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Fcrl5 and T-Bet Define Influenza-Specific Memory B Cells That Predict Long-Lived Antibody 2 Responses 3 4 Anoma Nellore1, Christopher D

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Fcrl5 and T-Bet Define Influenza-Specific Memory B Cells That Predict Long-Lived Antibody 2 Responses 3 4 Anoma Nellore1, Christopher D bioRxiv preprint doi: https://doi.org/10.1101/643973; this version posted May 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Fcrl5 and T-bet define influenza-specific memory B cells that predict long-lived antibody 2 responses 3 4 Anoma Nellore1, Christopher D. Scharer2, Rodney G. King3, Christopher M. Tipton4, Esther 5 Zumaquero3, Christopher Fucile3,5, Betty Mousseau3, John E. Bradley6, Kevin Macon3, Tian Mi2, 6 Paul A. Goepfert1,3, John F. Kearney,3 Jeremy M. Boss,2 Troy D. Randall6, Ignacio Sanz4, 7 Alexander Rosenberg3,5, Frances E. Lund3 8 9 10 11 1Dept. of Medicine, Division of Infectious Disease, 3Dept. of Microbiology, 5Informatics Institute, 12 6Dept of Medicine, Division of Clinical Immunology and Rheumatology, at The University of 13 Alabama at Birmingham, Birmingham, AL 35294 USA 14 15 2Dept. of Microbiology and Immunology and 4Department of Medicine, Division of 16 Rheumatology, at Emory University, Atlanta, GA 30322, USA 17 18 19 20 21 22 23 24 25 26 27 28 Lead Contact and to whom correspondence should be addressed: [email protected] 29 30 Mailing address: Frances E. Lund, PhD 31 Charles H. McCauley Professor and Chair 32 Dept of Microbiology 33 University of Alabama at Birmingham (UAB) 34 Dept of Microbiology 35 University of Alabama at Birmingham (UAB) 36 276 BBRB Box 11 37 1720 2nd Avenue South 38 Birmingham AL 35294-2170 39 40 SHORT RUNNING TITLE: Effector memory B cell development after influenza vaccination 41 bioRxiv preprint doi: https://doi.org/10.1101/643973; this version posted May 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 42 Abstract: Early surrogates for long-lived immunity after inactivated influenza vaccination (IIV) 43 are lacking. Antigen-specific memory B cells (Bmem) after IIV have been recently identified. We 44 show that the antigen-specific Bmem compartment after IIV is heterogenous and comprises a 45 clonotypically and transcriptionally distinct T-bethi subset that persists in circulation over time 46 after vaccination and exclusively correlates with the long-lived antibody response. We 47 demonstrate that this subset has an effector memory transcriptome and is epigenetically 48 remodeled to facilitate intracellular immunoglobulin production. Finally, via clonal sharing, we 49 show an enriched in vivo ontologic relationship between the secondary plasmablast response 50 that develops after vaccine boost and the T-bethi fraction of the flu-specific Bmem response that 51 forms after initial prime. Collectively, our data identify a novel biomarker of durable humoral 52 immunity after influenza vaccination. 53 54 55 Keywords: T-bet, memory B cells, plasmablasts, influenza vaccination, durable immunity 56 bioRxiv preprint doi: https://doi.org/10.1101/643973; this version posted May 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 57 Introduction: 58 After inactivated influenza vaccination (IIV), durable humoral immunity is mediated by 59 long-lived antibody (Ab) secreting cells (ASCs) and memory B cells (Bmem).1 In the influenza 60 experienced adult, inactivated influenza vaccination (IIV) mobilizes pre-existing hemagglutinin 61 (HA) antigen-specific memory B cell (Bmem) subsets.2 These responding Bmem may assume 62 different fates. They may proliferate and affinity mature in the germinal center (GC) response to 63 produce daughter cohorts of HA-specific Bmem; they may die immediately upon activation; they 64 may directly differentiate into ASCs; or they may persist as long-term memory. The early cues 65 that direct vaccine-elicited antigen-specific Bmem toward any of these various fates and the 66 relationship of these Bmem fate decisions to the development of durable humoral immunity are 67 not completely clear. 68 Traditionally defined according to expression of the canonical memory marker, CD27,3-6 69 human Bmem are now appreciated to be a heterogeneous subset by transcriptional 70 programming, differentiation capacity and responsiveness to antigen. For instance, a subset of 71 atypical Bmem that express the lineage defining transcription factor (TF), T-bet, have been 72 identified in the context of infection, vaccination, aging and autoimmunity.7 We recently reported 73 in a murine model of influenza infection that T-bet expression in flu-specific B cells as necessary 74 for the development of long-lived ASCs to primary influenza infection as well as for the 75 development of the secondary ASC (plasmablast or PB) in response to challenge infection.8 76 This finding led us to query the relationship between vaccine-elicited durable immunity and the 77 early vaccine-specific Bmem response after IIV of healthy human subjects. 