NAFLD causes selective CD4(+) T lymphocyte loss and promotes hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related death. Non-alcoholic fatty liver disease (NAFLD) affects a large proportion of the US population and is considered to be a metabolic predisposition to liver cancer. However, the role of adaptive immune responses in NAFLD-promoted HCC is largely unknown. Here we show, in mouse models and human samples, that dysregulation of lipid metabolism in NAFLD causes a selective loss of intrahepatic CD4(+) but not CD8(+) T lymphocytes, leading to accelerated hepatocarcinogenesis. We also demonstrate that CD4(+) T lymphocytes have greater mitochondrial mass than CD8(+) T lymphocytes and generate higher levels of mitochondrially derived reactive oxygen species (ROS). Disruption of mitochondrial function by linoleic acid, a fatty acid accumulated in NAFLD, causes more oxidative damage than other free fatty acids such as palmitic acid, and mediates selective loss of intrahepatic CD4(+) T lymphocytes. In vivo blockade of ROS reversed NAFLD-induced hepatic CD4(+) T lymphocyte decrease and delayed NAFLD-promoted HCC. Our results provide an unexpected link between lipid dysregulation and impaired anti-tumour surveillance.

Helios Deficiency Predisposes the Differentiation of CD4+Foxp3- T Cells into Peripherally Derived Regulatory T Cells.

The transcription factor Helios is expressed in a large percentage of Foxp3+ regulatory T (Treg) cells and is required for the maintenance of their suppressive phenotype, as mice with a selective deficiency of Helios in Treg cells spontaneously develop autoimmunity. However, mice with a deficiency of Helios in all T cells do not exhibit autoimmunity, despite the defect in the suppressor function of their Treg cell population, suggesting that Helios also functions in non-Treg cells. Although Helios is expressed in a small subset of CD4+Foxp3- and CD8+ T cells and its expression is upregulated upon T cell activation, its function in non-Treg cells remains unknown. To examine the function of Helios in CD4+Foxp3- T cells, we transferred Helios-sufficient or -deficient naive CD4+Foxp3- TCR transgenic T cells to normal recipients and examined their capacity to respond to their cognate Ag. Surprisingly, Helios-deficient CD4+ T cells expanded and differentiated into Th1 or Th2 cytokine-producing effectors in a manner similar to wild-type TCR transgenic CD4+ T cells. However, the primed Helios-deficient cells failed to expand upon secondary challenge with Ag. The tolerant state of the Helios-deficient memory T cells was not cell-intrinsic but was due to a small population of Helios-deficient naive T cells that had differentiated into Ag-specific peripheral Treg cells that suppressed the recall response in an Ag-specific manner. These findings demonstrate that Helios plays a role in the determination of CD4+ T cell fate.

Helios+ and Helios- Treg subpopulations are phenotypically and functionally distinct and express dissimilar TCR repertoires.

The transcription factor Helios is expressed in a large subset of Foxp3+ Tregs. We previously proposed that Helios is a marker of thymic derived Treg (tTreg), while Helios- Treg were induced from Foxp3- T conventional (Tconv) cells in the periphery (pTreg). To compare the two Treg subpopulations, we generated Helios-GFP reporter mice and crossed them to Foxp3-RFP reporter mice. The Helios+ Treg population expressed a more activated phenotype, had a slightly higher suppressive capacity in vitro and expressed a more highly demethylated TSDR but were equivalent in their ability to suppress inflammatory bowel disease in vivo. However, Helios+ Treg more effectively inhibited the proliferation of activated, autoreactive splenocytes from scurfy mice. When Helios+ and Helios- Treg were transferred to lymphoreplete mice, both populations maintained comparable Foxp3 expression, but Foxp3 expression was less stable in Helios- Treg when transferred to lymphopenic mice. Gene expression profiling demonstrated a large number of differentially expressed genes and showed that Helios- Treg expressed certain genes normally expressed in CD4+ Foxp3- T cells. TCR repertoire analysis indicated very little overlap between Helios+ and Helios- Treg. Thus, Helios+ and Helios- Treg subpopulations are phenotypically and functionally distinct and express dissimilar TCR repertoires.

