Active form of Notch members can enforce T lymphopoiesis on lymphoid progenitors in the monolayer culture specific for B cell development

The in vitro induction of T lymphopoiesis needs the precise stereoscopic structure of thymus tissues as seen in fetal thymus organ culture. In this study, we demonstrated for the first time that the introduction of the intracellular region of Notch1 can induce T cells expressing TCR without any thymic environment. In the coculture on the monolayer of OP-9, which was originally known to support B cell specific development, hemopoietic progenitors developed into Thy-1(+)CD25(+) T lineage cells if the progenitor cells were infected with the retrovirus containing Notch1 intracellular domains. The Thy-1(+) cells progressed to a further developmental stage, CD4 and CD8 double-positive cells expressing TCR on the cell surface, if they were further cultured on OP-9 or in the thymus. However, T cell induction by intracellular Notch1 failed unless both OP-9 and IL-7 were present. It is notable that Notch2 and Notch3 showed an effect on T lymphopoiesis similar to that of Notch1. These results indicate that in vitro T lymphopoiesis is inducible by signaling via Notch family members in a lineage-specific manner but shares other stroma-derived factors including IL-7 with B lymphopoiesis.     

Characterization of developmental pathway of natural killer cells from embryonic stem cells in vitro

In vitro differentiation of embryonic stem (ES) cells is often used to study hematopoiesis. However, the differentiation pathway of lymphocytes, in particular natural killer (NK) cells, from ES cells is still unclear. Here, we used a multi-step in vitro ES cell differentiation system to study lymphocyte development from ES cells, and to characterize NK developmental intermediates. We generated embryoid bodies (EBs) from ES cells, isolated CD34(+) EB cells and cultured them on OP9 stroma with a cocktail of cytokines to generate cells we termed ES-derived hematopoietic progenitors (ES-HPs). EB cell subsets, as well as ES-HPs derived from EBs, were tested for NK, T, B and myeloid lineage potentials using lineage specific cultures. ES-HPs derived from CD34(+) EBs differentiated into NK cells when cultured on OP9 stroma with IL-2 and IL-15, and into T cells on Delta-like 1-transduced OP9 (OP9-DL1) with IL-7 and Flt3-L. Among CD34(+) EB cells, NK and T cell potentials were detected in a CD45(-) subset, whereas CD45(+) EB cells had myeloid but not lymphoid potentials. Limiting dilution analysis of ES-HPs generated from CD34(+)CD45(-) EB cells showed that CD45(+)Mac-1(-)Ter119(-) ES-HPs are highly enriched for NK progenitors, but they also have T, B and myeloid potentials. We concluded that CD45(-)CD34(+) EB cells have lymphoid potential, and they differentiate into more mature CD45(+)Lin(-) hematopoietic progenitors that have lymphoid and myeloid potential. NK progenitors among ES-HPs are CD122(-) and they rapidly acquire CD122 as they differentiate along the NK lineage.     

Notch1 and IL-7 receptor interplay maintains proliferation of human thymic progenitors while suppressing non-T cell fates

Notch signaling is critical for T cell development of multipotent hemopoietic progenitors. Yet, how Notch regulates T cell fate specification during early thymopoiesis remains unclear. In this study, we have identified an early subset of CD34high c-kit+ flt3+ IL-7Ralpha+ cells in the human postnatal thymus, which includes primitive progenitors with combined lymphomyeloid potential. To assess the impact of Notch signaling in early T cell development, we expressed constitutively active Notch1 in such thymic lymphomyeloid precursors (TLMPs), or triggered their endogenous Notch pathway in the OP9-Delta-like1 stroma coculture. Our results show that proliferation vs differentiation is a critical decision influenced by Notch at the TLMP stage. We found that Notch signaling plays a prominent role in inhibiting non-T cell differentiation (i.e., macrophages, dendritic cells, and NK cells) of TLMPs, while sustaining the proliferation of undifferentiated thymocytes with T cell potential in response to unique IL-7 signals. However, Notch activation is not sufficient for inducing T-lineage progression of proliferating progenitors. Rather, stroma-derived signals are concurrently required. Moreover, while ectopic IL-7R expression cannot replace Notch for the maintenance and expansion of undifferentiated thymocytes, Notch signals sustain IL-7R expression in proliferating thymocytes and induce IL-7R up-regulation in a T cell line. Thus, IL-7R and Notch pathways cooperate to synchronize cell proliferation and suppression of non-T lineage choices in primitive intrathymic progenitors, which will be allowed to progress along the T cell pathway only upon interaction with an inductive stromal microenvironment. These data provide insight into a mechanism of Notch-regulated amplification of the intrathymic pool of early human T cell progenitors.     

