Co-Transplantation of Barcoded Lymphoid-Primed Multipotent (LMPP) and Common Lymphocyte (CLP) Progenitors Reveals a Major Contribution of LMPP to the Lymphoid Lineage.

T cells have the potential to maintain immunological memory and self-tolerance by recognizing antigens from pathogens or tumors. In pathological situations, failure to generate de novo T cells causes immunodeficiency resulting in acute infections and complications. Hematopoietic stem cells (HSC) transplantation constitutes a valuable option to restore proper immune function. However, delayed T cell reconstitution is observed compared to other lineages. To overcome this difficulty, we developed a new approach to identify populations with efficient lymphoid reconstitution properties. To this end, we use a DNA barcoding strategy based on the insertion into a cell chromosome of a lentivirus (LV) carrying a non-coding DNA fragment named barcode (BC). These will segregate through cell divisions and be present in cells' progeny. The remarkable characteristic of the method is that different cell types can be tracked simultaneously in the same mouse. Thus, we in vivo barcoded LMPP and CLP progenitors to test their ability to reconstitute the lymphoid lineage. Barcoded progenitors were co-grafted in immuno-compromised mice and their fate analyzed by evaluating the BC composition in transplanted mice. The results highlight the predominant role of LMPP progenitors for lymphoid generation and reveal valuable novel insights to be reconsidered in clinical transplantation assays.

Temporal Gene Expression Profiles Reflect the Dynamics of Lymphoid Differentiation.

Understanding the emergence of lymphoid committed cells from multipotent progenitors (MPP) is a great challenge in hematopoiesis. To gain deeper insight into the dynamic expression changes associated with these transitions, we report the quantitative transcriptome of two MPP subsets and the common lymphoid progenitor (CLP). While the transcriptome is rather stable between MPP2 and MPP3, expression changes increase with differentiation. Among those, we found that pioneer lymphoid genes such as Rag1, Mpeg1, and Dntt are expressed continuously from MPP2. Others, such as CD93, are CLP specific, suggesting their potential use as new markers to improve purification of lymphoid populations. Notably, a six-transcription factor network orchestrates the lymphoid differentiation program. Additionally, we pinpointed 24 long intergenic-non-coding RNA (lincRNA) differentially expressed through commitment and further identified seven novel forms. Collectively, our approach provides a comprehensive landscape of coding and non-coding transcriptomes expressed during lymphoid commitment.

Treatment of ongoing autoimmune encephalomyelitis with activated B-cell progenitors maturing into regulatory B cells.

The influence of signals perceived by immature B cells during their development in bone marrow on their subsequent functions as mature cells are poorly defined. Here, we show that bone marrow cells transiently stimulated in vivo or in vitro through the Toll-like receptor 9 generate proB cells (CpG-proBs) that interrupt experimental autoimmune encephalomyelitis (EAE) when transferred at the onset of clinical symptoms. Protection requires differentiation of CpG-proBs into mature B cells that home to reactive lymph nodes, where they trap T cells by releasing the CCR7 ligand, CCL19, and to inflamed central nervous system, where they locally limit immunopathogenesis through interleukin-10 production, thereby cooperatively inhibiting ongoing EAE. These data demonstrate that a transient inflammation at the environment, where proB cells develop, is sufficient to confer regulatory functions onto their mature B-cell progeny. In addition, these properties of CpG-proBs open interesting perspectives for cell therapy of autoimmune diseases.

Lymphoid Gene Upregulation on Circulating Progenitors Participates in Their T-Lineage Commitment.

