Due to increases in human life expectancy, aging is regarded as one of the biggest health issues worldwide. During aging, several functions of the immune system experience dramatic changes, which fundamentally affect health and survival. This process termed “immunosenescence” is complex and affects almost most cellular and humoral components of immunity. Clinically, these changes have noticeable consequences for the effectiveness of immune responses, the vulnerability to infection, the efficacy of vaccination, and reactivation of latent viruses in aging individuals. Together, these progressive deficits result in increased morbidity and mortality (Castle, 2000; Pawelec et al., 2010).

In particular, the T-cell pool undergoes a striking age-associated remodeling process. While peripheral cell numbers do not change, the relative composition of T-cell subpopulations is importantly affected. A major component of this change is represented by an inverted CD4⁺/CD8⁺ T-cell ratio which appears in parallel to a decline in naïve T-cell numbers and the accumulation of highly differentiated memory cells (Pawelec et al., 2010). Mostly, these cells are CD8-positive T-cells which lack the expression of the lymphoid homing receptor CCR7 and costimulatory molecules such as CD28 alongside re-expressing CD45RA, termed terminally differentiated effector memory (TEMRA) cells (Weinberger et al., 2007; Arnold et al., 2011). TEMRA cells are known to produce large amounts of pro-inflammatory cytokines suggesting that they are involved in the development of a systemic low grade inflammation during aging. The reasons for the progressive appearance of these cells are suggested to be the persistent exposure to pathogens, frequent reactivations of latent viruses, and increased levels of oxidative stress (Effros et al., 2003; Simpson et al., 2016). The concomitant increase of proinflammatory molecules during aging, particularly TNF-α (Bruunsgaard et al., 2003) and IL-6 (Cohen et al., 1997), is suggested to amplify T-cell differentiation and senescence (Di Mitri et al., 2011). Accordingly, critical age related T-cells changes are accelerated by the existence of metabolic risk factors like insulin resistance or type 2 diabetes (Ventura et al., 2017). In the end, TEMRA cells may show an impaired immune function due to a series of molecular changes and loss of co-stimulatory molecules (Goronzy et al., 2012). The general importance of the age associated changes in the T-cell repertoire is highlighted by their inclusion in the IRP. The IRP represents a cluster of immunological parameters, which was shown to be associated with a poor immune function in elderly and predictive of earlier mortality (Wikby et al., 1998).

Regular exercise has been shown to significantly improve several aspects of immune function that decline with ageing (Walsh et al., 2011). With regard to T-cells, exercise training in mice improved T-cell proliferation in response to an antigen encounter (Liu et al., 2017). In aging mice it was demonstrated that exercise restored the percentages of naïve and memory cells in the spleen toward that of young mice (Woods et al., 2003). Untrained humans exhibited higher proportions of late-stage differentiated CD4⁺ and CD8⁺ cells and lower proportions of naïve CD8⁺ cells compared to trained individuals (Brown et al., 2014). Therefore, it is proposed that regular training might delay the onset of T-cell ageing or even has rejuvenating effects. While in most therapeutic interventions concentric endurance exercise is used to stimulate patients’ immune system, eccentric endurance exercise has also been shown to affect T-cell immunity and metabolisms (Jensen and Richter, 2012; Peake et al., 2015). Compared to concentric endurance exercises such as uphill walking, eccentric endurance exercises such as downhill walking induce higher strain on the muscle while the metabolic cost remains relatively low (Johnson et al., 2002; Camillo et al., 2015). Thus, especially physically unfit subjects that one can find in a population of middle aged men suffering from pre-diabetes, could profit from an exercise form like downhill walking that is less demanding for the cardiovascular system than uphill walking (Drexel et al., 2008; Philippe et al., 2017).

The aim of the current study was to investigate the effects of a 3 weeks regular concentric (uphill walking) and eccentric (downhill walking) endurance exercise training in a real world setting (e.g., summer holidays in the Alps), on the composition of the T-cell compartment in pre-diabetic subjects. We hypothesize that exercise training induces an increase of naïve T-cells in parallel to a decrease of cells with a senescent phenotype. We further hypothesized that concentric and EE exhibit different effects.

We were not able to detect time x group (interaction) effects for any of the investigated parameters, i.e., the analysis of plasma cytokines (Table 2), and the analysis of T-cell subpopulations.However, time effects on cell type ratios and cell subsets proportions could be observed in both groups following eccentric and concentric training:

The ratios of CD4⁺/CD8⁺ (F_(1,13) = 5.725; p = 0.033) and CD4⁺/CD3⁺ (F_(1,13) = 7.725; p = 0.018) significantly increased after training, while the CD8⁺/CD3⁺ ratio (F_(1,13) = 6.815; p = 0.049) significantly decreased after training (Table 2).

