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The Journal of Immunology, 1999, 162: 711-717.
Copyright © 1999 by The American Association of Immunologists

Maturation of CD4+ Lymphocytes in the Aged Microenvironment Results in a Memory-Enriched Population1

Jenna A. Timm and Marilyn L. Thoman2

Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 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-{gamma}. In aged bone marrow-transplanted mice, the same phenotypic 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-{gamma} mRNA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Advancing age is accompanied by a variety of alterations in the immune system (1, 2, 3), notably changes in the composition of the CD4+ T lymphocyte population (3, 4, 5, 6, 7). While young mice possess a predominance of so-called naive or Ag-inexperienced cells with a phenotype CD44low CD45RBhigh Mel-14high, aged individuals have a majority of CD4+ T cells with the reciprocal phenotype, i.e., CD44high CD45RBlow Mel-14low, a phenotype associated with memory cells. Naive and memory cells differ from one another functionally as well as phenotypically, particularly in the spectrum of lymphokines produced upon activation (8, 9, 10, 11, 12, 13). While naive cells produce primarily IL-2, memory cells may produce IL-4, IL-5, IFN-{gamma}, and a host of additional cytokines.

It has been hypothesized that these compositional shifts in the CD4+ population occur gradually over the life span as a consequence of a reduction in naive T cell input and ongoing Ag-driven maturation of naive cells. The present studies were undertaken to assess the capacity of aged mice to reconstitute their T cell compartment following ablation and to determine whether such treatment regenerates a "youthful" T cell population, i.e., one that is enriched with naive CD4+ cells. Two experimental models were employed. In the first, the peripheral T cells were depleted by antiserum administration. Animals were allowed to recover, reconstituting the T cell pool from endogenous precursors. In the second model irradiated mice were reconstituted with Thy-congenic young bone marrow cells. The results indicate that the aged environment strongly influences the phenotypic distribution and functional attributes of the newly produced CD4+ lymphocytes. Rather than recover a more "young-like" T cell population, aged mice regenerate a population with the memory characteristics of CD4+ T cells from untreated aged animals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female C57BL/6JNNia mice were purchased from the National Institute on Aging’s colony through Charles Rivers (Wilmington, MA). Mice were 2 and 22 mo of age. Thy 1.1-congenic mice, B6PL Thy.1, 2 mo of age, were purchased from The Scripps Research Institute breeding colony (La Jolla, CA). All animals were housed in specific pathogen-free conditions.

Ab depletion of peripheral T cell model

Animals were administered by i.p. injection two doses, 60 µl each, of a 1/1 mixture of two antisera: rabbit anti-mouse thymocyte and rabbit anti-mouse brain (Accurate, Westbury, NY). The two doses were administered at a 2-day interval. Depletion of T lymphocytes was sequentially monitored by means of quantitating peripheral blood T cells. Animals were bled, and the samples were depleted of RBC by hypotonic lysis and stained with fluorescein-labeled anti-Thy 1.2. Thy 1.2+ cells were enumerated by flow cytometry.

Bone marrow chimeras

Host mice were prepared by exposure to a total of 1100 rad administered in two doses separated by 4 h. These animals were given 1–5 x 106 bone 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 with balanced salt solution (BSS)3 and 5% FCS. The cells were depleted of Thy 1.1+ cells by Ab- and complement-mediated lysis as previously described (14). For 3 wk following irradiation, animals were supplied with neomycin in the drinking water.

Flow cytometric phenotyping

Single cell suspensions of spleens and lymph nodes were prepared by mincing the organs with forceps. Cells (2 x 106) were stained with an appropriate quantity of antibody in a volume <100 µl. If necessary, after washing, a second staining step was performed. Cells were both stained and resuspended for analysis in FACS medium consisting of RPMI 1640 (deficient in biotin and phenol red; Irvine Scientific, Santa Ana, CA) supplemented 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).

