Stéphane J. C. Mancini

Notch regulation of lymphocyte development and function

Notch proteins regulate a broad spectrum of cell fate decisions and differentiation processes during fetal and postnatal development. Mammals have four Notch receptors that bind five different ligands. The function of Notch signaling during lymphopoiesis and T cell neoplasia, based on gain-of-function and conditional loss-of-function approaches for the Notch1 receptor, indicates Notch1 is essential in T cell lineage commitment. Recent studies have addressed the involvement of other Notch receptors and ligands as well as their downstream targets, demonstrating additional functions of Notch signaling in embryonic hematopoiesis, intrathymic T cell development, B cell development and peripheral T cell function.

β-Catenin Is Dispensable for Hematopoiesis and Lymphopoiesis

β-catenin–mediated Wnt signaling has been suggested to be critically involved in hematopoietic stem cell maintenance and development of T and B cells in the immune system. Unexpectedly, here we report that inducible Cre-loxP–mediated inactivation of the β-catenin gene in bone marrow progenitors does not impair their ability to self-renew and reconstitute all hematopoietic lineages (myeloid, erythroid, and lymphoid), even in competitive mixed chimeras. In addition, both thymocyte survival and antigen-induced proliferation of peripheral T cells is β-catenin independent. In contrast to earlier reports, these data exclude an essential role for β-catenin during hematopoiesis and lymphopoiesis.

Osteoclast activity modulates B-cell development in the bone marrow

B-cell development is dependent on the interactions between B-cell precursors and bone marrow stromal cells, but the role of osteoclasts (OCLs) in this process remains unknown. B lymphocytopenia is a characteristic of osteopetrosis, suggesting a modulation of B lymphopoiesis by OCL activity. To address this question, we first rescued OCL function in osteopetrotic oc/oc mice by dendritic cell transfer, leading to a restoration of both bone phenotype and B-cell development. To further explore the link between OCL activity and B lymphopoiesis, we induced osteopetrosis in normal mice by injections of zoledronic acid (ZA), an inhibitor of bone resorption. B-cell number decreased specifically in the bone marrow of ZA-treated mice. ZA did not directly affect B-cell differentiation, proliferation and apoptosis, but induced a decrease in the expression of CXCL12 and IL-7 by stromal cells, associated with reduced osteoblastic engagement. Equivalent low osteoblastic engagement in oc/oc mice confirmed that it resulted from the reduced OCL activity rather than from a direct effect of ZA on osteoblasts. These dramatic alterations of the bone microenvironment were disadvantageous for B lymphopoiesis, leading to retention of B-cell progenitors outside of their bone marrow niches in the ZA-induced osteopetrotic model. Altogether, our data revealed that OCLs modulate B-cell development in the bone marrow by controlling the bone microenvironment and the fate of osteoblasts. They provide novel basis for the regulation of the retention of B cells in their niche by OCL activity.

Impaired B-cell development at the pre-BII-cell stage in galectin-1-deficient mice due to inefficient pre-BII/stromal cell interactions.

Activation of the pre-B-cell receptor (pre-BCR) in the bone marrow depends on both tonic and ligand-induced signaling and leads to pre-BII-cell proliferation and differentiation. Using normal mouse bone marrow pre-BII cells, we demonstrate that the ligand-induced pre-BCR activation depends on pre-BCR/galectin-1/integrin interactions leading to pre-BCR clustering at the pre-BII/stromal cell synapse. In contrast, heparan sulfates, shown to be pre-BCR ligands in mice, are not implicated in pre-BCR relocalization. Inhibition of pre-BCR/galectin-1/integrin interactions has functional consequences, since pre-BII-cell proliferation and differentiation are impaired in an in vitro B-cell differentiation assay, without affecting cellular apoptosis. Most strikingly, although galectin-1-deficient mice do not show an apparent B-cell phenotype, the kinetics of de novo B-cell reconstitution after hydroxyurea treatment indicates a specific delay in pre-BII-cell recovery due to a decrease in pre-BII-cell differentiation and proliferation. Thus, although it remains possible that the pre-BCR interacts with other ligands, these results highlight the role played by the stromal cell-derived galectin-1 for the efficient development of normal pre-BII cells and suggest the existence of pre-BII-specific stromal cell niches in normal bone marrow.

Galectin-1 expressing stromal cells constitute a specific niche for pre-BII cell development in mouse bone marrow

In the bone marrow (BM), stromal cells constitute a supportive tissue indispensable for the generation of pro-B/pre-BI, pre-BII, and immature B lymphocytes. IL-7-producing stromal cells constitute a cellular niche for pro-B/pre-BI cells, but no specific stromal cell microenvironment was identified for pre-BII cells expressing a functional pre-B cell receptor (pre-BCR). However expression of the pre-BCR represents a crucial checkpoint during B-cell development. We recently demonstrated that the stromal cell derived-galectin1 (GAL1) is a ligand for the pre-BCR, involved in the proliferation and differentiation of normal mouse pre-BII cells. Here we show that nonhematopoietic osteoblasts and reticular cells in the BM express GAL1. We observed that pre-BII cells, unlike the other B-cell subsets, were specifically localized in close contact with GAL1(+) reticular cells. We also determined that IL-7(+) and GAL1(+) cells represent 2 distinct mesenchymal populations with different BM localization. These results demonstrate the existence of a pre-BII specific stromal cell niche and indicate that early B cells move from IL-7(+) to GAL1(+) supportive BM niches during their development.

