Memory-Enriched Population Aged Microenvironment Results in a Lymphocytes in the + Maturation Of

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

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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 CD4؉ Lymphocytes in the Aged Microenvironment Results in a Memory-Enriched Population1

Jenna A. Timm and Marilyn L. Thoman2

With advancing age the CD4؉ 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 CD4؉ T cells. In two models, aged and young mice were compared for the ability to reconstitute their peripheral CD4؉ 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 CD4؉ pool were CD44high CD45RBlow Mel-14low and upon activation with anti-CD3 -these CD4؉ T cells produced mRNA for IL-2, IL-4, IL-5, and IFN-␥. In aged bone marrow-transplanted mice, the same pheno typic profile and cytokine mRNA pattern were found in CD4؉ T cells of host and donor origin. In contrast, the majority of CD4؉ 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-␥ 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 CD4ϩ 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 CD4ϩ T lymphocyte population (3– regenerate a population with the memory characteristics of CD4ϩ 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 CD4ϩ 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-␥, 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 CD4ϩ population occur gradually over the life span as a conse- Animals were administered by i.p. injection two doses, 60 ␮l 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 ϩ ϩ 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 ϫ 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 1.1ϩ 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 ϫ 106) were stained with an with 18 U.S.C. Section 1734 solely to indicate this fact. appropriate quantity of antibody in a volume Ͻ100 ␮l. 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.

(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 CD4ϩ T lymphocytes Cell activation resistant to in vivo treatment with anti-thymocyte antisera. C57BL/6J mice (2–4 mo old) were administered 60 ␮l of a 1/1 mixture of anti-mouse Cells were activated for cytokine mRNA production as described previ- ϩ ϩ 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 CD4ϩ 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 ϫ 105/ml, 645 ␮l 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 ␮g/ml anti-CD28 (soluble). At the conclusion of this period, protection assay. These data are shown in Fig. 3. cells were recovered, washed, and restimulated with 10 ng/ml PMA, 500 ϩ ng/ml ionomycin, and 1.5 ␮M monensin for an additional 5 h. Cells were The CD4 T cells purified form sham-injected young and aged then incubated with various fluorochrome-labeled antibodies against sur- animals display characteristic and distinctive phenotypic patterns face markers for 30 min. After washing with 1% FCS/PBS, cells were fixed and lymphokine mRNA profiles. The cells derived from young http://www.jimmunol.org/ with 4% paraformaldehyde/PBS for 20 min on ice. Cells were resuspended animals are predominantly CD44low, Mel-14high, and CD45RBhigh in 0.1% saponin/PBS with FITC-conjugated rat anti-mouse cytokine or isotype-matched rat IgG control for 30 min. Cells were washed, resus- (Fig. 2 and Table I). The predominant phenotype of the aged ϩ pended in 4% paraformaldehyde/PBS, and run on a FACSCalibur (Becton CD4 T cells is the reciprocal, CD44high, Mel-14low, CD45RBlow. Dickinson, Mountain View, CA) flow cytometer. The cytokine mRNA profile is also age specific, as has been described by Hobbs et al. (9). Young CD4ϩ T cells produce mRNA RNase protection assay for TNF-␣ and -␤, IL-2, and low levels of IFN-␥. In contrast, the Cytokine mRNA profiles were generated exactly as previously described (9). aged show enhanced IFN-␥ message and, in addition, have IL-4 and IL-5 mRNA transcripts (Fig. 3). Frequently the IL-2 mRNA Statistical analysis signal is reduced in the aged samples. by guest on September 25, 2021 ϩ The Student-Newman-Keuls test for multiple comparisons was employed CD4 cells derived form Ab-treated animals displayed pheno- to test for significance. Differences were accepted as significant if they met typic and functional properties characteristic for the age of the a p Ͻ 0.05 criterion. animal. That is, in the aged reconstituted mice, cells were enriched for those having the CD44high (77%), Mel-14low (64%), Results CD45RBlow (72%) phenotype, while the reconstituted young Ab-depleted model CD4ϩ pool was predominantly CD44low (65%), Mel-14high (57%), C57BL/6J mice, either 2–3 or 22–24 mo of age, were injected with CD45RBhigh (63%). The entire dataset is tabulated in Table I, ϩ a mixture of anti-thymocyte and anti-lymphocyte sera. To monitor which shows the percentage of the splenic CD4 T cells express- the efficacy of depletion and to characterize the effect of antiserum ing high levels of CD44, CD45RB, and Mel-14. injection on the splenic lymphoid compartment, animals were sac- Likewise, the mRNA profile was similar to that of age-matched ϩ rificed at 1 wk after treatment. The fraction of splenic CD4ϩ T controls. As shown in Fig. 3, the CD4 cells from aged Ab-treated cells was determined and was found to be reduced by 85–90% as mice produced a lymphokine mRNA profile consistent with their a result of antiserum administration in both young and aged indi- phenotype, with the appearance of IL-4, IL-5, and large amounts of viduals. The phenotype of the residual T cells was determined by IFN-␥ message as well as IL-2. Similar populations from young means of multiparameter flow cytometry, and these data are shown animals lack IL-4 and IL-5 message and show greatly reduced in Fig. 1. The resistant cells are enriched for a population that IFN-␥ mRNA. differs from the controls in that CD44 expression is higher, and CD45RB expression is lower. This suggests that memory cells are Bone marrow chimera model more resistant to lysis with the antibody mixture than are the naive. To confirm and extend these findings that the environment influ- Two injections of anti-thymocyte and anti-lymphocyte sera result ences the differentiation of peripheral CD4ϩ T cells, bone marrow in Ͼ90% depletion of Thy 1.2ϩ cells in the peripheral blood and chimeras were constructed using C57BL/6 host animals of various spleen. ages reconstituted with bone marrow from B6.PL Thy 1.1 donors. The capacity of the aged animal to recover and reconstitute the This model allows the definitive identification of the source of peripheral T cell compartment was assessed 8–10 wk postablation. cells within the regenerated population. In the Ab depletion model, Spleen cells were recovered from antibody or BSS (sham)-injected it is possible that expansion of a limited number of Ab-resistant animals. T cells were enumerated and phenotypically characterized mature cells regenerates the aged T lymphocyte compartment in by multiparameter flow cytometry. Characteristic histograms are the absence of new T cell differentiation. Following irradiation and shown in Fig. 2, while the entire dataset is summarized in Table I. T cell-depleted bone marrow injection (1–3 ϫ 106 i.v.), peripheral Function was determined by activating highly purified CD4ϩ T Thy 1.1ϩ CD4ϩ cells were recovered at various times and ana- cell populations by exposure to immobilized anti-2C11 and assess- lyzed as described in the previous section. The Journal of Immunology 713