Extrathymic T cell precursors can be detected in many tissues and represent an immediately competent population for rapid T cell reconstitution in the event of immunodeficiencies. Blood T cell progenitors have been detected, but their source in the bone marrow (BM) remains unclear. Prospective purification of BM-resident and circulating progenitors, together with RT-PCR single-cell analysis, was used to evaluate and compare multipotent progenitors (MPPs) and common lymphoid progenitors (CLPs). Molecular analysis of circulating progenitors in comparison with BM-resident progenitors revealed that CCR9(+) progenitors are more abundant in the blood than CCR7(+) progenitors. Second, although Flt3(-) CLPs are less common in the BM, they are abundant in the blood and have reduced Cd25(+)-expressing cells and downregulated c-Kit and IL-7Rα intensities. Third, in contrast, stage 3 MPP (MPP3) cells, the unique circulating MPP subset, have upregulated Il7r, Gata3, and Notch1 in comparison with BM-resident counterparts. Evaluation of the populations' respective abilities to generate splenic T cell precursors (Lin(-)Thy1.2(+)CD25(+)IL7Rα(+)) after grafting recipient nude mice revealed that MPP3 cells were the most effective subset (relative to CLPs). Although several lymphoid genes are expressed by MPP3 cells and Flt3(-) CLPs, the latter only give rise to B cells in the spleen, and Notch1 expression level is not modulated in the blood, as for MPP3 cells. We conclude that CLPs have reached the point where they cannot be a Notch1 target, a limiting condition on the path to T cell engagement.

CXCR4-related increase of circulating human lymphoid progenitors after allogeneic hematopoietic stem cell transplantation.

Immune recovery after profound lymphopenia is a major challenge in many clinical situations, such as allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recovery depends, in a first step, on hematopoietic lymphoid progenitors production in the bone marrow (BM). In this study, we characterized CD34+Lin-CD10+ lymphoid progenitors in the peripheral blood of allo-HSCT patients. Our data demonstrate a strong recovery of this population 3 months after transplantation. This rebound was abolished in patients who developed acute graft-versus-host disease (aGVHD). A similar recovery profile was found for both CD24+ and CD24- progenitor subpopulations. CD34+lin-CD10+CD24- lymphoid progenitors sorted from allo-HSCT patients preserved their T cell potentiel according to in vitro T-cell differentiation assay and the expression profile of 22 genes involved in T-cell differentiation and homing. CD34+lin-CD10+CD24- cells from patients without aGVHD had reduced CXCR4 gene expression, consistent with an enhanced egress from the BM. CCR7 gene expression was reduced in patients after allo-HSCT, as were its ligands CCL21 and CCL19. This reduction was particularly marked in patients with aGVHD, suggesting a possible impact on thymic homing. Thus, the data presented here identify this population as an important early step in T cell reconstitution in humans and so, an important target when seeking to enhance immune reconstitution.

Innate pro-B-cell progenitors protect against type 1 diabetes by regulating autoimmune effector T cells.

Diverse hematopoietic progenitors, including myeloid populations arising in inflammatory and tumoral conditions and multipotent cells, mobilized by hematopoietic growth factors or emerging during parasitic infections, display tolerogenic properties. Innate immune stimuli confer regulatory functions to various mature B-cell subsets but immature B-cell progenitors endowed with suppressive properties per se or after differentiating into more mature regulatory B cells remain to be characterized. Herein we provide evidence for innate pro-B cells (CpG-proBs) that emerged within the bone marrow both in vitro and in vivo upon Toll-like receptor-9 activation and whose adoptive transfer protected nonobese diabetic mice against type 1 diabetes (T1D). These cells responded to IFN-γ released by activated effector T cells (Teffs), by up-regulating their Fas ligand (FasL) expression, which enabled them to kill Teffs through apoptosis. In turn, IFN-γ derived from CpG-proBs enhanced IFN-γ while dramatically reducing IL-21 production by Teffs. In keeping with the crucial pathogenic role played by IL-21 in T1D, adoptively transferred IFN-γ-deficient CpG-proBs did not prevent T1D development. Additionally, CpG-proBs matured in vivo into diverse pancreatic and splenic suppressive FasL(high) B-cell subsets. CpG-proBs may become instrumental in cell therapy of autoimmune diseases either on their own or as graft complement in autologous stem cell transplantation.

Thymocytes may persist and differentiate without any input from bone marrow progenitors.

