Sicilian semi- and supercentenarians: age-related Tγδ cell immunophenotype contributes to longevity trait definition

Abstract

The immune system of semi- (from ≥105 to <110 years old) and supercentenarians (≥110 years old), i.e. oldest centenarians, is thought to have characteristics that allow them to reach extreme longevity in relatively healthy status. Thus, we investigated variations of the two principal subsets of Tγδ, Vδ1, and Vδ2, and their functional subsets using the markers defining Tαβ cells, i.e. CD27, CD45RA, in a cohort of 28 women and 26 men (age range 19–110 years), including 11 long-living individuals (from >90 years old to<105 years old), and eight oldest centenarians (≥105 years old), all of them were previously analysed for Tαβ and NK cell immunophenotypes on the same blood sample collected on recruitment day. Naïve Vδ1 and Vδ2 cells showed an inverse relationship with age, particularly significant for Vδ1 cells. Terminally differentiated T subsets (T_EMRA) were significantly increased in Vδ1 but not in Vδ2, with higher values observed in the oldest centenarians, although a great heterogeneity was observed. Both naïve and T_EMRA Vδ1 and CD8+ Tαβ cell values from our previous study correlated highly significantly, which was not the case for CD4+ and Vδ2. Our findings on γδ T_EMRA suggest that these changes are not unfavourable for centenarians, including the oldest ones, supporting the hypothesis that immune ageing should be considered as a differential adaptation rather than a general immune alteration. The increase in T_EMRA Vδ1 and CD8+, as well as in NK, would represent immune mechanisms by which the oldest centenarians successfully adapt to a history of insults and achieve longevity.

The aim of the present study was to investigate the impact of ageing on the blood levels of the two main Tγδ populations, including various subsets, in a cohort of 54 Sicilian individuals aged between 19 and 110 years (including eight semi- and supercentenarians). This cohort had previously been extensively studied for Tαβ and NK subsets. The results regarding Tγδ, when combined with the previous findings on Tαβ and NK, lend support to the idea that immune ageing should be regarded as a specific adaptation rather than a general immune alteration. In this context, the increase in T_EMRA Vδ1 and CD8+ cells, along with NK CD3–CD56+CD16+, could represent immune mechanisms that enable the oldest centenarians to successfully adapt to a lifetime of antigenic challenges and achieve extreme longevity.

Graphical Abstract

Graphical Abstract.

Introduction

In the coming years, both developed and developing countries will experience a marked ageing of the population. This phenomenon poses an unprecedented challenge to healthcare systems since the increased human lifespan is accompanied by the increase in the prevalence of chronic diseases such as dementia, cardiovascular disease, frailty, and sarcopenia as well as cancer [1]. Changes in the composition of the immune system with ageing, such as a reduced amount of naïve T cells and an increased number of memory T cells, hallmarks of immunosenescence [2, 3], are thought to be important contributing factors to susceptibility and severity of the age-related diseases as well as of infectious diseases. In combination with ageing, latent Cytomegalovirus (CMV) infection is another major factor that drives the immune system toward immunosenescence [2, 3].

A large number of studies on T-cell immunosenescence have focussed on Tαβ cells, probably due to their prevalence in the blood. However, other minority T-cell subsets with ʻ innate-likeʼ properties less prevalent in blood, such as Tγδ cells (ranging from 1% to 10% of circulating T cells) have been underexplored, despite playing an important role against foreign pathogens and cancer. Tγδ cells are classified as an intermediate between the adaptive and innate immune systems due to their speed of response after exposure to new antigens, typical of the innate responses, and their ability to generate long-lived memory cells, typical of the adaptive responses. Moreover, antigen recognition by Tγδ cells is not entirely dependent on the major histocompatibility complex (MHC) as in the case of Tαβ cells [4–7].