FIGURE 2. Expression of CD44, CD45RB, and Mel-14 by CD4ϩ T lymphocytes derived from Ab- depleted or control young and aged mice. C57BL/6J mice (2–4 and 22–24 mo) were injected with either antisera or BSS. Twelve weeks after treatment spleen cells were stained and examined by ﬂow cytometry. Representative histograms are shown. A total of 12 mice of each type were examined (solid line, aged; dashed line, young). Downloaded from

To establish the degree and relative participation of donor and age-associated difference in the distribution of cells between the http://www.jimmunol.org/ host origin cells to the reconstitution, spleens and lymph nodes two size subsets. were examined at 8 wk following bone marrow transplantation. The CD44, Mel-14, and CD45RB profiles of CD4ϩ Thy 1.1ϩ The percentage of CD4ϩ cells was determined as well as the frac- splenic and lymph node lymphocytes were determined and, as tion of this population bearing Thy 1.1 (bone marrow donor phe- shown in Fig. 5 (characteristic histograms) and Tables I (complete notype). These data, shown in Table II, indicate that a substantial data set) and III, are age characteristic in pattern (as was found in proportion of the CD4ϩ T cell population has been regenerated the Ab-depleted animals). That is, Thy 1.1ϩ CD4ϩ T cells isolated within 8 wk of bone marrow transplantation in both the young and from aged host chimeras were enriched for high CD44 expression aged host animals. Relative contributions of host and donor origin and low Mel-14 and CD45RB display. In contrast, the young chi- cells vary depending on the tissue and age of host. The frequency meras had Thy 1.1ϩ CD4ϩ profiles characterized by CD44low, by guest on September 25, 2021 of Thy 1.1ϩ donor (bone marrow-derived) CD4ϩ cells is higher in CD45RBhigh and Mel-14high expression on the majority of cells. the lymph nodes than in the spleen with approximately 70% of the Cells isolated from both the spleen and lymph nodes displayed young and 47% of the aged being of this phenotype, while in the the same pattern (Table III). That is, a higher fraction of CD4ϩ spleen 42% of the young CD4ϩ cells and 32% of the aged were cells with “memory” levels of CD44 and CD45RB were identified derived from the transplanted bone marrow. The forward and side light scatter characteristics of the CD4ϩ T cells of both donor and host origin were compared, and no notable difference was seen. Fig. 4 illustrates the forward light scatter histograms characteristic of the splenic CD4ϩ populations in chimeric mice. These data indicate that in both the Thy 1.1ϩ and Thy 1.2ϩ subsets, the majority of cells were small (mean fluorescence intensity, 54) with a minority population having a higher forward scatter intensity (mean fluorescence intensity, 111). There was no

