T-cell Lineage Development Research Articles

The Function of E2A in B-Cell Development.

Helix-loop-helix (HLH) transcription factors (TFs) play a key role in various cellular differentiation and function through the regulation of enhancer activity. E2A, a member of the mammalian E-protein family (class I HLH protein), is well known to play an important role in hematopoiesis, especially in adaptive lymphocyte development. E2A instructs B- and T-cell lineage development through the regulation of enhancer activity for B- or T-cell signature gene expression, including Rag1 and Rag2 (Rag1/2) genes. In this chapter, we mainly focus on the function of E2A in B-cell development and on the roles of E2A in establishing the enhancer landscape through the recruitment of EP300/KAT3B, chromatin remodeling complex, mediator, cohesion, and TET proteins. Finally, we demonstrate how E2A orchestrates the assembly of the Rag1/2 gene super-enhancer (SE) formation by changing the chromatin conformation across the Rag gene locus.

Differentiation of BCMA-specific induced pluripotent stem cells into rejuvenated CD8αβ+ T cells targeting multiple myeloma

AbstractA major hurdle in adoptive T-cell therapy is cell exhaustion and failure to maintain antitumor responses. Here, we introduce an induced pluripotent stem cell (iPSC) strategy for reprogramming and revitalizing precursor exhausted B-cell maturation antigen (BCMA)-specific T cells to effectively target multiple myeloma (MM). Heteroclitic BCMA72-80 (YLMFLLRKI)–specific CD8+ memory cytotoxic T lymphocytes (CTL) were epigenetically reprogrammed to a pluripotent state, developed into hematopoietic progenitor cells (CD34+ CD43+/CD14− CD235a−), differentiated into the T-cell lineage and evaluated for their polyfunctional activities against MM. The final T-cell products demonstrated (1) mature CD8αβ+ memory phenotype, (2) high expression of activation or costimulatory molecules (CD38, CD28, and 41BB), (3) no expression of immune checkpoint and senescence markers (CTLA4, PD1, LAG3, and TIM3; CD57), and (4) robust proliferation and polyfunctional immune responses to MM. The BCMA-specific iPSC–T cells possessed a single T-cell receptor clonotype with cognate BCMA peptide recognition and specificity for targeting MM. RNA sequencing analyses revealed distinct genome-wide shifts and a distinctive transcriptional profile in selected iPSC clones, which can develop CD8αβ+ memory T cells. This includes a repertoire of gene regulators promoting T-cell lineage development, memory CTL activation, and immune response regulation (LCK, IL7R, 4-1BB, TRAIL, GZMB, FOXF1, and ITGA1). This study highlights the potential application of iPSC technology to an adaptive T-cell therapy protocol and identifies specific transcriptional patterns that could serve as a biomarker for selection of suitable iPSC clones for the successful development of antigen-specific CD8αβ+ memory T cells to improve the outcome in patients with MM.

Fibrotic liver microenvironment promotes Dll4 and SDF-1-dependent T-cell lineage development

The reconstitution of the T-cell repertoire and quantity is a major challenge in the clinical management of HIV infection/AIDS, cancer, and aging-associated diseases. We previously showed that autologous bone marrow transfusion (BMT) via the hepatic portal vein could effectively restore CD4+ T-cell count in AIDS patients also suffering from decompensated liver cirrhosis. In the current study, we characterized T-cell reconstitution in a mouse model of liver fibrosis induced by CCl4 and found that T-cell reconstitution after BMT via hepatic portal vein was also greatly enhanced. The expression of Dll4 (Delta-like 4), which plays an important role in T-cell progenitor expansion, was elevated in hepatocytes of fibrotic livers when compared to normal livers. This upregulation of Dll4 expression was found to be induced by TNFα in an NFκB-dependent manner. Liver fibroblasts transfected with Dll4 (LF-Dll4) also gained the capacity to promote T-cell lineage development from hematopoietic stem cells (HSCs), resulting in the generation of DN2 (CD4 and CD8 DN 2) and DN3 T-cell progenitors in vitro, which underwent a normal maturation program when adoptively transferred into Rag-2 deficient hosts. We also demonstrated a pivotal role of SDF-1 produced by primary liver fibroblasts (primary LF) in T-lineage differentiation from HSCs. These results suggest that Dll4 and SDF-1 in fibrotic liver microenvironment could promote extrathymic T-cell lineage development. These results expand our knowledge of T-cell development and reconstitution under pathological conditions.

