Pregnancy dedifferentiates memory CD8+ T cells into hypofunctional cells with exhaustion-enriched programs.

Alloreactive memory, unlike naïve, CD8+ T cells resist transplantation tolerance protocols and are a critical barrier to long-term graft acceptance in the clinic. We here show that semi-allogeneic pregnancy successfully reprogrammed memory fetus/graft-specific CD8+ T cells (TFGS) towards hypofunction. Female C57BL/6 mice harboring memory CD8+ T cells generated by the rejection of BALB/c skin grafts and then mated with BALB/c males achieved rates of pregnancy comparable to naive controls. Post-partum fetus/graft-specific CD8+ T cells (TFGS) from skin-sensitized dams upregulated expression of T cell exhaustion (TEX) markers (Tox, Eomes, PD-1, TIGIT, and Lag3). Transcriptional analysis corroborated an enrichment of canonical T exhaustion (TEX) genes in post-partum memory TFGS and additionally, revealed a downregulation of a subset of memory-associated transcripts. Strikingly, pregnancy induced extensive epigenetic modifications of exhaustion- and memory-associated genes in memory TFGS, whereas minimal epigenetic modifications were observed in naive TFGS cells. Finally, post-partum memory TFGS durably expressed the exhaustion-enriched phenotype, and their susceptibility to transplantation tolerance was significantly restored compared to memory TFGS. These findings advance the concept of pregnancy as an epigenetic modulator inducing hypofunction in memory CD8+ T cells that has relevance not only for pregnancy and transplantation tolerance, but also for tumor immunity and chronic infections.

Pregnancy programs epigenetic and transcriptional exhaustion in memory CD8+ T cells.

Alloreactive memory T cells, unlike naive T cells, fail to be restrained by transplantation tolerance protocols or regulatory T cells, and therefore represent a critical barrier to long-term graft acceptance. Using female mice sensitized by rejection of fully-mismatched paternal skin allografts, we show that subsequent semi-allogeneic pregnancy successfully reprograms memory fetus/graft-specific CD8+ T cells (TFGS) towards hypofunction in a manner that is mechanistically distinct from naive TFGS. Post-partum memory TFGS were durably hypofunctional, exhibiting enhanced susceptibility to transplantation tolerance induction. Furthermore, multi-omics studies revealed that pregnancy induced extensive phenotypic and transcriptional modifications in memory TFGS reminiscent of T cell exhaustion. Strikingly, at loci transcriptionally modified in both naive and memory TFGS during pregnancy, chromatin remodeling was observed exclusively in memory and not naive TFGS. These data reveal a novel link between T cell memory and hypofunction via exhaustion circuits and pregnancy-mediated epigenetic imprinting. This conceptual advance has immediate clinical relevance to pregnancy and transplantation tolerance.

Tcf-1 promotes genomic instability and T cell transformation in response to aberrant ß-catenin activation.

Understanding the mechanisms promoting chromosomal translocations of the rearranging receptor loci in leukemia and lymphoma remains incomplete. Here we show that leukemias induced by aberrant activation of ß-catenin in thymocytes, which bear recurrent Tcra/Myc-Pvt1 translocations, depend on Tcf-1. The DNA double strand breaks (DSBs) in the Tcra site of the translocation are Rag-generated, whereas the Myc-Pvt1 DSBs are not. Aberrantly activated ß-catenin redirects Tcf-1 binding to novel DNA sites to alter chromatin accessibility and down-regulate genome-stability pathways. Impaired homologous recombination (HR) DNA repair and replication checkpoints lead to retention of DSBs that promote translocations and transformation of double-positive (DP) thymocytes. The resulting lymphomas, which resemble human T cell acute lymphoblastic leukemia (T-ALL), are sensitive to PARP inhibitors (PARPis). Our findings indicate that aberrant ß-catenin signaling contributes to translocations in thymocytes by guiding Tcf-1 to promote the generation and retention of replication-induced DSBs allowing their coexistence with Rag-generated DSBs. Thus, PARPis could offer therapeutic options in hematologic malignancies with active Wnt/ß-catenin signaling.

TCF-1: a maverick in T cell development and function.

