Bertrand Huard

Identification of proteoglycans as the APRIL-specific binding partners

B cell activating factor of the tumor necrosis factor (TNF) family (BAFF) and a proliferation-inducing ligand (APRIL) are closely related ligands within the TNF superfamily that play important roles in B lymphocyte biology. Both ligands share two receptors—transmembrane activator and calcium signal–modulating cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA)—that are predominantly expressed on B cells. In addition, BAFF specifically binds BAFF receptor, whereas the nature of a postulated APRIL-specific receptor remains elusive. We show that the TNF homology domain of APRIL binds BCMA and TACI, whereas a basic amino acid sequence (QKQKKQ) close to the NH2 terminus of the mature protein is required for binding to the APRIL-specific “receptor.” This interactor was identified as negatively charged sulfated glycosaminoglycan side chains of proteoglycans. Although T cell lines bound little APRIL, the ectopic expression of glycosaminoglycan-rich syndecans or glypicans conferred on these cells a high binding capacity that was completely dependent on APRILs basic sequence. Moreover, syndecan-1–positive plasma cells and proteoglycan-rich nonhematopoietic cells displayed high specific, heparin-sensitive binding to APRIL. Inhibition of BAFF and APRIL, but not BAFF alone, prevented the survival and/or the migration of newly formed plasma cells to the bone marrow. In addition, costimulation of B cell proliferation by APRIL was only effective upon APRIL oligomerization. Therefore, we propose a model whereby APRIL binding to the extracellular matrix or to proteoglycan-positive cells induces APRIL oligomerization, which is the prerequisite for the triggering of TACI- and/or BCMA-mediated activation, migration, or survival signals.

APRIL is critical for plasmablast survival in the bone marrow and poorly expressed by early-life bone marrow stromal cells.

The persistence of serum IgG antibodies elicited in human infants is much shorter than when such responses are elicited later in life. The reasons for this rapid waning of antigen-specific antibodies elicited in infancy are yet unknown. We have recently shown that adoptively transferred tetanus toxoid (TT)-specific plasmablasts (PBs) efficiently reach the bone marrow (BM) of infant mice. However, TT-specific PBs fail to persist in the early-life BM, suggesting that they fail to receive the molecular signals that support their survival/differentiation. Using a proliferation-inducing ligand (APRIL)- and B-cell activating factor (BAFF) B-lymphocyte stimulator (BLyS)-deficient mice, we demonstrate here that APRIL is a critical factor for the establishment of the adult BM reservoir of anti-TT IgG-secreting cells. Through in vitro analyses of PB/plasma cell (PC) survival/differentiation, we show that APRIL induces the expression of Bcl-X(L) by a preferential binding to heparan sulfate proteoglycans at the surface of CD138(+) cells. Last, we identify BM-resident macrophages as the main cells that provide survival signals to PBs and show that this function is slowly acquired in early life, in parallel to a progressive acquisition of APRIL expression. Altogether, this identifies APRIL as a critical signal for PB survival that is poorly expressed in the early-life BM compartment.

APRIL secreted by neutrophils binds to heparan sulfate proteoglycans to create plasma cell niches in human mucosa

The bone marrow constitutes a favorable environment for long-lived antibody-secreting plasma cells, providing blood-circulating antibody. Plasma cells are also present in mucosa-associated lymphoid tissue (MALT) to mediate local frontline immunity, but how plasma cell survival there is regulated is not known. Here we report that a proliferation-inducing ligand (APRIL) promoted survival of human upper and lower MALT plasma cells by upregulating expression of the antiapoptotic proteins bcl-2, bcl-xL, and mcl-1. The in situ localization of APRIL was consistent with such a prosurvival role in MALT. In upper MALT, tonsillar epithelium produced APRIL. Upon infection, APRIL production increased considerably when APRIL-secreting neutrophils recruited from the blood infiltrated the crypt epithelium. Heparan sulfate proteoglycans (HSPGs) retained secreted APRIL in the subepithelium of the infected zone to create APRIL-rich niches, wherein IgG-producing plasma cells accumulated. In lower MALT, neutrophils were the unique source of APRIL, giving rise to similar niches for IgA-producing plasmocytes in villi of lamina propria. Furthermore, we found that mucosal humoral immunity in APRIL-deficient mice is less persistent than in WT mice. Hence, production of APRIL by inflammation-recruited neutrophils may create plasma cell niches in MALT to sustain a local antibody production.