The Vδ chains identify two major subsets of Tγδ cells, Vδ1 and Vδ2, and other two less common, and consequently less studied, δ3 and δ5. Vδ1 T cells are predominant in the thymus and peripheral tissues and recognize: (i) MHC Class I-related molecule (MIC) A, MICB, and UL16-binding proteins, expressed on stressed and cancer cells [8, 9]; (ii) glycolipid presented by MICB molecules CD1c and CD1d [10, 11]; (iii) unidentified ligands that engage natural cytotoxicity receptors, such as NKp30 and NKp44 [12]. These self-ligands are upregulated during oxidative stress and after malignant transformation [13]. Vδ2 cells account for the majority of Tγδ cells in the blood. In adults, their δ2 chain is almost always associated with the Vγ9 chain; they mainly respond against phospho-antigens that are elevated in cancer cells and phosphorylated non-peptide molecules which are metabolic intermediates of isoprenoid biosynthesis produced by bacteria and parasites [4, 7]. However, Vδ1 show highly focussed clonal expansion after viral and parasitic infections thus adopting a more adaptive immunobiology generating long-lived, T-cell receptor (TCR)- focussed, T effectors [14].

Moreover, Tγδ can attack target cells either directly through their cytotoxic activity or indirectly by activating other cells of the immune system. Collectively, recognition of stress antigens promotes cytokine production and regulates pathogen clearance, inflammation, and tissue homeostasis [4].

Activated Tγδ cells have a differentiation program like Tαβ and give rise to both central memory (T_CM), effector memory (T_EM), and terminally differentiated (T_EMRA) cells. T_CM γδ cells home to secondary lymphoid organs while T_EM and T_EMRA γδ cells migrate to sites of inflammation where they display effector functions such as cytokine production and cytotoxicity, respectively [15, 16].

Although age-related changes of Tγδ blood cells have been studied quite well [4–7], no studies have considered the extreme limit of ageing, represented by the semi- (from ≥105 years old to <110 years old) and supercentenarians (≥110 years old). Moreover, to the best of our knowledge, only two studies analysed blood levels of Tγδ in centenarians [17, 18].

Semi- and supercentenarians (in this paper, we refer to them as the oldest centenarians) are rare individuals who have survived two world wars and a plethora of environmental and microbial stress. Therefore, it is reasonable to infer that their immune system has peculiar characteristics that allow them to reach the extreme limits of human life [19–24].

In infections, Tγδ cells seem to respond earlier than Tαβ cells, suggesting that they are part of the ʻfirst line of defenceʼ and the initiation of an inflammatory response. Consistent with this notion, Tγδ cells are potent producers of pro-inflammatory cytokines [25]. Thus, understanding what happens to blood Tγδ in exceptional longevity should be quite important for a more complete understanding of the immune system of these exceptional individuals. In turn, a better understanding of their immune systems could provide insights into preventing infections and improving vaccine response in older people.

Therefore, the aim of the present paper was to study the impact of ageing and gender on the blood levels of the two main Tγδ populations, including the various subsets in a cohort of 54 Sicilians aged between 19 and 110. This cohort, including 11 long living individuals (LLIs, from >90 years old to <105 years old), and eight oldest centenarians, has been already extensively studied for Tαβ subsets, inflammatory markers, and CMV positivity [22]. In all individuals, both Tαβ and Tγδ analyses were performed in the same blood samples. Thus, we assessed the blood percentages of the two principal subsets of γδ lymphocytes identifying them by the expression of the δ1 or δ2 chain of the TCR. To analyse their phenotype and their subsets, we used the markers that we have used to define Tαβ cell subsets, i.e. CD27, CD45RA, and PD1. In addition, we correlated the values of naïve and T_EMRA Vδ2/Vδ1 subsets to those of CD4+/CD8+ subsets evaluated in our previous study, since Vδ1 naïve and effector subsets exhibit immunophenotypic similarities to conventional adaptive CD8+ naïve and T_EMRA subsets, respectively [26].