We found a significant increase of the relative proportion of CD4⁺/CCR7⁺/CD45RO⁺ central memory cells (F_(1,13) = 5.912; p = 0.030) and a significant decrease of CD4⁺/CCR7^-/CD45RO^- TEMRA cells (p = 0.012) and the relative proportion of CD16⁺ cells (F_(1,13) = 7.617; p = 0.016) after training (Table 1).

Training significantly increased the relative proportion of CD8⁺/CCR7⁺/CD45RO⁺ central memory cells (F_(1,13) = 9.282; p = 0.009) and CD8⁺/ CCR7⁺/CD45RO^- naïve cells (p = 0.047), and significantly decreased the relative proportion of CD8⁺/CCR7^-/CD45RO^- TEMRA cells (F_(1,13) = 7.286; p = 0.018; Table 1).

ANCOVA showed no significant group effect for the above mentioned parameter thus, we may assume that baseline differences between groups may not account for the observed time effects.

At baseline, 9 participants were CMV-positive and 7 participants were CMV-negative. Chi-squared test showed no difference in CMV serostatus (positive or negative) between CE and EE (p = 0.131). CMV serostatus neither correlated with any T-cell subpopulation before exercise nor with exercise induced changes of T-cell subpopulations. Changes of TNF-α plasma levels significantly positively correlated with changes of the CD16⁺ cells proportion (R = 0.533; p = 0.041).

The current study demonstrates that 3 weeks of concentric or eccentric endurance training were able to increase both, the CD4⁺/CD8⁺ ratio as well as the CD4⁺/CD3⁺ ratio in pre-diabetic subjects, indicating a relative increase in CD4⁺ T helper cells. The proportional increase of naïve and of central memory CD8⁺ cells after CE and EE indicates a shift to lower differentiated cell types. In parallel, the proportion of CD8⁺ TEMRA cells decreased indicating a relative reduction of highly differentiated T-cells. Surprisingly, despite CE and EE being different training modalities, inducing different stress on the contractile filaments of muscle and the muscle metabolism (Camillo et al., 2015; Philippe et al., 2017), we found no interaction effects regarding changes of cytokine plasma levels, cell ratios or CD4⁺ and CD8⁺ T-cell subset proportions.

A decreased CD4⁺/CD8⁺ ratio is an accepted hallmark of immunological ageing. In a series of longitudinal studies of octogenarians in exceptional health, and nonagenarians, this parameter was identified as part of the “Immune Risk Profile” (IRP) (Pawelec et al., 2010). Accordingly, it represents an important characteristic which predicts a greater all-cause mortality (Wikby et al., 1998). Chronic diseases which are accompanied by a systemic low grade inflammation, have been shown to amplify the decrease of the CD4⁺/CD8⁺ ratio, resulting in a compromised host protection (Richard et al., 2017). The current data demonstrate that 3 weeks of regular CE or EE were followed by an increase of the CD4⁺/CD8⁺ ratio. The concomitant increase of the CD4⁺/CD3⁺ ratio indicates that these changes are due to an increase of CD4⁺ T helper cells. The mechanisms for these changes are speculative. On the one hand, each bout of acute exercise mobilizes hematopoietic stem cells into blood which might travel to the thymus or extrathymic places for differentiation into naïve CD4⁺ cells (Krüger et al., 2015). On the other hand, exercise might mobilize more differentiated T-cells from the marginal pool and the spleen into the circulation (Krüger et al., 2008). Both mechanisms might contribute to altered CD4⁺ cell numbers. Regarding the slight decrease of the CD8/CD3 ratio, it is suggested that the relative number of CD8⁺ T-cells is lower after training. This assumption is supported by previous data from longitudinal training studies which have also reported declines in total CD8⁺ T-cells after 12 weeks and 6 months of endurance training (Baj et al., 1994; Gleeson et al., 2000).

Besides a general increase of CD4⁺ T helper cells, we also found changes in CD4⁺ T-cell subsets. While the proportion of CD4⁺ central memory cells increased, there was a decrease of CD4⁺ cells with TEMRA cell characteristics after training, without differences between CE and EE. These alterations represent a shift from more differentiated to less differentiated cells in the CD4⁺ T helper cell compartment. However, actually the concept of the CD4⁺ equivalent to TEMRA cells is intensively discussed and not clearly defined. Therefore, it is difficult to conclude any immunological impact of changes in this subpopulation (Larbi and Fulop, 2014).

