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SUMMARYChanges in lymphocyte production patterns occurring across human ontogeny remain poorly defined. In this study, we demonstrate that human lymphopoiesis is supported by three waves of embryonic, fetal, and postnatal multi-lymphoid progenitors (MLPs) differing in CD7 and CD10 expression and their output of CD127-/+early lymphoid progenitors (ELP). Our results reveal that, like the fetal-to-adult switch in erythropoiesis, transition to postnatal life coincides with a shift from multilineage to B lineage-biased lymphopoiesis and an increase in production of CD127+ELPs which persists until puberty. A further developmental transition is observed in elderly individuals where B-cell differentiation bypasses the CD127+compartment and branches directly from CD10+MLPs. Functional analyses indicate that these changes are determined at the level of the hematopoietic stem cell. Besides reconciling controversies about the identity and function of human MLPs, these results may shed light on the causes of age-related differences in the incidence of lymphoblastic leukemia.

SUMMARYThe developmental cartography of human lymphopoiesis remains incompletely understood. Here, we establish a multimodal map that extends the current view of lymphoid development. Our results demonstrate that lymphoid specification follows independent direct or stepwise differentiation pathways converging toward the emergence of CD117lomulti-lymphoid progenitors (MLPs) that undergo a proliferation arrest before entering the CD127-(T/NK/ILC) or CD127+(B) lymphoid pathways. While the emergence of CD127-early lymphoid progenitors is driven by Flt3 signaling, differentiation of their CD127+counterparts is regulated cell-intrinsically and depends exclusively on the divisional history of their precursors. Single-cell mapping of lymphoid differentiation trajectories reveals that a dissociation between proliferation and differentiation phases allows amplification of the precursor pools prior to the onset of antigen receptor rearrangement. Besides demonstrating that B and T/NK/ILC lineages are subjected to differential cell-autonomousversusFlt3-inducible regulation, our results go a long way to reconciling human and mouse models of lymphoid architecture.

Due to difficulties to access primary human bone marrow samples and age or donor effects, human hematopoiesis has long remained far less well characterized than in the mouse. Despite recent progresses in single-cell RNA profiling only little is known as to phenotype, function and developmental trajectories of human lymphomyeloid progenitors and precursors. This is especially true regarding the developmental architecture of the lymphoid lineage which has been the subject of persistent controversies over the past decades. Here, we describe an original approach of in vivo modeling of human fetal hematopoiesis immunodeficient NSG mice engrafted with neonatal CD34

Due to difficulties to access primary human bone marrow samples and age or donor effects, human hematopoiesis has long remained far less well characterized than in the mouse. Despite recent progresses in single-cell RNA profiling only little is known as to phenotype, function and developmental trajectories of human lymphomyeloid progenitors and precursors. This is especially true regarding the developmental architecture of the lymphoid lineage which has been the subject of persistent controversies over the past decades. Here, we describe an original approach of in vivo modeling of human fetal hematopoiesis immunodeficient NSG mice engrafted with neonatal CD34+ hematopoietic progenitor cells (HPCs) allowing for rapid identification and isolation of lymphomyeloid developmental intermediates.

Mechanisms driving acute graft-versus-host disease (aGVHD) onset in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) are still poorly understood. To provide a detailed characterization of tissue-infiltrating T lymphocytes (TL) and search for eventual site-specific specificities, we developed a xenogeneic model of aGVHD in immunodeficient mice. Phenotypic characterization of xenoreactive T lymphocytes (TL) in diseased mice disclosed a massive infiltration of GVHD target organs by an original CD4+CD8+ TL subset. Immunophenotypic and transcriptional profiling shows that CD4+CD8+ TL comprise a major PD1+CD62L−/+ transitional memory subset (>60%) characterized by low level expression of cytotoxicity-related transcripts. CD4+CD8+ TL produce high IL-10 and IL-13 levels, and low IL-2 and IFN-γ, suggestive of regulatory function. In vivo tracking of genetically labeled CD4+ or CD8+ TL subsequently found that CD4+CD8+ TL mainly originate from chronically activated cytotoxic TL (CTL). On the other hand, phenotypic profiling of CD3+ TL from blood, duodenum or rectal mucosa in a cohort of allo-HSCT patients failed to disclose abnormal expansion of CD4+CD8+ TL independent of aGVHD development. Collectively, our results show that acquisition of surface CD4 by xenoreactive CD8+ CTL is associated with functional diversion toward a regulatory phenotype, but rule out a central role of this subset in the pathogenesis of aGVHD in allo-HSCT patients.

