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. Author manuscript; available in PMC: 2010 Jan 15.
Published in final edited form as: J Immunol. 2009 Jan 15;182(2):784–792. doi: 10.4049/jimmunol.182.2.784

Clonal expansions and loss of receptor diversity in the naïve CD8 T cell repertoire of aged mice 1

Mushtaq Ahmed *,3, Kathleen G Lanzer *, Eric J Yager, Pamela S Adams *, Lawrence L Johnson *, Marcia A Blackman *,2
PMCID: PMC2724652  NIHMSID: NIHMS80408  PMID: 19124721

Abstract

There are well-characterized age-related changes in the peripheral repertoire of CD8 T cells, characterized by reductions in the ratio of naïve to memory T cells and the development of large clonal expansions in the memory pool. In addition, the TCR repertoire of naïve T cells is reduced with aging. As a diverse repertoire of naïve T cells is essential for a vigorous response to new infections and vaccinations, there is much interest in understanding mechanisms responsible for declining repertoire diversity. It has been proposed that one reason for declining repertoire diversity in the naïve T cell pool is an increasing dependence on homeostatic proliferation in the absence of new thymic emigrants for maintenance of the naïve peripheral pool. Here we have analyzed the naïve CD8 T cell repertoire in young and aged mice by DNA spectratype and sequence analysis. Our data show that naïve T cells from aged mice have perturbed spectratype profiles compared to the normally Gaussian spectratype profiles characteristic of naïve CD8 T cells from young mice. In addition, DNA sequence analysis formally demonstrated a loss of diversity associated with skewed spectratype profiles. Unexpectedly, we found multiple repeats of the same sequence in naïve T cells from aged but not young mice, consistent with clonal expansions previously described only in the memory T cell pool. Clonal expansions among naïve T cells suggests dysregulation in the normal homeostatic proliferative mechanisms that operate in young mice to maintain diversity in the naïve T cell repertoire.

Keywords: T cell receptor, T cells, Aging, Repertoire, Rodent

Introduction

Immune function declines with aging, characterized by an increased susceptibility to new infections and reduced responsiveness to vaccination (1–6). Because a diverse repertoire of naïve peripheral T lymphocytes is essential for efficient generation of immune responses to new infections and vaccines, it has been proposed that age-associated reductions in naïve repertoire diversity contribute to the characteristic decline in immune function in the elderly (7–11).

Naïve and memory T cells have been shown to occupy separate niches in the periphery and the ratio of naïve and memory peripheral T cells is relatively stable through adulthood. Furthermore, the separate niches are maintained by independent mechanisms. Whereas the memory pool is influenced by antigen experience, maintenance of the naïve T cell pool is independent of foreign antigen, and is controlled by thymic export and peripheral homeostasis (12–15). However, both in mouse and man, the peripheral repertoire becomes dramatically skewed with age in favor of memory T cells (16, 17). This is due to both thymic and peripheral events. Thymic involution as a consequence of aging results in decreased thymic output necessary to replenish the peripheral naïve T cell pool (3, 18–22). In addition, peripheral events, including the accumulating antigen experience of the host and the development of clonally expanded populations of CD8 T cells are thought to contribute to the relative increase in proportions of memory compared with naive T cells (23–27). Thus, the overall peripheral repertoire becomes progressively biased toward memory cells as fewer new T cells are produced and naïve T cells continue to be recruited into the memory pool as a consequence of antigen exposure.

Despite these pressures, naïve T cells persist with age and maintenance of the naïve pool in the face of decreasing thymic export becomes increasingly dependent on homeostatic proliferation (16, 28–30). Although T cell maturation takes place predominantly in the thymus, thymic emigrants undergo further phenotypic and functional maturation in the periphery after low affinity interactions with self peptide/MHC complexes (31–37). Consistent with this, naïve T cells have been shown to proliferate at a low level, although significantly less than the memory pool (38, 39). Whereas numbers of peripheral naïve T cells can be maintained by homeostatic proliferation, it has been suggested that peripheral homeostatic proliferation of naïve T cells in the face of declining thymic export will result in loss of repertoire diversity in aged mice (18).

