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Lymphocyte Differentiation Section

Nan-ping Weng, M.D., Ph.D., Senior Investigator

General and antigen-specific a/b TCR repertoires: diversity, distribution, and age-associated change: The T cell receptor provides a basis for specific interactions between T cells and antigen presenting cells/target cells. The diversity of the TCR repertoire plays an essential role in the competency of the adaptive immune response. However, the critical information regarding the size of TCR diversity, the distribution of specific TCR-bearing T cells in the T cell repertoire, and the degree of age-associated changes in both general and antigen-specific TCR repertoires are not well determined. We have applied a unique molecular identifier (UMI) marking TCR mRNA and high throughout sequencing method to directly measure the general and antigen-specific a/b TCR repertoires in human and mouse. In human, we have sequenced 1.9 x 108 T cells from 34 human subjects with over 3 x 109 sequence reads. We have identified over one million unique TCRa and TCRb sequences. Through longitudinal follow-up, we are able to track the TCR repertoire changes with age, which provides dynamics of TCR repertoires over the full-spectrum of adult life from 20s to 90s. For the antigen-specific TCR repertoires, we conducted comprehensive analysis of CD8+ TCR repertoires for two dominant viral epitopes: pp65495–503 (NLV) of cytomegalovirus and M1 58–66 (GIL) of influenza A virus. The highly-individualized repertoires (87–5,533 a or b clonotypes per subject) comprised thousands of unique TCRa and TCRb sequences and dozens of distinct CDR3a and CDR3b motifs. However, diversity is effectively restricted by preferential V-J combinations, CDR3 lengths, and CDR3a/CDR3b pairings. Structures of two GIL-specific TCRs bound to GIL–HLA-A2 provided a potential explanation for the lower diversity of GIL-specific than NLV-specific repertoires. These anti-viral TCRs occupied up to 3.4% of the CD8+ TCRb repertoire, ensuring broad T cell responses to single epitopes. Our portrait of two anti-viral TCR repertoires may provide information for development of predictors of immune protection. Our current focus is on 1) determining the size and specificity of general and antigen-specific a/b TCR repertoires in humans using a single cell approach, and 2) determining age-associated changes in antigen-specific TCR repertoires as a measure of the immune competency to the specific pathogen. Such information will serve as a measure of immune competency and a guide for clinical interventions such as vaccination in the elderly.

Epigenetic regulation of transcription, function, and aging of CD8+ T cells: Transcription and its epigenetic regulation plays a key role in CD8+ T cell generation, differentiation, and function. In the past, we have identified genes that are differentially expressed in naïve (TN) and memory (central, TCM, and effector, TEM) CD8+ T cells in the resting and activated states, with parallel gene specific changes in histone modifications (H3K4me3, H3K9ac, and H3K27me3). Such information provides a basis for elucidating the contribution of these differentially expressed genes in CD8+ T cell formation, maintenance, function and possibly aging. Currently, we focus our studies on two areas: 1) Epigenetic regulation of differential gene expression in CD8+ T cells and its change with aging. We analyze activation-induced proliferation and function of CD8+ T cells isolated from young and old humans as well as longitudinal follow-up of selected subjects in gene expression and histone methylation (H3K4me3 and H3K27me3) and 2) the role of histone methyltransferases in CD8+ T cell formation, activation/proliferation and maintenance. We use the various mouse models with T cell specific deletion of histone methyltransferase (Kmt2d, Kmt7, and Ezh2) to determine their target genes and roles in CD8+ T cell activation/proliferation and function. Our study will provide insight into the changes of histone methylation and gene expression after activation and the role of histone methylation in regulation and function of CD8+ T cells.

Roles of telomere and telomerase in human immune function: Telomeres are special structures at chromosomal ends and telomerase is an enzyme synthesizing telomeres. Our previous studies have shown that 1) telomere attrition occurs in lymphocytes with age in vivo based on a 5-year longitudinal analysis and the rates of telomere loss are individualized, 2) healthy old subjects with relatively shorter telomeres have overall weak T and B cell responses to influenza vaccine, and 3) telomerase activity is tightly regulated in lymphocyte development and differentiation, and is reduced in resting T and B cells in old individuals. Our current study focuses on 1) the 13-year longitudinal follow up of over 450 BLSA subjects in telomere length, circulating pro-inflammatory cytokines, and anti-CMV IgG titers and 2) regulation of telomerase expression by alternative splicing of telomerase reverse transcriptase (hTERT) in T cell subsets from young and old human subjects. Our study will provide information on several key biomarkers of the age-associated changes in vivo including telomere/telomerase, pro-inflammatory cytokines, and anti-CMV IgG titer, particularly the relationship these changes. Finally, understanding the role of hTERT alternative splicing in regulation of telomerase activity may enable the ability to manipulate telomerase activity by changing the pattern of alternative splicing of hTERT in lymphocytes as a potential counter measure to aging of the immune system in the elderly.

Portfolio/Research Areas

  • General and antigen-specific TCR repertoires: diversity, distribution, and age-associated change
  • Epigenetic regulation of transcription, function, and aging of CD8+ T cells
  • Roles of telomere and telomerase in human immune function

Findings and Publications

Weng, N-P, Araki, Y. and Subedi, K. The molecular basis of the memory T cell response: differential gene expression and its epigenetic regulation. Nature Reviews Immunology 12:306-315, 2012. PMCID: PMC2801888.

Najarro, KM, Nguyen, H., Chen, G., Xu, M., Sorokina, A., Alcorta, S., Zukley, L., Lin, Y., Li, H., Oelke, M., Metter, J. Xu., X., Link, S., Schneck, J., Longo, DL., Leng, S., Ferrucci, L., and Weng, N-P. Telomere length as an indicator of the robustness of B- and T-cell response to influenza in older adults. J. Infect. Disease 212:1261-1269, 2015. PMCID: PMC4577042.

Li, H-M, Hiroi, T., Zhang, Y., Shi, A., Chen, G., De, S., Metter, J., Wood, W., Sharov, A., Milner, J., Becker, K., Zhan, M., and Weng, N-P. TCRβ repertoire of CD4+ and CD8+ T cells is distinct in richness, distribution, and CDR3 amino acid composition. J. Leukoc. Biol. 99(3):505-513, 2015. PMCID: PMC5338248.

Lustig, A., Sheter, I., Geyer, S., Shi, A., Hu, Y., Morishita, Y., Nagamura, H., Sasaki, K., Maki, M., Hayashi, I., Furukawa, K., Yoshida, K., Kajimura, J., Kyoizumi, S., Kusunoki, Y., Ohishi, W., Nakachi, K.,Weng, N-P*., and Hayashi, T*. Long term effects of radiation exposure on telomere lengths of leukocytes and its associated biomarkers among atomic-bomb survivors. Oncotarget 7(6):38988-38998, 2016. PMCID: PMC5129908.

Chen, G., Yang, XB, Ko, A., Gao, MM., Sun,X., Shi, A., Zhang,Y., Mariuzza, R., and Weng, N-P. Sequence and structural analyses reveal distinct and highly diverse human CD8+ TCR repertoires to immunodominant viral antigens. Cell Reports (in press) 2017.