Helios: still behind the clouds.

Regulatory T (Treg) cells are a subset of CD4+ T cells that are critical for the maintenance of self-tolerance. The forkhead box transcription factor Foxp3 is a master regulator for the Treg phenotype and function and its expression is essential in Treg cells, as the loss of Foxp3 results in lethal autoimmunity. Two major subsets of Treg cells have been described in vivo; thymus-derived Treg (tTreg) cells that develop in the thymus and peripherally induced Treg (pTreg) cells that are derived from conventional CD4+  Foxp3- T cells and are converted in peripheral tissues to cells that express Foxp3 and acquire suppressive ability. The transcription factor Helios, a member of the Ikaros transcription factor family, is expressed in 60-70% of Treg cells in both mouse and man, and is believed to be a marker of tTreg cells. In this review, we discuss the role and function of Helios in Treg cells, the controversy surrounding the use of Helios as a marker of tTreg cells, and how Helios controls specific aspects of the Treg cell program.

Selective deletion of Eos (Ikzf4) in T-regulatory cells leads to loss of suppressive function and development of systemic autoimmunity.

Eos (lkzf4) is a member of the Ikaros family of transcription factors and is preferentially expressed in T-regulatory (Treg) cells. However, the role of Eos in Treg function is controversial. One study using siRNA knock down of Eos demonstrated that it was critical for Treg suppressor function. In contrast, Treg from mice with a global deficiency of Eos had normal Treg function in vitro and in vivo. To further dissect the function of Eos in Tregs, we generated mice with a conditional knock out of Eos in Treg cells (lkzf4fl/fl X Foxp3YFP-cre, Eos cKO). Deletion of Eos in Treg resulted in activation of CD4+Foxp3- and CD8+ T cells at the age of 3 months, cellular infiltration in non-lymphoid tissues, hyperglobulinemia, and anti-nuclear antibodies. While Tregs from Eos cKO mice displayed normal suppressive function in vitro, Eos cKO mice developed severe Experimental Autoimmune Encephalomyletis (EAE) following immunization with myelin oligodendrocyte glycoprotein (MOG) and Eos cKO Treg were unable to suppress Inflammatory Bowel Disease (IBD). Eos cKO mice had decreased growth of the transplantable murine adenocarcinoma MC38 tumor accompanied by enhanced IFN-γ/TNF-α production by CD8+ T cells in tumor draining lymph nodes. Mice with a global deficiency of Eos or a deficiency of Eos only in T cells developed autoimmunity at a much older age (12 months or 7-8 months, respectively). Taken together, Eos appears to play an essential role in multiple aspects of Treg suppressor function, but also plays an as yet unknown role in the function of CD4+Foxp3- and CD8+ T cells and potentially in non-T cells.

Helios Controls a Limited Subset of Regulatory T Cell Functions.

A subpopulation (60-70%) of Foxp3(+) regulatory T cells (Tregs) in both mouse and man expresses the transcription factor Helios, but its role in Treg function is still unknown. We generated Treg-specific Helios-deficient mice to examine the function of Helios in Tregs. We show that the selective deletion of Helios in Tregs leads to slow, progressive systemic immune activation, hypergammaglobulinemia, and enhanced germinal center formation in the absence of organ-specific autoimmunity. Helios-deficient Treg suppressor function was normal in vitro, as well as in an in vivo inflammatory bowel disease model. However, Helios-deficient Tregs failed to control the expansion of pathogenic T cells derived from scurfy mice, failed to mediate T follicular regulatory cell function, and failed to control both T follicular helper cell and Th1 effector cell responses. In competitive settings, Helios-deficient Tregs, particularly effector Tregs, were at a disadvantage, indicating that Helios regulates effector Treg fitness. Thus, we demonstrate that Helios controls certain aspects of Treg-suppressive function, differentiation, and survival.

tTregs, pTregs, and iTregs: similarities and differences.

