Augmentation in Expression of Activation-Induced Differentiates Memory from Naive CD4+ T Cells and Is a Molecular Mechanism for Enhanced Cellular This information is current as Response of Memory CD4+ T Cells of September 25, 2021. Kebin Liu, Yu Li, Vinayakumar Prabhu, Lynn Young, Kevin G. Becker, Peter J. Munson and Nan-ping Weng J Immunol 2001; 166:7335-7344; ; doi: 10.4049/jimmunol.166.12.7335 Downloaded from http://www.jimmunol.org/content/166/12/7335

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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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Augmentation in Expression of Activation-Induced Genes Differentiates Memory from Naive CD4؉ T Cells and Is a Molecular Mechanism for Enhanced Cellular Response of Memory CD4؉ T Cells

Kebin Liu,* Yu Li,* Vinayakumar Prabhu,† Lynn Young,† Kevin G. Becker,‡ Peter J. Munson,† and Nan-ping Weng1*

In an attempt to understand the molecular basis for the immunological memory response, we have used cDNA microarrays to measure expression of human memory and naive CD4؉ T cells at rest and after activation. Our analysis of 54,768 cDNA ؉

clones provides the first glimpse into patterns of memory and naive CD4 T cells at the genome-scale and reveals Downloaded from .several novel findings. First, memory and naive CD4؉ T cells expressed similar numbers of genes at rest and after activation Second, we have identified 14 cDNA clones that expressed higher levels of transcripts in memory cells than in naive cells. Third, we have identified 135 (130 known genes and 5 expressed sequence tags) up-regulated and 68 (42 known genes and 26 expressed -sequence tags) down-regulated cDNA clones in memory CD4؉ T after in vitro stimulation with anti-CD3 plus anti-CD28. Inter estingly, the increase in mRNA levels of up-regulated genes was greater in memory than in naive CD4؉ T cells after in vitro ؉ stimulation and was higher with anti-CD3 plus anti-CD28 than with anti-CD3 alone in both memory and naive CD4 T cells. http://www.jimmunol.org/ Finally, the changes in expression of actin and genes identified by cDNA microarrays were confirmed by Northern and analyses. Together, we have identified ϳ200 cDNA clones whose expression levels changed after activation and suggest that the level of expression of up-regulated genes is a molecular mechanism that differentiates the response of memory from naive .CD4؉ T cells. The Journal of Immunology, 2001, 166: 7335–7344

he phenomenon that the immune system can remember CD4ϩ T cells require the engagement not only of TCR as signal 1, the identity of a pathogen and respond to it at an accel- but also a costimulatory receptor, such as CD28, as signal 2 for a erated speed and with greater strength in a successive en- complete activation leading to proliferation and differentiation into

T by guest on September 25, 2021 counter is known as immunological memory and has been used as helper T cells (15, 17–19). Stimulation with signal 1 in the absence the basis for vaccination and/or immunization for over two centu- of signal 2 may lead naive T cells into an unresponsive or anergic ries (1). At the cellular level, immunological memory is carried by state (20). In contrast, memory CD4ϩ T cells appear to have a ϩ ϩ memory CD4 and CD8 T and B that are derived lower activation threshold than naive T cells. Depending on the in from Ag-naive lymphocytes after antigenic activation (2, 3). The vitro system, memory CD4ϩ T cells can be either fully activated function of memory CD4ϩ T cells, unlike memory B (4) and ϩ (13) or partially activated by signal 1 alone (15, 17, 21, 22). Once CD8 T cells (5), is to produce that in turn influence the activated, memory and naive CD4ϩ T cells display quite different functions of effector cells and the outcome of an immune response ϩ ϩ cellular responses (6, 23). Memory CD4 T cells produce a (6). Recent studies have identified memory and naive CD4 T broader spectrum and a greater quantity of cytokines including cells based on the differential expression of CD45 isoforms ␣ ␥ ␣ ϩ IL-1 , IL-2, IL-4, IL-5, IL-6, IL-17, IFN- , TNF- , and GM-CSF, (CD45RA and CD45R0 for naive and memory CD4 T cells, re- ϩ whereas naive CD4 T cells produce only IL-1␣, IL-2, IFN-␥, and spectively) and other markers (7–10) and have characterized sev- TNF-␣ (24–29). eral functional differences between these two subsets of CD4ϩ T At the molecular level, information regarding the difference be- cells (11, 12). ϩ tween memory and naive CD4 response is limited (30). The well-documented differences between human memory and ϩ With the development of DNA microarray technology, the sys- naive CD4 T cells include the requirements for activation and the tematic analysis of gene expression has become feasible (31, 32). magnitude of the subsequent cellular responses (13–16). Ag-naive Two recent reports have analyzed gene expression in murine T cells and human CD4ϩ T cell clones using DNA arrays (33, 34). *Laboratory of Immunology, National Institute on Aging, National Institutes of Teague et al. (33) studied murine T cells after activation with a Health, Baltimore, MD 21224; †Mathematical and Statistical Computing Laboratory, in vivo. They reported that resting T cells express Center of Information Technology, National Institutes of Health, Bethesda, MD 20892; and ‡DNA Array Unit, National Institute on Aging, National Institutes of about the same number of genes as activated T cells and that ac- Health, Baltimore, MD 21224 tivation via superantigen induced expression of 51–280 genes in ϩ Received for publication October 20, 2000. Accepted for publication April 12, 2000. murine T cells. Rogge et al. (34) analyzed human CD4 Th1 and The costs of publication of this article were defrayed in part by the payment of page Th2 clones in vitro and showed that Th1 and Th2 clones expressed charges. This article must therefore be hereby marked advertisement in accordance different sets of genes that could in turn modulate effector func- with 18 U.S.C. Section 1734 solely to indicate this fact. tions. However, analysis of the gene expression profile of memory 1 Address correspondence and reprint request to Dr. Nan-ping Weng, Laboratory of ϩ ϩ Immunology, National Institute on Aging, National Institutes of Health, 5600 Nathan CD4 T cells and its comparison with naive CD4 T cells at the Shock Drive, Box 21, Baltimore, MD 21224. E-mail address: [email protected] genome scale has not been reported.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 7336 GENE EXPRESSION IN MEMORY AND NAIVE CD4ϩ T CELLS

