The Loss of TET2 Promotes CD8+ T Cell Memory Differentiation Shannon A. Carty, Mercy Gohil, Lauren B. Banks, Renee M. Cotton, Matthew E. Johnson, Erietta Stelekati, Andrew D. This information is current as Wells, E. John Wherry, Gary A. Koretzky and Martha S. of September 29, 2021. Jordan J Immunol published online 17 November 2017 http://www.jimmunol.org/content/early/2017/11/17/jimmun
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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 17, 2017, doi:10.4049/jimmunol.1700559 The Journal of Immunology
The Loss of TET2 Promotes CD8+ T Cell Memory Differentiation
Shannon A. Carty,*,1 Mercy Gohil,† Lauren B. Banks,† Renee M. Cotton,‡ Matthew E. Johnson,x Erietta Stelekati,{,‖ Andrew D. Wells,‡,x,‖ E. John Wherry,{,‖ Gary A. Koretzky,†,# and Martha S. Jordan‡,‖
T cell differentiation requires appropriate regulation of DNA methylation. In this article, we demonstrate that the methylcytosine dioxygenase ten-eleven translocation (TET)2 regulates CD8+ T cell differentiation. In a murine model of acute viral infection, TET2 loss promotes early acquisition of a memory CD8+ T cell fate in a cell-intrinsic manner without disrupting Ag-driven cell expansion or effector function. Upon secondary recall, TET2-deficient memory CD8+ T cells demonstrate superior pathogen control. Genome-wide methylation analysis identified a number of differentially methylated regions in TET2-deficient versus wild- type CD8+ T cells. These differentially methylated regions did not occur at the loci of differentially expressed memory markers; Downloaded from rather, several hypermethylated regions were identified in known transcriptional regulators of CD8+ T cell memory fate. Together, these data demonstrate that TET2 is an important regulator of CD8+ T cell fate decisions. The Journal of Immunology, 2018, 200: 000–000.
n response to infection, naive CD8+ T cells proliferate and Several cell surface proteins can be used to identify cells with differentiate into a heterogeneous pool of Ag-specific cells differing memory potential. Ag-specific CD8+ T cells that are http://www.jimmunol.org/ having divergent cell fates. Following pathogen clearance, most CD127hi and KLRG1lo preferentially differentiate into long-lived I + Ag-specific CD8 T cells die, but a subset persists to become long- memory cells, whereas CD127lo and KLRG1hi cells are largely lived memory cells, which are able to rapidly respond to rechallenge. short-lived terminally differentiated effector cells (1–3). This program of CD8+ T cell differentiation is regulated through the integration of signals from the TCR, costimulatory/coinhibitory *Department of Medicine, Perelman School of Medicine, University of Pennsylvania, receptors, and inflammatory cytokines, which direct transcrip- Philadelphia, PA 19104; †Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; tional changes that control cell fate. Although it is evident that ‡
Department of Pathology and Laboratory Medicine, Perelman School of Medicine, particular transcription factors, such as T-bet, eomesodermin by guest on September 29, 2021 University of Pennsylvania, Philadelphia, PA 19104; xThe Children’s Hospital of { (Eomes), Blimp-1, Bcl-6, IRF4, and Runx3, are important in de- Philadelphia, Philadelphia, PA 19104; Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; ‖Institute termining the fate of activated cells (4–11), it is also becoming for Immunology, Perelman School of Medicine, University of Pennsylvania, clear that epigenetic programming plays a crucial role in T cell Philadelphia, PA 19104; and #Department of Medicine, Weill Cornell Medicine, New York, NY 10065 fate determination. DNA methylation is one epigenetic mechanism by which T cell 1Current address: Department of Medicine and University of Michigan Comprehen- sive Cancer Center, University of Michigan, Ann Arbor, MI. differentiation is regulated, and recent genome-wide studies have ORCIDs: 0000-0002-8727-9723 (M.G.); 0000-0003-4521-8911 (R.M.C.); 0000- identified coordinated epigenetic changes associated with tran- 0002-8684-5359 (M.E.J.); 0000-0003-4155-3202 (G.A.K.); 0000-0003-2359- scriptional programs during CD4+ or CD8+ T cell differentiation 3227 (M.S.J.). (12–19). It is now appreciated that DNA undergoes regulated Received for publication April 19, 2017. Accepted for publication October 10, 2017. demethylation. Recently, the ten-eleven translocation (TET) This work was supported by National Institutes of Health Grants K08 AI101008 (to family of methylcytosine dioxygenases was shown to mediate this S.A.C.), R37 GM053256 (to G.A.K.), AI105343, AI112521, AI082630, and AI115712 (to E.J.W.) and other support from R01AI082292. process by catalyzing the conversion of 5-methylcytosine (5mC) The sequencing data presented in this article have been submitted to the National to 5-hydroxymethylcytosine (5hmC) and, subsequently, to 5-for- Center for Biotechnology Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/ mylcytosine and 5-carboxylcytosine, critical enzymatic steps geo/query/acc.cgi?acc=GSE105176) under accession number GSE105176. necessary for generating unmodified cytosines (20–22). The TET Address correspondence and reprint requests to Dr. Martha S. Jordan or Dr. Gary A. family member TET2 is widely expressed in the hematopoietic Koretzky, University of Pennsylvania, BRB II/III, Room 507, 421 Curie Boulevard, Philadelphia, PA 19104 (M.S.J.) or 1300 York Avenue, A125, New York, NY 10065 system, and murine models reveal that TET2 loss leads to ex- (G.A.K.). E-mail addresses: [email protected] (M.S.J.) or pansion of hematopoietic stem cells (HSCs) and myeloid com- [email protected] (G.A.K.) partments (23–26). In T cells, TET2 contributes to CD4+ Th cell The online version of this article contains supplemental material. differentiation (27) and cooperates with TET3 to stabilize Foxp3 Abbreviations used in this article: AF, Alexa Fluor; DMC, differentially methylated expression in regulatory T cells (28). cytosine; DMR, differentially methylated region; Eomes, eomesodermin; ERRBS, + enhanced reduced representation bisulfite sequencing; 5hmC, 5-hydroxymethylcyto- However, the function of TET2 in CD8 T cell differentiation is sine; HSC, hematopoietic stem cell; IPA, Ingenuity Pathway Analysis; LCMV, lym- unknown. In this study, we investigated TET2’s role in directing phocytic choriomeningitis virus; Lm-gp33, Listeria monocytogenes that expresses the + LCMV gp33 epitope; 5mC, 5-methylcytosine; MPEC, memory precursor effector CD8 T cell fate following acute lymphocytic choriomeningitis cell; p.i., postinfection; SLEC, short-lived effector cell; TCM, central memory virus (LCMV) infection. We found that TCR signaling rapidly CD8+ T cell; TET, ten-eleven translocation; TET2cKO, TET2 conditional knockout; and dynamically regulates TET2 expression and TET activity. WT, wild-type. Although mice with selective loss of TET2 in T cells have no overt Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 thymic or peripheral T cell phenotypes at steady-state, following
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700559 2 TET2 REGULATES CD8+ T CELL MEMORY acute viral infection, CD8+ T cells preferentially adopt a memory performed using a Cytofix/Cytoperm kit (BD Biosciences) or a Foxp3/ phenotype in a cell-intrinsic manner and demonstrate superior Transcription Factor Staining Buffer Set (eBioscience), according to the pathogen control upon rechallenge. Methylation analysis of LCMV- manufacturer’s instructions. Discrimination of live cell populations was per- + formed using LIVE/DEAD Aqua stain (Invitrogen), according to the manu- specific CD8 T cells identified genomic loci that gained 5mC/5hmC facturer’s instructions. in TET2-deficient cells, including several transcriptional regulators For experiments involving measurement of intracellular 5hmC, T cells known to direct CD8+ T cell effector versus memory differentiation. were surface stained prior to fixation/permeabilization with a Cytofix/ Together, these data demonstrate a novel role for TET2 in directing Cytoperm kit (BD Biosciences), treated with DNase I (300 mg/ml in + PBS) at 37˚C for 1 h, and intracellularly stained with isotype or anti–5hmC CD8 T cell fates. (catalog number 39791; 1 mg/ml; Active Motif) Ab for 30 min, followed by fluorochrome-conjugated goat anti-rabbit secondary Ab (Invitrogen). Materials and Methods For experiments involving ex vivo stimulation, single-cell suspensions Mice were stimulated with 200 ng/ml gp33, gp276, or NP396 peptides in the presence of 1 mg/ml brefeldin A for 5 h and then analyzed for intracellular B6;129S-Tet2tm1.1Iaai/J (TET2fl/fl) mice, C57BL/6J mice, and CD4Cre+ cytokine staining. Data were acquired using a FACS LSR II (BD Biosci- mice were obtained from The Jackson Laboratory and bred at the Uni- ences) and analyzed with FlowJo software (TreeStar). versity of Pennsylvania or the University of Michigan. B6.SJL-Ptprca For experiments involving cell sorting, T cells were isolated and sorted (CD45.1+) mice were from Taconic. For the LCMV memory phenotyping on a FACSAria II (BD Biosciences). For isolation of naive CD8+ T cells, experiments, TET2fl/flCD4Cre+ mice were backcrossed twice to C57BL/6J CD8+ T cells from the spleen and lymph nodes were purified by negative mice. For all other experiments, TET2fl/flCD4Cre+ mice were backcrossed selection and magnetic separation (CD8a+ T Cell Isolation Kit II; Miltenyi 2 2 5–10 times to C57BL/6J mice. Control mice for experiments included age- Biotec) and then sorted for naive CD8+ T cells (TCRb+CD8+CD4 CD44 matched TET2fl/fl, TET2fl/+CD4Cre2, TET2+/+CD4Cre+, or C57BL/6J CD62L+). For sorting of gp33+CD8+ T cells, CD8+ T cells from the fl/fl + animals. P14 mice (29) were bred to TET2fl/flCD4Cre+ mice to generate spleens of control and TET2 CD4Cre mice were negatively isolated as Downloaded from 2 TET2fl/flCD4Cre+P14+ animals. All experiments were performed accord- above and sorted for gp33+CD8+CD4 CD44+ on a FACSAria II using ing to protocols approved by the Institutional Animal Care and Use appropriate biohazardous precautions. Committee of the University of Pennsylvania (numbers 803976 and 803071) and the University of Michigan (PRO00007214). Enhanced reduced representation bisulfite sequencing Infections Genomic DNA was bisulfite converted using an EZ DNA Methylation Kit (Zymo Research). Base-pair resolution DNA methylation was performed on 5 5 + + Mice were infected with 2 3 10 PFU LCMV-Armstrong i.p. or 0.5–2 3 10 control and TET2-deficient gp33 CD8 T cells (n = 4 per genotype), as http://www.jimmunol.org/ CFU Listeria monocytogenes that expresses the LCMV gp33 epitope described (32). Differentially methylated cytosines (DMCs) were identified (LM-gp33), as indicated. LM-gp33 was grown and used as previously using a 25% methylation difference threshold and q-value , 0.01 using described (30). LCMV viral titers and Listeria bacterial loads were measured methylKit (33); differentially methylated regions (DMRs) were determined as described (30, 31). using the bioinformatic algorithm eDMR (34). In vitro stimulation Pathway analysis Murine lymphocytes were isolated from spleen and lymph nodes, and Data were analyzed through the use of Ingenuity Pathways Analysis (IPA; T cells were purified by negative selection and magnetic separation (Pan Ingenuity Systems, Redwood City, CA; http://www.ingenuity.com) speci- T Cell Isolation Kit; Miltenyi Biotec). T cells were cultured in T cell media fied for “Mouse.” The differentially methylated genes containing DMRs
(10% FCS, 50 mM 2-ME, 2 mM L-glutamine/penicillin/streptomycin in that corresponded to at least one pathway annotation in the Ingenuity by guest on September 29, 2021 IMDM) and activated with plate-bound anti-CD3 (1 mg/ml; 2C11) and Knowledge Base were eligible for analysis. The p values associated with 5 mg/ml anti-CD28 (37.51; both from eBioscience) for the indicated times. pathways were calculated using the right-tailed Fisher exact test. Pharmacologic activation of T cells was performed using PMA at 10, 25, or 50 ng/ml and ionomycin at 100, 250, or 500 ng/ml. Data accessibility Lymphocyte isolation and adoptive transfer Enhanced reduced representation bisulfite sequencing (ERRBS) data have been deposited to the National Center for Biotechnology Information Gene Lymphoid and nonlymphoid organs were processed, and single-cell sus- Expression Omnibus under accession number GSE105176 (https://www. pensions were obtained. Peripheral blood was collected into 4% sodium ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE105176). citrate, purified with a Ficoll gradient (Ficoll-Paque Plus; GE Healthcare), and stained for flow cytometric analysis. CD8+ T cells (purified as de- Statistical analysis scribed above) from “memory” mice were injected into congenic hosts + + Statistical significance was calculated as noted in the figure legends. Prism so that 5000 or 7500 CD8 gp33 cells were transferred. For P14 adoptive- (GraphPad) was used for statistical analysis. transfer experiments, 2000 CD8+gp33+Va2+ cells isolated from the peripheral blood were transferred into congenic hosts. Results Flow cytometry and cell sorting TCR signaling dynamically regulates TET2 expression and Cells were isolated, washed, and stained with the indicated Abs. The following activity Abs were used (all from BD Biosciences unless otherwise noted): CD8a- Pacific Blue or Alexa Fluor (AF)700 (53-6.7; BioLegend); CD4 FITC If TET2 plays an important role in T cell differentiation, we (GK1.5; eBioscience), PE-Cy7 (RM4-5; BioLegend), or PE–Texas Red reasoned that its expression and/or function might be altered in (RM4-5; Invitrogen); TCRb allophycocyanin-e780 (H57-597; eBioscience); response to TCR ligation. We found that TET2 mRNA expression CD62L PE–Texas Red (MEL-14; Invitrogen) or Brilliant Violet e605NC (MEL-14; eBioscience); KLRG1 PE-Cy7, FITC or PerCP-e710 (2F1; eBio- was rapidly induced in murine T cells after TCR plus CD28 science), CD127 PE-Cy7 (A7R34; BioLegend) or Pacific Blue (A7R34; stimulation, with maximal expression at 2 h (Fig. 1A). This up- eBioscience); CD27 PE (LG.7F9; eBioscience); CXCR3 PerCP-Cy5.5 regulation was dependent on Ca2+ signaling, because TET2 (CXCR3-173; BioLegend), 2B4 FITC (eBio244F4; eBioscience; 2B4; BD), mRNA was induced by the Ca2+ ionophore ionomycin but not CD160 PE (7H1; BioLegend), CD45.1 PerCP-Cy5.5, PE-Cy5, PE-Cy7 (A20; PMA alone, suggesting that the protein kinase C pathway is not eBioscience) or AF700 (A20; BioLegend); CD45.2 AF700, allophycocyanin- e780 (104; eBioscience) or Pacific Blue (104; BioLegend); CD44 AF700 sufficient for TET2 induction (Fig. 1B). In addition to regulation (IM7; BioLegend); IFN-g–PerCP–Cy5.5 (XMG1.2; BioLegend); TNFa–Pa- of TET2 mRNA levels, TET2 protein is negatively regulated by cific Blue (MP6-XT22; eBioscience); IL-2 allophycocyanin (JES6-5H4); the CXXC-type zinc finger domain containing proteins IDAX and granzyme B PE-Cy7 (NGZB; eBioscience); CD107a FITC or PE (1D4B); CXXC5 (35). We evaluated their expression in primary murine human Ki67-FITC (B56); and Eomes AF647 (Dan11mag; eBioscience). Biotinylated monomers specific for H2-Db–restricted gp33–41 of LCMV T cells as a possible additional means by which TET2 could be were obtained from the National Institutes of Health Tetramer Core Facility regulated. Although IDAX was not detected in T cells, CXXC5 and tetramerized using their published protocol. Intracellular staining was was expressed in resting cells and was downregulated by TCR The Journal of Immunology 3 stimulation (Supplemental Fig. 1A); thus, CXXC5 could serve to the peak of the CD8+ T cell response (day 8), we found similar further regulate TET2 expression. We also assessed whether TCR frequencies and absolute numbers of gp33+CD8+ T cells in the stimulation directs TET activity, specifically the oxidation of 5mC spleens, as well as similar absolute numbers (although lower to 5hmC. Using flow cytometry, we found that 5hmC levels were frequencies) of gp33+CD8+ T cells among PBMCs of TET2cKO rapidly induced in murine T cells after TCR stimulation (Fig. 1C). mice compared with control mice (Fig. 2A). Additionally, a Together, these data indicate that TET2 gene expression and TET similar frequency of gp33+CD8+ T cells expressed the prolifera- function are regulated by TCR activation. tion marker Ki67 on day 8 postinfection (p.i.) in control and
