T-cell Lineage Specification Research Articles

Deciphering the TET3 interactome in primary thymic developing T cells

Ten-eleven translocation (TET) proteins are DNA dioxygenases that mediate active DNA demethylation. TET3 is the most highly expressed TET protein in thymic developing Tcells. TET3, either independently or in cooperation with TET1 or TET2, has been implicated in Tcell lineage specification by regulating DNA demethylation. However, TET-deficient mice exhibit complex phenotypes, suggesting that TET3 exerts multifaceted roles, potentially by interacting with other proteins. We performed liquid chromatography with tandem mass spectrometry in primary developing Tcells to identify TET3 interacting partners in endogenous, invivo conditions. We discover TET3 interacting partners. Our data establish that TET3 participates in a plethora of fundamental biological processes, such as transcriptional regulation, RNA polymerase elongation, splicing, DNA repair, and DNA replication. This resource brings in the spotlight emerging functions of TET3 and sets the stage for systematic studies to dissect the precise mechanistic contributions of TET3 in shaping Tcell biology.

The Wiskott-Aldrich syndrome protein is required for positive selection during T-cell lineage differentiation.

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immune deficiency caused by a mutation in the WAS gene. This leads to altered or absent WAS protein (WASp) expression and function resulting in thrombocytopenia, eczema, recurrent infections, and autoimmunity. In T cells, WASp is required for immune synapse formation. Patients with WAS show reduced numbers of peripheral blood T lymphocytes and an altered T-cell receptor repertoire. In vitro, their peripheral T cells show decreased proliferation and cytokine production upon aCD3/aCD28 stimulation. It is unclear whether these T-cell defects are acquired during peripheral activation or are, in part, generated during thymic development. Here, we assessed the role of WASp during T-cell differentiation using artificial thymic organoid cultures and in the thymus of humanized mice. Although CRISPR/Cas9 WAS knockout hematopoietic stem and progenitor cells (HSPCs) rearranged the T-cell receptor and differentiated to T-cell receptor (TCR)+ CD4+ CD8+ double-positive (DP) cells similar to wild-type HSPCs, a partial defect in the generation of CD8 single-positive (SP) cells was observed, suggesting that WASp is involved in their positive selection. TCR repertoire analysis of the DP and CD8+ SP population, however, showed a polyclonal repertoire with no bias toward autoreactivity. To our knowledge, this is the first study of the role of WASp in human T-cell differentiation and on TCR repertoire generation.

PHF6 Mutations Defines a Subgroup of Mixed Phenotype of Acute Leukemia with Aberrant T-Cell Differentiation