Materials and methods

Study cohort

This cohort includes Sicilians recruited for the ʻDiscovery of molecular and genetic/epigenetic signatures underlying resistance to age-related diseases and comorbidities (DESIGN, 20157ATSLF)ʼ project, funded by the Italian Ministry of Education, University and Research. Detailed study design and participant recruitment have been previously described [27]. The Ethics Committee of Palermo University Hospital approved the study protocols (Nutrition and Longevity, No. 032017). All studies were conducted in accordance with the Declaration of Helsinki and its amendments. All study participants (or their children) had given their written informed consent prior to enrolment.

For the present paper, we have studied 54 Sicilians aged between 19 and 110 years, enrolled between 2020 and 2022, selected because of the absence of health issues (LLIs and the oldest centenarians were relatively healthy). As shown in Table 1, the cohort included adult people (n = 20; 10 males and 10 females; years/months range 19.5–63.6); older people (n = 15; 8 M and 7 F; range 68.5–87.3); LLIs (n = 11; 7 M and 4 F; range 93.3–104.7); oldest centenarians (n = 8; 1 M and 7 F; range 105.7–110.3). For the experiments depicted in Figs. 6, 7a,b, and 8 concerning Vδ2 subsets, due to cell shortage, it was not possible to immunophenotype two men (104 and 108 years old) and three women (107, 107, and 109 years old). All LLIs and oldest centenarians were CMV-seropositive. In contrast, CMV seropositivity was observed in 78% of older and 63% of adults (Table 1) [22].

Table 1.

Gender, age, and CMV serological status of the 54 healthy Sicilians (age range 19.5–110.3) included in the study

	Adults	Older	LLI	Oldest cent.
	(n = 20)	(n = 15)	(n = 11)	(n = 8)
Gender (number and %)
Women	10 (50)	7 (46.6)	4 (36.4)	7 (87.5)
Men	10 (50)	8 (53.4)	7 (63.6)	1 (12.5)
Age (years and months)
Mean ± SD	39 ± 12.5	74.7 ± 6.2	99.8 ± 3.5	108.2 ± 1.6
Age range	19.5–63.6	68.5–87.3	93.3–104.7	105.7–110.3
CMV+ (number and %)	12 (63)	11 (78)	11 (100)	8 (100)
Men	5 (41.7)	6 (54.5)	7 (63.6)	1 (12.5)
Women	7 (58.3)	5 (45.5)	4 (36.4)	7 (87.5)
Anti-CMV IgG titre range (U/ml)	25.6->180	63.6->180	110.2->180	150->180

The serological status of one adult and one older are missing. LLI = long-lived individuals; n = number; SD = standard deviation. Oldest centenarians refer to semi- and supercentenarians. Modified from Ligotti et al. (2023).

Figure 6.

Correlation between Vδ2 T-cell subsets and age. Linear regression analysis illustrates the relationship between Vδ2 T-cell subsets and age. The data is presented for all individuals (n = 49) (black line), males (n = 24) (blue line), and females (n = 25) (pink line). Each point represents data from an individual healthy donor. The coefficient of determination and P‐values are shown on the graphs. ns = not significant; R² = R squared; T_EMRA, terminally differentiated effector memory

Figure 7.

Correlation between Vδ2/Vδ1 subsets and CD4+/CD8 + subsets. Each point represents data from an individual donor. The coefficient of determination and P‐values are shown on the graphs. ns = not significant; R² = R squared; T_EMRA, terminally differentiated effector memory. Linear regression analysis illustrates the correlation between Vδ2 T naïve and CD4+ naïve (a) and between Vδ2 T_EMRA and CD4+ T_EMRA (b) (n = 49). Linear regression analysis illustrates the correlation between Vδ1 T naïve and CD4+ naïve (c) and between Vδ2 T_EMRA and CD4+ T_EMRA (d) (n = 54)

Figure 8.

