Notch3 activation is sufficient but not required for inducing human T-lineage specification.

Although the role for the individual Notch receptors in early hematopoiesis have been thoroughly investigated in mouse, studies in human have been mostly limited to the use of pan-Notch inhibitors. However, such studies in human are important to predict potential side effects of specific Notch receptor blocking reagents because these are currently being considered as therapeutic tools to treat various Notch-dependent diseases. In this study, we studied the individual roles of Notch1 and Notch3 in early human hematopoietic lineage decisions, particularly during T-lineage specification. Although this process in mice is solely dependent on Notch1 activation, we recently reported Notch3 expression in human uncommitted thymocytes, raising the possibility that Notch3 mediates human T-lineage specification. Although expression of a constitutive activated form of Notch3 (ICN3) results in the induction of T-lineage specification in human CD34(+) hematopoietic progenitor cells, similar to ICN1 overexpression, loss-of-function studies using blocking Abs reveal that only Notch1, but not Notch3, is critical in this process. Blocking of Notch1 activation in OP9-DLL4 cocultures resulted in a complete block in T-lineage specification and induced monocytic and plasmacytoid dendritic cell differentiation instead. In fetal thymus organ cultures, impeded Notch1 activation resulted in B and dendritic cell development. In contrast, Notch3 blocking Abs only marginally affected T-lineage specification and hematopoietic differentiation with a slight increase in monocyte development. No induction of B or dendritic cell development was observed. Thus, our results unambiguously reveal a nonredundant role for Notch1 in human T-lineage specification, despite the expression of other Notch receptors.

Jagged2 acts as a Delta-like Notch ligand during early hematopoietic cell fate decisions.

Notch signaling critically mediates various hematopoietic lineage decisions and is induced in mammals by Notch ligands that are classified into 2 families, Delta-like (Delta-like-1, -3 and -4) and Jagged (Jagged1 and Jagged2), based on structural homology with both Drosophila ligands Delta and Serrate, respectively. Because the functional differences between mammalian Notch ligands were still unclear, we have investigated their influence on early human hematopoiesis and show that Jagged2 affects hematopoietic lineage decisions very similarly as Delta-like-1 and -4, but very different from Jagged1. OP9 coculture experiments revealed that Jagged2, like Delta-like ligands, induces T-lineage differentiation and inhibits B-cell and myeloid development. However, dose-dependent Notch activation studies, gene expression analysis, and promoter activation assays indicated that Jagged2 is a weaker Notch1-activator compared with the Delta-like ligands, revealing a Notch1 specific signal strength hierarchy for mammalian Notch ligands. Strikingly, Lunatic-Fringe- mediated glycosylation of Notch1 potentiated Notch signaling through Delta-like ligands and also Jagged2, in contrast to Jagged1. Thus, our results reveal a unique role for Jagged1 in preventing the induction of T-lineage differentiation in hematopoietic stem cells and show an unexpected functional similarity between Jagged2 and the Delta-like ligands.

Continuous CD27 triggering in vivo strongly reduces NK cell numbers.

NK cells are important mediators of the early defense. In mice, immature and mature NK (mNK) cells constitutively express the TNF receptor family member CD27; however, mNK cells eventually lose CD27 expression and become resting NK cells. Interaction of CD27 with its ligand, CD70, enhances proliferation and effector functions of NK cells. We used mice that constitutively express CD70 on B cells (CD70-Tg) to study the in vivo effects of continuous triggering of CD27 on NK cells. Continuous CD70-CD27 interaction resulted in strongly down-modulated CD27 expression on NK cells and gradually reduced absolute NK cell numbers. This reduction was most prominent in the mNK cell subpopulation and was at least partially due to increased apoptosis. Residual NK cells showed lower expression of activating Ly49 receptors and normal (liver) or decreased (spleen) IFN-gamma production. Nevertheless, NK cells from CD70-Tg mice displayed higher YAC-1 killing capacities. CD70-Tg NK cells exhibited up-regulated expression of NKG2D, which is in accordance with the increased YAC-1 lysis, as this is mainly NKG2D-dependent. Taken together, this study is the first to demonstrate that continuous CD70 triggering of CD27 on NK cells in vivo results in a severe reduction of NK cells. On a single cell basis, however, residual NK cells display enhanced cytotoxicity.