fl/fl + TET2cKO mice (data not shown). Together, these data suggest TET2 CD4Cre mice have intact thymic and peripheral that TET2 is not required for Ag-specific CD8+ T cell proliferation T cell populations and expansion following acute viral infection. TET2 deficiency in the hematopoietic compartment results in Next, we examined the functional ability of TET2cKO CD8+ expansion of HSCs with skewing toward the myeloid lineage T cells to respond to antigenic stimulation. Based on findings in (23–26, 36). To specifically study TET2 in T cells, we generated CD4+ T cells, in which TET2 deficiency led to reduced cytokine fl/fl + TET2 CD4Cre mice that lack TET2 in all mature ab Tcells production following TH1 and TH17 in vitro skewing (27), we (TET2 conditional knockout [TET2cKO] mice). These mice had predicted that TET2cKO CD8+ T cells would produce less cyto- similar frequencies and numbers of thymic and peripheral T cell kine. However, in contrast to our expectation, TET2cKO LCMV- populations compared with age-matched control mice, without ev- specific CD8+ T cells had enhanced IFN-g expression and higher idence of substantially altered homeostasis, as indicated by similar expression of CD107a, a surrogate marker for degranulation, in 2 absolute numbers of naive (CD44 CD62L+)andCD44+ Tcell response to gp33 and gp276 peptides, with percentages trending Downloaded from populations (Supplemental Fig. 1B–D). Moreover, we did not see higher in response to NP396 peptide stimulation (Fig. 2B, 2C). + changes in early activation markers (CD25, CD69, Bcl-xL)or There was no difference in “double producers” (i.e., IFN-g TNF- proliferation following CD3/CD28 stimulation (Supplemental Fig. a+) or IL-2–producing CD8+ T cells following LCMV–peptide 1E, 1F). Together, these data suggest that TET2 does not substan- stimulation, and control and TET2cKO mice were able to clear tially alter T cell homeostasis or regulate short-term TCR-induced virus by day 8 p.i. (data not shown). Together, these data dem- + responses in vitro. onstrate that TET2 loss enhances CD8 T cell effector function at http://www.jimmunol.org/ the peak of infection for certain TCR specificities. TET2cKO CD8+ T cells have intact effector function after acute viral infection TET2 loss promotes memory CD8+ T cell formation Because DNA methylation regulates the cell fate of T cells and After T cell expansion in response to infection, the majority of Ag- other cell types (37, 38), and TET2 is known to control HSC specific CD8+ T cells undergo cell death, whereas a small subset differentiation, we hypothesized that TET2 might influence CD8+ persists and differentiates into long-lived memory cells. Memory T cell differentiation. To test this hypothesis, we infected control CD8+ T cells have several stem cell–like properties, including and TET2cKO mice with LCMV-Armstrong and followed virus- long-term survival, self-renewal, and multipotent potential (39), + specific effector and memory CD8 T cell differentiation using as well as a shared transcriptional program (40). Given these by guest on September 29, 2021 tetramers specific for the gp33–41 (gp33) epitope of LCMV. At characteristics and the fact that TET2 loss in HSCs leads to an
FIGURE 1. TCR signaling regulates TET2. (A) Ex- pression of TET2, relative to b-actin, in cDNA generated from WT T cells stimulated with anti-CD3/CD28 for the indicated time periods, normalized to unstimulated cells (n = 5, two independent experiments). (B) TET2 expres- sion, relative to b-actin, in cDNA generated from WT T cells stimulated with increasing concentrations of PMA and/or ionomycin (n = 6, three independent experiments). (C) Representative line graphs of intracellular 5hmC (up- per left panel) or isotype control (lower left panel) in WT T cells stimulated with anti-CD3/CD28 for the indicated time periods. Fold change in median fluorescence intensity (MFI) of 5hmC compared with unstimulated cells (right panel; n = 7, three independent experiments). *p , 0.05, **p , 0.01, ***p , 0.001, Dunnett multiple-comparison test, repeated-measures one-way ANOVA. 4 TET2 REGULATES CD8+ T CELL MEMORY
FIGURE 2. TET2cKO T cells have intact effec- tor function in response to acute LCMV infection. LCMV-specific responses were assessed on day 8 p. i. in control and TET2cKO mice. (A) Representa- tive flow cytometric analysis of gp33 and CD44 on TCRb+CD8+ splenocytes and PBMCs from control and TET2cKO mice (left panels). Frequency and Downloaded from absolute number of gp33+CD8+ T cells per spleen and 104 PBMCs (right panels). Plots are gated on live CD8+TCRb+ lymphocytes. (B and C) Repre- sentative plots of intracellular TNF-a, IFN-g, and CD107a expression (upper panels) and frequencies (lower panels) of the indicated cell populations in +
CD8 splenocytes stimulated ex vivo with the in- http://www.jimmunol.org/ dicated LCMV-specific peptides. Data are repre- sentative of n = 9 control and n = 12 TET2cKO, three independent experiments (A)orn = 4 control and n = 8 TET2cKO, two independent experiments (B and C). *p , 0.05, **p , 0.01, unpaired t test. by guest on September 29, 2021
expanded stem/progenitor pool (23–26), we reasoned that TET2 MPEC population among control and TET2-deficient gp33+CD8+ deficiency might skew CD8+ T cells toward a memory phenotype T cells (Supplemental Fig. 2E). Together, these data suggest that at the expense of short-lived effector cells. We monitored cohorts TET2 loss directs an early decision toward a TCM fate. of mice for persistence of gp33+ CD8+ T cells in the peripheral To examine whether TET2 loss promotes “bona fide” memory blood of LCMV-infected animals. The frequency of control and formation, we examined control and TET2cKO mice on day TET2-deficient gp33+CD8+ T cells in the blood had a similar 45 p.i. In the spleens of TET2cKO mice, there was an increase in + + pattern of expansion, contraction, and persistence over time for the frequency and absolute number of TCM gp33 CD8 Tcells .80 d p.i. (Supplemental Fig. 2A). However, in the spleens on (defined by CD62L and CD44 expression; Fig. 3B). Addition- + + day 8, among TET2cKO gp33 CD8 T cells, the memory ally, we saw a significant increase in the expression of other TCM precursor effector cell (MPEC) population, identified by its markers, including CD27 and CXCR3 (Fig. 3C). Consistent with 2 + KLRG1 CD127 phenotype (1–3), was significantly augmented increased TCM formation, we found an increase in the absolute with regard to frequency and absolute number, with a concomitant number of gp33+CD8+ T cells in the lymph nodes of TET2cKO decrease in the KLRG1+CD1272 short-lived effector cell (SLEC) mice compared with control mice (Fig. 3D). To further charac- population (Fig. 3A). The phenotype was also evident among terize the TET2-deficient memory CD8+ Tcells,weexamined gp33+CD8+ T cells in the peripheral blood (Supplemental Fig. expression of transcription factors associated with CD8+ Tcell 2B). Additionally, during longitudinal monitoring of peripheral differentiation, including Eomes and T-bet. TET2-deficient blood following LCMV infection, compared with controls, more gp33+CD8+ T cells had higher expression of Eomes compared TET2cKO gp33+CD8+ T cells expressed the markers CD27, with control gp33+CD8+ T cells (Fig. 3E), but no significant CD62L, and CXCR3 (Supplemental Fig. 2C, 2D), which are differences were noted in T-bet expression (data not shown). + + highly expressed on central memory CD8 T cells (TCM) (41–43). Together, these data show that TET2 loss promotes CD8 Tcell Interestingly, the rate of increase over time was similar in the memory formation. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ FIGURE 3. TET2 loss enhances memory CD8+ T cell differentiation. Control and TET2cKO mice were infected with LCMV, and gp33+CD8+ T cells isolated from spleen and lymph nodes were evaluated at the indicated time points. (A) Frequency (left panel) and absolute number (right panel) of SLEC and MPEC populations among gp33+CD8+TCRb+ splenocytes from control and TET2cKO mice isolated on day 8 p.i. (B) Representative flow cytometric analysis of CD62L expression on gp33+CD8+ T cells (left panel). Frequency (middle panel) and absolute number (right panel) of CD62L2 effector memory (EM) and CD62L+ central memory (CM) populations among gp33+CD8+TCRb+ splenocytes from control and TET2cKO mice isolated on day 45 p.i. (C) Representative flow cytometric analysis of CD27 (upper panel) and CXCR3 (lower panel) expression on gp33+CD8+ T cells on day 45 p.i. (D) Absolute number of gp33+CD8+TCRb+ lymphocytes isolated from lymph nodes of control and TET2cKO mice on day 45 p.i. (E) Representative flow cytometric plots of intracellular Eomes expression in gp33+CD8+TCRb+ splenocytes isolated from control and TET2cKO mice on day 45 p.i. Plots are gated on live gp33+CD8+TCRb+ lymphocytes. Data are representative of n = 9 control and n = 12 TET2cKO, three independent experiments (A), n = 10 per genotype, two independent experiments (B–E). *p , 0.05, ***p , 0.001, ****p , 0.00001, unpaired t test. by guest on September 29, 2021