Foxp3(+) T-regulatory cells (Tregs) are primarily generated in the thymus (tTreg), but also may be generated extrathymically at peripheral sites (pTreg), or induced in cell culture (iTreg) in the presence of transforming growth factor ß (TGFß). A major unresolved issue is how these different populations of Tregs exert their suppressive function in vivo. We have developed novel systems in which the function of Tregs can be evaluated in vivo in normal mice. Our studies demonstrate that one prominent mechanism of action of polyclonal tTregs is to inhibit T-effector cell trafficking to the target organ, while antigen-specific iTregs primarily prevent T-cell priming by acting on antigen-presenting dendritic cells (DCs). Interleukin-10 (IL-10) plays an important role in the suppressive function of antigen-specific iTregs by controlling the expression of MARCH1 and CD83 on the DC. Activated tTregs may mediate infectious tolerance by delivery of cell surface-expressed TGFß to naive responder T cells to generate pTregs. Manipulation of Treg function will require the ability to differentiate tTregs from pTregs and iTregs. The expression of the transcription factor Helios has proven to be a useful marker for the identification of stable tTregs in both mouse and human.

Eos Is Redundant for Regulatory T Cell Function but Plays an Important Role in IL-2 and Th17 Production by CD4+ Conventional T Cells.

Eos belongs to the Ikaros family of transcription factors. It was reported to be a regulatory T cell (Treg) signature gene, to play a critical role in Treg suppressor functions, and to maintain Treg stability. We used mice with a global deficiency in Eos to re-examine the role of Eos expression in both Tregs and conventional T cells (Tconvs). Tregs from Eos-deficient (Eos(-/-)) mice developed normally, displayed a normal Treg phenotype, and exhibited normal suppressor function in vitro. Eos(-/-) Tregs were as effective as Tregs from wild-type (WT) mice in suppressing inflammation in a model of inflammatory bowel disease. Bone marrow (BM) from Eos(-/-) mice was as effective as that from WT mice in controlling T cell activation when used to reconstitute immunodeficient mice in the presence of scurfy fetal liver cells. Surprisingly, Eos was expressed in activated Tconvs and was required for IL-2 production, CD25 expression, and proliferation in vitro by CD4(+) Tconvs. Eos(-/-) mice developed more severe experimental autoimmune encephalomyelitis than WT mice, displayed increased numbers of effector T cells in the periphery and CNS, and amplified IL-17 production. In conclusion, our studies are not consistent with a role for Eos in Treg development and function but demonstrate that Eos plays an important role in the activation and differentiation of Tconvs.

Release of active TGF-ß1 from the latent TGF-ß1/GARP complex on T regulatory cells is mediated by integrin ß8.

Activated T regulatory cells (Tregs) express latent TGF-ß1 on their cell surface bound to GARP. Although integrins have been implicated in mediating the release of active TGF-ß1 from the complex of latent TGF-ß1 and latent TGF-ß1 binding protein, their role in processing latent TGF-ß1 from the latent TGF-ß1/GARP complex is unclear. Mouse CD4(+)Foxp3(+) Treg, but not CD4(+)Foxp3(-) T cells, expressed integrin ß8 (Itgb8) as detected by quantitative RT-PCR. Itgb8 expression was a marker of thymically derived (t)Treg, because it could not be detected on Foxp3(+)Helios(-) Tregs or on Foxp3(+) T cells induced in vitro. Tregs from Itgb8 conditional knockouts exhibited normal suppressor function in vitro and in vivo in a model of colitis but failed to provide TGF-ß1 to drive Th17 or induced Treg differentiation in vitro. In addition, Itgb8 knockout Tregs expressed higher levels of latent TGF-ß1 on their cell surface consistent with defective processing. Thus, integrin αvß8 is a marker of tTregs and functions in a cell intrinsic manner in mediating the processing of latent TGF-ß1 from the latent TGF-ß1/GARP complex on the surface of tTregs.