Cell activation

Cells were activated for cytokine mRNA production as described previously (9). Briefly, Thy 1.1+ (donor-derived) CD4+ T lymphocytes from bone marrow chimeras or CD4+ T cells from Ab-treated mice, purified by FACS were cultured at a density of 7.5 x 105/ml, 645 µl in 48-well plates. The wells were precoated with 145-3C11 (anti-CD3) or hamster Ig. Cells were harvested between 30–36 h for cytokine mRNA analysis.

Intracellular cytokine quantitation

Spleen cells were incubated for 24 h in the presence of immobilized anti-CD3 plus 5 µg/ml anti-CD28 (soluble). At the conclusion of this period, cells were recovered, washed, and restimulated with 10 ng/ml PMA, 500 ng/ml ionomycin, and 1.5 µM monensin for an additional 5 h. Cells were then incubated with various fluorochrome-labeled antibodies against surface markers for 30 min. After washing with 1% FCS/PBS, cells were fixed with 4% paraformaldehyde/PBS for 20 min on ice. Cells were resuspended in 0.1% saponin/PBS with FITC-conjugated rat anti-mouse cytokine or isotype-matched rat IgG control for 30 min. Cells were washed, resuspended in 4% paraformaldehyde/PBS, and run on a FACSCalibur (Becton Dickinson, Mountain View, CA) flow cytometer.

RNase protection assay

Cytokine mRNA profiles were generated exactly as previously described (9).

Statistical analysis

The Student-Newman-Keuls test for multiple comparisons was employed to test for significance. Differences were accepted as significant if they met a p < 0.05 criterion.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ab-depleted model

C57BL/6J mice, either 2–3 or 22–24 mo of age, were injected with a mixture of anti-thymocyte and anti-lymphocyte sera. To monitor the efficacy of depletion and to characterize the effect of antiserum injection on the splenic lymphoid compartment, animals were sacrificed at 1 wk after treatment. The fraction of splenic CD4+ T cells was determined and was found to be reduced by 85–90% as a result of antiserum administration in both young and aged individuals. The phenotype of the residual T cells was determined by means of multiparameter flow cytometry, and these data are shown in Fig. 1Go. The resistant cells are enriched for a population that differs from the controls in that CD44 expression is higher, and CD45RB expression is lower. This suggests that memory cells are more resistant to lysis with the antibody mixture than are the naive. Two injections of anti-thymocyte and anti-lymphocyte sera result in >90% depletion of Thy 1.2+ cells in the peripheral blood and spleen.



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  		FIGURE 1. CD44 and CD45RB expression by CD4+ T lymphocytes resistant to in vivo treatment with anti-thymocyte antisera. C57BL/6J mice (2–4 mo old) were administered 60 µl of a 1/1 mixture of anti-mouse thymocyte and anti-mouse brain antisera by i.p. injection on both days 2 and 0 or BSS as a control. On day 7, animals were sacrificed, and a single cell suspension was prepared from the spleens. Cells were stained and processed for flow cytometry. Four mice of each type were examined, the histograms shown are representative of the complete dataset.

 
The capacity of the aged animal to recover and reconstitute the peripheral T cell compartment was assessed 8–10 wk postablation. Spleen cells were recovered from antibody or BSS (sham)-injected animals. T cells were enumerated and phenotypically characterized by multiparameter flow cytometry. Characteristic histograms are shown in Fig. 2Go, while the entire dataset is summarized in Table IGo. Function was determined by activating highly purified CD4+ T cell populations by exposure to immobilized anti-2C11 and assessing cytokine mRNA profiles by means of a multiprobe RNase protection assay. These data are shown in Fig. 3Go.



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  		FIGURE 2. Expression of CD44, CD45RB, and Mel-14 by CD4+ 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).

 

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  		Table I. Phenotype of CD4+ T lymphocytes following depletion and recovery

 


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  		FIGURE 3. CD4+ T lymphocyte cytokine mRNA profile. C57BL/6J mice (2–4 and 22–24 mo old) were treated with Abs or irradiated and reconstituted with bone marrow. At 12 wk post-treatment, spleens were 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 culture with plate-bound 2C11 (anti-CD3) for 30 h. RNA was prepared and analyzed in a multiprobe RNase protection assay. RNA from an equal number of cells was applied to each lane. A minimum of six animals of each type were analyzed. Representative data are shown.

