfate-related structures associated with tumor cells promote the generation of functional Tregs in cancer. The discovery of this novel structural-functional relationship may open new avenues of research with important clinical implications in cancer treatment.

Context-Dependent Effects Explain Divergent Prognostic Roles of Tregs in Cancer.

Simple SummaryImmune cells play an important role in cancer, with regard to classification, diagnostic or prognostic matters. In particular, we focused on the prognostic value of Tregs in this meta-analysis. We took into account the local context and their heterogeneity in order to solve their apparent ambiguous role. We used three proxies to recapitulate the complexity of the context: the neighboring cell, the tissue and the quantification method; and we carefully dissected the regulatory population into existing subsets. We showed that CD45RO Tregs had a reproducible negative prognostic value across all five cancer types studied (breast, colorectal, gastric, lung and ovarian). It suggests that Tregs from an homogeneous context have a consistent prognostic role across cancer types.Assessing cancer prognosis is a challenging task, given the heterogeneity of the disease. Multiple features (clinical, environmental, genetic) have been used for such assessments. The tumor immune microenvironment (TIME) is a key feature, and describing the impact of its many components on cancer prognosis is an active field of research. The complexity of the tumor microenvironment context makes it difficult to use the TIME to assess prognosis, as demonstrated by the example of regulatory T cells (Tregs). The effect of Tregs on prognosis is ambiguous, with different studies considering them to be negative, positive or neutral. We focused on five different cancer types (breast, colorectal, gastric, lung and ovarian). We clarified the definition of Tregs and their utility for assessing cancer prognosis by taking the context into account via the following parameters: the Treg subset, the anatomical location of these cells, and the neighboring cells. With a meta-analysis on these three parameters, we were able to clarify the prognostic role of Tregs. We found that CD45RO Tregs had a reproducible negative effect on prognosis across cancer types, and we gained insight into the contributions of the anatomical location of Tregs and of their neighboring cells on their prognostic value. Our results suggest that Tregs play a similar prognostic role in all cancer types. We also establish guidelines for improving the design of future studies addressing the pathophysiological role of Tregs in cancer.

Functional Diversities of Regulatory T Cells in the Context of Cancer Immunotherapy.

Regulatory T cells (Tregs) are a subset of CD4+ T cells with their immunosuppressive activities to block abnormal or excessive immune responses to self and non-autoantigens. Tregs express the transcription factor Foxp3, maintain the immune homeostasis, and prevent the initiation of anti-tumor immune effects in various ways as their mechanisms to modulate tumor development. Recognition of different phenotypes and functions of intratumoral Tregs has offered the possibilities to develop therapeutic strategies by selectively targeting Tregs in cancers with the aim of alleviating their immunosuppressive activities from anti-tumor immune responses. Several Treg-based immunotherapeutic approaches have emerged to target cytotoxic T lymphocyte antigen-4, glucocorticoid-induced tumor necrosis factor receptor, CD25, indoleamine-2, 3-dioxygenase-1, and cytokines. These immunotherapies have yielded encouraging outcomes from preclinical studies and early-phase clinical trials. Further, dual therapy or combined therapy has been approved to be better choices than single immunotherapy, radiotherapy, or chemotherapy. In this short review article, we discuss our current understanding of the immunologic characteristics of Tregs, including Treg differentiation, development, therapeutic efficacy, and future potential of Treg-related therapies among the general cancer therapy.

TNFR2 pathways are fully active in cancer regulatory T cells.

Tumor necrosis factor receptor 2 (TNFR2), a membrane-bound tumor necrosis factor receptor expressed by regulatory T cells (Tregs), participates in Treg proliferation. Although a specific TNFR2 pathway has been reported, the signaling mechanism has not been completely elucidated. This study sought to clarify TNFR2 signaling in human Tregs using amplicon sequencing and single-cell RNA sequencing to assess Tregs treated with a TNFR2 agonist antibody. Pathway enrichment analysis based on differentially expressed genes highlighted tumor necrosis factor α signaling via nuclear factor kappa B, interleukin-2 signal transducer and activator of transcription 5 signaling, interferon-γ response, and cell proliferation-related pathways in Tregs after TNFR2 activation. TNFR2-high Treg-focused analysis found that these pathways were fully activated in cancer Tregs, showing high TNFR2 expression. Collectively, these findings suggest that TNFR2 orchestrates multiple pathways in cancer Tregs, which could help cancer cells escape immune surveillance, making TNFR2 signaling a potential anticancer therapy target.