Table I. Phenotype of CD4ϩ T lymphocytes following depletion and recovery

Mean % of Population Expressinga

Mice CD44high CD45RBhigh ME1-14high

Y control 24.8 Ϯ 4.4 66.3 Ϯ 8.8 63.8 Ϯ 11.5 A control 76.7 Ϯ 5.9 32.8 Ϯ 1.1 12.7 Ϯ 3.4 FIGURE 3. CD4ϩ T lymphocyte cytokine mRNA profile. C57BL/6J b Ϯ Ϯ Ϯ Y Ab-depleted 34.8 16.0 62.8 1.6 56.7 4.8 mice (2–4 and 22–24 mo old) were treated with Abs or irradiated and A Ab-depletedb 77.2 Ϯ 4.2 28.1 Ϯ 3.5 35.9 Ϯ 8.9 c reconstituted with bone marrow. At 12 wk post-treatment, spleens were Y chimera 24.9 Ϯ 9.5 61.9 Ϯ 6.9 59.0 Ϯ 1.2 ϩ ϩ ϩ A chimerac 57.8 Ϯ 7.9 35.7 Ϯ 8.1 34.7 Ϯ 14.4 removed, and CD4 T cells (Ab-depleted) or Thy 1.1 CD4 T cells (bone marrow chimeras) were purified by FACS. Purified cells were activated by a High expression was defined as described in Ref. 4 using histograms of control culture with plate-bound 2C11 (anti-CD3) for 30 h. RNA was prepared and animals. analyzed in a multiprobe RNase protection assay. RNA from an equal b Ab-treated (ATS) mice. Y ϭ 2–4 mo of age; A ϭ 22–24 mo. c Irradiation bone-marrow chimeras. Donor-derived Thy 1.1ϩ CD4ϩ T cells only number of cells was applied to each lane. A minimum of six animals of were included in this analysis. each type were analyzed. Representative data are shown. 714 MATURATION OF CD4ϩ T CELLS IN THE AGED MICROENVIRONMENT

Table II. Recovery of the CD4ϩ T lymphocyte population following bone marrow transplantationa

% Thy 1.1ϩ in Total CD4ϩ Host-Ageb Tissuec % CD4ϩ Populationd

Young SP 19.7 Ϯ 1.7 42.3 Ϯ 7.7 Aged SP 12.2 Ϯ 4.1 32.3 Ϯ 10.9 Young LN 37.2 Ϯ 1.0 68.9 Ϯ 2.9 Aged LN 25.2 Ϯ 4.6 47.1 Ϯ 10.1

a C57BL/6 (Thy 1.2ϩ) host animals were irradiated and administered 1–5 ϫ 106 T cell-depleted B6.PL (Thy 1.1ϩ) bone marrow. Eight weeks posttransplant tissues were recovered from four animals of each age group. Data are given as mean Ϯ SD. b Young ϭ 2–3 mo; Aged ϭ 20–22 mo. c SP, spleen; LN, lymph node. d The fraction of the CD4ϩ population that expressed Thy 1.1.

that was activated to produce higher amounts of mRNA specific ϩ FIGURE 4. Forward light scatter characteristics of splenic CD4 T for IL-4, IL-5, and IFN-␥ than those from young host animals. Downloaded from cells. C57BL/6J mice of 3–4 and 22–24 mo of age were transplanted with To obtain a more quantitative measure of cytokine-producing T-depleted bone marrow cells as described in Materials and Methods.At capacity, a second assay was performed in which the number of 8 wk post-transplant, spleens were removed, stained, and processed for cytokine-producing cells was enumerated by intracellular cytokine flow cytometric analysis. Forward light scatter histograms are shown for staining and flow cytometry. An activation protocol was employed the four CD4ϩ populations: A, donor origin, young host; B, donor origin, that used both anti-CD3 as well as anti-CD28. T cell-enriched aged host; C, host origin, young host; D, host origin, aged host.

populations were activated by culture for 24 h in the presence of http://www.jimmunol.org/ immobilized anti-CD3 as well as soluble anti-CD28. Following in aged hosts. In both young and aged hosts, a lower percentage of this period, cells were further activated by a 5-h pulse with iono- memory cells was identified in the lymph nodes compared with the mycin and PMA in the presence of monensin. Intracellular cyto- corresponding splenic populations. kines (IL-2, IL-4, and IFN-␥) were then detected by flow cytom- Statistical analyses indicate that the marker levels measured on etry in conjunction with cell surface staining for CD4 and Thy. The all the young control and experimental animals are significantly data are summarized in Table IV. As suggested by the RNase different (at the 95% confidence level) from the aged groups for all protection assay, Thy 1.1ϩ CD4ϩ T cells arising in aged host mice three differentiation Ags (Table I). There are no significant differ- more readily produce IL-4 and IFN-␥ than the same population ences between treatment groups of the same age, with the excep- arising in young host animals. The percentage of IFN-␥ containing by guest on September 25, 2021 tion of CD44 expression by aged chimeric mice, which is signif- Thy 1.1ϩ CD4ϩ T cells from aged hosts is fourfold greater than icantly different from both aged control and aged Ab-depleted that in young hosts, while a seven to eightfold higher percentage of animals. aged T cells produce IL-4 than young. In contrast, the fraction of The lymphokine mRNA profiles of Thy 1.1ϩ CD4ϩ cells were IL-2-producing Thy 1.1ϩ CD4ϩ cells is almost threefold higher in compared between those purified from aged with those purified the young compared with the aged host mice. from young host mice. As shown in Fig. 4, the profiles were con- The cytokine content of the host-derived Thy 1.2ϩ CD4ϩ pop- sistent with phenotype, that is, the aged host yielded a population ulation was also examined. A similar, but not identical, pattern of

