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Innate Pro–B-Cell Progenitors Protect Against Type 1 Diabetes By

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Innate Pro–B-Cell Progenitors Protect Against Type 1 Diabetes By Innate pro–B-cell progenitors protect against type 1 PNAS PLUS diabetes by regulating autoimmune effector T cells Ruddy Montandona,b,1, Sarantis Korniotisa,b,1, Esther Layseca-Espinosaa,b,2, Christophe Grasa,b, Jérôme Mégretc, Sophie Ezinea,d, Michel Dya,b, and Flora Zavalaa,b,3 aFaculté de Médecine Site Necker, Université Paris Descartes, bCentre National de la Recherche Scientifique Unité Mixte de Recherche 8147, 75015 Paris, France; cInstitut Fédératif de Recherche 94 Necker-Enfants Malades, 75015 Paris, France; and dInstitut National de la Santé et de la Recherche Médicale U1020, 75015 Paris, France Edited by Simon Fillatreau, Deutsches Rheuma-Forschungszentrum, Berlin, Germany, and accepted by the Editorial Board May 6, 2013 (received for review December 24, 2012) Diverse hematopoietic progenitors, including myeloid populations emergence of regulatory B cells (Bregs), along with acquired-type arising in inflammatory and tumoral conditions and multipotent stimulation, such as B-cell receptor (BCR) engagement concomi- cells, mobilized by hematopoietic growth factors or emerging during tant or not with CD40 activation (10, 11). Such induced regulatory parasitic infections, display tolerogenic properties. Innate immune B-cell functions are believed to be more robust than those ex- stimuli confer regulatory functions to various mature B-cell subsets pressed by naive and resting B cells, which can nevertheless tolerize but immature B-cell progenitors endowed with suppressive proper- naive T cells and induce regulatory T cells (Tregs) (12, 13). ties per se or after differentiating into more mature regulatory Bregs are a heterogeneous lymphocyte subset present among all B cells remain to be characterized. Herein we provide evidence for major B-cell populations (14–17). The rare so-called B10 cells innate pro-B cells (CpG-proBs) that emerged within the bone marrow identified by their CD19+CD1dhi CD5+ phenotype (18–20), peri- both in vitro and in vivo upon Toll-like receptor-9 activation and toneal CD5+ B1a cells (21, 22), large follicular B cells, and acti- whose adoptive transfer protected nonobese diabetic mice against vated transitional, marginal zone (MZ) B cells can all acquire type 1 diabetes (T1D). These cells responded to IFN-γ released by regulatory properties. The most immature Breg subset described so activated effector T cells (Teffs), by up-regulating their Fas ligand far is composed of B220+IgM+CD21lowCD93+CD23+ transitional (FasL) expression, which enabled them to kill Teffs through apopto- T2 MZ precursor B (T2 MZP-B) cells, which are continuously sis. In turn, IFN-γ derived from CpG-proBs enhanced IFN-γ while dra- produced in adult bone marrow and home to the MZ of the spleen, matically reducing IL-21 production by Teffs. In keeping with the where they differentiate into IgMhighCD1dhighCD21highCD23low crucial pathogenic role played by IL-21 in T1D, adoptively transferred MZ B cells (23, 24). IFN-γ–deficient CpG-proBs did not prevent T1D development. Addi- The differentiation pathways of the various Breg subsets remain tionally, CpG-proBs matured in vivo into diverse pancreatic and unknown. Only functional precursors, named “B10pro,” which are splenic suppressive FasLhigh B-cell subsets. CpG-proBs may become mature B cells requiring additional BCR-activating antigenic sig- instrumental in cell therapy of autoimmune diseases either on their nals to produce immunosuppressive IL-10 but cannot be distin- own or as graft complement in autologous stem cell transplantation. guished from B10 cells by phenotypic criteria, have hitherto been identified (25). It is unknown whether Bregs derive from one or immune therapy | peripheral tolerance | MyD88 signaling | several progenitors or solely from conventional B-cell subsets. B lymphocytes | T lymphocytes Moreover, an immature B-cell progenitor population endowed with suppressive properties per se or after differentiation into more growing body of evidence attests that immune cells with im- mature Bregs has not been demonstrated as yet. Amunoregulatory functions do not exclusively belong to mature Herein we describe a hematopoietic progenitor population that populations of diverse lineage, but also comprise several hemato- emerges transiently in vitro and in vivo in the bone marrow of poietic progenitor subsets. The first subset to be recognized com- prised myeloid progenitors that acquired suppressive properties in Significance tumoral and inflammatory environments (1) and played either detrimental or beneficial roles in different pathological situations. Immunoregulatory poperties have been principally ascribed to We have reported previously that mobilization with hematopoietic various mature immune cell types, including regulatory B cells. growth factors conferred tolerogenic properties to multipotent An immature B-cell progenitor population endowed with sup- hematopoietic progenitors at the multipotent progenitor (MPP2) pressive properties per se or after differentiation into more stage