CD27-deficient mice show normal NK-cell differentiation but impaired function upon stimulation.

Natural killer (NK) cells are part of the first line defense against tumors, parasites and virus-infected cells. Therefore, factors that control NK-cell numbers and their function are important. CD27 is constitutively expressed on NK cells and its expression correlates with sequential phases in NK-cell development, discriminating phenotypically and functionally different subsets within the NK-cell population. Although CD27 has been described to have an important regulatory role in effector and memory T and B lymphocytes, its role in NK-cell biology remains to be addressed. In this study, we used CD27(-/-) mice to investigate the role of CD27 in NK-cell development and function, both during the resting state and upon stimulation. The results show that NK-cell numbers are not impaired in CD27(-/-) mice. Moreover, CD27(-/-) NK cells reach full phenotypic maturity, evidenced by normal expression of CD49b, CD43 and CD11b. Expression of activating receptors is unaltered, whereas expression of several inhibitory receptors is increased. Cytotoxicity and interferon-γ production by NK cells from CD27(-/-) mice in the resting state are normal. However, upon in vivo anti-CD40- or poly-I:C-mediated activation, or in vitro interleukin-15 priming plus anti-NKp46 stimulation, the absence of CD27 results in decreased cytolytic activity and cytokine production by spleen and liver NK cells. In conclusion, this study demonstrates that CD27 is dispensable for the development of functional NK cells. However, upon stimulation of NK cells, CD27 displays an important role in their activation and functionality.

Notch signaling is required for proliferation but not for differentiation at a well-defined beta-selection checkpoint during human T-cell development.

Notch signaling is absolutely required for beta-selection during mouse T-cell development, both for differentiation and proliferation. In this report, we investigated whether Notch has an equally important role during human T-cell development. We show that human CD34(+) thymocytes can differentiate into CD4(+)CD8beta(+) double positive (DP) thymocytes in the absence of Notch signaling. While these DP cells phenotypically resemble human beta-selected cells, they lack a T-cell receptor (TCR)-beta chain. Therefore, we characterized the beta-selection checkpoint in human T-cell development, using CD28 as a differential marker at the immature single positive CD4(+)CD3(-)CD8alpha(-) stage. Through intracellular TCR-beta staining and gene expression analysis, we show that CD4(+)CD3(-)CD8alpha(-)CD28(+) thymocytes have passed the beta-selection checkpoint, in contrast to CD4(+)CD3(-)CD8alpha(-)CD28(-) cells. These CD4(+)CD3(-)CD8alpha(-)CD28(+) thymocytes can efficiently differentiate into CD3(+)TCRalphabeta(+) human T cells in the absence of Notch signaling. Importantly, preselection CD4(+)CD3(-)CD8alpha(-)CD28(-) thymocytes can also differentiate into CD3(+)TCRalphabeta(+) human T cells without Notch activation when provided with a rearranged TCR-beta chain. Proliferation of human thymocytes, however, is clearly Notch-dependent. Thus, we have characterized the beta-selection checkpoint during human T-cell development and show that human thymocytes require Notch signaling for proliferation but not for differentiation at this stage of development.

An early decrease in Notch activation is required for human TCR-alphabeta lineage differentiation at the expense of TCR-gammadelta T cells.