T cell specificity is determined by the TCR. The diversity of the TCR repertoire is generated in the thymus through imprecise assembly of V, D, J and C gene segments to form the α and β chains of the TCR (40). A major contribution to repertoire diversity is the third hypervariable region of the receptor chains, also referred to as the third complementarity-determining region (CDR3). As a consequence of the imprecise joins between the V, D and J regions, there is a range of CDR3 sizes in a population of T cells that vary in length by 10 or more amino acids (41). This diversity can be analyzed by T cell receptor spectratyping, which measures the sizes of the CDR3s in a pool of cells by analyzing Vβ-Cβ RT-PCR products that have been labeled with a run-off reaction on a sequencing gel (10, 42–44). Typically, a histogram of CDR3 lengths from a diverse population of T cells is symmetrical and bell-shaped, generally referred to as Gaussian. Deviations from a Gaussian distribution are often seen within CD44high (memory) T cells, a consequence of expansion of a particular component of the population, for example as a result of antigen exposure or age-associated clonal expansions. In young individuals, the naïve repertoire remains diverse and Gaussian. However, perturbed spectratype profiles have been demonstrated among naïve CD4 T cells of aged humans (29), and estimates of receptor diversity among naïve CD4 T cells showed a precipitous decline after 70 years of age (16). These data are consistent with the hypothesis that homeostatic proliferation in the absence of new thymic emigrants results in constraints in naïve T cell repertoire diversity (18).

In this study, we have characterized repertoire diversity of naïve CD8 T cells from young and aged mice. Spectratype profiles of naïve CD8 T cells from individual young mice showed a Gaussian distribution, indicative of a diverse repertoire, as expected. In stark contrast, there ware extensive perturbations in the spectratype profiles of naïve CD8 T cells from individual aged mice, suggestive of a skewed repertoire. We carried out sequence analysis of individual clones from selected peaks within the Vβ8.3-Cβ spectratypes of young and aged mice to directly assess repertoire diversity. Unexpectedly, sequence analysis revealed evidence for clonal expansions in the naïve CD8 T cell pool in aged mice. These data provide the first report of age-associated clonal expansions in naïve T cells and support the idea that in the absence of new thymic emigrants homeostatic proliferation results in unequal maintenance of individual clones and loss of repertoire diversity.

Materials and Methods

Mice

Aged female C57BL/6J mice (17 months old) were purchased from the National Institute of Aging and used at 19 months of age. Young female C57BL/6 mice were purchased from Jackson Laboratory or bred at Trudeau Institute and used at 6 months of age. Animals were housed under specific pathogen-free conditions. All animal procedures were performed in compliance with the Institutional Animal Care and Use Committee at Trudeau Institute.

Cell enrichment and sorting

Single-cell suspensions of splenocytes were lysed of erythrocytes and B cells were removed by direct panning with 100 μg/ml goat anti-mouse IgG. The remaining cells were stained with fluorochrome conjugated monoclonal antibodies specific for CD19, CD4, CD8α, and CD44 in the presence of Fc block. Samples were then sorted on a FACSVantageTM flow cytometer with DIVA options (BD Biosciences) into CD8+/CD44low (naïve) and CD8+/CD44high (memory) populations. Monoclonal antibodies were purchased from either BD Biosciences (San Jose, CA) or eBiosciences (San Diego, CA).

RNA extraction and cDNA synthesis

Total RNA was extracted from sorted cells (0.78–1.5×106 cells from young mice and 0.95–4.0 × 105 cells from aged mice) using the RNeasy Mini kit (Qiagen, Chatsworth, CA) and eluted in a volume of 30 μl of DEPC-treated water. Using the RETROscript kit (Applied Biosystems/Ambion, Austin, TX), cDNA was synthesized from equal quantities of total RNA (160 ng) in a 40 μl reaction following the manufacturer’s instructions.

DNA spectratype analysis

Spectratyping analysis was carried out with modifications of the protocol described by Pannetier et al (42). Primer sequences for mouse Vβ and Cβ segments were synthesized at Integrated DNA Technologies (Coralville, IA). 1μl of cDNA was added to a final 50μl mixture containing 5μl of GeneAmp 10X PCR Buffer II (Applied Biosystems, Foster City, CA), 0.2 mM of dNTP mix (Invitrogen, Carlsbad, CA), 1.5 mM of MgCl2 (Applied Biosystems, Foster City, CA ), 10 pmol of 5’ Vβ and Cβ primers (Pannetier et al (1993) (Table I), and 1.25 units of AmpliTAQ Gold DNA polymerase (Applied Biosystems, Foster City, CA). PCR was run as follows: 10 min at 95°C, 35 cycles of 94°C for 45sec, 60°C for 45sec and 72°C for 1 min, with a final extension at 72°C for 7 min. 2μl of the PCR products were then used as template for an elongation reaction (run-off reaction) using only a 6-FAM-labeled 3’ Cβ primer in a 50ul reaction. PCR was carried out as follows: 10 min at 95°C, 10 cycles of 94°C for 45sec, 62°C for 45sec and 72°C for 1 min, with a final extension at 72°C for 7 min. PCR products (1 μl) from run-off reactions were mixed with loading buffer containing GeneScan 500 ROX size standard (Applied Biosystems, Foster City, CA ) and denatured at 95°C for 2 min. Samples were then applied to an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). GeneScan software, version 3.1 (Applied Biosystems, Foster City, CA) was used to analyze the spectratype data.

