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The early stages of human lymphopoiesis are poorly characterized. Here, we compared the lymphoid potential of a novel umbilical cord blood CD34(+)CD45RA(hi)CD7(+) hematopoietic progenitor cell (HPC) population with that of CD34(+)CD45RA(hi)Lin(-)CD10(+) HPCs, previously proposed as candidate common lymphoid progenitors. Limiting-dilution and clonal analysis, fetal thymic organ cultures, and culture onto Notch ligand Delta-like-1-expressing OP9 cells, showed that although CD34(+)CD45RA(hi)CD7(+) HPCs could generate cells of the 3 lymphoid lineages, their potential was skewed toward the T/natural killer (T/NK) lineages. In contrast, CD34(+)CD45RA(hi)Lin(-)CD10(+) HPCs predominantly exhibited a B-cell potential. Gene expression profiling with DNA microarrays confirmed that CD34(+)CD45RA(hi)CD7(+) HPCs selectively expressed T-lymphoid and NK lineage-committed genes while retaining expression of genes affiliated to the granulomonocytic lineage, whereas CD34(+)CD45RA(hi)Lin(-)CD10(+) HPCs displayed a typical pro-B-cell transcription profile and essentially lacked genes unrelated to the B lineage. In addition, both populations could be generated in vitro from CD34(+)CD45RA(int)CD7(-) and CD34(+)CD45RA(hi)Lin(-) HPCs with mixed lymphomyeloid potential, from which they emerged independently with different growth/differentiation factor requirements. These findings indicate that CD34(+)CD45RA(hi)CD7(+) and CD34(+)CD45RA(hi)Lin(-)CD10(+) HPCs correspond to multipotent early lymphoid progenitors polarized toward either the T/NK or B lineage, respectively.

Dendritic cells (DC) are essential antigen-presenting cells that initiate and regulate adaptive immune responses. There are distinct DC populations of diverse origins, which develop from hematopoietic progenitors already committed to the lymphoid or the myeloid lineages and, in the latter case, even from terminally differentiated macrophages. One may assume that DC of lymphoid origin are dedicated to the adaptive immune system, along which they have phylogenetically co-evolved, whereas myeloid DC would be more involved as an interface between the innate and adaptive immune systems. However, any DC can ultimately present antigens in either an immunogenic or tolerogenic manner according to whether they are more or less or not at all activated towards maturation, depending on the condition under which they encountered antigen. Hence, DC either induce the appropriate immune response to pathogens or prevent autoimmune reactivity. Thus, besides default programming, which should be necessary to face the challenges of their usual setting, each type of DC can also display functions that are similar, in an instructive mode, to elicit immune responses deemed necessary for unexpected stimuli. In such a system, DC provide enough flexibility and sufficient redundancy to ensure that an essential function of the immune system, i.e., passing information from its innate to adaptive arms and affecting the latter's responses, occurs under optimal conditions. Working on the basis of such a unified theory of DC diversity should be useful for learning to adequately manipulate the immune system for the development of cellular immunotherapy.

International audience; The classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127- and CD127+ early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127- and CD127+ ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127- ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127+ ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis.

We examined the effect of interleukin (IL)-4 or CD40 ligation on the differentiation and maturation of CD1a+CD14– and CD1a–CD14+ dendritic cell (DC) precursors. Cord blood CD34+ cells were cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor α (TNF-α), to which stem cell factor and Flt-3 ligand were added for 5 days. Phenotypic analysis of DC precursors on culture day 7 showed that CD1a+CD14– cells expressed higher CD11c and CD80 levels and lower CD116/GM-CSFR and CCR-5 levels than their CD1a–CD14+ counterparts. Culturing CD1a+CD14– precursors with GM-CSF and TNF-α resulted in DC with heterogeneous CD1a, HLA;SMDR (DR), CD11b, and CD83 expression, 10% of which acquired CD14. IL-4 and CD40 ligation affected their differentiation in contrasting ways: IL-4 induced CD1ahiCD14– DRloCD11b+CD83–S100+ DC with reduced MLR-stimulating capacity, whereas CD40 ligation led to CD1alo/-CD14–CD40–DRhiCD11b–CD83+S100+/– DC with stronger MLR-stimulating capacity. Also, both IL-4 and CD40 ligation promoted RelB expression and nuclear translocation. When CD1a–CD14+ precursors were maintained in only the presence of GM-CSF and TNF-α, this led to mixed populations of adherent macrophages and nonadherent CD1a–CD14+ monocytes, and of CD1a+CD14– and CD1a+CD14+ DC, which were DRloCD11b+-CD83–S100–. IL-4 or CD40 ligation prevented their differentiation into macrophages and resulted in DC with phenotypes close to those issued from CD1a+CD14– precursors, with only a minority staying CD14+ but most being S100–; their MLR-stimulating capacity also increased but remained lower than that of DC differentiated from CD1a+CD14– precursors. Thus, IL-4 or CD40 ligation induced CD1a+CD14– and CD1a–CD14+ DC precursors to differentiate into phenotypically close but functionally different DC populations, suggesting that DC function is primarily determined by their origin. The heterogeneity of DC should then be related to different developmental pathways and to different stages of maturation/activation. J. Leukoc. Biol. 64: 235–244; 1998.

