The transcription factor Six1 is essential for induction of sensory cell fate and formation of auditory sensory epithelium, but how it activates gene expression programs to generate distinct cell-types remains unknown. Here, we perform genome-wide characterization of Six1 binding at different stages of auditory sensory epithelium development and find that Six1-binding to cis-regulatory elements changes dramatically at cell-state transitions. Intriguingly, Six1 pre-occupies enhancers of cell-type-specific regulators and effectors before their expression. We demonstrate in-vivo cell-type-specific activity of Six1-bound novel enhancers of Pbx1, Fgf8, Dusp6, Vangl2, the hair-cell master regulator Atoh1 and a cascade of Atoh1's downstream factors, including Pou4f3 and Gfi1. A subset of Six1-bound sites carry consensus-sequences for its downstream factors, including Atoh1, Gfi1, Pou4f3, Gata3 and Pbx1, all of which physically interact with Six1. Motif analysis identifies RFX/X-box as one of the most significantly enriched motifs in Six1-bound sites, and we demonstrate that Six1-RFX proteins cooperatively regulate gene expression through binding to SIX:RFX-motifs. Six1 targets a wide range of hair-bundle regulators and late Six1 deletion disrupts hair-bundle polarity. This study provides a mechanistic understanding of how Six1 cooperates with distinct cofactors in feedforward loops to control lineage-specific gene expression programs during progressive differentiation of the auditory sensory epithelium.

T-cell development in mammals is a model for lineage choice and differentiation from multipotent stem cells. Although T-cell fate choice is promoted by signaling in the thymus through one dominant pathway, the Notch pathway, it entails a complex set of gene regulatory network and chromatin state changes even before the cells begin to express their signature feature, the clonal-specific T-cell receptors (TCRs) for antigen. This review distinguishes three developmental modules for T-cell development, which correspond to cell type specification, TCR expression and selection, and the assignment of cells to different effector types. The first is based on transcriptional regulatory network events, the second is dominated by somatic gene rearrangement and mutation and cell selection, and the third corresponds to establishing a poised state of latent regulator priming through an unknown mechanism. Interestingly, in different lineages, the third module can be deployed at variable times relative to the completion of the first two modules. This review focuses on the gene regulatory network and chromatin-based kinetic constraints that determine activities of transcription factors TCF1, GATA3, PU.1, Bcl11b, Runx1, and E proteins in the primary establishment of T-cell identity.

Ovarian high-grade serous carcinoma (HGSC) is the most lethal gynecological malignancy. Prevailing evidences suggest that drug resistance and recurrence of ovarian HGSC are caused by the presence of cancer stem cells. Therefore, targeting cancer stems is appealing, however, all attempts to date, have failed. To circumvent this limit, we analyzed differential transcriptomes at early differentiation of ovarian HGSC stem cells and identified the developmental transcription factor GATA3 as highly expressed in stem, compared to progenitor cells. GATA3 expression associates with poor prognosis of ovarian HGSC patients, and was found to recruit the histone H3, lysine 27 (H3K27) demethylase, UTX, activate stemness markers, and promote stem-like phenotypes in ovarian HGSC cell lines. Targeting UTX by its inhibitor, GSKJ4, impeded GATA3-driven stemness phenotypes, and enhanced apoptosis of GATA3-expressing cancer cells. Combinations of gemcitabine or paclitaxel with GSKJ4, resulted in a synergistic cytotoxic effect. Our findings provide evidence for a new role for GATA3 in ovarian HGSC stemness, and demonstrate that GATA3 may serve as a biomarker for precision epigenetic therapy in the future.

GATA-binding protein 3 (GATA3) acts as the master transcription factor for type 2 T helper (Th2) cell differentiation and function. However, it is still elusive how GATA3 function is precisely regulated in Th2 cells. Here, we show that the transcription factor B cell lymphoma 11b (Bcl11b), a previously unknown component of GATA3 transcriptional complex, is involved in GATA3-mediated gene regulation. Bcl11b binds to GATA3 through protein-protein interaction, and they colocalize at many important cis-regulatory elements in Th2 cells. The expression of type 2 cytokines, including IL-4, IL-5, and IL-13, is up-regulated in Bcl11b-deficient Th2 cells both in vitro and in vivo; such up-regulation is completely GATA3 dependent. Genome-wide analyses of Bcl11b- and GATA3-regulated genes (from RNA sequencing), cobinding patterns (from chromatin immunoprecipitation sequencing), and Bcl11b-modulated epigenetic modification and gene accessibility suggest that GATA3/Bcl11b complex is involved in limiting Th2 gene expression, as well as in inhibiting non-Th2 gene expression. Thus, Bcl11b controls both GATA3-mediated gene activation and repression in Th2 cells.