Table I.

Oligonucleotide sequences of spectratyping primers

Vβ and Cβ primers Sequence 5’ to 3’ Size of primer (bp) Distance to CDR3 (bp)a
Vβ1 CT GAA TGC CCA GAC AGC TCC AAG C 24 83
Vβ5.1 CAT TAT GAT AAA ATG GAG AGA GAT 24 135
Vβ8.1 CAT TAC TCA TAT GTC GCT GAC 21 141
Vβ8.3 T GCT GGC AAC CTT CGA ATA GGA 22 127
Vβ9 TCT CTC TAC ATT GGC TCT GCA GGC 24 57
Vβ10 ATC AAG TCT GTA GAG CCG GAG GA 23 48
Vβ11 G CAC TCA ACT CTG AAG ATC CAG AGC 25 64
Vβ12 G ATG GTG GGG CTT TCA AGG ATC 22 117
Vβ16 C ACT CTG AAA ATC CAA CCC AC 21 58
Cβ CTT GGG TGG AGT CAC ATT TCT 21 60
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a

The length in bp for V primers is counted from the first base in the V gene corresponding to the 5’ end of the primer through to the last base of the V gene before the CDR3 region. The length in bp for the C primer are counted from the first base of the J gene after the CDR3 region through to the last base in the C gene corresponding to the 5’ end of the primer (42).

Repertoire analysis by isolation of TCR Vβ PCR products and CDR3 junctional sequencing

The TCR Vβ PCR products corresponding to selected Vβ8.3 spectratype profiles from one young and three aged mice were resolved on a 6% denaturing acrylamide gel. The PCR products were silver-stained using a Silver Sequence DNA Staining Kit (Promega, Madison, WI). DNA bands of interest were excised and purified using a QIAEX II kit (Qiagen, Chatsworth, CA). 1μl of eluted PCR products was re-amplified using the SuperTaq Plus Polymerase PCR kit (Applied Biosystems/Ambion Austin, TX). PCR was run as follows: 2 min at 94°C, 20 cycles of 94°C for 45 sec, 60°C for 45 sec and 72°C for 1 min, with a final extension at 72°C for 7 min. These PCR products were further resolved on a 15% non-denaturing acrylamide gel and subjected to DNA silver staining. The bands of interest were excised, purified and re-amplified by PCR, as described above. The amplification products were purified using a PureLink Gel Extraction Kit (Invitrogen, Carlsbad, CA). The purified PCR products were cloned into vector pCR2.1-TOPO using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Ligated products were transformed into One-Shot OmniMAX 2T1 Phage-Resistant Cells (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions, to achieve maximum transformation efficiency. By this approach a spectrum of clones representative of all band species within a spectraype peak was generated. DNA sequencing of the clones was performed at Genewiz Inc. (South Plainfield, NJ), using plasmid DNA, or directly on LacZ− colonies. TCR CDR3 sequences derived from each peak were aligned and analyzed using CLUSTALW2 program and multiple alignment tool at http://www.ebi.ac.uk/Tools/clustalw2/index.html.

Statistical analysis

Comparative analysis of spectratype profiles from young and aged mice

The normalized value of peak intensities for individual aged mice was compared peak by peak with the normalized mean ± 3 SD for young mice. The probability that a given peak within a spectratype would fall more than 3 standard deviations from the mean of the corresponding peak in the reference set is 0.01 under the null hypothesis that the spectratype was a random sample from the reference population. Under that hypothesis, the probability of finding at least one "skewed" peak within a given spectratype is 1-(probability of 0 skewed peaks) = 1 - 0.99(# peaks within that spectratype). Thus, the probability of an 8 peak spectratype drawn at random from the reference population being scored as "skewed" was calculated to be 1-0.998 = 0.077. The number of "skewed" spectra for each Vβ was noted for each aged mouse. Given the above probability (0.077) that a given spectratype of 8 peaks would be scored as "skewed" under the null hypothesis, we next determined the binomial probability of finding one or more skewed spectratypes within the set of all Vβ spectratypes. This probability is 1- (prob. of 0 skewed spectratypes) = 1-(1-0.077)6 = 0.381. From this, we calculated the probability that any mouse would have 3 or more skewed spectratypes simply by chance to be <0.01.