CD1a+ dendritic cells (DC) differentiate from a major population of nonadherent CD13(hi)lin-cells that appear when human cord blood CD34+ hematopoietic progenitor cells are cultured with stem-cell factor, granulocyte/macrophage (MA) colony-stimulating factor, and tumor necrosis factor-alpha (TNF-alpha) for 5 days. CD13hilin- cells, which also comprise MA and granulocyte precursors, are CD4+ and can thus be targets of human immunodeficiency virus (HIV). Low replication was noted when these day 5 cells were infected with lymphotropic HIV-1LA1 (p24: < or = 4 ng/mL on day 8 postinfection [PI]), while high virus production occurred with MA-tropic HIV-1Ba-L, HIV-1Ada, or HIV-1-m-n. (p24: 50 to > or = 1,000 ng/mL). Strong cytopathicity (CPE) was then observed in nonadherent cells as in adherent MA. However, FACS analysis on day 7 PI showed that HIV did not affect differentiation of DC that survived CPE: apart from CD4 downmodulation related to HIV production, overall expression of CD40, CD80, and CD86 costimulatory molecules, and of HLA-DR, was unchanged relative to controls. At that time, the capacity of DC from HIV-infected cultures to stimulate the mixed leukocyte reaction was only altered less than 10-fold. Immunocytochemistry on day 7 PI showed that most HIV-infected cells were included in syncytia that were stained by anti-CD1a, anti-S100, and anti-CD14 antibodies, indicating that syncytia consisted of DC and cells of the MA lineage. Polymerase chain reaction analysis of FACS- sorted CD1a+ cells confirmed that they harbored then HIV DNA. Viral DNA was also detected in CD1a+ DC from noninfected cultures that had been exposed to HIV only after sorting. Therefore, we examined whether in infected cultures DC precursors were infected at the onset or if virus spread later from other infected cells to differentiated DC. This was answered by showing that, 24 hours postexposure to HIV, viral DNA was preferentially detected in day 5 sorted CD13hilin-versus CD13hilin- cells, and that it was found in the CD1a+ progeny of CD13(hi)lin-cells 48 hours later. In addition, HIV replication did not affect myeloid clonogenic progenitors in day 0 to day 7 PI cultures, although viral DNA was detected in colony-forming unit-granulocyte/macrophage (CFU-GM)/CFU-M colonies derived from day 3 and 7 PI cultures. Thus, precursors of DC and their progeny are susceptible to HIV in vitro, but, apart from CPE, the effect of virus production on DC differentiation or function is limited.

The most effective antigen-presenting cells for T lymphocytes are dendritic cells (DCs), the differentiation pathway of which, however, is incompletely characterized. We examined here how DCs differentiated from human cord blood CD34+ progenitor cells cultured with granulocyte- macrophage colony-stimulating factor, tumor necrosis factor-alpha, and stem cell factor. After 5 days, 2 of 3 nonadherent cells were CD13hiHLA- DRhiCD4+, half of them were also CD14+, and < or = 10% were CD1a+. When day-5 sorted CD13hiCD1a- and CD13lo cells were further cultured, CD1a+ cells appeared in the already CD13hi population, whereas CD13hi cells, a minority of which rapidly became CD1a+, emerged from the CD13lo population. By day 12, still 66% of bulk cells in suspension were CD13hi, most of which displayed high forward and side scatters of large granular cells. Half of CD13hi cells were CD1a+. All CD13hi cells expressed to the same extent DR, CD4, costimulatory and adhesion molecules, and various amounts of CD14. CD1a+ cells stimulated allogeneic lymphocytes more than CD13hiCD1a- cells and, although they were CD14+, both cell types were nonspecific esterase-negative nonphagocytic cells and were stronger mixed leukocyte reaction stimulators than were their macrophage counterparts. Eventually, the percentage of CD1a+ cells decreased. However, typical CD1a+ DCs still emerged in culture of sorted day-12 CD13hiCD1a- cells, and adding interleukin-4 to bulk cultures at that time led to the persistence of the CD1a+ population while diminishing CD14 expression. Thus, this system results first in the differentiation of CD13hi precursors that strongly express DR and CD4, from which more mature CD1a+ DCs continuously differentiate all along the culture period.