FIGURE 5. Expression of CD44, CD45RB, and Mel-14 by Thy 1.1ϩ CD4ϩ T lymphocytes from chimeric and Thy 1.2ϩ CD4ϩ T cells from control mice. Young and aged bone marrow chimeric mice were sacriﬁced 12 wk following bone marrow injection. Spleen cells were stained and processed for ﬂow cytometric analysis. A minimum of 12 animals of each age group were analyzed (solid line, aged; dashed line, young). The histograms shown are representative of the total dataset. The Journal of Immunology 715

Table III. Lymph node CD4ϩ T cell phenotype

Agea Treatmentb % CD45RBhigh % CD44high

Young Control 72.5 Ϯ 5.5 26.5 Ϯ 3.0 Young BMT 73.3 Ϯ 12.8 23.7 Ϯ 3.6 Aged Control 33.7 Ϯ 11.5 62.3 Ϯ 6.9 Aged BMT 34.3 Ϯ 24.5 54.4 Ϯ 10.7

a Young ϭ 2–3 mo; Aged ϭ 22–24 mo. b Control, untreated C57BL/6J mice. Eight animals were examined. BMT, bone marrow-transplanted. C57BL/6J (Thy 1.2) host mice were irradiated and administered 1–5 ϫ 106 T cell-depleted B6.PL (Thy 1.1ϩ) bone marrow. Tissues were examined 8 wk posttransplant. Only Thy 1.1ϩ CD4ϩ cells are included in this analysis. Eight animals were examined. cytokine production was identified. As with the donor bone marrow-derived cells, IL-2 production is higher in the young host population, while IL-4 and IFN-␥ synthesis is lower than that in the aged host-derived cells. There are differences between the donor- and host-derived lymphocytes within the same host animal, most Downloaded from notably in the young host mice. In these animals, a greater fraction FIGURE 6. Distribution of memory and naive subsets in splenic Thy ϩ ϩ of the CD4ϩ cells of host origin produce IL-2 and IFN-␥ than 1.1 CD4 T lymphocytes. Bone marrow chimeras were constructed as described. Host animals were sacrificed at 4 or 8 wk post-transplant, and those of bone marrow donor origin. Differences in the aged pop- ϩ ulations are of much less magnitude (less than twofold). the distribution of donor (bone marrow) origin CD4 T cells into memory These data indicate that the aged host is capable of supporting and naive subsets was determined by the relative expression of CD44 and ϩ CD45RB as measured by multiparameter flow cytometry. Memory/effector differentiation of CD4 T lymphocytes from bone marrow precur- high low