Mechanistic studies have improved our understanding of molecular and cellular components involved in asthma and our ability to treat severe patients. An mAb directed against IgE (omalizumab) has become an established add-on therapy for patients with uncontrolled allergic asthma and mAbs specific for IL-5 (reslizumab, mepolizumab), IL-5R (benralizumab), and IL-4R (dupilumab) have been approved as add-on treatments for uncontrolled eosinophilic (type 2) asthma. While these medications have proven highly effective, some patients with severe allergic and/or eosinophilic asthma, as well as most patients with severe non-type-2 disease, have poorly controlled disease. Agents that have recently been evaluated in clinical trials include an antibody directed against thymic stromal lymphopoietin, small molecule antagonists to the chemoattractant receptor-homologous molecule expressed on T_HGATA3. Antibodies to IL-33 and its receptor, ST2, are being evaluated in ongoing clinical studies. In addition, a number of antagonists directed against other potential targets are under consideration for future trials, including IL-25, IL-6, TNF-like ligand 1A, CD6, and activated cell adhesion molecule (ALCAM). Clinical data from ongoing and future trials will be important in determining whether these new medications will offer benefits in place of or in addition to existing therapies for asthma.

The activation mechanism of chimeric antigen receptor (CAR)-engineered T cells may differ substantially from T cells carrying native T cell receptor, but this difference remains poorly understood. We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19/4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4⁺ helper T (T_H) cells and CD8⁺ cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of T_H1 and T_H2 signature cytokines, e.g., interferon γ, tumor necrotic factor α, interleukin 5 (IL5), and IL13, as confirmed by the expression of master transcription factor genes TBX21 and <em>GATA3</em>. However, rather than conforming to stringent T_H1 or T_H2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed T_H1/T_H2 function in the same cell. The regulatory T cell activity, although observed in a small fraction of activated cells, emerges from this hybrid T_H1/T_H2 population. Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status, although all major differentiation subsets such as naïve, central memory, effector memory, and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GM-CSF, supporting the notion that polyfunctional CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights into the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.

A member of winged-helix/forkhead transcription factors, Foxp1, is expressed in the developing spinal cord during mouse embryogenesis. To shed light on the potential role of Foxp1 in neurons of the developing spinal cord, we investigated the detailed expression pattern of Foxp1 between embryonic day (E) 9.5 and E17.5. At E10.25, some postmitotic neurons with strong expression of Foxp1 (Foxp1(high)) were first detected in the ventral half of the brachial spinal cord. By E11.5, Foxp1(high) neurons increased in the ventral spinal cord at the limb levels. All of Foxp1(high) neurons at the limb levels were Islet2(+)/Lhx3(-) motor neurons (MNs) of the lateral motor column and some neurons that expressed Foxp1 weakly (Foxp1(low)) at the thoracic level were MNs of the preganglionic motor column. Between E12.5 and E17.5, Foxp1(low) neurons were also observed in the intermediate zone throughout the ventral spinal cord, all of which were Pax2(+), En1(+), Evx1(-), Chx10(-), Gata3(-), and Lhx3(-) V1 interneurons. Interestingly, no colocalization of Foxp1 with Lhx3 was observed in the developing spinal cord. In addition, overexpression of Foxp1 markedly attenuated the endogenous expression of Lhx3 in a neuroendocrine cell line. Chromatin immunoprecipitation assays in a neuronal cell line and E13.5 spinal cords revealed an interaction between Foxp1 and the consensus motif in the Lhx3 promoter. These results suggest that Foxp1 may play some important roles in the determination of neuronal fates of the ventral spinal cord, possibly through the suppression of Lhx3 expression.