Although well characterized in the mouse, the role of Notch signaling in the human T-cell receptor alphabeta (TCR-alphabeta) versus TCR-gammadelta lineage decision is still unclear. Although it is clear in the mouse that TCR-gammadelta development is less Notch dependent compared with TCR-alphabeta differentiation, retroviral overexpression studies in human have suggested an opposing role for Notch during human T-cell development. Using the OP9-coculture system, we demonstrate that changes in Notch activation are differentially required during human T-cell development. High Notch activation promotes the generation of T-lineage precursors and gammadelta T cells but inhibits differentiation toward the alphabeta lineage. Reducing the amount of Notch activation rescues alphabeta-lineage differentiation, also at the single-cell level. Gene expression analysis suggests that this is mediated by differential sensitivities of Notch target genes in response to changes in Notch activation. High Notch activity increases DTX1, NRARP, and RUNX3 expression, genes that are down-regulated during alphabeta-lineage differentiation. Furthermore, increased interleukin-7 levels cannot compensate for the Notch dependent TCR-gammadelta development. Our results reveal stage-dependent molecular changes in Notch signaling that are critical for normal human T-cell development and reveal fundamental molecular differences between mouse and human.

T-lymphoid differentiation potential measured in vitro is higher in CD34+CD38-/lo hematopoietic stem cells from umbilical cord blood than from bone marrow and is an intrinsic property of the cells.

BACKGROUND: Human bone marrow and umbilical cord blood are sources of allogeneic hematopoietic stem cells for transplantation, which is a life-saving treatment in a variety of diseases but is burdened by delayed T-cell reconstitution. Observational studies evaluating T-cell reconstitution in post-transplant recipients suggest that cord blood hematopoietic stem cells have a more effective capacity for T-cell reconstitution. This study focuses on the comparison of the capacity of cord blood and bone marrow hematopoietic stem cells to generate T cells in vitro. DESIGN AND METHODS: Hematopoietic stem cells were cultured in OP9-delta-like-1 and OP9-green fluorescent protein co-cultures to estimate T and myeloid generation capacity, respectively. Phenotypic markers of T-lineage or myeloid differentiation were measured by flow cytometry and used to analyze their kinetics as a function of culture time. Hematopoietic stem cells were labeled with carboxyfluorescein diacetate succinamidyl ester and analyzed after culture to track their phenotypic progression in consecutive generations. Mixed OP9-delta-like-1 co-cultures were done with either carboxyfluorescein diacetate succinamidyl ester-labeled bone marrow and unlabeled cord blood hematopoietic stem cells, or vice versa, to evaluate their mutual influence on T-lineage differentiation. The T-cell potential of hematopoietic stem cells was addressed quantitatively by limiting dilution analysis. RESULTS: Bulk cultures showed faster and more extensive T-cell differentiation by cord blood hematopoietic stem cells. Furthermore, the T-lymphoid differentiation capacity of cord blood and bone marrow hematopoietic stem cells can be discriminated very early based on the coordinated expression of CD34 and CD7. Mixing experiments with cord blood hematopoietic stem cells and bone marrow hematopoietic stem cells showed that these differences are cell intrinsic. Quantitative clonal analyses demonstrated that CD34(+)CD38(-/lo) hematopoietic stem cells from cord blood contained a two-fold higher T-lineage generation capacity than CD34(+)CD38(-/lo) bone marrow hematopoietic stem cells, whereas the myeloid differentiation was similar. CONCLUSIONS: Our data shows that cord blood hematopoietic stem cells have higher T-lymphoid differentiation potential than bone marrow hematopoietic stem cells and that this property is cell autonomous.

Generation of T cells from human embryonic stem cell-derived hematopoietic zones.

Human embryonic stem cells (hESC) are pluripotent stem cells. A major challenge in the field of hESC is the establishment of specific differentiation protocols that drives hESC down a particular lineage fate. So far, attempts to generate T cells from hESC in vitro were unsuccessful. In this study, we show that T cells can be generated in vitro from hESC-derived hematopoietic precursor cells present in hematopoietic zones (HZs). These zones are morphologically similar to blood islands during embryonic development, and are formed when hESC are cultured on OP9 stromal cells. Upon subsequent transfer of these HZs on OP9 cells expressing high levels of Delta-like 1 and in the presence of growth factors, cells expand and differentiate to T cells. Furthermore, we show that T cells derive exclusively from a CD34(high)CD43(low) population, further substantiating the notion that hESC-derived CD34(high)CD43(low) cells are formed in HZs and are the only population containing multipotent hematopoietic precursor cells. Differentiation to T cells sequentially passes through the physiological intermediates: CD34(+)CD7(+) T/NK committed, CD7(+)CD4(+)CD8(-) immature single positive, CD4(+)CD8(+) double positive, and finally CD3(+)CD1(-)CD27(+) mature T cell stages. TCRalphabeta(+) and TCRgammadelta(+) T cells are generated. Mature T cells are polyclonal, proliferate, and secrete cytokines in response to mitogens. This protocol for the de novo generation of T cells from hESC could be clinically and scientifically relevant.

