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Changes in the B-cell repertoire with age
Vaccine 18 (2000) 1624±1628
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Changes in the B-cell repertoire with age Marc E. Weksler* Division of Geriatrics and Gerontology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA
Abstract Changes in the B-cell repertoire during aging include a shift in antibody speci®cities from foreign to autologous antigens associated with a decline in the activity of conventional B2 compared to B1 lymphocytes. The age-associated increase in B1 lymphocyte number and activity contribute to the increased serum concentration of autoantibodies and the B-cell clonal expansions that develop with age. Aging is also associated with a decreased diversity of the antibody response re¯ected in the preferential loss of IgG and high anity antibodies following immunization with a foreign antigen. Many of these changes can be traced to an impaired capacity of T cells to support isotype switching and somatic mutation in the periphery and the generation of a diverse B-cell repertoire from bone marrow B-cell precursors. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Aging; Immunology; Repertoire
1. Introduction The birth of modern humoral immunity has been dated to 1890 when von Behring and Kitasato reported that the transfer of serum anti-bacterial antibodies from immunized animals to naive animals protected the recipients from bacterial challenge [reviewed in 1]. Subsequently, Tilselius and Kabat demonstrated that antibodies were immunoglobulin molecules; Porter and Edelman established the structural basis of antibody activity; and Tonegawa and Baltimore described the molecular mechanism underlying the generation of antibody diversity. During this period, the complex interactions of B, T and APC required for the generation of antibodies were discovered. As knowledge of the cellular and molecular regulation of antibody production has grown, it has been possible to explore the eect of age on humoral immunity with increasing precision. These studies have shown that the eects of age on humoral immunity are due largely to changes in the B-cell repertoire. For this
* Tel.: +1-212-746-6478; fax: +1-212-746-6382. E-mail address: [email protected] (M.E. Weksler).
reason, we have chosen to use the phrase eect of age on the ``B-cell repertoire'' rather than on the ``antibody repertoire''. Aging in¯uences both the quantity and the quality of humoral immunity. Quantitative changes include an altered number of immunoglobulin (Ig) and antigenspeci®c Ig-secreting B-cells. Qualitative changes include shifts in the number and activity of B-cell subsets as well as shifts in the antibody repertoire with respect to speci®city, isotype, anity, and idiotype. A comprehensive review of aging and the immune system was published in the December, 1997 issue of Tranplantation Reviews [2]. This brief review includes some recent observations on the eect of age on the B-cell repertoire. 2. Age-associated changes in the B-cell repertoire The ®rst evidence that the humoral immunity changed with age was the report that the quantity of serum antibodies speci®c for foreign antigens declined with age [3]. Soon thereafter, aging was shown to aect the quality of the antibody response to bacteria produced by rabbits [4]. In this study, antibodies to bacteria
0264-410X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 9 9 ) 0 0 4 9 7 - 1
M.E. Weksler / Vaccine 18 (2000) 1624±1628
induced in old rabbits agglutinated the bacteria less rapidly than did antibodies induced in young rabbits. Speci®c antibody responses in humans to virtually all vaccines, including anti-tetanus toxin, anti-in¯uenza and anti-encephalitis viruses as well as salmonella and pneumococcus bacteria decrease with age [5]. In experimental animals the eect of age on the antibody response depends on the type of antigen [6]. Thus, the antibody response to T-dependent antigens produced by B2 lymphocytes is diminished by 90% in old mice. In contrast, the antibody response to T-independent antigens and antigens that stimulated B1 lymphocytes in old mice is equal to or greater than that in young mice. The fact that the antibody response to most foreign antigens was lower in old compared to young individuals led to a view that aging led to an immune de®ciency state. However, we found no evidence for a global loss of B-cell function as neither the total number of B-cells or of Ig-secreting B-cells were decreased with age [7]. This is explained by an increase in the number of B-cells secreting antibodies to autologous antigens. Thus, the decrease in antibody response to a foreign antigen re¯ects not a decrease in humoral immunity but a change in the B-cell repertoire with respect antigen speci®city. In addition to the shift in the repertoire with respect to antibody speci®cities from foreign to autologous antigens, we recognize that aging aects both the size and the diversity of the Bcell repertoire leading to changes in both the quantity and quality of the antibody produced. 2.1. Changes in the repertoire of B-cells during aging: self and non-self speci®cities The concentration of natural and antigen-induced antibodies speci®c for foreign antigens decline with age in both humans and experimental animals [5,6]. Furthermore, the number of B-cells that bind a foreign antigen did not decrease although the number of speci®c antibody-forming cells did [7]. These defects are probably related to impaired T cell help as the response to T-independent antigens in young and old mice are comparable [6]. Despite these defects in the antibody response to most foreign antigens, neither the level of serum immunoglobulins nor the number of Ig-secreting Bcells declines with age [5,8]. Furthermore, we demonstrated that the total number of antigen-induced Igproducing cells were not decreased in old compared to young mice [8]. This paradox was, at least in part, resolved by the ®nding that the serum concentration of autoantibodies increased with age [9]. We have shown that immunization of young mice with a foreign antigen stimulates a large number of Ig-secreting cells producing antibodies to the nominal antigen and a very