Modulation of Treg cells/T effector function by GITR signaling is context-dependent.

Treg cells express high levels of the glucocorticoid-induced tumor necrosis factor-related receptor (GITR), while resting conventional T (Tconv) cells express low levels that are increased upon activation. Manipulation of GITR/GITR-Ligand (GITR-L) interactions results in enhancement of immune responses, but it remains unclear whether this enhancement is secondary to costimulation of Tconv cells or to reversal of Treg-cell-mediated suppression. Here, we used a nondepleting Fc-GITR-L and combinations of WT and GITR KO Treg cells and Tconv cells to reexamine the effects of GITR stimulation on each subpopulation in both unmanipulated mice and mice with inflammatory bowel disease. Treatment of mice with Fc-GITR-L resulted in significant expansion of Treg cells and a modest expansion of Tconv cells. When RAG KO mice were reconstituted with Tconv cells alone, GITR-L resulted in Tconv-cell expansion and severe inflammatory bowel disease. The protective effect of Treg cells was lost in the presence of Fc-GITR-L, secondary to death of the Treg cells. When RAG KO mice were reconstituted with Treg cells alone, the transferred cells expanded normally, and Fc-GITR-L treatment resulted in a loss of Foxp3 expression, but the ex-Treg cells did not cause any pathology. The effects of GITR activation are complex and depend on the host environment and the activation state of the Treg cells and T effector cells.

Oligodeoxynucleotides stabilize Helios-expressing Foxp3+ human T regulatory cells during in vitro expansion.

Foxp3(+) regulatory T cells (Tregs) maintain self-tolerance and adoptive therapy, and using Foxp3(+) Tregs has been proposed as treatment for autoimmune diseases. The clinical use of Tregs will require large numbers of cells and methods for in vitro expansion of Tregs are being developed. Foxp3(+) Tregs can be divided into 2 subpopulations based on expression of the transcription factor, Helios. Foxp3(+)Helios(+) Tregs (70%) are thymic-derived, whereas Foxp3(+)Helios(-) Tregs (30%) are induced in the periphery. Foxp3(+)Helios(+) Tregs differ from Foxp3(+)Helios(-) Tregs in terms of epigenetic changes at the Foxp3 locus, their capacity to produce effector cytokines, and their stability of Foxp3 expression on days to weeks of expansion in vitro. Addition of a 25 mer DNA oligonucleotide of random composition for a short period during the expansion of Foxp3(+) Tregs in vitro results in prolonged stabilization of the Foxp3(+)Helios(+) subpopulation and yields an optimal population for use in cellular biotherapy.

A self-reactive TCR drives the development of Foxp3+ regulatory T cells that prevent autoimmune disease.

Although Foxp3(+) regulatory T cells (Tregs) are thought to express autoreactive TCRs, it is not clear how individual TCRs influence Treg development, phenotype, and function in vivo. We have generated TCR transgenic mice (termed SFZ70 mice) using Tcra and Tcrb genes cloned from an autoreactive CD4(+) T cell isolated from a Treg-deficient scurfy mouse. The SFZ70 TCR recognizes a cutaneous autoantigen and drives development of both conventional CD4(+) Foxp3(-) T cells (T(conv)) and Foxp3(+) Tregs. SFZ70 Tregs display an activated phenotype evidenced by robust proliferation and expression of skin-homing molecules such as CD103 and P-selectin ligand. Analysis of Foxp3-deficient SFZ70 mice demonstrates that Tregs inhibit T(conv) cell expression of tissue-homing receptors and their production of proinflammatory cytokines. In addition, Treg suppression of SFZ70 T(conv) cells can be overcome by nonspecific activation of APCs. These results provide new insights into the differentiation and function of tissue-specific Tregs in vivo and provide a tractable system for analyzing the molecular requirements of Treg-mediated tolerance toward a cutaneous autoantigen.

The deubiquitinase CYLD targets Smad7 protein to regulate transforming growth factor ß (TGF-ß) signaling and the development of regulatory T cells.

