Acquired hematopoietic stem cell defects determine B-cell repertoire changes associated with aging Lisa M. Guerrettaz*, Sara A. Johnson*, and John C. Cambier*† Department of Immunology, University of Colorado, Denver School of Medicine and National Jewish Health, Denver, CO 80206 Communicated by Philippa Marrack, National Jewish Medical and Research Center, Denver, CO, June 9, 2008 (received for review February 28, 2008) Aging is associated with an inability to mount protective antibody decreased ability of LT-HSCs from aged animals to replenish the responses to vaccines and infectious agents. This decline is asso- follicular compartment. Studies described in this report test this ciated with acquisition of defects in multiple cellular compart- hypothesis. ments, including B cells. While peripheral B-cell numbers do not A major obstacle to studying the effects of aging on the B-cell decline with aging, the composition of the compartment appears compartment is the difficulty of resolving B-cell repertoire to change, with loss of naı¨ve follicular B cells, accumulation of changes in wild type (WT) mice where B cells exhibit diverse Ig antigen-experienced cells, and alteration of the antibody reper- specificity. Therefore, to address the relationship between the toire. The underlying cause of this change is unknown. We tested age-associated decline in B lymphopoiesis and the peripheral the hypothesis that aging-associated repertoire changes can be B-cell repertoire, we used the 3-83 ␮␦ Ig transgenic (Tg) mouse attributed directly to decreased B lymphopoiesis. Using an Ig model. In young 3-83 ␮␦ mice, ϳ93% of B cells express transgene transgenic model to report changes in the B-cell repertoire, we encoded Igs (tgIg) specific for the MHC class I antigen H-2Kk/b, show that the reduced B-cell generative capacity of ‘‘aged’’ long- an exotic antigen in the H-2Kd mice (B10.D2) used in this study term reconstituting hematopoietic stem cells (LT-HSCs) alters the (15). The remaining B cells in these mice express B-cell receptors representation of antigen specificities in the peripheral B-cell encoded by endogenous Ig (enIg) genes. As reported previously, repertoire. Further, we show that reconstitution using suboptimal immature B cells in the bone marrow of aged 3-83 ␮␦ mice numbers of fully functional LT-HSCs results in the generation of a maintain this ratio of 93% tgIg:7% enIg (6). Importantly how- similarly altered B-cell repertoire. This may be an important factor ever, in these same aged mice the frequency of peripheral B cells to consider when deciding the number of bone marrow cells to (B cells in blood, spleen, and lymph nodes) expressing tgIg transplant in the clinical setting. In conclusion, when B lympho- decreases, and the compartment becomes dominated by cells poiesis is limited peripheral B-cell homeostasis is altered. This is expressing enIg that appear, on the basis of surface phenotype, ϩ reflected in reduced diversity of the B-cell repertoire, which likely to be antigen experienced (memory, marginal zone, and CD5 reduces the protective quality of the immune response. B1-like). By 18 months of age, ϳ90% of 3-83 ␮␦ mice express a B-cell compartment skewed in favor of enIg. In ϳ75% of these Ͼ ollicular (FO) lymphocytes are the largest B-cell subpopu- mice, 60% of B cells express enIg (6). Furthermore, this enIg Flation in peripheral lymphoid organs and display the maxi- population is highly enriched in cells specific for auto- and mum breadth of germline-encoded antibody specificity for for- environmental antigens, a hallmark of aging. Finally in aged 3-83 ␮␦ eign antigens. Because of the breadth of this repertoire, these mice, naive FO B cells are reduced in number and uniformly naı¨ve FO cells are uniquely suited to give rise to protective express transgene-encoded receptors. Thus, in this model system primary immune responses and to be selected for high-affinity a shift in the peripheral B-cell repertoire away from tgIg and antibody production. toward enIg expression is an easily measured indicator of Aging is associated with decreased efficacy of vaccination in immunologic aging. both humans and mice, increased susceptibility to infection, and Using this model, we show that although LT-HSCs increase in increased rate of cancer (1–4). In mice, where it can be exam- number as mice age, these LT-HSCs lose functionality, resulting ined, aging is associated with a reduction in FO B cells, but not in decreased ability to generate B cells. This decreased output of total B-cell numbers (5, 6). This reduction in FO B-cell numbers naive B cells from the BM determines the aged peripheral is further associated with a reduction in immunoglobulin (Ig) repertoire. Furthermore, artificial limitation of HSC