To model the developmental pattern of human prothymocytes and thymopoiesis, we used NOD-scid/γc−/− mice grafted with human umbilical cord blood CD34+ hematopoietic progenitor cells (HPCs). Human prothymocytes developed in the murine bone marrow (BM) from multipotent CD34++CD38lolineage− HPCs to CD34++CD7+CD2- pro-T1 cells that progressed in a Notch-dependent manner to CD34+CD7++CD2+ pro-T2 cells, which migrated to the thymus. BM prothymocyte numbers peaked 1 mo after graft, dropped at mo 2, and persisted at low levels thereafter, with only a few CD34+CD7lo prothymocytes with limited T potential being detected by mo 5. As a consequence, thymopoiesis in this xenogeneic setting began by weeks 4–6, peaked at mo 3, and decreased thenceforth. Analyzing mice grafted at 2, 4 or 8, mo of age showed that in an “older” BM, prothymocyte differentiation was perturbed and resulted in CD34+CD7lo prothymocytes with limited T potential. Whereas the early drop in BM thymopoietic activity was related to a Notch-independent loss of T potential by CD34++CD38lolineage− HPCs, the later age-dependent production decline of prothymocytes was linked to a more complex mix of cell-intrinsic and microenvironmental defects. Accordingly, and contrasting with what was observed with umbilical cord blood HPCs, CD34+ HPCs from human adult BM displayed only marginal thymopoietic activity when grafted into young 2-mo-old NOD-scid/γc−/− mice. These data demonstrate that the developmental pattern of BM prothymocytes during human late fetal and early postnatal life can be reproduced in humanized mice, and they suggest that onset of human thymus involution relates to decreased colonization by prothymocytes.

SummaryHere, we identify fetal bone marrow (BM)-derived CD34hiCD45RAhiCD7+ hematopoietic progenitors as thymus-colonizing cells. This population, virtually absent from the fetal liver (FL), emerges in the BM by development weeks 8–9, where it accumulates throughout the second trimester, to finally decline around birth. Based on phenotypic, molecular, and functional criteria, we demonstrate that CD34hiCD45RAhiCD7+ cells represent the direct precursors of the most immature CD34hiCD1a− fetal thymocytes that follow a similar dynamics pattern during fetal and early postnatal development. Histological analysis of fetal thymuses further reveals that early immigrants predominantly localize in the perivascular areas of the cortex, where they form a lymphostromal complex with thymic epithelial cells (TECs) driving their rapid specification toward the T lineage. Finally, using an ex vivo xenogeneic thymus-colonization assay, we show that BM-derived CD34hiCD45RAhiCD7+ progenitors are selectively recruited into the thymus parenchyma in the absence of exogenous cytokines, where they adopt a definitive T cell fate.