Here we report a genome-scale analysis of gene expression in RNA isolation and cDNA probe preparation ϩ human memory and naive CD4 T cells after analyzing 54,768 Total RNA was extracted from memory and naive CD4ϩ T cells using unique cDNA clones. We demonstrate that memory and naive STAT-60 RNA isolation solution (Tel-Test, Friendswood, TX). Messenger ϩ CD4 T cells have a similar pattern of gene expression at rest and RNA was isolated from the total RNA by oligo(dT) beads (Dynal) and used after in vitro stimulation with anti-CD3 mAb alone or with anti- for making cDNA probe. In general, mRNA (0.6 ␮g) was mixed with 1 ␮l 50 ␮M oligo(dT) (Research Genetics, Huntsville, AL), incubated at 70°C CD3 plus anti-CD28 (anti-CD3/CD28) mAbs. At the individual 18 for 10 min, frozen on dry ice, and lyophilized to dryness. The dried mRNA gene level, we have identified 14 cDNA clones that were highly ϩ ϩ and oligo(dT) mixtures were dissolved in reserve transcription solution expressed in memory CD4 T cells relative to naive CD4 T cells containing 1ϫ Moloney murine leukemia virus buffer, 571 ␮m each of and ϳ200 cDNA clones whose expression levels were changed in dATP, dGTP, and dTTP, 40 ␮Ci [␣-33P]dCTP (2000 Ci/mmol, 10 ␮Ci/␮l), memory CD4ϩ T cells after in vitro anti-CD3/CD28 stimulation. and 240 U of Moloney murine leukemia virus reverse transcriptase (Pro- mega, Madison, WI) and incubated at 42°C for 2 h. To degrade RNA, 39.5 Although the mRNA levels of the down-regulated genes are di- ␮l water and 12.5 ␮l 1 N NaOH were added to the reaction mixture and minished rather uniformly, the levels of up-regulated genes are incubated at 37°C for 10 min and then neutralized by adding 12.5 ␮l1M higher in memory than in naive cells and are higher after stimu- Tris-HCl (pH 8.0) and 10 ␮l 1 N HCl. The unincorporated nucleotides lations with anti-CD3/CD28 than with anti-CD3 alone. Taken to- were removed from the probe by a G-50 spin column (Bio-Rad, Hercules, gether, these results provide a general assessment of activation- CA). The labeled cDNA probes were heated at 100°C for 3 min, quickly cooled on ice, and used immediately. induced changes in gene expression in memory and naive CD4ϩ T cells and suggest that the increased expression of certain genes cDNA microarray experiments using commercial filters ϩ defines memory CD4 T cell response. The gene discovery array (GDA)2 human filters version 1.3 consisting of two 22 ϫ 22-cm and one 11 ϫ 22-cm nylon filter were purchased from Downloaded from Materials and Methods Incyte Genomics (St. Louis, MO). The filters contain a total of 45,878 ϩ nonredundant human cDNA clones spotted in duplicate. Hybridization was Isolation of memory and naive CD4 T cells from peripheral conducted according to manufacturer’s instruction. In brief, filters were blood prehybridized in 35 ml (for 22 ϫ 22-cm filters) or 17.5 ml (for 11 ϫ 22-cm Human memory and naive CD4ϩ T cells were isolated from peripheral filter) Quickhyb hybridization solution (Stratagene, La Jolla, CA) at 43°C for2hinroller bottles and hybridized in the presence of sheared salmon blood by immunomagnetic separation as previously described (12). In ␮ ␮ brief, blood was obtained from normal donors of the National Institutes of sperm DNA (115 g/ml) (Research Genetics), Cot1 DNA (0.1 g/ml) http://www.jimmunol.org/ Health Blood Bank. PBMC were isolated by Ficoll gradient centrifugation (Life Technologies), and cDNA probes at 43°C for 12 h. cDNA probes prepared from 1.8 and 0.9 ␮g mRNA were used for the 22 ϫ 22-cm and (Organon Teknika, Durham, NC), and then were incubated with a panel of ϫ ϫ mouse mAbs, including mAbs against CD8 (B9.8), CD19 (FMC63), 11 22-cm filters, respectively. Filters were washed twice with 200 ml 2 SSC/0.1% SDS at 22°C for 30 min, once with 1ϫ SSC/0.1% SDS at 65°C CD11b (NIH11b-1), CD14 (63D3), CD16 (3G8), MHC class II (IVA12), ϫ erythrocytes (glycophorin, 10F7) and platelets (37F9-E7). mAbs against for 30 min, and twice with 0.6 SSC/0.1% SDS at 65°C for 30 min. The CD45RA (FMC71) or CD45R0 (UCHL-1) were added reciprocally for filters were then exposed to PhosphorImager screens (Molecular Dynam- isolation of memory or naive CD4ϩ T cells. Ab-bound cells were subse- ics, Sunnyvale, CA) for 20 h, and the images were collected by a Phos- quently removed by incubation with anti-mouse IgG-conjugated magnetic phorImager scanner (Storm 860; Molecular Dynamics). The human Gene- beads (Polysciences, Warrington, PA). The purity of isolated memory and Filters (GF200, GF201, and GF202) containing a total of 16,056 cDNA naive CD4ϩ T cells was generally 90–95% determined by FACS analysis. clones in each filter were purchased from Research Genetics. Hybridization by guest on September 25, 2021 Among the contaminating cells, CD45RAϩCD45ROϩ made up 3–5%, and was conducted according to the manufacturer’s instruction and was under each of CD8ϩ, CD14ϩ, CD19ϩ, CD40ϩ, and CD83ϩ cells made up Ͻ1%. similar conditions to those described above. cDNA probes prepared from 0.6 ␮g mRNA were used for each hybridization based on the size of the ϩ filters. Stimulation of memory and naive CD4 T cells in vitro The procedures for stimulation of memory and naive CD4ϩ T cells were Custom-made cDNA microarray filters previously described (35). Anti-CD3 (OKT3) alone or anti-CD3/CD28 A total of 2,878 cDNA clones were selected for constructing the custom- (9.3) mAb-conjugated magnetic beads (Dynal, Lake Success, NY) were made cDNA microarray. cDNA clones were obtained from Incyte Genom- gifts from C. June (University of Pennsylvania, Philadelphia, PA). Memory ics and Research Genetics and were cloned in this laboratory. Plasmids ϩ ϫ 6 and naive CD4 T cells were resuspended at 2–5 10 cells/ml in RPMI were isolated from bacteria with an alkaline lysis (Edge BioSystems, 1640 medium (Life Technologies, Rockville, MD) supplemented with 10% Gaithersburg, MD). The insert of cDNA clones were amplified by PCR ϫ FBS (Gemini Biological Products, Calabasas, CA) and 1 penicillin-strep- using a pair of common primers, universal forward (5Ј-CTGCAAGGC tomycin (Life Technologies), mixed with Ab-conjugated beads at