Defective apoptosis of eosinophils, the main leukocyte in the pathogenesis of asthma, and delay in its removal lead to lung damage and loss of pulmonary function due to failure in the resolution of inflammation. Here, we investigated the ability of angiotensin-(1-7) [Ang-(1-7)], a pivotal peptide of the renin-angiotensin system, to promote resolution of an allergic lung inflammatory response. Balb/c mice were sensitized and challenged with ovalbumin and treated with Ang-(1-7) at the peak of the inflammatory process. Bronchoalveolar lavage (BAL) fluid and lungs were collected 24 h after treatment. Different lung lobes were processed for histology to evaluate inflammatory cell infiltration, airway and pulmonary remodeling, total collagen staining, and measurements of (i) collagen I and III mRNA expression by qRT-PCR; (ii) ERK1/2, IκB-α, and GATA3 protein levels by Western blotting; and (iii) eosinophilic peroxidase activity. Total number of inflammatory cells, proportion of apoptotic eosinophils and immunofluorescence for caspase 3 and NF-κB in leukocytes were evaluated in the BAL. Mas receptor immunostaining was evaluated in mouse and human eosinophils. Engulfment of human polimorphonuclear cells by macrophages, efferocytosis, was evaluated in vivo. Ang-(1-7) reduced eosinophils in the lung and in the BAL, increased the number of apoptotic eosinophils, shown by histology criteria and by increase in caspase 3 immunostaining. Furthermore, Ang-(1-7) decreased NF-kB immunostaining in eosinophils, reduced GATA3, ERK1/2, and IκB-α expression in the lung and decreased pulmonary remodeling and collagen deposition. Importantly, Ang-(1-7) increased efferocytosis. Our results demonstrate, for the first time, Ang-(1-7) activates events that are crucial for resolution of the inflammatory process of asthma and promotion of the return of lung homeostasis, indicating Ang-(1-7) as novel endogenous inflammation-resolving mediator.

BACKGROUND

-Sarcomatoid urothelial carcinoma (UCa) is a rare but aggressive variant of bladder cancer that can show diagnostic challenges even using ancillary techniques.

OBJECTIVE

-To examine immunohistochemical markers in the context of sarcomatoid UCa, including those associated with epithelial-to-mesenchymal transition.

METHODS

-Twenty-eight cases of sarcomatoid UCa were rereviewed. Clinical outcomes were obtained through database search. Immunohistochemistry for clinical and epithelial-to-mesenchymal transition markers was performed.

RESULTS

-All patients had biopsy-proven invasive UCa; 61% (17 of 28) had sarcomatoid UCa at initial diagnosis. A recognizable epithelial component(s) was present in 17 lesions. The sarcomatoid component accounted for 65% of the lesion (average), with heterologous elements present in 3 of 28 cases (11%). The morphologic spectrum of the sarcomatoid element included spindled not otherwise specified, myxoid, pseudoangiosarcomatous, and malignant fibrous histiocytoma-like undifferentiated features. The sarcomatoid component was immunoreactive for pancytokeratin (22 of 26; 85%), p63 (20 of 26; 77%), cytokeratin 903 (17 of 26; 65%), cytokeratin 7 (16 of 26; 62%), GATA3 (16 of 26; 62%), and cytokeratin 5/6 (16 of 26; 62%). STAT-6, CD31, CD34, and HMB45 were all nonreactive, whereas smooth muscle actin often showed at least focal immunoreactivity (22 of 26; 85%). Epithelial-to-mesenchymal transition markers were frequently expressed, including vimentin (26 of 26; 100%), FoxC2 (26 of 26; 100%), SNAIL (23 of 26; 88.5%), and ZEB1 (18 of 26; 69.2%). Follow-up was available for 24 patients (median, 7 months). Sixteen of 28 patients (57%) died of disease (overall mean survival, 9.1 months). The presence of myxoid or chordoid features was associated with reduced survival (P < .05).

CONCLUSIONS

-Sarcomatoid UCa is an aggressive form of UCa that frequently expresses epithelial-to-mesenchymal transition markers, suggesting a possible mechanism associated with aggressive tumor behavior.

Upon activation, naïve CD4⁺ T cells differentiate into a number of specialized T helper (Th) cell subsets. Th2 cells are central players in immunity to helminths and are implicated in mediating the inflammatory pathology associated with allergies. The differentiation of Th2 cells is dependent on transcription factors such as GATA3 and STAT6, which prime Th2 cells for the secretion of interleukin- (IL-) 4, IL-5, and IL-13. Several lines of work now suggest that differentiating Th2 cells in the lymph node are potent IL-4 cytokine producers, but do not become competent IL-5- and IL-13-producing cells until after receiving cues from non-lymphoid tissue. It is evident that Th2 cells that enter tissues undergo considerable changes in chromatin architecture and gene expression, and that over this time, the metabolic requirements of these cells change considerably. Herein, we discuss the metabolic requirements of Th2 cells during their early and late differentiation, focusing on the impact of glucose and lipid metabolism, mTOR activation, the nuclear receptor PPAR-γ and several metabolites.