cells were characterized as having the phenotype CD44 CD45RB , http://www.jimmunol.org/ sors. Despite the significant contribution of donor origin T cells to while CD44low CD45RBhigh cells were considered naive. the total population, the resulting compartment remains, in aged hosts, highly enriched for memory/activated cells, rather than re- generating a substantial pool of naive cells. To ascertain whether in the aged host animals, and now displays a balance shifted to- the aged microenvironment preferentially supported memory cell ward memory cells. Young host mice display a similar shift, but of expansion, chimeric mice were sacrificed at 4 wk post-transplant. much less magnitude than that in the aged hosts, which does not ϩ At this time point 16% of splenic CD4 T cells were of bone reverse the naive to memory ratio of CD4ϩ T cells. marrow origin in young hosts, while comprising 5% of the CD4 pool in aged hosts. Over the next 4 wk these proportions rise to 42 Discussion by guest on September 25, 2021 and 32% in young and aged hosts, respectively. The purpose of The aged mouse is able to regenerate its peripheral T cell com- this experiment was to determine the stability of the phenotypic ϩ partment from either endogenous or syngeneic bone marrow stem distribution of donor CD4 T lymphocytes over the period 4–8 cells following Ab or irradiation treatment. However, the newly wk after transplant. At 4 wk and again at 8 wk post-transplant, ϩ ϩ produced T lymphocytes do not rejuvenate a young-like CD4 one-half of the animals were sacrificed, and the splenic Thy 1.1 ϩ compartment, but, rather, the phenotype and lymphokine produc- CD4 was analyzed for simultaneous expression of CD44 and tion profile of the reconstituted CD4ϩ population is very similar to CD45RB. The naive population is defined as displaying the phe- that of the unmanipulated control animals, which in aged host mice notype, CD44low, CD45RBhigh, while the converse phenotype is is enriched for cells with a memory/effector phenotype. defined as the memory type. As shown in Fig. 6, there is a notable These results suggest that the aged environment influences the change in the relative proportions of naive and memory cells maturation state of the peripheral T cells. This may result from within the aged hosts between these time points. At 4 wk, the ratio ϩ ϩ alterations in thymopoiesis with age, causing the differentiation of of naive to memory splenic Thy 1.1 CD4 T cells in both the T cells with a memory-like phenotype and function, or post-thy- young and aged host mice is weighted toward the naive. At 8 wk, mically, naive thymic emigrants may be induced by the aged mi- in contrast, the fraction of naive phenotype cells is greatly reduced croenvironment to undergo expansion and maturation. The possibility that the intrathymic T cell differentiation path- Table IV. Production of cytokines by CD4ϩ T cells in chimeric mice way is altered with age has been suggested previously by Hiro- kawa and colleagues (15, 16). Using a thymus transplantation Cytokineb model in which thymic rudiments are placed under the kidney capsule of athymic, nude mice, these investigators have found that Origin of Cell Populationa IL-2 IL-4 IFN-␥ as the age of the thymus donor increases, the ratio of peripheral Donor-derived (Thy 1.1ϩ) CD4ϩ to CD8ϩ T cells produced in the nude mice changes. Fur- Young host 11.9 Ϯ 0.2 1.6 Ϯ 0.7 10.9 Ϯ 0.2 thermore, the CD4ϩ T lymphocytes produced in the animals trans- Aged host 4.4 Ϯ 0.2 14.9 Ϯ 0.4 43.4 Ϯ 0.6 ϩ planted with aged thymic lobes, displayed memory-like character- Host-derived (Thy 1.2 ) Young host 31.5 Ϯ 0.2 2.9 Ϯ 0.1 22.9 Ϯ 0.2 istics, expressing high levels of CD44 and producing high levels of Aged host 5.9 Ϯ 0.2 8.3 Ϯ 0.3 56.2 Ϯ 0.4 IL-4 upon activation. These data were interpreted as suggesting that the mature CD4ϩSP T cells produced by the aged thymus a Spleen cells were removed from bone marrow-transplanted C57BL/6 mice 8 wk posttransplant. Cells were cultured with activators as described in Materials and differ from those produced in the young. Methods prior to multi-parameter flow cytometric analysis of surface phenotype and If thymopoiesis were altered during the aging process in such a cytokine content. b Intracellular cytokine detected by flow cytometry. Data was given as the per- way as to result in the production of memory cells, it would be centage of the population containing cytokine. predicted that evidence of such a change could be detected in the 716 MATURATION OF CD4ϩ T CELLS IN THE AGED MICROENVIRONMENT