a 1:1 GATTAAGTTGGGTAAC-3Ј) and universal reverse (5Ј-GTGAGCG cell/bead ratio and were incubated for 16 h or other defined time points GATAACAATTTCACACAGGAAACAGC-3Ј), precipitated with ethanol before harvest. and resuspended in Tris-EDTA buffer. The PCR products were denatured by NaOH (0.1 N) and spotted onto nylon membrane (Nytran Supercharge Proliferation assay membrane; Schleicher & Schull, Keene, NH) by GMS 417 Arrayer (Affy- ␮ ϩ ϫ 5 metrix, Santa Clara, CA). The pin size (diameter) of the arrayer is 300 m, Memory and naive CD4 T cells were cultured at 5 10 cells/well in ␮ quadruplicate in 96-well flat-bottom plates (Falcon, Franklin Lake, NJ) in and the space between the centers of two spots is 665 m. Each cDNA 0.2 ml RPMI 1640 medium/10% FBS/1ϫ penicillin-streptomycin and un- clone was spotted in duplicate onto each membrane. The printed filters der the stimulation conditions as described above over a 5-day period. Then were treated with UV cross-linking (Stratagene) for immobilizing DNA to 1 ␮Ci of [3H]thymidine was added to each well at 0, 24, 48, 72, and 96 h the membrane. after stimulation. After incubation for another 20–22 h, the cells were then Analysis of microarray results by P-SCAN harvested at day 1, 2, 3, 4, and 5 by a 96-well plate harvester (Tomtec, Orange, CT), and the incorporation of [3H]thymidine into DNA was quan- Image files were collected from the PhosphorImager in “gel” format and tified using a liquid scintillation counter (Beckman Coulter, Fullerton, CA). were processed using the P-SCAN analysis software as described (36) and freely available at http://abs.cit.nih.gov/pscan. Briefly, the hybridization Analysis of cell division by cell tracking dye (CFSE) spots on the image of the microarray were located, and the average image intensity was determined for the pixels within a circle of fixed radius ϩ ␮ Memory and naive CD4 T cells were cultured with 10 M CFSE (Mo- around the spot. These numerical intensities were saved in a file along with ϫ 6 lecular Probes, Eugene, OR) in RPMI 1640 at 2 10 cells/ml at 37°C for array coordinates and merged with the “gene list.” The analysis steps in- 10 min. Cells were washed with 10 volumes of cold PBS/0.1%BSA, re- cluded a normalization of spot intensity by dividing by the median spot suspended in RPMI 1640 medium, and incubated at 37°C for 30 min before intensity for the entire image and the determination of relatively over- and stimulation with anti-CD3/CD28 as described above for 5 days. Cells were collected from a FACScan (BD Biosciences, San Jose, CA), and the CFSE profiles were analyzed by CellQuest (BD Biosciences) and ModFit (Verity 2 Abbreviations used in this paper: GDA, gene discovery array; EST, expressed se- Software House, Topsham, ME). quence tag; FI, fluorescence intensity. The Journal of Immunology 7337 underexpressed genes between two experimental conditions. For the GDA Results filters, a 2-fold cutoff was established to select interesting spots whose Experimental design for gene expression profiles of human intensities have changed between two images; one of which must also ϩ show an intensity above the background level (approximated by the median memory and naive CD4 T cells intensity). Resulting lists of spots were then reviewed graphically to verify Our strategy to assess the transcriptional nature of memory and that the apparent spot intensities had, in fact, changed between the two ϩ original images. naive CD4 T cells is shown in Fig. 1. We have applied the cDNA The custom-made cDNA microarrays consisting of three filters were microarray method with commercial filters for the initial genome- analyzed using the same methods as above but using the “custom array” scale screening and custom-made filters for detailed analysis of option of P-SCAN. Each array contained two spots corresponding to each activation-induced gene expression changes. We then used con- clone, an internal replicate that was used for analyzing the consistency of ventional methods (Northern and Western blots) for confirmation the results. A database of microarray data was created using Matlab (ver- sion 5.2; Mathworks, Natick, MA) for results of the custom-made microar- of array results. The commercial filters consisted of a total of ray. This database contained a GenBank identifier for each clone, the lo- 54,768 nonredundant cDNA clones that were obtained from two cation on the array of each clone, and the numerical measured intensity for sources: 1) GDA human filters (GDA version 1.3; Incyte Genom- each experiment using these microarrays. It also contained coded informa- ics; 45,968 clones), and 2) GeneFilters (GF200, GF201, and tion about the experiment type (experiment number, cell type, cell pre- treatment, time after treatment, replicate number, internal replicate number, GF202; Research Genetics; 16,056 clones including 7,166 over- etc.). Using this database, it was possible to quickly test a variety of nor- lapping clones with the GDA filters). The custom-made filter ar- malization strategies and to select subsets of clones that met certain criteria. rays consisted of 2,878 cDNA clones selected from either the com- An initial subset was determined by requiring that the median change in mercial filters or genes with known immunological functions that normalized intensity of each clone from two independent hybridization were not on the commercial filters. experiments was Ͼ1.6-fold compared with the unstimulated condition. ϩ Downloaded from These selected clones were then presented in a color-coded (red ϭ intensity Memory and naive CD4 T cells were isolated from peripheral increase, green ϭ intensity decrease, white ϭ no change) array, and in- blood of normal donors. The proliferative response of purified consistently colored clones (indicating inconsistent expression ratios) were memory and naive CD4ϩ T cells under in vitro stimulation con- removed from the study. ditions was assessed. As expected, anti-CD3 stimulation induced a low degree of proliferation in memory but not in naive CD4ϩ T Northern blot analysis cells, and anti-CD3/CD28 induced rapid and strong proliferation in