Mixed phenotype acute leukemia (MPAL) is a rare form of acute leukemia, and comprises 2% of all acute leukemia diagnoses. With the exception of a small subset harboring BCR/ABL1 or MLL translocations (around 15% in combination), the genetic aberrations of MPAL are largely unknown. PHF6 gene is located at Xq26-27 and encodes plant homeodomain finger 6, a zinc finger containing protein. Recurrent somatic PHF6 mutations are frequent in T-ALL (up to 30%) and rarely in AML (~3%). Here we report recurrent PHF6 mutations in approximately 20% of MPAL patients, making PHF6 the most common genetic aberrations identified in MPAL.From 2013 to 2016, a total of 1329 patients with myeloid neoplasms were sequenced at the MSKCC with a targeted next generation sequencing panel containing 30 genes that are frequently mutated in acute leukemia. We identified 38 PHF6 mutations with predicted pathologic significance in 33 patients (2.5%) spanning the whole coding sequence: 10 frameshift, 11 nonsense, 1 splicing site, and 16 missense (Figure 1). The most common co-mutant gene observed in cases with PHF6 mutations was RUNX1 (30%, 11/37). No TP53 mutations are detected within the PHF6-mutant cohort(Figure 2). The 33 patients with PHF6 mutations included patients with a diagnosis of MPAL (n=5), AML (n=19), MDS (n=5), and MPN (n=4).The disproportional distribution of PHF6 mutations in MPAL prompted us to further analyze these patients. During the same period of time, 24 MPAL patients were identified and confirmed based on WHO 2016 classification (excluding AML-MRC and t-AML). Within this cohort, 3 (12%) had BCR-ABL1 (p190) fusion, and 1 (4%) had MLL translocation, two distinct genetically defined subgroups. Importantly, 5 (21%) harbored PHF6 mutations, these mutations were mutually exclusive with BCR-ABL1 or MLL. Four of the 5 patients showed T-cell lineage differentiation, in contrast to MPAL with BCR-ABL1 or MLL translocations which were characterized by B/myeloid differentiation. The 2-year overall survival for the PHF6 mutation positive MPAL was markedly reduced compared to non-mutated cases (40% vs. 100% in PHF6 unmutated cases (p=0.005)).In the larger subset of AML cases, PHF6 mutations were nearly exclusively present in secondary AML (11 AML-MRC, 6 t-AML and only 1 de novo AML). In contrast to their rarity in de novo AML, PHF6 mutations were identified in 11% (11/88) of AML-MRC and 7.1% (6/84) of t-AML. Interestingly, T-cell markers (cytoplasmic CD3, CD2, CD5, CD7) were often expressed on the blasts of secondary AML cases regardless of PHF6 mutational status (56%, 9/16 vs. unmutated 51%, 36/70). In comparison to unmutated cases, the blasts were often negative for CD33 (69%, 11/16, vs 28%, 20/70, p=0.004 by Fisher's exact test) and CD38 (44%, 7/16, vs. 17%, 12/70, p=0.04 by Fisher's exact test) in PHF6 mutated AML, suggestive of a stemness phenotype with aberrant T-cell differentiation.In conclusion, PHF6 mutations are the most common somatic alteration identified in MPAL and define a subgroup of MPAL with altered T-cell differentiation and inferior outcome. Amongst AML, PHF6 mutations are almost exclusively seen in secondary AML and are also associated with T-cell antigen expression and a more primitive stem/progenitor immunophenotype. PHF6 mutations co-exist with RUNX1 mutations but are mutually exclusive to TP53 mutations. Notably, PHF6 mutations may provide a genetic and functional link between MPALs with poor outcome and secondary AML and provide insights into shared mechanisms of transformation. RNA sequencing studies on the sorted blast population are ongoing and will be presented at the ASH meeting. [Display omitted] DisclosuresArcila:Invivoscribe: Honoraria; Archer: Honoraria; Raindance Tecnologies: Honoraria. Levine:Celgene: Research Funding; Roche: Research Funding; Qiagen: Equity Ownership; Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership.

Deciphering the multifaceted roles of TET proteins in T‐cell lineage specification and malignant transformation

TET proteins are DNA demethylases that can oxidize 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC) and other oxidized mC bases (oxi-mCs). Importantly, TET proteins govern cell fate decisions during development of various cell types by activating a cell-specific gene expression program. In this review, we focus on the role of TET proteins in T-cell lineage specification. We explore the multifaceted roles of TET proteins in regulating gene expression in the contexts of T-cell development, lineage specification, function, and disease. Finally, we discuss the future directions and experimental strategies required to decipher the precise mechanisms employed by TET proteins to fine-tune gene expression and safeguard cell identity.

Twist2 promotes CD8+ T-cell differentiation by repressing ThPOK expression.

