Our results confirm and extend the observation that aging is associated with alterations in the phenotypes of T cells [45, 46], which likely underlie the deterioration of the aged immune system. Here we evaluated Naive, CM, EM and TEMRA subsets of TCD4 and TCD8 cells as defined by the expression of CCR7/CD45RA molecules, a widely accepted criterion for T-cell subsets definition . We compared healthy aging, COPD patients and a group composed of chronically tobacco-exposed individuals without clinical-laboratory evidence of pulmonary compromise. The three aged groups were additionally compared with a group of young adults (18–30 y-o). For each T cells subset, we analysed the expression of markers associated with differentiated, senescent and exhausted cells (CD27, CD28, CD57, KLRG1, and PD1).
As expected , we found a marked depletion of the Naive subset, with a shift toward phenotypes of advanced stages of differentiation on aged groups. Additionally, our results indicated that such shift was neither identical among the three aged groups, nor was the expression of the markers in each subset. Notably, imbalanced expression of these markers was already observed in Naive cells, particularly in COPD patients. These patients showed more intense alterations compared with the Youngs as well as with the Healthy aged in the CM, EM and TEMRA subsets of TCD4 and TCD8 cells. Surprisingly, Smokers displayed milder alterations than COPD patients, showing a profile similar to that of the Healthy aged group. Nonetheless, the three memory subsets in the aged groups were very disparate compared with the Youngs, presenting enhanced proportions of cells expressing markers of differentiation, senescence and/or exhaustion. The current findings, together with our previous report on altered telomerase length and telomerase activity , help to explain some of the immunological deficiencies observed in these aged groups.
Our data showed that COPD patients presented not only a drastically reduced pool of “truly” naive cells available for de novo immune responses, but also, paradoxically, increased fractions of naive cells with differentiation, senescence or exhaustion characteristics, likely impacting on their immunocompetence. In fact, the homogeneity of the naive cell compartment has recently been challenged. Data have been shown that they can be divided into truly naive cells (RTE and mature naive cells) and a small subset of “virtual memory” (VM) cells characterized by the expression of CD45RA and NK cell markers (KIR and/or NKG2A) . These cells have higher homeostatic proliferation (HP), higher response to cytokines and increased reactivity to autoantigens . However, data refer mainly to mice and the few human data described this subset only in TDC8 cells. Besides, the phenotype makers used to define naive/memory subsets in previous studies were often distinct from those we used: human TCD8 VM cells are typically RA+CD27− . In addition, there is evidence of functional impairment in elderly naive TCD8 and TCD4 cells such as reduced capacity of naive (CD45RA+CCR7+) TCD8 cells from healthy elderly (> 70 y-o) to be primed in vitro . This was linked to both quantitative deficits of the naive population, also verified here, and altered TCR signalling [51, 52]. Goronzy et al. reported that healthy elderly’s naive (CD45RA+) TCD4 cells also have a deficiency in TCR-associated intracellular signalling . Other authors observed that the TCD4 cell repertoire drastically contracted in the elderly, possibly due to increased homeostatic (non-T-cell receptor triggered) proliferation that follows aging . Interestingly, these alterations were observed in elderly over the age of 70, but not within 60–65 y-o, while the mean age of our three aged groups was ~ 65 y-o (83% were ≤ 70 years-old). Our findings show that (i) the phenotype alterations in the Naive subset were seen mainly in COPD patients yet they were rare in the Healthy aged or Smokers and, (ii) our COPD group showed alterations similar to a group of healthy elderly individuals at least 10 years older. Taken together, these observations indicate that COPD patients undergo premature immunosenescence, likewise it has been proposed to other pathological conditions (e.g., aids, autoimmunity) [31, 54, 55].
Pronounced phenotypic alterations were also evidenced in the three memory T-cell subsets of COPD patients. Compared with Healthy aged, Smokers and Youngs, the COPD CM TCD4 and TCD8 cells presented increased fractions of cells expressing highly differentiated, senescence or exhaustion markers. The CM TCD4 cells still retained a substantial fraction of non-exhausted/non-senescent and undifferentiated cells (e.g., CD27+CD28+), while in CM TCD8 cells these fractions were markedly reduced. Altogether, these data point to a dysfunctional CM compartment in COPD patients.