 
The CD4+ T cells purified form sham-injected young and aged animals display characteristic and distinctive phenotypic patterns and lymphokine mRNA profiles. The cells derived from young animals are predominantly CD44low, Mel-14high, and CD45RBhigh (Fig. 2Go and Table IGo). The predominant phenotype of the aged CD4+ T cells is the reciprocal, CD44high, Mel-14low, CD45RBlow. The cytokine mRNA profile is also age specific, as has been described by Hobbs et al. (9). Young CD4+ T cells produce mRNA for TNF-{alpha} and -ß, IL-2, and low levels of IFN-{gamma}. In contrast, the aged show enhanced IFN-{gamma} message and, in addition, have IL-4 and IL-5 mRNA transcripts (Fig. 3Go). Frequently the IL-2 mRNA signal is reduced in the aged samples.

CD4+ cells derived form Ab-treated animals displayed phenotypic and functional properties characteristic for the age of the animal. That is, in the aged reconstituted mice, cells were enriched for those having the CD44high (77%), Mel-14low (64%), CD45RBlow (72%) phenotype, while the reconstituted young CD4+ pool was predominantly CD44low (65%), Mel-14high (57%), CD45RBhigh (63%). The entire dataset is tabulated in Table IGo, which shows the percentage of the splenic CD4+ T cells expressing high levels of CD44, CD45RB, and Mel-14.

Likewise, the mRNA profile was similar to that of age-matched controls. As shown in Fig. 3Go, the CD4+ cells from aged Ab-treated mice produced a lymphokine mRNA profile consistent with their phenotype, with the appearance of IL-4, IL-5, and large amounts of IFN-{gamma} message as well as IL-2. Similar populations from young animals lack IL-4 and IL-5 message and show greatly reduced IFN-{gamma} mRNA.

Bone marrow chimera model

To confirm and extend these findings that the environment influences the differentiation of peripheral CD4+ T cells, bone marrow chimeras were constructed using C57BL/6 host animals of various ages reconstituted with bone marrow from B6.PL Thy 1.1 donors. This model allows the definitive identification of the source of cells within the regenerated population. In the Ab depletion model, it is possible that expansion of a limited number of Ab-resistant mature cells regenerates the aged T lymphocyte compartment in the absence of new T cell differentiation. Following irradiation and T cell-depleted bone marrow injection (1–3 x 106 i.v.), peripheral Thy 1.1+ CD4+ cells were recovered at various times and analyzed as described in the previous section.

To establish the degree and relative participation of donor and host origin cells to the reconstitution, spleens and lymph nodes were examined at 8 wk following bone marrow transplantation. The percentage of CD4+ cells was determined as well as the fraction of this population bearing Thy 1.1 (bone marrow donor phenotype). These data, shown in Table IIGo, indicate that a substantial proportion of the CD4+ T cell population has been regenerated within 8 wk of bone marrow transplantation in both the young and aged host animals. Relative contributions of host and donor origin cells vary depending on the tissue and age of host. The frequency of Thy 1.1+ donor (bone marrow-derived) CD4+ cells is higher in the lymph nodes than in the spleen with approximately 70% of the young and 47% of the aged being of this phenotype, while in the spleen 42% of the young CD4+ cells and 32% of the aged were derived from the transplanted bone marrow.


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  		Table II. Recovery of the CD4+ T lymphocyte population following bone marrow transplantationa

 
The forward and side light scatter characteristics of the CD4+ T cells of both donor and host origin were compared, and no notable difference was seen. Fig. 4Go illustrates the forward light scatter histograms characteristic of the splenic CD4+ populations in chimeric mice. These data indicate that in both the Thy 1.1+ and Thy 1.2+ 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 age-associated difference in the distribution of cells between the two size subsets.



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  		FIGURE 4. Forward light scatter characteristics of splenic CD4+ T cells. C57BL/6J mice of 3–4 and 22–24 mo of age were transplanted with T-depleted bone marrow cells as described in Materials and Methods. At 8 wk post-transplant, spleens were removed, stained, and processed for flow cytometric analysis. Forward light scatter histograms are shown for the four CD4+ populations: A, donor origin, young host; B, donor origin, aged host; C, host origin, young host; D, host origin, aged host.

