Virus-Specific Memory b lymphocytes Unique Requirements for Reactivation of
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- Material Supplementary 0.DC1 http://www.jimmunol.org/content/suppl/2010/08/25/jimmunol.100154 References
| of May 25, 2017. This information is current as Virus-Specific Memory B Lymphocytes Unique Requirements for Reactivation of Michael Mach and Thomas H. Winkler Jasmin U. Horlitz, Nico van Rooijen, Heinrich Korner, Florian J. Weisel, Uwe K. Appelt, Andrea M. Schneider, http://www.jimmunol.org/content/185/7/4011 doi: 10.4049/jimmunol.1001540 August 2010; 2010; 185:4011-4021; Prepublished online 25 J Immunol Material Supplementary 0.DC1 http://www.jimmunol.org/content/suppl/2010/08/25/jimmunol.100154 References http://www.jimmunol.org/content/185/7/4011.full#ref-list-1 , 17 of which you can access for free at:
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The Journal of Immunology Unique Requirements for Reactivation of Virus-Specific Memory B Lymphocytes Florian J. Weisel,* Uwe K. Appelt, † Andrea M. Schneider,* Jasmin U. Horlitz,* Nico van Rooijen, ‡ Heinrich Korner, x Michael Mach, { and Thomas H. Winkler* ,† Memory B cells (MBCs) are rapidly activated upon Ag re-exposure in vivo, but the precise requirements for this process are still elusive. To address these requirements, T cell-independent reactivation of MBCs against virus-like particles was analyzed. As few as 25 MBCs are sufficient for a measurable Ab response after adoptive transfer. We found that MBCs were reactivated upon antigenic challenge to normal levels after depletion of macrophages, CD11c + dendritic cells, and matured follicular dendritic cells. Fur- thermore, MBC responses were possible in TNF/lymphotoxin a double-deficient mice after partial normalization of lymphoid architecture by means of long-term reconstitution with wild-type bone marrow. Activation did not occur when chimeric mice, which still lack all lymph nodes and Peyer’s patches, were splenectomized prior to MBC transfer. Together with our finding that MBC responses are weak when Ag was administered within minutes after adoptive MBC transfer, these results strongly suggest that MBCs have to occupy specific niches within secondary lymphoid tissue to become fully Ag-responsive. We provide clear evidence that MBCs are not preferentially resident within the splenic marginal zones and show that impaired homing to lymphoid follicles resulted in significantly diminished activation, suggesting that reactivation of MBCs occurred inside lymphoid follicles. Furthermore, comparison of virus-specific MBC T cell-independent reactivation versus primary T cell-independent type II B cell activation revealed unique requirements of MBC activation. The Journal of Immunology, 2010, 185: 4011–4021. I mmunological memory, the ability to respond rapidly and effectively to Ag upon re-exposure after initial encounter, is the defining feature of adaptive immunity. Memory is an emergent property that extends in increased precursor frequencies of Ag-specific B and T cells, long-lived plasma cells (PCs), pre- existing Abs, as well as memory lymphocytes with functional properties different from those of their naive precursors (1, 2). Memory B cells (MBCs) and long-lived PCs arise from germinal center reactions and express somatically hypermutate Ig receptors of switched isotypes (3), although it was recently demonstrated that thymus-independent type II (TI-2) Ags can also generate MBCs (4). Humoral immunity is maintained by either long-lived PCs, which home to the bone marrow (BM) constitutively secreting Abs (5), or nonsecreting resting MBCs that are rapidly reactivated upon Ag re-encounter (6). It is still a matter of debate how longevity of MBCs is achieved and to what extent they sustain Ab titers. Dif- ferent concepts to explain persistence of Abs with a given speci- ficity are presently discussed (7, 8). We were interested in the reactivation requirements of MBCs, which are largely undefined. Recently, we have shown that reac- tivation of human CMV (hCMV)-specific murine MBCs can occur in the absence