Functionally mature CD4 and CD8 TCRalphabeta cells are generated in OP9-DL1 cultures from human CD34+ hematopoietic cells.

Human CD34(+) hematopoietic precursor cells cultured on delta-like ligand 1 expressing OP9 (OP9-DL1) stromal cells differentiate to T lineage cells. The nature of the T cells generated in these cultures has not been studied in detail. Since these cultures do not contain thymic epithelial cells which are the main cell type mediating positive selection in vivo, generation of conventional helper CD4(+) and cytotoxic CD8(+) TCRalphabeta cells is not expected. Phenotypically mature CD27(+)CD1(-) TCRgammadelta as well as TCRalphabeta cells were generated in OP9-DL1 cultures. CD8 and few mature CD4 single-positive TCRalphabeta cells were observed. Mature CD8 single-positive cells consisted of two subpopulations: one expressing mainly CD8alphabeta and one expressing CD8alphaalpha dimers. TCRalphabeta CD8alphaalpha and TCRgammadelta cells both expressed the IL2Rbeta receptor constitutively and proliferated on IL-15, a characteristic of unconventional T cells. CD8alphabeta(+) and CD4(+) TCRalphabeta cells were unresponsive to IL-15, but could be expanded upon TCR stimulation as mature CD8alphabeta(+) and CD4(+) T cells. These T cells had the characteristics of conventional T cells: CD4(+) cells expressed ThPOK, CD40L, and high levels of IL-2 and IL-4; CD8(+) cells expressed Eomes, Runx3, and high levels of granzyme, perforin, and IFN-gamma. Induction of murine or human MHC class I expression on OP9-DL1 cells had no influence on the differentiation of mature CD8(+) cells. Similarly, the presence of dendritic cells was not required for the generation of mature CD4(+) or CD8(+) T cells. These data suggest that positive selection of these cells is induced by interaction between T precursor cells.

Thymic selection generates a large T cell pool recognizing a self-peptide in humans.

The low frequency of self-peptide-specific T cells in the human preimmune repertoire has so far precluded their direct evaluation. Here, we report an unexpected high frequency of T cells specific for the self-antigen Melan-A/MART-1 in CD8 single-positive thymocytes from human histocompatibility leukocyte antigen-A2 healthy individuals, which is maintained in the peripheral blood of newborns and adults. Postthymic replicative history of Melan-A/MART-1-specific CD8 T cells was independently assessed by quantifying T cell receptor excision circles and telomere length ex vivo. We provide direct evidence that the large T cell pool specific for the self-antigen Melan-A/MART-1 is mostly generated by thymic output of a high number of precursors. This represents the only known naive self-peptide-specific T cell repertoire directly accessible in humans.

Endothelial outgrowth cells are not derived from CD133+ cells or CD45+ hematopoietic precursors.