Correlations between PD1 expression on Vδ1 and Vδ2 T cells and age. Linear regression analysis illustrates the relationship between percentages of Vδ1+ PD1+ (a, b) and Vδ2+ PD1+ (c, d) and age. The data is presented for all individuals (Vδ1+ PD1+ n = 54; Vδ2+ PD1+ n = 49) (black line), males (Vδ1+ PD1+ n = 26; Vδ2+ PD1+ n = ) (blue line) and females (Vδ1+ PD1+ n = 28; Vδ2+ PD1+ n = 25) (pink line). Each point represents data from an individual healthy donor. The coefficient of determination and P‐values are shown on the graphs. F = female; M = male; ns = not significant; PD-1 = programmed death-1; R² = R squared

Flow cytometric analysis of frequency, phenotype, and exhaustion marker of Tγδ cells

EDTA-anticoagulated whole blood samples were used to assess the ex vivo frequency of the various functional subsets of Tγδ. Whole blood samples were stained with specific fluorochrome-conjugated monoclonal antibodies (Table 2) and were incubated for 20 min at room temperature in the dark. We did not use a pan-γδ antibody and we determined the frequency of total Tγδ cells by adding the frequencies of Vδ1 and Vδ2 subsets, which are the subsets more presented in peripheral blood [28]. Following erythrocytes lysis with BD FACS™ Lysing solution (BD Biosciences, Franklin Lakes, NJ, USA) as manufacturing guidelines, the cells were washed with FACS buffer (PBS, 4% FBS, 2 mM EDTA), acquired using FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA) and analysed by FlowJo software (Tree Star, version 10.5.3, Singapore, Singapore). After setting the lymphocytes region in the SSC-A/FSC-A dot-plot, events were gated in the CD3/ Vδ1 and CD3/Vδ2 dot-plots to define both subsets. Based on the surface marker CD27 and CD45RA, Vδ1 and Vδ2 T cell subsets were further divided into CD27+/CD45RA+ Naïve, CD27+/CD45RA– T_CM, CD27–/CD45RA– T_EM, and CD27–/CD45RA+ T_EMRA. Moreover, PD1 expression was evaluated in both populations. The analyses were performed at the Central Laboratory of Advanced Diagnosis and Biomedical Research, ʻP. Giacconeʼ University Hospital in Palermo (see Fig. 1 for gating strategies).

Table 2.

List of antibodies used in the study

Antibody	Flurochrome	Clone
Anti-human CD3	FITC	REA613
Anti-human Vδ1	PE	REA173
Anti-human Vδ2	PE	REA771
Anti-human PD-1	APC	PD1.3.1.3
Anti-human CD27	PE-Vio615	REA499
Anti-human CD45RA	PerCP	T6D11

Purchased from Miltenyi Biotec, Bergisch Gladbach, Germany.

Figure 1.

Gating strategy for the identification of γδ T lymphocytes. Following lymphocyte identification on the side-scatter area (SSC-A) versus the forward-scatter area (FSC-A), double cells were excluded on the forward-scatter height (FSC-H) versus the forward-scatter area (FSC-A). Vδ1 and Vδ2 T cells were identified as Vδ1⁺CD3⁺ and Vδ2⁺CD3⁺ cells, respectively. A representative dot plot displays CD27 and CD45RA expression, gated on Vδ1 and Vδ2 T cells. A representative histogram displays PD-1 expression on both γδ T-lymphocyte subsets

Statistical analysis

To analyse the percentages of Tγδ cells statistical analysis was performed with GraphPad Prism version 9.3.1 (GraphPad Software, San Diego, CA, USA). Analysis of Vδ1, Vδ2, and total γδ T-cell percentages between age groups was performed by one-way ANOVA test. Correlation between the percentage of cells and age as well as between Tγδ and Tαβ subsets in all individuals and males and females was examined using simple linear regression analysis. Figures were plotted as scatter plots with a linear regression line and 95% confidence bands. For each statistical analysis, only P-values < 0.05 were considered significant.

Age was the denominator of Figs. 2–6 and 8. Regarding Fig. 1, from left to right, the denominators were FSC-a (2), CD3 (2), and PD-1 (2). Regarding Fig. 7: (i) CD4 naïve, (ii) CD4 T_EMRA, (iii) CD8 naïve, and (iv) CD8 T_EMRA.