1625
small number to autoantigens. The converse is true in old mice where few Ig-secreting producing antibodies bind the nominal antigen and a large number bind autoantigens [6,8]. Thus, aging is associated with alterations in the B-cell repertoire with respect to the ratio of antibodies speci®c for the nominal versus autologous antigen. The autoantibodies detected at increased concentrations in the serum of elderly as compared to young humans were speci®c for autoantigens, DNA, immunoglobulin, thyroglobulin, found at high levels in patients with systemic lupus erythematosis, rheumatoid arthritis or hypothyroidism [10]. For this reason, it was not clear whether autoantibodies in the elderly re¯ected subclinical autoimmune disease or the dysregulation of the immune response. To distinguish between these two possibilities, we measured the reactivity of serum from young and old mice to antigens in normal tissues that were not selected for their association with an autoimmune diseases [11]. This technique had shown that autoantibodies detectable in serum from normal, young mice bind to proteins in lysates from a variety of normal tissues. Furthermore, the IgM autoantibodies were not stimulated by foreign antigens as they were present in serum from antigen-free mice. We showed that autoantibodies that increase with age have the same binding spectra for antigens of the spleen, skin and muscle as natural autoantibodies present in young mice at lower concentration [12]. There was a single exception to this rule detected in autoantibodies in old mice: a single band was detected in old but not young mice to proteins present in brain extracts. Therefore, as a rule, the quantity but not the speci®city of serum autoantibodies dier in old and young mice. The stability of autoantibody reactivity during aging was also demonstrated in humans [13]. The reactivity of human serum obtained from the same ®ve individuals was measured in sera obtained over a 25-year interval. The ®rst serum specimen was obtained when the donors were, on average, 43 years old and the last when the donors were, on average 69 years old. IgG and IgM immunoblots were made with lysates of normal lung, kidney, stomach, and thymus tissue. The pro®les of self-reactivity of both serum IgM and IgG were strikingly similar in the sera obtained from the same individual. These results indicate that in both humans and mice autoantibodies in young and old individuals express similar speci®cities suggesting the autoreactivity of antibodies are not random but highly selected based on their recognition of a limited set of auto-antigens. Indirect evidence from these studies in humans also indicated that auto-anti-idiotypic antibodies increase with age. Previous studies in mice had shown that among the autoantibodies that increase in old mice following im-
1626
M.E. Weksler / Vaccine 18 (2000) 1624±1628
munization are auto-anti-idiotypic antibodies [14]. This class of autoantibodies is of interest because auto-antiidiotypic antibodies inhibit the secretion of antibodies to nominal antigen following immunization. Thus, the appearance of auto-anti-idiotypic antibodies in old mice not only re¯ects immune senescence but contributes to the impaired antibody response to the nominal antigen. Mixed transfer experiments demonstrated that the secretion of auto-anti-idiotypic antibodies in mice are stimulated by T cells from in old mice. Thus, like other defects in humoral immunity, the appearance of auto-anti-idiotypic antibodies with age re¯ect altered T-cell function. 2.2. Changes in the repertoire of B-cells during aging: appearance of monoclonal antibodies and clonal B-cell expansions In addition to the age-associated polyclonal increase in serum autoantibodies, there is an increasing frequency of serum monoclonal immunoglobulin (MIg) [15]. Sensitive analytic techniques reveal that as many as one-half of elderly humans and old mice have MIg in their serum. The age-associated shift in the speci®city of serum antibodies from foreign to autoantigens is also re¯ected in the speci®cities of serum MIg as approximately 50% of MIg react with autoantigens [16]. Indirect evidence suggests that cells that secrete MIg are drawn from the CD5+ B-cell subpopulation [17]. Our lifespan study of C57Bl/6Xid mice, which have less than 5% of the normal complement of CD5+ Bcells, revealed that these mice do not develop either autoantibodies or stable serum MIg during aging [18]. Thus, CD5+ B-cells appear to be the source of both the age-associated autoantibodies and serum MIg. We also have examined the B-cell populations for evidence of age-associated B-cell clonal expansions that might be the cellular basis of the serum MIg. To search for B-cell clonal expansions we used Ig heavy chain mRNA CDR3 size analysis [19]. We found that virtually all old mice, whether or not they had MIg in their serum, had splenic B-cell VHspeci®c expansions. Direct sequencing of the RT-PCR products yielded readable sequences con®rming the clonal nature of the B-cell expansion. Old mice frequently have more than one B-cell clonal expansion and these were detected in multiple lymphoid compartments including the spleen, lymph nodes, bone marrow, and thymus. The majority of the B-cell clonal expansions are derived from the CD5+ subset of Bcells. The B-cell clonal expansions develop in the peripheral immune compartments and not generated within B-cell precursor populations. Preliminary evidence suggests that B-cell clonal expansions do not develop in MHC class II-de®cient mice, which lack CD4 T