of differentiation that enabled them to promote the expan- mature regulatory B cells has not been demonstrated as yet. IMMUNOLOGY – sion of regulatory T cells (2, 3), thereby preventing spontaneous We now describe a pro B-cell progenitor population that emerged autoimmune type 1 diabetes (T1D) in the nonobese diabetic upon stimulation with the Toll-like receptor-9 ligand CpG and (NOD) mouse model. Moreover, parasitic infections were shown prevented disease upon adoptive transfer into autoimmune type to stimulate via IL-25 the emergence of Th2 cytokine-secreting 1 diabetes-prone mice. Effector T cells were the target of immu- MPPs (MPPTh2) that ultimately differentiated into mature cell noregulatory pro-B cells and of their mature progeny. Such pro- tective pro-B cells could be instrumental for cell therapy of types with pro-Th2 functions, thus contributing to parasitic autoimmune diseases. clearance (4). Direct interactions between pathogens and hematopoietic stem Author contributions: R.M., S.K., and F.Z. designed research; R.M., S.K., E.L.-E., C.G., and cells occur through Toll-like receptor (TLR) activation, driving F.Z. performed research; R.M., S.K., E.L.-E., C.G., J.M., S.E., and F.Z. analyzed data; and S.E., their differentiation along myeloid pathways to enforce anti- M.D., and F.Z. wrote the paper. infectious defenses (5, 6). TLR agonists also promote hematopoiesis The authors declare no conflict of interest. by enhancing the production of the hematopoietic growth factor This article is a PNAS Direct Submission. S.F. is a guest editor invited by the Editorial Board. G-CSF, with whom they synergize to mobilize hematopoietic 1R.M. and S.K. contributed equally to this work. stem cells from the bone marrow to the periphery (7). 2Present address: Department of Immunology, Medical Faculty, San Luis Potosi University, TLR-mediated innate-type stimulation by infectious (8) and San Luis Potosi, 78210, Mexico. parasitic (9) agents also plays a major role in promoting the 3To whom correspondence should be addressed. E-mail: fl[email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1222446110 PNAS | Published online May 28, 2013 | E2199–E2208 Downloaded by guest on September 26, 2021 NOD mice, after activation with the TLR-9 agonist CpG and whose and may play a major role in the durable protection against T1D adoptive transfer into NOD mice prevents T1D onset. These cells provided by the progenitors in vivo. − were c-kitlowSca-1lowCD127+B220+CD19+IgM CD1dintCD43+, Results a phenotype consistent with a pro–B-cell stage of differentia- low low + tion, except for their CD1d expression. The cells differentiated c-kit Sca-1 B220 Bone Marrow Progenitors Emerging upon Exposure to CpG Prevent T1D. Incubation of bone marrow cells from NOD in vivo exclusively into B lymphocytes, at various stages of mice with the oligonucleotide CpG 1668 (CpG-B), but not with maturation. control GpC oligonucleotide, led to the transient emergence of a Functionally, these TLR-induced hematopoietic progenitors low + c-kit Sca-1 cell population within 18 h. These cells were het- suppressed pathogenic effector T cells (Teffs) by reducing their erogeneous in terms of size, Sca-1 and B220 expression (Fig. 1A), − IL-21 production and by inducing their apoptosis via Fas ligand and could be further sorted into a small-size Sca-1lowB220+IgM − − (FasL). Additionally, the B-cell progeny of CpG-induced proBs and a large-size Sca-1highB220 IgM fraction. Adoptive transfer continued to express high levels of FasL and to suppress Teffs, to NOD mice at 6 wk of age revealed that only small-size CpG- − Fig. 1. Phenotypic characterization and prevention of T1D in vivo by a CpG-induced c-kitlowSca-1lowB220+IgM bone marrow subset. (A) Bone marrow (BM) cells, incubated for 18 h with CpG-B (1 μg/mL), were magnetically selected for c-kit+ cells, further stained for Sca-1, B220, and IgM and electronically sorted into large-size − − − (FSChighSSChigh)c-kitlowSca-1highB220 IgM and small-size (FSClowSSClow)c-kitlowSca-1lowB220+IgM cells. BM cells incubated with the control oligonucleotide GpC were electronically sorted as c-kit+Sca-1+ cells that were B220−IgM−.(B) Diabetes incidence in NOD mice injected with PBS or after intravenous transfer of the above sorted subsetsat6-wkofage,P = 0.0021 by Kaplan–Meier estimates when comparing cumulative incidence curves for controls and for CpG-induced c-kitlowSca-1lowB220+IgM− progenitor recipients, not significant for other groups. (C) Incidence of T1D in NOD mice injected intravenously either with PBS or with 60 × 103 CpG-induced c-kitlowSca- − 1lowB220+IgM progenitors at 16 wk of age, P = 0.0328. Results in B and C are pooled from two experiments. (D) Phenotypic characterization of the protective bone marrow progenitor subset by flow cytometry analysis of expression of CD19, CD127, CD43, IgM, CD1d, and CD5. Cells were stained with specific antibodies (open histograms) or control isotype antibodies (filled histograms) after FACS sorting. CD1d level on CpG-induced c-kitlowSca-1lowB220+IgM- progenitors (red histogram) was − compared with that measured on splenic follicular B cells (FoB, blue histogram) and MZ B cells (MZB, black histogram).
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