Thymocyte development requires a complex orchestration of multiple factors. Ablating either Tcf-1 or HEB in CD4+CD8+-thymocytes elicits similar developmental outcomes including increased proliferation, decreased survival, and reduced late Tcra rearrangements. Here, we provide a mechanistic explanation for these similarities by showing that Tcf-1 and HEB share ~7000 DNA binding-sites genome- wide and promote chromatin accessibility. The binding of both Tcf-1 and HEB is required at these shared sites for epigenetic and transcriptional gene regulation. Binding of Tcf-1 and HEB to their conserved motifs in enhancer regions of T-cell differentiation and survival genes promotes their expression. Binding to sites that lack conserved motifs in promoter regions of cell-cycle genes limits proliferation. Tcf-1 alone displaces nucleosomes to allow for chromatin accessibility. Importantly, Tcf-1 inhibits Notch-signaling to protect HEB from Notch mediated proteasomal degradation. Thus, Tcf-1 shifts nucleosomes and safeguards HEB to enable their cooperation in establishing the epigenetic and transcription profile of CD4+CD8+-thymocytes.

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Objectives Lung cancer is the leading cause of cancer-related death in the USA. Regulatory T cells (Tregs) normally function to temper immune responses and decrease inflammation. Previous research has demonstrated different subsets of Tregs with contrasting anti- or pro-inflammatory properties. This study aimed to determine Treg subset distributions and characteristics present in non-small cell lung cancer (NSCLC) patients. Methods Peripheral blood was collected from healthy controls (HC) and NSCLC patients preceding surgical resection, and mononuclear cells were isolated, stained, and analyzed by flow cytometry. Tregs were defined by expression of CD4 and CD25 and classified into CD45RA+Foxp3int (naïve, Fr. I) or CD45RA−Foxp3hi (activated Fr. II). Activated conventional T cells were CD4+CD45RA−Foxp3int (Fr. III). Results Samples from 23 HC and 26 NSCLC patients were collected. Tregs isolated from patients with NSCLC were found to have enhanced suppressive function on naive T cells. Cancer patients had significantly increased frequencies of activated Tregs (fraction II: FrII), 17.5 versus 3.2 % (P < 0.001). FrII Tregs demonstrated increased RORγt and IL17 expression and decreased IL10 expression compared to Tregs from HC, indicating pro-inflammatory characteristics. Conclusions This study demonstrates that a novel subset of Tregs with pro-inflammatory characteristics preferentially expand in NSCLC patients. This Treg subset appears identical to previously reported pro-inflammatory Tregs in human colon cancer patients and in mouse models of polyposis. We expect the pro-inflammatory Tregs in lung cancer to contribute to the immune pathogenesis of disease and propose that targeting this Treg subset may have protective benefits in NSCLC.

The nature of regulatory T cells (Tregs) in cancer is ambivalent. In Colon Cancer (CC) patients and animal models of CC, we previously described a distinct subset of Tregs that suppresses anti-tumor effector T cell functions but simultaneously maintains inflammation. These Tregs co-express the Th17 lineage transcription factor RORγt along with Foxp3. Moreover, we causatively linked the pro-inflammatory skewing of T cells to Wnt/β-catenin-signaling. To elucidate how Wnt/β-catenin signaling orchestrates the induction of pathologic properties in Tregs we analyzed inflammatory bowel disease (IBD) patients. As IBD can progress to CC, this disease presents a unique platform to study the mechanisms underlying the emergence of RORγt+/Foxp3+Tregs. Indeed, in a sub-population of IBD patients we were able to detect RORγt+/Foxp3+Tregs, expressing high levels of β-catenin in the patients’ blood and tissue. To further establish that activated Wnt/β-catenin signaling is responsible for the pro-inflammatory skewing of Foxp3+ Tregs we over-expressed β-catenin Treg-specifically in a conditional Foxp3-Cre driven mouse model. Mice expressing constitutively active β-catenin in Tregs show striking X-linked immune pathology. Males die 4 weeks after birth of a severe scurfy-like phenotype. Although their Tregs are highly activated, they fail to suppress the proliferation of effector T cells. In heterozygous Foxp3-Cre mice, representing natural chimeras harboring wildtype and β-catenin over-expressing Tregs, the relative fitness of genetically altered Tregs is decreased. By further elucidating how Wnt/β-catenin signaling mediates the pathogenic conversion of Tregs, we hope to reveal novel targets for cancer immuno-therapy.

The molecular and functional diversity of regulatory T-cells (Tregs) in health and in disease remains unclear. We previously described in colorectal cancer (CRC) patients a subpopulation of RORγt+ Tregs with elevated expression of β-catenin and pro-inflammatory properties. Here we observed progressive expansion of RORγt+ Tregs in inflammatory bowel disease (IBD) patients during inflammation and early dysplasia. Activating Wnt/β-catenin signaling in human and murine Tregs was sufficient to recapitulate the disease-associated increase in frequencies of RORγt+ Tregs expressing IL-17, IFN-γ, and TNFa. We found that binding of the β-catenin interacting partner, TCF-1, to DNA overlapped with Foxp3 binding at enhancer sites of pro-inflammatory pathway genes. Sustained Wnt/β-catenin activation induced newly accessible chromatin sites in these genes and upregulated their expression. These findings indicate that TCF-1 and Foxp3 together limit the expression of pro-inflammatory genes in Tregs. Activation of ꞵ-catenin signaling interferes with this function and promotes the disease-associated RORγt+ Treg phenotype.