CYLD is a lysine 63-deubiquitinating enzyme that inhibits NF-κB and JNK signaling. Here, we show that CYLD knock-out mice have markedly increased numbers of regulatory T cells (Tregs) in peripheral lymphoid organs but not in the thymus. In vitro stimulation of CYLD-deficient naive T cells with anti-CD3/28 in the presence of TGF-ß led to a marked increase in the number of Foxp3-expressing T cells when compared with stimulated naive control CD4(+) cells. Under endogenous conditions, CYLD formed a complex with Smad7 that facilitated CYLD deubiquitination of Smad7 at lysine 360 and 374 residues. Moreover, this site-specific ubiquitination of Smad7 was required for activation of TAK1 and p38 kinases. Finally, knockdown of Smad7 or inhibition of p38 activity in primary T cells impaired Treg differentiation. Together, our results show that CYLD regulates TGF-ß signaling function in T cells and the development of Tregs through deubiquitination of Smad7.

Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells.

Helios, a member of the Ikaros transcription factor family, is preferentially expressed at the mRNA level by regulatory T cells (Treg cells). We evaluated Helios protein expression using a newly generated mAb and demonstrated that it is expressed in all thymocytes at the double negative 2 stage of thymic development. Although Helios was expressed by 100% of CD4(+)CD8(-)Foxp3(+) thymocytes, its expression in peripheral lymphoid tissues was restricted to a subpopulation ( approximately 70%) of Foxp3(+) T cells in mice and humans. Neither mouse nor human naive T cells induced to express Foxp3 in vitro by TCR stimulation in the presence of TGF-beta expressed Helios. Ag-specific Foxp3(+) T cells induced in vivo by Ag feeding also failed to express Helios. Collectively, these results demonstrate that Helios is potentially a specific marker of thymic-derived Treg cells and raises the possibility that a significant percentage of Foxp3(+) Treg cells are generated extrathymically.

Helios represses megakaryocyte priming in hematopoietic stem and progenitor cells.

Our understanding of cell fate decisions in hematopoietic stem cells is incomplete. Here, we show that the transcription factor Helios is highly expressed in murine hematopoietic stem and progenitor cells (HSPCs), where it is required to suppress the separation of the platelet/megakaryocyte lineage from the HSPC pool. Helios acts mainly in quiescent cells, where it directly represses the megakaryocyte gene expression program in cells as early as the stem cell stage. Helios binding promotes chromatin compaction, notably at the regulatory regions of platelet-specific genes recognized by the Gata2 and Runx1 transcriptional activators, implicated in megakaryocyte priming. Helios null HSPCs are biased toward the megakaryocyte lineage at the expense of the lymphoid and partially resemble cells of aging animals. We propose that Helios acts as a guardian of HSPC pluripotency by continuously repressing the megakaryocyte fate, which in turn allows downstream lymphoid priming to take place. These results highlight the importance of negative and positive priming events in lineage commitment.

CD4(+)CD25(+) regulatory T cells can mediate suppressor function in the absence of transforming growth factor beta1 production and responsiveness.

CD4(+)CD25(+) regulatory T cells inhibit organ-specific autoimmune diseases induced by CD4(+)CD25(-) T cells and are potent suppressors of T cell activation in vitro. Their mechanism of suppression remains unknown, but most in vitro studies suggest that it is cell contact-dependent and cytokine independent. The role of TGF-beta1 in CD4(+)CD25(+) suppressor function remains unclear. While most studies have failed to reverse suppression with anti-transforming growth factor (TGF)-beta1 in vitro, one recent study has reported that CD4(+)CD25(+) T cells express cell surface TGF-beta1 and that suppression can be completely abrogated by high concentrations of anti-TGF-beta suggesting that cell-associated TGF-beta1 was the primary effector of CD4(+)CD25(+)-mediated suppression. Here, we have reevaluated the role of TGF-beta1 in CD4(+)CD25(+)-mediated suppression. Neutralization of TGF-beta1 with either monoclonal antibody (mAb) or soluble TGF-betaRII-Fc did not reverse in vitro suppression mediated by resting or activated CD4(+)CD25(+) T cells. Responder T cells from Smad3(-/-) or dominant-negative TGF-beta type RII transgenic (DNRIITg) mice, that are both unresponsive to TGF-beta1-induced growth arrest, were as susceptible to CD4(+)CD25(+)-mediated suppression as T cells from wild-type mice. Furthermore, CD4(+)CD25(+) T cells from neonatal TGF-beta1(-/-) mice were as suppressive as CD4(+)CD25(+) from TGF-beta1(+/+) mice. Collectively, these results demonstrate that CD4(+)CD25(+) suppressor function can occur independently of TGF-beta1.