 
The CD44, Mel-14, and CD45RB profiles of CD4+ Thy 1.1+ splenic and lymph node lymphocytes were determined and, as shown in Fig. 5Go (characteristic histograms) and Tables I (complete data set) and III, are age characteristic in pattern (as was found in the Ab-depleted animals). That is, Thy 1.1+ CD4+ T cells isolated from aged host chimeras were enriched for high CD44 expression and low Mel-14 and CD45RB display. In contrast, the young chimeras had Thy 1.1+ CD4+ profiles characterized by CD44low, CD45RBhigh and Mel-14high expression on the majority of cells.



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  		FIGURE 5. Expression of CD44, CD45RB, and Mel-14 by Thy 1.1+ CD4+ T lymphocytes from chimeric and Thy 1.2+ CD4+ 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.

 
Cells isolated from both the spleen and lymph nodes displayed the same pattern (Table IIIGo). That is, a higher fraction of CD4+ cells with "memory" levels of CD44 and CD45RB were identified in aged hosts. In both young and aged hosts, a lower percentage of memory cells was identified in the lymph nodes compared with the corresponding splenic populations.


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  		Table III. Lymph node CD4+ T cell phenotype

 
Statistical analyses indicate that the marker levels measured on all the young control and experimental animals are significantly different (at the 95% confidence level) from the aged groups for all three differentiation Ags (Table IGo). There are no significant differences between treatment groups of the same age, with the exception of CD44 expression by aged chimeric mice, which is significantly different from both aged control and aged Ab-depleted animals.

The lymphokine mRNA profiles of Thy 1.1+ CD4+ cells were compared between those purified from aged with those purified from young host mice. As shown in Fig. 4Go, the profiles were consistent with phenotype, that is, the aged host yielded a population that was activated to produce higher amounts of mRNA specific for IL-4, IL-5, and IFN-{gamma} than those from young host animals.

To obtain a more quantitative measure of cytokine-producing capacity, a second assay was performed in which the number of cytokine-producing cells was enumerated by intracellular cytokine staining and flow cytometry. An activation protocol was employed that used both anti-CD3 as well as anti-CD28. T cell-enriched populations were activated by culture for 24 h in the presence of immobilized anti-CD3 as well as soluble anti-CD28. Following this period, cells were further activated by a 5-h pulse with ionomycin and PMA in the presence of monensin. Intracellular cytokines (IL-2, IL-4, and IFN-{gamma}) were then detected by flow cytometry in conjunction with cell surface staining for CD4 and Thy. The data are summarized in Table IVGo. As suggested by the RNase protection assay, Thy 1.1+ CD4+ T cells arising in aged host mice more readily produce IL-4 and IFN-{gamma} than the same population arising in young host animals. The percentage of IFN-{gamma} containing Thy 1.1+ CD4+ T cells from aged hosts is fourfold greater than that in young hosts, while a seven to eightfold higher percentage of aged T cells produce IL-4 than young. In contrast, the fraction of IL-2-producing Thy 1.1+ CD4+ cells is almost threefold higher in the young compared with the aged host mice.


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  		Table IV. Production of cytokines by CD4+ T cells in chimeric mice

 
The cytokine content of the host-derived Thy 1.2+ CD4+ population was also examined. A similar, but not identical, pattern of 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-{gamma} 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 notably in the young host mice. In these animals, a greater fraction of the CD4+ cells of host origin produce IL-2 and IFN-{gamma} than those of bone marrow donor origin. Differences in the aged populations are of much less magnitude (less than twofold).

