From our array results Supplemental Fig
From our array results (Supplemental Fig. S2), we found that differential lncRNA profiling exhibited much greater disparity compared to differential gene profiling in CD28nullCD8+ T cells, indicating that differentially expressed lncRNA(s) would serve as ideal hallmarks. Moreover, we were fortunate that the annotated antisense lncRNA subgroup with the least lncRNAs provided a double-validation of the lncRNAs and their antisense target genes (in the present study, only the neighboring target gene was studied) to support investigation of the screened biomarkers. Finally, to maximize the integrative potential of the screened lncRNA biomarker(s), we focused on immunity-related target genes that corresponded to the age-accumulated lncRNAs that had undergone cross-validation. From our stepwise screening and validation in CD28nullCD8+T survivin (baculoviral IAP repeat-containing protein 5) (21-28) synthesis and CMVpp65CD8+T cells, we identified the lncRNA NRON as a candidate biomarker for age-accumulated CMV-enhanced T cell aging. We used two pathway-dependent integrative networks to predict how lncRNA NRON might function with its immunity-related target gene, NFAT1, in T cell aging. In the upregulated network for CD28nullCD8+ T cells, we saw that an enhanced IL-4-inhibited anti-inflammatory cascade decreased NFAT1-dependent T cell differentiation, which has been indicated to promote the loss of CD28 (Weng et al., 2009). In that scenario, we assumed that reduction of NRON would attenuate the expression of NFAT1 during the inflammatory cascade, which could be enhanced by the reactivation of CMV persistence. In the downregulated network for CD28nullCD8+ T cells, we observed an increase in the coupled inactivation of GSK3b bonding in the phosphorylation cascade downstream of the CD28 transmembrane protein, which would attenuate the inhibition of NFAT1-dependent differentiation of CD8+ T cells [reviewed by (Esensten et al., 2016)]. In that scenario, it remains unclear how NRON would attenuate NFAT1 signaling. The greatest challenge in the present study was the very low cell count from the CMVpp65CD8+ T cell subset in only three HLA-matched donors. We could not draw >15 mL of peripheral blood biannually from the participants over 80 years of age, based on recommendations from the ethics commission. We were also unable to “activate” these epitope-specific T cells ex vivo to obtain more cells of interest. Because our aim was to investigate natural T cell aging in vivo, these degenerating/decaying antigen-specific T cells realistically represent the milieu within which the T cell aging biomarkers act. Furthermore, because the CMVpp65CD8+ T cell counts were very low, we could only focus on the CD28-dichotomous T cell compartment without further compartmentalization, because more specificity in the sub-grouped CD8+ T cells would reduce our ability to detect potential biomarkers in overlapping cell types. When considering that virus persistence enhances T cell aging, it is notable that NRON has been found to modulate HIV latency by both NFAT-dependent (Imam et al., 2015) and NFAT-independent (Li et al., 2016) mechanisms. It is also intriguing that episodic reactivation of HIV latency is similarly accompanied by an expansion of CD28nullCD8+T cells (Borthwick et al., 1994), which has led to HIV persistence being considered as an model of accelerated immunity aging [reviewed by (Desai and Landay, 2010)]. Similar to elderly “silent” CMV carriers, the increasing number of elderly HIV subclinical carriers also present the dilemma of age-accumulated virus contributions during CD8+ T cell aging [reviewed by (Pathai et al., 2014)]. Interestingly, CMV-specific CD8+ T cells often exceed HIV-specific CD8+ T cells in these subclinical HIV carriers who are virtually always CMV co-carriers (Effros, 2016)]. These results support the idea that NRON is substantially involved in the process of extrinsic viruses repetitively enhancing age-accumulated T cell aging. Our study had two main shortcomings. First, the subclinical hallmarks of CMV carriers at very advanced age only represent a relatively small portion of the elderly population, because the majority of nonagenarians cannot be free from clinical manifestations in the long-term. Second, we obtained only one candidate lncRNA as an integrative biomarker, after our validation series. These issues are both reflections of the complexity of age-accumulated CMV-enhanced CD8+ T cell aging. We attempted to validate the expression of NRON in other episodes of CMV to HLA-restricted TCRs rather than CMVpp65-HLA-0201*; however, this might require single-cell techniques in our advanced-age cohort.