L’étude de l’hématopoïèse humaine a longtemps été limitée par l’accès aux prélèvements primaires de moelle osseuse. Afin de s’affranchir de cette contrainte, une approche originale de modélisation chez la souris immunodéficiente a été développée dans notre laboratoire. L’analyse de l’ensemble des populations cellulaires humaines générées dans la moelle osseuse des animaux greffés a permis d’établir une nouvelle cartographie de l’hématopoïèse humaine. Nous montrons que, contrairement aux prédictions du modèle canonique de l’hématopoïèse, la lymphopoïèse humaine présente une organisation bipartite, articulée autour de l’existence de deux familles de progéniteurs lymphoïdes identifiées par l’expression différentielle du récepteur de l’interleukine 7 (CD127). Outre leurs différences phénotypiques, les progéniteurs lymphoïdes CD127- et CD127+ se distinguent par leurs capacités de différenciation, leur dépendance vis-à-vis de facteurs de croissance, leurs modalités de restriction de potentiel, ainsi que par leurs signatures transcriptionnelles.

International audience; Human KIAA0922/TMEM131L encodes a transmembrane protein, TMEM131L, that regulates the canonical Wnt/b-catenin signaling pathway by eliciting the lysosome-dependent degradation of phosphorylated LRP6 co-receptor. Here, we use a heterospecific Drosophila transgenic model to examine the potential evolutionary conservation of TMEM131L function. Analysis of TMEM131L transgenic flies shows that TMEM131L interference with the Wnt pathway results primarily from a Notch-dependent decrease in Wingless production. Consistently, lentivirus-mediated overexpression of TMEM131L in human CD34 þ hematopoietic progenitor cells leads to decreased susceptibility to Notch1 ligation and defective commitment toward the T lineage. These results show that TMEM131L corresponds to an evolutionary conserved regulator of the Notch signaling pathway.

Dans cette etude, nous nous sommes tout d’abord interroges sur le concept de vie a travers une lecture croisee, celle du semioticien et du biologiste, des textes de la tradition philosophique occidentale depuis Platon et Aristote jusqu’a H. Bergson et G. Deleuze, ainsi que de deux textes cles de l’histoire de la biologie, La Philosophie zoologique de Lamark et L’Origine des especes de Darwin. Nous avons ensuite procede a une analyse critique des approches conventionnelles physicaliste, vitaliste et historiciste, du concept de vie, avant d’explorer de facon plus specifique les relations qui s’etablissent entre le vivant et le langage. Ce parcours nous conduit a formuler l’hypothese de l’existence d’une communaute de nature, c’est-a-dire d’une identite partagee, entre les processus biologiques elementaires qui prennent place a l’echelon cellulaire et moleculaire, et les modes de production et d’organisation du langage en acte mis en œuvre dans le discours du sujet parlant.

We analyzed the role of human immunodeficiency virus (HIV)-1 matrix protein (MA) during the virus replication afferent phase. Single-round infection of H9 T lymphocytes showed that the combined mutation of MA Lys residues 26–27 in MA reported nuclear localization signal (NLS)-1 [ Haffar, O.K., Popov, S., Dubrovsky, L., Agostini, I., Tang, H., Pushkarsky, T., Nadler, S.G., Bukrinsky, M., 2000. Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex. J. Mol. Biol. 299 (2): 359–368 ] impaired infectivity, abrogated 2-LTR-circle formation and significantly reduced integration. However, the mutation did not affect viral DNA docking to chromatin in either interphasic or mitotic cells, indicating that MA N-terminal basic domain should not represent a major determinant of HIV-1 nuclear import in T lymphocytes. These data point to a previously unreported role of MA in the late, post-chromatin-binding, afferent phase of HIV-1 replication cycle.

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.