Adoptive Chimeric Antigen Receptor T Cell (CAR-T) and Treg Cell-Based Immunotherapies: Frontier Therapeutic Aspects in Cancers

Based on this point that some cancers do not appropriately respond to conventional therapy, and there is the possibility of relapse, immunotherapy is currently under investigation. Cancer immunotherapies are widely recognized as transformational for several cancers and enable to move to the front-line therapy with few side effects. One of its new branches is treatment with T-cells that have been changed their receptor. The research on these cells is generally according to the design of a receptor against a specific tumor antigen. Also, manipulation of regulatory T-cell (Tregs), as the barriers to proper immune responses in the tumor microenvironment, will promote Tregs-targeted therapeutic opportunities and improve the efficacy of the current cancer treatment, such as radiation and chemotherapy. This review attempts to show novel insights into the roles of Tregs in cancer which can be considered a promising anticancer therapeutic strategy for targeting them and approaches for the generation of tumor antigen-specific T lymphocytes (AST) using chimeric antigen receptors.

High-dimensional profiling of tumor-infiltrating CD4+T cells in Human cancer: Are they all tumor-specific?

Abstract Tumor-specific T cells act as major actors in underpinning effective immune checkpoint-blockade therapies in cancer, but most of the studies focus on CD8+ tumor-infiltrating lymphocytes (TILs). Although roles of CD4+ T cells and especially Tregs in cancer have been established, the basis for their phenotypic heterogeneity in human tumor infiltrates is not well determined. Here we profile ex-vivounexpanded CD4+ TILs in human lung and colorectal cancer using single-cell and mass-cytometry analysis. Surprisingly, up to 80% of CD4+ TILs are composed of non-Treg cells (FoxP3−). Non-Treg CD4+ TILs are highly heterogeneous and consist of different populations of effector (CD45RO+), cytotoxic (Granzyme B+), senescent (CD57+) and exhausted cells at different levels (PD-1+/++, CTLA-4+/−, TIGIT+, CD39+/−). Recently, we showed that the surface marker CD39 can accurately discriminate between bystander (CD39−) and tumor-specific (CD39+) CD8+ TILs. Across all tumors tested, more than 90% of Tregs cells expressed CD39. However, frequencies of CD39+ among non-Treg CD4+ TILs varied dramatically from 0% to 90% and strongly correlated (r2=0.8) with the frequency of CD39+CD8+ TILs in our cohort of patients. These observations suggest the presence of bystander CD4+ TILs and a high degree of coordination between the tumor-specific CD4 and CD8 T cell responses. Using in-vitroassay, we aim to confirm the presence of these bystander CD4+ TILs specific for cancer-unrelated antigens (HCMV, EBV, Influenza). Overall, our findings could highlight that not all CD4+ TILs are tumor-specific and could suggest measuring CD39 expression as a straightforward way to quantify or isolate tumor-specific T cells, thus opening new diagnostic and therapeutic avenues.

Regulatory T Cells in Tumor Microenvironment and Approach for Anticancer Immunotherapy.

Tregs have a role in immunological tolerance and immune homeostasis by suppressing immune reactions, and its therapeutic potential is critical in autoimmune diseases and cancers. There have been multiple studies conducted on Tregs because of their roles in immune suppression and therapeutic potential. In tumor immunity, Tregs can promote the development and progression of tumors by preventing effective anti-tumor immune responses in tumor-bearing hosts. High infiltration of Tregs into tumor tissue results in poor survival in various types of cancer patients. Identifying factors specifically expressed in Tregs that affect the maintenance of stability and function of Tregs is important for understanding cancer pathogenesis and identifying therapeutic targets. Thus, manipulation of Tregs is a promising anticancer strategy, but finding markers for Treg-specific depletion and controlling these cells require fine-tuning and further research. Here, we discuss the role of Tregs in cancer and the development of Treg-targeted therapies to promote cancer immunotherapy.