CD4/CD8 T-cell lineage differentiation is a key process in immune system development; however, a defined regulator(s) that converts the signal from T-cell receptor and co-receptor complexes into lineage differentiation remains unclear. Here, we show that Twist2 is a critical factor in CD4/CD8 thymocyte differentiation. Twist2 expression is differentially regulated by T-cell receptor signaling, leading to differentiation into the CD4 or CD8 lineage. Forced Twist2 expression perturbed CD4+ thymocyte differentiation while enhancing CD8+ thymocyte differentiation. Furthermore, Twist2 expression produced mature CD8+ thymocytes in B2m-/- mice, while its deficiency significantly impaired CD8+ cells in MHC class-II-/- and TCR transgenic mice, favoring CD8 T-cell differentiation. During CD8 lineage differentiation, Twist2 interacted with Runx3 to bind to the silencer region of the ThPOK locus, thereby blocking ThPOK expression. These findings indicate that Twist2 is a part of the transcription factor network controlling CD8 lineage differentiation.

Rab4A inactivation in T cells blocks mTOR activation, pro-inflammatory lineage development, and disease pathogenesis in lupus-prone mice

Abstract Introduction Expression of Rab4A is increased in T cells of patients and mice with SLE. Overexpression of Rab4A forms a positive feedback loop with mTOR that can be blocked with therapeutic efficacy in SLE. To determine the impact of this gene on disease development, the triple-congenic lupus-prone Sle1.2.3 mouse strain (TC) have been backcrossed with C57Bl/6 wild-type (WT) mice that carry floxed Rab4AQ72L (TC-FL) alleles or lack Rab4A in T cells (TC-KO). Methods Proteinuria was assessed by the Bradford assay. Splenocytes were examined by flow cytometry. Autoantibody production was measured by ELISA. Results TC mice had increased proteinuria over age and sex-matched WT controls. Deletion of Rab4A in T cells reduced proteinuria ≥ 50% in female TC-KO mice relative to TC-FL mice at 21 or 40 weeks of age. Similar trends were noted in male mice. The production of antinuclear and antiphospholipid antibodies was reduced in TC-KO mice as compared to TC-FL and parental TC controls. Immunophenotyping unveiled a 45% depletion of CD4+ T cells and 50% expansion of CD8+ T cells in female TC-KO mice relative to TC-FL controls. CD38 expression was reduced on CD4+ T cells of TC-KO mice by 51% and 48% relative to TC and TC-FL controls. CD38 expression was also reduced on CD8+ T cells but not on CD19+ B cells. mTORC1 activity was reduced by 36% in CD4 T cells, but not in CD8 T cells or B cells of TC-KO mice. Along these lines, overexpression of Rab4A activated mTORC1, reduced expression of CD4, and increased expression of CD38 in Jurkat cells. Conclusion These findings reveal an opposite influence of Rab4A on endosomal recycling of CD4 and CD38 that facilitates mTORC1 activation and thus causes pro-inflammatory T-cell lineage specification and triggers autoimmunity in SLE.

Sirolimus in patients with clinically active systemic lupus erythematosus resistant to, or intolerant of, conventional medications: a single-arm, open-label, phase 1/2 trial

