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The Journal of Immunology, 2007, 178: 1321-1331.
Copyright © 2007 by The American Association of Immunologists, Inc.

Environmental and Intrinsic Factors Lead to Antigen Unresponsiveness in CD4+ Recent Thymic Emigrants from Aged Mice1

Karen Clise-Dwyer, Gail E. Huston, Amanda L. Buck, Debra K. Duso and Susan L. Swain2

Trudeau Institute, Saranac Lake, NY 12983


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Naive CD4 cells from aged mice respond inefficiently to Ag, but the factors that underlie the age-associated defects remain unclear. We have used two approaches to isolate recent thymic emigrants (RTE) in young and aged mice and have compared their capacity to respond to antigenic stimulation ex vivo. An in situ intrathymic CFSE injection labeled developing thymocytes and allowed the identification of RTE in secondary lymphoid tissues. Analysis of CFSE-labeled RTE and control unlabeled naive CD4 cells indicated that cells from aged mice were defective in their ability to increase intracellular Ca2+ concentration following TCR cross-linking. Aged naive and RTE CD4 also secreted less IL-2 and proliferated less than that of comparable young CD4 populations. Defects in effector generation in aged RTE were overcome by the addition of IL-2 to cultures. RTE from both polyclonal and TCR transgenic mice were compromised, indicating that defects were independent of TCR specificity. In the second model, the cotransfer of congenic marker-labeled young and aged BM cells into young and aged syngeneic hosts revealed that hyporesponsiveness in aged RTE was caused by a combination of defects intrinsic to CD4 progenitors and defects induced by the aged environment. Depletion of peripheral CD4 cells in aged mice led to production of new RTE that were not defective. The results of this study suggest that defects induced by environmental and lineage intrinsic factors act together to reduce responses to Ag in aged naive CD4 cells and that these defects can be overcome in aged CD4 cells produced during recovery from lymphopenia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Elderly populations suffer increased morbidity and mortality from infection due to decreased functional responses to cognate Ag recognition and altered lymphocyte homeostasis. It is critical to understand the genesis of these defects to develop strategies to boost immunity in aged populations. Age-associated changes in the composition of and loss of function in CD4 cell populations are well described, although the causative factors are unclear. In young animals, the majority of cells in the CD4 pool have a naive cell surface phenotype (CD44low CD62Lhigh) and stringent Ag and costimulatory requirements as well as a delay before the onset of proliferation after Ag recognition. A smaller cohort of CD4 cells results from previous exposure to Ag and has the reciprocal memory phenotype (CD44highCD62Llow). These memory cells respond more rapidly to challenge and have less stringent requirements for Ag and costimulatory factors than naive CD4 cells. Advancing age leads to a predominance of CD44highCD62Llow cells that are thought to accumulate as a result of a lifetime of exposure to Ags (1, 2), although some "memory" phenotype cells that arise with age may also be derived through homeostatic division or other Ag independent age-related changes, because they develop in TCR transgenic cells that have not been exposed to cognate Ag as well as in mice raised in germfree environments (3). Defects in response to Ag have been described in both CD44low CD62Lhigh cells and CD44highCD62Llow CD4 cells in aged mice and humans (4, 5, 6). The naive CD44lowCD62Lhigh CD4 T cells in aged animals proliferate and expand poorly in response to Ag due to the reduced synthesis of IL-2 (7, 8, 9, 10), resulting in reduced effector functions and reduced help to B lymphocytes (11).

The decline in the numbers of CD44lowCD62Lhigh CD4 cells in aged animals is in part due to reduced thymopoiesis in aged animals. Thymic output declines abruptly after puberty and thereafter the thymus undergoes progressive involution. Thymic involution is evident by a loss in overall thymic mass as well as by numerous changes in both the thymic microenvironment and thymic epithelial cells (12, 13). Recent studies in humans have shown that despite the alterations in the thymus with age, a low level of thymic output is sustained into old age (14, 15). In mice, signal-joint TCR excision circle levels, which indicate recent emigration from the thymus, remain constant in CD4+ splenocytes from 6 to 90 wk of age, suggesting that newly generated T cells continue to be incorporated into the CD4 population into old age (16). To produce new T cells, the thymus must be continuously seeded by early T cell progenitors derived from pluripotent hemopoietic stem cells in the bone marrow (BM)3 (17). T cell progenitor cells are reduced in number and frequency in aged mice and show a greatly diminished ability to repopulate fetal thymic lobes in organ cultures (18). Together, these studies suggest that T cells that develop in the aged thymus may come from defective progenitor cells and are subject to alterations in the environment in which they develop, which could decrease their competence.

To better understand the origin of the defects that are observed in aged naive CD4 cells, it is necessary to determine the functional capacity of aged recent thymic emigrant (RTE) in response to Ag. Because thymic output decreases in aged animals while peripheral CD4 cell numbers remain largely unchanged, it is likely that aged naive CD4 cells have been present in the aged environment for a longer period of time than comparable cells in young mice. It has been suggested that the development of age-related CD4 cell defects is associated with the post-thymic age of naive cells (19). If either increased life span or increased duration of exposure to the aged post-thymic environment was the sole cause of age-associated defects in naive CD4 cells, these defects would not be present in RTE from aged mice. A recent study from our laboratory sought to address this question by testing the functional capacities of CD4 cells developing in the aged environment following depletion and reconstitution (19). This study found that when CD4 cells were depleted from aged and young AND TCR transgenic (Tg) mice, they produced new CD4 cells with comparable in vitro Ag responses after reconstitution. However, the CD4 cells present in the reconstituted young and aged animals had likely undergone homeostatic division after their emergence from the thymus, and this may affect their ability to respond to Ags. Additionally, cell death associated with depletion could lead to inflammation and inflammatory mediators that are themselves known to enhance aged immune responses (20). These results left unanswered the question of whether newly generated CD4 cells developed in a nonlymphopenic aged environment would be able to respond efficiently to Ags.