To better characterize human dendritic cells (DCs) that originate from lymphoid progenitors, the authors examined the DC differentiation pathways from a novel CD7(+)CD45RA(+) progenitor population found among cord blood CD34(+) cells. Unlike CD7(-)CD45RA(+) and CD7(+)CD45RA(-) progenitors, this population displayed high natural killer (NK) cell differentiation capacity when cultured with stem cell factor (SCF), interleukin (IL)-2, IL-7, and IL-15, attesting to its lymphoid potential. In cultures with SCF, Flt3 ligand (FL), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-alpha (standard condition), CD7(+)CD45RA(+) progenitors expanded less (37- vs 155-fold) but yielded 2-fold higher CD1a(+) DC percentages than CD7(-)CD45RA(+) or CD7(+)CD45RA(-) progenitors. As reported for CD34(+)CD1a(-) thymocytes, cloning experiments demonstrated that CD7(+)CD45RA(+) cells comprised bipotent NK/DC progenitors. DCs differentiated from CD7(-)CD45RA(+) and CD7(+)CD45RA(+) progenitors differed as to E-cadherin CD123, CD116, and CD127 expression, but none of these was really discriminant. Only CD7(+)CD45RA(+) or thymic progenitors differentiated into Lag(+)S100(+) Langerhans cells in the absence of exogenous transforming growth factor (TGF)-beta 1. Analysis of the DC differentiation pathways showed that CD7(+)CD45RA(+) progenitors generated CD1a(+)CD14(-) precursors that were macrophage-colony stimulating factor (M-CSF) resistant and CD1a(-)CD14(+) precursors that readily differentiated into DCs under the standard condition. Accordingly, CD7(+)CD45RA(+) progenitor-derived mature DCs produced 2- to 4-fold more IL-6, IL-12, and TNF-alpha on CD40 ligation and elicited 3- to 6-fold higher allogeneic T-lymphocyte reactivity than CD7(-)CD45RA(+) progenitor-derived DCs. Altogether, these findings provide evidence that the DCs that differentiate from cord blood CD34(+)CD7(+)CD45RA(+) progenitors represent an original population for their developmental pathways and function. (Blood. 2000;96:3748-3756)

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.

Dendritic cells (DC) are the most potent antigen-presenting cells. Thus, ex vivo antigen-pulsed DC are a potentially powerful tool to induce in vivo immunity against tumor-associated or viral antigens. Therefore, culture methods to generate high numbers of DC from bone marrow or blood CD34+ hematopoietic progenitor cells have recently been developed. These methods, which use different combinations of growth factor--mainly granulocyte/macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-alpha and interleukin (IL)-4--make the characterization of DC obtained from CD34+ cells of different origins easier and allow to assess whether DC relate to a unique or distinct differentiation pathways. Monocytes and even macrophages can also directly differentiate into DC in the presence of GM-CSF and IL-4. This has to be reconciled with evidence supporting earlier branching off of the macrophage and DC lineages, and raises questions as to the identity of the latter lineage. Apart from DC of myeloid origin, DC may also originate from lymphoid progenitors. Because the capacity of DC to capture, process and present antigens is known to vary according to their differentiation stage, and lymphoid DC might behave differently from lymphoid DC in this respect, the definition of which type of DC to use for immunotherapy must be more precise, in order to avoid detrimental side effects or results. From a practical point of view, it is also necessary to define the most appropriate cytokine combinations and schedules thereof to optimize proliferation and differentiation of DC from different origins. These conditions should then be applied to generated DC for their efficient and safe use for clinical immunotherapy.

In this study, we identify transmembrane protein 131–like (TMEM131L) as a novel regulator of thymocyte proliferation and demonstrate that it corresponds to a not as yet reported inhibitor of Wnt signaling. Short hairpin RNA–mediated silencing of TMEM131L in human CD34+ hematopoietic progenitors, which were then grafted in NOD-SCID/IL-2rγnull mice, resulted in both thymocyte hyperproliferation and multiple pre– and post–β-selection intrathymic developmental defects. Consistent with deregulated Wnt signaling, TMEM131L-deficient thymocytes expressed Wnt target genes at abnormally high levels, and they displayed both constitutive phosphorylation of Wnt coreceptor LRP6 and β-catenin intranuclear accumulation. Using T cell factor reporter assays, we found that membrane-associated TMEM131L inhibited canonical Wnt/β-catenin signaling at the LRP6 coreceptor level. Whereas membrane-associated TMEM131L did not affect LRP6 expression under basal conditions, it triggered lysosome-dependent degradation of its active phosphorylated form following Wnt activation. Genetic mapping showed that phosphorylated LRP6 degradation did not depend on TMEM131L cytoplasmic part but rather on a conserved extracellular domain proximal to the membrane. Collectively, these data indicate that, during thymopoiesis, stage-specific surface translocation of TMEM131L may regulate immature single-positive thymocyte proliferation arrest by acting through mixed Wnt-dependent and -independent mechanisms.