ϩ http://www.jimmunol.org/ Northern blotting was conducted as described previously (37). The probes both memory and naive CD4 T cells as measured by the incor- were PCR products of selected cDNA clones and labeled by random prim- poration of [3H]thymidine (Fig. 2A). The profiles of the CFSE ing using Ready to Go DNA labeling beads (Amersham Pharmacia Bio- experiment showed that memory and naive CD4ϩ T cells divided tech, Piscataway, NJ) in the presence of [␣-32P]dCTP (New England Nu- clear, Boston, MA). Prehybridization and hybridization were conducted at 55°C for 1 and 16 h, respectively. The washing includes twice with 2ϫ SSC/0.1% SDS at 22°C for 15 min, once with 1ϫ SSC/0.1% SDS at 65°C for 30 min, and twice with 0.5ϫ SSC/0.1% at 65°C for 30 min. The filters were exposed to PhosphorImager screens, and the time of exposure was optimized for each gene. The images were collected from the Phosphor Imager scanner (Storm 860; Molecular Dynamics). by guest on September 25, 2021

Western blot analysis Cells were lysed in Kyriakis lysis buffer (20 mM HEPES, pH 7.4, 50 mM ␤-glycerophosphate, 10 mM sodium fluoride, 1% Triton X-100, 10% glyc- erol, 1 mM sodium orthovanadate, 10 ␮g/ml leupeptin, 10 ␮g/ml aprotinin, and 100 ␮g/ml [4-(2-aminoethyl)-benzenesulfonylfluoride hydrochlo- ride]). Lysates from ϳ1 ϫ 106 cells were loaded to each well and separated by SDS-PAGE. were transferred to Immobilon-P membranes (Millipore, Bedford, MA). The membranes were probed with anti-actin Ab (University of Iowa, Iowa City, IA), washed three times, and incubated with donkey anti-rabbit Ig HRP-linked whole Ab (Amersham Pharmacia Biotech). Signals were detected using the ECLPlus detection system (Am- ersham Pharmacia Biotech) according to manufacturer’s instructions. The membranes were stripped and probed again with anti-ZAP70 Ab (a gift from Ron Wange, National Institute on Aging, National Institutes of Health, Baltimore, MD).