Recent advances with Treg depleting fusion protein toxins for cancer immunotherapy.

T regulatory cells (Tregs) are an important T cell population for immune tolerance, prevention of autoimmune diseases and inhibition of antitumor immunity. The tumor-promoting role played by Tregs in cancer has prompted numerous approaches to develop immunotherapeutics targeting Tregs. One approach to depletion of Treg cells is retargeting the highly potent cytotoxic activity of bacterial toxins. These agents capitalize on the well-characterized bacterial toxins, diphtheria toxin and Pseudomonas aeruginosa exotoxin A-both of which harbor membrane translocation domains and enzymatic domains that catalytically halt protein synthesis within intoxicated eukaryotic cells and act at picomolar or subpicomolar concentrations. In this review, we summarize the preclinical and clinical development of several Treg-depleting cancer immunotherapies based on these two bacterial toxins.

Abstract 2713: Discovery and characterization of AZD8701, a high affinity antisense oligonucleotide targeting FOXP3 to relieve immunosuppression in cancer

Abstract Regulatory T cells (Treg) critically maintain immuno-suppression in the tumor microenvironment, representing an attractive immuno-oncology target. The Treg lineage is defined by expression of the FOXP3 transcription factor, which controls immune-suppressive functions. We have developed the clinical candidate AZD8701, a next-generation antisense oligonucleotide inhibitor of FOXP3 (utilizing the Ionis Gen 2.5 cEt-modified ASO platform). AZD8701 treatment knocked down FOXP3 in primary human Tregs via free uptake (IC50 65nM), which was also associated with modulation of known FOXP3 target genes including 25-50% reduction in CTLA4, ICOS, CCR8 and GITR. Tregs treated with AZD8701 further exhibited reduced suppressive functions in in vitro suppression assays, which confirmed the functional effects of FOXP3 modulation. Finally, AZD8701 promoted dose-dependent knockdown of FOXP3 in humanized mice, including >50% FOXP3 knockdown at doses that can be feasibly achieved with the Gen 2.5 ASO platform in the clinic. To support the importance of FOXP3 in immuno-oncological settings, we characterized murine surrogate FOXP3 ASOs in the context of syngeneic tumour bearing mice. Murine FOXP3 ASOs similarly promoted >50% FOXP3 knockdown in mice and were well tolerated with no overt toxicological findings at high doses, over a maximum of 12 weeks of treatment. Murine FOXP3 ASOs significantly attenuated tumour growth in A20 and ID8-VEGF syngeneic models, which was associated with some complete tumour regressions. Moreover, we found that mouse surrogate FOXP3 ASOs promoted additive/enhanced therapeutic effects when combined with immune checkpoint blockade. Collectively, FOXP3 ASOs represent a first-in-class strategy to target Tregs in cancer in a highly selective manner. The clinical application of AZD8701 may provide therapeutic benefit to patients either as a monotherapy or in combination with immune checkpoint blocking agents. Citation Format: Charles Sinclair, Alexey S. Revenko, R B. Johnson, Alison Peter, Molly A. Taylor, Lisa A. Hettrick, Stephanie Klein, Anisha Solanki, Melissa Chapman, James Yates, Helen K. Angell, Andrew Watt, Danielle Gattis, Brett P. Monia, Simon T. Barry, Paul Lyne, Mark Edbrooke, Frederick Goldberg, A R. Macleod. Discovery and characterization of AZD8701, a high affinity antisense oligonucleotide targeting FOXP3 to relieve immunosuppression in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2713.