RTE are cells that have just emerged from the thymus and have migrated to secondary lymphoid tissues to join the naive T cell pool. In both young and aged animals RTE represent the only input into the naive T cell population and maintain the diverse repertoire that allows a high-affinity response to newly encountered Ags. In mice and humans no definite cell surface phenotype has been determined to identify RTE, although in the rat CD4+ RTE are described as CD45RC–CD90+ (21). Intrathymic FITC labeling has been widely used to identify CD4 RTE in mice and other experimental animals and to establish that their Ag responsiveness and cell surface phenotype is comparable to that of unlabeled CD4 cells (22, 23, 24), but this technique has not previously been used in the context of research in aging. Two studies were recently published that examine RTE in young (25) and aged (26) mice transgenic for GFP under control of the RAG-2 promoter. The authors isolated GFP+ young and aged RTE and found them unresponsive to antigenic stimulation regardless of age. Functional defects in young RTE would be surprising because other groups have reported that CD4highCD8– thymocytes, the immediate precursors of CD4 RTE, are responsive to in vitro TCR stimulation even in the absence of exogenous IL-2 (27). The results described herein clearly demonstrate that aging leads to reduced Ag responses in RTE but that young RTE have functional properties similar to those of naive CD4 cells from young mice.

To determine whether CD4 RTE developed in an aged environment would be able to respond to Ag, we used intrathymic CSFE injection to identify RTE. The Ag responses of RTE from young and aged AND TCR Tg (7, 8, 20, 28, 29) and polyclonal inbred mice (30, 31, 32, 33, 34, 35) were examined by comparing the functional capacity of CFSE+ RTE and CFSE– naive CD4 cells. The results indicated that many of the functional defects apparent in aged CD4 cells are also apparent in newly generated RTE from aged mice. A second model confirmed and extended these results. BM transplants were performed using a low dose of radiation to facilitate engraftment without causing long-term immune ablation. Congenically labeled donor BM cells from young and aged mice were cotransferred into syngeneic young and aged hosts to differentiate between the influences of lineage intrinsic defects in aged CD4 progenitor cells and defects in the aged environment on the generation of hyporesponsive CD4 cells in aged mice. The results revealed that age-associated defects are not completely rescued when aged progenitors develop in a young environment, and that similar defects are induced in young progenitors that develop in an aged environment. Surprisingly, Ag-responsive RTE were produced by aged mice that were recovering from CD4 depletion. Together, these results support a model in which age-associated CD4 defects are caused by factors intrinsic to aged T cell progenitors in combination with the aged environment but that these defects can be overcome to allow aged animals to produce functional CD4 cells.


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

AND TCR Tg mice were bred onto C57BL/6, (C57BL/6 x B10.Br)F1, (C57BL/6 x PepBoy CD45.1+)F1, or (C57BL/6 x B6.PL CD90.1+)F1 genetic backgrounds and inbred B10.Br and C57BL/6 mice were bred and housed at the Trudeau Institute Animal Facility, where they were fed sterile standard diet ad libitum and housed in isolator cages under specific pathogen-free conditions. Mice referred to as young were 2–4 mo of age; aged mice were 16–24 mo old. Aged mice were inspected for gross pathology and animals exhibiting pathology were excluded from experiments. All experimental animal procedures were approved by the Trudeau Institute Institutional Animal Care and Use Committee.

Intrathymic dye labeling

Mice were anesthetized with sodium pentothal. An incision was made over the suprasternal notch and through the thoracic cage. Retractors were used to expose the thymus. Twenty microliters of 5 mM CFSE (Molecular Probes) was injected directly into the thymus. The wound was closed with 9-mm wound clips. Mice injected intrathymically with 0.1 µg CFSE had detectable CFSE labeling of thymocytes 24 h after the injection. Total cellularity of the thymus was not different in CFSE-labeled and unlabeled mice, indicating that the dye labeling procedure did not significantly disrupt thymopoiesis by damaging tissue. In all four major thymic developmental subsets, CFSE was of heterogeneous fluorescence intensity as expected for the dye labeling of tissue in situ similar to published reports using FITC intrathymic dye injection (23, 36, 37). Due to heterogeneous levels of dye labeling, cells in the thymus, spleen, or lymph nodes were considered CFSE+ if their CFSE fluorescence exceeded that of CD4–CD8– splenocytes of nonthymic origin, which were not CFSE labeled during thymic injection. The frequency of CFSE+ cells observed in each thymic subset declined in all subsets over time as labeled thymocytes underwent developmental cell divisions causing dye loss. CFSE+ thymocytes were no longer detectable at day 9 in young or aged mice. Analyses of CFSE-labeled RTE were conducted 10 days after intrathymic injection unless otherwise indicated.

BM transfer

BM cells were harvested from the femurs and tibia of young AND CD45.1+ and aged AND CD90.1+ mice by flushing into sterile RPMI 1640. Mature T cells were depleted by incubation with mAb anti-CD8 (3.155), anti-CD4 (RL172), and anti Thy1.2 (HO13.4 and F7D5) followed by treatment with complement. Cells were then counted and mixed at a 1:1 ratio of young to aged cells and 3 x 107 cells were transferred i.v. Adoptive hosts were young and aged C57BL/6 mice given 300 or 950 rad of whole body irradiation from a cesium source. Analyses of donor BM-derived RTE (BM-RTE) were executed four weeks after BM transfer unless otherwise indicated.