CD4ϩ SP population, as an increased number of cells bearing levels, thus influencing the ratio of the Th1/Th2 effector cells CD44highCD4RBlow characteristic of the memory subset. How- produced. ever, a direct examination of the CD4ϩ single-positive thymocytes The data presented here imply that reconstitution of a naive does not reveal such a population in the thymus of aged mice (17). population in the mature adult may not be easily accomplished by Alternatively, the memory-enriched population may arise pe- differentiation of a new CD4ϩ population. This conclusion may ripherally. The data presented here support this alternative. The have significant implication in clinical situations where immune fraction of splenic CD4ϩ population that is derived from donor system regeneration is anticipated, as in the case of bone marrow bone marrow stem cells increases more dramatically in aged vs transplantation following high dose chemotherapy or the proposed young host mice between 4 and 8 wk post-transplant. In young stem cell transfusions following CD4ϩ ablative treatments for mice there is a threefold increase in the representation of bone HIVϩ individuals. Numerous studies have indicated that immune marrow-derived T cells (16–48%), while in the aged host there is reconstitution in adult bone marrow transplant patients may be a fivefold increase during the same period (6 to Ͼ30%). Further- delayed or incomplete for many years (29, 41–43). Other work more, the Thy 1.1ϩ population at 4 wk in both aged and young hosts suggests that rather than new differentiation, T cells within the is enriched for naive cells. By 8 wk post-transplant, this ratio has been graft expand and are the major source of mature, peripheral T reversed in the aged hosts, with memory cells predominating. lymphocytes in the host (27, 44–46). The results reported here The post-thymic expansion potential of T cells is well docu- indicate that while the aged murine host retains the capacity for at mented. Miller and Stutman (18) estimated that peripheral T cells least a limited degree of thymic-dependent regeneration of the pe- can undergo 10,000-fold expansion, and sequential cell transfer ripheral CD4ϩ T cell population, the characteristics of the new ϩ experiments indicate that CD4 T cells have the capacity for 8 ϫ lymphocytes differ dramatically from those produced in young Downloaded from 105-fold expansion (19). animals. The intermitotic lifespan of naive T cells resident in normal young adult mice has been calculated to be quite long, on the order References of several months (20). Determining the lifespan of memory T 1. Miller, R. A. 1995. Immune system. In Handbook of Physiology, Sect. 2. cells has presented greater difficulty due to the lack of a defining E. J. Masoro, ed. Oxford University Press, Oxford, p. 555. 2. Thoman, M. L., and W. O. Weigle. 1989. The cellular and subcellular bases of phenotype that unambiguously distinguishes effector from memory http://www.jimmunol.org/ immunosenescence. Adv. Immunol. 46:221. T cell populations. Approximately 70% of the memory/effector 3. Chopra, R. K. 1990. Mechanisms of impaired T-cell function in the elderly. In phenotype T cell population incorporates DNA-specific label over Review of Biological Research in Aging, Vol. 4, M. Rothstein, ed. Wiley-Liss, a relatively short labeling period (4–6 wk). These results suggest New York, p. 83. 4. Ernst, D. N., M. V. Hobbs, B. E. Torbett, A. L. Glasebrook, M. A. Rehse, that the memory T cell life span may be heterogeneous, with both K. Bottomly, K. Hayakawa, R. R. Hardy, and W. O. Weigle. 1990. Differences short- and long-lived subsets (21–23). in the expression profiles of CD45RB, Pgp-1, and 3G11 membrane antigens and in the patterns of lymphokine secretion by splenic CD4ϩ T cells from young and These data reflect the situation in homeostatic balance. In cir- aged mice. J. Immunol. 145:1295. cumstances where the T cell pool is reduced, such as following 5. Utsuyama, M., K. Hirokawa, and C. Jurashima. 1992. Differential age-change in ϩ ϩ ϩ ϩ irradiation, residual T cells have a marked capacity for rapid cell the numbers of CD4 CD45RA and CD4 CD29 T cell subsets in human