Perturbations similar to those found in CM were observed in EM and TEMRA T cells, which also discriminated the COPD patients from Youngs, Smokers and Healthy aged. In COPDs, while the EM TCD4 cells subset was inflated, it was the TEMRA subset that was inflated in the TCD8 compartment, probably reflecting the TCD8 cells-enriched pulmonary infiltrates in COPD and the fact that TCD8 cells are more prone to undergo age and inflammation-driven detrimental effects than TCD4 cells . Increase in cell fractions expressing phenotypes associated with advanced differentiation was seen in both EM TCD4 and TCD8 cells and consisted mainly of cells that lost CD28 expression or gained PD1 expression, resulting in marked decrease in the number of non-exhausted/non-senescent cells. In TEMRA cells, a shift toward highly differentiated and likely dysfunctional cells was observed in both TCD4 and TCD8 compartments, but more intensely in the latter, as exemplified by the significantly increased fractions of partially and highly differentiated cells.
The age-associated shift from naive cells to differentiated cells can result from two distinct proliferation pathways, antigen-driven and homeostatic proliferation (HP). Classically, chronic or repeated antigen stimulation has strongly been associated with the generation of highly differentiated memory T cells with senescent or exhausted phenotypes . However, the role played by homeostatic proliferation in this shift has been increasingly recognized .
In HP, proliferation of naive and memory T-cells is driven by specific cytokines (e.g., IL-2/IL-7/IL-15, possibly IL-21) and/or tonic self-antigens-MHC signals to TCR [58,59,60]. However, there is evidence that HP can also generate memory cells with these phenotypes . It is yet unclear the relative contribution of each of the two pathways to the construction of either the adulthood physiologic memory subsets or the aging-associated alterations in T-cell phenotype and function. Studies on young adults thymectomized during childhood as a model of premature aging, showed that the subgroup of patients with more pronounced alterations in T cell phenotypes and repertoire diversity, similar to those found in individuals over 75 years old age, was the one with chronic CMV exposure. Those without CMV exposure presented only mild alterations . This suggests that chronic/repetitive Ag driven proliferation plays a more important role than HP in the acceleration of immunosenescence. Such reasoning likely applies to our COPD patients who, differently from the healthy aged, have repeated infections and a persistent inflammatory background due to the pulmonary airway architectural destruction [62, 63]. However, HP itself is up-regulated in aged individuals and eventually further unregulated in age-associated settings such as lymphopenia or enhanced inflammatory background (inflammaging) , generating senescence-associated T cells , thereby fueling the increase in senescent and exhausted T-cells.
On the other hand, the comorbidities presented by the aged groups (Supplementary Table 2), especially CMV infection, could also influence the shifts in T cell subsets observed in the aged groups. We analyzed these potential interferences by applying a linear regression test with independent categorical variables (to allow utilization of continuous variables, i.e., the percentages of TCD4 and TCD8 Naive, CM, EM, and TEMRA subsets). These analyses did not find a statistically significant influence of the possible confounder factors on the size of the respective TCD4 and TCD8 subsets (data not shown).
Patients with COPD are known for having a clinically significant immune dysfunction, resulting in enhanced disease severity, higher risk of exacerbations and lower humoral immune responses to vaccines such as influenza vaccine . The characteristics and the mechanisms conducive to COPD’s immune depression are certainly multiple and yet not fully characterized, hence the chronic lung inflammation is currently considered a major driving force of the disease .
A few studies showed increased number of Tregs, PD1+ TCD4 or CD28null cells, and TCD8+CD28− cells in COPD patients [65, 66]. Our study extends these findings by showing that COPD have cells expressing a full range of senescent or exhausted phenotypes encompassing all TCD4 and TCD8 subsets, consistent with a premature immunosenescence phenotype as it has been proposed for AIDS, autoimmune diseases and other pro-inflammatory diseases. The concept of accelerated aging in COPD has been proposed with respect to the lung alterations ; however, the present data suggest that the systemic effects of lung alterations are sufficient to cause a generalized state of premature senescence of all T cell compartments. Cho et al.  have proposed immunosenescence as a critical mechanism for the development of COPD. On the contrary, based on our findings, we argue that the alterations of COPD (that occur at lower intensity in cigarette smokers without COPD) is what leads to accelerated or premature immunosenescence.