Flt3 ligand expands lymphoid progenitors prior to recovery of thymopoiesis and accelerates T cell reconstitution after bone marrow transplantation

Deficient thymopoiesis and retarded recovery of newly developed CD4(+) T cells is one of the most important determinants of impaired immunocompetence after hemopoietic stem cell transplantation. Here we evaluated whether Fms-like tyrosine kinase 3 (Flt3) ligand (FL) alone or combined with IL-7 affects T cell recovery, thymopoiesis, and lymphoid progenitor expansion following bone marrow transplantation in immunodeficient mice. FL strongly accelerated and enhanced the recovery of peripheral T cells after transplantation of a low number of bone marrow cells. An additive effect on T cell recovery was not observed after coadministration of IL-7. Lineage(-)sca-1(+)c-kit(+)flt3(+) lymphoid progenitor cell numbers were significantly increased in bone marrow of FL-treated mice before recovery of thymopoiesis. Thymocyte differentiation was advanced to more mature stages after FL treatment. Improved T cell recovery resulted in better immunocompetence against a post-bone marrow transplantation murine CMV infection. Collectively, our data suggest that FL promotes T cell recovery by enhanced thymopoiesis and by expansion of lymphoid progenitors.     

Identification of an IL-7-dependent pre-T committed population in the spleen

Several extrathymic T cell progenitors have been described but their various contributions to the T cell lineage puzzle are unclear. In this study, we provide evidence for a splenic Lin(-)Thy1.2(+) T cell-committed population, rare in B6 mice, abundant in TCRalpha(-/-), CD3epsilon(-/-), and nude mice, and absent in IL-7- and Rag-2-deficient mice. Neither B nor myeloid cells are generated in vivo and in vitro. The incidence of these pre-T cells is under the control of thymus and/or mature T cells, as revealed by graft experiments. Indeed, IL-7 consumption by mature T cells inhibits the growth of these pre-T cells. Moreover, the nude spleen contains an additional Lin(-)Thy1.2(+)CD25(+) subset which is detected in B6 mice only after thymectomy. We establish that the full pre-T cell potential and proliferation capacity are only present in the c-kit(low) fraction of progenitors. We also show that most CCR9(+) progenitors are retained in the spleen of nude mice, but present in the blood of B6 mice. Thus, our data describe a new T cell lineage restricted subset that accumulates in the spleen before migration to the thymus.     

T cell development from kit-negative progenitors in the Foxn1Delta/Delta mutant thymus

Foxn1Delta is a hypomorphic allele of the nude gene that causes arrested thymic epithelial cell differentiation and abnormal thymic architecture lacking cortical and medullary domains. T cells develop in the Foxn1Delta/Delta adult thymus to the double- and single-positive stages, but in the apparent absence of double-negative 3 (DN3) cells; however, DN3 cells are present in the fetal thymus. To investigate the origin of this seemingly contradictory phenotype, we performed an analysis of fetal and adult DN cells in these mutants. Neither adult bone marrow-derived cells nor fetal liver cells from wild-type or Rag1-/- mice were able to differentiate to the DN2 or DN3 stage in the Foxn1Delta/Delta thymus. Our data suggest that thymopoiesis in the Foxn1Delta/Delta adult thymus proceeds from CD117- atypical progenitors, while CD117+ DN1a cells are absent or blocked in their ability to differentiate to the T lineage. Wild-type cells generated by this pathway in the postnatal thymus were exported to the periphery, demonstrating that these atypical cells contributed to the peripheral T cell pool. The Foxn1Delta/Delta adult (but not fetal) thymus also preferentially supports B cell development, specifically of the B-1 type, and this phenotype correlated with reduced Notch ligand expression in the adult stroma.     

IL-7 enhances the responsiveness of human T cells that develop in the bone marrow of athymic mice

The beige/nude/xid/human (bnx/hu) model of human hematopoiesis provides a unique opportunity to study extrathymic human T lymphocyte development in an in vivo system. Purified human hematopoietic stem cells develop into mature T lymphocytes and immature progenitors in the bone marrow of athymic bnx mice. The human T cells are all TCR alpha beta(+) and display a restricted TCRV beta repertoire. In the current studies, we examined the effects of systemic human IL-7 (huIL-7) administration on the phenotype and the activation status of the bnx/hu T cells. In the majority of the mice that did not have huIL-7 administration, a higher frequency of human CD3(+)/CD8(+) than CD3(+)/CD4(+) T cells developed in the bone marrow. This phenomenon is also frequently observed in human bone marrow transplant recipients. Extremely low levels of IL-2 were expressed by human CD3(+) cells isolated from these mice, in response to PMA plus ionomycin and to CD3 and CD28 cross-linking. IL-4 was not expressed by cells exposed to either stimulus, demonstrating a profound inability of the bnx/hu T cells to produce this cytokine. Systemic production of huIL-7 from engineered stromal cells transplanted into the mice increased the human CD4 to CD8 ratios, and increased the ratio of memory to naive CD4(+) and CD8(+) T cells. The human CD3(+) cells recovered from mice that had systemic huIL-7 and equivalent numbers of CD3(+)/CD4(+) and CD3(+)/CD8(+) cells in the marrow were still unable to produce IL-4 in response to any condition tested, but were capable of normal levels of IL-2 production following stimulation.     