Figure 2.

Mean of the percentages of Vδ1, Vδ2, and total Tγδ cells in each age group. Column bar graphs showing differences between the mean of the values of Vδ1 (a), Vδ2 (b), and total γδ (c) T percentages from each aged group obtained by one-way ANOVA test. The coefficient of determination and P-values are shown on the graphs. ^*P ≤ 0.05

Figure 3.

Age-related changes in Vδ1 and Vδ2 T-cell subsets. Linear regression analysis illustrates the relationship between Vδ1 and Vδ2 T-cell subsets and age. The data is presented for all individuals (n = 54) (black line), males (n = 26) (blue line), and females (n = 28) (pink line). Each point represents data from an individual healthy donor. The coefficient of determination and P‐values are shown on the graphs. F = female; M = male; ns = not significant; R² = R squared

Figure 4.

Correlations between Vδ2/Vδ1 T-cell ratio and age. Linear regression analysis illustrates the relationship between percentages of Vδ2/Vδ1 T cell ratio and age. The data is presented for all individuals (n = 54) (black line) (a), males (n = 26) (blue line) and females (n = 28) (pink line) (b). Each point represents data from an individual healthy donor. The coefficient of determination and P‐values are shown on the graphs. F = female; M = male; ns = not significant; R² = R squared

Figure 5.

Correlation between Vδ1 subsets and age. Linear regression analysis illustrates the relationship between Vδ1 T cell subsets and age. The data is presented for all individuals (n = 54) (black line), males (n = 26) (blue line), and females (n = 28) (pink line). Each point represents data from an individual healthy donor. The coefficient of determination and P‐values are shown on the graphs. ns = not significant; R² = R squared; T_EMRA, terminally differentiated effector memory

Results

We compared the mean of the percentages of Vδ1 and Vδ2 and total Tγδ cells between each aged group. Although no statistical significance was present, LLIs showed higher percentages of Tγδ cells (Fig. 2c), due to the increase in Vδ2 (Fig. 2b). Oldest centenarians, however, showed an increase in Vδ1, which was statistically significant when compared with adult and older people (Fig. 2a). Linear regression analysis regarding the relationship between Vδ1 and Vδ2 T-cell subsets and age in all individuals showed no significant age-related changes in the blood level percentages of both Vδ1 and Vδ2 T cells, also after gender stratification (Fig. 3), while there was a slight age-related increase of Vδ1 T cells in females. So, we did not observe significant age-related changes in the δ2/δ1 ratio in all individuals but there was a significant inversion in females (Fig. 4).

We next assessed Vδ1 and Vδ2 cell four subsets, based on the expression of CD45RA and CD27. The analysis showed a significant inverse correlation between age and naïve Vδ1 cell percentages in all individuals (R² = 0.278, P = 0.0001, Fig. 5a), confirmed only in the female group (R² = 0.418, P = 0.0005, Fig. 5b). Naïve Vδ2 cell percentages also showed a significant age-related decrease in all individuals (R² = 0.113, P = 0.0207, Fig. 6a) but not after gender stratification (Fig. 6b). In contrast to the Vδ1 population, where it was observed a significant age-related decrease in the TCM subset (R² = 0.108, P = 0.024, Fig. 5c), mostly due to the male component (R² = 0.189, P = 0.043, Fig. 5d), there was an age-related increase in TCM Vδ2 cells in males (R² = 0.022, P = 0.043, Fig. 6d) but not in females and in all individuals Fig. 6c and d). The TEM subset was unaffected by age and gender in Vδ1 cells (Fig. 5e and f), but increased with ageing in Vδ2 cells (R² = 0.091, P = 0.039, Fig. 6e), although this increase was not observed after gender stratification (Fig. 5f). Then, we found a significant increase in T_EMRA Vδ1 cell percentages with age (R² = 0.368, P < 0.0001, Fig. 5g), mainly due to the female group (R² = 0.548, P < 0.0001, Fig. 5h), while no changes were observed for T_EMRA Vδ2 cells with a wide variability between individuals (Fig. 6g and h).