Analysis of sequence diversity

Based on the concepts outlined by Jost (45), we chose the Shannon diversity index as an appropriate measure of diversity within the sequence sets. We then followed the procedure outlined in Hutcheson (46) to compare diversity indices for different sequence sets statistically by t-test. Thus, a set of N=50 total sequences was classified as n1 of type 1, n2 of type 2, ….,ns of type s, yielding s different sequences among the ni= N total sequences. The diversity index H for each aged and young sequence set was calculated by the well known formula

H=−∑(ni/N)ln(ni/N).

The variance of H (varH) was estimated from a series expansion as shown in Hutcheson (46). We compared two diversity indices by t-test, using the degrees of freedom (df) calculated by

df=(varH1+varH2)2/(varH12/N1+varH22/N2)andto=(H1−H2)/(varH1+varH2)1/2

We then compared to with tabulated tα for the calculated df to determine the significance level.

Results

Highly purified naïve CD8 T cells can be isolated from aged mice

The ratio of naïve to memory T cells in the periphery declines with increasing age (16, 17, 47). Indeed, using CD44 as a cell surface marker to distinguish naïve (CD44low) and activated/memory (CD44high) CD8 T cells (48), we found that the proportion of naïve CD8 T cells was greatly diminished in the periphery of aged (19 months old) compared with young (6 months old) mice (Figure 1). Despite the limited numbers of naïve CD8 T cells in aged mice, we were nevertheless able to obtain substantial numbers (>1 × 105) of highly purified naïve CD8 T cells from individual aged mice using flow cytometric sorting (Figure 1).

Figure 1. Highly purified populations of naïve CD8 T cells can be sorted from individual aged mice.

Figure 1

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Gated CD8 T cells from a representative young and two individual aged mice were analyzed for CD44 expression. The data show that the proportion of naïve (CD44low) relative to memory (CD44high) CD8 T cells varied in individual aged mice, but was consistently lower than in young mice. Despite the under-representation of naïve CD8 T cells in aged mice, highly purified populations for repertoire analysis could be obtained by flow cytometric sorting.

Spectratype profiles of naïve T cells from aged mice are skewed

We next analyzed the diversity of the TCR repertoire in naïve CD8 T cells from young and aged mice by Vβ spectratype analysis. To compare individual mice, RNA was prepared from similar numbers of FACS-sorted CD44low (naïve) CD8 T cells from each mouse, and cDNA was synthesized from 160 ng RNA from each mouse. Analysis of β-actin amplicons confirmed the quality and quantity of the cDNA from individual mice (data not shown).

The cDNA samples were subjected to spectratype analysis using 5’ Vβ primers for 9 different Vβ families and a 3’ Cβ primer (Table I). Spectratype analysis of naïve CD8 T cells from 5 individual young mice and 9 individual aged mice is shown in Figure 2. As expected for the young mice, for each Vβ family, a Gaussian pattern with an average of six to eight peaks, spaced by three nucleotides, was observed. However, the spectratype profiles for the aged samples showed perturbations for many Vβs, which varied within individual aged mice.

Figure 2. Spectratype analysis of naïve CD8 T cells from aged mice shows distortions in the Gaussian distribution characteristic of young mice.

Figure 2

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Spectratype analysis for selected Vβ gene families for 5 young (Y1 through Y5) and 9 aged (AG 2 through AG 10) mice is presented. CDR3 size is shown on the x axis and relative peak area is shown on the y axis. The peak corresponding to a CDR3 length of 10 amino acids is shown for each Vβ family.

To quantitate the degree of perturbation among the aged spectratype profiles, peak areas in each Vβ spectratype were compared against the distribution of a reference standard that was determined by averaging values obtained from the naïve CD8 T cell component of the 5 young mice. The intensities of the peaks within each Vβ spectratype for each young and aged mouse were normalized to a value of 1. The mean and standard deviation of each (normalized) peak for each Vβ was calculated for the set of young controls, and the mean + 3 standard deviations plotted for each peak (shown by bars in Figure 3). For each aged mouse (individual symbols in Figure 3), the spectratype for a given Vβ was compared peak by peak with the corresponding spectratype of averaged values for the young mice. A Vβ spectratype was scored as “skewed” for an aged mouse if one or more peaks within that spectratype fell outside 3 standard deviations of the corresponding young mouse reference peak. The data in Table II show that there was substantial skewing in all the Vβs except Vβ16, and that the patterns of skewing varied for individual aged mice. The lack of skewing in Vβ16 may be a consequence of the large standard deviation of the reference profiles for young mice seen uniquely for this Vβ (see Figure 3). Among individual aged mice there was a range of skewing among the Vβ families from less (skewing in 4 of 9 Vβs, observed in aged mouse #3) to more (skewing in 8 of 9 Vβs, observed in aged mice #5, #6 and #10). Thus, for each aged mouse, we rejected the null hypothesis that the presence of skewed spectratypes was random. All aged mice showed evidence of significant skewing of Vβ spectratypes (Table II).