RTE isolation

CD4+ cells were enriched before cell sorting using mouse CD4 (clone L3T4) conjugated MACS beads, LS columns, magnets, and protocols provided by Miltenyi Biotec. Cells were then stained to identify CD4 RTE and cell sorting was done on a FACSVantage SE flow cytometer equipped with the DIVA digital processing system (BD Biosciences). Staining for cell sorting was done in azide-free buffer. In some experiments, two to four young or aged mice were pooled before sorting for functional studies. Purity of all sorted populations was always ≥96%.

Flow cytometric analysis

For cytometric analysis, 106–107 cells suspended in PBS supplemented with 2% BSA and 0.1% NaN3 were incubated with fluorochrome-conjugated Abs for 20–30 min on ice and in darkness. Cells were either analyzed immediately or fixed in 1% paraformaldehyde. mAbs used for these studies were directed to CD4, CD8{alpha}, Vbeta3, V{alpha}11, CD44, and CD62L. Flow cytometric data were acquired on a Cytek-modified dual laser five-color FACScan (BD Biosciences) cytometer using CellQuest (BD Biosciences) and Rainbow (Cytek) software. Analysis of flow cytometric data was done using FlowJo version 6.1.1 software (Tree Star).

Ca2+ mobilization

Cells were stained for cell surface phenotype and then loaded with 12 µM indo-1 (Molecular Probes) in HBSS for 45 min at 37°C. Cells were then washed and stimulated with either anti-CD3 (clone 2C11; 10 µg/ml for 3 min) and goat anti-hamster IgG (20 µg/ml) or ionomycin (10 µg/ml) for 10 s following the initiation of acquisition on the FACSVantage SE equipped with a krypton UV laser. The median values for the ratio of Ca2+-bound indo-1 fluorescence to Ca2+-free dye, which are proportional to intracellular Ca2+ concentration, are shown over the course of 5 min. Although sometimes not shown, responses to ionomycin were comparable for all groups in each experiment, indicating that all samples were able to produce a strong Ca2+ flux.

Rhodamine 123 (Rh123) extrusion

Cells were stained for cell surface phenotype, incubated with 20 µg/ml Rh123 (Molecular Probes) in PBS for 10 min at 37°C, and resuspended in fresh PBS for 45 min at 37°C. Cells were then put on ice and analyzed immediately.

Cell cultures

AND TCR Tg cells were cultured with 5 µM pigeon cytochrome c fragment and mitomycin C-treated DCEK-ICAM cells, a fibroblast line that expresses CD80, ICAM-1 and I-Ek as APCs. B10.Br cells were cultured on anti-CD3 (clone 2C11)-coated plates with soluble anti-CD28. Cultures were initiated in sterile RPMI 1640 (7.5% FBS) with CD4 cells at an initial density of 2 x 105/ml and a 2:1 ratio of T cells to APCs. Recombinant mouse IL-2, obtained from the culture supernatant of X63.Ag8–653 cells transfected with murine IL-2 cDNA, was added at a final concentration of 11 ng/ml as indicated.

Cytokine analysis

Cytokine analysis of culture supernatants was performed on a Luminex instrument using Beadmate kits and protocols (Upstate Biotechnology).

Proliferation assays

Purified T cells were added to 96-well plates at 5000 cell/well with pigeon cytochrome c fragment/APC or anti-CD3 and anti-CD28 with or without IL-2. Cells were pulsed with [3H]thymidine 18 h before the indicated day of culture and then harvested onto glass fiber filters. Counts per minute were determined by scintillation counter. Although not shown, IL-2 added to cultures always restored aged T cell proliferation, abrogating differences between groups.

In vivo CD4 depletion

Mice were given 200 µg of ant- CD4 (GK1.5) or isotype control rat IgG2b (clone LTF-2) in PBS i.p. CD4 T cell numbers were monitored every 2 wk in the spleen, lymph nodes, thymus, and blood to assure complete depletion and assess reconstitution.

Statistical analysis

Differences were determined by paired two-tailed Student’s t test, assuming unequal variance. Values of p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Detection of CFSE-labeled RTE in secondary lymphoid tissues

To study aged RTE we developed a method to reproducibly label thymocytes in situ with CFSE, a fluorescent dye that has longer persistence than FITC, which has been used in previous studies (23, 36). We analyzed the appearance of CFSE+ cells in splenic CD4+ T cells in thymically injected AND Tg (Fig. 1A) and polyclonal inbred (Fig. 1B) mice. CFSE-labeled cells were readily apparent within the splenic CD4+ population. No difference was detected in the frequency of CFSE+/CD4+ cells between spleen and lymph node cells (data not shown). Labeling was not observed in CD4–CD8– cells in spleen and lymph node cells, confirming that the CFSE labeling was specific to thymus-derived cells. The fraction of peripheral CFSE+ RTE increased in frequency until day 10 and declined to near baseline levels by day 14 in young and aged mice, regardless of the presence of the TCR transgene (Fig. 1, A and B). The increase in the frequency of CFSE+ RTE correlated with the loss of the CFSE label in the thymus (data not shown). The loss of CFSE+ RTE after 10 days may be due to RTE cell division as is suggested in a recent study (26) or, alternatively, to the turnover of CFSE labeled proteins or cell death in the RTE population. As previously reported (26), aged mice produced significantly fewer CFSE+ thymic emigrants.