Analysis of F-actin by flow cytometry Freshly isolated and stimulated memory and naive CD4ϩ T cells were fixed in 3.7% formaldehyde in PBS at 4°C overnight (or up to 1 wk). The fixed cells were washed twice in PBS at room temperature and incubated with 0.1% Triton X-100 in PBS for 5 min, then washed three times with PBS, and resuspended in 500 ␮l PBS containing 1% BSA (ICN Biomedicals, Costa Mesa, CA). To quantify the polymerized actin (F-actin) level, cells were stained with Alexa Fluor 488 phalloidin (Molecular Probes) and an- alyzed by flow cytometry

Cytokine assays Supernatants were collected from in vitro-stimulated memory and naive CD4ϩ T cells. IL-2, IL-4, IL-5, IFN-␥, TNF-␣, and TNF-␤ concentrations were determined using ELISA immunoassay kit (BioSource International, Camarillo, CA, and R&D Systems, Minneapolis, MN) according to the FIGURE 1. Experimental scheme of gene expression analysis of human manufacturer’s instructions. memory and naive CD4ϩ T cells by cDNA microarray. 7338 GENE EXPRESSION IN MEMORY AND NAIVE CD4ϩ T CELLS Downloaded from

FIGURE 2. Profiles of cell proliferation and activation marker (CD69 and CD25) expression in memory and naive CD4ϩ T cells after stimulation with ϩ anti-CD3 or with anti-CD3/CD28. A, Profiles of [3H]thymidine incorporation of memory and naive CD4 T cells after in vitro stimulation. B, Percentages http://www.jimmunol.org/ of memory and naive CD4ϩ T cells at a particular number of divisions after 5 days stimulation with anti-CD3/CD28. Memory and naive CD4ϩ T cells were stimulated in the presence of CFSE for 5 days. The CFSE profiles were collected by FACS, and analyzed by ModFit. The cell division times are indicated at the top and the percentage of cells that had divided certain times is indicated at the bottom of each figure. C, The percentage of memory and naive CD4ϩ T cells that express CD69 or CD25 after stimulation. D, Fluorescence intensity (FI) of CD69- or CD25-expressing memory and naive CD4ϩ T cells. The FI of CD69-expressing cells stimulated with both anti-CD3 and anti-CD3/CD28 for 16 h and CD25-expressing cells stimulated with anti-CD3/CD28 for 16 h are presented. The open area represents the FI of memory CD4ϩ T cells, and the filled area represents the FI of naive CD4ϩ T cells. All experiments were conducted with cells from three different donors, and the representative results from one donor are shown. by guest on September 25, 2021 a maximum of six times following anti-CD3/CD28 stimulation for that the expressed gene must be at least 2-fold higher than the median 5 days (Fig. 2B). Almost all memory (98.6%) and naive cells intensity of all clones on the filter and clearly visible over the back- (99.8%) entered cell cycle after stimulation. However, memory ground. The results were derived from two independent experiments cells had a greater number of cells in division 5 and 6 than naive withquadruplicatedataforeachcloneandarelistedinhttp://www.grc.nia. cells (Fig. 2B). nih.gov/branches/li/weng/arraydata.htm. Comparative analyses of The optimal time after in vitro stimulation for gene expression anal- gene expression using commercial and subsequently custom-made ysis was determined by the level of induced expression of two acti- filters revealed that the difference in gene expression between freshly vation markers (CD69 and CD25) in memory and naive CD4ϩ T isolated memory and naive CD4ϩ T cells was very small and that cells. After anti-CD3 stimulation, the percentage of CD69ϩ memory significant changes of gene expression were found after stimulation in and naive CD4ϩ T cells reached plateau around 16–48 h. However, both memory and naive CD4ϩ T cells. memory cells had a higher percentage of CD69-expressing cells ϩ (76.6%) than in naive (50.5%) populations at their respective peak Genes highly expressed in memory CD4 T cells (Fig. 2C). No CD25 expression was detected in either memory or After a sequential analysis of gene expression using the commer- ϩ naive CD4 T cells after anti-CD3 stimulation (data not shown). Af- cial and the custom-made filters, we identified 14 cDNA clones (11 ter anti-CD3/CD28 stimulation, the kinetics of CD69 expression was known genes and 3 expressed sequence tags (ESTs)) that were similar to that after anti-CD3 stimulation, and the percentage of CD25 expressed at consistently higher levels in memory cells than in expressing cells approached a plateau 16 h after stimulation (Fig. 2C). naive cells (Fig. 3A). Among the 11 known genes, CD69, cyclin- Therefore, we selected 16 h after stimulation for the initial analysis of dependent kinase inhibitor 1B, and protein 4 are ϩ activation-induced genes in memory CD4 T cells. Interestingly, al- involved in T cell activation and cell cycle regulation (38–40). ϩ ␤ though memory and naive CD4 T cells exhibited a similar percent- Integrin 5, proteoglycan 1, and annexin A1 function in cell-cell age of surface expression of CD69 and CD25 after stimulation, the and cell-matrix interaction, which could contribute to intensity levels of CD69 and CD25 were higher in memory than in migration and activation processes (41–43). The functional impor- ϩ naive CD4 T cells (Fig. 2D). tance of the other genes and ESTs in memory cell function is not clear and will require further study. Upon in vitro stimulation with Genome-scale gene expression profiles of memory and naive ϩ anti-CD3/CD28 for 16 h, the expression of four cDNA clones was CD4 T cells at rest and after in vitro stimulation up-regulated (proteoglycan 1, protein kinase inhibitor, cytochrome At the genome-scale level, memory and naive CD4ϩ T cells ex- c, and one EST) (Figs. 3B and 4A) and three clones were down- pressed similar numbers of genes at rest (22 and 20%), after stimu- regulated (annexin A1, cyclin-dependent kinase inhibitor 1B, and lation with anti-CD3 alone (28 and 27%), and with anti-CD3/CD28 one EST) (Figs. 3B and 4B) in both memory and naive CD4ϩ T (19 and 20% of total cDNA clones) based on a conservative estimate cells. The remaining seven clones did not change their expression The Journal of Immunology 7339