Figure 3. Skewing of the spectratype profiles for aged compared with young mice.

Figure 3

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The areas of individual peaks were normalized to a value of 1. The mean and standard deviation of each normalized peak for each Vβ was calculated for the set of young controls and the mean ± 3 SD plotted for each peak (shown by the bars). The normalized peak values for individual aged animals are plotted as distinct symbols.

Table II.

Skewed Vβ spectratypes in aged compared with young mice

Vβ1 Vβ5.1 Vβ8.1 Vβ8.3 Vβ9 Vβ10 Vβ11 Vβ12 Vβ16 Number of skewed profilesb Probability of skewnessc
AG 2 2 a 2 5 1 1 0 6 1 0 7/9 <0.0001
AG 3 2 0 7 0 1 0 6 0 0 4/9 <0.0001
AG 4 4 6 6 6 3 6 7 0 0 7/9 <0.0001
AG 5 1 6 7 1 1 1 6 3 0 8/9 <0.0001
AG 6 3 6 4 4 4 1 6 2 0 8/9 <0.0001
AG 7 3 1 5 3 3 0 5 5 0 7/9 <0.0001
AG 8 5 5 2 2 0 4 3 2 0 7/9 <0.0001
AG 9 2 6 1 1 0 0 5 1 0 6/9 <0.0001
AG 10 3 6 3 1 1 1 3 2 0 8/9 <0.0001
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a

Number of skewed peaks within each Vβ-Cβ spectratype profile

b

Number of skewed Vβ-Cβ spectratype profiles

c

Probability under the null hypothesis (refer to Materials and Methods) that Vβ spectratypes do not differ from those of young adults

The relationship between naïve and memory CD8 T cell spectratype profiles for the same Vβ varied between individual mice and also varied within different Vβ spectratype profiles for the same mouse. The data demonstrated four distinct patterns (Figure 4). In some cases individual Vβ spectratype profiles were skewed for naïve cells but not memory cells (Figure 4A) and in other cases, they were skewed for memory cells but not the corresponding naïve cells (Figure 4B, 4C and 4F). In another case there was skewing in both naïve and memory cells, the patterns were different (Figure 4I). Finally, there was an example where there was a similar pattern of skewing in both the naïve and memory cells (Figure 4E). This latter pattern could not be attributed to contamination of the sorted population of naïve cells with memory cells because this pattern was not consistent for all the Vβ spectratype profiles from this mouse. For example, although the patterns for the naïve and memory cells were similar for Vβ8.3 in aged mouse 4 (Figure 4E), the patterns were very distinct for Vβ8.1 (Figure 4D) and Vβ12 (Figure 4F). This latter pattern in which skewing is similar for both the naïve and memory CD8 T cell pool raises the possibility that, at least in some cases, clonal expansions in the memory pool may stem directly from perturbations in the naïve pool. This possible mechanism merits further investigation. Importantly, however, this was only seen in one of nine cases examined. These results, taken together with published data (49–52), show that clonal expansions that are initiated in the naïve pool don’t appear to be a primary source of age-associated clonal expansions in the memory pool.

Figure 4. Lack of similarity between Vβ-specific spectratype profiles in naïve and memory cells from individual aged mice.

Figure 4

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Spectratype analysis was carried out for FACS-sorted naïve (CD44low) and memory (CD44high) CD8 T cells for selected Vβ gene families (Vβ8.1, Vβ8.3 and Vβ12) for 3 aged mice (AG 3, AG 4 and AG 8). CDR3 size is shown on the x axis and relative peak area is shown on the y axis. The peak corresponding to a CDR3 length of 10 amino acids is shown for each spectratype.

Sequence analysis of individual clones from selected peaks within the Vβ8.3 spectratype for naïve cells from young and aged mice

As a Gaussian spectratype profile in naïve T cells is indicative of a diverse repertoire, we considered the possibility that the perturbations observed in naïve T cells from aged mice were suggestive of reduced diversity. In order to formally link a skewed spectratype pattern with reduced diversity, we undertook sequence analysis of the specificities within an isolated band on a gel representing a spectratype peak of a given CDR3 size, as carried out previously to determine the diversity of the naïve repertoire in young mice (53). Each band in a spectratype peak, while uniform in size, is heterogeneous with respect to the nucleotide sequence composition (53, 54). Therefore, to determine whether the skewed profiles of aged mice are indicative of reduced repertoire diversity, we cloned and sequenced DNA isolated from selected single peaks from spectratype profiles of young and aged mice to estimate repertoire diversity.

