CD70 contributes to age-associated T cell defects and overwhelming inflammatory responses

Aging is associated with immune dysregulation, especially T cell disorders, which result in increased susceptibility to various diseases. Previous studies have shown that loss of co-stimulatory receptors or accumulation of co-inhibitory molecules play important roles in T cell aging. In the present study, CD70, which was generally regarded as a costimulatory molecule, was found to be upregulated on CD4+ and CD8+ T cells of elderly individuals. Aged CD70+ T cells displayed a phenotype of over-activation, and expressed enhanced levels of numerous inhibitory receptors including PD-1, 2B4 and LAG-3. CD70+ T cells from elderly individuals exhibited increased susceptibility to apoptosis and high levels of inflammatory cytokines. Importantly, the functional dysregulation of CD70+ T cells associated with aging was reversed by blocking CD70. Collectively, this study demonstrated CD70 as a prominent regulator involved in immunosenescence, which led to defects and overwhelming inflammatory responses of T cells during aging. These findings provide a strong rationale for targeting CD70 to prevent dysregulation related to immunosenescence.


INTRODUCTION
Aging is accompanied by dysregulated immune responses that result in high susceptibility to various diseases [1][2][3]. It is characterized as immunosenescence, which involves a gradual deterioration of immunity as well as enhanced inflammatory responses [4,5]. In particular, T cell aging is considered to be a prominent contributor to age-associated-immune dysregulation [6,7].
Co-stimulatory and co-inhibitory molecules are crucial for regulating T cell activation, differentiation, effector function and survival [8]. Loss of some co-stimulatory receptors, such as CD28 and CD27, is one of the most consistent immunological markers of T cell aging [9,10]. Co-inhibitory molecules also play important roles in T cell aging. In murine models as well as in humans, programmed death-1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and tyrosine-based inhibitory motif (ITIM) domain (TIGIT) were found to be upregulated during aging [11][12][13][14]. These findings suggest that suppressive pathways contribute to immunosenescence. However, the mechanism that regulates enhanced inflammatory responses of T cells has not been established. Additionally, the effects of newly identified cosignaling molecules during aging needs to be investigated.

AGING
CD70 is the sole ligand for co-stimulatory receptor CD27 [15]. Thus, it was generally considered as a stimulatory molecule. It is expressed on antigen presenting cells (APCs), epithelial cells, mature dendritic cells, and many types of tumor cells [16][17][18][19][20]. However, recent studies indicated T cell-derived CD70 as an inhibitory molecule in patients with B-cell non-Hodgkin's lymphoma and murine models of inflammatory bowel disease or allogeneic graftversus-host disease [21,22]. Herein, we assessed the role of CD70 in T cell immunosenescence using blood samples from healthy individuals. Overall, this study suggests that CD70 upregulation is an important process associated with T cell aging, which leads to defects and overwhelming inflammatory responses of T cells.

Age-related CD70 accumulation in CD4 + and CD8 + T cells
To investigate the potential role of CD70 signaling in T-cell aging, we examined the expression of CD70 on T cells from 217 healthy adults using flow cytometry ( Table 1). The results showed that CD70-expressing CD4 + and CD8 + T cells accumulated with aging. The frequencies of CD70 + fractions among CD4 + and CD8 + T cells from the elderly (61-80 years) were significantly higher than those from young (21-30 and 31-40 years) and middle-aged individuals (41-50 and 51-60 years; Figure 1A-1C). Additionally, middleaged individuals showed higher expression of CD70 as compared to young individuals. Correlation analysis showed a strong correlation of CD70 expression on CD4 + and CD8 + T cells with age (CD4: r = 0.5118, p < 0.0001; CD8: r = 0.6244, p < 0.0001; Figure 1D-1E).