cells), and –0.296 Ϯ 0.008 (anti-CD3/CD28-treated memory cells). Functional characteristics of up-regulated genes in memory and naive CD4ϩ T cells To further analyze the function of genes that were up-regulated after anti-CD3/CD28 stimulation, we divided them into five func- tional groups and a “miscellaneous” group: 1) transcriptional reg- ulation, 2) receptor and signal transduction, 3) cytokines and re- ceptors, 4) cell cycle, and 5) structure and metabolism (Fig. 4A). There are several distinct factors that contribute to the transcrip- tional regulation in memory and naive CD4ϩ T cells. The basic transcription factors, including transcription factor 6-like 1, LSF, and SL1, may regulate the basal level of transcription during lym- FIGURE 3. Identification of cDNA clones that are highly expressed in ϩ phocyte activation. Histone deacetylase 2 and nonhistone chromo- freshly isolated memory CD4 T cells. A, List of 14 cDNA clones that somal proteins HMG14 and HMG17 are known for regulating express at higher levels in memory cells than in naive cells. Each colored chromatin structure during transcription (44), which has been re- square represents the ratio of memory over naive CD4ϩ T cell from one ported in the regulation of cytokine gene expression (45). v-Myc, measurement of one cDNA clone. B, The changes of the expression levels

␬ Downloaded from of these 14 cDNA clones (shown in A) in naive and memory CD4ϩ T cells IL enhancer binding factor 2 (NF-ATp45), and NF- B are in- after anti-CD3/CD28 stimulation for 16 h. Each colored square represents volved in mediating signal transduction and cell proliferation (46) the ratio of stimulated memory over resting memory CD4ϩ T cells (right as well as in regulating transcription of IL-2, IL-4, and other genes panel) or stimulated naive over resting naive CD4ϩ T cells (left panel) involved in T cell activation and differentiation (47, 48). from one measurement of one cDNA clone. The results were derived from The engagement of TCR and costimulatory receptors that acti- two independent hybridization experiments conducted with a total of six vate kinases and their downstream targets does not require independent replicate measurement of each cDNA clone. The scale of the new gene expression (49). However, a sustained activation event http://www.jimmunol.org/ intensity ratio is –1:1 in logarithm value (10-fold down-regulation to 10- does require transcription of these signaling-related genes. Indeed, fold up-regulation) with green color indicating a decrease and red indicat- many activation up-regulated genes appeared to function in sig- ing an increase. The GenBank accession number and gene names are in- naling, such as H-RYK receptor-like tyrosine kinase and HAX-1, dicated at the right. a protein associated with the Src family tyrosine kinase substrate HS1 that functions in promoting cell survival in activated CD4ϩ T cells (Fig. 4A) (50). In addition, a large group of genes, including RhoC, nucleolar GTPase, Ras-related GTP-binding protein levels significantly after stimulation in both memory and naive ϩ RAGA, GTP-binding protein NGB, guanine nucleotide exchange