peripheral blood. Mech. Ageing Dev. 63:57. by guest on September 25, 2021 cycle. Transfer of purified T cell populations into T-deficient host 6. Cossarizza, A., C. Ortolani, and R. Pagnelli. 1992. Age-related imbalance of mice results in significant expansion of the donor cells (19, 20, virgin (CD45RAϩ) and memory (CD45ROϩ) cells between CD4ϩ and CD8ϩ T 24–26). This expansion is almost exclusively reported to be of lymphocytes in humans: a study from newborns to centenarians. J. Immunol. Res. 4:118. memory cells (26–28). Ag-dependent expansion of mature mem- 7. Hobbs, M. V., D. N. Ernst, B. E. Torbett, A. L. Glasebrook, M. A. Rehse, ory T cells is reported to occur following bone marrow transplan- D. N. McQuitty, M. L. Thoman, K. Bottomly, A. L. Rothermel, D. J. Noonan, et al. 1991. Cell proliferation and cytokine production by CD4ϩ cells from old mice. tation of thymic-deficient hosts (29–30). However, there are re- J. Cell. Biochem. 46:312. ports indicating increases in naive T cells in the absence of specific 8. Lerner, A., T. Yamada, and R. A. Miller. 1989. Pgp-high T lymphocytes accu- or environmental Ag stimulus (31), suggesting that under some mulate with age in mice and respond poorly to concanavalin A. Eur. J. Immunol. 19:977. conditions, this population may be induced to proliferate by 9. Hobbs, M. V., W. O. Weigle, D. J. Noonan, B. E. Torbett, R. J. McEvilly, non-Ag activation. R. J. Koch, G. J. Cardenas, and D. N. Ernst. 1993. Patterns of cytokine gene ϩ The data presented here suggest that in bone marrow-trans- expression by CD4 T cells from young and old mice. J. Immunol. 159:3602. 10. Nagelkerken, L., A. Hertogh, Huijbregts, R. Dobber, and A. Drager. 1991. Age- planted mice, naive T cells, recently emigrating from the thymus, related changes in lymphokine production related to a decreased number of encounter an environment in the periphery that drives expansion CD45RBhigh CD4ϩ T cells. Eur. J. Immunol. 21:272. 11. Engwerda, C. R., B. S. Fox, and B. S. Handwerger. 1996. Cytokine production by and maturation to the memory state. The extent of this process is T lymphocytes from young and aged mice. J. Immunol. 156:3621. greater in the aged than in the young host. What might be the 12. Swain, S. L., L. M. Bradley, M. Croft, S. Tonkonogy, G. Atkins, A. D. Weinberg, triggering stimuli? The capacity of the host environment to present D. D. Duncan, S. M. Hedrick, R. W. Dutton, and G. Huston. 1991. Helper T-cell subsets: phenotype, function and the role of lymphokines in regulating their de- Ag appears to play a role in this process (21). It is possible that velopment. Immunol. Rev. 123:115. characteristics of APCs such as lymphokine production or co- 13. Hayakawa, K., and R. R. Hardy. 1991. Murine CD4ϩ T-cell subsets. Immunol. stimulatory molecule expression might differ with age and thereby Rev. 123:145. 14. Thoman, M. L., and W. O. Weigle. 1980. Cellular requirements for generation of differentially affect T cell homeostasis. A wealth of data supports thymocyte stimulatory factor and characterization of its target cell. J. Immunol. the role of lymphokine networks in driving CD4ϩ T cell matura- 124:1093. 15. Utsuyama, M., M. Kasai, C. Kurashima, and K. Hirokawa. 1991. Age influence tion (reviewed recently in Refs. 31–33). It is clear that the relative on the thymic capacity to promote differentiation of T cells: induction of different abundance of IL-4, IFN-␥, and IL-12 plays a critical role in de- composition of T cell subsets by aging thymus. Mech. Ageing Dev. 58:267. termining the outcome of T cell activation. The costimulatory mol- 16. Hirokawa, K., M. Utsuyama, M. Kasai, C. Kurashima, S. Ishijima, and Y.-X. Zeng. 1994. Understanding the mechanism of the age-change of thymic function ecules, B7-1 and B7-2, appear to be differentially involved with to promote T cell differentiation. Immunol. Lett. 40:269. maturation of Th1 and Th2 effector cells (34–40). Blocking the 17. Thoman, M. 1997. Effects of the aged microenvironment on CD4ϩ T cell mat- B7-1 interaction increases IL-4 production in vitro, while in- uration. Mech. Ageing Dev. 96:75. 18. Miller, R. A., and O. Stutman. 1984. T cell repopulation from functionally re- creased IFN-␥ production results from blocking B7-2 interaction stricted splenic progenitors: 10,000-fold expansion documented by using limiting with CD4ϩ T cells. These costimulatory proteins are expressed in dilution analyses. J. Immunol. 133:2925. 19. Rocha, B., N. Dautigny, and P. Pereira. 1989. Peripheral T lymphocytes: expan- different amounts by various accessory cells (39–40) and may sion potential and homeostatic regulation of pool sizes and CD4/CD8 ratios in therefore be present within the microenvironment in very different vivo. Eur. J. Immunol. 19:905. The Journal of Immunology 717