Neuroendocrine breast carcinomas (NBCs) account for 2-5% of all invasive breast cancers, and are histologically similar to neuroendocrine tumours from other sites. They typically express oestrogen receptor (ER), and are HER2-negative and of luminal 'intrinsic' subtype. Here, we sought to define the mutational profile of NBCs, and to investigate whether NBCs and common forms of luminal (ER+ /HER2- ) breast carcinoma show distinct repertoires of somatic mutations. Eighteen ER+ /HER2- NBCs, defined as harbouring >50% of tumour cells expressing chromogranin A and/or synaptophysin, and matched normal tissues were microdissected and subjected to massively parallel sequencing targeting all exons of 254 genes most frequently mutated in breast carcinomas and/or related to DNA repair. Their mutational repertoire was compared with that of ER+ /HER2- breast carcinomas (n = 240), PAM50-defined luminal breast carcinomas (luminal A, n = 209; luminal B, n = 111) and invasive lobular carcinomas (n = 127) from The Cancer Genome Atlas. NBCs were found to harbour a median of 4.5 (range 1-11) somatic mutations, similar to that of luminal B breast carcinomas (median = 3, range 0-17) but significantly higher than that of luminal A breast carcinomas (median = 3, range 0-18, p = 0.02). The most frequently mutated genes were GATA3, FOXA1, TBX3, and ARID1A (3/18, 17%), and PIK3CA, AKT1, and CDH1 (2/18, 11%). NBCs less frequently harboured PIK3CA mutations than common forms of ER+ /HER2- , luminal A and invasive lobular carcinomas (p < 0.05), and showed a significantly higher frequency of somatic mutations affecting ARID1A (17% versus 2%, p < 0.05) and the transcription factor-encoding genes FOXA1 (17% versus 2%, p = 0.01) and TBX3 (17% versus 3%, p < 0.05) than common-type ER+ /HER2- breast carcinomas. No TP53 somatic mutations were detected in NBCs. As compared with common forms of luminal breast carcinomas, NBCs show a distinctive repertoire of somatic mutations featuring lower frequencies of TP53 and PIK3CA mutations, enrichment for FOXA1 and TBX3 mutations, and, akin to neuroendocrine tumours from other sites, ARID1A mutations. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Type 2 cytokines are usually predominant in tumor patients and associated with tumor progression. To explore whether reversing of type 2 predominance could be a promising strategy in tumor immunotherapy, PBMC of 35 lung cancer patients and 19 healthy subjects were prepared and subjected to be examined for cytokine secretion and gene expression. Tetra-Methylpyrazine (TTMP), extracted from a traditional Chinese medicinal herb which has been used in clinic to reverse the Th2 status of cancer patients in China, was added to PBMC culture. Determined by RT-PCR, the positive percentages of mRNA expression of type 1 cytokines (8.6% for IFN-gamma and 11.4% for IL-2) were lower than those of type 2 cytokines (71.4% for IL-4, 60% for IL-6 and 80% for IL-10) in patients' PBMCs. The potential of gene expressing (measured as relative intensity to the ratio of beta-actin) in the patients for type 1 cytokines was also in a low level (0.111 for IFN-gamma, 0.119 for IL-2) in comparison with a relative high level for type 2 cytokines (0.319 for IL-4, 0.303 for IL-6 and 0.377 for IL-10). Meanwhile, both positive percentage and relative intensity of gene expression were lower for a type 1 cytokine-related transcription factor T-bet (31.4% and 0.142, respectively) than those for type 2 cytokine-related GATA3 (85.7% and 0.378, respectively). The blood serum levels of IFN-gamma and IL-2 in the patients were slightly lower but not significantly when compared with healthy control. In contrast, the levels IL-4 and IL-6 in patients were significantly higher than those in healthy subjects by ELISA analysis. TTMP could enhance supernatant concentration and gene expression levels of IFN-gamma, IL-2 and T-bet, but reduce those of type 2 cytokines. These results demonstrate that the lung cancer patients had a predominant expression of type 2 cytokines and TTMP could reverse the type 2 dominant status, which might offer an alternative therapeutic regime for lung cancer patients.