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.

It is not known whether impaired hematopoiesis noted during human immunodeficiency virus (HIV) infection results from infection of stem/progenitor cells or of cells of the bone marrow microenvironment. Normal adherent primary stromal layers were exposed to HIV to determine which of this mixture of endothelial cells, fibroblasts, and macrophages are susceptible to the virus. Viral p24 in supernatants was noted with monocytotropic HIV-1Ada, HIV-1Ba-L, and HIV-1JR-FL but not with lymphotropic HIV-1LAI nor HIV-1MN strain, and only stromal macrophages expressed the viral antigens. Coculture of the layers with PHA-activated normal lymphocytes failed to rescue lymphotropic virus. No p24 was produced when macrophagedepleted stromal cells were exposed to either HIV-1Ba-L or HIV-1LAI; proviral DNA was then amplified by PCR in cells exposed to either virus, though coculture with lymphocytes rescued only HIV-IBa-L. Altogether, these data indicate that macrophages are the major targets of HIV in cultured stromal layers. As virus replication in macrophages did not affect the profile of major cytokines involved in regulating hematopoiesis, HIV infection could alter hematopoiesis by other as yet unspecified mechanisms.

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.

This review gives a general overview of the recent advances in the field of fetal hematopoiesis and T-cell development. Although it is now well recognized that early lymphoid progenitors first emerge in the fetal liver (FL) where active B lymphopoiesis take places, the identity of early T-cell precursors, as well as the mechanisms of thymus colonization, has long been controversial. Here we discuss the experimental evidence supporting the concept of prothymocyte in both human and mouse species, as well as its major implications regarding lineage relationships among the immune system and general hematopoietic organization.

The C-type lectin DC-SIGN expressed on immature dendritic cells (DCs) captures human immunodeficiency virus (HIV) particles and enhances the infection of CD4 + T cells. This process, known as trans -enhancement of T-cell infection, has been related to HIV endocytosis. It has been proposed that DC-SIGN targets HIV to a nondegradative compartment within DCs and DC-SIGN-expressing cells, allowing incoming virus to persist for several days before infecting target cells. In this study, we provide several lines of evidence suggesting that intracellular storage of intact virions does not contribute to HIV transmission. We show that endocytosis-defective DC-SIGN molecules enhance T-cell infection as efficiently as their wild-type counterparts, indicating that DC-SIGN-mediated HIV internalization is dispensable for trans -enhancement. Furthermore, using immature DCs that are genetically resistant to infection, we demonstrate that several days after viral uptake, HIV transfer from DCs to T cells requires viral fusion and occurs exclusively through DC infection and transmission of newly synthesized viral particles. Importantly, our results suggest that DC-SIGN participates in this process by cooperating with the HIV entry receptors to facilitate cis -infection of immature DCs and subsequent viral transfer to T cells. We suggest that such a mechanism, rather than intracellular storage of incoming virus, accounts for the long-term transfer of HIV to CD4 + T cells and may contribute to the spread of infection by DCs.

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 examined the influence of mitosis on the kinetics of human immunodeficiency virus type 1 integration in T cells. Single-round infection of cells arrested in G1b or allowed to synchronously proceed through division showed that mitosis delays virus integration until 18–24 h postinfection, whereas integration reaches maximum levels by 15 h in G1b-arrested cells. Subcellular fractionation of metaphase-arrested cells indicated that, while nuclear envelope disassembly facilitates docking of viral DNA to chromatin, chromosome condensation directly antagonizes and therefore delays integration. As a result of the balance between the two effects, virus integration efficiency is eventually up to threefold greater in dividing cells. At the single-cell level, using a green fluorescent protein-expressing reporter virus, we found that passage through mitosis leads to prominent asymmetric segregation of the viral genome in daughter cells without interfering with provirus expression.

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.

Cells from the dendritic lineage presumably play an important role in the pathogenesis of human immunodeficiency virus (HIV) infection1,2. Langerhans cells (LC), are probably among the first targets for HIV in genital mucosae3,4. Later, in T-cell dependent areas of lymphoid organs, dendritic cells (DC) may also be involved in both virus transmission to CD4+ T cells and the generation of an effective anti-HIV immune response2. There is now agreement that mature DC/LC can take up the virus, reverse transcribe its RNA, cluster to and form syncytia with CD4+ T cells, but the real degree of their permissivity to HIV still remains debated1,5,6. A precise knowledge of the relationships of cells of the DC lineage with HIV is important for understanding AIDS pathogenesis as well as for delineating possible new immunotherapy strategies. Studying their interactions with HIV in different models is therefore warranted.