Figure 1
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  		FIGURE 1. Expression of memory cell markers in aged RTE is similar to that in the bulk of the aged CD4 pool. Three to 14 days following CFSE labeling, splenocytes were stained to identify CFSE-labeled RTE and examined by flow cytometry. A and B, Gate identifies CD4 RTE in young and aged AND TCR Tg (A) and polyclonal (B) mice. Cytometric plots are gated on CD4+CD8– lymphocytes. Numbers in plots indicate the frequency of CFSE+ cells within the CD4+CD8– splenic lymphocyte population. Aged mice produce fewer CD4+ RTE than young mice. C and D, Expression of CD44 is elevated on aged RTE from AND TCR Tg (C) and polyclonal (D) mice, whereas CD62L expression is decreased on aged RTE. CD44 remains higher and CD62L remains lower on aged RTE compared with young RTE throughout the interval in which RTE can be detected. Right bar graph panels, Average CD44 (left) and CD62L (right) mean fluorescence intensity (MFI) values for 2–5 mice per group over 14 days following CFSE labeling, C, Histograms are gated on CD4+CD8–Vbeta3+ cells. D, Histograms are gated on CD4+CD8– cells. {plussim}, p < 0.5 in comparison of mean fluorescence intensity in young and aged CD4 RTE. Results representative of three to eight independent experiments.

 
It has been reported that small numbers of mature peripheral T cells can migrate to thymic tissue and could thus be present in our CFSE+ populations (26, 38). To determine whether mature T cells were labeled in the thymus of either young or aged mice, 107 purified CD4+ T cells from syngeneic CD45.1+ polyclonal mice containing 15% memory phenotype cells were transferred to young and aged AND mice one day before intrathymic CFSE labeling. If mature CD4 cells were a significant fraction of cells in the young or aged thymus or if the CFSE injection itself recruited mature cells to the thymus, we would expect to observe obvious CFSE labeling in the donor cell cohort. When donor cells were examined after 10 days, <0.5% of donor cells fell into the CFSE+ gate compared with 3–10% of host CD4 cells (data not shown). No age-associated difference was detected in the frequency of donor cells that fell into the CFSE+ gate, indicating that intrathymic CFSE labeling was confined primarily to thymocytes in both young and aged animals. This result confirmed that the CFSE-labeled cells detected in the secondary lymphoid tissues in the period following dye labeling of both young and aged mice were RTE.

Cell surface phenotype of young and aged RTE

We examined the expression of a number of activation markers on RTE and control CD4+ cells from young and aged mice. The CD4 population shifts from a predominance of CD44lowCD62Lhigh CD4 cells in young mice to predominantly CD44highCD62Llow CD4 cells in aged animals. This shift is often considered to be due to the accumulation of memory cells after a lifetime of antigenic exposure. However, other studies suggest that at least part of this change in the phenotype of the CD4 pool is Ag independent and may be due to fundamental age-related changes in T cell homeostasis and that it correlates with the reduced capacity of aged CD4 cells to respond to antigenic stimulation (39). We found that CD4 RTE from aged mice differed significantly in their phenotype from young RTE (Fig. 1, C and D). RTE from young mice had the characteristic naive phenotype found in young CD4 cells as has been observed previously (40). A noticeable fraction of RTE from aged mice were CD44highCD62Llow, similar to the bulk of CD4 cells in an aged animal. These phenotypic differences between young and aged RTE were apparent as early as 3 days after intrathymic dye labeling and were sustained but did not increase for at least 2 weeks. These data suggest that the T cell phenotype may not correlate with Ag exposure in aged animals. Although the causes of these phenotypic changes are unclear, the similarity between aged RTE and the bulk of the aged CD4 population led to the hypothesis that aged RTE may already be hyporesponsive to antigenic stimulation like aged naive CD4 cells.

Functional defects of aged RTE in response to in vitro antigenic stimulation

The initial step in CD4 T cell response to Ag is ligation and signal transduction through the TCR. TCR cross-linking leads to a dramatic increase in intracellular Ca2+ concentration ([Ca2+]i). This transient elevation of [Ca2+]i can also be induced in vitro by treatment with cell permeable Ca2+ ionophores such as ionomycin. The rapid increase in [Ca2+]i in response to TCR ligation has been correlated with the ability of aged T cells to secrete IL-2 in effector cultures (41, 42, 43). Examination of the Ca2+ mobilization abilities of RTE and control CD4 cells from young and aged mice revealed a reduced [Ca2+]i flux in response to TCR cross-linking in both aged RTE and naive CD4 cells (Fig. 2, A and B, upper panels). In contrast, young RTE responded as robustly as young CD4 cells. Stimulation with ionomycin rescued the defect in aged cells (Fig. 2, A and B, lower panels). These data indicate that there are defects in the ability of aged CD4 RTE to respond to TCR ligation that could lead to reduced effector differentiation in aged CD4 cells.


Figure 2
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  		FIGURE 2. Weak responses to antigenic stimulation in aged RTE and naive CD4 cells. A and B, Reduced TCR-dependent Ca2+ flux in aged naive CD4 and RTE from AND TCR Tg (A) and polyclonal inbred (B) mice. Ten days after CFSE injection, spleen and lymph node cells were stained to identify RTE, labeled with indo-1, and stimulated with either anti-CD3 and a cross-linking 20 Ab or ionomycin. MFI, mean fluorescence intensity. C, Reduced proliferation in purified aged AND TCR Tg RTE (CD4+CD8–Vb3+CFSE+) and naive CD4 cells (CD4+CD8–Vb3+CD44lowCFSE–) cultured with APCs and cognate peptide. D, Purified polyclonal RTE (CD4+CD8–CFSE+) and naive CD4 cells (CD4+CD8–CD44lowCFSE–) from aged mice do not proliferate strongly when cultured with plate-bound anti-CD3 and soluble anti-CD28. E and F, AND TCR Tg (E) and polyclonal (F) RTE and naive CD4 from aged mice synthesize less IL-2 in effector generation cultures than CD4 populations from young mice. IL-2 was measured in the culture supernatants. *, p < 0.05 compared with young naive cells. Results representative of three to eight independent experiments.