Thymus transplants can correct deficiencies of the thymus epithelium caused by the complete DiGeorge syndrome or FOXN1 mutations. However, thymus transplants were never used to correct T cell-intrinsic deficiencies because it is generally believed that thymocytes have short intrinsic lifespans. This notion is based on thymus transplantation experiments where it was shown that thymus-resident cells were rapidly replaced by progenitors originating in the bone marrow. In contrast, here we show that neonatal thymi transplanted into interleukin 7 receptor-deficient hosts harbor populations with extensive capacity to self-renew, and maintain continuous thymocyte generation and export. These thymus transplants reconstitute the full diversity of peripheral T cell repertoires one month after surgery, which is the earliest time point studied. Moreover, transplantation experiments performed across major histocompatibility barriers show that allogeneic transplanted thymi are not rejected, and allogeneic cells do not induce graft-versus-host disease; transplants induced partial or total protection to infection. These results challenge the current dogma that thymocytes cannot self-renew, and indicate a potential use of neonatal thymus transplants to correct T cell-intrinsic deficiencies. Finally, as found with mature T cells, they show that thymocyte survival is determined by the competition between incoming progenitors and resident cells.

Gene coexpression analysis in single cells indicates lymphomyeloid copriming in short-term hematopoietic stem cells and multipotent progenitors.

Progressive restriction to a differentiation pathway results from both activation and silencing of particular gene expression programs. To identify the coexpression and the expression levels of regulatory genes during hematopoietic stem cell (HSC) differentiation toward the T cell branch, we applied a new single-cell RT-PCR technique to analyze the simultaneous expression of 13 genes in 9 functionally purified populations from the bone marrow and the thymus. We report in this paper that Lin(-)Sca1(+)ckit(+) HSCs display, at the single-cell level, a homogeneous and high transcriptional activity as do early thymic progenitors. Moreover, the coexpression of lymphoid and myeloid genes is an early event detected in approximately 30% of short-term HSC and most multipotent progenitors, suggesting novel sources for the generation of early thymic progenitors, common lymphoid progenitors (CLPs), and common myeloid progenitors. Loss of multipotency in Lin(-)Sca1(+)ckit(+) cells directed to the lymphoid branch is characterized by Lmo2 and Gata2 gene expression downregulation. Indeed, highest levels of Gata2 expression are detected only in long-term and short-term HSC populations. Complete shutdown of Pu1 gene expression in all triple-negative (TN)3 stage thymic pre-T cells is indicative of total T cell commitment. Interestingly, this is also observed in 30% of TN2 cells and 25% of CLP in the bone marrow, suggesting a possible initiation of T cell engagement in TN2 and CLP. Also, our strategy highlights similar gene patterns among HSCs and intrathymic progenitors, proposing, therefore, that identical activation signals are maintained until further maturation and generation of CD4 and CD8 coreceptors bearing thymocytes.

Human and murine amniotic fluid c-Kit+Lin- cells display hematopoietic activity.

We have isolated c-Kit(+)Lin(-) cells from both human and murine amniotic fluid (AF) and investigated their hematopoietic potential. In vitro, the c-Kit(+)Lin(-) population in both species displayed a multilineage hematopoietic potential, as demonstrated by the generation of erythroid, myeloid, and lymphoid cells. In vivo, cells belonging to all 3 hematopoietic lineages were found after primary and secondary transplantation of murine c-Kit(+)Lin(-) cells into immunocompromised hosts, thus demonstrating the ability of these cells to self-renew. Gene expression analysis of c-Kit(+) cells isolated from murine AF confirmed these results. The presence of cells with similar characteristics in the surrounding amnion indicates the possible origin of AF c-Kit(+)Lin(-) cells. This is the first report showing that cells isolated from the AF do have hematopoietic potential; our results support the idea that AF may be a new source of stem cells for therapeutic applications.

Hierarchy of Notch-Delta interactions promoting T cell lineage commitment and maturation.

