Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be noncoding, including 5' UTRs and long noncoding RNAs (lncRNAs). Apparent ribosome footprints outside of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs). Here, we show hallmarks of translation in these footprints: copurification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including the induction of immune responses following human cytomegalovirus (HCMV) infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts at understanding how cells manage and exploit its consequences.

Repeated antigenic encounter drives proliferation and differentiation of memory T cell pools. An important question is whether certain specific T cells may be driven eventually to exhaustion in elderly individuals since the human life expectancy is increasing. We found that CMV-specific CD4⁺ T cells were significantly expanded in healthy young and old carriers compared with purified protein derivative-, varicella zoster virus-, EBV-, and HSV-specific populations. These CMV-specific CD4⁺ T cells exhibited a late differentiated phenotype since they were largely CD27 and CD28 negative and had shorter telomeres. Interestingly, in elderly CMV-seropositive subjects, CD4⁺ T cells of different specificities were significantly more differentiated than the same cells in CMV-seronegative individuals. This suggested the involvement of bystander-secreted, differentiation-inducing factors during CMV infection. One candidate was IFN-α, which induced loss of costimulatory receptors and inhibited telomerase in activated CD4⁺ T cells and was secreted at high levels by CMV-stimulated plasmacytoid dendritic cells (PDC). The CMV-specific CD4⁺ T cells in elderly subjects had severely restricted replicative capacity. This is the first description of a human memory T cell population that is susceptible to being lost through end-stage differentiation due to the combined effects of lifelong virus reactivation in the presence of bystander differentiation-inducing factors.

Highly differentiated CD8⁺CD28⁻CD27⁻ T cells have short telomeres, defective telomerase activity, and reduced capacity for proliferation, indicating that they are close to replicative senescence. In addition, these cells express increased levels of the senescence-associated inhibitory receptor KLRG1 and have poor capacity for IL-2 synthesis and defective Akt (ser⁴⁷³) phosphorylation after activation. It is not known whether signaling via KLRG1 contributes to any of the attenuated differentiation-related functional changes in CD8⁺ T cells. To address this, we blocked KLRG1 signaling during T-cell receptor activation using antibodies against its major ligand, E-cadherin. This resulted in a significant enhancement of Akt (ser⁴⁷³) phosphorylation and T-cell receptor–induced proliferative activity of CD8⁺CD28⁻CD27⁻ T cells. Furthermore, the increase of proliferation was directly linked to the Akt-mediated induction of cyclin D and E and reduction in the cyclin inhibitor p27 expression. In contrast, the reduced telomerase activity in highly differentiated CD8⁺CD28⁻CD27⁻ T cells was not altered by KLRG1 blockade, indicating the involvement of other mechanisms. This is the first demonstration of a functional role for KLRG1 in primary human CD8⁺ T cells and highlights that certain functional defects that arise during progressive T-cell differentiation toward replicative senescence are maintained actively by inhibitory receptor signaling.

Although human naturally occurring regulatory T cells (Tregs) may express either CD45RA or CD45RO, we find in agreement with previous reports that the (∼80%) majority of natural Tregs in adults are CD45RO⁺. The proportion of CD45RA⁺ Tregs decreases, whereas CD45RO⁺ Tregs increase significantly with age. Nevertheless, a small proportion of CD45RA⁺ Tregs are found even in old (>80 y) adults and a proportion of these express CD31, a marker for recent thymic emigrants. We found that CD45RO⁺ Tregs were highly proliferative compared with their CD45RA⁺ counterparts. This was due in part to the conversion of CD45RA Tregs to CD45RO expression after activation. Another difference between these two Treg populations was their preferential migration to different tissues in vivo. Whereas CD45RA⁺ Tregs were preferentially located in the bone marrow, associated with increased CXCR4 expression, CD45RO⁺ Tregs were preferentially located in the skin, and this was associated with their increased expression of CLA and CCR4. Our studies therefore show that proliferation features strongly in maintenance of the adult Treg pool in humans and that the thymus may make a minor contribution to the maintenance of the peripheral pool of these cells, even in older adults. Furthermore, the different tissue compartmentalization of these cells suggests that different Treg niches exist in vivo, which may have important roles for their maturation and function.

Short telomeres of circulating leukocytes are a risk factor for age-related diseases, such as atherosclerosis, but the exact mechanisms generating variations in telomere length are unknown. We hypothesized that induction of differentiated T cells during chronic CMV infection would affect T cell telomere length. To test this, we measured the amount of differentiated T cells and telomere length of lymphocytes during primary CMV infection as well as CMV-seropositive and -seronegative healthy individuals. After primary CMV infection, we observed an increase in highly differentiated cells that coincided with a steep drop in telomere length. Moreover, we found in a cohort of 159 healthy individuals that telomere shortening was more rapid in CMV-seropositive individuals and correlated with the amount of differentiated T cells in both CD4⁺ T cells and CD8⁺ T cells. Finally, we found that telomere length measured in blood leukocytes is correlated with lymphocyte telomere length. Thus, CMV infection induces a strong decrease in T cell telomere length, which can be explained by changes in the composition of the circulating lymphocyte pool.