cells, although mice de®cient in MHC class I antigens and CD8 T cells develop B-cell clonal expansions at the same frequency as wild type mice. 2.3. Age-associated changes in antibody speci®city: isotype and anity In mice, the age-associated increase in serum immunoglobulin concentration is associated with a marked increase in the number of Ig-secreting cells [7]. In humans, the serum concentration of IgM, IgA and IgG also increases with age although the concentration of IgD decreases during aging [20]. In contrast to the increase in the steady state level of serum IgG and IgM with age, the number of antigen-speci®c IgG- and IgM-producing cells stimulated by immunization is decreased in old compared to young mice [21]. These studies showed that there was a greater loss of IgGproducing compared to IgM-producing cells with age. The preferential loss of IgG antigen-speci®c antibody re¯ects an age-associated defect in isotype switching probably as a consequence of impaired T-cell function required for the generation of germinal centers and for isotype switching. Old mice, in addition to impaired isotype switching following immunization with DNP-BGG, produce a restricted repertoire of anti-DNP antibodies with respect to anity. The limited diversity of haptenspeci®c antibodies is due to the limited production of high anity antibodies. The production of high anity antibodies depends upon the process of somatic mutation and this process like isotype switching takes place within germinal centers. The age-associated impairment in both processes has been shown to be due to an age-associated defect in T-cell function [14]. We have traced the defect back to thymocytes [22]. Mixed cell transfer experiments in which the capacity of thymocytes from mice of dierent ages to support the production of high anity antibodies were performed. We transferred thymocytes from syngeneic mice of dierent ages with fetal liver cells into irradiated young mice and measured the anity of the antibody response in immunized recipients. High anity antibodies were produced by recipients of thymocytes from 2 to 4 month old mice aged while high anity antibodies were not seen in recipients of thymocytes from 12 to 24 month old mice. The preferential loss during aging of IgG and high anity antibody, the most protective antibodies against bacterial and viral diseases, contribute to the increased susceptibility and severity of infectious disease in the elderly as well as the lower protection aorded by vaccines in old compared to young humans from infection. While epidemiological studies have shown that the magnitude antibody response following vaccination is a good indication of protective
M.E. Weksler / Vaccine 18 (2000) 1624±1628
immunity, evidence now exists that antibody-mediated protection depends on the anity and isotype as well as the quantity of antibody [23]. Anti-pneumococcal antibody resulting from the immunization of old mice was less protective when transferred to young naive mice than antibody from young mice. The greater protection of the antibody preparation from young mice was correlated with its higher anity for the pneumococcus. 2.4. Age-associated changes in the B-cell repertoire: VH utilization and B-cell subsets In contrast to the 3 ' VH gene family preference and few N additions in the CDR3 region seen in antibodies from fetal and neonatal as compared to adult mice, old mice show no similar global changes in VH utilization or N additions [24]. However, there are subtle alterations in VH gene usage with age. We demonstrated that VH11 and Q52 variable gene families are used to a greater extent by spleen cells of old compared to young mice. These gene families are used preferentially by CD5+ B-cells and their increased usage can be explained by the 3-fold increase in CD5+ Bcells in the spleens of old compared to young mice. Individual old mice overutilize VH gene families [25]. This results from B-cell clonal expansions which can be detected in the spleen as well as in other lymphoid compartments in virtually all mice over 18 months of age [19]. The majority of the B-cell clonal expansions are found in the CD5+ B-cell subset and many express the VH11 gene family. The sequence of the Ig CDR3 region in one such clonal B-cell expansion was identical to that previously reported for an anti-phosphatidylcholine antibody produced by CD5+ B-cells [24]. Another age-associated change in VH gene utilization by B-cells was seen following immunization with phosphorylcholine (PC) [26]. Virtually all B-cells in young BALB/c mice that produce anti-PC antibody use the S107 gene family. In contrast, only 30% of anti-PC antibodies produced by old mice use the S107 gene family. These shifts in VH gene family utilization provide the structural basis for the age-associated shift in the idiotypic repertoire that has been observed during aging [27]. 3. Clinical consequences of age-associated changes in the B-cell repertoire The changes in the quality and quantity of antibody during aging contributes importantly to the increased susceptibility and sensitivity of the elderly to infection. Furthermore, these same factors explain the impaired ecacy of vaccines in protecting elderly from infection.
