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Maturation of CD4+ Lymphocytes in the Aged Microenvironment Results in a Memory-Enriched Population This information is current as Jenna A. Timm and Marilyn L. Thoman of September 25, 2021. J Immunol 1999; 162:711-717; ; http://www.jimmunol.org/content/162/2/711 Downloaded from References This article cites 44 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/162/2/711.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 25, 2021 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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Maturation of CD41 Lymphocytes in the Aged Microenvironment Results in a Memory-Enriched Population1 Jenna A. Timm and Marilyn L. Thoman2 With advancing age the CD41 T lymphocyte compartment becomes enriched for memory cells in both humans and experimental animals. Although it has been assumed that the shift from a naive to a memory-dominant population is due to a lifetime of antigenic exposure and selection as well as a loss of naive cell input due to reduced thymopoiesis, the present data suggest that the aged microenvironment influences the maturation of newly produced CD41 T cells. In two models, aged and young mice were compared for the ability to reconstitute their peripheral CD41 T cell pools following depletion, and both age groups were found to be competent to renew this population. However, the phenotype and lymphokine profile of populations arising in aged animals were distinctly different from those in the young mice. In contrast to the expectation that depletion and reconstitution might give rise to a naive-dominant T cell pool, aged mice reconstituted a population nearly indistinguishable from that found in control Downloaded from age-matched individuals. The majority of the CD41 pool were CD44high CD45RBlow Mel-14low and upon activation with anti-CD3 these CD41 T cells produced mRNA for IL-2, IL-4, IL-5, and IFN-g. In aged bone marrow-transplanted mice, the same phenotypic profile and cytokine mRNA pattern were found in CD41 T cells of host and donor origin. In contrast, the majority of CD41 T cells in young reconstituted mice were CD44low CD45RBhigh Mel-14high. These lymphocytes, when activated, produced high levels of mRNA for IL-2, with little or no IL-4, IL-5, or IFN-g mRNA. The Journal of Immunology, 1999, 162: 711–717. http://www.jimmunol.org/ dvancing age is accompanied by a variety of alterations tional attributes of the newly produced CD41 lymphocytes. Rather in the immune system (1–3), notably changes in the than recover a more “young-like” T cell population, aged mice A composition of the CD41 T lymphocyte population (3– regenerate a population with the memory characteristics of CD41 7). While young mice possess a predominance of so-called naive T cells from untreated aged animals. or Ag-inexperienced cells with a phenotype CD44low CD45RBhigh Mel-14high, aged individuals have a majority of CD41 T cells with Materials and Methods the reciprocal phenotype, i.e., CD44high CD45RBlow Mel-14low,a Mice phenotype associated with memory cells. Naive and memory cells Female C57BL/6JNNia mice were purchased from the National Institute differ from one another functionally as well as phenotypically, par- on Aging’s colony through Charles Rivers (Wilmington, MA). Mice were by guest on September 25, 2021 ticularly in the spectrum of lymphokines produced upon activation 2 and 22 mo of age. Thy 1.1-congenic mice, B6PL Thy.1, 2 mo of age, (8–13). While naive cells produce primarily IL-2, memory cells were purchased from The Scripps Research Institute breeding colony (La may produce IL-4, IL-5, IFN-g, and a host of additional cytokines. Jolla, CA). All animals were housed in specific pathogen-free conditions. It has been hypothesized that these compositional shifts in the Ab depletion of peripheral T cell model CD41 population occur gradually over the life span as a conse- Animals were administered by i.p. injection two doses, 60 ml each, of a 1/1 quence of a reduction in naive T cell input and ongoing Ag-driven mixture of two antisera: rabbit anti-mouse thymocyte and rabbit anti- maturation of naive cells. The present studies were undertaken to mouse brain (Accurate, Westbury, NY). The two doses were administered assess the capacity of aged mice to reconstitute their T cell com- at a 2-day interval. Depletion of T lymphocytes was sequentially monitored partment following ablation and to determine whether