of cognate or bystander T cell help (9). Interestingly, our results indicated that homing to intact, compartmentalized sec- ondary lymphoid tissue is required for proper T cell-independent MBC responses (9). We established sorting of single Ag-specific MBCs, thus enabling us to analyze the reactivation of these cells qualitatively as well as quantitatively. We show that not only T lymphocytes but also macrophages (Mfs), CD11c + dendritic cells (DCs), and follicular DCs (FDCs) are not essential for the reactivation of MBCs, and we provide evidence that reactivation of MBCs takes place within follicles of secondary lymphoid organs. Comparison of T cell-independent virus-specific MBC activation to primary immune reactions against TI-2 Ags revealed unique re- quirements of murine MBC activation. Materials and Methods Mice, BM transplantation, and splenectomy C57BL/6 (B6) mice were obtained from Charles River Laboratories (Sulzfeld, Germany). B6-IgH a congenic (B6.PL-Thy1a/CyJ) and B6- TCR 2/2
mice were obtained from The Jackson Laboratory (Bar Harbor, ME). B6-CD11c-diphtheria toxin receptor (DTR)/GFP transgenic (tg) mice (10) were a gift from U. Schleicher (University of Erlangen). B6-TNF/ lymphotoxin a (LTa) 2/2 (11), Ly5.1 congenic B6 mice and B6-RAG1 2/2 mice (12) and all other strains of mice were maintained under specific pathogen-free conditions and used between 8 and 12 wk of age. All experiments were conducted in accordance with international guidelines for animal care and use and in accordance with the guidelines of the Animal Care and Use Committee of the Government of Bavaria and the institutional guidelines of the University of Erlangen–Nuremberg. For BM reconstitution, BM cells from donor mice were harvested by flushing femurs and tibias with sterile PBS. BM cells (2–5 3 10
6 ) were
*Hematopoiesis Unit, Department of Biology, † Nikolaus-Fiebiger Center for Molec- ular Medicine, University Erlangen; { Institute for Clinical and Molecular Virology, University Hospital Erlangen, Erlangen, Germany; ‡ Department of Molecular Cell Biology, Free University, Amsterdam, The Netherlands; and x Comparative Genomics Centre, Cook University, Townsville, Queensland, Australia Received for publication May 10, 2010. Accepted for publication July 22, 2010. This work was supported in part by the Deutsche Forschungsgemeinschaft through Sonderforschungsbereich 643 (to M.M. and T.H.W.). Address correspondence and reprint requests to Dr. Thomas H. Winkler, Hematopoi- esis Unit, Department of Biology, Nikolaus-Fiebiger Center for Molecular Medi- cine, Glueckstrasse 6, 91054 Erlangen, Germany. E-mail address: uni-erlangen.de">twinkler@molmed.
The online version of this article contains supplemental material. Abbreviations used in this paper: B6, C57BL/6; BM, bone marrow; CL, clodronate liposome; DTR, diphtheria toxin receptor; DTx, diphtheria toxin; FDC, follicular dendritic cell; FO, follicular; FSC, follicular stromal cell; gB, glycoprotein B; hCMV, human CMV; LTa, lymphotoxin a; Mf, macrophage; MBC, memory B cell; MZ, marginal zone; PC, plasma cell; PP, Peyer’s patch; RU, relative unit; tg, transgenic; TI- 2, thymus-independent type II; TNP, 2,4,6-trinitrophenyl; VLP, virus-like particle; wt, wild-type. Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001540 by guest on May 25, 2017 http://www.jimmunol.org/ Downloaded from
injected into the tail veins of recipient mice within 24 h after lethal gamma irradiation (11 Gy). Recipients were rested for at least 50 d. For splenectomy, mice were anesthetized i.p. with Ketavet (100 mg/kg; Pharmacia, Karlsruhe, Germany) and Rompun (20 mg/kg; Bayer Health- Care, Leverkusen, Germany). The spleen was removed after appropriate blood vessel ligation, and peritoneum and skin were closed in separate layers using sterile absorbable sutures. Ags, immunizations, and in vivo treatment hCMV strain AD169 was propagated in primary human foreskin fibroblasts. Dense bodies (noninfectious enveloped particles that mainly consist of tegu- ment proteins contained in a complete viral envelope with all glycoproteins embedded, referred