A Tie2-Notch1 signaling axis regulates regeneration of the endothelial bone marrow niche

Loss-of-function studies have determined that Notch signaling is essential for hematopoietic and endothelial development. By deleting a single allele of the Notch1 transcriptional activation domain we generated viable, post-natal mice exhibiting hypomorphic Notch signaling. These heterozygous mice, which lack only one copy of the transcriptional activation domain, appear normal and have no endothelial or hematopoietic phenotype, apart from an inherent, cell-autonomous defect in T-cell lineage development. Following chemotherapy, these hypomorphs exhibited severe pancytopenia, weight loss and morbidity. This phenotype was confirmed in an endothelial-specific, loss-of-function Notch1 model system. Ang1, secreted by hematopoietic progenitors after damage, activated endothelial Tie2 signaling, which in turn enhanced expression of Notch ligands and potentiated Notch1 receptor activation. In our heterozygous, hypomorphic model system, the mutant protein that lacks the Notch1 transcriptional activation domain accumulated in endothelial cells and interfered with optimal activity of the wildtype Notch1 transcriptional complex. Failure of the hypomorphic mutant to efficiently drive transcription of key gene targets such as Hes1 and Myc prolonged apoptosis and limited regeneration of the bone marrow niche. Thus, basal Notch1 signaling is sufficient for niche development, but robust Notch activity is required for regeneration of the bone marrow endothelial niche and hematopoietic recovery.

A Meta-Analysis Of Hodgkin Lymphoma Reveals 19p13.3 (TCF3) As a Novel Susceptibility Loc

BackgroundRecent genome wide association studies (GWAS) of Hodgkin lymphoma (HL) have identified several associations at both HLA and non-HLA loci. However, much of HL heritability remains unexplained. MethodsTo identify novel risk loci, we performed a meta-analysis of 3 HL GWAS including a total of 1,810 cases and 7,879 controls. Results were replicated in an independent set of 1,163 cases and 2,580 controls, for a total of 3,097 and 11,097 cases and controls combined, respectively. participants in discovery and replication stages were of European descent. quality control and imputation we conducted a meta-analysis addressing 1,004,829 variants (λ= 1.10, λ1000= 1.03).Associations between SNP genotypes and HL risk were evaluated under a log-additive model of inheritance adjusting for sex, study center and significant principal components to control for population stratification. We performed an analysis with all HL cases and then conducted stratified analyses by histological subtype (classical, nodular sclerosis and mixed cellularity), age at diagnosis (nodular sclerosis among those diagnosed at 15- 35 years in all studies, and those diagnosed at 35 and older years in the European Study only) and EBV tumor status (negative and positive). We then used a bioinformatic approach (FunciSNP) to identify potential functional variants associated with HD risk correlated with risk loci of interest. We extracted publically available ENCODE data on biofeatures to identify potential functional motifs associated with the index SNP or correlated SNPs. Finally, we measured expression levels of the two alternative mRNA transcripts in lymphoblastoid cell lines (LCLs) from 49 post-therapy HL patients and 25 unaffected controls. RT-PCR was carried out in triplicate. Relative expression levels were calculated relative to TBP as housekeeping gene. Linear models were used to assess correlation between genotype and TCF3expression levels. ResultsThe meta-analysis identified a novel susceptibility variant at chromosome 19p13.3 (rs1860661) associated with HL risk (Odds Ratio [OR]= 0.78, P=2.0*10-8, I2=0%). variant is located in intron 2 of TCF3 (also known as E2A), a regulator of B- and T-cell lineage commitment. was also significantly associated with HL (OR= 0.85, P=0.002) in the replication series of 1,281 cases and 3,218 controls. the combined analysis consisting of the discovery and replication sets, rs1860661was strongly associated with HL (OR=0.81,=3.5*10-10), with no evidence of heterogeneity between contributing studies (Phom=0.41, I2=0%).The number of G alleles defined by rs1860661 was significantly associated with a reduced risk of each HL subtype except EBV positive HL. rs1860661 and two correlated SNPs, rs10413888 (r2=0.90) and rs8103453 (r2=0.89) identified by FunciSNP analysis map in or near marks of open chromatin and in DNAse hypersensitivity sites in TCF3 in CD20+ B cell lines., the protective minor alleles of these SNPs as defined by the G-G-G haplotype map to the binding sites for ZBTB7a (rs10413888 and rs1860661) and (rs8103453) transcription factors, likely improving the binding efficiency to the sites which may result in increased transcription rates of TCF3. TCF3 is encoded by two alternative transcripts (E12 and E47). Higher expression levels of TCF3-E47, whose transcription start site is located close to rs1860661, was associated with the rs1860661-G allele in controls (P=0.02), but not in HL patients (P=0.22). Conclusion/DiscussionTCF3 is essential for the commitment of lymphoid progenitors to both B-cell and T-cell lineage development. A molecular and phenotypic hallmark of classical HL is the loss of the B-cell phenotype in HRS cells, including lack of demonstrable B-cell receptor and most B-cell specific markers such as CD19 or CD20. HRS cells have a low level of TCF3, particularly homodimers of the isoform E47, due to expression of the ABF-1 and ID2 inhibitors that bind to TCF3. Thus, higher TCF3 levels in HRS precursor cells may lead to enhanced retention of the B cell phenotype, thereby conferring a protective effect. These data suggest a link between the 19p13.3 locus including TCF3 and HL risk, indicating that TCF3 could be relevant to HL etiology and pathogenesis. Disclosures:Link:Genentech: Consultancy; Millenium: Consultancy; Pharmacyclics: Consultancy; Spectrum: Consultancy.