RIP140 is a transcriptional coregulator involved in energy homeostasis, ovulation, and mammary gland development. Although conclusive evidence is lacking, reports have implicated a role for RIP140 in breast cancer. Here, we explored the mechanistic role of RIP140 in breast cancer and its involvement in estrogen receptor α (ERα) transcriptional regulation of gene expression. Using ChIP-seq analysis, we demonstrate that RIP140 shares more than 80% of its binding sites with ERα, colocalizing with its interaction partners FOXA1, GATA3, p300, CBP, and p160 family members at H3K4me1-demarcated enhancer regions. RIP140 is required for ERα-complex formation, ERα-mediated gene expression, and ERα-dependent breast cancer cell proliferation. Genes affected following RIP140 silencing could be used to stratify tamoxifen-treated breast cancer cohorts, based on clinical outcome. Importantly, this gene signature was only effective in endocrine-treated conditions. Cumulatively, our data suggest that RIP140 plays an important role in ERα-mediated transcriptional regulation in breast cancer and response to tamoxifen treatment.

Nonimmunological connective tissue phenotypes in humans are common among some congenital and acquired allergic diseases. Several of these congenital disorders have been associated with either increased TGF-β activity or impaired STAT3 activation, suggesting that these pathways might intersect and that their disruption may contribute to atopy. In this study, we show that STAT3 negatively regulates TGF-β signaling via ERBB2-interacting protein (ERBIN), a SMAD anchor for receptor activation and SMAD2/3 binding protein. Individuals with dominant-negative STAT3 mutations (STAT3mut ) or a loss-of-function mutation in ERBB2IP (ERBB2IPmut ) have evidence of deregulated TGF-β signaling with increased regulatory T cells and total FOXP3 expression. These naturally occurring mutations, recapitulated in vitro, impair STAT3-ERBIN-SMAD2/3 complex formation and fail to constrain nuclear pSMAD2/3 in response to TGF-β. In turn, cell-intrinsic deregulation of TGF-β signaling is associated with increased functional IL-4Rα expression on naive lymphocytes and can induce expression and activation of the IL-4/IL-4Rα/GATA3 axis in vitro. These findings link increased TGF-β pathway activation in ERBB2IPmut and STAT3mut patient lymphocytes with increased T helper type 2 cytokine expression and elevated IgE.

Previously we have screened out Insulin-like Growth Factor Binding Protein 7 (IGFBP7) as a differentially expressed gene in post-implantation uterus versus pre-implantation uterus by suppressive subtractive hybridation. However its function in uterus was not clearly identified. In this research, the expression and function of IGFBP7 during post-implantation were studied. We found that IGFBP7 was mainly located in the glandular epithelium and the stroma, and was upregulated after embryo implantation. The vector pCR3.1-IGFBP7-t expressing partial IGFBP7 was constructed. Inhibition of IGFBP7 by specific DNA immunization induced significant reduction of implanted embryos and pregnancy rate. The number of implanted embryos (5.68 ± 0.46) was significantly reduced after immunization with pCR3.1-IGFBP7-t, as compared with that of the mice immunized with the control vector (12.29 ± 0.36) or saline (14.58 ± 0.40) (p<0.01). After specific inhibition of IGFBP7, the T helper type 1 (Th1) cytokine IFNγ, was significantly elevated (p<0.05) and the Th2 cytokines IL-4 and IL-10, were reduced in uteri (p<0.05). The increase of Tbet and the decrease of Gata3 were found in mice peripheral lymphocytes by flow cytometry. The expression of decidualization marker IGFBP1 and angiogenesis regulator VEGF were declined in uteri (p<0.05). The expression of apoptosis-associated proteins, caspase3 and Bcl-2, were also declined (p<0.05). These results showed that inhibition of IGFBP7 induced pregnancy failure by shifting uterine cytokines to Th1 type dominance and repressing uterine decidualization.