to as virus-like particles [VLPs] throughout this work) were purified from cell culture supernatant via glycerol-tartrate gradient centrifugation as described (13). Highly purified, endotoxin-free, recombi- nant hCMV-glycoprotein B (gB) was a gift from Sanofi Pasteur (Lyon, France). Mice were immunized twice with 5–10 mg VLPs or soluble gB in Imject Alum (Pierce, Rockford, IL) at intervals of 6 wk and with 2 mg VLPs or gB in PBS i.v. 6 wk later. Mice were rested for at least 6 wk after the last immunization. For analysis of TI-2 immune reactions, mice were i.p. injected once with 15 mg of 2,4,6-trinitrophenyl (TNP)-80-Ficoll (Biosearch Tech- nologies, Novato, CA) in PBS without adjuvant. To investigate the distri- bution of blood-borne Ags, mice were injected with 1 3 10 8
conjugated Escherichia coli (K-12 strain) BioParticles in PBS (Invitrogen, Karlsruhe, Germany) 15 min prior to analysis. For depletion of Mfs or CD11c +
treated i.v. with 50 mg clodronate (clodronate was a gift of Roche Diag- nostics, Mannheim, Germany; it was encapsulated in liposomes as described in Ref. 14) in 200 ml PBS or i.p. with 4 ng/g bodyweight diphtheria toxin (DTx; Sigma-Aldrich, St. Louis, MO) in 200 ml PBS, respectively. Re- location of B cells from the marginal zones (MZs) into the follicles was achieved by repetitive i.p. treatment of mice with 2.5 mg/kg bodyweight FTY720 (Cayman Chemical, Ann Arbor, MI) in DMSO. In vivo Ab labeling was performed by i.v. injection of 2.5 mg PE-conjugated anti-Ly5.1 Ab (clone A20; BD Biosciences, Heidelberg, Germany) in 200 ml PBS. To impair B cell homing to lymphoid follicles, mice were i.v. injected with 200 mg anti-CXCL13 Ab (clone 143614; R&D Systems, Wiesbaden, Germany) in PBS. GB was conjugated to biotin by the use of No-Weigh sulfo-NHS- biotin (Pierce) followed by purification with Pall Nanosep centrifugal devices with Omega membrane 30K (Sigma-Aldrich). Particularization of gB-biotin on inert streptavidin microbeads was achieved by using a mMACS streptavidin kit (Miltenyi Biotec, Bergisch Gladbach, Ger- many) according to the manufacturer’s instructions. Mouse and human monoclonal anti-HCMV gB Abs generated in our laboratory (unpublished data) were used to measure and equilibrate the amount of gB on beads and VLPs by ELISA. Flow cytometry, cell sorting, and adoptive transfer of B lymphocytes Spleens were harvested and, in case of DC analysis, digested in 5 ml Hank’s buffer supplemented with 1 mg/ml collagenase D (Roche Diagnostics, Mannheim, Germany) and 200 U/ml DNaseI (Roche Diagnostics) for 30 min at 37˚C prior to single-cell suspension. After erythrocyte lysis (5 min in 0.15 M NH 4
anti-mouse CD16/CD32; clone 93, eBioscience, Frankfurt, Germany), splenocytes were incubated in PBS, 2% FCS, 2 mM EDTA for 30 min at 4˚C with varying combinations of the following Abs (if not listed otherwise, Abs were obtained from BD Biosciences): rat anti-mouse CD19 (clone 1D3, PE- or allophycocyanin-conjugated), rat anti-mouse IgG1 (clone A85-1, FITC- conjugated), rat anti-mouse IgG2a (clone R19-15, FITC-conjugated), rat anti-mouse IgG2b (clone R12-3, FITC-conjugated), rat anti-mouse CD11b (clone M1/70, PE-conjugated), Armenian hamster anti-mouse CD11c (clone N418, AF-647-conjugated; eBioscience), rat anti-mouse CD23 (clone B3B4, PE- or biotin-conjugated), rat anti-mouse CD21 (clone 7G6, FITC- conjugated), mouse anti-mouse Ly5.1 (clone A20, PE-conjugated), and mouse anti-mouse Ly5.2 (clone 104, FITC-conjugated). Cells were washed once in PBS, 2% FCS, 2 mM EDTA between incubation steps with listed primary Abs and streptavidin-conjugated PerCP to detect biotinylated pri- mary Abs. Expression of cell surface markers was analyzed using a FACS- Calibur running CellQuest software (BD Biosciences), with data analysis performed using FlowJo (Tree Star, Ashland, OR). For adoptive transfer, single-cell suspensions of splenocytes were depleted of T cells by incubation with supernatants of rat IgM anti-mouse CD4 (174.2)- and rat IgM anti- mouse CD8 (31-68.1)-producing cell lines (15) and the addition of Low- Tox rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada) followed by Ficoll gradient purification. CD19 + B lymphocytes were further enriched by MACS (Miltenyi Biotec). In general, a purity $99.8% was achieved by this procedure. B cells were either adoptively transferred into the tail vein of recipient mice or further purified by two rounds of cell sorting using a MoFlo cell sorter (Dako, Glostrup, Denmark) to isolate IgG + gB-binding MBCs (Supplemental Fig. 1). Therefore, 5 3 10 7 B cells/ml were stained with anti-CD19 (PE-conjugated), anti-IgG1 (FITC-conjugated), anti- IgG2a (FITC-conjugated), and anti-IgG2b (FITC-conjugated) and were ex- posed to 5 mg/ml gB, which was fluorescently labeled by the use of a Cy5 Ab labeling kit (Amersham Biosciences, Freiburg, Germany) according to the manufacturer’s instructions. Detection of specific Abs Sera were analyzed by ELISA to measure virus-specific IgG Abs. Soluble gB (1 mg/ml) was used throughout this work as coating Ag for the de- tection of specific Abs to yield high specifity and overcome limited sen- sitivity because of insufficient Ag density on VLPs. Sera were applied as 2-fold serial dilutions (ranging from 1/50 to 1/51,200) in PBS, 2% FCS, 0.05% Tween 20 (Sigma-Aldrich) and were compared with a 2-fold di- lution series (ranging from 1 mg/ml to 1 ng/ml) of a murine, gB-specific mAb (mAb 27-287; a gift from W. Britt, Birmingham, AL), which was included on each individual ELISA plate to generate standard curves with the value 1000 relative units (RUs) allotted. For detection of allotype- specific gB-specific serum IgG1 a/b
and IgG2a a/b
, samples were compared with sera from VLP hyperimmune B6 and IgH a congenic B6 mice, which were included on each individual ELISA plate to generate standard curves with the value 1000 RUs allotted. These hyperimmune sera were equili- brated for gB-specific IgG1 or IgG2a titers, respectively. Equilibration was achieved by the use of subclass-specific, biotinylated secondary Abs (BD Biosciences) and HRP-coupled streptavidin (Amersham Biosciences). De- termination of allotype-specific serum IgG was also performed by the use of allotype-specific, biotinylated secondary Abs (BD Biosciences). GB- specific serum IgG was detected by Fcg-specific goat anti-mouse IgG Abs (Dianova, Hamburg, Germany) coupled with HRP. For the detection of TNP-specific Ab titers, ELISA plates were coated with 10 mg/ml TNP 14 -
with standard curves generated from sera of TNP-Ficoll immune wild-type (wt) mice. Sera from naive mice served as control in all experiments, and resulting RUs were depicted as hatched areas in the graphs if the RUs were
.1. Data presentation was performed with GraphPad Prism (GraphPad Software, San Diego, CA), and results of unpaired Student t tests were shown as comparisons between indicated experimental groups: pp , 0.05; ppp , 0.01; pppp , 0.001. Immunofluorescence microscopy Spleens were embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA) and stored at 280˚C. Cryostat sections (9 mm thick) were thawed on SuperFrost Plus slides (Menzel Gla¨ser, Braunschweig, Ger- many), air dried, fixed for 10 min in acetone at 220˚C, and outlined with a liquid repellent slide marker pen (Science Services, Munich, Germany). After rehydration in PBS for 5 min, nonspecific binding sites were blocked for 30 min at room temperature with PBS, 10% FCS, 0,1% BSA, 2% rat serum (eBioscience), and 5 mg/ml rat anti-mouse CD16/CD32 (clone 93; eBioscience) Abs. Cryosections were incubated in PBS, 2% FCS, 0.05% Tween 20 for 30 min at room temperature in the dark with varying com- binations of the following Abs (if not listed otherwise, Abs were obtained from BD Biosciences): polyclonal goat anti-mouse IgM (FITC-conjugated; Dianova), rat anti-mouse IgD (clone 11-26c, biotin-conjugated; eBio- science), rabbit anti-mouse pan-laminin (455, unconjugated; a gift from L. Sorokin, Muenster University), rat anti-mouse metallophilic Mf (clone MOMA-1, biotin-conjugated; BMA, Augst, Switzerland), rat anti-mouse MZ Mf (clone ER-TR9, biotin-conjugated; Acris Antibodies, Hidden- hausen, Germany), rat anti-mouse B220 (clone