The origin of mesoderm in phoronids

Integrin alphaIIb is a cell adhesion molecule expressed in association with beta3 by cells of the megakaryocytic lineage, from committed progenitors to platelets. While it is clear that lymphohemopoietic cells differentiating along other lineages do not express this molecule, it has been questioned whether mammalian hemopoietic stem cells (HSC) and various progenitor cells express it. In this study, we detected alphaIIb expression in midgestation embryo in sites of HSC generation, such as the yolk sac blood islands and the hemopoietic clusters lining the walls of the major arteries, and in sites of HSC migration, such as the fetal liver. Since c-Kit, which plays an essential role in the early stages of hemopoiesis, is expressed by HSC, we studied the expression of the alphaIIb antigen in the c-Kit-positive population from fetal liver and adult bone marrow differentiating in vitro and in vivo into erythromyeloid and lymphocyte lineages. Erythroid and myeloid progenitor activities were found in vitro in the c-Kit(+)alphaIIb(+) cell populations from both origins. On the other hand, a T cell developmental potential has never been considered for c-Kit(+)alphaIIb(+) progenitors, except in the avian model. Using organ cultures of embryonic thymus followed by grafting into athymic nude recipients, we demonstrate herein that populations from murine fetal liver and adult bone marrow contain T lymphocyte progenitors. Migration and maturation of T cells occurred, as shown by the development of both CD4(+)CD8- and CD4-CD8(+) peripheral T cells. Multilineage differentiation, including the B lymphoid lineage, of c-Kit(+)alphaIIb(+) progenitor cells was also shown in vivo in an assay using lethally irradiated congenic recipients. Taken together, these data demonstrate that murine c-Kit(+)alphaIIb(+) progenitor cells have several lineage potentialities since erythroid, myeloid, and lymphoid lineages can be generated.     

Human thymic epithelial cells inhibit IL-15- and IL-2-driven differentiation of NK cells from the early human thymic progenitors

T/NK progenitors are present in the thymus; however, the thymus predominantly promotes T cell development. In this study, we demonstrated that human thymic epithelial cells (TEC) inhibit NK cell development. Most ex vivo human thymocytes express CD1a, indicating that thymic progenitors are predominantly committed to the T cell lineage. In contrast, the CD1a(-)CD3(-)CD56(+) NK population comprises only 0.2% (n = 7) of thymocytes. However, we observed increases in the percentage (20- to 25-fold) and absolute number (13- to 71-fold) of NK cells when thymocytes were cultured with mixtures of either IL-2, IL-7, and stem cell factor or IL-15, IL-7, and stem cell factor. TEC, when present in the cultures, inhibited the increases in the percentage (3- to 10-fold) and absolute number (3- to 25-fold) of NK cells. Furthermore, we show that TEC-derived soluble factors inhibit generation of NK-CFU and inhibit IL15- or IL2-driven NK cell differentiation from thymic CD34(+) triple-negative thymocytes. The inhibitory activity was found to be associated with a 8,000- to 30,000 Da fraction. Thus, our data demonstrate that TEC inhibit NK cell development from T/NK CD34(+) triple negative progenitors via soluble factor(s), suggesting that the human thymic microenvironment not only actively promotes T cell maturation but also controls the development of non-T lineage cells such as the NK lineage.     