The T cell-specific DNA-binding protein TCF-1 is a central regulator of T cell development and function along multiple stages and lineages. Because it interacts with ß-catenin, TCF-1 has been classically viewed as a downstream effector of canonical Wnt signaling, although there is strong evidence for ß-catenin-independent TCF-1 functions. TCF-1 co-binds accessible regulatory regions containing or lacking its conserved motif and cooperates with other nuclear factors to establish context-dependent epigenetic and transcription programs that are essential for T cell development and for regulating immune responses to infection, autoimmunity and cancer. Although it has mostly been associated with positive regulation of chromatin accessibility and gene expression, TCF-1 has the potential to reduce chromatin accessibility and thereby suppress gene expression. In addition, the binding of TCF-1 bends the DNA and affects the chromatin conformation genome wide. This Review discusses the current understanding of the multiple roles of TCF-1 in T cell development and function and their mechanistic underpinnings.

TCF-1 controls Treg cell functions that regulate inflammation, CD8+ T cell cytotoxicity and severity of colon cancer.

The transcription factor TCF-1 is essential for the development and function of regulatory T (Treg) cells; however, its function is poorly understood. Here, we show that TCF-1 primarily suppresses transcription of genes that are co-bound by Foxp3. Single-cell RNA-sequencing analysis identified effector memory T cells and central memory Treg cells with differential expression of Klf2 and memory and activation markers. TCF-1 deficiency did not change the core Treg cell transcriptional signature, but promoted alternative signaling pathways whereby Treg cells became activated and gained gut-homing properties and characteristics of the TH17 subset of helper T cells. TCF-1-deficient Treg cells strongly suppressed T cell proliferation and cytotoxicity, but were compromised in controlling CD4+ T cell polarization and inflammation. In mice with polyposis, Treg cell-specific TCF-1 deficiency promoted tumor growth. Consistently, tumor-infiltrating Treg cells of patients with colorectal cancer showed lower TCF-1 expression and increased TH17 expression signatures compared to adjacent normal tissue and circulating T cells. Thus, Treg cell-specific TCF-1 expression differentially regulates TH17-mediated inflammation and T cell cytotoxicity, and can determine colorectal cancer outcome.

Wnt-ß-catenin activation epigenetically reprograms Treg cells in inflammatory bowel disease and dysplastic progression.

The diversity of regulatory T (Treg) cells in health and in disease remains unclear. Individuals with colorectal cancer harbor a subpopulation of RORγt+ Treg cells with elevated expression of ß-catenin and pro-inflammatory properties. Here we show progressive expansion of RORγt+ Treg cells in individuals with inflammatory bowel disease during inflammation and early dysplasia. Activating Wnt-ß-catenin signaling in human and murine Treg cells was sufficient to recapitulate the disease-associated increase in the frequency of RORγt+ Treg cells coexpressing multiple pro-inflammatory cytokines. Binding of the ß-catenin interacting partner, TCF-1, to DNA overlapped with Foxp3 binding at enhancer sites of pro-inflammatory pathway genes. Sustained Wnt-ß-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 Treg cells. Activation of ß-catenin signaling interferes with this function and promotes the disease-associated RORγt+ Treg phenotype.

DP1 Activation Reverses Age-Related Hypertension Via NEDD4L-Mediated T-Bet Degradation in T Cells.

BACKGROUND: Blood pressure often rises with aging, but exact mechanisms are still not completely understood. With aging, the level of proinflammatory cytokines increases in T lymphocytes. Prostaglandin D2, a proresolution mediator, suppresses Type 1 T helper (Th1) cytokines through D-prostanoid receptor 1 (DP1). In this study, we aimed to investigate the role of the prostaglandin D2/DP1 axis in T cells on age-related hypertension. METHODS: To clarify the physiological and pathophysiological roles of DP1 in T cells with aging, peripheral blood samples were collected from young and older male participants, and CD4+ T cells were sorted for gene expression, prostaglandin production, and Western blot assays. Mice blood pressure was quantified by invasive telemetric monitor. RESULTS: The prostaglandin D2/DP1 axis was downregulated in CD4+ T cells from older humans and aged mice. DP1 deletion in CD4+ T cells augmented age-related hypertension in aged male mice by enhancing Th1 cytokine secretion, vascular remodeling, CD4+ T cells infiltration, and superoxide production in vasculature and kidneys. Conversely, forced expression of exogenous DP1 in T cells retarded age-associated hypertension in mice by reducing Th1 cytokine secretion. Tumor necrosis factor α neutralization or interferon γ deletion ameliorated the age-related hypertension in DP1 deletion in CD4+ T cells mice. Mechanistically, DP1 inhibited Th1 activity via the PKA (protein kinase A)/p-Sp1 (phosphorylated specificity protein 1)/neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) pathway-mediated T-box-expressed-in-T-cells (T-bet) ubiquitination. T-bet deletion or forced NEDD4L expression in CD4+ T cells attenuated age-related hypertension in CD4+ T cell-specific DP1-deficient mice. DP1 receptor activation by BW245C prevented age-associated blood pressure elevation and reduced vascular/renal superoxide production in male mice. CONCLUSIONS: The prostaglandin D2/DP1 axis suppresses age-related Th1 activation and subsequent hypertensive response in male mice through increase of NEDD4L-mediated T-bet degradation by ubiquitination. Therefore, the T cell DP1 receptor may be an attractive therapeutic target for age-related hypertension.