OBJECTIVE: Two types of endothelial progenitor cells (EPCs), early EPCs and late EPCs (also called endothelial outgrowth cells [EOCs]), were described in vitro previously. In this report, we dissect the phenotype of the precursor(s) that generate these cell types with focus on the markers CD34, CD133, and vascular endothelial growth factor receptor-2 (VEGFR2) that have been used to identify putative circulating endothelial precursors. We also included CD45 in the analysis to assess the relation between CD34+ hematopoietic progenitors (HPC), CD34+ endothelial precursors, and both in vitro generated EPC types. Addressing this issue might lead to a better understanding of the lineage and phenotype of the precursor(s) that give rise to both cell types in vitro and may contribute to a consensus on their flowcytometric enumeration. METHODS AND RESULTS: Using cell sorting of human cord blood (UCB) and bone marrow (BM) cells, we demonstrate that EOC generating precursors are confined to a small CD34+ CD45- cell fraction, but not to the CD34+ CD45+ HPC fraction, nor any other CD45+ subpopulation. CD34+ CD45+ HPC generated monocytic cells that displayed characteristics typical for early EPCs. Phenotypic analysis showed that EOC generating CD34+ CD45- cells express VEGFR2 but not CD133, whereas CD34+ CD45+ HPC express CD133 as expected, but not VEGFR2. CONCLUSION: EOCs are not derived from CD133+ cells or CD45+ hematopoietic precursors.

Tumor necrosis factor promotes T-cell at the expense of B-cell lymphoid development from cultured human CD34+ cord blood cells.

OBJECTIVE: Human CD34+ cord blood (CB) cells are hematopoietic progenitors useful for stem cell transplantation, even after ex vivo expansion. We investigated the effect of tumor necrosis factor (TNF) on lymphoid development from cultured CD34+ CB cells. MATERIALS AND METHODS: Human CD34+ CB cells were cultured in cytokine mixes with or without TNF. Preculture during 60 hours was followed by in vitro differentiation assays, including fetal thymus organ culture and coculture on murine stromal MS-5 cells. In a next step, experiments were extended to CD34+CD38- and CD34+CD38+ CB cells and prolonged preculture. RESULTS: Preculture in the presence of TNF improved differentiation into T cells and diminished the ability to generate B cells, while NK potential and myeloid development were unaffected. Sorted CD34+CD38- CB cells were more potent T-cell precursors after preculture in TNF, compared to CD34+CD38+ CB cells. In precultured CD34+CD38- CB cells, TNF increased GATA3 but decreased EBF1 expression, in line with the skewed lymphoid differentiation induced by TNF. However, when preculture in the presence of TNF was extended to 1 week, T-cell precursors were lost. CONCLUSION: After short-term culture of CD34+ CB cells in the presence of TNF, T-cell generation is stimulated at the expense of B-cell generation. T-cell progenitors are enriched in the CD34+CD38- fraction. These results have implications on the culture conditions to be used for CB CD34+ cells prior to transplantation.

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.

Specific Notch receptor-ligand interactions control human TCR-αß/γδ development by inducing differential Notch signal strength.

In humans, high Notch activation promotes γδ T cell development, whereas lower levels promote αß-lineage differentiation. How these different Notch signals are generated has remained unclear. We show that differential Notch receptor-ligand interactions mediate this process. Whereas Delta-like 4 supports both TCR-αß and -γδ development, Jagged1 induces mainly αß-lineage differentiation. In contrast, Jagged2-mediated Notch activation primarily results in γδ T cell development and represses αß-lineage differentiation by inhibiting TCR-ß formation. Consistently, TCR-αß T cell development is rescued through transduction of a TCR-ß transgene. Jagged2 induces the strongest Notch signal through interactions with both Notch1 and Notch3, whereas Delta-like 4 primarily binds Notch1. In agreement, Notch3 is a stronger Notch activator and only supports γδ T cell development, whereas Notch1 is a weaker activator supporting both TCR-αß and -γδ development. Fetal thymus organ cultures in JAG2-deficient thymic lobes or with Notch3-blocking antibodies confirm the importance of Jagged2/Notch3 signaling in human TCR-γδ differentiation. Our findings reveal that differential Notch receptor-ligand interactions mediate human TCR-αß and -γδ T cell differentiation and provide a mechanistic insight into the high Notch dependency of human γδ T cell development.

In vitro expanded cells contributing to rapid severe combined immunodeficient repopulation activity are CD34+38-33+90+45RA-.