These data indicate that the aged host is capable of supporting differentiation of CD4+ T lymphocytes from bone marrow precursors. Despite the significant contribution of donor origin T cells to the total population, the resulting compartment remains, in aged hosts, highly enriched for memory/activated cells, rather than regenerating a substantial pool of naive cells. To ascertain whether the aged microenvironment preferentially supported memory cell expansion, chimeric mice were sacrificed at 4 wk post-transplant. At this time point 16% of splenic CD4+ T cells were of bone 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 and 32% in young and aged hosts, respectively. The purpose of this experiment was to determine the stability of the phenotypic distribution of donor CD4+ T lymphocytes over the period 4–8 wk after transplant. At 4 wk and again at 8 wk post-transplant, one-half of the animals were sacrificed, and the splenic Thy 1.1+ CD4+ was analyzed for simultaneous expression of CD44 and CD45RB. The naive population is defined as displaying the phenotype, CD44low, CD45RBhigh, while the converse phenotype is defined as the memory type. As shown in Fig. 6Go, there is a notable change in the relative proportions of naive and memory cells within the aged hosts between these time points. At 4 wk, the ratio of naive to memory splenic Thy 1.1+ CD4+ T cells in both the young and aged host mice is weighted toward the naive. At 8 wk, in contrast, the fraction of naive phenotype cells is greatly reduced in the aged host animals, and now displays a balance shifted toward memory cells. Young host mice display a similar shift, but of much less magnitude than that in the aged hosts, which does not reverse the naive to memory ratio of CD4+ T cells.



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  		FIGURE 6. Distribution of memory and naive subsets in splenic Thy 1.1+ CD4+ T lymphocytes. Bone marrow chimeras were constructed as described. Host animals were sacrificed at 4 or 8 wk post-transplant, and the distribution of donor (bone marrow) origin CD4+ T cells into memory and naive subsets was determined by the relative expression of CD44 and CD45RB as measured by multiparameter flow cytometry. Memory/effector cells were characterized as having the phenotype CD44high CD45RBlow, while CD44low CD45RBhigh cells were considered naive.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aged mouse is able to regenerate its peripheral T cell compartment from either endogenous or syngeneic bone marrow stem cells following Ab or irradiation treatment. However, the newly produced T lymphocytes do not rejuvenate a young-like CD4+ compartment, but, rather, the phenotype and lymphokine production profile of the reconstituted CD4+ population is very similar to that of the unmanipulated control animals, which in aged host mice is enriched for cells with a memory/effector phenotype.

These results suggest that the aged environment influences the maturation state of the peripheral T cells. This may result from alterations in thymopoiesis with age, causing the differentiation of T cells with a memory-like phenotype and function, or post-thymically, naive thymic emigrants may be induced by the aged microenvironment to undergo expansion and maturation.

The possibility that the intrathymic T cell differentiation pathway is altered with age has been suggested previously by Hirokawa and colleagues (15, 16). Using a thymus transplantation model in which thymic rudiments are placed under the kidney capsule of athymic, nude mice, these investigators have found that as the age of the thymus donor increases, the ratio of peripheral CD4+ to CD8+ T cells produced in the nude mice changes. Furthermore, the CD4+ T lymphocytes produced in the animals transplanted with aged thymic lobes, displayed memory-like characteristics, expressing high levels of CD44 and producing high levels of IL-4 upon activation. These data were interpreted as suggesting that the mature CD4+SP T cells produced by the aged thymus differ from those produced in the young.

If thymopoiesis were altered during the aging process in such a way as to result in the production of memory cells, it would be predicted that evidence of such a change could be detected in the CD4+ SP population, as an increased number of cells bearing CD44highCD4RBlow characteristic of the memory subset. However, a direct examination of the CD4+ single-positive thymocytes does not reveal such a population in the thymus of aged mice (17).

Alternatively, the memory-enriched population may arise peripherally. The data presented here support this alternative. The fraction of splenic CD4+ population that is derived from donor bone marrow stem cells increases more dramatically in aged vs young host mice between 4 and 8 wk post-transplant. In young mice there is a threefold increase in the representation of bone marrow-derived T cells (16–48%), while in the aged host there is a fivefold increase during the same period (6 to >30%). Furthermore, the Thy 1.1+ population at 4 wk in both aged and young hosts is enriched for naive cells. By 8 wk post-transplant, this ratio has been reversed in the aged hosts, with memory cells predominating.