20. von Boehmer, H., and K. Hafen. 1993. The lifespan of naive ␣/␤ T cells in molecules activate differentially the Th1/Th2 developmental pathways: applica- secondary lymphoid organs. J. Exp. Med. 177:891. tion to autoimmune disease therapy. Cell 80:707. 21. Sprent, J., and D. Tough. 1994. Lymphocyte life-span and memory. Science 36. Lenschow, D. J., S. C. Ho, H. Sattar, L. Rhee, G. Gray, N. Nabavi, K. C. Herold, 265:1395. and J. A. Bluestone. 1995. Differential effects of anti-B7-1 and B7-2 monoclonal 22. Ahmed, R., and D. Gray. 1996. Immunological memory and protective immunity: antibody treatment on the development of diabetes in the non-obese diabetic understanding their relation. Science 272:54. mouse. J. Exp. Med. 181:1145. 23. Tough, D. F., and J. Sprent. 1994. Turnover of naive and memory phenotype T 37. Lanier, L. L., S. O’Fallon, C. Somoza, J. H. Philipps, P. S. Linsley, K. Okumura, cells. J. Exp. Med. 179:1127. D. Ito, and M. Azuma. 1995. CD80 (B7-1) and CD86 (B7-2) provide similar 24. Bell, E. B., S. Sparshott, M. Drayson, and W. Ford. 1987. The stable and per- costimulatory signals for T cell proliferation, cytokine production, and generation manent expansion of functional T lymphocytes in athymic nude rats after a single of CTL. J. Immunol. 154:97. injection of mature T cells. J. Immunol. 139:1379. 38. Thompson, C. B. 1995. Distinct roles for the costimulatory ligands B7-1 and 25. Mackall, C., F. Hakim, and R. Gress. 1997. Restoration of T-cell homeostasis B7-2 in T helper cell differentiation? Cell 81:979. after T-cell depletion. Semin. Immunol. 9:339. 39. Inaba, K., M. Witmer-Pack, M. Inaba, S. Hathcock, H. Sakuta, M. Azuma, 26. Swain, S. L. 1994. Generation and in vivo persistence of polarized Th1 and Th2 H. Yagita, K. Okumura, P. S. Linsley, S. Ikehara, et al. 1994. The tissue distri- memory cells. Immunity 1:543. bution of the B7-2 costimulator in mice: abundant expression on dendritic cells 27. Mackall, C. L., F. T. Hakim, and R. E. Gress. 1997. T cell regeneration: all in situ and during maturation in vitro. J. Exp. Med. 180:1849. repertoires are not created equal. Immunol. Today 18:245. 40. Larsen, C. P., S. C. Ritchie, R. Hendrix, P. S. Linsley, K. S. Hathcock, 28. Tanchot, C., F. Lemonnier, B. Perarnau, A. Freitos, and B. Rocha. 1997. Differ- R. J. Hodes, R. P. Lowry, and T. C. Pearson. 1994. Regulation of immunostimu- ential requirements for survival and proliferation of CD8 naive or memory T latory function and costimulatory molecule (B7-1 and B7-2) expression on mu- cells. Science 276:2057. rine dendritic cells. J. Immunol. 152:5208. 29. Mackall, C. L., L. Granger, M. A. Sheard, R. Cepeda and R. E. Gress. 1993. 41. Schneider, L. C., J. H. Antin, H. Weinstein, J. S. Abrams, M. K. Pearce, T-cell regeneration after bone marrow transplantation: differential CD45 isoform R. S. Geha, and D. Vercelli. 1991. Lymphokine proﬁle in bone marrow transplant expression on thymic-derived versus thymic-independent progeny. Blood recipients. Blood 78:3076. 82:2585. 42. Miller, R. A., J. Daley, R. Ghalie, and H. Kaizer. 1991. Clonal analysis of T-cell 30. Mackall, C. L., C. V. Bare, L. A. Granger, S. O. Sharrow, J. A. Titus, and deﬁciencies in autotransplant recipients. Blood 77:1845. R. E. Gress. 1996. Thymic-independent T cell regeneration occurs via antigen- 43. Mackall, C. L., T. A. Fleisher, M. R. Brown, M. P. Andrich, C. C. Chen, Downloaded from driven expansion of peripheral T cells resulting in a repertoire that is limited in I. M. Feuerstein, M. E. Horowitz, I. T. Magrath, A. T. Shad, S. M. Steinberg, et diversity and prone to skewing. J. Immunol. 156:4609. al. 1995. Age, thymopoiesis, and CD4ϩ T-lymphocyte regeneration after inten- 31. Bell, E., and S. Sparshott. 1997. The peripheral T-cell pool: regulation by non- sive chemotherapy. N. Engl. J. Med. 332:143. antigen-induced proliferation? Semin. Immunol. 9:347. 44. Moses, R. D., S. O. Sharrow, D. A. Stephany, K. S. Orr, and R. E. Gress. 1989. 32. Seder, R. A., and W. E. Paul. 1994. Acquisition of lymphokine-producing phe- Cardiac allograft survival across major histocompatibility complex barriers in the notype by CD4ϩ T cells. Annu. Rev. Immunol. 12:635. rhesus monkey following T lymphocyte-depleted autologous marrow transplan- 33. Paul, W. E., and R. A. Seder. 1994. Lymphocyte responses and cytyokines. Cell tation. IV. Immune reconstitution. Transplantation 48:774.

76:241. 45. Andrews, R. G., E. M. Bryant, S. H. Bartelmez, D. Y. Juirhead, G. H. Knitter, http://www.jimmunol.org/ 34. Freeman, G. J., V. A. Boussiotis, A. Anumanthan, G. M. Bernstein, X.-Y. Ke, W. Bensinger, D. M. Strong, and I. D. Bernstein. 1992. CD34ϩ marrow cells, P. D. Rennert, G. S. Gray, J. G. Gribben, and L. M. Nadler. 1995. B7-1 and B7-2 devoid of T and B lymphocytes, reconstitute stable lymphopoiesis and myelo- do not deliver identical costimulatory signals, since B7-2 but not B7-1 preferen- poiesis in lethally irradiated allogeneic baboons. Blood 80:1693. tially costimulates the initial production of IL-4. Immunity 2:523. 46. Chomczynski, P., and N. Sacchi. 1987. Single-strep method of RNA isolation by 35. Kuchroo, V. K., M. P. Das, J. A. Brown, A. M. Ranger, S. S. Zamvil, R. A. Sobel, acid quanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: H. L. Weiner, N. Nabavi, and L. H. Glimcher. 1995. B7-1 and B7-2 costimulatory 156. by guest on September 25, 2021