T-cells and Aging. Jörg J Goronzy, M.D., Ph.D. Stanford University School of Medicine

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1 T-cells and Aging Jörg J Goronzy, M.D., Ph.D. Stanford University School of Medicine

2 Cells Kinetics of T cell responses after vaccination Ag Pre-existing T cell repertoire Differentiation Clonal expansion > 13 divisions TCR signaling Effector function B cell help Clonal contraction Apoptosis of effector cells Days Survival long-lived memory T cells

3 Age and Vaccine Responses What are the critical defects with aging that need to be prevented or repaired to improve vaccine responses? Lack of players ( # naïve T cells, # of antigen-specific memory T cells, T cell receptor diversity) Failure of T cell activation/signaling Failure to clonal expand and develop effector cells Failure to develop long-lived memory T cells

4 Estimation of T Cell Diversity by Nexgen Sequencing Naive and memory CD4 and CD8 T cells from young and elderly adults Five aliquots of 1x10 6 cells each Illumina MiSeq sequencing (150bp) Estimates of richness (number of different TCR sequences): Incidence-based non-parametric analysis using Chao 2 estimator Estimates of clonality (unevenness in clonal size distribution): Lymphclon (Liu, Olshen, Fire, Boyd)

5 Age and T Cell Diversity The naive T cell repertoire in older individuals continues to be very diverse CD4 naive CD8 naive Nucleotide sequence Amino acid sequence Qi Q et al. PNAS 2014;111:

6 Age and T Cell Diversity Richness in the memory T cell repertoire does not change with age CD4 memory CD8 memory Nucleotide sequence Amino acid sequence Qi Q et al. PNAS 2014;111:

7 Log ( VZV-specific antibody titers) Age and Herpes Zoster Reactivation VZV-specific antibodies do not decline with age Age (years)

8 Log (frequency IFNg+ VZV-specific T cells) Age and Herpes Zoster Reactivation Decline in VZV-specific CD4 T cells with age Age (years)

9 Repertoire analysis of VZV-specific T cells Experimental Design CFSE-labelled PBMC stimulated with VZV lysate for 8 days in triplicate Triplicate samples of CSFE-low cells sequenced TCR considered specific for VZV if present in 2 replicates and enriched by at least 2-fold compared with total CD4

10 Age and Vaccine Responses What are the critical defects with aging that need to be prevented or repaired to improve vaccine responses? Lack of players ( # naive T cells, # of antigen-specific memory T cells, T cell receptor diversity) Failure of T cell activation/signaling Failure to clonal expand and develop effector cells Failure to develop long-lived memory T cells

11 MFI of pperk Functionality of CD4 T cells with age T cell receptor signaling: defective ERK phosphorylation Naive CD4 T cells Memory CD4 T cells P< Time (min) Nature Medicine 18, (2012) yrs yrs

12 DUSP6 (Relative intensity) Aging and naive CD4 T cell activation Increased expression of DUSP6 Naive CD4 T cells R = Age (years) Nature Medicine 18, (2012)

13 mir-181a (AU) Aging and naive CD4 T cell activation Age-related decrease in mir-181a expression P= yrs yrs 0.0 Naive CD4 T cells Memory Nature Medicine 18, (2012)

14 mir-181a is required for T cell clonal expansion and effector differentiation

15 DUSP6 ERK Activation T cell mir181a/b SIRT1 NFkB c-jun DUSP4 Effector function T cell AMPK mtorc1

16 Cluster analysis of T cell responses after varicella zoster vaccination

17 Age-associated defects in T cell responses Increased cell loss during clonal contraction Expansion Contraction Net Long-lived

18 Gene expression in activated CD4 T cells correlates with contraction and generation of long-lived memory T cells o n C o n tr a c ti D a y 1 4 D a y 8 e r L o n g t m L o n g t D a y 1 4 e r m o n D a y 8 C o n tr a c ti o n D a y 0 C o n tr a c ti D a y 0 L o n g t e r m

19 Count Count Increased expression of CD39 in CD4 T cell responses from older individuals Naive CM EM TEMRA 33y 2.84% 17.6% 28.2% 7.24% 66y 3.31% 33.8% 47.2% 8.77% CD39 Cell Reports, in press

20 Increased apoptosis rates in activated CD39+ CD4+ T cells Percentage (%) CD39+CD25+ CD39-CD25+ 0

21 Apoptotic CD39 T cells (%) Inhibition of CD39 ATPase activity improves effector T cell survival 40 p = p < p < ARL (um) POM-1 (um) Cell Reports, in press

22 IFN-g+ Flu-specificTcells log (fold change) IFN-g+ VZV-specificTcells log ( fold change) Promoter polymorphisms of the ATPase ENTPD1 (CD39) correlates with generation of memory T cells in vaccine responses p=0.097 p= AA AG GG ENTPD1 SNP A/California/07/2009 A/Victoria/361/2011 B/Wisconsin/1/2010 AA AG GG p=0.016 p=0.019 AA AG GG ENTPD1 SNP p=0.004 p=0.17 AA AG GG

23 Age-associated increased CD39 expression impairs generation of long-lived memory T cells Cell Reports, in press

24 Conclusions The naïve T cell receptor repertoire contracts with age, but remains very rich making holes in the repertoire unlikely. The breadth of the VZV-specific repertoire differs markedly between individuals irrespective of age and genetic make-up. The diversity of VZV-specific CD4 T cell repertoire increases after vaccination due to the expansion of infrequent clones, new recruitment of naïve T cells into the memory repertoire and the exhaustion of previously dominant clones. Since vaccination expands preferentially expands small VZVspecific T cells, their clonal sizes remain small even after vaccination.

25 Conclusions Activation of naive T cells in older individuals is compromised due to the loss of age-associated loss of mir181a and associated attenuation of signaling pathways. The major age-associated defect in VZV-specific CD4 T cell recall responses is a failure of effector cells to survive and develop into long-lived memory T cells. The propensity of effector T cells to survive and differentiate into long-lived memory cells is determined by the ecto- ATPase CD39 and purinergic signaling. Expression of CD39 is increased in T cell responses from older individuals leading to increased loss of antigen-specific T cells.

26 Acknowledgements Mary Cavanagh Fengqin Fang Bin Hu Claire Gustafson Chulwoo Kim Sabine Le Saux WonWoo Lee Guangjin Li Qian Qi Rolando Yanes Mingcan Yu David Zhang Stanford University Scott Boyd Yi Liu Mark Davis Cornelia Dekker Holden Maecker Richard Olshen Lu Tian Cornelia Weyand Emory University Rafi Ahmed Rama Akondy Rustom Antia Philip Johnson Andrew Yates Supported by NIH U19 CCHI and HIPC grants and by RO1 grants from NIAID and from NIA