TET2cKO mice lack TET2 in mature CD4+ and CD8+ T cells; functional memory properties, we evaluated the ability of therefore, we sought to determine whether the preferential CD8+ TET2cKO memory CD8+ T cells to mount an effective memory memory differentiation in TET2cKO mice was intrinsic to TET2 de- response. Purified CD8+ T cells from control and TET2cKO mice ficiencyintheCD8+ T cell population or dependent on an altered previously infected with LCMV (.100 d p.i.) were transferred environment due to TET2 loss in all T cells. To address this issue, we into WT congenic hosts, such that equal numbers of gp33+CD8+ used two complementary approaches. In one approach, we transferred T cells were injected. Hosts were subsequently infected with LM- CD8+ T cells isolated from congenic (CD45.1+CD45.2+)mice gp33. Five days following LM-gp33 infection, more gp33+ expressing the P14 TCR transgene, which is specific for the LCMV TET2cKO cells than control cells were present in the peripheral gp33 epitope presented by H-2Db, into control and TET2cKO mice blood of infected hosts (Fig. 5A). Comparable gp33+CD8+ T cell and subsequently infected these mice with LCMV (Fig. 4A). As ex- numbers were found in the spleen, and frequencies within both pected, endogenous host (CD45.2+) TET2cKO gp33+CD8+ T cells organs were similar (Fig. 5A, Supplemental Fig. 3A). To assess rapidly differentiated into MPECsattheexpenseofSLECformation the function of these memory populations, bacterial burden was (Fig. 4B). In contrast, similar frequencies of SLEC and MPEC pop- measured. Mice receiving TET2cKO memory CD8+ T cells had ulations derived from P14 cells were present in control and TET2cKO lower Listeria burden in their livers at 5 d p.i. (Fig. 5B), indicating mice (Fig. 4B; CD45.1+CD45.2+ gate). Additionally, the transferred that TET2-deficient memory cells exhibit greater pathogen con- P14 cells expressed CD62L at similar frequencies in control and trol. This enhanced bacterial control was not accompanied by TET2cKO mice (data not shown). As a second approach, we trans- increased production of IFN-g, TNF-a, or CD107a (Supplemental ferred equal numbers of wild-type (WT) or TET2-deficient P14 CD8+ Fig. 3B) but rather was associated with higher pretransfer ex- + T cells into congenic (CD45.1 ) hosts and infected them with LCMV- pression of CD62L and CXCR3 (Supplemental Fig. 3C), two TCM Armstrong (Fig. 4C). Consistent with prior results, a higher frequency receptors that have been associated with more efficient recall re- of TET2-deficient P14 cells differentiated into MPECs compared with sponses (42–44). Together, these data demonstrate that loss of WT P14 cells, which predominantly differentiated into SLECs TET2 favors a more effective memory immune response. (Fig. 4D). Together, these data demonstrate that TET2 regulates CD8+ TET2 loss leads to DNA hypermethylation T cell memory differentiation in a cell-intrinsic manner. We posited that TET2 loss promotes CD8+ Tcellmemorydiffer- Loss of TET2 enhances pathogen control after rechallenge entiation through altered epigenetic regulation of gene expression. Thus far, we have shown that TET2 loss promotes a central memory To examine cytosine methylation differences in control and phenotype. To determine whether TET2cKO cells displayed enhanced TET2cKO T cells, we performed ERRBS, a method that allows for 6 TET2 REGULATES CD8+ T CELL MEMORY Downloaded from
FIGURE 4. TET2 regulates CD8+ T cell differentiation in a CD8+ T cell–intrinsic manner. (A) Experimental schema: congenically marked P14 CD8+ T cells were adoptively transferred into control or TET2cKO mice that were subsequently infected with LCMV-Armstrong, and PBMCs were analyzed on day 15 p.i. (B) Representative flow cytometric analysis of CD127 and KLRG1 expression on host-derived and P14-derived gp33+CD8+TCRb+ PBMCs (left panel). Frequencies of the indicated populations (right panel). (C) Experimental schema: Equal numbers of P14 control and TET2-deficient P14 CD8+ D T cells were transferred into congenic mice that were subsequently infected with LCMV-Armstrong, and PBMCs were analyzed on day 15 p.i. ( ) http://www.jimmunol.org/ Representative flow cytometric analysis of CD127 and KLRG1 expression on P14-derived gp33+CD8+TCRb+ PBMCs (left panel). Frequencies of SLEC (CD1272KLRG1+) and MPEC (CD127+KLRG12) populations among adoptively transferred P14 CD8+ T cells (right panel). Data are representative of n = 6 control and n = 4 TET2cKO, two independent experiments (A and B), n = 10 per genotype, two independent experiments (C and D). **p , 0.01, ****p , 0.0001, unpaired t test. quantitative genome-wide single–base pair resolution analysis of genic (35%) and intronic (47%) regions (data not shown). Next, cytosine methylation, with coverage within and outside of CpG is- we grouped DMCs into DMRs, in which multiple adjacent CpG lands (32, 45). Similar to bisulfite sequencing, ERRBS does not sites have differential methylation ($25% difference in all control distinguish between 5mC and 5hmC, reading both as a methylated versus TET2cKO samples), using the bioinformatic algorithm cytosine residue and, thus, referred to as 5mC/5hmC to indicate eDMR (34). Three hundred and fifty-five DMRs mapped to 184 by guest on September 29, 2021 detection of either. unique ReqSeq genes (mm 9; Supplemental Table I). The identi- We isolated gp33+CD8+ T cells from control and TET2cKO fied DMRs were mapped to different genomic regions, with the mice on day 8 p.i. with LCMV. Genomic DNA was digested with a majority located in introns and coding sequences (Fig. 6B), and methylation-insensitive restriction enzyme and subjected to stan- most commonly occurred in genes involved in cellular growth dard next-generation sequencing library preparation. Unmethy- and proliferation, cellular development, cell death and survival, lated cytosines underwent bisulfite conversion prior to final and gene expression, according to IPA (Fig. 6C). amplification and sequencing. The ERRBS analysis covered an Several proteins that were differentially expressed on TET2cKO average of 1.54 3 106 individual CpGs with minimum 103 versus control gp33+CD8+ T cells, including KLRG1 (15), CD127 coverage of the mouse genome. Analysis of DMCs (.25% (46), CD27 (47), CXCR3 (15, 48) and CD62L (15), are known to methylation difference) demonstrated that the majority gained be regulated by DNA methylation. Thus, mechanistically, we 5mC/5hmC in TET2cKO LCMV-specific CD8+ T cells compared reasoned that TET2 could individually regulate the methylation with control (Fig. 6A). These DMCs occurred primarily in inter- status of the loci of differentially expressed memory markers.
FIGURE 5. LCMV-specific TET2cKO memory CD8+ T cells reduce LM-gp33 bacterial loads. Equal numbers of gp33+CD8+ T cells from LCMV-immune (.100 d p.i.) control and TET2cKO mice were transferred into congenic hosts that were subsequently infected with LM-gp33. gp33-specific memory responses were assessed 5 d later. (A) Representative flow plots of gp33 and CD44 expression on CD8+ T cells from peripheral blood or spleen on day 5 p.i. with LM-gp33 (left panel). Absolute number of transferred gp33+ of CD8+TCRb+ PBMCs and splenocytes (middle and right panels). (B) Bacterial load (CFU) in liver on day 5 p.i. LM-gp33 of congenic hosts that received control or TET2cKO gp33+ memory CD8+ T cells. Data are representative of n =12 control and n =14 TET2cKO, three independent experiments. *p , 0.05, unpaired t test. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021
FIGURE 6. TET2 loss leads to genomic hypermethylation in LCMV-specific CD8+ T cells. Genomic DNA from control and TET2cKO gp33+CD8+ T cells on day 8 p.i. with LCMV was subjected to ERRBS methylation analysis. (A) Stacked bar plot showing frequency of DMCs of all covered CpGs per autosomal chromosome. Magenta represents gain of 5mC/5hmC and green represents loss of 5mC/5hmC in TET2cKO versus control samples. (B) Pie chart representing genomic location of annotated DMRs. (C) Top four molecular and cellular functions of 182 genes containing DMRs analyzed by IPA software with associated p values and number of differentially methylated genes grouped into functional categories. (D) Integrated Genomics Viewer browser visualization of loss of 5hmC/5mC methylation measured by ERRBS at the indicated genomic loci from representative control (blue) and TET2-deficient (red) samples. DMRs are outlined in red. (E) PRDM1 and Bcl6 expression (relative to b-actin), Bcl-6/PRDM1 ratio, and Runx3 expression in cDNA generated from control or TET2cKO CD8+ T cells activated for 3 d with plate-bound anti-CD3/CD28 and IL-2. (F) Representative flow cytometric analysis of intracellular IRF4 expression (left panel) and median fluorescence intensity (MFI; right panel) in control and TET2cKO CD8+ T cells activated for 3 d with plate-bound anti-CD3/CD28 and IL-2. Data are representative of n = 9 per genotype, three independent experiments (E)orn = 5 per genotype, two independent experiments (F). *p , 0.05, **p , 0.01, unpaired t test.