Expansion of hematopoietic stem cells could be used clinically to shorten the prolonged aplastic phase after umbilical cord blood (UCB) transplantation. In this report, we investigated rapid severe combined immunodeficient (SCID) repopulating activity (rSRA) 2 weeks after transplantation of CD34(+) UCB cells cultured with serum on MS5 stromal cells and in serum- and stroma-free cultures. Various subpopulations obtained after culture were studied for rSRA. CD34(+) expansion cultures resulted in vast expansion of CD45(+) and CD34(+) cells. Independent of the culture method, only the CD34(+)33(+)38(-) fraction of the cultured cells contained rSRA. Subsequently, we subfractionated the CD34(+)38(-) fraction using stem cell markers CD45RA and CD90. In vitro differentiation cultures showed CD34(+) expansion in both CD45RA(-) and CD90(+) cultures, whereas little increase in CD34(+) cells was observed in both CD45RA(+) and CD90(-) cultures. By four-color flow cytometry, we could demonstrate that CD34(+)38(-)45RA(-) and CD34(+)38(-)90(+) cell populations were largely overlapping. Both populations were able to reconstitute SCID/nonobese diabetic mice at 2 weeks, indicating that these cells contained rSRA activity. In contrast, CD34(+)38(-)45RA(+) or CD34(+)38(-)90(-) cells contributed only marginally to rSRA. Similar results were obtained when cells were injected intrafemorally, suggesting that the lack of reconstitution was not due to homing defects. In conclusion, we show that after in vitro expansion, rSRA is mediated by CD34(+)38(-)90(+)45RA(-) cells. All other cell fractions have limited reconstitutive potential, mainly because the cells have lost stem cell activity rather than because of homing defects. These findings can be used clinically to assess the rSRA of cultured stem cells.

Notch signaling induces cytoplasmic CD3 epsilon expression in human differentiating NK cells.

It has been proposed that heterogeneity in natural killer (NK)-cell phenotype and function can be achieved through distinct thymic and bone marrow pathways of NK-cell development. Here, we show a link between Notch signaling and the generation of intracellular CD3epsilon (cyCD3)-expressing NK cells, a cell population that can be detected in vivo. Differentiation of human CD34(+) cord blood progenitors in IL-15-supplemented fetal thymus organ culture or OP9-Delta-like 1 (DL1) coculture resulted in a high percentage of cyCD3(+) NK cells that was blocked by the gamma-secretase inhibitor DAPT. The requirement for Notch signaling to generate cyCD3(+) NK cells was further illustrated by transduction of CD34(+) cord blood (CB) cells with either the active intracellular part of Notch or the dominant-negative mutant of mastermind-like protein 1 that resulted in the generation of NK cells with respectively high or low frequencies of cyCD3. Human thymic CD34(+) progenitor cells displayed the potential to generate cyCD3(+) NK cells, even in the absence of Notch/DL1 signaling. Peripheral blood NK cells were unable to induce cyCD3 expression after DL1 exposure, indicating that Notch-dependent cyCD3 expression can only be achieved during the early phase of NK-cell differentiation.

Overexpression of HES-1 is not sufficient to impose T-cell differentiation on human hematopoietic stem cells.

By retroviral overexpression of the Notch-1 intracellular domain (ICN) in human CD34+ hematopoietic stem cells (HSCs), we have shown previously that Notch-1 signaling promotes the T-cell fate and inhibits the monocyte and B-cell fate in several in vitro and in vivo differentiation assays. Here, we investigated whether the effects of constitutively active Notch-1 can be mimicked by overexpression of its downstream target gene HES1. Upon HES-1 retroviral transduction, human CD34+ stem cells had a different outcome in the differentiation assays as compared to ICN-transduced cells. Although HES-1 induced a partial block in B-cell development, it did not inhibit monocyte development and did not promote T/NK-cell-lineage differentiation. On the contrary, a higher percentage of HES-1-transduced stem cells remained CD34+. These experiments indicate that HES-1 alone is not able to substitute for Notch-1 signaling to induce T-cell differentiation of human CD34+ hematopoietic stem cells.

Ex vivo characterization of allo-MHC-restricted T cells specific for a single MHC-peptide complex.