T-Cell Receptor Gene Deletions Are Associated with High Risk Features and Worse Outcome In Childhood Precursor B-Cell Acute Lymphoblastic Leukemia (ALL)

AbstractAbstract 275T-cell receptor (TCR) gene rearrangements occur in 50–80% of childhood precursor B-cell ALL and have been used for minimal residual disease (MRD) detection. However, TCR rearrangement has never been examined for its contribution to disease classification and outcome. We used Molecular Inversion Probe technology (Affymetrix) to analyze precursor B-cell ALL samples (n=48) from the University of Utah for genome-wide copy number changes, and evaluated the relationship between focal TCR deletions (indicating rearrangement) and clinical features. Copy number deletions were detected by Nexus Copy Number (BioDiscovery, Inc.) and were based on 5 consecutive probes having a value of <1.5 (diploid copy number=2). We then validated our findings in the diagnostic B-precursor ALL samples (n=221) from the Children's Oncology Group (COG) High Risk (HR)-ALL TARGET (Therapeutically Applicable Research to Generate Effective Targets) Project which includes 500K SNP array (Affymetrix) data from patients enrolled on COG P9906. In the Utah cohort, we observed the following TCR deletions: TCR-α (14q11.2)=23/48 (48%), TCR-β (7q34)=18/48 (38%), TCR-γ (7p14.1)=29/48 (60%), TCR-δ (14q11.2)=23/48 (48%), or any TCR deletion (TCR-'any')=36/48 (75%). Utah samples contained 37.5% NCI-Rome designated “High Risk” patients, and 15/18 (83%) of these patients had TCR-'any' deletions. Fisher's Exact Test (two-tailed) was used to look for associations in the Utah cohort between TCR-α,-β,-γ,-δ, or -‘any’ deletions and high risk clinical features. TCR-α deletion was significantly associated with Day 28 MRD Positivity (p=0.0486); there was significant association between TCR-β deletion and Day 7 M3 Marrow (>25% blasts; p=0.019). TCR-δ deletion was also significantly associated with Day 28 MRD Positivity (p=0.0206). Due to the small sample size and insufficient follow-up, outcome analyses were not done in this cohort. The COG HR-ALL TARGET cohort had the following TCR deletions: TCR-α=53/221 (24%), TCR-β=24/221 (11%), TCR-γ=55/221 (25%), TCR-δ=53/221 (24%), or TCR-'any'=96/221 (43%). Fisher's Exact test (two-tailed) was used to look for associations between TCR deletions and clinical features; outcome analyses were conducted using the Kaplan-Meier method and the log-rank test. In the TARGET cohort, we found TCR-α, TCR-γ, and TCR-δ deletions were significantly associated with occurrence of relapse and/or death (p=0.0209, p=0.0143, and p=0.0209, respectively). TCR-β deletion was associated with age at diagnosis ≥ 10 yo (p=0.0225), a high risk feature, and Day 29 MRD Positivity (p=0.0084). In the high-risk TARGET validation cohort, 43 of the 78 children (55%) who experienced a relapse or died contained TCR deletions vs. 53 of the 143 children (37%) who were event-free with TCR deletions (p=0.0109). There was a significant difference in event-free survival (EFS) between patients with TCR-'any' deletions vs. those without the deletions (p=0.013), with the largest difference in survival occurring beyond two years of diagnosis (Figure 1). TCR deletions were uncommon in Hyperdiploid (0/4, 0%), E2A-PBX1 (n=1/25, 4%), and MLL (n=4/19, 21%) subtypes in the TARGET cohort, but were more frequent in TEL-AML1 (n=2/3, 67%) and Other (n=89/170, 52%) subtypes. In summary, both the Utah discovery cohort and the larger COG HR-ALL TARGET validation cohort showed focal TCR gene deletions that were linked to high risk clinical features, as well as associated with poor prognostic attributes such as end-of-induction MRD Positivity. Additionally, the larger TARGET cohort demonstrated that deletion of any TCR region was significantly associated with worse outcome. TCR gene deletions may indicate a more immature state of developmental arrest and could imply initial abortive T-cell lineage development prior to B-cell commitment and transformation. This represents the first study to reveal an association between TCR gene deletion status and clinical outcome in childhood B-precursor ALL, and these findings will need validation in additional pediatric and adult cohorts. Ultimately, focal TCR gene deletions may prove to be a valuable addition to the molecular risk classification schema currently applied to B-precursor ALL. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Cell-Type-Specific Activation and Repression of PU.1 by a Complex of Discrete, Functionally Specialized cis-Regulatory Elements

The transcription factor PU.1 is critical for multiple hematopoietic lineages, but different leukocyte types require strictly distinct patterns of PU.1 regulation. PU.1 is required early for T-cell lineage development but then must be repressed by a stage-specific mechanism correlated with commitment. Other lineages require steady, low expression or upregulation. Until now, only the promoter plus a distal upstream regulatory element (URE) could be invoked to explain nearly all Sfpi1 (PU.1) activation and repression, including bifunctional effects of Runx1. However, the URE is dispensable for most Sfpi1 downregulation in early T cells, and we show that it retains enhancer activity in immature T-lineage cells even where endogenous Sfpi1 is repressed. We now present evidence for another complex of conserved noncoding elements that mediate discrete, cell-type-specific regulatory features of Sfpi1, including a myeloid cell-specific activating element and a separate, pro-T-cell-specific silencer element. These elements yield opposite, cell-type-specific responses to Runx1. T-cell-specific repression requires Runx1 acting through multiple nonconsensus sites in the silencer core. These newly characterized sites recruit Runx1 binding in early T cells in vivo and define a functionally specific scaffold for dose-dependent, Runx-mediated repression.