such treat- by means of quantitating peripheral blood T cells. Animals were bled, and the samples were depleted of RBC by hypotonic lysis and stained with ment regenerates a “youthful” T cell population, i.e., one that is 1 1 fluorescein-labeled anti-Thy 1.2. Thy 1.2 cells were enumerated by flow enriched with naive CD4 cells. Two experimental models were cytometry. employed. In the first, the peripheral T cells were depleted by antiserum administration. Animals were allowed to recover, recon- Bone marrow chimeras stituting the T cell pool from endogenous precursors. In the second Host mice were prepared by exposure to a total of 1100 rad administered model irradiated mice were reconstituted with Thy-congenic in two doses separated by 4 h. These animals were given 1–5 3 106 bone young bone marrow cells. The results indicate that the aged envi- marrow cells by i.v. injection prepared from B6.PL-Thy 1.1 mice. Bone marrow cells were flushed from the femurs of 2- to 4-mo-old donor animals ronment strongly influences the phenotypic distribution and func- with balanced salt solution (BSS)3 and 5% FCS. The cells were depleted of Thy 1.11 cells by Ab- and complement-mediated lysis as previously described (14). For 3 wk following irradiation, animals were supplied with Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037 neomycin in the drinking water. Received for publication August 28, 1998. Accepted for publication September 28, 1998. Flow cytometric phenotyping The costs of publication of this article were defrayed in part by the payment of page Single cell suspensions of spleens and lymph nodes were prepared by charges. This article must therefore be hereby marked advertisement in accordance mincing the organs with forceps. Cells (2 3 106) were stained with an with 18 U.S.C. Section 1734 solely to indicate this fact. appropriate quantity of antibody in a volume ,100 ml. If necessary, after 1 This is publication 11061-IMM from the Department of Immunology, The Scripps washing, a second staining step was performed. Cells were both stained Research Institute (La Jolla, CA). This work was supported by U.S. Public Health and resuspended for analysis in FACS medium consisting of RPMI 1640 Service Grant R01AG09948. 2 Address correspondence and reprint requests to Dr. Marilyn L. Thoman, Sidney Kimmel Cancer Center, Altman Row, San Diego, CA 92121. 3 Abbreviation used in this paper: BSS, balanced salt solution. Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 712 MATURATION OF CD41 T CELLS IN THE AGED MICROENVIRONMENT (deficient in biotin and phenol red; Irvine Scientific, Santa Ana, CA) sup- plemented with FCS, 0.1 M HEPES, and azide. Antibodies The following Abs and other fluorescent reagents were used: anti-CD4-tricolor (clone YTS 191.1, Caltag, South San Francisco, CA), anti-CD8a- phycoerythrin (53-6.7, PharMingen, San Diego, CA), anti-CD44-fluorescein or phycoerythrin (IM7.8.1, PharMingen), anti-CD45RB-fluorescein or biotin (23G2, PharMingen), avidin-fluorescein (Life Technologies, Grand Island, NY), streptavidin-phycoerythrin (Biomedia, Foster City, CA), streptavidin-tricolor (Caltag), anti-Thy 1.2-biotin (52-8, Caltag), and anti- Thy 1.1-biotin (Ox-7, PharMingen). FIGURE 1. CD44 and CD45RB expression by CD41 T lymphocytes Cell activation resistant to in vivo treatment with anti-thymocyte antisera. C57BL/6J mice (2–4 mo old) were administered 60 ml of a 1/1 mixture of anti-mouse Cells were activated for cytokine mRNA production as described previ- 1 1 thymocyte and anti-mouse brain antisera by i.p. injection on both days 2 ously (9). Briefly, Thy 1.1 (donor-derived) CD4 T lymphocytes from bone marrow chimeras or CD41 T cells from Ab-treated mice, purified by and 0 or BSS as a control. On day 7, animals were sacrificed, and a single FACS were cultured at a density of 7.5 3 105/ml, 645 ml in 48-well plates. cell suspension was prepared from the spleens. Cells were stained and The wells were precoated with 145-3C11 (anti-CD3) or hamster Ig. Cells processed for flow cytometry. Four mice of each type were examined, the were harvested between 30–36 h for cytokine mRNA analysis. histograms shown are representative of the complete dataset. Intracellular cytokine quantitation Downloaded from Spleen cells were incubated for 24 h in the presence of immobilized anti- ing cytokine mRNA profiles by means of a multiprobe RNase CD3 plus 5 mg/ml anti-CD28 (soluble).

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