We selected profiles generated from Vβ8.3-Cβ spectratyping from one young and three aged mice for further analysis (Figure 5). As expected, the spectratype profile for the young mouse was not skewed, whereas the Vβ8.3 spectratype profiles for the aged mice showed variable skewing. For example, as indicated in Table II, aged mouse #3 (AG 3) did not have a skewed spectratype profile for Vβ8.3. In contrast, the overall Vβ8.3 spectratype profile for aged mouse #4 (AG 4) was highly skewed (6/9 peaks skewed) and was moderately skewed for aged mouse #8 (AG 8) (2/9 peaks skewed). The DNA from the 211 and 220 base pair peaks of individual mice was cloned, and between 47 and 100 individual clones were sequenced. Analysis of diversity was based on the first (approximately) 50 sequences examined in each peak.

Figure 5. Isolation of individual Vβ8.3 spectratype peaks from young and aged mice.

Figure 5

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DNA from the 211 and the 220 bp spectratype peaks from 1 young (Y 3) and 3 aged (AG 4, AG 8 and AG 3) mice was separated by acrylamide gel electrophoresis and visualized by silver staining, to allow isolation of DNA representative of a single CDR3 size prior to cloning and sequencing.

The sequence data are summarized in the histograms in Panels A and B of Figure 6 showing the number of occurrences (y axis) of distinct sequences assigned identification numbers “1”, “2”, etc (x axis). Sequences that are not repeated are designated “unique” on the histogram. Analysis of both the 211 bp peak (Panel A) and 220 bp peak (Panel B) from the young mouse (Y 3) revealed that the sequences were mostly unique, and only a minority of the sequences were repeated 2–3 times. In contrast, sometimes a single sequence from aged mice occurred as many as 25–35 times (e.g. 211 bp sequences numbered “1” from AG 4 and 220 bp sequences numbered “1” from AG 4 and AG 8.) These data indicated the presence of clonal expansions in the naïve Vβ8.3+ CD8 T cell population of aged mice.

Figure 6. Over-representation of individual sequences reduces the diversity of TCR sequence usage among Vβ8.3+ naïve CD8 T cells in aged mice.

Figure 6

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DNA extracted from individual spectratype peaks (211 bp, closed bars or 220 bp, open bars) of the Vβ8.3 spectratype profiles of one young and three aged mice, isolated as shown in Figure 4, was cloned and sequenced. The data show the number of occurrences of individual base sequences (out of ~50 total sequences) from the 211 bp peak (Panel A) or 220 bp peak (Panel B) isolated from spectratypes derived from young (Y) or aged (AG) mice. The numbers along the x axis represent different DNA sequences and the number of times the same sequence occurs is plotted on the y axis. Sequences which occurred only once are presented to the right of the hatches on the x axis, and are designated unique. Panel C. The sequence frequency data, as shown in the histograms in panels A and B, were used to calculate diversity indices with their standard deviations, as described in Materials and Methods. Bars from aged mice marked with an * indicate diversity indices significantly reduced (p<0.001) compared with those of young mice with the same bp length.

In order to determine a quantitative measure of diversity among individual mice as members of a population we used a data analysis previously developed in disparate fields such as information science and ecology. We calculated measures of sequence diversity (diversity index) among sets of base sequences from young and aged mice and compared the indices statistically by t-test. Thus, the sequence frequency data shown in Panels A and B of Figure 6 were used to calculate diversity indices, as described in Materials and Methods, which are plotted (along with SD) in Panel C. Bars marked with an asterisk indicate diversity indices significantly (p<0.001) reduced compared with those of young control mice with the same bp length. The analysis shows that repertoire diversity in mice from which histograms are dominated by a single sequence occurring many times, indicative of clonal expansions, is significantly reduced compared to mice with histograms in which most sequences are unique.

Taking together the spectratype and sequence diversity analysis, the three aged mice examined fell into three patterns. One aged mouse (AG 3) showed minimal repertoire perturbation in terms of spectratype skewing (only 4 of 9 Vβ spectratype profiles showed skewing, and there were no skewed peaks in the Vβ8.3 spectratype profile, Figures 3 and 5) and demonstrated comparable sequence diversity of the 211 Vβ8.3 peak to the young mouse (Figure 6C). A second aged mouse (AG 4) was skewed in terms of spectratype profiles (7 of 9 Vβ spectratype profiles showed skewing and there were 6 skewed peaks in the Vβ8.3 spectratype profile) and showed limited sequence diversity in both Vβ8.3 peaks that were analyzed (Figure 6C). A third aged mouse (AG 8) was skewed in terms of spectratype profiles comparably to mouse AG 4 (7 of 9 Vβ spectratype profiles showed skewing), although there were only 2 skewed peaks in the Vβ8.3 spectratype profile of AG 8. One peak (211 bp) demonstrated high sequence diversity comparable to that of the young mice and the second peak (220 bp) showed limited diversity (Figure 6C). The overall conclusion is that skewing of the spectratype profile correlated with reduced repertoire diversity and evidence for clonal expansions. Thus, the overall tendency toward skewed spectratype profiles in aged mice (Figures 2 and 3, Table II) reflects reduced repertoire diversity within the aged naïve CD8 T cell pool (Figure 6).