Notch1 (N1) receptor signaling is essential and sufficient for T cell development, and recently developed in vitro culture systems point to members of the Delta family as being the physiological N1 ligands. We explored the ability of Delta1 (DL1) and DL4 to induce T cell lineage commitment and/or maturation in vitro and in vivo from bone marrow (BM) precursors conditionally gene targeted for N1 and/or N2. In vitro DL1 can trigger T cell lineage commitment via either N1 or N2. N1- or N2-mediated T cell lineage commitment can also occur in the spleen after short-term BM transplantation. However, N2-DL1-mediated signaling does not allow further T cell maturation beyond the CD25(+) stage due to a lack of T cell receptor beta expression. In contrast to DL1, DL4 induces and supports T cell commitment and maturation in vitro and in vivo exclusively via specific interaction with N1. Moreover, comparative binding studies show preferential interaction of DL4 with N1, whereas binding of DL1 to N1 is weak. Interestingly, preferential N1-DL4 binding reflects reduced dependence of this interaction on Lunatic fringe, a glycosyl transferase that generally enhances the avidity of Notch receptors for Delta ligands. Collectively, our results establish a hierarchy of Notch-Delta interactions in which N1-DL4 exhibits the greatest capacity to induce and support T cell development.

A thymic pathway of mouse natural killer cell development characterized by expression of GATA-3 and CD127.

Natural killer (NK) cell development is thought to occur in the bone marrow. Here we identify the transcription factor GATA-3 and CD127 (IL-7R alpha) as molecular markers of a pathway of mouse NK cell development that originates in the thymus. Thymus-derived CD127+ NK cells repopulated peripheral lymphoid organs, and their homeostasis was strictly dependent on GATA-3 and interleukin 7. The CD127+ NK cells had a distinct phenotype (CD11b(lo) CD16- CD69(hi) Ly49(lo)) and unusual functional attributes, including reduced cytotoxicity but considerable cytokine production. Those characteristics are reminiscent of human CD56(hi) CD16- NK cells, which we found expressed CD127 and had more GATA-3 expression than human CD56+ CD16+ NK cells. We propose that bone marrow and thymic NK cell pathways generate distinct mouse NK cells with properties similar to those of the two human CD56 NK cell subsets.

The thymus exports long-lived fully committed T cell precursors that can colonize primary lymphoid organs.

Thymic export of cells is believed to be restricted to mature T cells. Here we show that the thymus also exports fully committed T cell precursors that colonize primary lymphoid organs. These precursor cells exited the thymus before T cell receptor rearrangements and colonized lymphoid organs such as the thymus and the gut. Migration of the thymic T cell-committed precursors led to permanent colonization of the gut precursor compartment, improved the capacity of gut precursors to further differentiate into T cells and was sufficient for the generation of 'euthymic like' CD8alphaalpha(+) intraepithelial lymphocytes. These data demonstrate a new function for the thymus in peripheral seeding with T cell precursors that become long lived after thymus export.

Neoplastic transformation and angiogenesis in the thymus of transgenic mice expressing SV40 T and t antigen under an L-pyruvate kinase promoter (SV12 mice).

Using several techniques, we have assessed morphological characteristics of a malignant thymic tumour in SV12 transgenic (Tg) mice expressing SV40 T and t antigens under control of an L-PK promoter. We describe the development of a carcinoma originating from thymic hyperplasia and followed by the formation of a benign tumour composed chiefly of medullary epithelial cells expressing the transgene and of lymphocytes, a pathology very rarely reported in mice. Our study of the SV12 Tg mice represents the first description of a model of a pure malignant thymic tumour associated with extensive angiogenesis maintained in numerous descendants. The formation of a large tumoral neovascular network, observed here, has never been described in human and/or experimental thymic tumours. Tumoral transformation and angiogenesis are demonstrated by immunolabelling with antibodies against various cytokeratins (CKs) of different molecular weights, vascular endothelial cell markers and VEGF/receptor-2 (Flk-1) present on the neovascular endothelial cells. Different points raised by the originality of this model are discussed. These include the medullary nature of the cells expressing the SV40 transgene and their relationship with the tumoral development. The subset of different molecular weight CK components and their modifications are also considered, as well as the presence of type IV epithelial cells, progenitors of medullary epithelial cells. Finally, the cell signals involved in angiogenesis and the possible action of an angiogenic factor, probably secreted by the tumoral cells themselves, are discussed.