Alternatively, TET2 could control the expression of key tran- could not be evaluated. However, gene loci encoding KLRG1, scription factors and, thus, promote a CD8+ T cell memory tran- CD27, and CXCR3 were appropriately represented. Interestingly, scriptional program. Some differentially expressed molecules, these loci did not contain DMRs, suggesting that TET2 does not including CD127 and CD62L, did not have 103 ERRBS coverage regulate expression of these genes through direct demethylation. in all eight samples (our cutoff for assessing DMCs) and, thus, Therefore, we evaluated DMRs associated with transcription 8 TET2 REGULATES CD8+ T CELL MEMORY factors known to direct CD8+ T cell effector versus memory dif- contribute to these changes was not explored. However, based on a ferentiation, including Tbx21 (encoding T-bet), Eomes, PRDM1 study in which diminished cytokine production was noted in (encoding Blimp-1), Bcl6, Runx3, and IRF4 (4, 6–10, 49). Bcl6 TET2-deficient TH1 and TH17 cells (27), and our unpublished and Eomes did not contain DMRs, whereas Tbx21, PRDM1, IRF4, results (L.B. Banks, S.A. Carty, and M.S. Jordan, unpublished and Runx3 had associated DMRs that gained 5mC/5hmC in observations) indicating a similar finding in CD4+ cells, we pre- TET2cKO gp33+CD8+ T cells (Fig. 6D). Taken together, these dicted that TET2-deficient CD8+ T cells would have decreased data suggest that acquisition of a memory fate is not due to TET2- cytokine production and/or effector molecule expression follow- mediated loss of 5mC/5hmC at each locus encoding effector/ ing viral infection. To our surprise, we found increased cytokine memory markers (i.e., KLRG1, CD27, CXCR3) but rather TET2 production and effector molecule expression in TET2-deficient may regulate transcriptional drivers of effector or memory cell fate. CD8+ T cells in vivo during acute viral infection. These data in- Because we observed early differences in CD8+ T cell differ- dicate that there are alternative mechanisms regulating the re- entiation in TET2cKO CD8+ T cells, we questioned whether moval of repressive DNA methylation at these loci, which could methylation differences seen at this time point were a cause or include removal by other TET family members or by DNMT1 consequence of altered differentiation. Therefore, to evaluate early inhibition. The lack of DMRs associated with cytokine, effector, gene expression changes, we isolated control and TET2cKO naive and individual memory marker gene loci was contrasted with the CD8+ T cells, activated them in vitro with anti-CD3/CD28 and several DMRs found in known transcriptional regulators of CD8+ IL-2 for 3 d, and examined gene expression using RT-PCR and T cell differentiation. In this study, we noted that, rather than flow cytometry. Activated TET2cKO CD8+ T cells had diminished occurring at individual loci of differentially expressed memory
Blimp-1 mRNA, although this did not reach statistical signi- markers (such as KLRG1, CD27, and CXCR3), the DMRs iden- Downloaded from ficance. Because Blimp-1 and Bcl-6 negatively regulate one an- tified occur in known transcriptional regulators of CD8+ T cell other and have opposing effects on CD8+ T cell effector versus fate. To evaluate whether these methylation changes altered gene memory development (6, 7, 9), we also assessed Bcl-6 mRNA. We expression, we evaluated gene and protein expression in control found that TET2 loss promotes Bcl-6 expression and increased the and TET2-deficient CD8+ T cells activated in vitro. We found that Bcl-6/Blimp-1 ratio (Fig. 6E). Additionally, we noted a modest there were modest increases in Runx3 mRNA, as well as IRF4
increase in Runx3 mRNA and IRF4 protein (Fig. 6E, 6F) but no protein expression, which may contribute to the increase in IFN-g http://www.jimmunol.org/ significant difference in expression of T-bet or Eomes by flow and cytolytic potential seen early during infection (8, 55). More- cytometry at this early time point (data not shown). over, we found an alteration of the Bcl-6/PRDM1 ratio in a manner that would be predicted to promote memory CD8+ T cell Discussion differentiation. Although some of these expression changes were The TET family of methylcytosine dioxygenases mediates active modest, they could be synergistic in promoting memory CD8+ DNA demethylation. In multiple cell types, including embryonic T cell formation while maintaining strong effector responses. stem cells (20, 50), HSCs (23–26), and CD4+ T cells (27), TET Taken together, these findings suggest that TET2 may direct CD8+ activity has been shown to regulate cellular differentiation. It is T cell fate via regulation of transcriptional programs. known that DNA methylation dynamically changes in CD8+ Tcells Following LCMV infection, Ag-specific TET2-deficient CD8+ by guest on September 29, 2021 following acute viral infection, with both gain and loss of DNA T cells rapidly acquired surface markers associated with memory methylation at individual loci and across the genome (15); however, potential in a cell-intrinsic manner. These differences occurred the role that TET enzymes and active DNA demethylation play in early and correlated with more TET2-deficient TCM. Interestingly, CD8+ T cell fate decisions has not been fully explored. In this study, a recent report identified DNMT3A, another enzyme involved in we identify TET2 as a novel regulator of cell fate choice between DNA methylation, as a repressor of CD8+ T cell effector differ- + terminally differentiated effector CD8 TcellsandTCM formation. entiation, demonstrating that mice with a T cell–specific deletion TET expression and activity are regulated through multiple of DNMT3A have enhanced TCM differentiation following acute mechanisms in nonlymphoid cells, including protein stability via infection (56). The