Patients with systemic lupus erythematosus have T-cell dysfunction that has been attributed to the activation of the mammalian target of rapamycin (mTOR). Rapamycin inhibits antigen-induced T-cell proliferation and has been developed as a medication under the generic designation of sirolimus. We assessed safety, tolerance, and efficacy of sirolimus in a prospective, biomarker-driven, open-label clinical trial. We did a single-arm, open-label, phase 1/2 trial of sirolimus in patients with active systemic lupus erythematosus disease unresponsive to, or intolerant of, conventional medications at the State University of New York Upstate Medical University (Syracuse, NY, USA). Eligible participants (aged ≥18 years) had active systemic lupus erythematosus fulfilling four or more of 11 diagnostic criteria defined by the American College of Rheumatology. We excluded patients with allergy or intolerance to sirolimus, patients with life-threatening manifestations of systemic lupus erythematosus, proteinuria, a urine protein to creatinine ratio higher than 0·5, anaemia, leucopenia, or thrombocytopenia. Patients received oral sirolimus at a starting dose of 2 mg per day, with dose adjusted according to tolerance and to maintain a therapeutic range of 6-15 ng/mL. Patients were treated with sirolimus for 12 months. Safety outcomes included tolerance as assessed by the occurrence of common side-effects. The primary efficacy endpoint was decrease in disease activity, assessed using the British Isles Lupus Assessment Group (BILAG) index and the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Blood samples of 56 matched healthy individuals were obtained as controls for immunobiological outcomes monitored at each visit. The primary efficacy endpoint was assessed in all patients who completed 12 months of treatment, and all patients who received at least one dose of treatment were included in the safety analyses. This trial is registered with ClinicalTrials.gov, number NCT00779194. Between March 9, 2009, and Dec 8, 2014, 43 patients were enrolled, three of whom did not meet eligibility criteria. 11 of the 40 eligible patients discontinued study treatment because of intolerance (n=2) or non-compliance (n=9). SLEDAI and BILAG disease activity scores were reduced during 12 months of treatment in 16 (55%) of 29 patients who completed treatment. Mean SLEDAI score decreased from 10·2 (SD 5·6) at enrolment to 4·8 (4·5) after 12 months of treatment (p<0·001) and the mean total BILAG index score decreased from 28·4 (12·4) at enrolment to 17·4 (10·7) after 12 months of treatment (p<0·001). The mean daily dose of prednisone required to control disease activity decreased from 23·7 mg (SD 9·6) to 7·2 mg (2·3; p<0·001) after 12 months of treatment. Sirolimus expanded CD4+CD25+FoxP3+ regulatory T cells and CD8+ memory T-cell populations and inhibited interleukin-4 and interleukin-17 production by CD4+ and CD4-CD8- double-negative T cells after 12 months. CD8+ memory T cells were selectively expanded in SRI-responders. Patient liver function and lymphocyte counts were unchanged. Although HDL-cholesterol (Z=-2·50, p=0·012), neutrophil counts (Z=-1·92, p=0·054), and haemoglobin (Z=-2·83, p=0·005) were moderately reduced during treatment, all changes occurred within a range that was considered safe. Platelet counts were slightly elevated during treatment (Z=2·06, p=0·0400). These data show that a progressive improvement in disease activity is associated with correction of pro-inflammatory T-cell lineage specification in patients with active systemic lupus erythematosus during 12 months of sirolimus treatment. Follow-up placebo-controlled clinical trials in diverse patient populations are warranted to further define the role of mTOR blockade in treatment of systemic lupus erythematosus. Pfizer, the National Institutes of Health, and the Central New York Community Foundation.

GATA3 induces human T-cell commitment by restraining Notch activity and repressing NK-cell fate.

The gradual reprogramming of haematopoietic precursors into the T-cell fate is characterized by at least two sequential developmental stages. Following Notch1-dependent T-cell lineage specification during which the first T-cell lineage genes are expressed and myeloid and dendritic cell potential is lost, T-cell specific transcription factors subsequently induce T-cell commitment by repressing residual natural killer (NK)-cell potential. How these processes are regulated in human is poorly understood, especially since efficient T-cell lineage commitment requires a reduction in Notch signalling activity following T-cell specification. Here, we show that GATA3, in contrast to TCF1, controls human T-cell lineage commitment through direct regulation of three distinct processes: repression of NK-cell fate, upregulation of T-cell lineage genes to promote further differentiation and restraint of Notch activity. Repression of the Notch1 target gene DTX1 hereby is essential to prevent NK-cell differentiation. Thus, GATA3-mediated positive and negative feedback mechanisms control human T-cell lineage commitment.

Activation of mTOR (mechanistic target of rapamycin) in rheumatic diseases.