Disparate findings have been reported as to whether human immunodeficiency virus (HIV) affects cytokine production by macrophages (MA). We investigated production of different cytokines and of macrophage inflammatory protein (MIP)-1alpha by HIV-1Ba-L-or HIV-1Ada-infected blood-derived MA. Relative to controls, only MIP-1alpha levels increased twofold to > 10-fold in supernatants 2 to 3 weeks postinfection (PI), at the time of maximum virus production; levels of the other chemokines (RANTES, interleukin (IL)-8) and cytokines (IL-1alpha, IL-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1) investigated were not affected. MIP-1alpha mRNA signal assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) was, however, only occasionally greater in cells from infected cultures relative to controls. MIP-1alpha levels in supernatants remained in the same range as in control cultures when more than 10 mmol/L Zidovudine was added 24 hours PI, which indicates involvement of virus replication in the effect. Anti-MIP-1alpha antibody labeling identified a 10% to 25% subset of MA, strongly expressing HLA-DR and CD4, and also stained by anti-IL-6 and anti-TNF-alpha antibodies. Two weeks PI, dual staining showed that the majority of the 5% to 20% cells that were p24+ belonged to the MIP-1alpha+ population, which may define a MA subset capable to better sustain HIV replication. MIP-1alpha induced by HIV replication in MA might play a role in the pathophysiology of HIV infection; in impaired hematopoiesis; or as a CD4+ and CD8+ lymphocyte chemoattractant, by recruiting either or both HIV-susceptible and cytotoxic T lymphocytes to virus replication sites.

The Dielmo project, initiated in 1990, consisted of long-term investigations on host-parasite relationships and the mechanisms of protective immunity in the 247 residents of a Senegalese village in which malaria is holoendemic. Anopheles gambiae s.l. and An. funestus constituted more than 98% of 11,685 anophelines collected and were present all year round. Inoculation rates of Plasmodium falciparum, P. malariae, and P. ovale averaged respectively 0.51, 0.10, and 0.04 infective bites per person per night. During a four-month period of intensive parasitologic and clinical monitoring, Plasmodium falciparum, P. malariae, and P. ovale were observed in 72.0%, 21.1% and 6.0%, respectively, of the 8,539 thick smears examined. Individual longitudinal data revealed that 98.6% of the villagers harbored trophozoites of P. falciparum at least once during the period of the study. Infections by P. malariae and P. ovale were both observed in individuals of all age groups and their cumulative prevalences reached 50.5% and 40.3%, respectively. Malaria was responsible for 162 (60.9%) of 266 febrile episodes; 159 of these attacks were due to P. falciparum, three to P. ovale, and none to P. malariae. The incidence of malaria attacks was 40 times higher in children 0–4 years of age than in adults more than 40 years old. Our findings suggest that sterile immunity and clinical protection are never fully achieved in humans continuously exposed since birth to intense transmission.