similarity of these phenotypes was somewhat IDAX and CXXC5 (35), ascorbic acid (51–53), and acetylation surprising at first, because DNMT3A catalyzes the de novo (54). In this study, we find that TET2 gene expression is rapidly methylation of cytosines (57), and TET2 oxidizes 5mC to 5hmC. and transiently upregulated in primary murine T cells by TCR However, loss of both genes affects HSC differentiation and self- signaling. This upregulation occurs in a Ca2+-dependent manner renewal in a similar (although not identical) manner (23–26, 58). and coincides with a decrease in CXXC5 gene expression, a Moreover, mutations in both TET2 and DNMT3A occur in an known inhibitor of TET2 protein stability (35). We also found that overlapping set of human myeloid and T cell neoplasms (59, 60), TCR signals regulate TET enzymatic activity, although the timing as well as age-related clonal hematopoiesis (61–63). Recent work of changes in 5hmC levels appeared to occur faster than changes with TET2 and DNMT3A single- and double-deficient HSCs in TET2 mRNA expression. The discordant timing of 5hmC in- identified methylated regions that were independently and inter- duction versus TET2 gene expression may be reflective of the dependently regulated (64). These observations suggest that loss sensitivity of the respective assays or indicate that TCR signals of DNMT3A or TET2 may deregulate similar pathways or an regulate TET2 transcription and TET enzymatic activity inde- overlapping set of targets. pendently of each other. Together, these data suggest that TET2 We also found that TET2-deficient memory cells have intact expression and TET activity are tightly controlled in T cells, likely expansion and effector responses and provide enhanced pathogen through multiple mechanisms, following T cell activation. clearance upon rechallenge. The increase in the TCM population in Studies of DNA methylation in T cells have focused primarily on TET2cKO mice could potentially explain the improved pathogen regulation of cytokine and effector molecule gene loci. Previous control upon antigenic rechallenge. At day 45 following LCMV- genome-wide analyses of DNA methylation during CD8+ T cell Armstrong infection, there were more central memory gp33+CD8+ differentiation revealed decreased methylation and increased gene T cells with high CD62L, CD27, and CXCR3 expression in expression at effector gene loci, including Gzmb (granzyme B) TET2cKO mice compared with WT mice. Prior studies have + + and Ifng (IFN-g), in day-8 effector CD8 T cells following LCMV demonstrated that the TCM subset (defined by CD62L ) has en- infection compared with naive T cells (15). How TET2 might hanced pathogen clearance compared with the effector memory The Journal of Immunology 9 subset (42). Additionally, TET2-deficient memory CD8+ T cells 3. Joshi, N. S., W. Cui, A. Chandele, H. K. Lee, D. R. Urso, J. Hagman, L. Gapin, and S. M. Kaech. 2007. 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Two recent studies have demonstrated a role 10. Ichii, H., A. Sakamoto, Y. Kuroda, and T. Tokuhisa. 2004. Bcl6 acts as an Downloaded from for TET2 in maintaining hypomethylation at enhancer regions amplifier for the generation and proliferative capacity of central memory CD8+ T cells. J. Immunol. 173: 883–891. (67, 68), suggesting that its action at enhancer regions can modulate 11. Huber, M., and M. Lohoff. 2014. IRF4 at the crossroads of effector T-cell fate gene expression. However, in a separate study examining TET2- decision. Eur. J. Immunol. 44: 1886–1895. knockdown cells, a similar number of genes were found to be up- 12. Wei, G., L. Wei, J. Zhu, C. Zang, J. Hu-Li, Z. Yao, K. Cui, Y. Kanno, T.-Y. Roh, W. T. Watford, et al. 2009. Global mapping of H3K4me3 and H3K27me3 reveals regulated and downregulated (66). Taken together, these studies specificity and plasticity in lineage fate determination of differentiating CD4+ highlight the complexity of TET2-regulated gene expression. 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),),) #-.)-,5-112.1)# -,5-112.0 3.,1- -- .2'35-05325 3 0 .'-053(.4 5'.4,3-(.4 ),.-/55) %#! ),),) #-.)-,5-112.1)# -,5-112.0 3.,1- -- /.'/20-/,.2 4 3 0'1.5(/2 /'/,.22(/0 ),,-,3544/) %#! ),),) #-.)-,5-112.1)# -,5-112.0 04.14 -- .2'35-05325 3 0 .'-053(.4 5'.4,3-(.4 ),,-,3544/) %#! )-),) #-.)-,5.,0,54)# -,5.,0,53 ./134 -- /.'/20-/,.2 4 3 0'1.5(/2 /'/,.22(/0 ),,530/)$)-),) #.)-1.211-2/)# -1.211-2. 121 . - ./'1.,,-,/. -- 1 -'24,1(.. /'03,34(.. ),4,3,4) %#! )-0),) #1)531,/32/) 531,/32. -.,,, . 01'/34.12.3 2 2 2'/,14(// 3'03.0.(/. ),/,-34) %#! ),),) #2)--/.13//.) --/.13//- .022 - .5'53/...0. -3 -- 4'.4/4(/- 1'.,,3-(/, )-31044) %#! )3),) #-,)5/,.425-) 5/,.425, -3-5 /4 0/'0101-5.5 1 1 .'1,51(// /'.-042(/. ),,-,4,4,4) %#! )3),) #1)--2-30322)# --2-30321 2/22 20 .0'4,/,00.3 3 0 3',.0-(.4 .'0-.20(.3 ),.224-) %#! ).2),) #-.)-,....-4,)# -,....-35 /4/.1 44 /1'5,,23012 3 2 .'40,-(// /'0522.(/. ),,33-5)$),),) #--)55,,2.3.)# 55,,2.3- -,34 3 /,',-4.0.3 -- 5 -'41,3(./ 0'-53/4(./ ),,33-5)&%#/),),) #--)55,,11-/)# 55,,11-. 32, 3 /,',-4.0.3 -- 5 -'41,3(./ 0'-53/4(./ ),,541.) %#! )--),) #-5)-,4305,/)# -,4305,. .5/52 2 /,'--505322 2 2 -',,15(/, 2'-5.-2(/, ),,-,/34,-) %#! )-,),) #-5)-,4305,/)# -,4305,. .5/52 2 /,'--505322 2 2 -',,15(/, 2'-5.-2(/, ),,--.//42)$)-),) #-/)/15,3334) /15,3333 224 .1'3.31/1-0 3 0 -'314/(/, -',2405(.5 ),,544-)$)0),) #-/)/15,3334) /15,3333 224 .1'3.31/1-0 3 0 -'314/(/, -',2405(.5 ),,-,//0-5)$)1),) #3).,002014)# .,002013 00 -2 .-'0/,0/0-. 1 / /'202/(-4 1'2.,22(-4 ),,-,//0-5) %#! )0),) #3).,000.44)# .,000.43 .-3- -2 .-'0/,0/0-. 1 / /'202/(-4 1'2.,22(-4 ),,-,//0-5)&%#1)1),) #3).,0021,-)# .,0021,, -05 -2 .-'0/,0/0-. 1 / /'202/(-4 1'2.,22(-4 ),,-,//0-5)&")1,,,) #3).,002205)# .,002204 1,,- -2 .-'0/,0/0-. 1 / /'202/(-4 1'2.,22(-4 ),,-.33-05) %#! )/),) #0)43.42-/) 43.42-. /242 3 /5',,2-,14. 