CD70 was up-regulated on each subset of circulating T cells during aging
Since previous studies including ours reported an expansion of antigen-experienced T cells in the elderly population [14,23], we investigated whether heterogeneous T cell subsets expressed different levels of CD70 in this study. Based on the expression of CD45RA and CCR7, the T cells were divided into four subsets: naïve T cells (TN, CCR7 + CD45RA + ), central memory T cells (TCM, CCR7 + CD45RA -), effector memory T cells (TEM, CCR7 -CD45RA -) and terminally differentiated effector cells (TEMRA, CCR7 -CD45RA + ). Consist with previous studies, the frequencies of CD4 + and CD8 + TN cells were remarkably decreased with age, along with a dramatic increase in the frequencies of CD4 + TCM cells or CD8 + TCM, TEM and TEMRA cells (Supplementary Figure 1). The TCM, TEM, and TEMRA subsets of both CD4 + and CD8 + T cells, known as antigen-experienced T cells, expressed higher levels of CD70 than TN cells regardless of age ( Figure 2). Also, CD70 expression was substantially increased in each T cell subset of CD4 + and CD8 + cells from older subjects as compared to young and middle-aged subjects ( Figure  2). Thus, an elevated proportion of CD70 + fractions among CD4 + and CD8 + cells in elderly individuals was not only a result of the higher number of antigenencountered T cells, but also the age-related increase of CD70 expression. Collectively, these results showed that CD70 up-regulation is a common characteristic of T cell immunosenescence.

Aged CD70 + T cells displayed a phenotype of overactivation and exhaustion
In order to characterize the phenotype of CD70 + T cells in elderly individuals, we examined the expression levels of multiple activation markers, co-stimulatory and co-inhibitory molecules on the CD70 + and CD70fractions of T cells. Overall, these results indicated an over-activated and consequently exhausted status of aged CD70 + T cells. A total of 217 healthy adults were recruited, including 94 males and 123 females. Their median age was 45, and 34-60 adults were in every group. The Chi-square test demonstrated that the gender was balanced among all the groups (P = 0.5433). Age was described by median and interquartile rage (IQR) and analyzed using Kruskal-Wallis test.

CD70 + T cells in elderly individuals displayed increased sensitivity to apoptosis, which could be reversed by blocking CD70
To assess the functional status of CD70 + T cells from the elderly, we detected susceptibility of these cells to apoptosis by measuring percentage of apoptotic cells (Annexin V + 7AAD -) and expression of CD95 (Fas). Percentage of apoptotic cells and CD95 expression were significantly elevated on CD70 + CD4 + and CD8 + T cells ( Figure  AGING of Annexin V + 7AADcells and CD95 expression in T cells were strongly correlated with the percentage of activated HLA-DR + CD38 hi T cells, implying the role of activation-induced cell death (AICD) in CD70 associated T cell aging ( Figure 5E and 5F).
The specific role of CD70 in the induction of T-cell apoptosis was examined by blocking CD70 using an anti-CD70 neutralizing antibody. In the presence of the neutralizing antibody, percentage of Annexin V + 7AADcells and CD95 expression were decreased in aged CD4 + T cells ( Figure 5G-5J). This data indicated the important suppressive role of CD70 in the regulation of T-cell function in elderly individuals.

Aged CD70 + T cells showed increased levels of inflammatory cytokines and intracellular proteins
Senescent cells can secrete numerous inflammatory cytokines and chemokines, which act together to generate a proinflammatory environment [7,24]. To determine whether CD70 is involved in senescenceassociated inflammatory responses in T cells, we tested cytokine release after in vitro stimulation with anti-CD3 and anti-CD28. The results showed significantly increased levels of TNF-α, IFN-γ, and IL-2 in CD70 + CD4 + T cells as compared to CD70 -CD4 + T cells ( Figure 6A, 6C, 6E). Slight elevations of these cytokines were observed in CD70 + CD8 + T cells from elderly individuals ( Figure 6B, 6D, 6F). Similar results were observed in CD70 + CD4 + and CD8 + T cells from the young and middle-aged groups (Supplementary Figure 6A-6C). Importantly, TNF-α and IL-2 secretion was significantly decreased in aged CD4 + T cells after CD70 blocking (Figure 6G-6L). Moreover, CD70 + CD8 + T cells from all age groups showed elevated expression of perforin and Granzyme B, suggesting a greater non-specific killing potential. Interestingly, increased levels of perforin and Granzyme B were also observed in CD70 + CD4 +  AGING 6D and 6E). Additionally, CD70 + T cells exhibited significantly higher levels of Ki-67 than CD70 -T cells regardless of age (Supplementary Figure 6F).