 
Numerous effector generation defects have been reported in aged naive CD4 T cells (7, 8, 9, 10, 35). These defects include reduced proliferation and IL-2 synthesis in response to Ag, and can be largely rescued by the addition of exogenous IL-2 (7). To determine the functional capabilities of aged RTE in response to antigenic stimulation, we isolated CFSE+ CD4 RTE and, as a control, naive phenotype CFSE– CD4 cells from the spleens and peripheral lymph nodes of young and aged mice. Highly purified (>96%) control or RTE populations were placed into in vitro culture with or without added IL-2. Proliferation in cultures free of exogenous cytokines was significantly reduced for aged RTE as well as control naive CD4 cells in comparison to their young counterparts (Fig. 2, C and D). Reduced proliferation also led to a reduction in viable cells recovered in the cultures of aged cells (data not shown). Proliferation defects were apparent in aged CD4 cells regardless of TCR specificity and the type of stimulation that was used, e.g., peptide-pulsed APCs for the AND Tg cells or plate-bound anti-CD3 with soluble anti-CD28 for the polyclonal B10.Br cells. Defects in proliferation of aged control naive and RTE CD4 were rescued in cultures with added IL-2 (data not shown), as has been previously observed for aged AND Tg naive CD4 T cells (7, 8).

To determine whether aged RTE produced less IL-2, we measured IL-2 synthesis in cultures of TCR Tg and polyclonal CD4 cells. Cultures of aged CD4 RTE and naive T cells produced significantly less IL-2 in the absence of exogenous cytokines than young cells (Fig. 2, E and F). Results demonstrate a comparable reduction in IL-2 synthesis and proliferation by aged RTE and naive CD4 cells throughout effector differentiation, evident in both polyclonal and TCR Tg mice. As has been previously reported for aged naive CD4 cells (8), intracellular IL-2 staining indicated that the addition of exogenous IL-2 to cultures rescued the ability of aged RTE and control naive CD4 cells to produce IL-2 in response to antigenic stimulation (data not shown). These results indicate that substantial defects in CD4 T cells from aged mice are already evident at the time of release from the thymus.

CD4 depletion partially rescues age-associated defects in RTE

A recent study from our laboratory showed that when aged AND TCR Tg mice were treated with anti-CD4 mAb to deplete CD4 cells and then allowed to reconstitute the CD4 cell pool for 59 days, they produced new CD4 T cells with normal functional responsiveness to Ag (19). This could indicate that defects in aged cells were caused by extended cellular life span as compared with a young naive CD4 population. To better understand the causes of functional defects in aged CD4, it was important to reconcile the observations of loss of function in aged RTE that we observed here with the normal Ag responses in CD4 cells that reconstituted CD4-depleted mice in the previous study. We treated young and aged AND TCR Tg mice with anti-CD4 or isotype control Ab and, after the CD4 pool was replenished by thymic production of new T cells (50 days), we injected their thymi with CFSE. On day 59 following mAb treatment, we examined the functional and phenotypic characteristics of the CFSE+ RTE and CFSE– naive CD4 cells from the CD4-depleted and control mice. CD4 cells were not detected 2 days following anti-CD4 treatment in either young or aged mice, confirming that CD4 cells were completely depleted by the Ab. No difference was seen in CD4 cell numbers between anti-CD4-treated and isotype-treated mice by day 59, indicating that complete reconstitution of the CD4 population had occurred. In vivo CD4 depletion allowed repopulation of the Tg+ CD4 pool with cells that were uniformly CD44low in both the CFSE– and RTE populations of aged mice (Fig. 3A), suggesting that the reconstituted population may be functionally similar to young naive CD4. Indeed, the functional response to antigenic stimulation was essentially restored in the aged CD4-depleted mice as evidenced by the ability to increase [Ca2+]i, proliferate, and synthesize IL-2 in in vitro culture (Fig. 3, B–D). CD4 RTE and naive cells from aged isotype control Ab-treated mice failed to respond robustly to Ag like the cells from aged animals that received no Ab treatment in the previous experiments. Thus, CD4 depletion in aged mice leads to repopulation with peripheral CD4 cells with normal responsiveness to Ag.


Figure 3
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  		FIGURE 3. CD4 depletion restores production of Ag responsive T cells in aged mice. Young and aged AND TCR Tg mice were treated with anti-CD4 to deplete CD4 cells or isotype control Ab. Fifty days later, CFSE was injected into the thymus. Ten days after CFSE injection, spleen and lymph node cells were stained to identify RTE and their phenotype and response to Ag was examined. A, CD4 depletion prevents the generation of CD44high aged RTE. Left panels, Control CD4 (CD4+CD8–Vbeta3+CFSE–); right panels, RTE (CD4+CD8–Vbeta3+CFSE+). B, Strong TCR-dependent Ca2+ flux in RTE and naive CD4 from aged mice that are recovering from CD4 depletion. Left panels, Control naive CD4 (CD4+CD8– Vbeta3+CD44lowCFSE–); right panels, RTE (CD4+CD8–Vbeta3+CFSE+). MFI, Mean fluorescence intensity. C and D, Proliferation (C) and IL-2 synthesis (D) defects are rescued in aged RTE (CD4+CD8– Vbeta3+CFSE+) and naive CD4 cells (CD4+CD8–Vbeta3+CD44lowCFSE–) during recovery from CD4 lymphopenia. *, p < 0.05 in comparison to naive cells from young isotype control mAb-treated mice.