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Finally, it is likely that the B-cell clonal expansions that occur in mid-life are the precursors of late-life Bcell neoplasms as benign colonic adenomas are precursors of colonic carcinomas. Acknowledgements Supported in part by NIH grants AG 08707, AG 14669, the Gladys and Roland Harriman Foundation, and the DeWitt Wallace Foundation.
References [1] Silverstein AM. A history of immunology. New York: Academic Press, 1989. [2] Hodes RJ. Aging and the immune system. Transplant Reviews 1997;160:5±184. [3] Thomsen O, Kettel K. Die starke der menschlichen Isoagglutninne und entsperchenden Blutkorperperchenrezeptoren im vershiedenenen Lebensaltern. Z Immunitatsforsch 1929;63:67±93. [4] Baumgartner L. Age and antibody production. Qualitative changes in antisera associated with aging. J Immunol 1934;27:407±16. [5] Schwab R, Walters CA, Weksler ME. Host defense mechanisms and aging. Semin Oncol 1989;16:20±7. [6] Hu A, Ehleiter D, Ben-Yehuda A, et al. Eect of age on the expressed B-cell repertoire: role of B-cell subsets. Int Immunol 1993;5:1035±9. [7] Zhao KS, Wang YF, Gueret R, Weksler ME. Dysregulation of the humoral immune response in old mice. Int Immunol 1995;7:929±34. [8] Callard RE, Basten A, Waters LK. Immune function in aged mice. II. B-cell function. Cell Immunol 1977;31:26±36. [9] Rowley MJ, Buchanan H, Mackay IR. Reciprocal change with age in antibody to extrinsic and intrinsic antigens. Lancet 1968;2:24±6. [10] Hallgren HM, Buckley III CE, Gilbertsen VA, Yunis EJ. Lymphocyte phytohemagglutinin responsiveness, immunoglobulins and autoantibodies in aging humans. J Immunol 1973;111:1101±7. [11] Haury M, Sundblad A, Grandien A, Barreau C, Coutinho A, Nobrega A. The repertoire of serum IgM in normal mice is largely independent of external antigenic contact. Eur J Immunol 1997;27:1557±63. [12] Nobrega A, Haury M, Gueret R, Coutinho A, Weksler ME. The age-associated increase in autoreactive immunoglobulins re¯ects a quantitative increase in speci®cities detectable at lower concentrations in young mice. Scand J Immunol 1996;44:437± 43. [13] Lacroix-Desmazes S, Mouthon L, Kaveri SV, Kazatchkine MD, Weksler ME. Stability of natural self-reactive antibody repertoires during aging. J Clin Immunol 1999;19:26±34. [14] Weksler ME, Russo C, Siskind GW. Peripheral T cells select the B-cell repertoire in old mice. Immunol Rev 1989;110:173±85. [15] Radl J. Age-related monoclonal gammapathies: clinical lessons from the aging C57BL mouse. Immunol Today 1990;11:234±6. [16] Merlini G, Farhangi M, Osserman EF. Monoclonal immunoglobulins with antibody activity in myeloma, macroglobulinemia and related plasma cell dyscrasias. Semin Oncol 1986;13:350±65. [17] van Arkel C, Hopstaken CM, Zurcher C, et al. Monoclonal
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[18]
[19] [20]
[21]
[22]
M.E. Weksler / Vaccine 18 (2000) 1624±1628 gammopathies in aging mu, kappa-transgenic mice: involvement of the B-1 cell lineage. Eur J Immunol 1997;27:2436±40. Gueret R, Zhao K-S, Weksler ME, Age-related changes in serum immunoglobulin levels, autoantibodies and monoclonal immunoglobulins in xid and C57BL/6 mice, Aging, Immunol. Infect. Dis. 6;177±87. LeMaoult J, Manavalan JS, Dyall R, Szabo P, Nikolic-Zugic J, Weksler ME. Cellular basis of B-cell clonal populations in old mice. J Immunol 1999;162:6384±91. De Greef GE, Van Tol MJ, Van Den Berg JW, et al. Serum immunoglobulin class and IgG subclass levels and the occurrence of homogeneous immunoglobulins during the course of ageing in humans. Mech Ageing Dev 1992;66:29±44. Goidl EA, Innes JB, Weksler ME. Immunological studies of aging. II. Loss of IgG and high avidity plaque-forming cells and increased suppressor cell activity in aging mice. J Exp Med 1976;144:1037±48. Szewczuk MR, DeKruy RH, Goidl EA, Weksler ME, Siskind GW. Ontogeny of B lymphocyte function. VIII. Failure of thymus cells from aged donors to induce the functional maturation of B lymphocytes from immature donors. Eur J Immunol 1980;10:918±23.