Invariant NKT cells inhibit development of the Th 17 lineage

T cells differentiate into functionally distinct effector subsets in response to pathogen encounter. Cells of the innate immune system direct this process; CD1d-restricted invariant natural killer T (iNKT) cells, for example, can either promote or inhibit Th(1) and Th(2) responses. Recently, a new subset of CD4(+) T helper cells, called Th(17), was identified that is implicated in mucosal immunity and autoimmune disorders. To investigate the influence of iNKT cells on the differentiation of naïve T cells we used an adoptive transfer model of traceable antigen-specific CD4(+) T cells. Transferred naïve CD25(-)CD62L(+) CD4(+) T cells were primed by antigen immunization of the recipient mice, permitting their expansion and Th(17) differentiation. This study establishes that in vivo activation of iNKT cells during T-cell priming impedes the commitment of naïve T cells to the Th(17) lineage. In vivo cytokine neutralization experiments revealed a role for IL-4, IL-10, and IFN-gamma in the iNKT-cell-mediated regulation of T-cell lineage development. Moreover, by comparing IL-17 production by antigen-experienced T cells from unmanipulated wild-type mice and iNKT-cell-deficient mice, we demonstrate an enhanced Th(17) response in mice lacking iNKT cells. This invigorated Th(17) response reverts to physiological levels when iNKT cells are introduced into Jalpha18(-/-) mice by adoptive transfer, indicating that iNKT cells control the Th(17) compartment at steady state. We conclude that iNKT cells play an important role in limiting development of the Th(17) lineage and suggest that iNKT cells provide a natural barrier against Th(17) responses.

The Interaction of the Wnt and Notch Pathways Modulates NK vs. T Cell Commitment.

The Wnt and Notch signaling pathways have critical roles in cell fate decisions. However, the interaction of these pathways is poorly understood. Using highly purified Wnt3a and immobilized Notch ligand, Delta1ext-IgG, we investigated the mechanisms involved in Wnt and Notch signaling interactions during hematopoietic stem cell differentiation. When CD34+CD38- cord blood stem cells were cultured for 2 to 3 weeks with five growth factors (SCF 300ng/ml, Flt-3L 300ng/ml, TPO 100ng/ml, IL-6 100ng/ml, and IL-3 10ng/ml), most precursor cells lost CD34 expression and differentiated into mature cells, most of which were monocytes. However, as we previously reported, when cells were cultured with Delta1ext-IgG, we found an increased percentage of lymphoid progenitors (CD34+CD7+) and more mature lymphoid precursors (CD34-CD7+) in the cell population. The addition of purified Wnt3a (100ng/ml) alone without Delta1ext-IgG did not significantly change the percentage of CD7+ cells (1% vs. 3%). However, when both Wnt3a and Delta1ext-IgG were added, we saw an increased percentage of CD7+ cells (11% with Delta1ext-IgG alone to 27% with both) (Fig. 1A). Wnt3a also enhanced gene expression of CD3ε and preTα induced by Delta1ext-IgG. These results suggest that Wnt3a enhances the effect of Notch signaling on T-cell lineage development. To test the role of endogenous Wnt signaling, we added Dickkopf1, an inhibitor of Wnt signaling. When CD34+CD38- cells were cultured with Dickkopf1 (300ng/ml) alone for a 2 week, the percentage and number of CD56+ NK cells was unaffected. However, when cultured with Dickkopf1 and Delta1ext-IgG, the percentage and number of CD56+ NK precursor cells were increased (2% with Delta1ext-IgG alone vs. 17% with Dickkopf1 and Delta1ext-IgG; 0.2 ×105 vs. 1.5×105, p<0.01) (Fig. 1B). This result shows that decreased endogenous Wnt signaling enhances the generation of NK cells in the presence of Notch signaling. To address whether Wnt signaling affects Notch signaling by modulating protein levels, we assessed the amount of activated Notch1 intracellular domain with or without Wnt 3a by Western blot (Fig. 2A). After stimulation for 24 h, Wnt3a increased the amount of activated Notch1 intracellular domain induced by Delta1ext-IgG. Wnt3a also enhanced the expression of a primary target gene of Notch signaling, Hes1, determined by quantitative RT-PCR (Fig. 2B). In contrast, Dickkopf1 reduced Delta1ext-IgG-induced Hes1 expression. These results suggest that Wnt signaling directly modulates Notch signaling. Thus, these studies suggest that Wnt signaling is a key factor in cell fate determination at the point of NK/T cell commitment that is mediated via an interaction with Notch signaling. These studies also suggest that Wnt directly regulates Notch signaling by modulating protein turnover. [Display omitted]

Ex Vivo Expanded HOXB4-Transduced Bone Marrow Cells Enhance Allogeneic Repopulation in a Sublethal Irradiation Model of BMT.