 
Impact of age-associated environmental and lineage dependent changes on RTE

Because the above results indicated that RTE from aged mice were defective, we examined whether the defect was present in BM CD4 precursors or was induced by development of T cells in the aged, involuted thymic environment. We transferred donor T-depleted BM cells from young CD45.1+ and aged CD90.1+ AND mice into syngeneic young and aged C57BL/6 (CD45.1–CD90.1–) mice. These experiments were performed as cotransfers in which young and aged donor BM cells develop into CD4 cells in the same environment and are examined within ~1 wk of their migration to the secondary lymphoid tissues, as donor BM-RTE. Sublethal irradiation at 300 rad was used to simultaneously obtain sufficient numbers of BM-RTE to study and minimize the complicating impacts of lymphopenia and inflammation on these studies. Normal splenic cellularity was observed in young and aged mice given 300 Rad of whole body irradiation after 2 wk. Donor BM-RTE were initially detected in the spleens of young and aged recipients at 3 wk following donor cell transfer and continued to accumulate (Fig. 4A). Comparison of numbers of young and aged donor BM-RTE that developed in young hosts confirmed that age induces defects in T cell progenitors, and a comparison of young BM-RTE that develop in young or aged hosts indicated that age-related environmental changes also contributed to reduced production of new T cells. CD4 progenitor age and thymic environment appear to act additively to reduce T cell maturation with the result that aged BM-RTE that develop in aged hosts are the least frequent.


Figure 4
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  		FIGURE 4. Environmental and lineage intrinsic factors in T cell development act together to induce the reduced response to Ag in aged RTE. Bone marrow was harvested from young CD45.1+ and aged CD90.1+ AND TCR Tg mice and mature T cells were depleted. Young and aged cells were then mixed together and adoptively transferred to lightly irradiated (300 rad) young and aged CD45.1–/CD90.1– syngeneic host mice. The numbers, phenotypes, and function of congenically labeled BM-RTE were analyzed. A, Donor BM-RTE begin to arise in the spleen at 3 wk after transfer and continue to accumulate. Young donor BM-RTE that develop in young hosts are the most numerous. Both the development of donor BM in an aged mouse and the age of the donor BM negatively impact the number of BM-RTE that are produced, with the result that aged donor BM-RTE that develop in aged hosts are the least frequent. B, The generation of CD44high BM-RTE requires both aged donor BM and an aged host environment. Left panels, CD44 expression; right panels, expression of the p-glycoprotein pump detected by Rh123 dye extrusion; shaded histograms, young donor BM-RTE (CD4+CD8–Vbeta3+CD45.1+CD90.1–); open histograms, aged donor BM-RTE (CD4+CD8–Vbeta3+CD45.1–CD90.1+). C, Partial TCR signal propagation defects are apparent in aged donor BM-RTE that develop in young hosts and in young donor BM-RTE that develop in aged hosts compared with young donor BM-RTE that develop in young hosts, which exhibit a robust Ca2+ response, and aged donor BM-RTE that develop in aged hosts, which make a very weak Ca2+ flux. MFI, Mean fluorescence intensity. D, Ag responses of purified young donor BM-RTE (CD4+CD8–Vbeta3+CD45.1+CD90.1–) and aged donor BM-RTE (CD4+CD8–Vbeta3+CD45.1–CD90.1+). Partial proliferation and IL-2 synthesis defects are observed in aged donor BM-RTE that develop in young hosts and in young donor BM-RTE that develop in aged hosts compared with the strong responses of young donor BM-RTE that develop in young hosts. *, p < 0.05 in comparison of all groups to young BM-RTE in young hosts. Results representative of three to five independent experiments.

 
Because we had observed reduced Ag responses and up-regulation of CD44 in RTE from aged mice and demonstrated that CD44low RTE in both young mice (Fig. 1, C and D) and aged CD4-depleted mice (Fig. 3A) were responsive to Ag, we wondered if there was a correlation between RTE phenotype and function. Phenotypic analysis of BM-RTE revealed that up-regulation of CD44 was only observed on aged BM-RTE that had matured in aged hosts (Fig. 4B, left panels). We also examined the ability of BM-RTE to extrude the fluorescent dye Rh123 via the p-glycoprotein pump. Extrusion of Rh123 by CD4 T cells occurs with increasing frequency as animals age, and Rh123-extruding cells are reported to have reduced responses to Ag (39, 44, 45). Comparison of all BM-RTE populations indicated that only aged BM-RTE that developed in aged hosts extruded Rh123 (Fig. 4B, right panels). Therefore, both an aged progenitor cell and an aged environment are required to lead to an age-associated RTE phenotype.

Next, we analyzed Ag responses of young or aged donor BM-RTE that developed to antigenic stimulation. Ca2+ flux initiated by TCR ligation was reduced in aged BM-RTE that developed in young hosts and in young BM-RTE that developed in aged hosts compared with the strong Ca2+ flux observed in young BM-RTE that matured in young hosts. All of these groups increased [Ca2+]i more than aged BM-RTE that developed in aged hosts (Fig. 4C). When BM-RTE were purified and tested in effector cultures, both T cell progenitor intrinsic and environmental defects were apparent (Fig. 4D). Aged BM-RTE that developed in young hosts proliferated significantly less and produced significantly less IL-2 than their young competitors, demonstrating defects intrinsic to aged T cell progenitors. Interestingly, a similar reduction in IL-2 and proliferation was induced in young BM-RTE that developed in aged hosts compared with those developed in young hosts, suggesting that development of the aged environment comprises T cell function. As in all experimental systems, addition of IL-2 to the cultures restored these responses so that no difference was observed between the groups of BM-RTE (data not shown). These data demonstrate that lineage intrinsic defects in aged T cell progenitors act together with factors in the altered aged environment to produce hyporesponsive CD4 RTE in aged mice.