With regard to CD8⁺ cells, changes in T-cell subsets were found after training, without differences between CE and EE. For naïve and central memory CD8⁺ T-cells, a proportional increase was found. Both cell types are known to be effective in response to invading antigens by having a high capacity to expand and proliferate (Sallusto et al., 2004). With regard to immunosenescence these changes seem to be important because decreased numbers of naïve CD8 T-cells represent an important biomarker of an ageing immune system (Appay and Sauce, 2014). The age-related reduction of naïve CD8 cells is suggested to be due to reduced T-cell maturation as a result of thymic involution, but also to reduced generation of lymphoid progenitors and to a higher susceptibility to death receptor-mediated apoptosis, triggered by peripheral inflammatory cytokines like TNF-α (Messaoudi et al., 2004; Emmons et al., 2017). Exercise is suggested to effectively affect all these processes. Accordingly, each acute bout of exercise mobilizes hematopoietic progenitor cells into blood (Krüger et al., 2015). It was further shown that T-cells of trained subjects exhibit an increased resistance to apoptosis (Krüger and Mooren, 2014). Finally, exercise training has anti-inflammatory properties resulting in reduced systemic TNF-α levels (Pedersen and Bruunsgaard, 2003). However, since we did not find a significant reduction of either, TNF-α or IL-6 after training, this mechanism is unlikely.

After CE and EE, a decrease of CD4⁺/CCR7^-/CD45RO^- and CD8⁺/CCR7^-/CD45RO^- cells was found, implicating a proportional reduction of TEMRA cells. An accumulation of TEMRA cells, specifically CD8⁺ T-cells, is an important feature of immune aging since these cells produce large amounts of pro-inflammatory cytokines after activation (Zhang et al., 2001). In addition, CD8 TEMRA cells have only a reduced capacity to proliferate, suggesting that an increase of these cells reduces an organism’s capacity to defend itself against invading pathogens. The age-related increase of TEMRA cells is even more pronounced in the T-cell pool of CMV-seropositive elderly compared to CMV-seronegative elderly persons (Simpson et al., 2016). Therefore, the CMV serostatus was analyzed, but could be excluded as a significant confounder in the present study. The reduced proportion of CD8⁺TEMRA cells after exercise is speculated to be the result of at least two different processes. On the one hand, the relative increase of other CD8 positive T-cell subpopulations might reduce the relative proportion of TEMRA cells. On the other hand, it was shown that acute bouts of exercise induces apoptosis in T-cells which was more frequently observed in more differentiated cells (Krüger et al., 2016). Integrating the exercise-induced increase of naïve T-cells and decrease of TEMRA T-cells in a holistic model, there might exist a negative feedback loop between cell death and mobilization of progenitor cells. Such a connection has been demonstrated in mice, where application of apoptotic bodies evoked the mobilization of hematopoietic progenitors. Accordingly, the loss of senescent cells by apoptosis might create space for the expansion of naïve T-cells (Simpson, 2011; Mooren and Krüger, 2015). However, since we did not analyze either apoptosis or progenitor cell mobilization, these mechanisms have to be addressed in future projects.

CD16⁺ represent a subgroup of lymphocytes which include NK cells, gamma-delta T-cells and subsets of CD8⁺ T-cells. The correlation analyses showed a significant positive correlation between changes of TNF-α and changes of CD16⁺ cells. Thus, the participants with the highest reductions of CD16⁺ cells exhibited the most important decreases of TNF-α plasma levels. While a direct link between these processes is speculative, both processes might reflect the anti-inflammatory effect of exercise training. Consequently, especially persons suffering from pre-diabetes may benefit from these alterations as TNF-α mediated low-grade inflammation is co-responsible for the development of insulin resistance (Pedersen, 2017).

Despite the lower estimated energy expenditure of the EE group compared to the CE group all observed effects were not different between CE and EE. It has previously been shown that despite the lower energy cost, EE had equal or even superior positive effects on glucose and lipid metabolism and inflammation (Drexel et al., 2008; Paschalis et al., 2010). Our results indicate that these findings may also be true regarding positive alterations of immunosenescence. Thus, uphill and downhill walking may equally be recommended for older persons and especially for subjects suffering from pre-diabetes.

In conclusion, the current data implicate that 3 weeks of either concentric or eccentric endurance training increases the proportion of CD4⁺ T-cells and of naïve CD8⁺ T-cells and central memory CD4⁺ and CD8⁺ T-cells, and reduces the proportion of CD4⁺ and CD8⁺ TEMRA cells in pre-diabetic subjects. These changes might be favorable for patients since they represent an opposing trend against immunosenescence and might stimulate host defense against invading pathogens.

All participants gave their written informed consent to participate in the study, which was performed according to the declaration of Helsinki and approved by the ethics committee of the Medical University of Innsbruck (Protocol ID: AN5029; ClinicalTrials.gov ID: NCT01890876).

MP, MB, and BG-L conceived and designed the research. MP, HG, BW, MB, and MK performed the experiments. MP, HG, MK, BW, and KK analyzed the data. KK and MP drafted the manuscript. MP, HG, MK, KA, JF, MB, BG-L, and KK edited and revised the manuscript. MP, HG, MK, BW, KA, JF, MB, BG-L, and KK approved final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.