Cutting Edge: Endogenous IFN-β Regulates Survival and Development of Transitional B Cells This information is current as Jennie A. Hamilton, Qi Wu, PingAr Yang, Bao Luo, of September 28, 2021. Shanrun Liu, Huixian Hong, Jun Li, Mark R. Walter, Eleanor N. Fish, Hui-Chen Hsu and John D. Mountz J Immunol 2017; 199:2618-2623; Prepublished online 13 September 2017; doi: 10.4049/jimmunol.1700888 Downloaded from http://www.jimmunol.org/content/199/8/2618 Supplementary http://www.jimmunol.org/content/suppl/2017/09/13/jimmunol.170088 Material 8.DCSupplemental http://www.jimmunol.org/ References This article cites 25 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/199/8/2618.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 28, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Cutting Edge Immunology Cutting Edge: Endogenous IFN-b Regulates Survival and Development of Transitional B Cells Jennie A. Hamilton,* Qi Wu,* PingAr Yang,* Bao Luo,* Shanrun Liu,* † ‡,x Huixian Hong,* Jun Li,* Mark R. Walter,{ Eleanor N. Fish, Hui-Chen Hsu,* and John D. Mountz*, The transitional stage of B cell development is a forma- and extrinsic factors that regulate the abnormal survival re- tive stage in the spleen where autoreactive specificities sponses of T1 B cells in SLE are poorly understood. are censored as B cells gain immune competence, but Most studies into the differential survival responses of the intrinsic and extrinsic factors regulating survival transitional B cells, both in normal B cell development and in of transitional stage 1 (T1) B cells are unknown. We autoimmunity, have focused on the interactions between cell- report that B cell expression of IFN-b is required for surface Ag receptors and costimulatory molecules as well as the Downloaded from optimal survival and TLR7 responses of transitional role of factors present in the extracellular environment, in a B cells in the spleen and was overexpressed in T1 particular IFN- and TLR ligands (5). The responsiveness of B cells to IFN-b has been less well studied compared with B cells from BXD2 lupus-prone mice. Single-cell gene a b expression analysis of B6 Ifnb+/+ versus B6 Ifnb–⁄– T1 IFN- , although it has been shown that IFN- has a higher affinity for IFNARs than IFN-a (6, 7). It has been proposed B cells revealed heterogeneous expression of Ifnb in http://www.jimmunol.org/ that IFN-b acts as an initial signal that potentiates subsequent wild-type B cells and distinct gene expression patterns b signaling by other type I IFNs and cytokines (8), and a role associated with endogenous IFN- . Single-cell analysis for IFN-b in the promotion of TLR signaling has been dem- of BXD2 T1 B cells revealed that Ifnb is expressed in onstrated (9, 10). early T1 B cell development with subsequent upregu- In this study, we report a mechanistic model in which lation of Tlr7 and Ifna1. Together, these data suggest b b endogenous expression of IFN- is central to the survival that T1 B cell expression of IFN- plays a key role in responses of new immigrant T1 B cells. Endogenous IFN-b is regulating responsiveness to external factors. The an important regulator of TLR7 responses during T1 B cell Journal of Immunology, 2017, 199: 2618–2623. development and promotes their development into immune- by guest on September 28, 2021 competent B cells (3). he survival responses of transitional B cells play a key role in shaping the development of mature, Ab- Materials and Methods T producing B cells. Transitional stage 1 (T1) B cells Mice are the initial immigrants in the spleen and are highly sus- Ifnb-deficient C57BL/6J mice were provided by Dr. E. Fish, University of ceptible to negative selection following strong BCR ligand Toronto, Canada (11). Rag1-deficient and B6 Cd45.1 and Cd45.2 mice were engagement (1, 2). T1 B cells that survive this negative se- purchased from the Jackson Laboratory. BXD2 GFP mice were generated by crossing of BXD2 mice with B6 GFP mice for .15 generations. lection become transitional stage 2 (T2) or mature B cells that are competent to respond to immune challenges (3). Failures Bone marrow transplantation in this selection checkpoint are associated with aberrant ac- Bone marrow (BM) cells (1 3 107) from the indicated donors were transferred tivation and development of polyreactive self-antigen–reactive 2 2 or mixed at a 1:1 ratio of Cd45.1 B6/Cd45.2 B6-Ifnb / or a 1:1:1 ratio of 2 2 mature B cells in systemic lupus erythematous (SLE) and the Cd45.1 B6/Cd45.2 B6-Ifnb / /GFP+ BXD2, and injected i.v. into recipient development of anti-nuclear autoantibodies (4). The intrinsic mice as previously described (12). *Division of Clinical Immunology and Rheumatology, Department of Medicine, Uni- Research Award (to M.R.W. and H.