Fas receptor signaling is requisite for B cell differentiation.

The Fas/Fas ligand (FasL) pathway has been largely implicated in the homeostasis of mature cells. However, it is still unclear whether it plays a role at the progenitor level. To address this issue, we created chimeric mice by transferring C57BL/6 bone marrow (BM) cells of the lpr (Fas-FasL+) or gld (Fas+FasL-) genotype into Rag-2-/- hosts of the same genetic background. In this model, the consequences of a deficient Fas/FasL pathway on lymphoid differentiation could be evaluated without endogenous competition. Analysis of the chimerism revealed a differential sensitivity of hematopoietic lineages to the lack of Fas receptor signaling. While donor-derived myelo-monocytic cells were similarly distributed in all chimeric mice, mature B cells were deleted in the BM and the spleen of lpr chimera, leading to the absence of the marginal zone (MZ) as detected by immunohistology. In contrast, B cell hematopoiesis was complete in gld chimera but MZ macrophages undetectable. These defects suggest a direct and determinant dual role of FasL regulation in negative selection of B cells and in maintenance of the MZ.

Extrathymic hemopoietic progenitors committed to T cell differentiation in the adult mouse.

The role of the thymus in T cell commitment of hemopoietic precursor is yet controversial. We previously identified a major T cell progenitor activity in precursor cells isolated from bone marrow-derived spleen colonies. In this study, we characterize the properties of these pre-T cells. We demonstrate that they have unique phenotype and can be generated in a total absence of any thymic influence. Indeed, even when studied at the single-cell level, extrathymic T cell-committed precursors express T cell-specific genes. Moreover, these cells are not committed to a particular T cell differentiation pathway because they can generate both extrathymic CD8alphaalpha+ intraepithelial lymphocytes and thymus-derived conventional thymocytes. We also compared these pre-T cells with fully T cell-committed thymic progenitors. When tested in vitro or by direct intrathymic transfer, these cells have a low clonogenic activity. However, after i.v. transfer, thymus repopulation is efficient and these precursors generate very high numbers of peripheral T cells. These results suggest the existence of extra steps of pre-T cell maturation that improve thymus reconstitution capacity and that can be delivered even after full T cell commitment. Consequently, our studies identify a source of extrathymic progenitors that will be helpful in defining the role of the thymus in the earliest steps of T cell differentiation.

Listeria monocytogenes-infected bone marrow myeloid cells promote bacterial invasion of the central nervous system.

Listeria monocytogenes is a facultative intracellular pathogen that is able to invade the central nervous system causing meningoencephalitis and brain abscesses. The mechanisms allowing bacteria to cross the blood-brain barrier are poorly understood. In this work, we used an experimental model of acute listeriosis in the mouse inducing a reproducible invasion of the central nervous system. At the early phase of infection, we find that bacteria invade and rapidly grow in bone marrow cells identified as bone marrow myelomonocytic cells expressing the phenotype CD31pos:Ly-6Cpos:CD11b(pos):LY-6Glow. We demonstrate that central nervous system invasion is facilitated by injecting L. monocytogenes-infected bone marrow cells in comparison with free bacteria or infected spleen cells. In mice transplanted with bone marrow cells from transgenic donor mice expressing the green fluorescent protein (GFP), we show that infected myeloid GFP+ cells adhere to activated brain endothelial cells, accumulate in brain vessels and participate to the pathogenesis of meningoencephalitis and brain abscesses. Our results demonstrate that bone marrow, the main haematopoietic tissue, is a previously unrecognized reservoir of L. monocytogenes-infected myeloid cells, which can play a crucial role in the pathophysiology of meningoencephalitis by releasing infected cells into the circulation that ultimately invade the central nervous system.