78 Here we study the clonotype, transcriptome and epigenome of circulating human Bmem 79 after IIV strictly classified according to antigen reactivity and T-bet expression. We find that the 80 BCR inhibitory signaling molecule, FcRL5,9 accurately identifies HA-specific Bmem by T-bet 81 expression. We also find HApos T-bethi Bmem are transcriptionally remodeled as effector 82 memory with increased accessibility at an Xbp1 enhancer locus that aligns with an established 83 T-bet binding site.10 We show that the early HApos T-bethi subset predicts the development of 84 long-lived Ab response to the vaccine. Finally, we demonstrate that HApos T-bethi Bmem clones 85 are preferentially recalled into the early antigen-specific PB repertoire after re-vaccination. 86 Results: 87 I. T-bet parses phenotypically distinct HA-specific humoral immune readouts after IIV 88 After inactivated influenza vaccination (IIV), there is an early expansion of the circulating 89 follicular helper T cell (cTfh, CD4+CXCR5+PD1+ICOS+ T cell)11,12 and plasmablast fractions bioRxiv preprint doi: https://doi.org/10.1101/643973; this version posted May 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 90 (CD27posCD38hi, PB) within 5-10 days of vaccine receipt. 13,14 The plasmablast fraction has 91 been extensively studied as a measure of vaccine induced antigen-specific humoral immunity.15- 92 18 However, plasmablasts are rapidly cycling, short-lived cells and therefore do not reflect 93 longitudinal immune protection after vaccination. Using fluorochrome labeled tetramer or 94 monomer reagents, hemaggulutinin (HA)-specific Bmems have been identified within one month 95 after IIV. 19-21 This antigen-specific Bmem compartment after IIV has been described as 96 heterogeneous, comprising typical and atypical memory B cells.19-21 We wanted to assess the 97 phenotype and kinetics of the HA-specific Bmem compartment after IIV and the relationship of 98 this compartment to the plasmablast and long-lived antibody (Ab) response. We hypothesized 99 that the HA-specific Bmem and plasmablast humoral immune responses are distinct vaccine 100 outputs. In order to test this hypothesis, we administered the 2015 IIV to 19 healthy subjects 101 and drew blood at sequential proximal time points (day 0, 7, 14, 21, 28) as well as distant time 102 points from vaccine receipt (day 120). Detailed vaccination and infectious histories from 103 subjects were not recorded. Gating strategies are shown in Supplemental Fig 1 A-C. Specificity 104 of our HA tetramer has been described22 and, unless specifically indicated, HA tetramer 105 matches relevant vaccine antigen strain (H1 or H3). Our 2015 cohort responded to the vaccine 106 as evidenced by a significant increase in the circulating cTfh and PB fraction from day 0 to day 7 107 of IIV (Supplemental Figure 1 B,1C). Also within 7 days of IIV, two phenotypically unique 108 IgDnegCD38med/lo HA-specific (H1) Bmem populations were identified in circulation. These two 109 populations are shown in representative FACS plots (Figure 1A) and are phenotypically distinct 110 from CD38hi B cells and from each other according to HA (H1)-tetramer, Ki-67, T-bet expression 111 and SSC-A parameters. These HA-specific Bmem subsets were further phenotyped as shown 112 in Supplemental Figure 1D. Both subsets were enriched in the expression of the canonical 113 memory B cell marker, CD27 (Supplemental Figure 1D). Consistent with prior description of 114 atypical T-bethi B cells,8,19,20,23-29 we found day 7 HApos T-bet hi Bmem to have increased 115 expression of the integrin CD11c and the inhibitory B cell receptor (BCR) signaling molecule 116 FcRL5 as well as decreased expression of the chemokine receptor CXCR5 and the complement 117 receptor CD21. Underscoring these phenotypic differences, we interrogated the morphology of 118 H1pos T-bethi Bmem using ImageStream and found them to be larger and more granular than 119 H1pos T-betlo Bmem (Supplemental Fig 1E). 120 Ki67 is not a binary marker of proliferation and its expression at any individual timepoint 121 does not necessarily reflect ongoing or future proliferative potential but may rather reflective 122 prior replicative history.30,31 Thus we evaluated for phenotypic changes in Ki-67 expression in 123 the HA-specific Bmem compartment over time after IIV. We found that Ki-67 expression was bioRxiv preprint doi: https://doi.org/10.1101/643973; this version posted May 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 124 not sustained over time in the circulating HApos T-bethi Bmem compartment nor did the 125 circulating HApos T-betlo compartment develop significant Ki-67 expression over time after 126 vaccination (Figure 1B).
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