Langerhans cell histiocytosis (LCH) is a neoplasm of myeloid origin characterized by a clonal proliferation of CD1a(+)/CD207(+) dendritic cells. Recurrent BRAF V600E mutation has been reported in LCH. In the present report, we confirm the feasibility of the high-specificity monoclonal antibody VE1 for detecting BRAF V600E mutation in 36/97 (37.1%) retrospectively enrolled patients with LCH; concordant immunohistochemistry and Sanger sequencing results were seen in 94.8% of cases. We then assessed the tumor immune microenvironment status in LCH, and found that the GATA binding protein 3 (GATA3)(+)/T-bet(+) ratio could distinguish between clinical multi-system/single-system (SS) multifocal and SS unifocal LCH. Notably, we found that BRAF V600E mutation is significantly correlated with increased programmed cell death 1 ligand 1 (PDL1) expression and forkhead box protein 3 (FOXP3)(+) regulatory T cells (p < 0.001, 0.009, respectively). Moreover, Cox multivariate survival analysis showed that BRAF V600E mutation and PDL1 were independent prognostic factors of poor disease-free survival (DFS) in LCH (hazard ratio [HR] = 2.38, 95% confidence interval [CI] 1.02-5.56, p = 0.044; HR = 3.06, 95%CI 1.14-7.14, p = 0.025, respectively), and the superiority of PDL1 in sensitivity and specificity as biomarker for DFS in LCH was demonstrated by receiver operator characteristic (ROC) curves when compared with BRAF V600E and risk category. Collectively, this study identifies for the first time relationship between BRAF V600E mutation and a suppressive tumor immune microenvironment in LCH, resulting in disruption of host-tumor immune surveillance, which is DFS. Our findings may provide a rationale for combining immunotherapy and BRAF-targeted therapy for treating patients with BRAF V600E mutant LCH.

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.

T lymphocytes are the most numerous immune cells in tumor-associated infiltrates and include several subpopulations of either anticancer or pro-tumorigenic functions. However, the associations between levels of different T cell subsets and breast cancer molecular subtypes as well as other prognostic factors have not been fully established yet. We performed immunohistochemistry for CD8 (cytotoxic T cells (CTL)), FOXP3 (regulatory T cells (Tregs)), and GATA3 (Th2 cells) in 106 formalin-fixed paraffin-embedded invasive breast cancer tissue samples and analyzed both the numbers and percentages of investigated cells in tumor-associated infiltrates. We observed that triple-negative breast cancer (TNBC) and HER2+ non-luminal breast tumors were associated with more numerous CTLs and Tregs and a higher Treg/Th2 cell ratio as compared with luminal A subtype. A higher Treg percentage was related to a decreased hormone receptor expression, an increase in the Ki67 level, a greater tumor size of luminal tumors, and the presence of lymph node metastases. Moreover, differences in the composition of T cell infiltrates were associated with HER2 status and histologic grade and type, and a distinct immune pattern was observed in tumors of different phenotypes regarding pT stage and nodal status. The results of our work show the diversity of T cell infiltrates in primary invasive breast cancers of different phenotypes and suggest that progression of luminal or non-luminal tumors is related to distinct tumor-associated T cell composition.

CD4(+) T cells are essential for defenses against pathogens and affect the functions of most cells involved in the immune response. Although CD4(+) T cells generally recognize peptide antigens bound to MHC-II molecules, important subsets are restricted by other MHC or MHC-like molecules, including CD1d-restricted "invariant" iNK T cells. This review discusses recently identified nodes in the transcriptional circuits that are involved in controlling CD4(+) T-cell differentiation, notably the commitment factor Thpok and its interplay with Runx transcriptional regulators, and focuses on how transcription factors acting upstream of Thpok, including Gata3, Tox and E-box proteins, promote the emergence of CD4-lineage-specific gene expression patterns.

Tree shrew has increasingly become an attractive experimental animal model for human diseases, particularly for breast cancer due to spontaneous breast tumours and their close relationship to primates and by extension to humans. However, neither normal mammary glands nor breast tumours have been well characterised in the Chinese tree shrew (Tupaia belangeri chinensis). In this study, normal mammary glands from four different developmental stages and 18 spontaneous breast tumours were analysed. Haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) showed that normal mammary gland morphology and structures of tree shrews were quite similar to those found in humans. Spontaneous breast tumours of tree shrews were identified as being intraductal papilloma, papillary carcinoma, and invasive ductal carcinoma with or without lung metastasis. To further analyse breast cancer tumours among tree shrews, 40 3-4 month-old female tree shrews were orally administrated 20 mg 7,12-dimethylbenz(a)anthracene (DMBA) or peanut oil thrice, and then, 15 of these DMBA administrated tree shrews were implanted with medroxyprogesterone acetate (MPA) pellets. DMBA was shown to induce breast tumours (12%) while the addition of MPA increased the tumour incidence (50%). Of these, three induced breast tumours were intraductal papillary carcinomas and one was invasive ductal carcinoma (IDC). The PTEN/PIK3CA (phosphatase and tensin homologue/phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha), but not TP53 and GATA3, genes are frequently mutated in breast tumours, and the PTEN/PIK3CA gene mutation status correlated with the expression of pAKT in tree shrew breast tumours. These results suggest that tree shrews may be a promising animal model for a subset of human breast cancers with PTEN/PIK3CA gene mutations.