Cell Intrinsic Deregulated ß-Catenin Signaling Promotes Expansion of Bone Marrow Derived Connective Tissue Type Mast Cells, Systemic Inflammation, and Colon Cancer.

Mast cells constitutively express ß-catenin and expand in solid tumors such as colon and skin cancer. However, the role of ß-catenin signaling in mast cells and the cause or effect of mast cell expansion and tumor growth has yet to be established. In earlier studies we used mast cell depletion and protease staining approaches, to provide evidence for a causative role of mast cells in small bowel polyposis, and related specific phenotypes and distributions of tumor infiltrating mast cells to stages of tumor growth. Here we report that, stabilization of ß-catenin expands mast cells to promote high incidence of colon polyposis and infrequent small bowel polyps and skin cancer. Expression of a dominant acting ß-catenin in mast cells (5CreCAT) stimulated maturation and expression of granule stored proteases. Both mucosal and connective tissue type mast cells accumulated in colonic small bowel polyps independent of gender, and mice developed chronic systemic inflammation with splenomegaly. Reconstitution of polyposis-prone mice with bone marrow from 5CreCAT mice resulted in focal expansion of connective tissue like mast cells, which are normally rare in benign polyps and characteristically expand during adenoma-to-carcinoma transition. Our findings highlight a hitherto unknown contribution of ß-catenin signaling in mast cells to their maturation and to increased risk of colon cancer.

CXCR4 signaling directs Igk recombination and the molecular mechanisms of late B lymphopoiesis.

In B lymphopoiesis, activation of the pre-B cell antigen receptor (pre-BCR) is associated with both cell cycle exit and Igk recombination. Yet how the pre-BCR mediates these functions remains unclear. Here, we demonstrate that the pre-BCR initiates a feed-forward amplification loop mediated by the transcription factor interferon regulatory factor 4 and the chemokine receptor C-X-C motif chemokine receptor 4 (CXCR4). CXCR4 ligation by C-X-C motif chemokine ligand 12 activates the mitogen-activated protein kinase extracellular-signal-regulated kinase, which then directs the development of small pre- and immature B cells, including orchestrating cell cycle exit, pre-BCR repression, Igk recombination and BCR expression. In contrast, pre-BCR expression and escape from interleukin-7 have only modest effects on B cell developmental transcriptional and epigenetic programs. These data show a direct and central role for CXCR4 in orchestrating late B cell lymphopoiesis. Furthermore, in the context of previous findings, our data provide a three-receptor system sufficient to recapitulate the essential features of B lymphopoiesis in vitro.

Chromatin restriction by the nucleosome remodeler Mi-2ß and functional interplay with lineage-specific transcription regulators control B-cell differentiation.

Coordinated induction, but also repression, of genes are key to normal differentiation. Although the role of lineage-specific transcription regulators has been studied extensively, their functional integration with chromatin remodelers, one of the key enzymatic machineries that control chromatin accessibility, remains ill-defined. Here we investigate the role of Mi-2ß, a SNF-2-like nucleosome remodeler and key component of the nucleosome remodeling and histone deacetylase (NuRD) complex in early B cells. Inactivation of Mi-2ß arrested differentiation at the large pre-B-cell stage and caused derepression of cell adhesion and cell migration signaling factors by increasing chromatin access at poised enhancers and chromosome architectural elements. Mi-2ß also supported IL-7R signaling, survival, and proliferation by repressing negative effectors of this pathway. Importantly, overexpression of Bcl2, a mitochondrial prosurvival gene and target of IL-7R signaling, partly rescued the differentiation block caused by Mi-2ß loss. Mi-2ß stably associated with chromatin sites that harbor binding motifs for IKAROS and EBF1 and physically associated with these transcription factors both on and off chromatin. Notably, Mi-2ß shared loss-of-function cellular and molecular phenotypes with IKAROS and EBF1, albeit in a distinct fashion. Thus, the nucleosome remodeler Mi-2ß promotes pre-B-cell differentiation by providing repression capabilities to distinct lineage-specific transcription factor-based regulatory networks.