Effect of T cell depletion via high dose irradiation on BM-RTE function

We showed that acute CD4 depletion with mAb led to generation of RTE in aged mice that did not exhibit the defects of RTE in unmanipulated aged mice (Fig. 2). To further investigate the conditions under which T cell depletion increases the responsiveness of BM-RTE to Ag, we examined the effects of radiation doses on the BM-RTE described above (Fig. 4). Young and aged AND TCR Tg BM cells were cotransferred to young and aged syngeneic hosts that received either 300 rad, a low dose that does not lead to lymphopenia, or 950 rad, a high dose that is lethal without BM transfer, of whole body irradiation. As described previously, T cells originating from donor BM were examined 4 wk after transfer within 7–10 days following emigration from the thymus. Effects of lymphopenia-inducing high dose irradiation on RTE phenotype and function were immediately apparent. CD44high aged donor BM-RTE were not generated in aged host mice when a high dose of radiation was administered (Fig. 5A). We isolated the four groups of BM-RTE from the young and aged host mice and tested their responses to Ag in in vitro cultures. We observed no defects in proliferation or IL-2 synthesis in BM-RTE when progenitor cells developed in mice that had been depleted of mature T cells by high dose irradiation (Fig. 5, B and C). Together, these data suggest that both progenitor intrinsic and environmental defects can be rescued by lethal irradiation, likely by the same mechanism by which anti-CD4 depletion rescued RTE function.


Figure 5
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  		FIGURE 5. T cell depletion via high dose irradiation rescues function in aged RTE. Comparison of BM-RTE generated with a low dose (300 rad) or a high dose (950 rad) of irradiation. A, The up-regulation of CD44 in aged BM-RTE that develop in an aged host does not occur when T cells are depleted by lethal irradiation. B and C, High dose irradiation rescues both lineage intrinsic and environmental proliferation (B) and IL-2 synthesis (C) defects in young donor BM-RTE (CD4+CD8–Vbeta3+CD45.1+CD90.1–) and aged donor BM-RTE (CD4+CD8–Vbeta3+CD45.1–CD90.1+) isolated from young and aged hosts 4 wk following BM transfer. *, p < 0.05 in comparison of all groups to young BM-RTE in young hosts.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Recent evidence suggests that the poor function of naive CD4 T cells in aged animals is largely responsible for the poor cellular and humoral responses of the aged, resulting in their increased susceptibility to infection and their lack of protective responses to vaccination (46, 47, 48, 49). In this study we show that defects equivalent in magnitude to those of aged naive CD4 cells in TCR signal propagation, in vitro IL-2 production, and proliferation as well as an age-related phenotype shift are found in aged RTE. RTE and naive CD4 cells that develop from aged BM cells in young adoptive hosts exhibit less severe functional defects, indicating that age-associated defects are partially due to intrinsic changes in T cell progenitors. Interestingly, age-associated functional defects are induced in young progenitor cells that develop in aged hosts, indicating that development in the aged milieu is also partially responsible for the reduced function of aged CD4 cells. These results support a model in which age-associated CD4 defects are caused by events early in T cell development and their severity intensifies with further differentiation in an aged environment.

The functional capacity of aged RTE is indistinguishable from control aged naive CD4 cells and is markedly reduced in comparison with young RTE and naive CD4. Aged RTE and control aged naive CD4 are unable to efficiently mobilize Ca2+ in response to TCR cross-linking. A previous study indicated that inability to flux Ca2+ correlates with reduced IL-2 production (41). Our results also indicated that aged RTE had defects in proliferation and IL-2 synthesis similar to the well-characterized defects of aged naive CD4 (8, 28). Proliferation and IL-2 synthesis were reduced when either the T cell progenitor or the environment was aged, indicating that both defects in progenitor cells and alterations in thymopoietic environment can contribute to age-associated defects in CD4 cells. Whether the defects in aged T cell progenitors are themselves due to the duration of exposure of stem cells to the aged BM microenvironment, the accumulation of genetic mutations, or other factors is not clear.

Our results on the functional response to Ag in RTE stand in contrast to published reports from the Fink laboratory in which neither young nor aged RTE proliferated or produced IL-2 efficiently (25, 26). Reduced function in young RTE would be surprising, because thymocytes at the final stage of development have been shown to be responsive to Ag even in the absence of IL-2 (27). Importantly, we find no difference in the magnitude of Ag responses between young RTE and young naive CD4 and, thus, our results do not support a functional maturation (25) of RTE following thymic migration. Additionally, in the recent study of aged RTE (26), no difference was detected in the antigenic responses of young and aged control CD4 cells. We and others have consistently found that aged naive CD4 cells proliferate less and synthesize less IL-2 than young naive CD4 cells when cultured in the absence of exogenous cytokines (2, 6, 7, 8, 9, 10, 19, 20, 28) independent of TCR specificity, genetic background, and means of stimulus. We hypothesize that the discrepancy between their results and those described here may be due to peculiarities inherent in the RAG-2 reporter NG-BAC Tg mice used to identify RTE. Regardless of the source of this discrepancy, the age-associated defects in effector responses of CD4 cells are well established and serve as a methodological control in all of our aging studies.