[23] Nicoletti C, Yang X, Cerny J. Repertoire diversity of antibody response to bacterial antigens in aged mice. III. Phosphorylcholine antibody from young and aged mice dier in structure and protective activity against infection with Streptococcus pneumoniae. J Immunol 1993;150:543±9. [24] Ben-Yehuda A, Szabo P, LeMaoult J, Manavalan JS, Weksler ME. Increased VH 11 and VH Q52 gene use by splenic B-cells in old mice associated with oligoclonal expansions of CD5+Bcells. Mech Ageing Dev 1998;103:111±21. [25] Viale AC, Chies JA, Huetz F, et al. VH-gene family dominance in ageing mice. Scand J Immunol 1994;39:184±8. [26] Riley SC, Froscher BG, Linton PJ, Zharhary D, Marcu K, Klinman NR. Altered VH gene segment utilization in the response to phosphorylcholine by aged mice. J Immunol 1989;143:3798±805. [27] Goidl EA, Thorbecke GJ, Weksler ME, Siskind GW. Production of auto-anti-idiotypic antibody during the normal immune response: changes in the auto-anti-idiotypic antibody response and the idiotype repertoire associated with aging. Proc Natl Acad Sci USA 1980;77:6788±92.
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Changes in the B-cell repertoire with age Marc E. Weksler* Division of Geriatrics and Gerontology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA
Abstract Changes in the B-cell repertoire during aging include a shift in antibody speci®cities from foreign to autologous antigens associated with a decline in the activity of conventional B2 compared to B1 lymphocytes. The age-associated increase in B1 lymphocyte number and activity contribute to the increased serum concentration of autoantibodies and the B-cell clonal expansions that develop with age. Aging is also associated with a decreased diversity of the antibody response re¯ected in the preferential loss of IgG and high anity antibodies following immunization with a foreign antigen. Many of these changes can be traced to an impaired capacity of T cells to support isotype switching and somatic mutation in the periphery and the generation of a diverse B-cell repertoire from bone marrow B-cell precursors. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Aging; Immunology; Repertoire
1. Introduction The birth of modern humoral immunity has been dated to 1890 when von Behring and Kitasato reported that the transfer of serum anti-bacterial antibodies from immunized animals to naive animals protected the recipients from bacterial challenge [reviewed in 1]. Subsequently, Tilselius and Kabat demonstrated that antibodies were immunoglobulin molecules; Porter and Edelman established the structural basis of antibody activity; and Tonegawa and Baltimore described the molecular mechanism underlying the generation of antibody diversity. During this period, the complex interactions of B, T and APC required for the generation of antibodies were discovered. As knowledge of the cellular and molecular regulation of antibody production has grown, it has been possible to explore the eect of age on humoral immunity with increasing precision. These studies have shown that the eects of age on humoral immunity are due largely to changes in the B-cell repertoire. For this
* Tel.: +1-212-746-6478; fax: +1-212-746-6382. E-mail address: [email protected] (M.E. Weksler).