Major T cell progenitor activity in bone marrow-derived spleen colonies.

Common lymphoid progenitors (CLP) are generated in adult bone marrow (BM), but the intermediate steps leading to T cell commitment are unknown, and so is the site at which this commitment occurs. Here, we show that colonies arising in the spleen 12 days after BM injection harbor T cell precursors that are undetectable in BM. These precursors did not generate myeloid cells in vivo but repopulated the thymus and the peripheral T cell compartment much faster than did CLP. Two lineage negative (Lin(-)) subpopulations were distinguished, namely CD44(+) Thy1(-) cells still capable of natural killer generation and transient low-level B cell generation, and T cell-restricted CD44(-) Thy1(+) cells. At a molecular level, frequency of CD3epsilon and preTalpha mRNA was very different in each subset. Furthermore, only the CD44(-) Thy1(+) subset have initiated rearrangements in the T cell receptor beta locus. Thus, this study identifies extramedullary T cell progenitors and will allow easy approach to T cell commitment studies.

G-CSF therapy of ongoing experimental allergic encephalomyelitis via chemokine- and cytokine-based immune deviation.

Converging evidence that G-CSF, the hemopoietic growth factor of the myeloid lineage, also exerts anti-inflammatory and pro-Th2 effects, prompted us to evaluate its direct therapeutic potential in autoimmune diseases. Here we report a novel activity of G-CSF in experimental allergic encephalomyelitis, a murine model for multiple sclerosis, driven by Th1-oriented autoaggressive cells. A short 7-day treatment with G-CSF, initiated at the onset of clinical signs, provided durable protection from experimental autoimmune encephalomyelitis. G-CSF-treated mice displayed limited demyelination, reduced recruitment of T cells to the CNS, and very discrete autoimmune inflammation, as well as barely detectable CNS mRNA levels of cytokines and chemokines. In the periphery, G-CSF treatment triggered an imbalance in the production by macrophages as well as autoreactive splenocytes of macrophage inflammatory protein-1alpha and monocyte chemoattractant protein-1, the prototypical pro-Th1 and pro-Th2 CC chemokines, respectively. This chemokine imbalance was associated with an immune deviation of the autoreactive response, with reduced IFN-gamma and increased IL-4 and TGF-beta1 levels. Moreover, G-CSF limited the production of TNF-alpha, a cytokine also associated with early CNS infiltration and neurological deficit. These findings support the potential application of G-CSF in the treatment of human autoimmune diseases such as multiple sclerosis, taking advantage of the wide clinical favorable experience with this molecule.

Characterization of T cell differentiation in the murine gut.

Gut intraepithelial CD8 T lymphocytes (T-IEL) are distinct from thymus-derived cells and are thought to derive locally from cryptopatch (CP) precursors. The intermediate stages of differentiation between CP and mature T-IEL were not identified, and the local differentiation process was not characterized. We identified and characterized six phenotypically distinct lineage-negative populations in the CP and the gut epithelium: (a) we determined the kinetics of their generation from bone marrow precursors; (b) we quantified CD3-epsilon, recombination activating gene (Rag)-1, and pre-Talpha mRNAs expression at single cell level; (c) we characterized TCR-beta, -gamma, and -alpha locus rearrangements; and (d) we studied the impact of different mutations on the local differentiation. These data allowed us to establish a sequence of T cell precursor differentiation in the gut. We also observed that the gut differentiation varied from that of the thymus by a very low frequency of pre-Talpha chain mRNA expression, a different kinetics of Rag-1 mRNA expression, and a much higher impact of CD3 epsilon/delta and pre-Talpha deficiencies. Finally, only 3% of CP cells were clearly involved in T cell differentiation, suggesting that these structures may have additional physiological roles in the gut.