Germinal center reentries of BCL2-overexpressing B cells drive follicular lymphoma progression

It has recently been demonstrated that memory B cells can reenter and reengage germinal center (GC) reactions, opening the possibility that multi-hit lymphomagenesis gradually occurs throughout life during successive immunological challenges. Here, we investigated this scenario in follicular lymphoma (FL), an indolent GC-derived malignancy. We developed a mouse model that recapitulates the FL hallmark t(14;18) translocation, which results in constitutive activation of antiapoptotic protein B cell lymphoma 2 (BCL2) in a subset of B cells, and applied a combination of molecular and immunofluorescence approaches to track normal and t(14;18)(+) memory B cells in human and BCL2-overexpressing B cells in murine lymphoid tissues. BCL2-overexpressing B cells required multiple GC transits before acquiring FL-associated developmental arrest and presenting as GC B cells with constitutive activation-induced cytidine deaminase (AID) mutator activity. Moreover, multiple reentries into the GC were necessary for the progression to advanced precursor stages of FL. Together, our results demonstrate that protracted subversion of immune dynamics contributes to early dissemination and progression of t(14;18)(+) precursors and shapes the systemic presentation of FL patients.

JAM-B regulates maintenance of hematopoietic stem cells in the bone marrow

In adult mammals, hematopoietic stem cells (HSCs) reside in the bone marrow (BM) and are maintained in a quiescent and undifferentiated state through adhesive interactions with specialized microenvironmental niches. Although junctional adhesion molecule-C (JAM-C) is expressed by HSCs, its function in adult hematopoiesis remains elusive. Here, we show that HSCs adhere to JAM-B expressed by BM stromal cells in a JAM-C dependent manner. The interaction regulates the interplay between HSCs and BM stromal cells as illustrated by the decreased pool of quiescent HSCs observed in jam-b deficient mice. We further show that this is probably because of alterations of BM stromal compartments and changes in SDF-1α BM content in jam-b(-/-) mice, suggesting that JAM-B is an active player in the maintenance of the BM stromal microenvironment.

TCRγ Silencing during αβ T Cell Development Depends upon Pre-TCR-Induced Proliferation

During thymus development, immature T cells become committed to two distinct lineages based upon expression of αβ or γδ TCR. In the αβ lineage, developing thymocytes progressively extinguish transcription of the TCRγ genes by a poorly understood process known as γ silencing. We show that αβ lineage thymocytes in mice lacking a functional pre-TCR undergo limited proliferation and fail to silence TCRγ genes during development. Stimulation of pre-TCR-deficient immature thymocytes with anti-CD3 Abs does not directly down-regulate TCRγ transcription but restores TCRγ silencing following proliferation. Collectively our data reveal an important role for pre-TCR induced proliferation in activating the TCRγ silencer in αβ lineage thymocytes, a process that may reinforce αβ or γδ lineage commitment.

Galectin 1 Modulates Plasma Cell Homeostasis and Regulates the Humoral Immune Response

Galectin-1 (GAL1) is an S-type lectin with multiple functions, including the control of B cell homeostasis. GAL1 expression was reported to be under the control of the plasma cell master regulator BLIMP-1. GAL1 was detected at the protein level in LPS-stimulated B cells and was shown to promote Ig secretion in vitro. However, the pattern of GAL1 expression and function of GAL1 in B cells in vivo are still unclear. In this study, we show that, among B cells, GAL1 is only expressed by differentiating plasma cells following T-dependent or T-independent immunization. Using GAL1-deficient mice we demonstrate that GAL1 expression is required for the maintenance of Ag-specific Ig titers and Ab-secreting cell numbers. Using an in vitro differentiation assay we find that GAL1-deficient plasmablasts can develop normally but die rapidly, through caspase 8 activation, under serum starvation–induced death conditions. TUNEL assays show that in vivo–generated GAL1-deficient plasma cells exhibit an increased sensitivity to apoptosis. Taken together, our data indicate that endogenous GAL1 supports plasma cell survival and participates in the regulation of the humoral immune response.

Adhesion receptors involved in HSC and early-B cell interactions with bone marrow microenvironment

Hematopoiesis takes place in the bone marrow of adult mammals and is the process by which blood cells are replenished every day throughout life. Differentiation of hematopoietic cells occurs in a stepwise manner through intermediates of differentiation that could be phenotypically identified. This has allowed establishing hematopoietic cell classification with hematopoietic stem cells (HSCs) at the top of the hierarchy. HSCs are mostly quiescent and serve as a reservoir for maintenance of lifelong hematopoiesis. Over recent years, it has become increasingly clear that HSC quiescence is not only due to intrinsic properties, but is also mediated by cognate interactions between HSCs and surrounding cells within micro-anatomical sites called “niches”. This hematopoietic/stromal crosstalk model also applies to more mature progenitors such as B cell progenitors, which are thought to reside in distinct “niches”. This prompted many research teams to search for specific molecular mechanisms supporting leuko-stromal crosstalk in the bone marrow and acting at specific stage of differentiation to regulate hematopoietic homeostasis. Here, we review recent data on adhesion mechanisms involved in HSCs and B cell progenitors interactions with surrounding bone marrow stromal cells.