TCF-1 and HEB cooperate to establish the epigenetic and transcription profiles of CD4+CD8+ thymocytes.

Thymocyte development requires a complex orchestration of multiple transcription factors. Ablating either TCF-1 or HEB in CD4+CD8+ thymocytes elicits similar developmental outcomes including increased proliferation, decreased survival, and fewer late Tcra rearrangements. Here, we provide a mechanistic explanation for these similarities by showing that TCF-1 and HEB share ~7,000 DNA-binding sites genome wide and promote chromatin accessibility. The binding of both TCF-1 and HEB was required at these shared sites for epigenetic and transcriptional gene regulation. Binding of TCF-1 and HEB to their conserved motifs in the enhancer regions of genes associated with T cell differentiation promoted their expression. Binding to sites lacking conserved motifs in the promoter regions of cell-cycle-associated genes limited proliferation. TCF-1 displaced nucleosomes, allowing for chromatin accessibility. Importantly, TCF-1 inhibited Notch signaling and consequently protected HEB from Notch-mediated proteasomal degradation. Thus, TCF-1 shifts nucleosomes and safeguards HEB, thereby enabling their cooperation in establishing the epigenetic and transcription profiles of CD4+CD8+ thymocytes.

Mast cells promote small bowel cancer in a tumor stage-specific and cytokine-dependent manner.

Mast cells (MCs) are tissue resident sentinels that mature and orchestrate inflammation in response to infection and allergy. While they are also frequently observed in tumors, the contribution of MCs to carcinogenesis remains unclear. Here, we show that sequential oncogenic events in gut epithelia expand different types of MCs in a temporal-, spatial-, and cytokine-dependent manner. The first wave of MCs expands focally in benign adenomatous polyps, which have elevated levels of IL-10, IL-13, and IL-33, and are rich in type-2 innate lymphoid cells (ILC2s). These vanguard MCs adhere to the transformed epithelial cells and express murine mast cell protease 2 (mMCP2; a typical mucosal MC protease) and, to a lesser extent, the connective tissue mast cell (CTMC) protease mMCP6. Persistence of MCs is strictly dependent on T cell-derived IL-10, and their loss in the absence of IL-10-expressing T cells markedly delays small bowel (SB) polyposis. MCs expand profusely in polyposis-prone mice when T cells overexpress IL-10. The frequency of polyp-associated MCs is unaltered in response to broad-spectrum antibiotics, arguing against a microbial component driving their recruitment. Intriguingly, when polyps become invasive, a second wave of mMCP5+/mMCP6+ CTMCs expands in the tumor stroma and at invasive tumor borders. Ablation of mMCP6 expression attenuates polyposis, but invasive properties of the remaining lesions remain intact. Our findings argue for a multistep process in SB carcinogenesis in which distinct MC subsets, and their elaborated proteases, guide disease progression.

The STAT3 inhibitor pyrimethamine displays anti-cancer and immune stimulatory effects in murine models of breast cancer.

The transcription factor signal activator and transducer or transcription (STAT3), which regulates genes controlling proliferation, survival, and invasion, is activated inappropriately in many human cancers, including breast cancer. Activation of STAT3 can lead to both malignant cellular behavior and suppression of immune cell function in the tumor microenvironment. Through a chemical-biology screen, pyrimethamine (PYR), an FDA approved anti-microbial drug, was identified as an inhibitor of STAT3 function at concentrations known to be achieved safely in humans. We report that PYR shows therapeutic activity in two independent mouse models of breast cancer, with both direct tumor inhibitory and immune stimulatory effects. PYR-inhibited STAT3 activity in TUBO and TM40D-MB metastatic breast cancer cells in vitro and inhibited tumor cell proliferation and invasion into Matrigel basement membrane matrix. In tumor-transplanted mice, PYR had both direct and indirect tumor inhibitory effects. Tumor-bearing mice treated with PYR showed reduced STAT3 activation in tumor cells, attenuated tumor growth, and reduced tumor-associated inflammation. In addition, expression of Lamp1 by tumor infiltrating CD8+ T cells was elevated, indicating enhanced release of cytotoxic granules. These findings suggest that PYR may have beneficial effects in the treatment of breast cancer.

Superenhancer reprogramming drives a B-cell-epithelial transition and high-risk leukemia.