KIR expression on self-reactive CD8+ T cells is controlled by T-cell receptor engagement

Natural killer cell tolerance is maintained by the interaction of killer inhibitory receptors (KIRs) with self-major histocompatibility complex class I gene products. A subset of T cells also expresses inhibitory receptors, but the functional significance of these receptors on T cells is unclear. Here we show that, in the absence of T-cell receptor (TCR) engagement, KIRs expressed on CD8+ T cells are slowly downregulated by KIR ligands expressed on antigen-presenting cells. The resulting expression levels of KIR are no longer able to inhibit T-cell function. In contrast, TCR engagement sustains KIR expression, and re-induces functional levels of KIR expression after ligand-induced downregulation of KIR. Our data indicate that KIR expression on CD8+ T cells in vivo may be maintained through continuous encounters with antigen. As KIR-mediated inhibition of T-cell activation can be bypassed at high antigen concentrations, dynamic KIR expression may mediate T-cell tolerance to self-antigens by sparing self-reactive T cells, thus enabling them to mediate potentially useful immune functions to quantitatively or qualitatively different antigens.

Production of the plasma-cell survival factor a proliferation-inducing ligand (APRIL) peaks in myeloid precursor cells from human bone marrow

The bone marrow (BM) is an organ extremely efficient in mediating long-term survival of plasma cells (PCs), ensuring an immune humoral memory. This implies that the BM must provide continuously key PC survival factors. Our results show that the BM is an organ constitutively rich in a proliferation-inducing ligand (APRIL), a member of the tumor necrosis factor superfamily implicated in PC survival. APRIL production is induced during hematopoiesis in myeloid cells by non-lineage-committing factors such as stem cell factor, thrombopoietin, IL-3, and FMS-like tyrosine kinase 3 ligand. Notably, APRIL production, both in the human and mouse systems, peaks in myeloid precursor cells, before dropping in fully mature granulocytes. Myeloid cells secrete APRIL that circulates freely in BM plasma to act on PCs, usually at distance from APRIL production sites. Selective APRIL in vivo antagonism and in vitro coculture experiments further demonstrated that myeloid precursor cells mediates PC survival in an APRIL-dependent manner Thus, APRIL production by myeloid precursor cells shows that the 2 main BM functions, hematopoiesis and long-term PC survival, are linked. Such constitutive and high APRIL production may explain why BM mediates long-term PC survival.

Activating CD94:NKG2C and inhibitory CD94:NKG2A receptors are expressed by distinct subsets of committed CD8+ TCR αβ lymphocytes

A subset of CD8+ T cells express the natural killer cell receptors CD94:NKG2A or CD94:NKG2C. We found that although many CD8+ T cells transcribe CD94 and NKG2C, expression of a functional CD94:NKG2C receptor is restricted to highly differentiated effector cells. CD94:NKG2A is expressed by a different subset consisting of CCR7+ memory cells and CCR7– effector cells. Since NKG2A can only be induced on naive CD8+ T cells while CD94– memory cells are refractory, it is likely that commitment to the CD94:NKG2A+ subset occurs during the first encounter with antigen. CCR7+CD94:NKG2A+ T cells recirculate through lymph nodes where upon activation, they produce large quantities of IFN‐γ. These cells occur as a separate CD94:NKG2A+ T cell lineage with a distinct TCR repertoire that differs from that of the other CD8+CD94– T cells activated in situ.