The successful development of molecular strategies to improve the competitive capacity of donor hematopoietic cells would have major implications for bone marrow transplantation (BMT). The forced expression of HOXB4 has been previously shown to increase the competitive capacity of donor marrow in congenic competitive repopulation assays. However, a major clinical objective is to improve allogeneic engraftment after BMT utilizing reduced intensity conditioning regimens. We hypothesized that expansion of HSCs resulting from forced expression of HOXB4 would enhance stable allogenic engraftment after sublethal myeloablative conditioning.BM mononuclear cells (BMMNC) isolated from B6(H2Kb) donor mice treated with 5-FU (150mg/Kg body weight) were prestimulated with cytokines (IL3, IL6 and SCF) and transduced with an MSCV based retroviral vector expressing either HOXB4-ires-GFP or only GFP. Following incubation with viral supernatants, BMMNC were expanded in the absence of virus, examined for transduction efficiency, and phenotyped by flow cytometry prior to administration of 2.5 x 106 cells to Balb/c (H2Kd) sublethally irradiated (450cGy) mice. Data is expressed as mean ± SEM. Transduction efficiency with both vectors was 45–50% as measured by GFP expression. Phenotypic analysis of transduced cultures showed no significant difference in surface phenotype between the HOXB4 or GFP transduced BM cells. The majority of the cells were CD45R/B220 positive (70%) and/or CD11b positive (55%). As early as 4 weeks post-transplantation, GFP and H2Kb positive donor derived cells were present in the peripheral blood of recipients. At 4 weeks, HOXB4 transduced donor cells engrafted at a higher rate 55% (5 out of 9 animals; donor cells in PB = 81.92 ± 10.25%; n = 5) compared to 33% engraftment (3 out of 9 animals) with GFP transduced cells (donor cells in PB = 39.11 ± 20.66; n = 3). Long term donor cell chimerism (16 weeks) could be measured in 3 of 9 HOXB4 transduced recipients while GFP engrafted recipients failed to survive beyond 6 weeks. Multilineage analysis of donor derived phenotypes in long term HOXB4 engrafted mice in PB, BM and spleen are shown in the table. Analysis of thymus in one of the HOXB4 engrafted recipients showed CD4 +/GFP+ H2Kb (76.49%) or CD8+/GFP+ H2Kb (76.95%) cells, indicating the development of T-cell lineages from donor derived HOXB4 transduced HSCs.Our results indicate engraftment and long term donor chimerism throughout the hematopoietic compartment in a murine allogenic model. However, forced HOXB4 expression in HSCs also resulted in increased myeloid lineage expression in chimeric animals, consistent with earlier observations in congenic transplants (Antonchuk et al, 2002). Though the mechanisms of HOXB4 induced HSC self renewal and multilineage hematopoiesis are still not clear, its capability to contribute to enhanced engraftment across major histocompatibility complex barriers supports its potential utility for allogeneic BMT.Lineage Engraftment (%) of Donor Cells (GFP+/H2Kb+) in Hematopoietic TissuesTissueCD3CD11bCD19CD45PB4.14 ± 0.3983.77 ± 10.1411.25 ± 11.7389.47 ± 8.17BM1.56 ± 1.165.81 ± 9.85.76 ± 2.980.54 ± 6.2Spleen12.15 ± 10.145.92 ±25.8124.15 ± 21.585.87 ± 5.3

Differential regulation of a Thy-1 gene in transgenic mice.

We have generated Thy-1.1-transgenic Thy-1.2 mice to study the developmental expression of the Thy-1 gene in detail by transcriptional and immunological methods. In brain, the expression of the injected gene was identical to that of the endogenous gene in a tissue- and development-specific manner. In lymphoid tissue, the transferred gene was also expressed correctly in the early phases of T-cell lineage development; however, as the T cells matured, the transcription of the transferred gene, but not the endogenous gene, was suppressed. This result shows that different regulatory elements are used to express the Thy-1 gene in early and late lymphoid development.