1 1 .'1,51(// /'.-042(/. ),,52,-)&")1,,,) #--)252,500,)# 252,50/5 1,,- - ..'/15/3..3 5 0 2'--21(-5 5'434,4(-5 ),.5112) %#! ),),) #-0)-..14-,,0) -..14-,,/ -.502/ .5',4-/2--1 1 / -',4,.(., -'514,3(., ),,--2/.2.) %#! ),),) #4)--534-/,.) --534-/,- --5230 .5'.2005./ 5 0 -'44.1(./ 0'.05,0(./ ),,--2/.2.) %#! ),),) #4)--534-/,.) --534-/,- --5230 /.',5.532-0 3 1 .'14,4(// /'.2.-0(/. ),,--204,0) %#! )-,),) #0)02131524)# 02131523 /43.. . .2'4.0405/0 -- 0 .',5,5(/. .'/,04/(/- ),,-,.10/.) %#! )-0),) #-2)0-,5.-5)# 0-,5.-4 1112 .2'/.43,1.. 1 / 1'4321(.4 .',//-4(.3 ).,-/10) %#! ).),) #-4)312/-231)# 312/-230 033-5 /1'-1-,345. 1 0 -'/520(.3 0'22-/4(.3 ).,-/10) %#! )1),) #-4)313.03/1)# 313.03/0 0,104 /,'-,1002.1 2 0 0'5.55(-0 2',41/5(-0 )-54,-/) %#! ),),) #--)435-.402) 435-.401 3,... - 10'2/./.2-0 -, -, /'0-/2(/2 /'/,.22(/0 )-//243) %#! ),),) #-4)/154520-) /154520, .40,3 1 .,'23.-4330 5 0 -',.,1(-/ -'.0230(-/ ),.4,2-)&%#/),),) #/)-,3554245)# -,3554244 /00. 12-- ./'02/-0/54 4 0 -',-22(.- -'54/,/(.- ),,-,4-/.,)&%#/),),) #/)-,3554245)# -,3554244 /00. 12-- ./'02/-0/54 4 0 -',-22(.- -'54/,/(.- ),,-.1.0/4)$).),) #-2)341034.-)# 341034., 1-3 -203. //',40,5/11 4 3 0'1.5(/2 /'/,.22(/0 ),,-.1.00,)$).),) #-2)341034.-)# 341034., 1-3 -203. //',40,5/11 4 3 0'1.5(/2 /'/,.22(/0 ),,-.1.0/5)$).),) #-2)341034.-)# 341034., 1-3 -203. //',40,5/11 4 3 0'1.5(/2 /'/,.22(/0 ),.1523)$).),) #-2)341034.-)# 341034., 1-3 -203. //',40,5/11 4 3 0'1.5(/2 /'/,.22(/0 ),,-,///03)&%#/),),) #.)-,/54/./.)# -,/54/./- 13,3 0/,,0-,1 0.'413.343. -, -, -'/255(/2 /'/,.22(/0 ),01404)&%#1).),) #3).150---/) .150---. 1/4 5/,,.4 -0 ..'--241-13 5 / 4'051.(.0 -'5441-(./ ),01403)&%#1).),) #3).150---/) .150---. 1/4 5/,,.4 -0 ..'--241-13 5 / 4'051.(.0 -'5441-(./ )-54--0) %#! )-0),) #-5)-,//5142)# -,//5141 0,5. 0,'3/-1,5/2 1 1 .'1,51(// /'.-042(/. ),--5/.) %#! )1),) #/)-/32-.30.)# -/32-.30- --221 - /2'523/540- 1 0 /'0,2(.2 5'5/051(.2 )-5354.)$)1),) #4)42.012//) 42.012/. -., /5 ./'2525.111 -2 3 /'/23(.. 2'35-/4(.. )-5354.) %#! )0),) #4)42.01,0,) 42.01,/5 150 /5 ./'2525.111 -2 3 /'/23(.. 2'35-/4(.. )-5354.) %#! )1),) #4)42.0131.) 42.0131- /34 /5 ./'2525.111 -2 3 /'/23(.. 2'35-/4(.. ),-24--) %#! )..),) #-,)-.4-30.,-)# -.4-30.,, 221. .0'04,-.-,1 2 / 4'-2,2(-3 -'-2242(-2 ),,-,4-.,2) %#! )./),) #2)/3,,,.1/)# /3,,,.1. 3--, .3'.5.-4/0/ 2 / /'1452(.3 -'-/4--(.2 ),///30) %#! )03),) #--)/0101,45)# /0101,44 311 . //'2402,-.3 2 0 5',/,5(.0 .',44--(./ )-/0,30) %#! )-3),) #-0)-.-5504/-)# -.-5504/, .,/0 5 ..'.022/.0- -, 2 1'25./(-1 3'/,.-4(-1 ),,--.4/,3) %#! ).,),) #-0)-.-5504/-)# -.-5504/, .,/0 5 ..'.022/.0- -, 2 1'25./(-1 3'/,.-4(-1 ),,--.4/,4) %#! )-4),) #-0)-.-5504/-)# -.-5504/, .,/0 5 ..'.022/.0- -, 2 1'25./(-1 3'/,.-4(-1 ),,-,4-,/5) %#! )-4),) #-0)-.-5504/-)# -.-5504/, .,/0 5 ..'.022/.0- -, 2 1'25./(-1 3'/,.-4(-1 ),.2410) %#! )-),) #-4)0542,,0.)# 0542,,0- ././1 . 02'520,3/,2 2 2 -'.,44(/0 .'35055(// ),,--3,52,) %#! )-),) #-4)0542,,0.)# 0542,,0- .354. . 02'520,3/,2 2 2 -'.,44(/0 .'35055(// ),..,-5) %#! )-),) #-)-4142-1/.) -4142-1/- /-.,, -, 0,'-1-34,53 2 0 3'2,0(.0 -'34430(./ ),,-,41/5,)$).),) #-5)1/2--503) 1/2--502 ..- 1 /4'//1,113 1 1 2'052(/, /'2..01(.5 ),,3444)&")1,,,) #--)254,5-.4) 254,5-.3 1,,- . 01'134243/- 1 1 .'0.55(// /'.-042(/. )-55/,3) %#! ),),) #0)-/30-4.53) -/30-4.52 0.245 - .,'10-/00-- -- 1 -',.1(-2 -'01.02(-2 )-03-1-)&")1,,,) #-3)/1,/,000) /1,/,00/ 1,,- . .1'/10225.1 /, -0 -',13/(-2 -'05150(-2 )-014/,)&")1,,,) #-3)/1,/,5-5) /1,/,5-4 1,,- . .1'/10225.1 /, -0 -',13/(-2 -'05150(-2 ),,44-/)$)1),) #-,).0/3-,1-)# .0/3-,1, -1/ - 0-'2/5-,-/3 2 2 -'.,44(/0 .'35055(// ),,--02,01) %#! ),),) #-/)534/3.34) 534/3.33 .0052 -25 .4'405/.244 -- 3 2'/,,-(.3 -'5.,0(.2 ),,--,,014) %#! ),),) #-/)5340-223) 5340-222 .,-,3 -25 .4'405/.244 -- 3 2'/,,-(.3 -'5.,0(.2 ),,-,4,3,4) %#! )-3),) #.)-234.,034)# -234.,033 ..23 21 .-'./3,4,.2 -, / 2'/233(.5 /'-33-1(.4 ),-25.1) %#! )-2),) #4)-.14-,01.)# -.14-,01- -1-, 00'-1-024-0 4 3 2'41-3(// 4',-5,4(/. ),,-.34,31)$)/),) #-)3401-/.5)# 3401-/.4 --5 /,',1.50/// 2 0 -'0.32(.3 0'30420(.3 ),--4--)$).),) #-)3401-/.5)# 3401-/.4 --5 /,',1.50/// 2 0 -'0.32(.3 0'30420(.3 ),,-.34,31) %#! ).),) #-)3400,/22)# 3400,/21 -,520 /,',1.50/// 2 0 -'0.32(.3 0'30420(.3 ),,-.34,31) %#! )/),) #-)3401-003)# 3401-002 /250 /,',1.50/// 2 0 -'0.32(.3 0'30420(.3 ),--4--) %#! )-),) #-)3400,/22)# 3400,/21 -,520 /,',1.50/// 2 0 -'0.32(.3 0'30420(.3 ),--4--) %#! ).),) #-)3401-003)# 3401-002 /250 /,',1.50/// 2 0 -'0.32(.3 0'30420(.3 )-,.313) %#! )-),) #-)3400,/22)# 3400,/21 -,520 /,',1.50/// 2 0 -'0.32(.3 0'30420(.3 )-,.313) %#! ).),) #-)3401-003)# 3401-002 /250 /,',1.50/// 2 0 -'0.32(.3 0'30420(.3 )-,.314) %#! )-),) #-)3400,/22)# 3400,/21 -,520 /,',1.50/// 2 0 -'0.32(.3 0'30420(.3 )-,.314) %#! ).),) #-)3401-003)# 3401-002 /250 /,',1.50/// 2 0 -'0.32(.3 0'30420(.3 )-,.313)&%#/).),) #-)3401-/.5)# 3401-/.4 --5 /,',1.50/// 2 0 -'0.32(.3 0'30420(.3 )-,.314)&%#/).),) #-)3401-/.5)# 3401-/.4 --5 /,',1.50/// 2 0 -'0.32(.3 0'30420(.3 )-31./0) %#! ),),) #0).-414533) .-414532 04,0 02'/311231. 2 2 -'.,44(/0 .'35055(// ),-,-54)$),),) #--)252--3,0)# 252--3,/ 3. -- ..'/15/3..3 5 0 2'--21(-5 5'434,4(-5 ),,--33204) %#! )-1),) #.)/,52,434)# /,52,433 /20.. - .0'412535.0 2 / .'/-,.(.5 -'.,552(.4 ),,--33205) %#! )-/),) #.)/,52,434)# /,52,433 /20.. - .0'412535.0 2 / .'/-,.(.5 -'.,552(.4 ),-50,2) %#! )3),) #.)/,52,434)# /,52,433 /20.. - .0'412535.0 2 / .'/-,.(.5 -'.,552(.4 ),,-,/43,,) %#! )-/),) #.)/,52,434)# /,52,433 /20.. - .0'412535.0 2 / .'/-,.(.5 -'.,552(.4 )-015.3) %#! )-),) #-.)3352/1/4) 3352/1/3 -,4.2 1-'30.1,211 2 2 -'.,44(/0 .'35055(// ),-,./1)$)-),) #-5)101,-2,) 101,-15 -5/ - .0'.5,/-3 2 / 2'0-4-(-, 2'5/322(-, )-55,24) %#! )-),) #1)-0.5--.4-) -0.5--.4, -//2- - /4'2032-1/2 5 3 .'-2-3(// /'.-042(/. )-4/-42) %#! ).),) #-.)-,,1.51-.)# -,,1.51-- 1,-.- / 0,'2-3..340 3 2 -',521(// .'-2112(/. ),1/.,.) %#! )-/),) #2)5452222,)# 54522215 15-45 - //'4412.2-- 1 0 4'0034(.2 .'