Phenotypic characteristics of aged CD4 + CD28 null T lymphocytes are determined by changes in the whole-genome DNA methylation pattern
2016; Wiley; Volume: 16; Issue: 2 Linguagem: Inglês
10.1111/acel.12552
ISSN1474-9726
AutoresBeatriz Suárez-Álvarez, Ramón María Alvargonzález Rodríguez, Karin Schlangen, Aroa Baragaño Raneros, Leonardo Márquez‐Kisinousky, Agustín F. Fernández, Carmen Díaz‐Corte, Ana M. Aransay, Carlos López‐Larrea,
Tópico(s)Epigenetics and DNA Methylation
ResumoAging CellVolume 16, Issue 2 p. 293-303 Original ArticleOpen Access Phenotypic characteristics of aged CD4+ CD28null T lymphocytes are determined by changes in the whole-genome DNA methylation pattern Beatriz Suarez-Álvarez, Beatriz Suarez-Álvarez Department of Immunology, Hospital Universitario Central de Asturias, Oviedo, SpainSearch for more papers by this authorRamón M. Rodríguez, Ramón M. Rodríguez Department of Immunology, Hospital Universitario Central de Asturias, Oviedo, SpainSearch for more papers by this authorKarin Schlangen, Karin Schlangen Genome Analysis Platform, CIC bioGUNE, Bizkaia Technological Technology Park, Derio, SpainSearch for more papers by this authorAroa Baragaño Raneros, Aroa Baragaño Raneros Department of Immunology, Hospital Universitario Central de Asturias, Oviedo, SpainSearch for more papers by this authorLeonardo Márquez-Kisinousky, Leonardo Márquez-Kisinousky Department of Immunology, Hospital Universitario Central de Asturias, Oviedo, SpainSearch for more papers by this authorAgustín F. Fernández, Agustín F. Fernández Cancer Epigenetics Laboratory, Institute of Oncology of Asturias (IUOPA), Hospital Universitario Central de Asturias, Oviedo, SpainSearch for more papers by this authorCarmen Díaz-Corte, Carmen Díaz-Corte Department of Nephrology, Hospital Universitario Central de Asturias, Oviedo, SpainSearch for more papers by this authorAna M. Aransay, Ana M. Aransay Genome Analysis Platform, CIC bioGUNE, Bizkaia Technological Technology Park, Derio, Spain CIBERhedSearch for more papers by this authorCarlos López-Larrea, Corresponding Author Carlos López-Larrea inmuno@hca.es Department of Immunology, Hospital Universitario Central de Asturias, Oviedo, Spain Correspondence Carlos López-Larrea, Department of Immunology, Hospital Universitario Central de Asturias, Avenida de Roma s/n, 33011 Oviedo, Asturias, Spain. Tel.: ++34 985 106130; fax: ++34 985 106095; e-mail:inmuno@hca.esSearch for more papers by this author Beatriz Suarez-Álvarez, Beatriz Suarez-Álvarez Department of Immunology, Hospital Universitario Central de Asturias, Oviedo, SpainSearch for more papers by this authorRamón M. Rodríguez, Ramón M. Rodríguez Department of Immunology, Hospital Universitario Central de Asturias, Oviedo, SpainSearch for more papers by this authorKarin Schlangen, Karin Schlangen Genome Analysis Platform, CIC bioGUNE, Bizkaia Technological Technology Park, Derio, SpainSearch for more papers by this authorAroa Baragaño Raneros, Aroa Baragaño Raneros Department of Immunology, Hospital Universitario Central de Asturias, Oviedo, SpainSearch for more papers by this authorLeonardo Márquez-Kisinousky, Leonardo Márquez-Kisinousky Department of Immunology, Hospital Universitario Central de Asturias, Oviedo, SpainSearch for more papers by this authorAgustín F. Fernández, Agustín F. Fernández Cancer Epigenetics Laboratory, Institute of Oncology of Asturias (IUOPA), Hospital Universitario Central de Asturias, Oviedo, SpainSearch for more papers by this authorCarmen Díaz-Corte, Carmen Díaz-Corte Department of Nephrology, Hospital Universitario Central de Asturias, Oviedo, SpainSearch for more papers by this authorAna M. Aransay, Ana M. Aransay Genome Analysis Platform, CIC bioGUNE, Bizkaia Technological Technology Park, Derio, Spain CIBERhedSearch for more papers by this authorCarlos López-Larrea, Corresponding Author Carlos López-Larrea inmuno@hca.es Department of Immunology, Hospital Universitario Central de Asturias, Oviedo, Spain Correspondence Carlos López-Larrea, Department of Immunology, Hospital Universitario Central de Asturias, Avenida de Roma s/n, 33011 Oviedo, Asturias, Spain. Tel.: ++34 985 106130; fax: ++34 985 106095; e-mail:inmuno@hca.esSearch for more papers by this author First published: 27 December 2016 https://doi.org/10.1111/acel.12552Citations: 29AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Summary Aging is associated with a progressive loss of the CD28 costimulatory molecule in CD4+ lymphocytes (CD28null T cells), which is accompanied by the acquisition of new biological and functional properties that give rise to an impaired immune response. The regulatory mechanisms that govern the appearance and function of this cell subset during aging and in several associated inflammatory disorders remain controversial. Here, we present the whole-genome DNA methylation and gene expression profiles of CD28null T cells and its CD28+ counterpart. A comparative analysis revealed that 296 genes are differentially methylated between the two cell subsets. A total of 160 genes associated with cytotoxicity (e.g. GRZB, TYROBP, and RUNX3) and cytokine/chemokine signaling (e.g. CX3CR1, CD27, and IL-1R) are demethylated in CD28null T cells, while 136 de novo-methylated genes matched defects in the TCR signaling pathway (e.g. ITK, TXK, CD3G, and LCK). TCR-landscape analysis confirmed that CD28null T cells have an oligo/monoclonal expansion over the polyclonal background of CD28+ T cells, but feature a Vβ family repertoire specific to each individual. We reported that CD28null T cells show a preactivation state characterized by a higher level of expression of inflammasome-related genes that leads to the release of IL-1β when activated. Overall, our results demonstrate that CD28null T cells have a unique DNA methylation landscape, which is associated with differences in gene expression, contributing to the functionality of these cells. Understanding these epigenetic regulatory mechanisms could suggest novel therapeutic strategies to prevent the accumulation and activation of these cells during aging. Introduction During differentiation and homeostatic proliferation of CD4+ T lymphocytes, some cells lose expression of the CD28 costimulatory molecule, which is fundamental to the activation, proliferation and survival of CD4+ T cells (Vallejo et al., 2002). CD4+ CD28null cells (now known as CD28null T cells) acquire new phenotypic and functional characteristics compared with those of conventional CD4+CD28+ (named as CD28+) T cells (Maly & Schirmer, 2015). An increase in the percentage of CD4+ CD28null T cells with age has been reported not only in healthy donors (Weng et al., 2009), but also in patients with infections (CMV, HCV, HIV), inflammatory disorders, autoimmune diseases (rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis), and cardiovascular diseases, or in solid organ transplantation (Scarsi et al., 2011; Broux et al., 2012; Téo et al., 2013; Shabir et al., 2016). The appearance of this subset during aging can compromise the CD4+ T-cell compartment, leading to a reduced immune response to pathogens and vaccines in the elderly. By contrast, this CD28null subset is only detectable in a low percentage of young and middle-aged individuals, although it is sometimes highly enriched in the CD4+ T-cell compartment. The nature of the stimuli that trigger expansion of these CD28null T cells is not fully understood. Some studies suggest that they are generated in response to repeated specific auto-antigen stimulation, while others show that they respond to ubiquitous antigens such as heat-shock proteins (Hsp 60) and viral antigens (proteins from CMV), thereby acquiring an innate T-cell-like phenotype (Zal et al., 2004; van Bergen et al., 2009). Some therapeutic approaches have been proposed with the purpose of reducing the percentage of CD28null T cells, such as activation in the presence of IL-12 or treatments with statins, abatacept, or ATG (Warrington et al., 2003; Link et al., 2011; Scarsi et al., 2011; Duftner et al., 2012). However, the basic mechanisms leading to the generation of CD28null T cells remain unknown. Knowledge of them might suggest new therapeutic options to prevent its accumulation and function. Aging has been associated with gradual DNA methylation changes over the lifespan, marked by global hypomethylation and specific hypermethylation in promoter-associated CpG islands (Martin, 2005; Heyn et al., 2012). Some studies have linked these changes to alterations in the expression levels of DNMT1 and DNMT3B regardless of nutritional habits, lifestyle, or clinical parameters (Ciccarone et al., 2016). In this way, age-associated 'epigenetic drift' can potentially restrict the plasticity of T cells while supporting new phenotypes, such as exhausted or senescent T cells (Fraga et al., 2005; Issa, 2014). Moreover, it has recently been shown that changes in DNA methylation levels in certain CpG sites are closely associated with the age of individuals (Florath et al., 2014). This 'epigenetic clock' demonstrates that using a small number of these sites can accurately predict the chronological age of an individual. DNA methylation is a key process in hematopoietic differentiation and the maintenance of subset-specific T lymphocyte gene expression (Calvanese et al., 2012; Rodriguez et al., 2015). The regulatory regions of some specific genes and transcription factors are demethylated, and therefore, the genes are overexpressed in certain T subsets (e.g. IFNG in Th1 cells), while others are repressed by de novo methylation at regulatory regions (e.g. IFNG and FOXP3 in Th2 cells; Suárez-Álvarez et al., 2013). However, these methylation patterns can be altered in response to intrinsic or external stimuli, causing changes in gene expression (Agrawal et al., 2010; Suarez-Alvarez et al., 2012). In CD28null T lymphocytes, low levels of Dnmt1 and Dnmt3a lead to a loss of methylation and overexpression of CD70, perforin, and KIR2DL4 (Liu et al., 2009). Here, we examined the whole-genome DNA methylation and expression profiles of CD28null and CD28+ T-cell subsets. We identified 296 differentially methylated genes related to defects in TCR signaling, cell death pathways, and cytotoxicity ability. Alterations in the expression of genes related to the inflammasome pathway suggest the presence of a preactivated inflammatory phenotype in CD28null T cells. Our results indicate that specific changes in DNA methylation dynamics in conventional CD4+ T cells contribute to the acquisition and maintenance of the CD28null phenotype that may predispose individuals to age-associated decline in their immune response. Results Overall gain of gene expression in CD28null T cells is associated with changes in immune response and programmed cell death We analyzed the differential gene expression between CD28null T cells and their CD28+ counterparts isolated from healthy donors at different ages to avoid changes due to pathologies. To minimize the differences among individuals, samples were grouped into two pools of 12 donors each (range: 45–75 years; average: 62.1 ± 9.9 years). We identified 1978 differentially expressed genes (DEGs; adjusted P < 0.01 and FC ≥ 1.5 or ≤ −1.5), of which 1205 (60.9%) were upregulated and 773 (39.1%) were downregulated in CD28null relative to their CD28+ counterparts (Table S1, Supporting information). An overall gain of gene expression was observed in the CD28null T cells regardless of the fold-change criteria used (Fig. S1A, Supporting information). To study the biological functions involving these genes, we performed GO analysis using more severe criteria (adjusted P < 0.01 and FC ≥ 3 or ≤ −3; 545 upregulated and 125 downregulated genes). We observed that GO terms related to immune response (GO: 0006955), regulation of programmed cell death (GO: 0043067), defense response (GO: 0006952), and cell activation (GO: 0001775) were markedly enriched in CD28null T cells (Fig. S1B and Table S2, Supporting information). Genes related to immune and defense responses were upregulated in CD28null cells, while loss of expression was associated with a wider range of functions, such as response to stress, the macromolecule catabolic process, chromatin modifications, and cell death (Fig. S2 and Table S2, Supporting information). KEGG analysis showed that DEGs are enriched in pathways related to autoimmune diseases, infections or allograft rejection, in which the functionality of these CD28null T cells had been previously demonstrated (Fig. S1C and Table S2, Supporting information). We then analyzed in detail the two most significantly altered functional categories—immune response and regulation of programmed cell death—by considering all the DEGs within each category (P < 0.01 and FC ≥ 1.5 or ≤ −1.5). Our results showed that most of the DEGs related to the immune system (94 of 112 genes, 84%) were overexpressed in CD28null T cells, suggesting a gain of immune functions in these cells (Fig. 1). Some of these genes correspond to receptors that are strongly expressed in NK cells (LILRB3, LILRB2), chemokines (CCR6, CCL20), cytokines (IL-10, IL-18, IL-6R, IL-1R2), and adhesion molecules (CD14, CD1C, CD86, TNFSF8, TNFSF13B, TNFSF14), which regulate the migration and recruitment of other cell types. The genes involved in the interferon (IFN) pathway, such as IRF8 transcription factor (TF), were also more strongly expressed. This gene regulates expression of LST1 protein, which inhibits lymphocyte proliferation. Other autoimmunity-related TFs, such as ETS-1 and AIRE, were also upregulated. Conversely, the loss of CD40LG gene expression in CD28null T cells could involve defects in the interaction and activation of B cells. Figure 1Open in figure viewerPowerPoint Volcano plots of genes associated with the most representative categories, immune response and regulation of programmed cell death, and differentially expressed in CD28null T cells. Gray dots indicate all DEGs between CD28+ and CD28null T cells. Genes associated with the immune response (GO:0006955) and programmed cell death (GO: 0043067) categories are highlighted in blue and red, respectively. For this analysis, the criteria of adjusted P < 0.01 and ≥ 1.5-fold change in size were used. Numbers in the corners show the number of genes upregulated and downregulated in CD28null T cells in each functional category. One of the main characteristics of CD28null cells is their resistance to programmed cell death (Kovalcsik et al., 2015). We observed a clear deregulation of the expression levels of the Bcl-2 (B-cell lymphoma-2) family members. CD28null T cells expressed high levels of the antiapoptotic genes BCL2, BCL2L1 (encoding Bcl-x), and MCL-1, while the pro-apoptotic genes BAX and BCL2L11 (BIM) were significantly downregulated (Fig. 1). These data correlates with the low levels of Annexin V staining observed in CD28null T cells before and after staurosporine-induced apoptosis and in comparison with their CD28+ counterparts (Fig. S3, Supporting information). Moreover, expression of negative apoptosis regulators such as CFLAR (also known as FLIP or CASPER), BIRC2 (cIAP-1) and BIRC3 (cIAP-2) was stronger in these cells, in contrast to the downregulation of the positive regulator DIABLO, which is involved in caspase activation. Other genes related to necroptosis, such as RIPK1 and MLKL, were also underexpressed in CD28null cells. CD28null T cells overexpress genes involved in the inflammasome pathway We found that all genes comprising the NLRP3-inflammasome complex were overexpressed in CD28null cells (Fig. 2A). These included the NOD-like receptor (NLR) pyrin domain containing 3 (NLRP3), the ASC adaptor protein encoded by the PYCARD gene, the caspase recruitment domain family member 8 (CARD8), and the inactive pro-form caspase-1 (CASP1). Together, they form a multimolecular protein complex, which is essential for the cleavage of caspase-1 into its active form. The pro-forms of the IL-1B and IL-18 genes were also significantly more strongly expressed in CD28null T cells. To corroborate these findings, we analyzed the expression of these genes in paired CD28null/CD28+ T-cell samples isolated individually from 10 healthy donors. We confirmed that the expression levels of NLRP3, PYCARD (ASC), IL-1B, and IL-18 were higher in all CD28null T-cell samples (Fig. 2B). Nonetheless, the inactive form of caspase-1 was only more strongly expressed in four of ten (40%) samples, and the CARD8 gene expression levels were often downregulated in CD28null T cells. We also observed that in the baseline state, CD28null T cells showed higher active caspase-1 levels than their CD28+ counterparts (Fig. 2C), and under nigericin stimulation, they were able to release an active form of the pro-inflammatory IL-1β cytokine (Fig. 2D). Nigericin alone, but not TNF-α, was sufficient to activate caspase 1 and induce the release of IL-1β in CD28null T cells, suggesting a basal preactivating state in these cells. Figure 2Open in figure viewerPowerPoint Overexpression of the inflammasome pathway in CD28null T cells. (A) Difference in the expression of genes related to the inflammasome in CD28null T cells compared with CD28+ T cells based on whole-genome expression array data. (B) RT–PCR analysis of inflammasome genes (pro-IL-1B, pro-IL-18, CARD8, CASP-1, NLRP3, and PYCARD) in CD28+ and CD28null T-cell subsets isolated individually from 10 healthy donors. (C) Histograms showing the expression level of the active form of caspase-1 at baseline in CD28+ and CD28null T cells isolated from three healthy donors. For this purpose, the FLICA probe, which binds only to the active form of caspase, was added to the cell culture and further detected by flow cytometry. (D) IL-1β levels quantified by ELISA in the cell supernatants of CD28null and CD28+ T cells before and after overnight stimulation with TNF-α (5 ng mL−1), nigericin (10 μm), or both. Histograms are representative of three independent experiments and assayed by duplicates. * P <0.05 versus CD28+ T cells. Changes in the DNA methylation profile in CD28null T cells contribute to altered TCR signaling and cytotoxicity ability We examined whether changes in gene expression of CD28null T cells were due to alterations in the whole-genome methylation profile, using DNA isolated from the same pools as before. Unsupervised clustering analysis and scatterplots revealed the reproducibility of the two pools in both cell types (Fig. S4, Supporting information). We detected 317 probes or DMRs between CD28null and CD28+ T-cell subsets (Table S3, Supporting information). Of these, 170 probes (160 genes) corresponded to demethylated regions in CD28null T cells, and 147 probes (136 genes) were de novo-methylated regions (Fig. 3A). Most regions that lost DNA methylation were located outside CpG islands, while gain of methylation was associated with CpG islands (Fig. 3B). Figure 3Open in figure viewerPowerPoint Overview of the differentially methylated regions in CD28null T cells and their functional implication. (A) Heat map showing regions differentially methylated (DMRs) between CD28+ and CD28null T cells. A set of 317 probes (296 genes) differed between the T-cell subsets. Datasets were generated from two biological replicates per cell type (#1 and #2), obtained from a pool of 12 healthy donors each, to minimize the differences among individuals. Samples were pooled using the same DNA quantity per donor. The methylation levels vary from unmethylated (β = 0, red) to fully methylated (β = 1, green). (B) Distribution of DMRs in CD28null T cells according to localization in a CpG island or not. (C) Gene ontology (GO) analysis of all DMRs in CD28null T cells (160 genes demethylated and 136 genes de novo-methylated) with adjusted P < 0.01. The ten most significant categories and the number of genes in each are shown. (D) Genes associated with the immune response (cytotoxic molecules, costimulatory molecules, cytokines, and chemokines) and differentially methylated in CD28null T cells. (E) Illustration of the altered T-cell receptor (TCR) signaling pathway in CD28null T cells. De novo-methylated genes are highlighted in red; demethylated genes are shown in green. To determine the functional relevance of these changes, we performed GO analysis (Fig. 3C), which revealed an enrichment of cellular functions related to the functionality of CD4+ T lymphocytes, such as defense response, immune response, cell adhesion, response to wounding, cell activation, and inflammatory response. A detailed analysis of these categories is shown in Fig. S5 and Table S4 (Supporting information). Differentially methylated genes were associated with costimulatory molecules (ICOS, PDCD1, CD27, CD226), cytokines (IL-19, IL-27, IL-24, IL-32, IL-21R, LTA), chemokines (CCL21, CX3CR1, CXCL1, CCL4, CXCR6), adhesion molecules (ITGAL, KLRG1, LY9), apoptosis-related molecules (BCL2, FASLG), T-cell activation (WAS, LCK, SLA-2), and cytotoxicity ability (TYROBP, GZMB, GZMH, LYZ, CD244, CD59, NKG7, RUNX3; Fig. 3D). KEGG analysis revealed that de novo-methylated genes in CD28null T cells were mainly associated with the T-cell receptor (TCR) signaling pathway, whereas demethylated genes were mainly involved in NK-mediated cell cytotoxicity and cytokine/chemokine signaling (Table 1). Table 1. KEGG pathway of differentially methylated genes in CD28null T cells Pathway P Genes De novo-methylated genes hsa04660 T-cell receptor signaling pathway 2.25E-04 ITK, CD3G, CD3D, ICOS, LCK, GRAP2, PDCD1 hsa04060 Cytokine–cytokine receptor interaction 0.0194 CXCL1, CXCR6, FASLG, IL-24, BMPR1B, LTA, EPO hsa05340 Primary immunodeficiency 0.0333 CD3D, ICOS, LCK Demethylated genes hsa04062 Chemokine signaling pathway 0.0016 FGR, CCL21, NCF1, PREX1, CX3CR1, CCL8, WAS, CCL4 hsa04060 Cytokine–cytokine receptor interaction 0.0027 LTBR, CCL21, IL-19, IL-21R, CX3CR1, CCL8, CSF3R, IFNGR2, CCL4 hsa04650 Natural killer cell-mediated cytotoxicity 0.0358 CD244, GZMB, FCGR3B, IFNGR2, TYROBP Senescent CD28null T cells are not able to mount a robust proliferative response to stimulation (Chou & Effros, 2013). We found many genes associated with TCR activation and signaling to be de novo-methylated in CD28null cells (Fig. 3E), for example, the CD3 complex (CD3G and CD3D), protein tyrosine kinase (PTK) genes (LCK, ITK, TXK), the Grb-2-related adaptor protein 2 (GRAP2), and the adapter proteins SIT1 (signaling threshold regulating transmembrane adaptor 1) and SLA2 (Scr-like adaptor 2), which negatively regulate TCR signaling. Only the gene encoding the FYN-binding protein, FYB, was demethylated in CD28null T cells. Additionally, we examined T-cell diversity and selection by analyzing TCR (β-chain) usage biases between the CD28+ and CD28null T-cell subsets (Fig. 4A). The Vβ transcription profile revealed higher Vβ/HPRT ratios in CD28null T cells, corresponding to specific families in each sample (Vβ6 and Vβ21 in the first individual; Vβ8 and Vβ13 in the second), which accumulate a high percentage of alterations, as shown by the dark red color. The length distribution of CDR3 in CD28+ T cells shows a Gaussian profile (polyclonal), while CD28null T cells show a specific CDR3 length distribution, resembling an oligo/monoclonal expansion (Fig. 4B). Figure 4Open in figure viewerPowerPoint Alterations of the TCRVB repertoire between CD28+ and CD28null T cells. (A) Quantitative and qualitative analysis of TCR repertoire was performed in CD28+ and CD28null T cells isolated from two healthy donors. Results are displayed as a three-dimensional TcLandscape® in which the X, Y, and Z axes represent the 26 human Vβ families, the Vβ/HPRT (nonregulated gene) ratios, and the CDR3 lengths, respectively. The percentages of CDR3-LD alterations are represented as a color code from deep blue (−30%) to dark red (+30%). (B) Histograms showing the spectratype analysis of CDR3 lengths for Vβ families with the highest Vβ/HPRT ratio in the CD28null T-cell subset for each donor. CpG methylation is inversely correlated with gene expression in CD28null T cells Changes in DNA methylation are directly related to modifications in chromatin structure and gene expression. We analyzed the association between differentially methylated and expressed genes in CD28+ and CD28null T cells based on array data. We took data from 170 genes, of which 31 (18.23%; LILRB3, CX3CR1, GZMB, BCL2, or TYROBP among others) were demethylated and had a higher level of expression in CD28null T cells, and 13 (7.64%) genes (such as ITK, LY9, SLA2, or LTA) were de novo-methylated and downregulated in CD28null T lymphocytes (Fig. S6, Supporting information). Additionally, we corroborate that CD28null T cells show a lesser expression of Lck that leads to a diminished phosphorylation of ZAP70 (Fig. S7, Supporting information). All other differentially methylated genes without changes in gene expression may be primed for further alteration upon T-cell activation. We selected six genes on the basis of their immunological relevance to validate and replicate the array-based results. Methylation was analyzed by bisulfite pyrosequencing, and expression levels were evaluated by flow cytometry or qRT–PCR in CD28+ and CD28null T-cell subsets isolated from 10 healthy donors (range: 48–92 years; average 70.2 ± 11.6 years). There was a strong correlation between the methylation level and expression status (Fig. 5), for example, the chemokine CX3CR1, which is involved in cellular migration, was demethylated and highly expressed on the cell surface of CD28null T cells. Similar results were obtained for the adapter molecule TYROBP (also known as DAP12) and the cytotoxic molecule GZMB. Both molecules had a higher level of expression in CD28null T cells due to demethylation, although the degree of demethylation in GZMB was dependent on each donor. Demethylation of the DAP12 locus could facilitate its expression in CD28null cells, thereby acting as an activating signal transduction element and enhancing its cytotoxicity ability. Conversely, the costimulatory molecule TNFRSF7 (CD27) was highly methylated in CD28null cells. Although the gene expression array did not confirm the loss of CD27 expression in CD28null T cells compared with their CD28+ counterparts, flow cytometry corroborated that all CD28null T cells lacked this costimulatory molecule. The PTK genes, ITK and TXK, were de novo-methylated in CD28null T cells, causing their expression to be downregulated. Figure 5Open in figure viewerPowerPoint Validation of differentially methylated and expressed genes in CD28+ and CD28null T-cell subsets isolated individually from ten healthy donors. For each gene is shown (from left to right): percentage of methylation based on data from the Illumina Infinium HumanMethylation27 BeadChip array; methylation level determined by bisulfite pyrosequencing analysis in CD28+ and CD28null T-cell subsets isolated individually from ten healthy donors; mRNA expression levels based on the Illumina expression array; gene expression analyzed by flow cytometry or qRT–PCR in CD28+; and CD28null T-cell subsets isolated individually from ten healthy donors. Dot plots are representative of one of the ten experiments. Box plots show the average expression levels of ten donors.* P < 0.05 versus CD28+ T cells. Discussion One of the most consistent age-associated changes in human T cells that contribute to a decline in immune function is the accumulation of CD28null T cells. A high frequency of CD28null T cells (up to 50% of total CD4+ T lymphocytes) has been described in aging and several pathologies associated with chronic inflammatory processes, but also in some healthy young and middle-aged individuals (Weng et al., 2009). The generation of this subset has been mostly attributed to pro-inflammatory environments and repeated antigen stimulation, but the molecular mechanisms that contribute to its accumulation and maintenance remain to be elucidated. To date, most studies have been carried out with CD28null T cells isolated from patients whose disease etiology, treatments, and concomitant effects differed substantially, increasing the variability associated with this cell subset. Our study, carried out with healthy donors at different ages, demonstrates that DNA methylation contribute to the functionality and characteristics of CD28null T lymphocytes. This is the first report of whole-genome DNA methylation and expression profiles of CD28null and its CD28+ counterpart. Changes in DNA methylation levels in the promoter regions of specific genes related to the immune response, TCR signaling, and cell death pathways reveal epigenetic control during differentiation to CD28null T cells. DNA methylation is an essential regulatory mechanism of key processes of T lymphocytes, such as differentiation to effector cells or the establishment of a unique phenotype in the memory cells (Li et al., 2009; Suarez-Alvarez et al., 2012; Komori et al., 2015). Previous studies have observed global DNA demethylation with aging associated with a lower level of DNMT1 and DNMT3A/B expression. There is a linear decrease in DNMT3B expression with age, while the levels of DNMT1 only gradually decreased up to the age of 64 years (Ciccarone et al., 2016). Moreover, Li et al. (2009) reported that in 'senescent' CD28null T cells, decreased DNMT1 and DNMT3A expression causes demethylation and overexpression of CD70, PRF, and KIR2DL4 genes. Here, we show that changes in the global DNA methylation profile in CD28null T cells are equally associated with gain and loss of methylation in specific genes. We observed that demethylated regions are mainly associated with genes related to immune function, such as cytotoxicity response, cytokine/chemokine signaling, or costimulation. Resu
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