In our effort to understand the causes of age-associated decline in CD4 T cell function, we have observed that aged RTE are both phenotypically and functionally distinct from their counterparts in young animals. Aged RTE have a cell surface phenotype that is characteristic of the aged CD4 T cell pool, while RTE in young animals have a uniformly naive phenotype. Some component of the age-associated phenotype shift in CD4 T cells is likely to be independent of exposure to cognate Ag, because aged mice housed in germ-free conditions exhibit a shift to predominance of "memory" phenotype cells (3). It is unlikely that the up-regulation of CD44 in aged RTE is a result of antigenic exposure, because it is observed in RTE from both TCR Tg and polyclonal inbred mice. Homeostatic division is the only reported mechanism for CD4 cells to shift to a CD44high phenotype in the absence of Ag (50) but seems unlikely in this case because the up-regulation of CD44 was observed as soon as RTE were detected, and the frequency of CD44high RTE did not increase during the time that RTE could be detected. Additionally, in our BM-RTE model young donor BM-RTE that developed in an aged host did not develop an age-associated phenotype, suggesting that the aged environment cannot be solely responsible for CD44 up-regulation. Development of the age-associated phenotype required both an aged environment and an aged CD4 progenitor cell and was reflected also in Rh123 extrusion, which increases with age and has been directly correlated with reduced function (39, 44). With the finding that RTE from depleted mice are CD44low, we have established a correlation between low expression levels of CD44 on RTE and strong antigenic responses. These observations are striking in that they raise questions regarding the antigenic history of aged CD4 cells and whether the accumulation of long-lived memory cells over a lifetime is the sole cause of the age-related shift to the predominance of "memory" phenotype cells.

We had hypothesized, based on an earlier study, that increased post-thymic life span of aged naive CD4 cells might be largely responsible for defects associated with aging (19). If that hypothesis were correct, newly generated RTE would not be expected to exhibit age-associated defects. Because we found that aged RTE do show a group of defects like those seen with aged naive CD4 cells, we must reconsider this hypothesis. Together, these data suggest that defects in early TCR signal propagation, proliferation, and IL-2 production are the consequences of a complex process and cannot be attributed solely to increased post-thymic life span of the naive CD4 population. We considered the possibility that CD4 T cells with increased postthymic life span in aged mice may have an inhibitory affect on effector generation in aged RTE but dismissed this as unlikely because for all experiments described herein, each T cell subset is observed following isolation. In addition, we do not find increased functional responses in aged naive CD4 that are adoptively transferred to CD4 knockout hosts, so we do not think that removing old T cells alone will rescue age-associated defects in naive CD4. Therefore, we revise our understanding of the genesis of defects in aged CD4 and conclude that at least those defects that we describe here develop in newly generated RTE. We are in the process of examining further age-associated defects, including help for B cells and memory generation, to assess whether RTE also show such defects.

This leaves the question of why T cell depletion and reconstitution might rescue the aging defect. There are several possibilities. First, it is clear from earlier studies (8) that IL-2 reverses the age-associated defects in effector generation, including proliferation and IL-2 production. Indeed, we found that IL-2 reversed the defects seen here in both RTE and naive CD4 cells. IL-7 and other {gamma}c receptor-binding cytokines also restored proliferation but did not support the generation of fully competent effectors (7). It is thus possible that, in the T- or CD4-depleted situation, levels of IL-7 are elevated (51) and/or competition for IL-7 is reduced, resulting in an IL-7 mediated rescue. Alternately, T cell depletion requires large amounts of cell death, which may lead to inflammation. We have shown that the inflammatory cytokines IL-1, IL-6, and TNF-{alpha} can dramatically improve responses of aged naive cells (20). It is possible that these or other cytokines may be expressed at increased levels in mice recovering from T cell depletion and may create an environment that restores the development of functional CD4 cells. Finally, lymphopenia increases homeostatic proliferation, which is likely to affect T cell function. Regardless of the mechanism leading to the restoration of CD4 immunity, it is important that multiple extrinsic factors may be able to augment responses in aged CD4 cells. Further studies will be needed to identify the specific factors involved and to determine whether rescue can lead to functional effector and memory responses.

It is clear that immunological aging is a complicated, multifactorial process. Although thymic output declines, CD4 T cell numbers remain fairly constant into old age in mice and humans. CD4 T cells that comprise the peripheral pool in aged animals are believed to compensate for the reduced input of RTE through longer life span and peripheral Ag-independent proliferation. Because the current study focuses on the comparison between young and aged RTE, these results do not negate the hypothesis that the extension of life span and the peripheral expansion of T cell populations also contribute to functional defects in aged CD4 cells. Decreased T cell helper function in the elderly is likely due to a combination of defects in thymopoiesis and exposure of cells to altered aged environments in the primary and secondary lymphoid tissues as well as changes in the homeostasis of peripheral T cells, culminating in a severely compromised response to primary antigenic challenge and inadequate memory cells. Therapies designed to increase the rate of thymic output such as IL-7 treatment (52, 53, 54, 55, 56, 57) may ameliorate CD4 repertoire deficiencies in older individuals and, in combination with strategies to increase RTE function, might have the potential to improve functional immunity.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AG025805 and AG021600 and the Trudeau Institute. Back

2 Address correspondence and reprint requests to Dr. Susan L. Swain, Trudeau Institute, 154 Algonquin Avenue, Saranac Lake, NY 12983. E-mail address: sswain{at}northnet.org Back

3 Abbreviations used in this paper: BM, bone marrow; RTE, recent thymic emigrant; BM-RTE, BM-derived RTE; [Ca2+]i, intracellular Ca2+; Rh123, rhodamine 123; Tg, transgenic. Back

Received for publication July 11, 2006. Accepted for publication November 15, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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