Mechanistic target of rapamycin (mTOR, also known as mammalian target of rapamycin) is a ubiquitous serine/threonine kinase that regulates cell growth, proliferation and survival. These effects are cell-type-specific, and are elicited in response to stimulation by growth factors, hormones and cytokines, as well as to internal and external metabolic cues. Rapamycin was initially developed as an inhibitor of T-cell proliferation and allograft rejection in the organ transplant setting. Subsequently, its molecular target (mTOR) was identified as a component of two interacting complexes, mTORC1 and mTORC2, that regulate T-cell lineage specification and macrophage differentiation. mTORC1 drives the proinflammatory expansion of T helper (TH) type 1, TH17, and CD4(-)CD8(-) (double-negative, DN) T cells. Both mTORC1 and mTORC2 inhibit the development of CD4(+)CD25(+)FoxP3(+) T regulatory (TREG) cells and, indirectly, mTORC2 favours the expansion of T follicular helper (TFH) cells which, similarly to DN T cells, promote B-cell activation and autoantibody production. In contrast to this proinflammatory effect of mTORC2, mTORC1 favours, to some extent, an anti-inflammatory macrophage polarization that is protective against infections and tissue inflammation. Outside the immune system, mTORC1 controls fibroblast proliferation and chondrocyte survival, with implications for tissue fibrosis and osteoarthritis, respectively. Rapamycin (which primarily inhibits mTORC1), ATP-competitive, dual mTORC1/mTORC2 inhibitors and upstream regulators of the mTOR pathway are being developed to treat autoimmune, hyperproliferative and degenerative diseases. In this regard, mTOR blockade promises to increase life expectancy through treatment and prevention of rheumatic diseases.

Dendritic cell SIRT1–HIF1α axis programs the differentiation of CD4+ T cells through IL-12 and TGF-β1

The differentiation of naive CD4(+) T cells into distinct lineages plays critical roles in mediating adaptive immunity or maintaining immune tolerance. In addition to being a first line of defense, the innate immune system also actively instructs adaptive immunity through antigen presentation and immunoregulatory cytokine production. Here we found that sirtuin 1 (SIRT1), a type III histone deacetylase, plays an essential role in mediating proinflammatory signaling in dendritic cells (DCs), consequentially modulating the balance of proinflammatory T helper type 1 (TH1) cells and antiinflammatory Foxp3(+) regulatory T cells (T(reg) cells). Genetic deletion of SIRT1 in DCs restrained the generation of T(reg) cells while driving TH1 development, resulting in an enhanced T-cell-mediated inflammation against microbial responses. Beyond this finding, SIRT1 signaled through a hypoxia-inducible factor-1 alpha (HIF1α)-dependent pathway, orchestrating the reciprocal TH1 and T(reg) lineage commitment through DC-derived IL-12 and TGF-β1. Our studies implicates a DC-based SIRT1-HIF1α metabolic checkpoint in controlling T-cell lineage specification.

S100A4-neutralizing antibody suppresses spontaneous tumor progression, pre-metastatic niche formation and alters T-cell polarization balance.

BackgroundThe tumor microenvironment plays a determinative role in stimulating tumor progression and metastasis. Notably, tumor-stroma signals affect the pattern of infiltrated immune cells and the profile of tumor-released cytokines. Among the known molecules that are engaged in stimulating the metastatic spread of tumor cells is the S100A4 protein. S100A4 is known as an inducer of inflammatory processes and has been shown to attract T-cells to the primary tumor and to the pre-metastatic niche. The present study aims to examine the immunomodulatory role of S100A4 in vivo and in vitro and assess the mode of action of 6B12, a S100A4 neutralizing antibody.MethodsThe therapeutic effect of the 6B12 antibody was evaluated in two different mouse models. First, in a model of spontaneous breast cancer we assessed the dynamics of tumor growth and metastasis. Second, in a model of metastatic niche formation we determined the expression of metastatic niche markers. The levels of cytokine expression were assessed using antibody as well as PCR arrays and the results confirmed by qRT-PCR and ELISA. T-cell phenotyping and in vitro differentiation analyses were performed by flow cytometry.ResultsWe show that the S100A4 protein alters the expression of transcription factor and signal transduction pathway genes involved in the T-cell lineage differentiation. T-cells challenged with S100A4 demonstrated reduced proportion of Th1-polarized cells shifting the Th1/Th2 balance towards the Th2 pro-tumorigenic phenotype. The 6B12 antibody restored the Th1/Th2 balance. Furthermore, we provide evidence that the 6B12 antibody deploys its anti-metastatic effect, by suppressing the attraction of T-cells to the site of primary tumor and pre-metastatic niche. This was associated with delayed primary tumor growth, decreased vessel density and inhibition of metastases.ConclusionThe S100A4 blocking antibody (6B12) reduces tumor growth and metastasis in a model of spontaneous breast cancer. The 6B12 antibody treatment inhibits T cell accumulation at the primary and pre-metastatic tumor sites. The 6B12 antibody acts as an immunomodulatory agent and thus supports the view that the 6B12 antibody is a promising therapeutic candidate to fight cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1034-2) contains supplementary material, which is available to authorized users.