Low-Dose IL-2 Signaling Is Specifically Impaired in Regulatory T Cells from T1D Patients

Background: The response to interleukin-2 (IL-2) is essential for the stability and function of regulatory T cells (Tregs) in vivo. Impaired IL-2 receptor signaling might play a crucial role in the decreased function of Tregs in autoimmunity but might also contribute to the increased suppressive capacity of Tregs in cancer. In this study we aimed to examine differences in the phosphorylation of STAT5 as a downstream measure of IL-2 signaling in type 1 diabetes (T1D), rheumatoid arthritis (RA), colo-rectal cancer (CRC) and healthy controls. Material and Methods: Phosphorylation of STAT5 was quantified in 10 T1D, 10 RA, 10 CRC and 10 healthy control samples. Freshly drawn whole blood was stimulated with increasing amounts of IL-2 [0, 0.5, 1, 5, 10 U/ml] for 30 minutes. The expression of phosphorylated STAT5 (pSTAT5) in Tregs was quantified by flow cytometry. Results: Tregs from T1D patients showed significantly decreased levels of pSTAT5 when compared to healthy controls after stimulation with 0.5 U IL-2 (mean fluorescence intensity, MFI: 896 ± 502 vs. 3435 ± 1035, p<0.001) and after stimulation with 1 U IL-2/ml (MFI: 1095 ± 942 vs. 3835 ± 1415, p=0.004). Tregs from RA patients showed decreased levels after stimulation with 0.5 U IL-2/ml (p=0.043) but not after stimulation with 1U IL-2/ml when compared to healthy controls. No significant differences between cells from CRC patients and controls were found. Stimulation with 5 or 10 U IL-2/ml led to comparable levels of pSTAT5 in all investigated samples. Conclusion: The response towards low doses of IL-2 is significantly decreased in Tregs from T1D and RA patients. This might contribute to the loss of tolerance and increased autoimmunity in those diseases. However, low dose IL2 signaling does not seem to be involved in the increased suppressive capacity of Tregs in CRC patients. Disclosure B. Prietl: None. S. Kofler: None. S. Stanzer: None. B.M. Obermayer-Pietsch: None. T.R. Pieber: Consultant; Self; Arecor, AstraZeneca, Eli Lilly and Company, Novo Nordisk A/S, Sanofi. Employee; Self; CBmed. Research Support; Self; Novo Nordisk A/S, AstraZeneca. H. Sourij: Speaker's Bureau; Self; Boehringer Ingelheim GmbH, Novo Nordisk A/S, Amgen Inc., Sanofi, MSD K.K.. Research Support; Self; AstraZeneca, Boehringer Ingelheim GmbH, MSD K.K., GI Dynamics Inc..

Targeting EZH2 Reprograms Intratumoral Regulatory T Cells to Enhance Cancer Immunity

SUMMARYRegulatory T cells (Tregs) are critical for maintaining immune homeostasis, but their presence in tumor tissues impairs anti-tumor immunity and portends poor prognoses in cancer patients. Here, we reveal a mechanism to selectively target and reprogram the function of tumor-infiltrating Tregs (TI-Tregs) by exploiting their dependency on the histone H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) in tumors. Disruption of EZH2 activity in Tregs, either pharmacologically or genetically, drove the acquisition of pro-inflammatory functions in TI-Tregs, remodeling the tumor microenvironment and enhancing the recruitment and function of CD8+ and CD4+ effector T cells that eliminate tumors. Moreover, abolishing EZH2 function in Tregs was mechanistically distinct from, more potent than, and less toxic than a generalized Treg depletion approach. This study reveals a strategy to target Tregs in cancer that mitigates autoimmunity by reprogramming their function in tumors to enhance anti-cancer immunity.In BriefEZH2 plays an intrinsic role in neoplastic cells as an oncogene, prompting the development of EZH2 inhibitors for cancer therapy. Wang et al. show that disrupting EZH2 function also has immunomodulatory activities and, when blocked in Tregs, promotes potent cancer immunity.