reason, we have chosen to use the phrase eect of age on the ``B-cell repertoire'' rather than on the ``antibody repertoire''. Aging in¯uences both the quantity and the quality of humoral immunity. Quantitative changes include an altered number of immunoglobulin (Ig) and antigenspeci®c Ig-secreting B-cells. Qualitative changes include shifts in the number and activity of B-cell subsets as well as shifts in the antibody repertoire with respect to speci®city, isotype, anity, and idiotype. A comprehensive review of aging and the immune system was published in the December, 1997 issue of Tranplantation Reviews [2]. This brief review includes some recent observations on the eect of age on the B-cell repertoire. 2. Age-associated changes in the B-cell repertoire The ®rst evidence that the humoral immunity changed with age was the report that the quantity of serum antibodies speci®c for foreign antigens declined with age [3]. Soon thereafter, aging was shown to aect the quality of the antibody response to bacteria produced by rabbits [4]. In this study, antibodies to bacteria
0264-410X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 9 9 ) 0 0 4 9 7 - 1
M.E. Weksler / Vaccine 18 (2000) 1624±1628
induced in old rabbits agglutinated the bacteria less rapidly than did antibodies induced in young rabbits. Speci®c antibody responses in humans to virtually all vaccines, including anti-tetanus toxin, anti-in¯uenza and anti-encephalitis viruses as well as salmonella and pneumococcus bacteria decrease with age [5]. In experimental animals the eect of age on the antibody response depends on the type of antigen [6]. Thus, the antibody response to T-dependent antigens produced by B2 lymphocytes is diminished by 90% in old mice. In contrast, the antibody response to T-independent antigens and antigens that stimulated B1 lymphocytes in old mice is equal to or greater than that in young mice. The fact that the antibody response to most foreign antigens was lower in old compared to young individuals led to a view that aging led to an immune de®ciency state. However, we found no evidence for a global loss of B-cell function as neither the total number of B-cells or of Ig-secreting B-cells were decreased with age [7]. This is explained by an increase in the number of B-cells secreting antibodies to autologous antigens. Thus, the decrease in antibody response to a foreign antigen re¯ects not a decrease in humoral immunity but a change in the B-cell repertoire with respect antigen speci®city. In addition to the shift in the repertoire with respect to antibody speci®cities from foreign to autologous antigens, we recognize that aging aects both the size and the diversity of the Bcell repertoire leading to changes in both the quantity and quality of the antibody produced. 2.1. Changes in the repertoire of B-cells during aging: self and non-self speci®cities The concentration of natural and antigen-induced antibodies speci®c for foreign antigens decline with age in both humans and experimental animals [5,6]. Furthermore, the number of B-cells that bind a foreign antigen did not decrease although the number of speci®c antibody-forming cells did [7]. These defects are probably related to impaired T cell help as the response to T-independent antigens in young and old mice are comparable [6]. Despite these defects in the antibody response to most foreign antigens, neither the level of serum immunoglobulins nor the number of Ig-secreting Bcells declines with age [5,8]. Furthermore, we demonstrated that the total number of antigen-induced Igproducing cells were not decreased in old compared to young mice [8]. This paradox was, at least in part, resolved by the ®nding that the serum concentration of autoantibodies increased with age [9]. We have shown that immunization of young mice with a foreign antigen stimulates a large number of Ig-secreting cells producing antibodies to the nominal antigen and a very
1625
small number to autoantigens. The converse is true in old mice where few Ig-secreting producing antibodies bind the nominal antigen and a large number bind autoantigens [6,8]. Thus, aging is associated with alterations in the B-cell repertoire with respect to the ratio of antibodies speci®c for the nominal versus autologous antigen. The autoantibodies detected at increased concentrations in the serum of elderly as compared to young humans were speci®c for autoantigens, DNA, immunoglobulin, thyroglobulin, found at high levels in patients with systemic lupus erythematosis, rheumatoid arthritis or hypothyroidism [10]. For this reason, it was not clear whether autoantibodies in the elderly re¯ected subclinical autoimmune disease or the dysregulation of the immune response. To distinguish between these two possibilities, we measured the reactivity of serum from young and old mice to antigens in normal tissues that were not selected for their association with an autoimmune diseases [11]. This technique had shown that autoantibodies detectable in serum from normal, young mice bind to proteins in lysates from a variety of normal tissues. Furthermore, the IgM autoantibodies were not stimulated by foreign antigens as they were present in serum from antigen-free mice. We showed that autoantibodies that increase with age have the same binding spectra for antigens of the spleen, skin and muscle as natural autoantibodies present in young mice at lower concentration [12]. There