T cells (Supplementary Figure
Taken together, these results indicated that aged CD70 + T cells produced high levels of inflammatory cytokines and intracellular Granzyme B and perforin.

DISCUSSION
It has been demonstrated that down-regulation of some co-stimulatory molecules and up-regulation of some coinhibitory molecules are key features of T cell aging.
However, this study showed a significant age-related accumulation of CD70, which was generally regarded as a co-stimulatory molecule, on both CD4 + and CD8 + T cells. Consistent with its contradictory role in T cell activation, which was reported by recent studies, the findings of this study highlighted an important role of CD70 in T cell aging. CD70 + T cells from elderly individuals displayed phenotypic features of exhaustion and high susceptibility to apoptosis. In contrast, aged CD70 + T cells also produced higher levels of proinflammatory cytokines and expressed more intracellular Granzyme B and perforin, which was consistent with an important feature of senescent cells known as the AGING senescence-associated secretory phenotype (SASP) [24,25]. These data indicated that CD70 is a biomarker of T cell aging and elucidated a potential mechanism of aging.
To the best of our knowledge, this is the first evidence for the involvement of CD70 in immunosenescence.
Since aged CD70 + T cells expressed numerous coinhibitory molecules, suggesting a phenotype of exhaustion, we compared senescent and exhausted T cells. Both cells were dysfunctional in some aspects. However, they differed from each other in inflammatory AGING cytokine and intracellular protein expression. Recent studies indicated that senescent cells are metabolically active rather than dormant. They expressed numerous cytokines, chemokines, growth factors, and proteases, which was characterized as SASP [24,26]. However, previous studies including ours indicated that upregulation of co-inhibitory molecules such as PD-1, TIM-3, or TIGIT on aged T cells induced defective cytokine production, suggesting exhaustion rather than senescence [10,14,27]. In the present study, CD70 + T cells from elderly individuals showed increased pro-inflammatory cytokines such as TNF-α and IFN-γ, and higher levels of intracellular Granzyme B and perforin. These findings supported the notion that senescence was associated with enhanced chronic inflammation. Notably, once chronic inflammation was established, it often induced the immune system to produce more cytokines through positive feedback loops [25]. Thus, it was thought to underlie the increased incidence of autoimmune diseases in the elderly. Previous studies reported that CD70 was overexpressed on human proinflammatory Th1 and Th17 cells, which contributed to pathogenesis of multiple sclerosis [28]. Increased CD70 + CD4 + T lymphocytes were also observed in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients [29][30][31], further confirming the hypothesis.
More importantly, increased inflammatory cytokines from aged CD70 + T cells induced a persistent over-activated status that finally led to apoptosis. It was characterized as AICD, which might account for CD70-associated immunosenescence. AICD is a critical pre-programmed death pathway that plays a central role in the aging process [32,33]. Higher AICD rate was observed during replicative senescence in vitro, and was more pronounced in T cells from the elderly than young individuals [34]. A previous study showed that chronic immune stimulation induced overexpression of death receptors on aged T cells, leading to up-regulation of AICD [35,36]. Cultured PBMCs from elderly individuals expressed higher CD95 levels than those from young individuals upon activation [34,37,38]. Consistently, the present study showed an increased activated HLA-DR + CD38 hi population as well as up-regulation of percentage of apoptotic cells and CD95 expression on aged CD70 + T cells. Moreover, blocking CD70 decreased apoptosis levels in aged CD4 + T cells. These results were further confirmed by close correlation between percentage of apoptotic cells and CD95 expression, and percentage of activated HLA-DR + CD38 hi T cells.
Several surface markers were reported to track the "age" of human circulating T cells, such as loss of CD27 and CD28, or gain of CD57 and killer cell lectin-like receptor sub family G (KLRG-1) [10,[39][40][41]. CD4 + and CD8 + T cells share some phenotypic changes during aging, however, age-related changes occur more frequently in CD8 + T cells than in CD4 + T cells. For instance, CD8 + T cells lost CD28 more rapidly than CD4 + cells during aging [42]. CD57 + CD8 + T cells and KLRG1 + CD8 + T cells were recruited with age [43,44]. Our previous study showed that TIGIT contributed to CD8 + T cell aging. However, CD4 + T cells were less sensitive to age, with a greater homeostatic stability when compared to CD8 + T cells [40,45]. The present study showed that CD70 was involved in aging of both CD4 + and CD8 + T cells. Of note, CD4 + T cells exhibited more significant upregulation of CD70 during aging than CD8 + T cells.
Collectively, these data suggested CD70 as a biomarker associated with T cell aging, especially for CD4 + T cells.
In summary, the present study demonstrated CD70 as a prominent regulator involved in immunosenescence, which led to defects and overwhelming inflammatory responses of T cells during aging. These findings may be beneficial in the treatment of age-related comorbidities.

Isolation of peripheral blood mononuclear cells (PBMCs)
Peripheral blood samples were collected from healthy donors and PBMCs were purified using standard Ficoll-Paque gradient centrifugation according to the manufacturer's instructions (Amersham Pharmacia Biotech, Sweden). Cells were cryopreserved in fetal bovine serum (GIBCO, Grand Island, NY, USA) supplemented with 10% DMSO, and stored in liquid nitrogen.

Analysis of T-cell apoptosis
Apoptosis rates were measured using an APC Annexin V apoptosis detection kit (BioLegend) as per the manufacturer's instructions, in combination with markers for T cells. Samples were analyzed by flow cytometry.

Cell separation and CD70 blockage
CD4 + and CD8 + T cells were isolated from PBMCs by positive selection using EasySep™ human CD4 and CD8 positive selection kit (StemCell Technologies, Vancouver, Canada). Purified cells were cultured at a concentration of 1 × 10 6 cells/mL in a 96 well tissue culture plate and 10 µg/ml anti-human CD70 antibody (clone 113-16; BioLegend) or isotype control was added to the culture medium. After 24 h of culture, Annexin V staining and cytokine production were measured by flow cytometry.

Statistical analysis
The data are expressed as the mean ± standard deviation (SD). GraphPad7 (GraphPad Software, La Jolla, CA, USA) or SPSS (IBM Corporation, New York, NY, USA) were used for statistical analyses. The normality of each variable was evaluated using the Kolmogorov-Smirnov test. For normally distributed data, the comparison of two variables was performed using unpaired, or paired where specified, two-tailed Student's t-tests for unpaired and paired data, respectively. One-way ANOVA followed by Tukey's multiple comparisons test was used for comparing two or more independent samples. When the data were not normally distributed, the comparison of variables was performed with a Mann-Whitney U test or a Wilcoxon matched-pairs signed rank test for unpaired and paired data, respectively. For comparing two or more independent samples, a Kruskal-Wallis test followed by Dunn's multiple comparisons test was used. Participant characteristics were compared using Chi-square test (categorical variables) or Kruskal-Wallis test (continuous variables). Pearson's or Spearman's correlation coefficients were used to evaluate correlations for normally or non-normally distributed data, respectively. For all analyses, p-values <0.05 were considered statistically significant.