Canonical Wnt signaling in Treg contributes to colon cancer

Abstract Regulatory T-cells (Tregs) play a dual role in colon cancer, promoting immune tolerance and suppressing inflammation. Two types of Tregs infiltrate the gut. Thymic derived Helios+CD4+Foxp3+ Tregs have high affinity for self-antigens, including tumor-antigens and protect the tumor, while Helios−CD4+Foxp3+ extra-thymic Tregs that are generated in the gut have microbial specificity and suppress harmful inflammation. In colon cancer, there is a preferential expansion of a population of Helios−CD4+Foxp3+RORγt+ Tregs with enhanced T-cell suppressive properties and pro-inflammatory properties that promote tumor growth. We showed that this population of Tregs in cancer patients expresses elevated levels of β-catenin. To test the role of canonical Wnt signaling in the generation of pro-inflammatory Tregs, we stabilized β-catenin or deleted its DNA binding partner TCF-1 in Tregs. In both instances expression of RORγt was upregulated. While the stabilization of β-catenin led to loss of Treg functions, ablation of TCF-1 reproduced the phenotype and functional characteristics of Tregs in colon cancer. The TCF-1 deficient Tregs were pro-inflammatory, potent T-cell suppressive, and tumor promoting.

Targeting EZH2 Selectively Alters Intratumoral Regulatory T Cells to Enhance Cancer Immunity

Abstract Immunosuppressive regulatory T cells (Tregs) are critical for maintaining immune homeostasis, but their presence in tumor tissues impairs anti-tumor immunity and portends poor prognoses in cancer patients. Targeting Tregs may be a powerful means to unleash more potent immune responses against cancer, but targeting these cells is challenging because their generalized inactivation may incite severe autoimmune toxicities. To selectively target Tregs in tumors, we investigated the role of the H3K27 methyltransferase EZH2 in Tregs and determined that its enhanced activity at tumor sites in mice and humans leads to more robust and stable Tregs. We demonstrate that blocking EZH2 activity, both pharmacologically and genetically, selectively reprograms the function of tumor-infiltrating Tregs without systemically altering Treg function. Genetic disruption of EZH2 in tumor-resident Tregs led to their acquisition of pro-inflammatory functions that remodeled the tumor microenvironment and enhanced the recruitment and function of effector T cells, leading to the complete elimination of tumors. Moreover, abolishing EZH2 function in Tregs was mechanistically distinct from, more potent than, and less toxic than a generalized Treg depletion approach. This study reveals a novel strategy to target Tregs in cancer that mitigates autoimmunity by reprogramming their function in tumors to enhance anti-cancer immunity.

The Dual Role of Treg in Cancer.

Regulatory T cells (Tregs) represent a small subpopulation of CD4+ cells. Tregs are characterized by the expression of transcription factor Forkhead box protein 3 (FoxP3), also known as scurfin. Tregs are modulators of adaptive immune responses and play an important role in maintaining tolerance to self-antigens, providing thesuppression associated with tumour microenvironment as well. These immunomodulatory properties are the main reason for the development of numerous therapeutic strategies, designed to inhibit the activity of cancer cells. However, due to Treg subpopulation diversity and its many functional pathways, the role of these cells in the cancer development and progression is still not fully understood.

An increased CD25-positive intestinal regulatory T lymphocyte population is dependent upon Cox-2 activity in the Apcmin/+ model.

Only mismatch repair (MMR)-deficient colorectal cancer (CRC) appears to respond well to programmed death (PD)-1 inhibition at the present time. Emerging evidence suggests a role for micro-environmental factors such as CD25+ cells modulating response to PD-1 inhibition. In the ApcMin/+ model of familial adenomatous polyposis (MMR-proficient CRC), increased Cyclooxygenase-2 (Cox-2) expression by cells which include alternatively activated mononuclear phagocytes promotes intestinal tumorigenesis by mechanisms which may include immune suppression. To gain insight into this, we compared regulatory T cell (Treg ) populations between ApcMin/+ and wild-type mice prior to and after the phase of increased intestinal Cox-2-dependent prostaglandin E2 (PGE2 ) production. There was no difference in systemic Treg function or numbers between ApcMin/+ and wild-type mice. However, increased numbers of small intestinal CD25+ Tregs were observed with increased Cox-2 activity in the absence of any difference in the expression of Tgf-β or Tslp between ApcMin/+ and wild-type mice. Cox-2 inhibitor therapy (Celecoxib) reversed the increase in ApcMin/+ intestinal CD25+ Treg numbers, without decreasing numbers of CD25+ systemic Tregs . Forkhead box protein 3 (FoxP3+ ) and Cox-2+ cells were co-localized to the interstitium of adenomas of Apcmin/+ mice. These results suggest selective dependence of an 'activated Treg ' phenotype on paracrine Cox-2 activity in ApcMin/+ small intestine. For therapeutic potential, further studies are required to evaluate the relevance of these findings to human cancer as well as the functional significance of CD25+ intestinal Tregs in cancer.