CD4 T cells (Fig. 3B). by guest on September 25, 2021 factor, and GTP-binding protein RAB-2, were significantly in- duced by TCR stimulation. Interestingly, calcium/calmodulin ki- nase II (CAMKII), which plays a critical role in T cell activation General characteristics of activation induced changes in gene ϩ and in generation of memory cells (51), was significantly increased expression between memory and naive CD4 T cells after activation. After the sequential analyses using the commercial and custom- Although the majority of memory and naive CD4ϩ T cells have made filters, we have identified ϳ200 cDNA clones whose ex- not entered the S phase of cell cycle after 16 h of anti-CD3/CD28 ϩ pressions are changed in memory CD4 T cells after anti-CD3/ stimulation measured by [3H]thymidine incorporation, genes in- CD28 stimulation (Fig. 4, A, B, and D). The reproducibility of the volved in cell division were already up-regulated when compared changes of these selected clones were analyzed between two in- with resting cells. These induced genes include cyclins, cyclin- dependent experiments (Fig. 4C) and were confirmed by Northern dependent kinases, DNA repair enzymes, and chromosomal pro- analysis from a randomly selected 16 cDNA clones (data not teins involved in DNA replication and segregation shown). Among the up-regulated cDNA clones, 130 are known (Fig. 4A). In addition, genes related to cell cytoskeleton, including genes and 5 are ESTs (Fig. 4A). In general, memory cells ex- actin, actin-related protein 3, and ␤-tubulin, were also significantly pressed higher levels of transcripts of these up-regulated genes increased along with genes involved in basic cellular functions and than did naive cells, and cells stimulated with anti-CD3/CD28 ex- energy metabolism such as GAPDH, glycogen synthase, ATP syn- pressed higher levels of these same transcripts than cells stimu- thase, and ribosomal proteins (Fig. 4A). lated with anti-CD3 alone (Fig. 4D). The average log-ratios of The ultimate consequence of activation of memory and naive these up-regulated known genes (ϮSE) are 0.065 Ϯ 0.005 (anti- CD4ϩ T cells is the production of cytokines, which influence the CD3-treated naive cells), 0.133 Ϯ 0.005 (anti-CD3-treated mem- outcome of an immune response. Remarkably, we observed that ory cells), 0.260 Ϯ 0.007 (anti-CD3/CD28-treated naive cells), and both memory and naive CD4ϩ T cells expressed a large group of 0.339 Ϯ 0.006 (anti-CD3/CD28-treated memory cells). cytokine genes, including IL-2, IL-9, IFN-␥, pro- colony- Among the 68 activation induced down-regulated cDNA clones, enhancing factor, and lymphotoxin ␣ (TNF-␤) (Fig. 4A). The ex- 42 are known genes and 26 are ESTs (Fig. 4, B and D). In contrast pression of IL-4 and IL-5 was extremely low and was detected by to up-regulated genes, the majority of the activation down-regu- Northern blot in activated memory CD4ϩ T cells (see Fig. 6). In lated genes (70%) had a similar degree of reduction in mRNA addition, we have observed enhanced expression of IL-1R and its levels in both memory and naive CD4ϩ T cells stimulated with accessory protein and IL-15R in both memory and naive CD4ϩ T either anti-CD3 or anti-CD3/CD28 (Fig. 4D). The average log- cells after activation. Because some common cytokine receptors ratios (ϮSE) of these down-regulated genes are –0.219 Ϯ 0.007 such as CD25 and IL-4R were not on the commercial or custom- (anti-CD3-treated naive cells), –0.206 Ϯ 0.010 (anti-CD3-treated made filters, we were not able to assess the expression status of memory cells), –0.295 Ϯ 0.007 (anti-CD3/CD28-treated naive these genes. 7340 GENE EXPRESSION IN MEMORY AND NAIVE CD4ϩ T CELLS Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Gene expression profiles in memory and naive CD4ϩ T cells after in vitro stimulation with anti-CD3 or anti-CD3/CD28. Genes that were up-regulated (A) or down-regulated (B) after in vitro stimulation with anti-CD3 alone or with anti-CD3/CD28 for 16 h are shown. Each colored square represents the ratio of stimulated over resting memory or naive CD4ϩ T cells from one measurement of one cDNA clone. Two independent hybridization experiments were conducted with a total of six independent replicate measurement of each cDNA clone. The scale of the intensity ratio is Ϫ1:1 in logarithm value (10-fold down-regulation to 10-fold up-regulation) with green color indicating a decrease and red indicating an increase. The GenBank accession number and gene names are indicated at the right. C, The scatter plot comparing the reproducibility of up- and down-regulated cDNA clones from two independent hybridization experiments. Each axis represents the mean log-ratio value of each clone (blue dot) in memory CD4ϩ T cells at rest and after stimulation with anti-CD3/CD28 from each experiment. The center blue line indicates an identical ratio value for the two independent experiments. The up-regulated clones shown in A plus ESTs (data not shown) are marked in red and the down-regulated clones shown in B plus ESTs (data not shown) The Journal of Immunology 7341