Thymopentin enhances the generation of T-cell lineage derived from human embryonic stem cells in vitro

Thymopentin is a group of biologically active peptide secreted mainly by the epithelial cells of thymic cortex and medulla. Whether it promotes T cells production from human embryonic stem cells(hESCs) in vitro remains an elusive issue. In the present study, we develop a novel strategy that enhances T-cell lineage differentiation of hESCs in collagen matrix culture by sequential cytokine cocktails treatment combined with thymopentin stimulation. We observed that approximately 30.75% cells expressed CD34 on day 14 of the cultures and expressed the surface markers of erythroid, lymphoid and myeloid lineages. The results of colony assays and gene expressions by RT-PCR analysis also demonstrated that hematopoietic progenitor cells (HPCs) derived from hESCs were capable of multi-lineage differentiation. Further study revealed that culturing with thymopentin treatment, the CD34+CD45RA+CD7+ cells sorted from HPCs expressed T-cell-related genes, IKAROS, DNTT, TCRγ and TCRβ, and T-cell surface markers, CD3, cytoplasmic CD3, CD5, CD27, TCRγδ, CD4 and CD8. The differentiated cells produced the cytokines including IFN-γ, IL-2 and TNF-α in response to stimulation, providing the evidence for T-cell function of these cells. In conclusion, thymopentin enhances T-cell lineage differentiation from hESCs in vitro by mimicking thymus peptide environment in vivo.

Impact of 5‐aza‐2′‐deoxycytidine and epigallocatechin‐3‐gallate for induction of human regulatory T cells

The epigenetic regulation of transcription factor genes is critical for T-cell lineage specification. A specific methylation pattern within a conserved region of the lineage specifying transcription factor gene FOXP3, the Treg-specific demethylated region (TSDR), is restricted to regulatory T (Treg) cells and is required for stable expression of FOXP3 and suppressive function. We analysed the impact of hypomethylating agents 5-aza-2'-deoxycytidine and epigallocatechin-3-gallate on human CD4(+) CD25(-) T cells for generating demethylation within FOXP3-TSDR and inducing functional Treg cells. Gene expression, including lineage-specifying transcription factors of the major T-cell lineages and their leading cytokines, functional properties and global transcriptome changes were analysed. The FOXP3-TSDR methylation pattern was determined by using deep amplicon bisulphite sequencing. 5-aza-2'-deoxycytidine induced FOXP3-TSDR hypomethylation and expression of the Treg-cell-specific genes FOXP3 and LRRC32. Proliferation of 5-aza-2'-deoxycytidine-treated cells was reduced, but the cells did not show suppressive function. Hypomethylation was not restricted to FOXP3-TSDR and expression of master transcription factors and leading cytokines of T helper type 1 and type 17 cells were induced. Epigallocatechin-3-gallate induced global DNA hypomethylation to a lesser extent than 5-aza-2'-deoxycitidine, but no relevant hypomethylation within FOXP3-TSDR or expression of Treg-cell-specific genes. Neither of the DNA methyltransferase inhibitors induced fully functional human Treg cells. 5-aza-2'-deoxycitidine-treated cells resembled Treg cells, but they did not suppress proliferation of responder cells, which is an essential capability to be used for Treg cell transfer therapy. Using a recently developed targeted demethylation technology might be a more promising approach for the generation of functional Treg cells.