Salt Sensing by Serum/Glucocorticoid-Regulated Kinase 1 Promotes Th17-like Inflammatory Adaptation of Foxp3+ Regulatory T Cells.

Regulatory T (Treg) cells integrate diverse environmental signals to modulate their function for optimal suppression. Translational regulation represents a favorable mechanism for Treg cell environmental sensing and adaptation. In this study, we carry out an unbiased screen of the Treg cell translatome and identify serum/glucocorticoid-regulated kinase 1 (SGK1), a known salt sensor in T cells, as being preferentially translated in activated Treg cells. We show that high salt (HS) drives thymic Treg cells to adopt a T helper type 17 (Th17)-like phenotype and enhances generation of Th17-like induced Treg cells in a SGK1-dependent manner, all the while maintaining suppressive function. Salt-mediated Th17-like differentiation of Treg cells was evident in mice fed with HS diet or injected with HS-preconditioned T cells. Overall, SGK1 enables Treg cells to adapt their function in response to environmental cues. By understanding these environmental-sensing mechanisms, we envision targeted approaches to fine-tune Treg cell function for better control of inflammation.

Overview and Preparation Guide for Academic Careers in Pediatric Pharmacy, Part 1: Career as a Pediatric Pharmacy Practice Faculty Member.

Pediatric clinical pharmacy is a growing and evolving field with an increasing number of pediatric clinical pharmacists in academia. In 2017, pediatric practice faculty members represented approximately 7.6% of all pharmacy practice faculty in the United States. The benefits of practicing in an academic environment are many, including, but not limited to, the ability to shape the future of pharmacy practice through the training of the next generation of pharmacists, contributing to science through research and scholarly activities for the care of pediatric patients, and positively impacting patient care for the most vulnerable of patients. Part one of this two-part series describes careers in academic pediatric pharmacy, as well as faculty roles and responsibilities, and provides information and advice related to the preparation and transition into careers in academic pediatric pharmacy.

IKZF2 Drives Leukemia Stem Cell Self-Renewal and Inhibits Myeloid Differentiation.

Leukemias exhibit a dysregulated developmental program mediated through both genetic and epigenetic mechanisms. Although IKZF2 is expressed in hematopoietic stem cells (HSCs), we found that it is dispensable for mouse and human HSC function. In contrast to its role as a tumor suppressor in hypodiploid B-acute lymphoblastic leukemia, we found that IKZF2 is required for myeloid leukemia. IKZF2 is highly expressed in leukemic stem cells (LSCs), and its deficiency results in defective LSC function. IKZF2 depletion in acute myeloid leukemia (AML) cells reduced colony formation, increased differentiation and apoptosis, and delayed leukemogenesis. Gene expression, chromatin accessibility, and direct IKZF2 binding in MLL-AF9 LSCs demonstrate that IKZF2 regulates a HOXA9 self-renewal gene expression program and inhibits a C/EBP-driven differentiation program. Ectopic HOXA9 expression and CEBPE depletion rescued the effects of IKZF2 depletion. Thus, our study shows that IKZF2 regulates the AML LSC program and provides a rationale to therapeutically target IKZF2 in myeloid leukemia.