In Fig. 7, we correlated the values of naïve and T_EMRA Vδ2/Vδ1 subsets to those of CD4+/CD8+ subsets. Both naïve and T_EMRA Vδ1 and CD8+ Tαβ cells values from our previous study correlated highly significantly (P < 0.0001 for naïve, P = 0.001 for T_EMRA) which was not the case for CD4+ and Vδ2 (only P = 0.046 for naïve). In each subject, the analyses had been performed in the same blood sample.

The analysis of exhaustion marker expression of both Vδ1 and Vδ2 cells showed a positive relation between Vδ2 PD1+ T-cell percentage with age (R² = 0.202, P = 0.001, Fig. 8c), with a major contribution from oldest female centenarians (R² = 0.280, P = 0.009, Fig. 8c). The increment in PD1 expression observed in the Vδ1 population was, instead, not significant, also after gender stratification (Fig. 8a and b).

Discussion

In previous papers, we analysed different haematological, immunological, and inflammatory markers in this cohort of 54 Sicilians, including eight semi- and supercentenarians (mean age 108.2 years), focussing on Tαβ and NK immunophenotypes [22, 24]. In the present paper, we focussed our attention on Tγδ cells. Oldest centenarians showed the lowest percentages of naïve Vδ1 and Vδ2 cells, due to their age, and higher percentages of Vδ1 T_EMRA (but not of Vδ2 T_EMRA), due to their age and their CMV-seropositivity, according to literature data [25, 29]. Some of them showed values comparable to those of the younger subjects, thus exhibiting a significant heterogeneity.

Concerning the observed sex-related variations, both sex (a biological attribute linked to hormonal differences or the presence of two X chromosomes) and gender (a complex social construct encompassing identity, roles, relationships, and institutionalized aspects of gender) can influence immune response [3, 30]. It has been recently demonstrated that, especially after the age of 65, men exhibit higher genomic activity for monocytes and inflammation, while older women have greater genomic activity for the acquired arm of the immune response. Therefore, men have a greater innate and pro-inflammatory activity and a lower adaptive activity [31].

So, it is not surprising that there may be age-related differences in the varying lymphocyte percentages observed in the comparison between male and female donors in this study, as well as in our previous studies [21, 22, 24]. In particular, we found a very significant age-related increase in the percentage of CD3–CD16+CD56+ NK cells in the same individuals studied in this paper. After gender stratification, the significant age-related increase was confirmed in both sexes, but with a greater significance in males [24], in line with data suggesting that activated NK cells were in greater numbers in men than in women [32]. All of this suggests a lasting effect, likely linked to epigenetics [3]. We could also hypothesize that these differences might be related to CMV infection since gender could influence the response to CMV infection. It is known that women are more resistant to infections [3]. However, the data on CMV reported in Table 1 do not support this possibility.

However, it is important to note that, in the study cohort, there was only one male semi- and supercentenarian; it is known, indeed, that women are more resilient and statistically more likely to achieve exceptional longevity [20, 30]. Therefore, regarding the role of gender in the blood levels of Tγδ cells, it should be emphasized that gender disparity in favour of the female oldest centenarians may have contributed to some of these differences. Thus, caution should be taken when interpreting all gender data.

Similarly to Tαβ [3, 21, 22], age-associated alterations in Tγδ cells are mainly present in individuals with CMV-seropositivity [25]. As well as in Tαβ cells, where CMV has a stronger effect on CD8+ functional subsets than in CD4+ ones, in Tγδ cells Vδ1 cells functional subsets are more affected than Vδ2 [29]. However, in our sample, it was not possible to distinguish the role of CMV since almost 80% of the subjects were CMV-positive.