RA3-6B2, biotin- or FITC- conjugated), mouse anti-mouse CD157 (clone BP-3, PE-conjugated), rat anti-mouse IgG1 (clone A85-1, FITC-conjugated), rat anti-mouse IgG2a (clone R19-15, FITC-conjugated), rat anti-mouse IgG2b (clone R12-3, FITC-conjugated), rat anti-mouse CD4 (clone RM4-5, AF488-conjugated; Caltag Laboratories, Hamburg, Germany), rat anti-mouse CD8 (clone 5H10, AF488-conjugated; Caltag Laboratories), and rat anti-mouse MAdCAM-1 (clone MECA-89, biotin-conjugated). Cryosections were washed for 10 min in PBS, 0.05% Tween 20 twice between incubation steps with depicted primary and secondary donkey anti-rabbit IgG (Cy3- or Cy5-conjugated; Dianova) Abs to detect pan-laminin or streptavidin-conjugated Cy3 (Amer- sham Biosciences) to detect biotinylated primary Abs. Slides were mounted in the anti-fading reagent Mowiol and sections were analyzed using an im- munofluorescence microscope (Axioplan 2; Carl Zeiss, Jena, Germany) equipped with a high-sensitivity grayscale digital camera (OpenLab imaging 4012
REQUIREMENTS FOR VIRUS-SPECIFIC MEMORY B CELL ACTIVATION by guest on May 25, 2017 http://www.jimmunol.org/ Downloaded from system; Improvision, Lexington, MA). Separate images were taken for each section, analyzed, and merged afterward. Results Frequency of virus-specific MBCs and quantitative analysis of secondary immune response Noninfectious enveloped hCMV particles, referred to as VLPs in this study, were chosen as immunizing Ag to generate and study virus-specific MBCs. To generate hCMV-specific MBCs, B6 mice were immunized three times and rested for at least 6 wk after the last immunization. Throughout this work hCMV gB was used for the detection of virus-specific MBC Ab responses, as this Ab specificity is immunodominant in mice (9). We were interested to know how many MBCs are necessary to obtain a measurable gB Ab response in adoptive transfer experiments. Toward this end, isotype-switched (IgG + ) CD19 + B cells binding to fluorescently labeled gB were isolated by cell sorting, and defined cell numbers were adoptively transferred into individual RAG1 2/2 mice (Sup- plemental Fig. 1). Challenge with VLPs resulted in strong gB- specific serum IgG in RAG1 2/2 recipients, receiving as few as 25 gB-specific MBCs (Fig. 1), demonstrating the immense immuno- logical power of MBCs. Homing of MBCs to specific niches within secondary lymphoid tissue is required for T cell-independent reactivation Previous findings of our group suggested that MBCs need to mi- grate to specific lymphoid compartments for strong T cell- independent activation by recurrent Ag (9). As depicted in Fig. 2A, MBCs are not fully responsive to Ag challenge within the first few hours after adoptive B cell transfer. Only weak gB-specific serum IgG titers, indicating suboptimal MBC reactivation, were detected in recipient mice when Ag was administered within minutes after adoptive B cell transfer. Strongest serum IgG titers were obtained when VLPs were injected between 24 h and 7 d after MBC transfer. Secondary application of Ag led to significant increase in serum IgG titers of recipients that received the first Ag challenge simulta- neously with B cell transfer, indicating that MBCs were not fully responsive to the first challenge but survived at least 14 d and could readily be reactivated. Together with our findings that MBCs cannot be reactivated in TNF/LTa 2/2
recipients (Fig. 2B) (9), which display complex immune abnormalities including disorga- nization of lymphoid tissue (11), these observations point to the Kataloq: content -> jimmunol content -> Dear Editor content -> Sample Letter for Contributions of $250 or More to ncw content -> [insert your firm’s name here] content -> Nat 74548-10. 2016 Fact sheet for Individuals content -> Əlavə d ətraf Mühitin və Sosial Sahənin İdarə Edilməsi və Monitorinqi Planı cqbk nin Genişləndirilməsi Layihəsi, Azərbaycan Ətraf Mühitə və Sosial Sahəyə Təsirin Qiymətləndirilməsinin Əlavəsi Yekun variant MÜNDƏRİcat jimmunol -> Strains Mycobacterium tuberculosis Yüklə 1,93 Mb. Dostları ilə paylaş:
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