Human lymphopoiesis remains poorly characterized compared to its murine counterpart. This is due to the lack of adequate experimental models and difficulties in accessing primary bone marrow samples. Thanks to the fine characterization of the populations of lymphoid progenitors and precursors in the bone marrow of transplanted mice, we have recently demonstrated that human lymphopoiesis proceeds along independent CD127- or CD127+ pathways that generate both common and specific lymphoid subsets. In this report, we developed a novel ex vivo diversification assay to analyze their developmental relationships and investigate the mechanisms driving the emergence of the CD127- or CD127+ early lymphoid progenitors (ELPs) from their common lympho-mono-dendritic (LMDP) ancestors. Time-course analyses show that CD127- or CD127+ ELPs emerge sequentially from “early” versus “late” LMDPs. Functional screening of a series of 30 receptor/ligand pairs show in addition that the CD127- or CD127+ ELPs are subjected to a differential regulation. Whereas Flt3L, IL-27 and Notch1 signaling impose a veto on CD127+ ELP emergence and polarize lymphoid differentiation toward exclusive CD127- pathway, the emergence of the CD127+ ELPs is instead favored by limiting cytokine conditions and low cell expansion rates. These new findings indicate that CD127- and CD127+ ELPs are subjected to distinct cell-extrinsic versus cell-intrinsic regulatory mechanisms thereby reinforcing the previously proposed dichotomous architecture of human lymphopoiesis. In as much as they also argue for multi-layered regulation of lympho-myeloid differentiation, our new results challenge the emerging continuous model of hematopoiesis.; La lymphopoïèse humaine reste mal caractérisée comparativement à son homologue murin. Ceci est dû à l’absence de modèles expérimentaux adéquats et aux difficultés d’accès aux prélèvements primaires de moelle osseuse humaine. Grâce à la caractérisation fine des populations de progéniteurs et précurseurs lymphoïdes présentes dans la moelle osseuse d’animaux greffés, nous avons récemment démontré que la lymphopoïèse humaine suit des voies CD127- ou CD127+ indépendantes qui génèrent une descendance lymphoïde commune et spécifique. Dans cette étude, nous avons mis au point un nouveau test de diversification ex vivo pour analyser leurs relations développementales et étudier les mécanismes qui favorisent l’émergence des progéniteurs lymphoïdes précoces (ELP) CD127- ou CD127+ à partir de leurs ancêtres lympho-mono-dendritiques (LMDP) communs. Les analyses cinétiques montrent que les ELP CD127- ou CD127+ émergent de manière séquentielle des LMDP "précoces" par rapport aux LMDP "tardifs". Le criblage fonctionnel d’une série de 30 récepteur/ligand montre en outre que les ELP CD127- ou CD127+ sont soumis à une régulation différentielle. Alors que les signaux Flt3L, IL27 et Notch1 bloquent l’émergence des ELP CD127+ et polarisent la différenciation lymphoïde vers la voie exclusive CD127-, l’émergence des ELP CD127+ est au contraire favorisée par des conditions de limitation cytokinique et de faibles taux d’expansion cellulaire. Ces résultats indiquent que les ELP CD127- et CD127+ sont soumises à des mécanismes de régulation distincts, selon qu’ils soient extrinsèques ou intrinsèques aux cellules, renforçant ainsi l’architecture dichotomique de la lymphopoïèse humaine proposée précédemment. Ces résultats plaident également en faveur d’une régulation multicouche de la différenciation lympho-myéloïde, remettant en question le modèle continu émergeant de l’hématopoïèse.

Human lymphopoiesis remains poorly characterized compared to its murine counterpart. This is due to the lack of adequate experimental models and difficulties in accessing primary bone marrow samples. Thanks to the fine characterization of the populations of lymphoid progenitors and precursors in the bone marrow of transplanted mice, we have recently demonstrated that human lymphopoiesis proceeds along independent CD127- or CD127+ pathways that generate both common and specific lymphoid subsets. In this report, we developed a novel ex vivo diversification assay to analyze their developmental relationships and investigate the mechanisms driving the emergence of the CD127- or CD127+ early lymphoid progenitors (ELPs) from their common lympho-mono-dendritic (LMDP) ancestors. Time-course analyses show that CD127- or CD127+ ELPs emerge sequentially from “early” versus “late” LMDPs. Functional screening of a series of 30 receptor/ligand pairs show in addition that the CD127- or CD127+ ELPs are subjected to a differential regulation. Whereas Flt3L, IL-27 and Notch1 signaling impose a veto on CD127+ ELP emergence and polarize lymphoid differentiation toward exclusive CD127- pathway, the emergence of the CD127+ ELPs is instead favored by limiting cytokine conditions and low cell expansion rates. These new findings indicate that CD127- and CD127+ ELPs are subjected to distinct cell-extrinsic versus cell-intrinsic regulatory mechanisms thereby reinforcing the previously proposed dichotomous architecture of human lymphopoiesis. In as much as they also argue for multi-layered regulation of lympho-myeloid differentiation, our new results challenge the emerging continuous model of hematopoiesis., La lymphopoïèse humaine reste mal caractérisée comparativement à son homologue murin. Ceci est dû à l’absence de modèles expérimentaux adéquats et aux difficultés d’accès aux prélèvements primaires de moelle osseuse humaine. Grâce à la caractérisation fine des populations de progéniteurs et précurseurs lymphoïdes présentes dans la moelle osseuse d’animaux greffés, nous avons récemment démontré que la lymphopoïèse humaine suit des voies CD127- ou CD127+ indépendantes qui génèrent une descendance lymphoïde commune et spécifique. Dans cette étude, nous avons mis au point un nouveau test de diversification ex vivo pour analyser leurs relations développementales et étudier les mécanismes qui favorisent l’émergence des progéniteurs lymphoïdes précoces (ELP) CD127- ou CD127+ à partir de leurs ancêtres lympho-mono-dendritiques (LMDP) communs. Les analyses cinétiques montrent que les ELP CD127- ou CD127+ émergent de manière séquentielle des LMDP "précoces" par rapport aux LMDP "tardifs". Le criblage fonctionnel d’une série de 30 récepteur/ligand montre en outre que les ELP CD127- ou CD127+ sont soumis à une régulation différentielle. Alors que les signaux Flt3L, IL27 et Notch1 bloquent l’émergence des ELP CD127+ et polarisent la différenciation lymphoïde vers la voie exclusive CD127-, l’émergence des ELP CD127+ est au contraire favorisée par des conditions de limitation cytokinique et de faibles taux d’expansion cellulaire. Ces résultats indiquent que les ELP CD127- et CD127+ sont soumises à des mécanismes de régulation distincts, selon qu’ils soient extrinsèques ou intrinsèques aux cellules, renforçant ainsi l’architecture dichotomique de la lymphopoïèse humaine proposée précédemment. Ces résultats plaident également en faveur d’une régulation multicouche de la différenciation lympho-myéloïde, remettant en question le modèle continu émergeant de l’hématopoïèse.