In our study, linear regression analysis regarding the relationship between age and Vδ1 and Vδ2 T cells showed no significant changes in blood level percentages both before and after gender stratification. In all individuals, however, the oldest centenarians showed an increase in Vδ1, but not in Vδ2, which was statistically significant when compared to adults and old people. There are conflicting results concerning the impact of ageing on blood levels of Vδ1 and Vδ2 subsets [7, 21, 24, 33–36]. Nonetheless, both studies focussing on centenarians reported a significant decrease in the absolute number of Tγδ cells in older people and centenarians [17, 18]. Therefore, it was unclear how the levels of these cells and their various functional subsets change in the extreme longevity (see below).

Twin studies suggest a higher heritability of the Vδ2Vγ9 population, while the diversity of Vδ1 T cells depends more on environmental factors [37]. These data, combined with studies performed by Xu et al. [5, 6], suggest that Vδ2 exhibits ʻinnate-likeʼ behaviour influenced by heritability, while Vδ1 are more mouldable by stressors encountered during life. This might explain the different results obtained in our study and in those mentioned above, as well as the increase only of Vδ1 cells but not of Vδ2 cells. This increase is linked to life-long stimulation by stressors, particularly CMV, resulting in a very significant increase in T_EMRA, which is responsible for the overall increase of Vδ1 cells in oldest centenarians.

In the present study, we have observed significant changes in age and gender-related in the different Vδ1 and Vδ2 functional subsets. Over the years, several studies have analysed the functional and numerical alterations of Tγδ and their subsets during ageing, often relating them to Tαβ [5, 25, 38–40]. The memory/effector differentiation pattern of Tγδ shows a comparable pattern to the distribution of the memory phenotype in the CD8+ compartment. In CMV seropositive older subjects, higher frequencies of late-differentiated cells and lower frequencies of early-differentiated cells were demonstrated in the Vδ1 + and Vδ1-Vδ2- populations, but not in the Vδ2 populations [5, 25, 39]. These findings confirm the association of these Vδ2 negative cells with CMV immunosurveillance. The memory differentiation pattern in the Vδ1 compartment is, as in CD8+, markedly changed by age and amplified by the presence of CMV [5, 25, 39]. All these studies have been performed in cohorts including older people but not LLIs and oldest centenarians.

The decrease in naïve T cells and increase in memory/effector T cells are among the major hallmarks of immunosenescence. These changes result from long-life antigenic stimulation, particularly due to CMV infection [2, 3, 21, 22]. In our study, both naïve Vδ1 and Vδ2 cells showed an inverse relationship with age. The decrease was more significant in Vδ1, and, when we analysed gender differences, this significance was present only in women. The results aligned with those observed in the same subjects in Tαβ [22]. Among the various memory/effector subpopulations, the most striking data concerns T_EMRA, which significantly increased in Vδ1 (in all individuals and in women) but not in Vδ2, with higher values observed in the never-studied semi- and supercentenarians. It is noteworthy that all LLIs and oldest centenarians were CMV-positive. These findings are consistent with what was observed in Tαβ where the most significant increase concerned CD8+, especially in women [22].

Consistent with previous statements, both naïve and T_EMRA Vδ1 and CD8+ Tαβ cell values from our previous study correlated significantly. Adaptive-like Tγδ subsets, such as Vδ1 [25], undergo a pathogen-driven differentiation process analogous to CD8+ Tαβ T cells. This guides the inflammatory and cytotoxic responses of Tγδ towards specific antigenic stimuli in various biological contexts. It is plausible that these responses include beneficial responses to pathogen infection, including herpes viruses like CMV, thus complementing Tαβ immunity in microenvironmental niches with compromised MHC expression resulting from viral immune evasion [14].

We also analysed the expression of PD1+ on Tγδ cells, which were expressed in fewer numbers on fresh isolated Tγδ but induced following antigenic stimulation [41]. As already observed on CD4+ and CD8+ [23], PD1+ expression exhibited wide variability among individuals, although the overall trend was an increase in Vδ2+PD1+with ageing. When analysed by gender, this significance was observed only in women. Vδ2 does not reach the senescence stage due to life-long stressors, unlike Vδ1 cells, which are influenced by these long-term stressors [5, 42]. This could explain why we have observed significant age-related exhaustion in Vδ2 but not in the Vδ1 subset.