Surprisingly, our Smokers showed a profile closer to the Healthy aged than COPD patients. They exhibited the usual age-associated shift of naive to EM TCD4 and TCD8 cells, but not to CM or TEMRA T-cells as in COPD patients, and the resultant marked reduction of the pool of T-cells able to respond to new antigens. Nonetheless, the phenotypes displayed by their naive cells were in general similar to those of the Youngs and Healthy aged, suggesting a less aggressive phenotypic change of this subset, which is in fact, not an unexpected observation for Healthy aged under 70 years-old . Regarding the TCD4 and TCD8 CM, EM and TEMRA subsets, their phenotype distribution in Smokers was also close to those of the Healthy aged but disparate with respect to that of Youngs. Interestingly, on several occasions there was even a trend for a less altered profile of the memory cells phenotype than the COPD patients and eventually Youngs, especially for TCD8 cells: either lower proportions of highly differentiated, senescent cells or higher proportions of non-exhausted/non-senescent cells. These results are consistent with our previous study on TA and TL of T cells from aged groups that were very similar to the groups studied here. T cells’ TA in Healthy aged and COPD, but not Smokers, was decreased compared with Youngs. This was probably linked to the unexpected observation of similar TCD4 and TCD8 cells’ TL between Smokers and Youngs, while Healthy aged and COPD had significantly reduced TL .
In fact, the impact of cigarette exposure on the immune system is still a matter of debate. A recent review concluded that, although smoking plays a harmful role in overall human health, it is yet unknown why smoking is deleterious, since it exerts dual effects on immune responses . Smokers who did not develop COPD showed evidence of a milder inflammatory status than COPD patients [69,70,71,72]. Among the putative immune effects is the enhancement of T cell memory in adults. Studies showed more vigorous T-cell proliferation in response to mitogens in non-COPD smokers than non-smokers  as well as elevated number of circulating memory T cells (CD3+CD45RO+, CD4+CD45RO+) and class-switched memory B cells in human peripheral blood of smokers . Proliferation and resistance to apoptosis of TCD8 cells were augmented in healthy smokers as compared with COPD patients and healthy non-smokers .
Recently it has been shown that nicotine exposure converts human TCD8 cells to a non-exhausted, functionally active phenotype (PD1−) with high expression of IL7R, favoring proliferation and survival of these T-cells . In fact, most studies in humans show that smoking increases the number of TCD8 cells and their activation and function  while, paradoxically, smokers may have weakened immunity against infections. To our knowledge no extended phenotyping analysis has been done within TCD4 and TCD8 naïve and memory subsets. Our findings are consistent with and extend these previous observations to better defined subsets. It is interesting to note that our COPD patients are ex-smokers, having ceased the addiction for at least 10 years. Thus, taking into account that the immune effects of tobacco exposure tend to disappear shortly after smoking cessation , we hypothesize that, compared with smokers with preserved pulmonary function, COPD patients not only undergo the systemic deleterious effects of the chronic inflammatory process of their smoke-damaged lungs, but have lost the putative immune-activatng effects of tobacco exposure.
Interestingly, we detected small fractions (~ 1%) of cells co-expressing CD57, KLRG1 and PD1 within EM and TEMRA TCD8 cells of the aged groups. However, in TCD4 cells, only COPD patients exhibited ~ 1% of these triple positive cells, out of a total of 40% EM cells. Although senescent and exhausted T cells result from different pathways and are functionally distinct, it has been hypothesized that T cells could be both senescent and exhausted [41, 78]. Our data indicate that, albeit at small numbers, these cells arise during physiological and pathological aging.
In conclusion, our results suggest that moderate to heavy chronic cigarette smoking may not accelerate immunosenescence when compared with aged non-smokers, provided the smokers escaped developing functional manifestations of lung damage even after many years of exposure. On the contrary, smokers who did not develop loss of lung function by age 65, may have undergone some delay in the deleterious immune effects of tobacco exposure. This retardment likely resulted in the more prolonged survival of already terminally differentiated cells. However, prolongation of the survival of highly differentiated cells is also related to mechanisms leading to cancer .
Besides that, the present results are consistent with the hypothesis that major immune defects do not appear to be the inevitable consequence of healthy aging , but that this does not hold true with pathological aging. We hypothesize that healthy aging is associated with alterations in T-cell subsets distribution as a consequence of a lifelong balanced exogenous Ag-driven and homeostatic proliferation, with the predominance of the latter. In this case, functionality of the immune system is relatively preserved, despite the shrinkage of the naive T-cells compartment. By contrast, in pathological aging, such as in COPD patients, an imbalanced Ag-driven proliferation would take over homeostatic proliferation, leading to enhanced accumulation of senescent and/or exhausted T cells and opening the way to infections, autoimmunity and other clinical complications.
It is important to note that our study has some limitations, such as the relatively small number of individuals within each group and the lack of functional studies of the putative senescent/exhausted T-cell subsets. Also, we observed some variability of the results within each study group, denoting those factors other than COPD or smoking influence the T-cell subsets distribution and phenotype. Finally, our results of the selected non-COPD smokers’ group, showing a rather physiological immunological ageing, indicates the need for further studies to unveil the complex immune effects of tobacco exposure.