Expression of alpha(4)beta(7) integrin defines a distinct pathway of lymphoid progenitors committed to T cells, fetal intestinal lymphotoxin producer, NK, and dendritic cells

During embryogenesis, the Peyer's patch anlagen are induced by a cell population that produces lymphotoxin (LT) alpha(1)beta(2) following stimulation of IL-7Ralpha. In this study, we show that the LT-producing cell is localized within the IL-7Ralpha(+) and integrin alpha(4)beta(7) (alpha(4)beta(7))(+) population in the embryonic intestine. Lineage commitment to the LT producer phenotype in the fetal liver coincides with expression of alpha(4)beta(7). Before expression of alpha(4)beta(7), the potential of IL-7Ralpha(+) population to generate B cells is lost. However, the progenitors for T cells and LT producer cells reside in the IL-7Ralpha(+)alpha(4)beta(7)(+) cells, but during subsequent differentiation, the potential to give rise to T cells is lost. This IL-7Ralpha(+)alpha(4)beta(7)(+) population migrates to the intestine, where it induces the Peyer's patch anlagen. When stimulated with IL-15 or IL-3 and TNF, the intestinal IL-7Ralpha(+)alpha(4)beta(7)(+) population can differentiate into fully competent NK1.1(+) NK cells or CD11c(+) APCs. Expression of alpha(4)beta(7) is lost during differentiation of both lineages; IL-7Ralpha expression is lost during NK1.1(+) cells differentiation. A newly discovered lineage(-)IL-7Ralpha(+)c-Kit(+)alpha(4)beta(7)(+) population in the fetal liver is committed to T, NK, dendritic, and fetal intestinal LT producer lineage, the latter being an intermediate stage during differentiation of NK and dendritic cells.     

Clonal analysis reveals uniformity in the molecular profile and lineage potential of CCR9(+) and CCR9(-) thymus-settling progenitors

The entry of T cell progenitors to the thymus marks the beginning of a multistage developmental process that culminates in the generation of self-MHC-restricted CD4(+) and CD8(+) T cells. Although multiple factors including the chemokine receptors CCR7 and CCR9 are now defined as important mediators of progenitor recruitment and colonization in both the fetal and adult thymi, the heterogeneity of thymus-colonizing cells that contribute to development of the T cell pool is complex and poorly understood. In this study, in conjunction with lineage potential assays, we perform phenotypic and genetic analyses on thymus-settling progenitors (TSP) isolated from the embryonic mouse thymus anlagen and surrounding perithymic mesenchyme, including simultaneous gene expression analysis of 14 hemopoietic regulators using single-cell multiplex RT-PCR. We show that, despite the known importance of CCL25-CCR9 mediated thymic recruitment of T cell progenitors, embryonic PIR(+)c-Kit(+) TSP can be subdivided into CCR9(+) and CCR9(-) subsets that differ in their requirements for a functional thymic microenvironment for thymus homing. Despite these differences, lineage potential studies of purified CCR9(+) and CCR9(-) TSP reveal a common bias toward T cell-committed progenitors, and clonal gene expression analysis reveals a genetic consensus that is evident between and within single CCR9(+) and CCR9(-) TSP. Collectively, our data suggest that although the earliest T cell progenitors may display heterogeneity with regard to their requirements for thymus colonization, they represent a developmentally homogeneous progenitor pool that ensures the efficient generation of the first cohorts of T cells during thymus development.     

Langerhans-type dendritic cells genetically modified to express full-length antigen optimally stimulate CTLs in a CD4-dependent manner

Oncoretroviral vectors encoding either full-length Ag or a corresponding immunodominant peptide were expressed in Langerhans-type dendritic cells (LCs) differentiated from CD34(+) progenitors. We used human CMV as a model Ag restricted by HLA-A*0201 to define parameters for eventual expression of cancer Ags by LCs for active immunization against tumors. Stimulation by CMVpp65(495-503)-pulsed LCs, CMVpp65(495-503)-transduced LCs, and full-length CMVpp65-transduced LCs respectively increased tetramer-reactive T cells with an effector memory phenotype by 10 +/- 11, 34 +/- 21, and 51 +/- 24-fold (p < 0.05) from CMV-seropositive donors. CMV-specific CD8(+) CTLs achieved respective frequencies of 231 +/- 102, 583 +/- 219, and 714 +/- 281 spot-forming cells per 10(5) input cells (p < 0.01) in ELISPOT assays for IFN-gamma secretion. LCs expressing full-length Ag stimulated greater lytic activity than either peptide-transduced or peptide-pulsed LCs (p < 0.05), all in the absence of exogenous cytokines. pp65-transduced LCs presenting class I and II MHC-restricted epitopes expanded IFN-gamma-secreting CD4(+) T cells, whereas pp65(495-503)-transduced LCs did not. CD4(+) T cell numbers even declined after stimulation by pp65(495-503) peptide-pulsed LCs. CD4(+) T cell depletion confirmed their contribution to the more robust CTL responses. LCs, transduced with a retroviral vector encoding full-length Ag, stimulate potent CTLs directed against multiple epitopes in a CD4(+) Th cell-dependent manner.     