function in diversity within the B-cell pool and an increase in the frequency young animals mimics aging, generating a B-cell repertoire of antigen-experienced cells, including marginal zone, B1, and skewed toward nonfollicular cell types. memory cells. In young animals the sizes of these compartments Results and Discussion is independently regulated (7). Thus, it seems plausible that the humoral immune defects seen in aging may be the consequence Aging-Associated B-Cell Repertoire Changes Are Caused by Stem-Cell of reduced frequency of FO B cells bearing high-affinity recep- Defects. To assess the stem-cell contribution to aging-associated tors for offensive pathogens. changes in the peripheral B-cell repertoire, we analyzed the ability of bulk HSC populations from prescreened immunologically aged, FO B cells are short-lived (estimates ranging from 40 to 120 d) Ͼ ␮␦ and nonself-renewing and therefore must be constantly replen- i.e., mice in which 60% of B cells express enIg, 3-83 mice to ished by new B cells produced in hematopoietic organs, e.g., recapitulate, in young lethally irradiated WT recipients, the reper- adult bone marrow (BM) (8, 9). For reasons that are unclear, transplanted bone marrow stem cells from older adult human Author contributions: L.M.G., S.A.J., and J.C.C. designed research; L.M.G. and S.A.J. per- donors often do not give rise to B cells in recipients (10). formed research; L.M.G., S.A.J., and J.C.C. analyzed data; and L.M.G., S.A.J., and J.C.C. wrote Similarly, LT-HSCs from aged mice, while more numerous than the paper. in young animals, are selectively impaired in the ability to The authors declare no conflict of interest. reconstitute the B-cell compartment of irradiated recipients (11, Freely available online through the PNAS open access option. 12). Finally, autoreconstitution of B cells following lymphoab- *These authors contributed equally to this work. lation is impaired in aged mice (13, 14). On the basis of these †To whom correspondence should be addressed at: Department of Immunology, K803A, findings we hypothesize that the poor quality of the primary National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. antibody response seen in the aged results from a decline in naive E-mail: [email protected]. follicular B-cell numbers, which is in turn the result of the © 2008 by The National Academy of Sciences of the USA 11898–11902 ͉ PNAS ͉ August 19, 2008 ͉ vol. 105 ͉ no. 33 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805498105 Downloaded by guest on September 26, 2021 B10.D2 Recipient A Young Aged Young Aged 3.45 96.1 2.94 96.5 2.11 6.67 Young cKit 3-83µδ 0.37 0.037 0.38 0.15 Sca-1 Donor BM 89.3 10.4 90.7 9.17 B Young B10.D2 Recipients 89.6 0.29 27.3 40 Aged Aged µδ 0.27 0.012 0.097 0 3-83 10.1 0 31.6 1.1 B220 LT-HSC 5.92 92.2 22.4 38 Idiotype Donor Fig. 1. Aging-associated decline in B lymphopoiesis is B-cell/stem cell auton- Young omous. Young or aged WT (B10.D2) mice were lethally irradiated, and lym- 1.65 0.21 38.8 0.73 phocyte-depleted bone marrow cells from young or prescreened immunolog- ically aged 3-83␮␦ animals were transferred by i.v. injection. Donor BM was β pooled from multiple animals before depletion, and BM equivalents were IgM TCR transferred at a ratio of one donor equivalent to four recipients. Reconstitu- Idiotype CD4 tion of the peripheral B-cell compartment was assessed 6 weeks posttransfer. Splenocytes were gated on live, CD19ϩ B cells, and 3-83␮␦ idiotype and total p<.0001* C 100 IgM expression was evaluated. Data are representative of analyses of a Young Lindep BM minimum of three individual animals in each category. Young 5FU BM 80 Young LT-HSC dep B cells 60 Aged Lin BM toire changes seen in the HSC donors. Furthermore, to assess the + Aged 5FU BM role played by the BM microenvironment in the development of the Aged LT-HSC aged B-cell repertoire, the converse experiment, in which bulk 40 HSCs from young 3-83 ␮␦ mice were transferred into aged lethally 20 irradiated recipients, was also performed. B-cell reconstitution was % Idiotype assessed 6 weeks posttransfer by flow cytometric analysis of the 0 splenic B-cell compartment. As shown in Fig. 1, lymphocyte- Young Aged depleted BM from young 3-83 ␮␦ mice reconstituted a young-like (predominantly tgIg, idiotype positive) repertoire in both young and Donors old recipients (top panels), whereas lymphocyte-depleted BM from ␮␦ Fig. 2. LT-HSCs from aged animals exhibit reduced B-cell generative capacity aged 3-83 mice reconstituted an aged-like (predominantly enIg, associated with repertoire alteration. Untreated whole BM was harvested idiotype negative) repertoire in both young and old recipients from four young or four aged mice, RBCs were lysed, and LT-HSCs purified as (bottom panels).