IKAROS is required for the differentiation of highly proliferative pre-B-cell precursors, and loss of IKAROS function indicates poor prognosis in precursor B-cell acute lymphoblastic leukemia (B-ALL). Here we show that IKAROS regulates this developmental stage by positive and negative regulation of superenhancers with distinct lineage affiliations. IKAROS defines superenhancers at pre-B-cell differentiation genes together with B-cell master regulators such as PAX5, EBF1, and IRF4 but is required for a highly permissive chromatin environment, a function that cannot be compensated for by the other transcription factors. IKAROS is also highly enriched at inactive enhancers of genes normally expressed in stem-epithelial cells. Upon IKAROS loss, expression of pre-B-cell differentiation genes is attenuated, while a group of extralineage transcription factors that are directly repressed by IKAROS and depend on EBF1 relocalization at their enhancers for expression is induced. LHX2, LMO2, and TEAD-YAP1, normally kept separate from native B-cell transcription regulators by IKAROS, now cooperate directly with them in a de novo superenhancer network with its own feed-forward transcriptional reinforcement. Induction of de novo superenhancers antagonizes Polycomb repression and superimposes aberrant stem-epithelial cell properties in a B-cell precursor. This dual mechanism of IKAROS regulation promotes differentiation while safeguarding against a hybrid stem-epithelial-B-cell phenotype that underlies high-risk B-ALL.

Preferential expansion of pro-inflammatory Tregs in human non-small cell lung cancer.

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(+)Foxp3(int) (naïve, Fr. I) or CD45RA(-)Foxp3(hi) (activated Fr. II). Activated conventional T cells were CD4(+)CD45RA(-)Foxp3(int) (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.

T-cell Expression of IL10 Is Essential for Tumor Immune Surveillance in the Small Intestine.

IL10 is attributed with immune-suppressive and anti-inflammatory properties, which could promote or suppress cancer in the gastrointestinal tract. Loss of IL10 exacerbates colonic inflammation, leading to colitis and cancer. Consistent with this, transfer of IL10-competent regulatory T cells (Treg) into mice with colitis or hereditary polyposis protects against disease, while IL10-deficient mice are predisposed to polyposis with increased colon polyp load. Little is known about the protective or pathogenic function of IL10 in cancers of the small intestine. We found CD4(+) T cells and CD4(+) Foxp3(+) Tregs to be the major sources of IL10 in the small intestine and responsible for the increase in IL10 during polyposis in the APC(Δ468) mouse model of hereditary polyposis. Targeted ablation of IL10 in T cells caused severe IL10 deficiency and delayed polyp growth. However, these polyps progressively lost cytotoxic activity and eventually progressed to cancer. Several observations suggested that the effect was due to the loss of IFNγ-dependent immune surveillance. IL10-incompetent CD4(+) T cells failed to secrete IFNγ when stimulated with polyp antigens and were inefficient in T-helper-1 (TH1) commitment. By contrast, the TH17 commitment was unaffected. These findings were validated using mice whose T cells overexpress IL10. In these mice, we observed high intra-polyp cytotoxic activity and attenuation of polyposis. Thus, expression of IL10 by T cells is protective and required for immune surveillance in the small intestine.

Repression of Ccr9 transcription in mouse T lymphocyte progenitors by the Notch signaling pathway.

The chemokine receptor CCR9 controls the immigration of multipotent hematopoietic progenitor cells into the thymus to sustain T cell development. Postimmigration, thymocytes downregulate CCR9 and migrate toward the subcapsular zone where they recombine their TCR ß-chain and γ-chain gene loci. CCR9 is subsequently upregulated and participates in the localization of thymocytes during their selection for self-tolerant receptor specificities. Although the dynamic regulation of CCR9 is essential for early T cell development, the mechanisms controlling CCR9 expression have not been determined. In this article, we show that key regulators of T cell development, Notch1 and the E protein transcription factors E2A and HEB, coordinately control the expression of Ccr9. E2A and HEB bind at two putative enhancers upstream of Ccr9 and positively regulate CCR9 expression at multiple stages of T cell development. In contrast, the canonical Notch signaling pathway prevents the recruitment of p300 to the putative Ccr9 enhancers, resulting in decreased acetylation of histone H3 and a failure to recruit RNA polymerase II to the Ccr9 promoter. Although Notch signaling modestly modulates the binding of E proteins to one of the two Ccr9 enhancers, we found that Notch signaling represses Ccr9 in T cell lymphoma lines in which Ccr9 transcription is independent of E protein function. Our data support the hypothesis that activation of Notch1 has a dominant-negative effect on Ccr9 transcription and that Notch1 and E proteins control the dynamic expression of Ccr9 during T cell development.