So, extreme longevity is associated with a highly significant reduction in naïve both CD8+ and Vδ1 cells and a highly significant increase in T_EMRA in both CD8+ and Vδ1 cells. In addition, extreme age is not associated with an increase of Vδ1 exhausted cells as measured by the PD1 marker.

While Tγδ cells are a rare population in peripheral blood, they play an important role in the immunological homeostasis of the organism [4–7]. These cells are involved in the immune response against infections and possess important anticancer functions, making them a promising candidate for cancer immunotherapy [43]. Notably, in vivo, the expansion of Tγδ T_EMRA cells has been linked to a reduced risk of cancer onset and leukaemia recurrence. It has also been associated with the clearance of CMV infection in allogeneic stem cell recipients and kidney transplant patients, respectively [40].

Our study also highlights the significant variability in various Tγδ parameters, particularly among centenarians, as previously observed for Tαβ in Ligotti et al. [22]. This variability can be attributed to individual differences in immunological history including variations in genetic susceptibility to CMV infection as well as to other factors influencing the immune system, such as lifestyle factors, education, socioeconomic status, and the microbiota [3, 22].

Nonetheless, our observations regarding age-related variations of naïve and T_EMRA Tγδ, as well as CMV seropositivity in centenarians, when considered alongside the data on Tαβ [22] and NK [24], suggest that these changes may not be unfavourable for centenarians, especially the oldest among them.

However, our study has some limitations: (i) since nearly the entire Sicilian population over 90 years old is CMV positive, it is impossible to separate the effects of age from those of CMV; (ii) the cross-sectional nature of the data as is the case with most immunophenotypic studies in the literature. Another potential limitation is the absence of functional data for different subsets such as the expression of perforin or granzyme or cytokine production. Nevertheless, the aim of this study was to investigate Tγδ changes in a unique group of old individuals, complementing our previous study on Tαβ changes [22].

In conclusion, our findings on Tαβ [22] and NK [24], combined with the results presented in this study, further support the idea that immune ageing should be seen as a specific adaptation rather than a generalized immune alteration. In this context, the increase in T_EMRA CD8+ and Vδ1 cells, along with NK CD3–CD56+CD16+, could represent immune mechanisms that enable the oldest centenarians to successfully adapt to a lifetime of challenges and achieve extreme longevity.

Glossary

Abbreviations

CMV

Cytomegalovirus

LLIs

long living individuals

MHC

major histocompatibility complex

MIC

MHC class I-related molecule

TCR

T-cell receptor

TCM

central memory T subset

TEM

effector memory T subset

T _EMRA

terminally differentiated T subset.

Funding

Financial support granted by the Italian Association of Anti-Ageing Physicians directed by Doctor Damiano Galimberti in the recruitment of participants as well as the support granted by Alessandro Delucchi http://www.supercentenariditalia.it in the identification of semi- and super-centenarians are also warmly acknowledged. Original work performed by authors from the Laboratory of Immunopathology and Immunosenescence in the field of longevity and immunosenescence was funded by the 20157ATSLF project (Discovery of molecular, and genetic/epigenetic signatures underlying resistance to age-related diseases and comorbidities), granted by the Italian Ministry of Education, University, and Research and the project Improved Vaccination Strategies for Older Adults granted by European Commission (Horizon 2020 ID 848).

Conflict of interests

The authors declare that they have no conflict of interest.

Ethical approval

The Institutional Ethics Committee (abstract “Paolo Giaccone”, University Hospital) approved the DESIGN study protocol (Nutrition and Longevity, No. 032017). All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from the participants or by their children.

Data availability

The data sets generated and/or analysed during the current study are not publicly available due to privacy reasons, but are available in anonymized form from the authors on reasonable request.