was a single exception to this rule detected in autoantibodies in old mice: a single band was detected in old but not young mice to proteins present in brain extracts. Therefore, as a rule, the quantity but not the speci®city of serum autoantibodies dier in old and young mice. The stability of autoantibody reactivity during aging was also demonstrated in humans [13]. The reactivity of human serum obtained from the same ®ve individuals was measured in sera obtained over a 25-year interval. The ®rst serum specimen was obtained when the donors were, on average, 43 years old and the last when the donors were, on average 69 years old. IgG and IgM immunoblots were made with lysates of normal lung, kidney, stomach, and thymus tissue. The pro®les of self-reactivity of both serum IgM and IgG were strikingly similar in the sera obtained from the same individual. These results indicate that in both humans and mice autoantibodies in young and old individuals express similar speci®cities suggesting the autoreactivity of antibodies are not random but highly selected based on their recognition of a limited set of auto-antigens. Indirect evidence from these studies in humans also indicated that auto-anti-idiotypic antibodies increase with age. Previous studies in mice had shown that among the autoantibodies that increase in old mice following im-
1626
M.E. Weksler / Vaccine 18 (2000) 1624±1628
munization are auto-anti-idiotypic antibodies [14]. This class of autoantibodies is of interest because auto-antiidiotypic antibodies inhibit the secretion of antibodies to nominal antigen following immunization. Thus, the appearance of auto-anti-idiotypic antibodies in old mice not only re¯ects immune senescence but contributes to the impaired antibody response to the nominal antigen. Mixed transfer experiments demonstrated that the secretion of auto-anti-idiotypic antibodies in mice are stimulated by T cells from in old mice. Thus, like other defects in humoral immunity, the appearance of auto-anti-idiotypic antibodies with age re¯ect altered T-cell function. 2.2. Changes in the repertoire of B-cells during aging: appearance of monoclonal antibodies and clonal B-cell expansions In addition to the age-associated polyclonal increase in serum autoantibodies, there is an increasing frequency of serum monoclonal immunoglobulin (MIg) [15]. Sensitive analytic techniques reveal that as many as one-half of elderly humans and old mice have MIg in their serum. The age-associated shift in the speci®city of serum antibodies from foreign to autoantigens is also re¯ected in the speci®cities of serum MIg as approximately 50% of MIg react with autoantigens [16]. Indirect evidence suggests that cells that secrete MIg are drawn from the CD5+ B-cell subpopulation [17]. Our lifespan study of C57Bl/6Xid mice, which have less than 5% of the normal complement of CD5+ Bcells, revealed that these mice do not develop either autoantibodies or stable serum MIg during aging [18]. Thus, CD5+ B-cells appear to be the source of both the age-associated autoantibodies and serum MIg. We also have examined the B-cell populations for evidence of age-associated B-cell clonal expansions that might be the cellular basis of the serum MIg. To search for B-cell clonal expansions we used Ig heavy chain mRNA CDR3 size analysis [19]. We found that virtually all old mice, whether or not they had MIg in their serum, had splenic B-cell VHspeci®c expansions. Direct sequencing of the RT-PCR products yielded readable sequences con®rming the clonal nature of the B-cell expansion. Old mice frequently have more than one B-cell clonal expansion and these were detected in multiple lymphoid compartments including the spleen, lymph nodes, bone marrow, and thymus. The majority of the B-cell clonal expansions are derived from the CD5+ subset of Bcells. The B-cell clonal expansions develop in the peripheral immune compartments and not generated within B-cell precursor populations. Preliminary evidence suggests that B-cell clonal expansions do not develop in MHC class II-de®cient mice, which lack CD4 T
cells, although mice de®cient in MHC class I antigens and CD8 T cells develop B-cell clonal expansions at the same frequency as wild type mice. 2.3. Age-associated changes in antibody speci®city: isotype and anity In mice, the age-associated increase in serum immunoglobulin concentration is associated with a marked increase in the number of Ig-secreting cells [7]. In humans, the serum concentration of IgM, IgA and IgG also increases with age although the concentration of IgD decreases during aging [20]. In contrast to the increase in the steady state level of serum IgG and IgM with age, the number of antigen-speci®c IgG- and IgM-producing cells stimulated by immunization is decreased in old compared to young mice [21]. These studies showed that there was a greater loss of IgGproducing compared to IgM-producing cells with age. The preferential loss of IgG antigen-speci®c antibody re¯ects an age-associated defect in isotype switching probably as a consequence of impaired T-cell function required for the generation of germinal centers and for isotype switching. Old mice, in addition to impaired isotype switching following immunization with DNP-BGG, produce a restricted repertoire of anti-DNP antibodies with respect to anity. The limited diversity of haptenspeci®c antibodies is due to the limited production of high anity