Discussion

We have formally evaluated the diversity of the naïve CD8 T cell repertoire in aged compared with young mice. Spectratype profiles of naïve T cells isolated from young mice typically show a Gaussian distribution, indicative of a diverse repertoire. However, our analysis of naïve CD8 T cells from aged mice shows extensive perturbations in the normally Gaussian Vβ spectratype profiles, suggestive of oligoclonal expansions and reduced repertoire diversity. Furthermore, DNA sequence analysis of clones obtained from isolated spectratype bands has revealed the presence of clonally-expanded populations of naïve CD8 T cells in aged mice, formally establishing a link between skewed spectratype profiles and reduced repertoire diversity. The detection of clonal expansions in the naïve population of CD8 T cells is a novel observation indicative of age-associated perturbations in mechanisms of homeostatic proliferation.

These data are consistent with previous reports describing an age-associated loss of repertoire diversity in naïve human T cells. In one study, spectratype analysis of naïve peripheral human CD4 T cells suggested highly restricted oligoclonal T cell repertoires (29). In other studies, analysis of repertoire diversity showed a dramatic decline after age 70 (16), and naïve T cells from elderly individuals greater than 65 years of age were shown to have highly skewed spectratype profiles (55). Our data confirm and extend these observations for naïve CD8 T cells from aged mice. Importantly, we have formally established a link between skewed spectratype profiles and clonal expansions. Whereas there was no evidence of clonal expansions in spectratype peaks from young mice or unskewed peaks from aged mice, at least some peaks from skewed, aged spectratype profiles were shown to contain clonal expansions.

These data support the premise that with decreased thymic export associated with aging, maintenance of the pool of naïve T cells by homeostatic proliferation results in reduced repertoire diversity. In further support of this scenario, it has previously been shown that there is increased turnover of naïve T cells with aging (16, 56). We propose that the clonal expansions we have identified in naïve T cells are a direct consequence of homeostatic proliferation in an environment of severely reduced thymic export. Repertoire diversity has thus become compromised because of pressures to maintain the size of the pool (18).

Our data raise the question of how clonally-expanded cells can maintain a naïve phenotype. The response to survival signals results in homeostatic proliferation but not in activation, so naïve T cells undergoing homeostatic proliferation typically do not express cell surface markers associated with activation, such as CD44 (57). Naïve T cells can transiently acquire a memory-like phenotype during homeostasis-driven proliferation, but show no change in other activation markers, such as CD62L, CD25, or CD49d, and in a normal, non-lymphopenic environment, the cells revert to a CD44low phenotype after homeostatic proliferation (58–60). Thus, although homeostatic proliferation may sometimes be associated with up-regulation of activation/memory markers (CD44), this is typically followed by reversion to a naïve phenotype. An exception has been reported in extreme cases such as intense homeostatic proliferation associated with repopulation of an empty host in which it has been shown that naïve T cells can permanently convert to a CD44high phenotype (60). It has also been suggested that intense homeostatic proliferation of naïve cells in the elderly, perhaps to a self peptide, may serve to drive naïve T cells into the memory pool, further contributing to the shift in the naïve to memory phenotype ratio (56).

We find no evidence for a consistent relationship between the skewed repertoire in naïve and memory CD8 T cells from individual aged mice. This argues against both a mechanism by which clonally-expanded naïve CD8 T cells are driven into the memory pool and a mechanism by which clonally expanded memory cells lose their memory phenotype and inappropriately appear to be naïve. Thus, the data support our hypothesis that the expansions we see in the naïve pool are a consequence of dysregulated homeostatic proliferation. Failure to find evidence for cross-over between the naïve and memory pools re-enforces the idea that the naïve and memory cells occupy separate niches in the periphery and are regulated independently.