Paracrine promotion of tumor development by the TNF ligand APRIL in Hodgkin's Disease

A proliferation-inducing ligand (APRIL) from the tumor necrosis factor (TNF) superfamily co-stimulates B-cell activation. When overexpressed in mice, APRIL induces B-cell neoplasia.1 The promoting activity of APRIL for B-cell derived tumors has been recently reported in vitro for human Hodgkins lymphoma (HL) cell lines.2 In this work, we sought to ascertain whether APRIL plays a role in the in situ development of HL with a particular emphasis on the pathway(s) involved.

Extralymphatic Tumors Prepare Draining Lymph Nodes to Invasion via a T-Cell Cross-Tolerance Process

Metastases often develop in lymphoid organs. However, the immunologic mechanism allowing such invasion is not known because these organs are considered to be hostile to tumor cells. Here, we analyzed the interactions between tumor cells and CD8(+) T cells in such lymphoid organs. Tumor cells implanted into lymph nodes were able to induce tumor-specific cytotoxic CD8(+) T-cell responses, conducting to tumor rejection, in contrast to primary extralymphatic tumors rapidly anergizing T cells in draining lymph nodes (DLN) via a cross-presentation process. This abortive CD8(+) T-cell response to extralymphatic tumor is independent of the subcellular localization of antigen in tumor cells and is consistent with a lack of CD4(+) T-cell help. Notably, this anergy was potent enough to allow successful DLN implantation of tumor cells. Such distant cross-tolerization of tumor-specific CD8(+) T cells may be a determinant permissive event leading to invasion of DLN. In this situation, metastatic tumor cells do not need to down-regulate their immunogenicity to spread.

Synovial tissues concentrate secreted APRIL

IntroductionA proliferation-inducing ligand (APRIL) from the TNF family, owing to its role in the generation and survival of plasma cells (PCs), is currently targeted for rheumatoid arthritis (RA) treatment. However, little is known about APRIL expression in RA lesions, hampering our understanding of the way APRIL may modulate this autoimmune disease.MethodsWe performed immunological staining of human normal, non-RA and RA synovial tissues with a pair of antibodies specifically recognizing APRIL-producing cells and secreted APRIL.ResultsWe detected significant amounts of secreted APRIL in normal synovium mostly concentrated around blood vessels and at the lining layer, but no cells producing APRIL. Meanwhile, we observed that blood neutrophils constitutively secrete APRIL, indicating that blood APRIL may diffuse into the synovium via its fenestrated vessels. Synovium from non-RA and RA patients retained similarly secreted APRIL, but in this case APRIL-producing cells, including neutrophils and macrophages, were present in the tissue. Notably, PCs - when present in RA synovium - accumulated in areas of APRIL retention, spreading from blood vessels towards the lining layer.ConclusionsPCs accumulate in synovial zones rich in secreted APRIL, consistent with a pro-survival role of APRIL for PCs in RA. The concentration of APRIL by normal synovium indicates that this tissue may constitute a proper environment for PCs even before RA onset.

Role of the tumour necrosis family ligand APRIL in solid tumour development: Retrospective studies in bladder, ovarian and head and neck carcinomas

A proliferation inducing ligand (APRIL) from the tumour necrosis family (TNF) promotes the natural development of solid tumours in pre-clinical models. Here, we studied the role of APRIL in patients with urothelial bladder, epithelial surface ovarian, and head and neck squamous carcinomas. By using immunohistochemistry, we revealed an upregulation of APRIL expression in lesions from a significant subset of patients compared to corresponding healthy tissues. APRIL upregulation was not due to autocrine production by tumour cells, but rather originated from infiltration of APRIL-producing neutrophils. Heparan sulphate proteoglycan (HSPG) efficiently concentrated secreted APRIL in lesions. Despite this retention, in situ APRIL upregulation did not significantly alter disease-free and overall survivals of carcinoma patients in retrospective studies. This indicates that APRIL is not potent enough to promote the development of solid tumour cells under the pressure of chemotherapy.