Epigenetics and prenatal influences on asthma and allergic airways disease

Educational aims Describe how genetic studies have explained a small portion of asthma heritability Discuss how epigenetic marks are influenced by the environment and are important in T-cell lineage differentiation Describe animal studies of the role of DNA methylation in the development of allergic airway disease Summarise human studies on the role of DNA methylation in asthma and allergy

Induction of T-cell development by Delta-like 4-expressing fibroblasts

The thymus provides a unique environment for the induction of T-cell lineage commitment and differentiation, which is predicted by specific Notch ligand-receptor interactions on epithelial cells and lymphoid progenitors, respectively. Accordingly, a bone marrow-derived stromal cell line (OP9) ectopically expressing the Notch ligand Delta-like 1 (Dll1) or Dll4 (OP9-DL1 and OP9-DL4, respectively) gains the ability to recapitulate thymus-like function, supporting T-cell differentiation of both mouse and human progenitors. In this study, we extend these findings by demonstrating that, unlike the NIH3T3 cell line, mouse primary fibroblasts made to express Dll4 (mFibro-DL4) acquire the capacity to promote and support T-cell development from hematopoietic stem cells (HSCs) into TCRαβ(+), CD4(+) and CD8(+) T-lineage cells. However, mFibro-DL4 cells showed a lower efficiency of T-cell generation than OP9-DL4 cells did. Nevertheless, progenitor T-cells (CD117(+) CD44(+) CD25(+)) generated in HSC/mFibro-DL4 co-cultures, when intravenously transferred into immunodeficient (Rag2(-/-) γc(-/-)) mice, home to the thymus, undergo differentiation, and give rise to mature T-cells that go on to populate the periphery. Surprisingly, primary human fibroblast cells expressing Dll4 showed very low efficiency in supporting human T-lineage differentiation, which could not be improved by blocking myelopoiesis. Nevertheless, mFibro-DL4 cells could support human T-cell lineage differentiation. Our results provide a functional framework for the induction of T-cell development using easily accessible fibroblasts made to express Dll4. These cells, which are amenable for in vitro applications, can be further utilized in the design of individualized systems for T-cell production, with implications for the treatment of immunodeficiencies.

Genome-wide identification of Ikaros targets elucidates its contribution to mouse B-cell lineage specification and pre-B–cell differentiation

AbstractIkaros family DNA-binding proteins are critical regulators of B-cell development. Because the current knowledge of Ikaros targets in B-cell progenitors is limited, we have identified genes that are bound and regulated by Ikaros in pre-B cells. To elucidate the role of Ikaros in B-cell lineage specification and differentiation, we analyzed the differential expression of Ikaros targets during the progression of multipotent to lymphoid-restricted progenitors, B- and T-cell lineage specification, and progression along the B-cell lineage. Ikaros targets accounted for one-half of all genes up-regulated during B-cell lineage specification in vivo, explaining the essential role of Ikaros in this process. Expression of the Ikaros paralogs Ikzf1 and Ikzf3 increases incrementally during B-cell progenitor differentiation, and, remarkably, inducible Ikaros expression in cycling pre-B cells was sufficient to drive transcriptional changes resembling the differentiation of cycling to resting pre-B cells in vivo. The data suggest that Ikaros transcription factor dosage drives the progression of progenitors along a predetermined lineage by regulating multiple targets in key pathways, including pre-B–cell receptor signaling, cell cycle progression, and lymphocyte receptor rearrangement. Our approach may be of general use to map the contribution of transcription factors to cell lineage commitment and differentiation.