Existing animal models for the study of human leukemia are not accurate for the proper development of innovative, targeted therapies. The aim of this project, which contains both a fundamental and an industrial perspective, therefore was to develop a new, versatile model of human leukemogenesis in the BRGS (BALB/c Rag2-/- IL-2Rγc-/- SIRPα.NOD) humanized mouse. Animals are grafted with hematopoietic progenitors transduced with lentivirals vectors to allow overexpression of MYC and BCL2 proteins under the control of an ubiquitous promotor (EF1α or SFFV). Longitudinal monitoring of the animals over five months shows that only the SFFV/Myc-T2A-Bcl2 construction induces the transformation of humans hematopoietics progenitors. Between 12 and 14 weeks post-transplantation, more than 90% of the animals develop pro-B lympho-proliferations (CD19+CD10+CD9+CD20-cytIgM-), with tumor cells being mainly found in the spleen, the bone marrow and in blood. Tumor transferability is achievable through secondary transplantation in immunodeficient mouse recipients. In vitro culture of bone marrow T cell progenitors suggest that the blasts arise from these cells after reactivation of a latent B cell program with blockade of their T cell development. In parallel, we have also developed an autologous tumor model. Altogether, these results validate the human leukemogenesis model constructed here in humanized BRGS mice and provide attractive prospects regarding the functional characterization of leukemogenesis and a preclinical validation of new anti-tumor strategies., Les modèles animaux actuellement disponibles pour l’étude des leucémies humaines ne sont pas adaptés pour le développement optimal de nouvelles thérapies ciblées. Au cours de ce projet, qui s’inscrit dans une double perspective fondamentale et industrielle, nous avons cherché à générer un modèle versatile de leucémogénèse humaine chez la souris humanisée BRGS (BALB/c Rag2-/- IL-2Rγc-/- SIRPα.NOD). Les animaux sont greffés avec des progéniteurs hématopoïétiques transduits par des vecteurs lentiviraux surexprimant les oncogènes MYC et BCL2 et placés sous le contrôle d’un promoteur ubiquitaire (EF1α ou SFFV). Le suivi longitudinal des animaux sur une période de 5 mois montre que seule la construction SFFV/Myc-T2A-Bcl2 entraîne la transformation des progéniteurs hématopoïétiques. Entre 12 à 14 semaines post-greffe, >90% des animaux développent des lympho-proliférations de type pro-B (CD19+CD10+CD9+CD20-cytIgM-) infiltrant principalement la rate et la moelle osseuse, et circulant en abondance dans le sang. Le caractère transmissible des tumeurs est validé par des greffes secondaires de tumeurs spléniques. Les cultures in vitro de progéniteurs hématopoïétiques suggèrent que l’émergence des blastes est liée à la réactivation d’un programme B latent dans les précurseurs T, dont le développement est bloqué. Nous avons développé en parallèle un modèle de tumeur autologue. L’ensemble de ces résultats valide le modèle de leucémogénèse humaine développé chez la souris humanisée BRGS et ouvre des perspectives pour la caractérisation fonctionnelle des mécanismes de leucémogénèse, et la validation préclinique de nouvelles stratégies anti-tumorales.