-C.H.), and a Tier 1 Canada Research Chair grant versity of Alabama at Birmingham, Birmingham, AL 35294; †Department of Microbi- (to E.N.F). ology, University of Alabama at Birmingham, Birmingham, AL 35294; ‡Toronto Address correspondence and reprint requests to Dr. John D. Mountz, Division of General Research Institute, University Health Network, Toronto, Ontario M5G 2C4, x Clinical Immunology and Rheumatology, Department of Medicine, University of Ala- Canada; Department of Immunology, University of Toronto, Toronto, Ontario M5G { bama at Birmingham, Shelby Interdisciplinary Biomed Research Building, Room SHEL 2M1, Canada; and Birmingham Veterans Administration Medical Center, Birming- 307, 1825 University Boulevard, Birmingham, AL 35294-2182. E-mail address: ham, AL 35233 [email protected] Received for publication June 22, 2017. Accepted for publication August 21, 2017. The online version of this article contains supplemental material. This work was supported by National Institutes of Health (NIH) Grant R01-AI-071110 Abbreviations used in this article: BM, bone marrow; C , cycle threshold; pDC, plas- and Veterans Administration Merit Review Grant 1I01BX000600 (to J.D.M.), NIH T macytoid dendritic cell; SLE, systemic lupus erythematous; T1, transitional stage 1; WT, Grant R01-AI-083705 (to H.-C.H.), NIH Immunology T32 Training Grant wild-type. 2T32AI007051-39 (to J.A.H.), NIH Grant P30-AR-048311/Pilot and Feasibility Study Project (to J.L.), NIH Grants P30-AR-048311 and P30-AI-027767, a Lupus Founda- Ó tion of America Finzi Summer Fellowship (to J.A.H.), a Lupus Research Institute Novel Copyright 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700888 The Journal of Immunology 2619 In vitro stimulation and type I IFN neutralization relatively lower on T1 B cells (16) (Fig. 1B). Stimulation of b Purified B cells were stimulated with mouse IFN-a or IFN-b (a gift from the sorted B cells in vitro confirmed that high-affinity IFN- Dr. V. Ghanta, CytImmune), 2 mg/ml TLR7 agonist CL264 (InvivoGen), or exhibited increased ability to stimulate all B cell subsets, CL264 + a polyclonal anti-mouse IgM (1 mg/ml; Jackson ImmunoResearch) compared with IFN-a (Fig. 1C). or nonspecific rat-IgG isotype control. For specific neutralization of type I b IFNs, cells were preincubated with 50 mg/ml anti-IFNAR (clone MAR1-5A3; To explore whether IFN- plays a role in the survival and b development of T1 B cells in vivo, we reconstituted irradiated Bio X Cell) or 500 IU/ml anti–IFN- (rabbit IgG, protein A purified, PBL ⁄ ⁄ Assay Science). Rag1– – recipients with BM from WT B6 Ifnb+ + (CD45.1) ⁄ mice and Ifnb– – (CD45.2) mice. This chimeric approach Real-time quantitative RT-PCR enabled distinction of the effects of endogenous production of RNA isolation, cDNA synthesis, and real-time PCR reactions were carried out IFN-b from exogenous influences. Confocal imaging of as described previously (12). spleens during B cell repopulation (Fig. 1D) confirmed that –⁄– Single-cell quantitative RT-PCR B cells derived from the BM of both WT and Ifnb mice were capable of seeding the same region of the spleen; how- For single-cell analyses, single T1 B cells were obtained from the spleens of 2 2 + CD45.1 B6: CD45.2 B6 Ifnb / BM chimeras or 4 mo old female BXD2 ever, IgM B cells of WT origin were significantly increased –⁄– mice. Analyses were performed using the BioMark Real-Time quantitative relative to those derived from Ifnb BM. Consistent with PCR system (Fluidigm, South San Francisco, CA) using the standard Flu- this, there was a significantly higher percentage of Annexin V+ idigm protocols. Primer sets amplifying the mRNAs of the relevant genes are apoptotic cells in transitional B cells derived from the B6 presented in Supplemental Table I. The averaged cycle threshold (CT) values –⁄– were calculated from the system software (BioMark Real-Time PCR Analysis; Ifnb BM compared with those derived from the WT BM Downloaded from Fluidigm). (Fig. 1E). The highest rates of apoptosis were observed in the ‒ΔC Gene expression values were calculated using the 2 T value.