antibodies. The production of high anity antibodies depends upon the process of somatic mutation and this process like isotype switching takes place within germinal centers. The age-associated impairment in both processes has been shown to be due to an age-associated defect in T-cell function [14]. We have traced the defect back to thymocytes [22]. Mixed cell transfer experiments in which the capacity of thymocytes from mice of dierent ages to support the production of high anity antibodies were performed. We transferred thymocytes from syngeneic mice of dierent ages with fetal liver cells into irradiated young mice and measured the anity of the antibody response in immunized recipients. High anity antibodies were produced by recipients of thymocytes from 2 to 4 month old mice aged while high anity antibodies were not seen in recipients of thymocytes from 12 to 24 month old mice. The preferential loss during aging of IgG and high anity antibody, the most protective antibodies against bacterial and viral diseases, contribute to the increased susceptibility and severity of infectious disease in the elderly as well as the lower protection aorded by vaccines in old compared to young humans from infection. While epidemiological studies have shown that the magnitude antibody response following vaccination is a good indication of protective
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immunity, evidence now exists that antibody-mediated protection depends on the anity and isotype as well as the quantity of antibody [23]. Anti-pneumococcal antibody resulting from the immunization of old mice was less protective when transferred to young naive mice than antibody from young mice. The greater protection of the antibody preparation from young mice was correlated with its higher anity for the pneumococcus. 2.4. Age-associated changes in the B-cell repertoire: VH utilization and B-cell subsets In contrast to the 3 ' VH gene family preference and few N additions in the CDR3 region seen in antibodies from fetal and neonatal as compared to adult mice, old mice show no similar global changes in VH utilization or N additions [24]. However, there are subtle alterations in VH gene usage with age. We demonstrated that VH11 and Q52 variable gene families are used to a greater extent by spleen cells of old compared to young mice. These gene families are used preferentially by CD5+ B-cells and their increased usage can be explained by the 3-fold increase in CD5+ Bcells in the spleens of old compared to young mice. Individual old mice overutilize VH gene families [25]. This results from B-cell clonal expansions which can be detected in the spleen as well as in other lymphoid compartments in virtually all mice over 18 months of age [19]. The majority of the B-cell clonal expansions are found in the CD5+ B-cell subset and many express the VH11 gene family. The sequence of the Ig CDR3 region in one such clonal B-cell expansion was identical to that previously reported for an anti-phosphatidylcholine antibody produced by CD5+ B-cells [24]. Another age-associated change in VH gene utilization by B-cells was seen following immunization with phosphorylcholine (PC) [26]. Virtually all B-cells in young BALB/c mice that produce anti-PC antibody use the S107 gene family. In contrast, only 30% of anti-PC antibodies produced by old mice use the S107 gene family. These shifts in VH gene family utilization provide the structural basis for the age-associated shift in the idiotypic repertoire that has been observed during aging [27]. 3. Clinical consequences of age-associated changes in the B-cell repertoire The changes in the quality and quantity of antibody during aging contributes importantly to the increased susceptibility and sensitivity of the elderly to infection. Furthermore, these same factors explain the impaired ecacy of vaccines in protecting elderly from infection.
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Finally, it is likely that the B-cell clonal expansions that occur in mid-life are the precursors of late-life Bcell neoplasms as benign colonic adenomas are precursors of colonic carcinomas. Acknowledgements Supported in part by NIH grants AG 08707, AG 14669, the Gladys and Roland Harriman Foundation, and the DeWitt Wallace Foundation.
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[23] Nicoletti C, Yang X, Cerny J. Repertoire diversity of antibody response to bacterial antigens in aged mice. III. Phosphorylcholine antibody from young and aged mice dier in structure and protective activity against infection with Streptococcus pneumoniae. J Immunol 1993;150:543±9. [24] Ben-Yehuda A, Szabo P, LeMaoult J, Manavalan JS, Weksler ME. Increased VH 11 and VH Q52 gene use by splenic B-cells in old mice associated with oligoclonal expansions of CD5+Bcells. Mech Ageing Dev 1998;103:111±21. [25] Viale AC, Chies JA, Huetz F, et al. VH-gene family dominance in ageing mice. Scand J Immunol 1994;39:184±8. [26] Riley SC, Froscher BG, Linton PJ, Zharhary D, Marcu K, Klinman NR. Altered VH gene segment utilization in the response to phosphorylcholine by aged mice. J Immunol 1989;143:3798±805. [27] Goidl EA, Thorbecke GJ, Weksler ME, Siskind GW. Production of auto-anti-idiotypic antibody during the normal immune response: changes in the auto-anti-idiotypic antibody response and the idiotype repertoire associated with aging. Proc Natl Acad Sci USA 1980;77:6788±92.