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Comparative Study
. 2009 Jan 15;182(2):784-92.
doi: 10.4049/jimmunol.182.2.784.

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

Mushtaq Ahmed  1 , Kathleen G Lanzer, Eric J Yager, Pamela S Adams, Lawrence L Johnson, Marcia A Blackman
Affiliations
Comparative Study

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

Mushtaq Ahmed et al. J Immunol. .

Abstract

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

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Figures

Figure 1
Figure 1. Highly purified populations of naïve CD8 T cells can be sorted from individual aged mice
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.
Figure 2
Figure 2. Spectratype analysis of naïve CD8 T cells from aged mice shows distortions in the Gaussian distribution characteristic of young mice
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.
Figure 3
Figure 3. Skewing of the spectratype profiles for aged compared with young mice
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.
Figure 4
Figure 4. Lack of similarity between Vβ-specific spectratype profiles in naïve and memory cells from individual aged mice
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.
Figure 5
Figure 5. Isolation of individual Vβ8.3 spectratype peaks from young and aged mice
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.
Figure 6
Figure 6. Over-representation of individual sequences reduces the diversity of TCR sequence usage among Vβ8.3+ naïve CD8 T cells in aged mice
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.
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