Homeodomain transcription factors serve important functions in organogenesis and tissue differentiation, particularly with respect to the positional identity of individual cells. The Nkx6 subfamily controls tissue differentiation in the developing central nervous system where they function as transcriptional repressor proteins. Recent work indicates that Nkx6.3 is expressed in hindbrain V2 interneurons that co-express Nkx6.1, suggesting the possibility of functional redundancy. Here, we report that Nkx6.3 expression is specific to Chx10+ V2a interneurons but not to Gata3+ V2b interneurons of the hindbrain, and that Nkx6.3 expression appears to mark cells of the prospective medullary reticular formation. Molecular analysis of Nkx6.3 null embryonic mouse hindbrain did not reveal detectable defects in progenitor markers, motor neuron or V2 interneuron sub-types. Forced expression of Nkx6.3 and Nkx6.1 promote V2 interneuron differentiation in the developing chick hindbrain. These findings indicate Nkx6.3 function is dispensable for CNS development and lead to the proposal that absence of overt defects is due to functional compensation from a related homeodomain transcription factor.

Upon activation in vitro, only a fraction of the bulk human T helper cell cultures secret the hallmark Th1/2 cytokines (IFN-gamma for Th1 and IL-4 for Th2, respectively). It is uncertain whether these IFN-gamma-/IL-4- cells are differentiated Th1 or Th2 cells. Here, we have characterized live IFN-gamma+, IL-4+ and IFN-gamma-/IL-4- cells isolated from Th cell cultures treated under Th1 or Th2 polarizing conditions by employing affinity matrix capture technology. RNA samples from the sorted cells were analyzed by real time RT-PCR and microarrays. The double negative cells from either Th1 or Th2 cultures expressed lower levels of Th1/Th2 marker cytokine genes (IFNgamma, IL4, and IL5). However, they were comparable with the IFN-gamma+ or IL-4+ cells in the expression levels of other Th1/Th2 marker genes (GATA3, Tbet, and IL12Rbeta2). Most importantly, these double negative cells were already committed in their Th1/Th2 lineages. Gene expression profiling analysis showed that very few previously identified Th1/Th2 marker genes were differentially expressed between the IFN-gamma or IL-4 producers and the non-producers, further underscoring the similarity between these two groups.

T lymphocyte development and differentiation is a multi-step process that begins in the thymus and completed in the periphery. Sequential development of thymocytes is dependent on T cell receptor (TCR) signaling and an array of transcription factors. In this study we show that special AT-rich binding protein 1 (SATB1), a T lineage-enriched chromatin organizer and regulator, is induced in response to TCR signaling during early thymocyte development. SATB1 expression profile coincides with T lineage commitment and upregulation of SATB1 correlates with positive selection of thymocytes. CD4 thymocytes exhibit a characteristic bimodal expression pattern that corresponds to immature and mature CD4 thymocytes. We also demonstrate that GATA3, the key transcriptional regulator of αβ T cells positively regulates SATB1 expression in thymocytes suggesting an important role for SATB1 during T cell development.

We report on a Japanese girl with HDR (hypoparathyroidism, sensorineural deafness, and renal dysplasia) syndrome who developed diabetes mellitus (DM) at three years of age (blood glucose 713 mg/dL, HbA(1c) 8.0%) in the absence of anti-glutamic acid decarboxylase autoantibodies. Mutation analysis revealed a de novo heterozygous two base pair deletion at exon 6 of the GATA3 gene (c.1200_1201delCA; p.H400fsX506). GATA3 expression was identified by PCR amplification for human pancreas cDNA, and mouse Gata3 was weekly but unequivocally expressed in pancreatic beta cells. The results, in conjunction with the previous findings indicating the critical role of GATA3 in lymphocyte function, GATA3 haploinsufficiency may affect the function of beta cells and/or lymphocytes, leading to the development of DM in relatively exceptional patients with high susceptibility to DM.