Alloreactive T cells are thought to be a potentially rich source of high-avidity T cells with therapeutic potential since tolerance to self-Ags is restricted to self-MHC recognition. Given the particularly high frequency of alloreactive T cells in the peripheral immune system, we used numerous MHC class I multimers to directly visualize and isolate viral and tumor Ag-specific alloreactive CD8 T cells. In fact, all but one specificities screened were undetectable in ex vivo labeling. In this study, we report the occurrence of CD8 T cells specifically labeled with allo-HLA-A*0201/Melan-A/MART-1(26-35) multimers at frequencies that are in the range of 10(-4) CD8 T cells and are thus detectable ex vivo by flow cytometry. We report the thymic generation and shaping of tumor Ag-specific, alloreactive T cells as well as their fate once seeded in the periphery. We show that these cells resemble their counterparts in HLA-A*0201-positive individuals, based on their structural and functional attributes.

Different thresholds of Notch signaling bias human precursor cells toward B-, NK-, monocytic/dendritic-, or T-cell lineage in thymus microenvironment.

Notch receptors are involved in lineage decisions in multiple developmental scenarios, including hematopoiesis. Here, we treated hybrid human-mouse fetal thymus organ culture with the gamma-secretase inhibitor 7 (N-[N-(3,5-difluorophenyl)-l-alanyl]-S-phenyl-glycine t-butyl ester) (DAPT) to establish the role of Notch signaling in human hematopoietic lineage decisions. The effect of inhibition of Notch signaling was studied starting from cord blood CD34(+) or thymic CD34(+)CD1(-), CD34(+)CD1(+), or CD4ISP progenitors. Treatment of cord blood CD34(+) cells with low DAPT concentrations results in aberrant CD4ISP and CD4/CD8 double-positive (DP) thymocytes, which are negative for intracellular T-cell receptor beta (TCRbeta). On culture with intermediate and high DAPT concentrations, thymic CD34(+)CD1(-) cells still generate aberrant intracellular TCRbeta(-) DP cells that have undergone DJ but not VDJ recombination. Inhibition of Notch signaling shifts differentiation into non-T cells in a thymic microenvironment, depending on the starting progenitor cells: thymic CD34(+)CD1(+) cells do not generate non-T cells, thymic CD34(+)CD1(-) cells generate NK cells and monocytic/dendritic cells, and cord blood CD34(+)Lin(-) cells generate B, NK, and monocytic/dendritic cells in the presence of DAPT. Our data indicate that Notch signaling is crucial to direct human progenitor cells into the T-cell lineage, whereas it has a negative impact on B, NK, and monocytic/dendritic cell generation in a dose-dependent fashion.

Negative thymocyte selection to HERV-K18 superantigens in humans.

An experimental system to explore central tolerance in humans is unavailable. However, the human endogenous retrovirus K-18 (HERV-K18) region on chromosome 1 provides an excellent model: HERV-K18 encodes a superantigen (SAg) stimulating Vbeta7CD4 T cells that is implicated in type 1 diabetes and Epstein-Barr virus persistence. In this study, we have addressed thymic HERV-K18 SAg expression, the capacity of SAg to induce negative selection, and the consequences of this for peripheral tolerance compared with SAg reactivity. We demonstrate that thymic HERV-K18 SAg expression is constitutive and is restricted in time and space such that it can induce negative selection. We developed an in vitro assay capable of detecting negative human thymocyte selection by bacterial SAgs presented on extrathymic antigen-presenting cells (APCs). Using this assay, the HERV-K18 SAg is necessary and sufficient for negative selection of immature or semimature Vbeta7CD4 thymocytes. Decreases of SAg reactive Vbeta7CD4 T cells generated in the thymus predict low or absent SAg reactivity. Therefore, these results indicate that negative thymic selection to HERV-K18 SAgs constitutes a first checkpoint controlling peripheral tolerance compared with SAg reactivity. This study now offers a framework to dissect negative selection and its interplay with viral persistence and autoimmunity in humans.