Type I IFN promotes IL-10 production from Th17 cells to suppress autoimmune inflammation (123.17)

Abstract Recent studies reveal a considerable degree of heterogeneity and flexibility in T-cell lineage differentiation, particularly in the development of Th17 cells. However, how the immune system regulates the plasticity of CD4 T cells in vivo during pathogen infection or autoimmunity is poorly defined. In this study, we investigated the direct effects of type I IFN (IFNα/β) on Th17 cell differentiation and plasticity. In this study, we demonstrate that the plasticity of Th17 cells could be influenced by IFNα/β. Our data show that IFNβ treatment of T cells cultured under Th17 polarizing conditions resulted in reduced production of IL-17, but increased production of IL-10. Furthermore, type I IFN induced IL-10 production by antigen specific T cells derived from immunized mice. We also found that IFNβ treatment leads to shift from Th17 phenotype to Th1 phenotypes with upregulation of IFN-gamma in antigen-specific T cells in vivo. IFN-treated Th17 cells were able to inhibit EAE in vivo via adaptive transfer models. Together, our current findings demonstrate the direct effects of type I IFN on Th17 cells and reveal that Th cell plasticity is influenced by type I IFN during immune response.

T‐cell lineage determination

T cells originate from hematopoietic stem cells (HSCs) in the bone marrow but complete their development in the thymus. HSCs give rise to a variety of non-renewing hematopoietic progenitors, among which a rare subset migrates to the thymus via the bloodstream. The earliest T-cell progenitors identified in the thymus are not T-lineage restricted but possess the ability to give rise to cells of many different lineages. Alternative lineage potentials are gradually lost as progenitors progress toward later developmental stages. Here, we review the early developmental events that might be involved in T-cell lineage fate determination, including the properties of possible thymus-settling progenitors, their homing into the thymus, and their T-cell lineage specification and commitment.

The structural basis for autonomous dimerization of the pre-T-cell antigen receptor

The pre-T-cell antigen receptor (pre-TCR), expressed by immature thymocytes, has a pivotal role in early T-cell development, including TCR β-selection, survival and proliferation of CD4(-)CD8(-) double-negative thymocytes, and subsequent αβ T-cell lineage differentiation. Whereas αβTCR ligation by the peptide-loaded major histocompatibility complex initiates T-cell signalling, pre-TCR-induced signalling occurs by means of a ligand-independent dimerization event. The pre-TCR comprises an invariant α-chain (pre-Tα) that pairs with any TCR β-chain (TCRβ) following successful TCR β-gene rearrangement. Here we provide the basis of pre-Tα-TCRβ assembly and pre-TCR dimerization. The pre-Tα chain comprised a single immunoglobulin-like domain that is structurally distinct from the constant (C) domain of the TCR α-chain; nevertheless, the mode of association between pre-Tα and TCRβ mirrored that mediated by the Cα-Cβ domains of the αβTCR. The pre-TCR had a propensity to dimerize in solution, and the molecular envelope of the pre-TCR dimer correlated well with the observed head-to-tail pre-TCR dimer. This mode of pre-TCR dimerization enabled the pre-Tα domain to interact with the variable (V) β domain through residues that are highly conserved across the Vβ and joining (J) β gene families, thus mimicking the interactions at the core of the αβTCR's Vα-Vβ interface. Disruption of this pre-Tα-Vβ dimer interface abrogated pre-TCR dimerization in solution and impaired pre-TCR expression on the cell surface. Accordingly, we provide a mechanism of pre-TCR self-association that allows the pre-Tα chain to simultaneously 'sample' the correct folding of both the V and C domains of any TCR β-chain, regardless of its ultimate specificity, which represents a critical checkpoint in T-cell development. This unusual dual-chaperone-like sensing function of pre-Tα represents a unique mechanism in nature whereby developmental quality control regulates the expression and signalling of an integral membrane receptor complex.

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system

Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Talpha, RAG-1, and T-cell receptor-Vbeta genes; and 3) produced interferon-gamma in response to T-cell receptor stimulation. These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation.