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Age-associated DNA methylation in blood cells convey information on health status. However, the mechanisms that drive these changes in circulating cells and their relationships to gene regulation are unknown. We identified age-associated DNA methylation sites in six purified blood borne immune cell types (naïve B, naïve CD4+and CD8+T cells, granulocytes, monocytes and NK cells) collected from healthy individuals interspersed over a wide age range. Of the thousand of age-associated sites, only 350 sites were differentially methylated in the same direction in all cell types and validated in an independent longitudinal cohort. Genes close to age-associated hypomethylated sites were enriched for collagen biosynthesis and complement cascade pathways, while genes close to hypermethylated sites mapped to neuronal pathways. In-silico analyses showed that in most cell types, the age-associated hypo- and hypermethylated sites were enriched for ARNT (HIF1β) and REST transcription factor motifs respectively, which are both master regulators of hypoxia response. To conclude, despite spatial heterogeneity, there is a commonality in the putative regulatory role with respect to transcription factor motifs and histone modifications at and around these sites. These features suggest that DNA methylation changes in healthy aging may be adaptive responses to fluctuations of oxygen availability.

Thymic regulatory T cells (tTreg) are critical in the maintenance of normal T cell immunity and tolerance. The role of TCR in tTreg selection remains incompletely understood. In this study, we assessed TCRα and TCRβ sequences of mouse tTreg and thymic conventional CD4+ T cells (Tconv) by high-throughput sequencing. We identified αβ TCR sequences that were unique to either tTreg or Tconv and found that these were distinct as recognized by machine learning algorithm and by preferentially used amino acid trimers in αβ CDR3 of tTreg. In addition, a proportion of αβ TCR sequences expressed by tTreg were also found in Tconv, and machine learning classified the great majority of these shared αβ TCR sequences as characteristic of Tconv and not tTreg. These findings identify two populations of tTreg, one in which the regulatory T cell fate is associated with unique properties of the TCR and another with TCR properties characteristic of Tconv for which tTreg fate is determined by factors beyond TCR sequence.

OBJECTIVE The present study investigates whether associations between telomere length (TL) and cognitive performance across multiple domains are moderated by poverty status and race. METHOD Participants were 325 African American and White urban-dwelling adults (M age = 47.9 years; 49.5% African American; 50.2% female; 48.9% living in poverty) from the Healthy Aging in Neighborhoods of Diversity across the Life Span study. TL was assayed from peripheral blood mononuclear cells using quantitative polymerase chain reactions. Multivariable regression analyses examined interactions of TL, poverty status, and race with performance on the following cognitive tests: Trail-Making Test Parts A and B, Digit Span Forward and Backward, semantic verbal fluency, Brief Test of Attention, Benton Visual Retention Test (BVRT), and California Verbal Learning Test-II total learning, short-delay free recall, and long-delay free recall scores. Analyses adjusted for age, sex, and high school-or-greater educational attainment. RESULTS Significant three-way interactions of TL × Poverty Status × Race revealed that, among White participants living in poverty, shorter TL was associated with worse performance on Digit Span Forward and Backward (ps

The rapidity of SARS-CoV-2 vaccination around the world has substantially reduced the number of new cases of COVID-19 and their severity in highly vaccinated countries. The unanticipated efficacy of SARS-CoV-2 vaccines in older adults has been very encouraging but the longevity of vaccine immunity is currently unknown and protection against emerging variants may be lower. Adoptive immunotherapy with neutralizing mAb may offer an alternative for poor vaccine responders, while the mechanisms underlying failure to respond are still unclear. Further studies of B and T cell responses and their regulation particularly in older populations will provide a more solid foundation to develop suitable approaches to optimize vaccine responses of older adults who fail to mount a durable response.

Epigenetic reprogramming underlies specification of immune cell lineages, but patterns that uniquely define immune cell types and the mechanisms by which they are established remain unclear. Here, we identified lineage-specific DNA methylation signatures of six immune cell types from human peripheral blood and determined their relationship to other epigenetic and transcriptomic patterns. Sites of lineage-specific hypomethylation were associated with distinct combinations of transcription factors in each cell type. By contrast, sites of lineage-specific hypermethylation were restricted mostly to adaptive immune cells. PU.1 binding sites were associated with lineage-specific hypo- and hypermethylation in different cell types, suggesting that it regulates DNA methylation in a context-dependent manner. These observations indicate that innate and adaptive immune lineages are specified by distinct epigenetic mechanisms via combinatorial and context-dependent use of key transcription factors. The cell-specific epigenomics and transcriptional patterns identified serve as a foundation for future studies on immune dysregulation in diseases and aging.

Stimulation of human primary T cells with immobilized anti-CD3 and anti-CD28 Abs in vitro provide a system to study T cell activation and proliferation and an avenue for expanding T cells for immunotherapy. Magnetic beads conjugated with anti-CD3 and anti-CD28 Abs (Dynabeads Human T-Activator [D-TCA]) have been a golden standard for stimulating human primary T cells in vitro. In this study, we report that an application using anti-CD3 and anti-CD28 Abs conjugated on lipid microbubbles (microbubble-based human T cell activator [MB-TCA]) to stimulate primary human naive T cells resulted in expansion superior to D-TCA. In 56-d cultures with three repeated stimulation cycles (14 d per stimulation), we found that 1) MB-TCA induced significantly better expansion (20- and 10-fold increase) of naive CD4+ and CD8+ T cells than did D-TCA; 2) MB-TCA– and D-TCA–stimulated T cells had a similar number of initial cell divisions, but MB-TCA had significantly lower activation-induced cell death than D-TCA; 3) MB-TCA–stimulated T cells produced less TNF-α than did D-TCA; and 4) blocking TNF-α action via adding an Ab against TNF-αR (TNFRSF1A) significantly improved expansion of T cells activated by D-TCA in vitro. Together, we demonstrated that the MB-TCA induces a better expansion of human naive T cells in vitro and offers advantages in both basic and clinical applications in which the outcome depends on the number of T cells.

COVID-19, an infectious disease caused by SARS-CoV-2, has given rise to the current global pandemic. Despite the number of infections nearing 300 million people worldwide, CD8+ T cells against SARS-CoV-2 in COVID-19 convalescent, vaccinated, and healthy adults and their changes with age have not been fully characterized. Here, we report the frequency, phenotype, and in vitro expansion capacity of circulating CD8+ T cells recognizing twenty-two epitopes from five SARS-CoV-2 proteins (S, N, M, ORF1ab, and ORF3a). Through multi-color flow cytometry using antigen-specific tetramers, we found that the frequencies of circulating CD8+ T cells ranged from undetectable to 1% in healthy, convalescent, and vaccinated individuals. Analysis of healthy adults ranging from 17 to 98 years of age revealed that the frequency of CD8+ T cells recognizing the epitope N-LLL (N222–230, LLLDRLNQL) is negatively correlated with age, particularly in the frequency of naive N-LLL-specific CD8+ T cells. Furthermore, the percentage of donors which displayed expansion of N-LLL-specific CD8+ T cells in response to in vitro peptide stimulation was significantly higher in convalescent patients than in healthy donors. These preliminary results suggest that there is an age-associated reduction of N-LLL-specific CD8+ T cells and exposure to SARS-CoV-2 primes these cells for robust expansion in response to stimulation with the N-LLL epitope. This work is still ongoing, and we will present the final results during the meeting.

Influenza A virus (IAV) is a major human pathogen that causes seasonal infections worldwide. IAV-specific CD8+ T-cell response provides an essential part of immune protection against IAV infections. In HLA-A2+ humans, the dominant epitope on IAV is matrix protein M1 residues 58–66 (GILGFVFTL, referred to as GIL). Here, we investigated the TCR repertoire size, predictability, and signaling strength of GIL-specific CD8+ T cells healthy adults. We analyzed over 40,000 GIL-tetramer positive CD8+ T cells by scRNAseq with TCR sequences and identified over 10,000 unique abTCRs sequences. Next, we developed a machine learning algorithm (an ensemble random forest models) to further predict the probability of GIL-binding TCR (ranging from 0.1 to 1 with 1 being highest binding confidence) using reported GIL-TCRs. To confirm the predicting accuracy, we expressed 72 TCR with different RF predicting score and showed that TCR with a RF score greater than 0.8 showed 100% GIL-binding (30 different TCRs). Using RF model and filtering, we identified over 4,000 unique GIL-specific TCRs with less than 800 were previously reported. Overall, our study revealed TCR repertoire size of GIL-specific CD8+ TCRs and indicated the features of GIL-binding TCRs are identified by our ML model to accurately predict this TCR specificity, which paces way to investigate frequency and quality of antigen specific TCR repertoire size and function in the general T cell population.

Long-term immunity against infections and effective response to vaccination are dependent on remarkable and unique feature of lymphocytes referred as “memory generation”. Despite having multiple mice studies focused on naïve to memory transition of lymphocytes our understanding of human naïve and memory properties is limited. In this study we explored epigenetics (DNA methylation and accessible chromatin), transcriptomics and activation induced gene expression of FACS sorted naïve and memory subsets of B, CD4 T and CD8 T lymphocytes of healthy donors from GESTALT cohort. Shared and specific methylomes between B and T lymphocyte subsets were identified. The relationship between DNA methylation, chromatin accessibility and transcriptome through ATAC-Seq and RNA-Seq data from the same donors is established. Through in-silico analyses and by utilizing data from Roadmap Epigenomics project and ChromHMM, we further identified differentiation-specific connections between DNA methylation, chromatin accessibility, and histone modifications. Transcription factor motif analyses identifies presence of ARNT, OCT and NF-κB motifs around sites hypomethylated in B memory while ETS motifs are in memory-associated hyper-methylated sites. Interestingly, OCT motifs were also significantly enriched in B memory specific accessible chromatin. To understand the extent to which these epigenetic changes effected lymphocytes, we activated naïve and memory subsets of B and CD4+ T lymphocytes in vitro and assayed early and late gene expression changes. These findings will provide the foundation for future studies related to dysregulation of memory cell differentiation in aging and infections. Supported by Intramural Research Program of the National Institute on Aging.

Previous research has demonstrated inverse associations between experiences of interpersonal discrimination and telomere length, a marker of cellular aging. Here, we investigate within-race interactions between multiple indices of interpersonal discrimination and sociodemographic characteristics in relation to telomere length in African American and White adults. Participants were from the Healthy Aging in Neighborhoods of Diversity across the Life Span study (Baltimore, Maryland). Ages ranged from 30–64 years old and all self-identified as either African American (n = 176) or White (n = 165). Using linear regression, three patterns were observed within African Americans and one within Whites. Among African Americans, (1) women reporting greater lifetime burden (p = .02), racial (p = .03) and gender (p = .01) discrimination; (2) those with higher socioeconomic status reporting greater lifetime burden (p = .03) and racial discrimination (p = .02); and (3) younger adults reporting greater exposure to multiple sources of discrimination (p = .03) had shorter telomere length. Among Whites, younger and older men reporting greater racial discrimination had shorter and longer telomeres, respectively (p = .02). Findings demonstrate within-race patterns of interpersonal discrimination and cellular aging, which may contribute to racial health disparities.

A diverse TCR repertoire is considered as an essential property of T cell immunity to efficiently contain and eliminate viral invasion. However, limited information is available regarding the characteristics of TCR repertoire to a defined viral antigen peptide in human. Here, we report a comprehensive analysis of CD8 TCR repertoires, consensus binding motifs in CDR3, affinities of selected TCR/pMHCs, and crystal structures of two dominant viral antigens (CMV pp65-NLV and IAV M1-GIL) from 8 healthy adults. A few thousands of distinct TCR alpha and beta chains for NLV and GIL were identified containing highly selected public TCRs. The repertoire of NLV-specific TCR was substantially more diverse than that of GIL-specific TCR for both alpha and beta chains. Further analysis of CDR3 amino acid sequences revealed dozens of distinct consensus motifs in alpha and beta chains of both antigen specific TCRs. Single cell analysis showed preference of the CDR3 motifs paring between TCR alpha and beta. The affinity of TCR/pMHC ranged more widely for GIL (Kd, 1.8–191 μM) than for NLV (4.7–28 μM). Structural analysis exhibited similar patterns of three TCR-GIL-HLA-A2 interactions, while two distinct binding patterns among three TCR-NLV-HLA-A2 interactions. Our findings revealed highly diverse viral antigen-specific CD8 TCR repertoire, the consensus binding motifs in CDR3, binding affinity of TCR and pMHC, and different patterns of TCR-pMHC interactions. Such information will be essential to understand the make-up of T cell immunity and to guide the potential immune based clinical intervention such as cell therapy.

Antinuclear antibodies (ANA), a marker of self-reactivity to DNA and other nuclear antigens, are present in several autoimmune diseases and have been observed in healthy persons in the absence of autoimmune disease. ANA prevalence is higher in women and older adults, but the health implications of ANA in middle- to older-aged adults are unknown. Immune system differences by sex may further result in sex-specific susceptibility to morbidity. In a cross-sectional analysis of data from the Baltimore Longitudinal Study of Aging, we examined the sex-specific relationship between age and ANA as well as the associations (odds ratios and 95% confidence intervals) between ANA and type-2 diabetes and multimorbidity (2 or more chronic diseases), stratified by sex and controlling for age and race. ANA was measured in a 1:160 dilution of sera by immunofluorescence using HEp-2 cells (seropositive = 3 or 4). Overall ANA seroprevalence was 12% (15.1% in women, 8.8% in men). We observed a non-linear relationship between age and ANA that varied by sex (interaction p-value

Human aging is associated with low grade chronic inflammation termed, inflammaging, which is a significant risk factor for morbidity and mortality in elderly. The innate immune system is master regulator of this process via regulating expression of cytokines and other key mediators. We assayed chromatin landscape of the human monocytes during healthy aging with an aim to predict the inflammation associated contemporary and imminent states of monocytes. Our deep sequencing ATAC-seq data from healthy donors of the GESTALT cohort identified, chromatin landscape signatures defining heterogeneity of monocytes. Differentially open chromatin (DOC) clustered old individual monocytes in two distinct groups, while young donors demonstrated ethnicity-based differences. DOC’s heterogeneity of old donors was characterized by presence of intergenic enhancer-like elements dominated by the DNA binding motif of the transcription factor NF-κB. We identified the relationships of age-associated DOC with DNA methylation, gene expression and proteome of monocytes and serum. Finally, the intracellular cytokine staining analysis using multi-parameter flow cytometry demonstrated the DOC’s influence on the monocyte’s response to LPS stimulation. Our data revels the age associated heterogenous functional states of monocytes which can help us understanding decision making mechanisms during an immune response.

COVID-19, an infectious disease caused by SARS-CoV-2, has caused the current global pandemic. Despite the number of infections nearing 100 million people worldwide, the CD8+ T cells against SARS-CoV-2 in healthy adults as well as in convalescent COVID-19 patients are not fully characterized. Here, we report an analysis of the frequency and phenotype of circulating CD8+ T cells recognizing seventeen epitopes from three SARS-CoV-2 proteins (S, N, and M) in HLA-A2+ healthy adults and COVID-19 convalescent patients by multi-color flow cytometry with antigen-specific tetramers. Through the analysis of 65 healthy adults ranging between 17 to 98 years of age, we found that the frequencies of CD8+ T cells recognizing SARS-CoV-2 ranged from 10−2 to undetectable. We did not find a generalized age-related change in the frequency of SARS-CoV-2-recognizing CD8+ T cells. Interestingly, we also did not find increased frequencies of SARS-CoV-2-recognizing CD8+ T cells from convalescent patients compared to the healthy controls. Phenotypical analysis showed that SARS-CoV-2-recognizing CD8+ T cells had a mixed naive and memory phenotype in healthy adults whereas convalescent patients mainly had a memory phenotype. Currently, we are identifying the TCRs of these SARS-CoV-2-recognizing CD8+ T cells. Taken together, our findings have identified the frequency and phenotype of CD8+ T cells which recognize SARS-CoV-2-specific epitopes in humans, and future TCR research will further reveal the binding specificity and functionality of these SARS-CoV-2-specific CD8+ T cells.

A competent T-cell receptor (TCR) repertoire contains TCRs recognizing both novel and experienced antigens. It is widely believed that the human TCR repertoire declines with age, but its precise change in size and clonal distribution with age has not been fully determined. Here, we investigated how the size and content of TCR repertoire changes with age by analyzing the TCR repertoire of human CD4+ and CD8+ T cells and their naïve and memory subsets from 30 healthy adults aged from 25 to 85 at first visit and an average of 9-year follow-up as second visit by RNAseq. First, we calculated abTCR repertoire changes with age and found a profound reduction of the TCRa and TCRb richness in naïve CD8+ T cells and an increase of clonal expansion especially in memory CD8 T cells compared to CD4+ T cells. We also study the degree of overlap of TCRa/b repertoire between naïve and memory T cells in each subject at the same visit and found little overlap of TCRa/b unique sequences between naïve and memory CD4 T cells and ~40% of the total TCRs in CD8 T cells. Moreover, we showed elevated overlap of TCR sequences between two visits in both CD4 and CD8 T cells with memory CD8 T cells as the highest. Finally, we determined the degree of TCR clonotypes shares among different subjects via the analysis of the sharing of TCR sequences in the 30 healthy adults. As results, we observed increased sharing of identical TCR between CD4 and CD8 T cells suggesting that the public clonotypes may share with the common pathogens shared in the population, particularly in the old population. Our findings showed more profound reduction in the CD8+TCR repertoire with age compared to CD4+ TCR repertoire. All together provide evidence of ab TCR sequence-based age-associated changes of T cells and their subsets.

Maintenance of telomeres is essential for preserving T cell proliferative responses yet the precise role of telomerase in human T cell differentiation, function and aging is not fully understood. Here we analyzed human telomerase reverse transcriptase (hTERT) expression and telomerase activity in six T cell subsets from 111 human adults and found that levels of hTERT mRNA and telomerase activity had an ordered decrease from naive (TN) to central memory (TCM) to effector memory (TEM) cells and were higher in CD4+ than their corresponding CD8+ subsets. This differentiation-related reduction of hTERT mRNA and telomerase activity was preserved after activation. Furthermore, the levels of hTERT mRNA and telomerase activity were positively correlated with the degree of activation-induced proliferation and survival of T cells in vitro. Partial knockdown of hTERT by an anti-sense oligo in naive CD4+ cells led to a modest but significant reduction of cell proliferation and increase of apoptosis. Finally, we found that activation-induced levels of hTERT mRNA and telomerase activity in naive CD4+ T cells were significantly lower in old than in young subjects. These findings reveal that hTERT/telomerase expression declines during T cell differentiation and age-associated reduction of activation-induced expression of hTERT/telomerase mainly affect naive CD4+ T cells and suggest that enhancing telomerase activity could be a strategy to improve T cell function in the elderly.

Anti-CD3 and anti-CD28 antibodies are a method of choice for general and physiological activation of human primary T cells in vitro. Here we report a comparison of the growth, cell death, and cytokine production of anti-CD3 and anti-CD28 antibodies conjugated with lipid microbubbles (MB-TCA) and the standard Dynal beads (D-TCA). In 56-day cultures with three repeated stimulation cycles (14 days per stimulation), we found that MB-TCA induced a significantly better expansion (~20-fold increase) of naïve CD4 and CD8 T cells than did D-TCA. We found the main difference between MB-TCA and D-TCA was not the speed of activation-induced initial T cell proliferation but rather the survival of activated T cells. Furthermore, we found that MB-TCA activated T cells had a better telomere length maintenance and produced less TNFα and other cytokines than did D-TCA. Finally, blocking TNFα action via antibody against TNFRSF1A significantly improved expansion of T cells activated by D-TCA in vitro. Together, we demonstrated that MB-TCA offers a better expansion of primary human T cells in vitro and will be a useful method for long term culture of primary human T cells for both basic and clinical applications.

Human aging is associated with low grade chronic inflammation termed, inflammaging, which is a significant risk factor for morbidity and mortality in elderly population. The innate immune system is implicated as master regulator of this process via expression of cytokines and other key mediators. We used ATAC-seq to identify chromatin signatures in monocytes obtained from healthy donors of the GESTALT cohort. State of differentially open chromatin (DOC) between young (60y) donors were identified. Older donors segregated in two clusters, young-like and distinct old-like cluster. Old-like DOC’s enriched for intergenic enhancer-like elements dominated by the DNA binding motif of the transcription factor NF-κB. The relationships of age-associated DOC to gene expression and DNA methylation reveled enrichment of cytokine mediated inflammatory pathways. Ongoing studies of cytokine expression using high-dimensional flowcytometry will be used to determine the functional consequences of chromatin structural changes associated with age.

Objective Studies have linked self-reported discrimination to telomere attrition, a biological marker of accelerated cellular aging. However, it is unknown whether intersections between social categories—race, socioeconomic status (SES), sex, and age—influence the association of varying forms of discrimination with telomere length. We examined these associations in a socioeconomically and racially/ethnically diverse urban sample. Methods Cross-sectional data were from 341 middle-aged (30–64 years) African American and White, community participants in the Healthy Aging in Neighborhoods of Diversity across the Life Span Study (HANDLS). Multiple regression models examined up to 3-way interactions between a discrimination measure (i.e., everyday, racial, gender, lifetime burden, and frequency of discrimination across sources) and two social categories. Results After adjusting for depressive symptoms, waist circumference, and lifetime substance use, two themes emerged: 1) among women with higher SES, a) greater lifetime discrimination burden (b = -0.23, p = .011), gender discrimination (b = -0.29, p = .040), and racial discrimination (b = -0.24, p = 0.023) and 2) among younger adults, irrespective of race and sex, greater frequency of discrimination across sources (b = 0.002, p = .008) was associated with shorter telomeres. Conclusions Irrespective of race, women with higher SES and younger adults reporting greater discrimination may be at particular risk for accelerated aging. Telomere attrition promotes and accelerates chronic health conditions for which there are health disparities. Future research explicating intersections among specific discrimination indices and social categories is warranted.

Previous studies suggest significant age-associated phenotypic remodeling of circulating immune cells (T and B clymphocytes and monocytes) in older adults, a key feature of immunosenescence. However, longitudinal evidence supporting such remodeling for older individuals is scarce. This study aimed to investigate longitudinal trajectories of circulating immune cell phenotyes in community-dwelling older women. Included in this study were WHAS II participants (n=107) aged 70–79 years at baseline with peripheral Blood Mononuclear Cells (PBMCs) collected and cryopreserved from each participant at 3–5 different study visits between 1994/1995 – 2007/2008. Immune cell phenotypes were determined through flow cytometry analyses using the following surface markers: CD3, CD4, CD8, CCR7, CD28, CD45RA, CD38 for T cells; CD24, CD38, CD27, CD19, CD20, IgD for B cells; and CD14, CD16 for monocytes. Their longitudinal trajectories were evaluated using linear mixed model. The results showed that most phenotypes had no cross-sectional association with age at baseline. While most T- and B-cell phenotypes showed significant decrease over time, central memory (CCR7+CD45RA-) CD4+ (0.8% per year, p

Transition from resting to cell cycle in response to antigenic stimulation is an essential step for naive CD8+ T cells to differentiate to effector and memory cells. Leaving the resting state requires dramatic changes of chromatin status in the key cell cycle inhibitors but the details of these concerted events are not fully elucidated. Here, we showed that Ezh2, an enzymatic component of polycomb repressive complex 2 (PRC2) catalyzing the trimethylation of lysine 27 on histone 3 (H3K27me3), regulates activation induced naive CD8+ T cells proliferation and apoptosis. Upon deletion of Ezh2 during thymocyte development (Ezh2fl/flCD4Cre+ mice), naive CD8+ T cells displayed impaired proliferation and increased apoptosis in response to antigen stimulation. However, naive CD8+ T cells only had impaired proliferation but no increase in apoptosis when Ezh2 was deleted after activation (Ezh2fl/flGzmBCre+ mice), suggesting cell cycle and apoptosis are temporally separable events controlled by Ezh2. We then showed that deletion of Ezh2 resulted in the increase in expression of cyclin-dependent kinase inhibitors Cdkn2a (p16 and Arf) and Cdkn1c (p57) in activated naive CD8+ T cells as the consequence of reduced levels of H3K27me3 at these two gene loci. Finally, with real time imaging, we observed prolonged cell division times of naive CD8+ T cells in the absence of Ezh2 post in vitro stimulation. Together, these findings reveal that repression of Cdkn1c and Cdkn2a by Ezh2 plays an essential role in execution of activation-induced CD8+ T cell proliferation.

The TCR repertoire serves as a reservoir of TCRs for recognizing all potential pathogens. Two major types of T cells, CD4+ and CD8+, that use the same genetic elements and process to generate a functional TCR differ in their recognition of peptide bound to MHC class II and I, respectively. However, it is currently unclear to what extent the TCR repertoire of CD4+ and CD8+ T cells is different. Here, we report a comparative analysis of the TCRβ repertoires of CD4+ and CD8+ T cells by use of a 5′ rapid amplification of cDNA ends–PCR–sequencing method. We found that TCRβ richness of CD4+ T cells ranges from 1.2 to 9.8 × 104 and is approximately 5 times greater, on average, than that of CD8+ T cells in each study subject. Furthermore, there was little overlap in TCRβ sequences between CD4+ (0.3%) and CD8+ (1.3%) T cells. Further analysis showed that CD4+ and CD8+ T cells exhibited distinct preferences for certain amino acids in the CDR3, and this was confirmed further by a support vector machine classifier, suggesting that there are distinct and discernible differences between TCRβ CDR3 in CD4+ and CD8+ T cells. Finally, we identified 5–12% of the unique TCRβs that share an identical CDR3 with different variable genes. Together, our findings reveal the distinct features of the TCRβ repertoire between CD4+ and CD8+ T cells and could potentially be used to evaluate the competency of T cell immunity.

Background: Norepinephrine (NE) is one of the primary catecholamines of the sympathetic nervous system released during a stress response and plays an important role in modulating immune function. NE binds to the adrenergic receptors on immune cells, including T cells, resulting in either suppressed or enhanced function depending on the type of cell, activation status of the cell, duration of NE exposure and concentration of NE. Here, we aim to analyze the effects of NE on the functionality of naive (Tn), central memory (Tcm) and effector memory (Tem) CD8 T cells. Methods: We isolated CD8 T cell subsets from healthy human adults and treated cells in vitro with NE (1 × 10 −6 M) for 16 h; we then stimulated NE treated and untreated CD8 T cell subsets with antibodies for CD3 and CD28 for 24 and 72 h. We assessed the level of beta-2 adrenergic receptor (ADRB2) expression in these cells as well as global gene expression changes in NE treated Tcm cells by microarray analysis. Altered expressed genes after NE treatment were identified and further confirmed by RT-qPCR, and by ELISA for protein changes. We further determined whether the observed NE effects on memory CD8 T cells are mediated by ADRB2 using specific adrenergic receptor agonist and antagonists. Finally, we examined the levels of mRNA and protein of the NE-induced genes in healthy adults with high serum levels of NE (>150 pg/mL) compared to low levels ( Results: We found that memory (Tcm and Tem) CD8 T cells expressed a significantly higher level of ADRB2 compared to naive cells. Consequently, memory CD8 T cells were significantly more sensitive than naive cells to NE induced changes in gene expressions in vitro . Global gene expression analysis revealed that NE induced an elevated expression of inflammatory cytokines and chemokines in resting and activated memory CD8 T cells in addition to a reduced expression of growth-related cytokines. The effects of NE on memory CD8 T cells were primarily mediated by ADRB2 as confirmed by the adrenergic receptor agonist and antagonist assays. Finally, individuals with high serum levels of NE had similar elevated gene expressions observed in vitro compared to the low NE group. Conclusions: Our results demonstrate that NE preferentially modulates the functions of memory CD8 T cells by inducing inflammatory cytokine production and reducing activation-induced memory CD8 T cell expansion.

Chemokines play a pivotal role in regulating the immune response through a tightly controlled expression. Elevated levels of inflammatory chemokines commonly occur with aging but the mechanism underlying this age-associated change is not fully understood. Here, we report the role of microRNA-125b (miR-125b) in regulating inflammatory CC chemokine 4 (CCL4) expression in human immune cells and its altered expression with aging. We first analyzed the mRNA level of CCL4 in eight different types of immune cells including CD4 and CD8 T-cell subsets (naive, central and effector memory), B cells and monocytes in blood from both young (≤42 years) and old (≥70 years) adults. We observed that monocytes and naive CD8 T cells expressed higher levels of CCL4 and exhibited an age-related increase in CCL4. We then found the level of miR-125b was inversely correlated with the level of CCL4 in these cells, and the level of miR-125b was reduced in monocytes and naive CD8 T cells of the old compared to the young adults. Knock-down of miR-125b by shRNA in monocytes and naive CD8 T cells led to an increase of CCL4 protein, whereas enhanced miR-125b expression by transfection in naive CD8 T cells resulted in a reduction of the CCL4 mRNA and protein in response to stimulation. Finally, we demonstrated that miR-125b action requires the ‘seed’ sequence in 3′UTR of CCL4. Together these findings demonstrated that miR-125b is a negative regulator of CCL4 and its reduction is partially responsible for the age-related increase of CCL4.

Influenza A virus (IAV) causes an acute infection in humans that is normally eliminated by CD8+ cytotoxic T lymphocytes. Individuals expressing the MHC class I molecule HLA-A2 produce cytotoxic T lymphocytes bearing T-cell receptors (TCRs) that recognize the immunodominant IAV epitope GILGFVFTL (GIL). Most GIL-specific TCRs utilize α/β chain pairs encoded by the TRAV27/TRBV19 gene combination to recognize this relatively featureless peptide epitope (canonical TCRs). However, ∼40% of GIL-specific TCRs express a wide variety of other TRAV/TRBV combinations (non-canonical TCRs). To investigate the structural underpinnings of this remarkable diversity, we determined the crystal structure of a non-canonical GIL-specific TCR (F50) expressing the TRAV13-1/TRBV27 gene combination bound to GIL–HLA-A2 to 1.7 Å resolution. Comparison of the F50–GIL–HLA-A2 complex with the previously published complex formed by a canonical TCR (JM22) revealed that F50 and JM22 engage GIL–HLA-A2 in markedly different orientations. These orientations are distinguished by crossing angles of TCR to peptide–MHC of 29° for F50 versus 69° for JM22 and by a focus by F50 on the C terminus rather than the center of the MHC α1 helix for JM22. In addition, F50, unlike JM22, uses a tryptophan instead of an arginine to fill a critical notch between GIL and the HLA-A2 α2 helix. The F50–GIL–HLA-A2 complex shows that there are multiple structurally distinct solutions to recognizing an identical peptide–MHC ligand with sufficient affinity to elicit a broad anti-IAV response that protects against viral escape and T-cell clonal loss.