We have previously shown that thymic CD34 1 cells have a very limited myeloid differentia- tion capacity and differentiate in vitro mostly into CD1a 1 -derived but not CD14 1 -derived dendritic cells (DC). Herein we characterized the human neo- natal thymic DC extracted from the organ in rela- tionship with the DC generated from CD34 1 cells in situ. We show that in vivo thymic DC express E cad- herin, CLA, CD4, CD38, CD40, CD44, and granu- locyte-macrophage colony-stimulating factor-R (GM- CSF-R; CD116) but no CD1a. According to their morphology, functions, and surface staining they could be separated into two distinct subpopulations: mature HLA-DR hi , mostly interleukin-3-R (CD123)- negative cells, associated with thymocytes, some apoptotic, and expressed myeloid and activation markers but no lymphoid markers. In contrast, immature HLA-DR 1 CD123 hi CD36 1 cells with monocytoid morphology lacked activation and my- eloid antigens but expressed lymphoid antigens. The latter express pTa mRNA, which is also found in CD34 1 thymocytes and in blood CD123 hi DC further linking this subset to lymphoid DC. How- ever, the DC generated from CD34 1 thymic pro- genitors under standard conditions were pTa-neg- ative. Thymic lymphoid DC showed similar pheno- type and cytokine production profile as blood/ tonsillar lymphoid DC but responded to GM-CSF, and at variance with them produced no or little type I interferon upon infection with viruses and did not induce a strict polarization of naive T cells into TH2 cells. Their function in the thymus re- mains therefore to be elucidated. J. Leukoc. Biol. 68: 836-844; 2000.

Dendritic cells (DC) were sorted on day 8 from cultures of CD34+ cells with stem cell factor/Flt-3 ligand/ granulocyte-macrophage colony-stimulating factor (GM-CSF)/tumor necrosis factor- (TNF-)/interleukin-4 (IL-4). Exposing immature CCR5+CXCR4lo/− DC to CCR5-dependent human immunodeficiency virus (HIV)-1Ba-L led to productive and cytopathic infection, whereas only low virus production occurred in CXCR4-dependent HIV-1LAI–exposed DC. PCR analysis of the DC 48 hours postinfection showed efficient entry of HIV-1Ba-L but not of HIV-1LAI. CD40 ligand- or monocyte-conditioned medium-induced maturation of HIV-1Ba-L–infected DC reduced virus production by about 1 Log, while cells became CCR5−. However, HIV-1Ba-L–exposed mature DC harbored 15-fold more viral DNA than their immature counterparts, ruling out inhibition of virus entry. Simultaneously, CXCR4 upregulation by mature DC coincided with highly efficient entry of HIV-1LAI which, nonetheless, replicated at the same low level in mature as in immature DC. In line with these findings, coculture of HIV-1Ba-L–infected immature DC with CD3 monoclonal antibody–activated autologous CD4+ T lymphocytes in the presence of AZT decreased virus production by the DC. Finally, whether they originated from CD1a+CD14− or CD1a−CD14+ precursors, DC did not differ as regards permissivity to HIV, although CD1a+CD14− precursor-derived immature DC could produce higher HIV-1Ba-L amounts than their CD1a−CD14+ counterparts. Thus, both DC permissivity to, and capacity to support replication of, HIV is primarily determined by their maturation stage.

We analyzed the effect of tumor necrosis factor (TNF)-alpha on the differentiation and viability of dendritic cells (DC) generated from cord blood CD34+ progenitors cultured for five days with GM-CSF, Flt-3 ligand (FL), and stem cell factor (SCF), and then with GM-CSF only [TNF(-) cultures]. Adding TNF-alpha from the start [TNF(+) cultures] potentiated progenitor cell proliferation and promoted early differentiation of CD1a+ DC precursors without affecting differentiation of CD14+ cells, which comprise bipotent precursors of DC and macrophages, nor of CD15+ granulocytic cells. Use of TNF-alpha was associated with increased cell mortality, which peaked on culture day 10 and mainly involved CD1a+ DC. Selective apoptosis of CD1a+ DC precursors was confirmed by showing that survival of day-7-sorted CD1a+CD14- cells from TNF(+) cultures was lower than that of CD1a-CD14+ cells. That similar findings were noted for sorted CD1a+CD14- cells of TNF(-) cultures, further cultured with GM-CSF without or with TNF-alpha, indicates that apoptosis of CD1a+ DC precursors was not induced by TNF-alpha. Apoptosis of CD1a+ DC precursors occurred after the cells had lost the capacity to incorporate bromodeoxyuridin. Finally, using higher GM-CSF concentrations or adding interleukin 3 (IL-3) improved viability of CD1a+ cells. Other cytokines, such as IL-4 and transforming growth factor (TGF)-beta1, were ineffective in this respect, though they promoted differentiation of CD1a+ DC. These results indicate that TNF-alpha promotes the differentiation of CD1a+ DC precursors, which display a high susceptibility to apoptosis that can be prevented by high concentrations of GM-CSF or use of IL-3, without affecting the differentiation of the CD14+ DC precursors.