The post-thymic expansion potential of T cells is well documented. Miller and Stutman (18) estimated that peripheral T cells can undergo 10,000-fold expansion, and sequential cell transfer experiments indicate that CD4+ T cells have the capacity for 8 x 105-fold expansion (19).

The intermitotic lifespan of naive T cells resident in normal young adult mice has been calculated to be quite long, on the order of several months (20). Determining the lifespan of memory T cells has presented greater difficulty due to the lack of a defining phenotype that unambiguously distinguishes effector from memory T cell populations. Approximately 70% of the memory/effector phenotype T cell population incorporates DNA-specific label over a relatively short labeling period (4–6 wk). These results suggest that the memory T cell life span may be heterogeneous, with both short- and long-lived subsets (21, 22, 23).

These data reflect the situation in homeostatic balance. In circumstances where the T cell pool is reduced, such as following irradiation, residual T cells have a marked capacity for rapid cell cycle. Transfer of purified T cell populations into T-deficient host mice results in significant expansion of the donor cells (19, 20, 24, 25, 26). This expansion is almost exclusively reported to be of memory cells (26, 27, 28). Ag-dependent expansion of mature memory T cells is reported to occur following bone marrow transplantation of thymic-deficient hosts (29, 30). However, there are reports indicating increases in naive T cells in the absence of specific or environmental Ag stimulus (31), suggesting that under some conditions, this population may be induced to proliferate by non-Ag activation.

The data presented here suggest that in bone marrow-transplanted mice, naive T cells, recently emigrating from the thymus, encounter an environment in the periphery that drives expansion and maturation to the memory state. The extent of this process is greater in the aged than in the young host. What might be the triggering stimuli? The capacity of the host environment to present Ag appears to play a role in this process (21). It is possible that characteristics of APCs such as lymphokine production or costimulatory molecule expression might differ with age and thereby differentially affect T cell homeostasis. A wealth of data supports the role of lymphokine networks in driving CD4+ T cell maturation (reviewed recently in Refs. 31–33). It is clear that the relative abundance of IL-4, IFN-{gamma}, and IL-12 plays a critical role in determining the outcome of T cell activation. The costimulatory molecules, B7-1 and B7-2, appear to be differentially involved with maturation of Th1 and Th2 effector cells (34, 35, 36, 37, 38, 39, 40). Blocking the B7-1 interaction increases IL-4 production in vitro, while increased IFN-{gamma} production results from blocking B7-2 interaction with CD4+ T cells. These costimulatory proteins are expressed in different amounts by various accessory cells (39, 40) and may therefore be present within the microenvironment in very different levels, thus influencing the ratio of the Th1/Th2 effector cells produced.

The data presented here imply that reconstitution of a naive population in the mature adult may not be easily accomplished by differentiation of a new CD4+ population. This conclusion may have significant implication in clinical situations where immune system regeneration is anticipated, as in the case of bone marrow transplantation following high dose chemotherapy or the proposed stem cell transfusions following CD4+ ablative treatments for HIV+ individuals. Numerous studies have indicated that immune reconstitution in adult bone marrow transplant patients may be delayed or incomplete for many years (29, 41, 42, 43). Other work suggests that rather than new differentiation, T cells within the graft expand and are the major source of mature, peripheral T lymphocytes in the host (27, 44, 45, 46). The results reported here indicate that while the aged murine host retains the capacity for at least a limited degree of thymic-dependent regeneration of the peripheral CD4+ T cell population, the characteristics of the new lymphocytes differ dramatically from those produced in young animals.


   Footnotes
 
1 This is publication 11061-IMM from the Department of Immunology, The Scripps Research Institute (La Jolla, CA). This work was supported by U.S. Public Health Service Grant R01AG09948. Back

2 Address correspondence and reprint requests to Dr. Marilyn L. Thoman, Sidney Kimmel Cancer Center, Altman Row, San Diego, CA 92121. Back

3 Abbreviation used in this paper: BSS, balanced salt solution. Back

Received for publication August 28, 1998. Accepted for publication September 28, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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