Lineage tracing reveals the dynamic contribution of Hes1+ cells to the developing and adult pancreas

Notch signaling regulates numerous developmental processes, often acting either to promote one cell fate over another or else to inhibit differentiation altogether. In the embryonic pancreas, Notch and its target gene Hes1 are thought to inhibit endocrine and exocrine specification. Although differentiated cells appear to downregulate Hes1, it is unknown whether Hes1 expression marks multipotent progenitors, or else lineage-restricted precursors. Moreover, although rare cells of the adult pancreas express Hes1, it is unknown whether these represent a specialized progenitor-like population. To address these issues, we developed a mouse Hes1(CreERT2) knock-in allele to inducibly mark Hes1(+) cells and their descendants. We find that Hes1 expression in the early embryonic pancreas identifies multipotent, Notch-responsive progenitors, differentiation of which is blocked by activated Notch. In later embryogenesis, Hes1 marks exocrine-restricted progenitors, in which activated Notch promotes ductal differentiation. In the adult pancreas, Hes1 expression persists in rare differentiated cells, particularly terminal duct or centroacinar cells. Although we find that Hes1(+) cells in the resting or injured pancreas do not behave as adult stem cells for insulin-producing beta (β)-cells, Hes1 expression does identify stem cells throughout the small and large intestine. Together, these studies clarify the roles of Notch and Hes1 in the developing and adult pancreas, and open new avenues to study Notch signaling in this and other tissues.     

Bone marrow-derived hemopoietic precursors commit to the T cell lineage only after arrival in the thymic microenvironment

T lymphocytes develop in the thymus from hemopoietic precursors that commit to the T cell lineage under the influence of Notch signals. In this study, we show by single cell analyses that the most immature hemopoietic precursors in the adult mouse thymus are uncommitted and specify to the T cell lineage only after their arrival in the thymus. These precursors express high levels of surface Notch receptors and rapidly lose B cell potential upon the provision of Notch signals. Using a novel culture system with complexed, soluble Notch ligands that allows the titration of T cell lineage commitment, we find that these precursors are highly sensitive to both Delta and Jagged ligands. In contrast, their phenotypical and functional counterparts in the bone marrow are resistant to Notch signals that efficiently induce T cell lineage commitment in thymic precursors. Mechanistically, this is not due to differences in receptor expression, because early T lineage precursors, bone marrow lineage marker-negative, Sca-1-positive, c-Kit-positive and common lymphoid progenitor cells, express comparable amounts of surface Notch receptors. Our data demonstrate that the sensitivity to Notch-mediated T lineage commitment is stage-dependent and argue against the bone marrow as the site of T cell lineage commitment.     

Notch signaling augments T cell responsiveness by enhancing CD25 expression

Notch receptors signal through a highly conserved pathway to influence cell fate decisions. Notch1 is required for T lineage commitment; however, a role for Notch signaling has not been clearly defined for the peripheral T cell response. Notch gene expression is induced, and Notch1 is activated in primary CD4(+) T cells following specific peptide-Ag stimulation. Notch activity contributes to the peripheral T cell response, as inhibition of endogenous Notch activation decreases the proliferation of activated T cells in a manner associated with the diminished production of IL-2 and the expression of the high affinity IL-2R (CD25). Conversely, forced expression of a constitutively active Notch1 in primary T cells results in increased surface expression of CD25, and renders these cells more sensitive to both cognate Ag and IL-2, as measured by cell division. These data suggest an important role for Notch signaling during CD4(+) T cell responses, which operates through augmenting a positive feedback loop involving IL-2 and its high affinity receptor.     

Thymic anlage is colonized by progenitors restricted to T, NK, and dendritic cell lineages

It remains controversial whether the thymus-colonizing progenitors are committed to the T cell lineage. A major problem that has impeded the characterization of thymic immigrants has been that the earliest intrathymic progenitors thus far identified do not necessarily represent the genuine thymic immigrants, because their developmental potential should have been influenced by contact with the thymic microenvironment. In the present study, we examined the developmental potential of the ontogenically earliest thymic progenitors of day 11 murine fetus. These cells reside in the surrounding mesenchymal region and have not encountered thymic epithelial components. Flow cytometric and immunohistochemical analyses demonstrated that these cells are exclusively Lin(-)c-kit(+)IL-7R(+). Limiting dilution analyses disclosed that the progenitors with T cell potential were abundant, while those with B cell potential were virtually absent in the region of day 11 thymic anlage. Clonal analyses reveled that they are restricted to T, NK, and dendritic cell lineages. Each progenitor was capable of forming a large number of precursors that may clonally accommodate highly diverse TCRbeta chains. These results provide direct evidence that the progenitors restricted to the T/NK/dendritic cell lineage selectively immigrate into the thymus.