Regulatory T-cell heterogeneity and the cancer immune response.

The frequency of circulating or tumour-infiltrating regulatory T cells (Tregs) has been associated with poor patient survival in many cancers including breast, melanoma and lung. It has been hypothesised that Tregs impact the anti-tumour function of effector T cells, resulting in worse outcomes for patients. However, high infiltrates of Tregs have been associated with a positive outcome of patients in a minority of cancers including colorectal, bladder and oesophageal. In addition, many studies have shown no impact of Tregs in patient outcome. Traditionally, research has identified Tregs as forkhead box P3 (FOXP3+) T cells in order to make such associations. Recently, it has become evident that regulatory populations are very heterogeneous, and this heterogeneity is essential for Treg function. Treg heterogeneity likely affects predictions of patient outcome, and different Treg populations may have different influences on tumours. The study of Tregs in cancer must include a better definition of the cells analysed. This review will focus primarily on colorectal cancer in humans, due to mixed data on the impact of Tregs on patient outcome in this disease.

Transcriptional regulation of FOXP3 requires integrated activation of both promoter and CNS regions in tumor-induced CD8+ Treg cells

T-regulatory cells are an upsurge in the tumor microenvironment and induce immune-evasion. CD4+ Treg cells are well characterized whereas the role of CD8+ Tregs in cancer has recently started to crease attention. Here, we report an augmentation CD8+FOXP3+ Tregs in breast tumor microenvironment. FOXP3, the lineage-specific transcription factor, is a dominant regulator of Treg cell development and function. FOXP3 is induced preferentially by divergent signaling in CD4+ Treg cells. But how FOXP3 is induced and maintained in tumor-CD8+ Tregs is the Cinderella of the investigation. We observed that RUNX3, a CD8+ lineage-specific transcription factor, binds at the FOXP3-promoter to induce its transcription. In addition to promoter activation, involvement of cis-elements CNS1 and CNS2 in the transcriptional regulation of FOXP3 was also evident in these cells. SMAD3 binds to CNS1 region and acts as transcription inducer, whereas GATA3 plays a temporal role in the FOXP3 transcription by differential chromatin modification in CNS regions. In CNS1 region, GATA3 acts as a repressor for FOXP3 in naïve CD8+ T cells. Whereas in CD8+ Tregs, GATA3 binds directly at CNS2 region and persuaded the maintenance of FOXP3. Therefore, the intervention of these concerted transcriptional machinery may have a therapeutic potential in immunotherapy of cancer.

Regulatory T Cells and Their Prognostic Relevance in Hematologic Malignancies.

Regulatory T cells (Tregs) have a fundamental function in monitoring the immune homeostasis in healthy individuals. In cancer and, in particular, in hematological malignancies, Tregs exert a major immunosuppressive activity, thus playing a critical role in tumor cell growth, proliferation, and survival. Here, we summarize published data on the prognostic significance of Tregs in hematological malignancies and show that they are highly conflicting. The heterogeneity of the experimental approaches that were used explains—at least in part—the discordant results reported by different groups that have investigated the role of Tregs in cancer. In fact, different tissues have been studied (i.e., peripheral blood, bone marrow, and lymph node), applying different methods (i.e., flow cytometry versus immunohistochemistry, whole blood versus isolated peripheral blood mononuclear cells versus depletion of CD25+ cells, various panels of monoclonal antibodies, techniques of fixation and permeabilization, and gating strategies). This is of relevance in order to stress the need to apply standardized approaches in the study of Tregs in hematological malignancies and in cancer in general.

<em>In Vitro</em> Differentiation of Human CD4<sup>+</sup>FOXP3<sup>+</sup> Induced Regulatory T Cells (iTregs) from Naïve CD4<sup>+</sup> T Cells Using a TGF-β-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human naïve T cells using a range of Treg-inducing factors (TGF-β plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-β plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.