/2.0(.1 ),,--53/.-) %#! )-/),) #2)5452222,)# 54522215 15-45 - //'4412.2-- 1 0 4'0034(.2 .'/2.0(.1 ),,--53/..)&")1,,,) #2)54534.5/)# 54534.5. 1,,- - //'4412.2-- 1 0 4'0034(.2 .'/2.0(.1 )-03..-) %#! )/),) #0)-,3.0--,3) -,3.0--,2 0203- - .3'/35030,. -, 2 -'.5,5(.1 /'103.0(.1 ),////.) %#! ),),) #--)1/15541.)# 1/15541- .3..3 -..-2 ./'-2501,,0 5 0 -'53/5(-5 /'.33-0(-5 )-4-0.-)$)3.),) #1)-.-4-/31.) -.-4-/31- -21 -14,, /-'3-03/11- 2 / 3'/-.4(.0 -'3.01-(./ ),,--012/3) %#! )1),) #0)--2444-23)# --2444-22 -.3// -22- /0'000-202/ 2 1 .'/41(/. .'15442(/- ),,-,//330) %#! ),),) #0)--2444-23)# --2444-22 -.3// -22- /0'000-202/ 2 1 .'/41(/. .'15442(/- ),01/..) %#! ),),) #-5)03143054) 03143053 .12 -5113 .-'.,-21435 4 0 /',352(,4 /'...0-(,4 ),01/..)&%#1),),) #-5)0314303.) 0314303- .3 -5113 .-'.,-21435 4 0 /',352(,4 /'...0-(,4 ),,-,,0-20) %#! )-),) #-,)4342/04/) 4342/04. ---35 /1'-.4352/. 4 3 .',21.(/1 5'34451(/0 )-3.324)&")1,,,) #5)0,.2//1,)# 0,.2//05 1,,- - .3'4--0/1.4 2 0 -'.-23(.3 0'--512(.3 ),-,/21) %#! )-,),) #1)-/03.-,1/)# -/03.-,1. -/.4 . .4'210.4,.- 2 0 -'.,44(/0 .'35055(// ),,-,4,303) %#! )-,),) #1)-/03.-,1/)# -/03.-,1. -/.4 . .4'210.4,.- 2 0 -'.,44(/0 .'35055(// ),,-,4,302) %#! )-,),) #1)-/03.-,1/)# -/03.-,1. -/.4 . .4'210.4,.- 2 0 -'.,44(/0 .'35055(// ),,-,4,305) %#! )-,),) #1)-/03.-,1/)# -/03.-,1. -/.4 . .4'210.4,.- 2 0 -'.,44(/0 .'35055(// ),,-,4,304) %#! )-,),) #1)-/03.-,1/)# -/03.-,1. -/.4 . .4'210.4,.- 2 0 -'.,44(/0 .'35055(// ),-,/24)$)0),) #1)-/,03/21,)# -/,03/205 -04 ..'3,/1530 -- 0 -',414(-0 -'/30/-(-0 ),-,/24)$)1),) #1)-/,03/443)# -/,03/442 -4, ..'3,/1530 -- 0 -',414(-0 -'/30/-(-0 ),-,/24) %#! )/),) #1)-/,03-0,.)# -/,03-0,- ..05 ..'3,/1530 -- 0 -',414(-0 -'/30/-(-0 ),-,/24) %#! )0),) #1)-/,03/353)# -/,03/352 5- ..'3,/1530 -- 0 -',414(-0 -'/30/-(-0 ),,-.,0.34) %#! )..),) #-1)5320.,2,)# 5320.,15 /.034 3 .4'0/113/24 3 0 -'.23-(.. .'21321(.. ),,-.,0.35) %#! )..),) #-1)5320.,2,)# 5320.,15 /.034 3 .4'0/113/24 3 0 -'.23-(.. .'21321(.. ),,-.,0.4,) %#! ).,),) #-1)5320.,2,)# 5320.,15 /.034 3 .4'0/113/24 3 0 -'.23-(.. .'21321(.. ),,-.,0.33) %#! )./),) #-1)5320.,2,)# 5320.,15 -53/- 3 .4'0/113/24 3 0 -'.23-(.. .'21321(.. ),,-.,0.32) %#! )./),) #-1)5320.,2,)# 5320.,15 -1-,3 3 .4'0/113/24 3 0 -'.23-(.. .'21321(.. ),,-.,0.31) %#! )./),) #-1)5320.,2,)# 5320.,15 /.034 3 .4'0/113/24 3 0 -'.23-(.. .'21321(.. ),-513.) %#! )..),) #-1)5320.,2,)# 5320.,15 -53/- 3 .4'0/113/24 3 0 -'.23-(.. .'21321(.. ),,-.,0.4-)&")1,,,) #-1)53201.51)# 53201.50 1,,- 3 .4'0/113/24 3 0 -'.23-(.. .'21321(.. )-02,,-) %#! ).5),) #1)-/15/2/0-)# -/15/2/0, 40023 - .,'-3.510.. 4 / .',-5.(.3 2'1-,53(.3 ),-,0/3) %#! ),),) #-,)-/2421,0) -/2421,/ 55442 . /2'4123.11- 2 1 -'4/0/(/- -'2,,14(/, ),-/4.,) %#! )-1),) #2)4.255//.)# 4.255//- -,323 . .1',0-...// 4 0 /'.2--(.4 -'-1342(.3 ),-/4.,) %#! )-2),) #2)4.3-,.2-)# 4.3-,.2, -/21. . 01'31..4,13 2 2 -'---2(/0 .'35055(// ),.31.-)$)-),) #-,)3504/0/1) 3504/0/0 -01 - ./'102-5/14 1 / -'/3-1(-3 .',031.(-3 ),,--0.3,-)$).),) #-,)3504/0/1) 3504/0/0 -01 - ./'102-5/14 1 / -'/3-1(-3 .',031.(-3 ),.31.-) %#! )-),) #-,)3504/135) 3504/134 --4 - ./'102-5/14 1 / -'/3-1(-3 .',031.(-3 ),,--0.3,-) %#! ).),) #-,)3504/135) 3504/134 --4 - ./'102-5/14 1 / -'/3-1(-3 .',031.(-3 )-/0-,5) %#! ).),) #-2)/23,53-1) /23,53-0 143/ - .-'/,,0/-/5 -, 0 /'1,-3(/- .'.3/25(/, )-3.-2-)$)0),) #2)--/2..3//) --/2..3/. .,. . //'/15./21. 2 / .'.2/-(.1 2',5041(.1 )-3.-2-)$)1),) #2)--/2./,-3) --/2./,-2 22 . //'/15./21. 2 / .'.2/-(.1 2',5041(.1 ),,---/11/)$)/),) #2)--/2..3//) --/2..3/. .,. . //'/15./21. 2 / .'.2/-(.1 2',5041(.1 ),,---/11/)$)0),) #2)--/2./,-3) --/2./,-2 22 . //'/15./21. 2 / .'.2/-(.1 2',5041(.1 )-3.-2-) %#! 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)0),) #1)-/51.233-)# -/51.233, --3/4 - .3'10,,513 1 0 /'-15-(-1 0'-.1,5(-1 )-3.22-) %#! ),),) #.)/.,,24-.) /.,,24-- /--23 . 0-'.,324/20 4 4 0'1.5(/2 /'/,.22(/0 ),,--152/0) %#! ),),) #.)/.,,24-.) /.,,24-- /--23 . 0-'.,324/20 4 4 0'1.5(/2 /'/,.22(/0 ),--.,-)&")1,,,) #.)-2331.4.3) -2331.4.2 1,,- - 02'5-0//0.. 2 2 -'.,-5(/0 .'35055(// )-33/31)$)3),) #-3).0225-13)# .0225-12 --. .2 /.'/,4113-0 3 2 0'-3/.(/1 -'44/5.(// )-33/31) %#! )2),) #-3).022333/)# .022333. -/41 .2 /.'/,4113-0 3 2 0'-3/.(/1 -'44/5.(// ),,--22/55)$)/),) #4)3/.4-,/3) 3/.4-,/2 -.2 / ./'-,011,4- 2 / -'425.(.0 0'2434(.0 ),,5,,-)$)/),) #4)3/.4-,/3) 3/.4-,/2 -.2 / ./'-,011,4- 2 / -'425.(.0 0'2434(.0 ),,--22/55) %#! ).),) #4)3/.4,021) 3/.4,020 13/ / ./'-,011,4- 2 / -'425.(.0 0'2434(.0 ),,5,,-) %#! ).),) #4)3/.4,021) 3/.4,020 13/ / ./'-,011,4- 2 / -'425.(.0 0'2434(.0 ),,5,-0) %#! )2),) #-.)4,0.4/5.) 4,0.4/5- //,0., 1- 02'2/-05,54 1 1 .'1,51(// /'.-042(/. ),,5,-0) %#! )2),) #-.)4,0.4/5.) 4,0.4/5- //,0., 1- 0/',35/32/. 2 2 -'.,44(/0 .'35055(// ),,-.1.12.) %#! )2),) #-.)4,0.4/5.) 4,0.4/5- -4,/,1 1- 02'2/-05,54 1 1 .'1,51(// /'.-042(/. )-31.--) %#! )--),) #.)//,/,,25)# //,/,,24 415/, - /0',/2--1/2 1 1 .'.5/.(// /'.-042(/. ),--.23)&%#/)0),) #-)-1115,542) -1115,541 -2-0 -2 .1'5102.4,3 1 0 .'0543(// /'.-042(/. )-3.13.) %#! )-3),) #--)--203-350)# --203-35/ -2.1. . /5'1/2,24/4 3 1 -'./20(.3 0'-3--.(.3 ),,--2324,) %#! )-3),) #--)--203-350)# --203-35/ -/255 . /5'1/2,24/4 3 1 -'./20(.3 0'-3--.(.3 ),--44.)&")1,,,) #-)-1115-112) -1115-111 1,,- .1'5102.4,3 1 0 .'0543(// /'.-042(/. )-//5//)$)2),) #2)44,1,2./) 44,1,2.. -0, - .,'.3505.0- -, 0 .'24/1(-1 /'1./1.(-1 ),.4.00)$)-,),) #-3)/.-5.,1,) /.-5.,05 .- - .3'.0,10,3 3 0 4'-224(.. -'2,515(.- ),,--2/3/0)$)3),) #-3)/.-5.,1,) /.-5.,05 .- - .3'.0,10,3 3 0 4'-224(.. -'2,515(.- ),.4.00) %#! )5),) #-3)/.-5,5,,) /.-5,455 --1- - .3'.0,10,3 3 0 4'-224(.. -'2,515(.- ),.4.00) %#! )-,),) #-3)/.-5.,3,) /.-5.,25 343 - .3'.0,10,3 3 0 4'-224(.. -'2,515(.- ),,--2/3/0) %#! )2),) #-3)/.-5,5,,) /.-5,455 --1- - .3'.0,10,3 3 0 4'-224(.. -'2,515(.- ),,--2/3/0) %#! 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