The development of clonal expansions suggests a selective mechanism for preferential homeostatic proliferation of specific clones. Individual T cell clones may have different requirements for their maintenance, probably depending on TCR avidity and the availability of selecting ligands (57, 61). In adoptive transfer experiments, it was shown that in the absence of cognate antigen there was differential proliferation among transferred T cells. In addition, there was a hierarchy of thymic and peripheral selection in which some lymphocytes were more easily replaced by competitors than others. As mentioned above, in the elderly, preferential homeostatic proliferation may be largely driven by self antigen, resulting in the development of autoreactivity. In support of this idea, it has been shown that as thymic output declines, the number of self-reactive T cells that may invade the peripheral pools increases (14). This is further supported by emergence of autoimmune diseases after thymectomy, and the age-related increase in self-reactive lymphocytes (62, 63).

An important issue, especially with regard to therapeutic intervention and attempts to restore repertoire diversity, is to determine the kinetics of the age-associated decline of repertoire diversity. In humans, there is a major involution of the thymus during adolescence and thymic output declines dramatically between 25 and 60 years of age. However, cycling remains steady, the proportions of naïve and memory T cells are maintained beyond age 65, and loss of repertoire diversity does not become apparent until approximately age 75 (16). Eventually, the reduced rate of thymic export cannot adequately replenish the peripheral pool and maintenance of pool size becomes almost completely dependent on homeostatic proliferation. Thus, after age 70, homeostatic proliferation doubles, repertoire diversity contracts dramatically, and the phenotypic distinction between naïve and memory CD4 T cells becomes unclear (16). In our study, kinetic analyses were not performed. Our analysis of individual 19 month old mice showed that at this age the mice exhibit aged repertoires in terms of naïve/memory distribution and the impaired ability to respond to influenza virus, yet have not developed a significant degree of clonal expansions in the memory pool (11) (data not shown). It will be important to determine at what age mice show evidence for loss of diversity among naïve T cells in terms of skewed spectratype profiles and clonal expansions.

Age-associated declining repertoire diversity of naïve CD8 T cells is thought to correlate with impaired immunity (7–11, 64). In support of this, we have previously shown an age-associated decline in reactivity to influenza virus-specific epitopes of low naïve precursor frequency and restricted diversity. In some cases, the decline in repertoire diversity was so extreme that it resulted in “holes in the repertoire” of aged mice to normally immunodominant epitopes. Importantly, the age-associated decline in repertoire diversity and loss of reactivity to the immunodominant epitope correlated both with impaired cellular immunity to de novo influenza virus infection and a compromised recall response to heterologous infection (11). More recently, it has become possible to accurately determine the precursor frequency of naïve CD4 and CD8 T cells of a given specificity. In one study, the number of peptide-specific CD4 precursors ranged between 20 and 200 (65) and, in another study, the number of peptide-specific CD8 precursors ranged between 80–1200 cells per mouse (66). Importantly, the CD4 precursor frequency predicted the size and TCR diversity of the primary CD4 T cell response following peptide immunization. The CD8 study showed that the Vβ bias in responding CD8 T cells was determined by a bias in precursor frequency and that the initial precursor frequency regulated the kinetics and immunodominance of the primary response, and also controlled the development of the memory population. Whereas age-associated loss in diversity may not always result in a “hole” in the repertoire, as in our previous studies (11), it is now possible to formally study the impact of age-associated declines in repertoire diversity to particular epitopes on responsiveness in individual mice.

In conclusion, at least one contributing factor to the well-characterized age-associated decline in immune function is reduced naïve T cell repertoire diversity due to loss of thymic function. Thus an important goal is the development of treatments to maintain or restore the size of thymus and/or to enhance thymic export in elderly individuals (67–69). A key point is whether, with restoration of high thymic output, the diversity of the repertoire can be restored (70, 71). A variety of therapeutic approaches are being tested, including thymic transplants (72) and restoration of thymic progenitor cells (73, 74) or haematopoietic stem cells (75). In addition, methods such as chemical or physical castration to lower circulating levels of sex hormones (76–78) or treatment with IL-7, alone, or in combination with LHRH agonists (67, 79, 80), have been shown to increase the rate of export from the existing, but atrophied, thymus. Finally, growth hormones to increase thymic mass (81) should be tested, as it has been shown that thymic output correlates with the size of the thymus and is independent of age (22). The mouse provides a good experimental model for determining whether T cell repertoire diversity can be restored in the aged, and whether this will result in improved immunity to new infections and vaccines.

Acknowledgments

We would like to thank Drs. David Woodland and Laura Haynes for critical evaluation of both the science and the manuscript. We acknowledge the early efforts of Drs. In-Jeong Kim and Stephen Judice in setting up the methodology for spectratype analysis.

We acknowledge assistance of Simon Monard and Brandon Sells with FACS sorting and Mark Lamere and Amy Moquin from the Trudeau Institute Molecular Biology Core for assistance with spectratype analysis.

Footnotes

1

This work was supported by the NIH grants (AG022175 and AG021600) and the Trudeau Institute to M. A. B

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