Functional characteristics of activation down-regulated genes in memory and naive CD4ϩ T cells The functional roles of genes that are highly expressed in the rest- ing T cells and down-regulated after activation have begun to be understood (52). Their functions range from prohibiting cellular proliferation to preventing apoptosis (33, 53). To facilitate further analysis, we have separated these down-regulated genes into four groups: 1) transcriptional regulation, 2) receptor and signal trans- duction, 3) antiproliferation, and 4) cell survival with the remain- ing genes placed in a “miscellaneous” group (Fig. 4B). We identified several genes that function in prohibiting cellular proliferation, including B cell translocation gene 1 (54), IFN-in- duced transmembrane protein 1 (55), RhoGDP dissociation inhib- itor (56), and latent TGF ␤ binding protein 1 (57). We also iden- tified genes that are involved in cell survival and antiapoptosis, such as ubiquination factor E4, which is known to maintain cell viability and survival under stress conditions (58), and neuronal apoptosis inhibitory protein, which is capable of protecting motor- neuron from apoptosis (59). The expression of two important IL Downloaded from receptors, IL-7␣ (60) and IL-10␤ (61), were decreased as well. In addition to these functionally grouped activation down-regulated genes, a gene involved in DNA recombination and repair (Ku Ag) was also identified (Fig. 4B). ϩ FIGURE 5. Actin expression profiles in memory and naive CD4 T cells after anti-CD3 and anti-CD3/CD28 stimulation. Northern blot (A) and Increase of actin expression in activated memory and naive ϩ http://www.jimmunol.org/ ϩ Western blot (B) analysis of Rho and actin in memory and naive CD4 T CD4 T cells cells at rest and after stimulation with anti-CD3 or anti-CD3/CD28 for 16 h Actin polymerization plays a critical role in TCR signaling (62). are shown. ADP-ribosylation factor-like 3 is used as a loading standard for To assess changes in actin expression, we have performed North- the Northern blots, and ZAP70 is used as a loading standard for the West- ern analysis and confirmed that mRNA levels of actin and its up- ern blots. The locations of Rho and actin are indicated at the left. C, Quan- titative analysis of actin polymerization in resting and stimulated memory and stream regulator, the small guanosine triphosphatase RhoC (63), ϩ increased in both memory and naive CD4ϩ T cells after stimula- naive CD4 T cells by FACS. Cells were stained with Alexa Fluor 488 phal- loidin that interacts specifically with polymerized actin (F-actin). The levels of tion (Fig. 5A). To further characterize actin changes at the protein polymerized actin in cells were measured by FACS analysis. The polymerized ϩ level, we examined the actin monomer and polymers in memory actin intensity in resting naive CD4 T cells is arbitrarily set at 1. by guest on September 25, 2021 and naive CD4ϩ T cells after in vitro stimulation. Both monomer and polymerized actin were significantly increased after stimula- tion with anti-CD3 alone or with anti-CD3/CD28 (Fig. 5, B and C). secreted IL-2, IL-4, IL-5, IFN-␥, TNF-␣, and TNF␤ were also In agreement with the levels of mRNA, memory cells expressed quite different between the two subsets of T cells (Fig. 6, B and C). higher levels of actin protein than naive cells under both stimula- After anti-CD3 stimulation, all six cytokines were undetectable in tion conditions, and anti-CD3/CD28 induced higher levels of actin the supernatant of naive cells, but low levels of IL-2, IFN-␥, than did anti-CD3 alone (Fig. 5, B and C). TNF-␣, and TNF␤ were detected in the supernatant of memory

ϩ cells (Fig. 6B). After anti-CD3/CD28 stimulation, both naive and Memory CD4 T cells secrete higher levels of IL-2, IL-4, IL-5, memory cells secreted high levels of IL-2, IFN-␥, TNF-␣, and ␥ ␣ ␤ ϩ IFN- , TNF- , and TNF proteins than naive CD4 T cells TNF␤ (Fig. 6C). However, memory cells secreted significantly The essential function of CD4ϩ T cells is to produce cytokines and greater amounts of these cytokines than did naive cells over a to “help” the effector cells in an immune response. Our cDNA 5-day period. Interestingly, IL-4 and IL-5 were only detected from ϩ microarray and/or Northern blot analyses showed that several lym- memory cells but not naive cells (Fig. 6C). Thus, memory CD4 phokine genes, including IL-2, IL-4, IL-5, IFN-␥, TNF-␣, and T cells should induce a stronger immune response through pro- TNF␤, were up-regulated differentially in memory and naive duction of a larger quantity and greater variety of cytokines than ϩ CD4ϩ T cells after stimulation (Figs. 4A and 6A). To test whether naive CD4 T cells. changes in the mRNA content resulted in corresponding changes in protein levels, and to determine the kinetics of cytokine pro- Discussion duction, we measured IL-2, IL-4, IL-5, IFN-␥, TNF-␣, and TNF␤ We have analyzed the gene expression profiles in memory and proteins in the supernatants of stimulated memory and naive naive CD4ϩ T cells at rest and after stimulation using cDNA mi- CD4ϩ T cells over a 5-day period. Consistent with the mRNA croarray technology. Here, we report an identification of 14 cDNA expression, IL-2, IL-4, IL-5, IFN-␥, TNF-␣, and TNF␤ proteins clones that are highly expressed in memory CD4ϩ T cells and of were not detected in freshly isolated lymphocytes but were signif- ϳ200 cDNA clones whose expression was changed after in vitro icantly induced after stimulation (Fig. 6, B and C). The kinetics of stimulation with anti-CD3/CD28. We present results showing that

are in green. D, The ratio plot of up- and down-regulated genes after in vitro stimulation in memory and naive CD4ϩ T cells. The intensity ratios of up-regulated genes/ESTs (n ϭ 135, red) and of down-regulated genes/ESTs (n ϭ 68, green) of memory and naive CD4ϩ T cells stimulated with anti-CD3 and anti-CD3/CD28 were averaged from six measurements in two independent experiments. 7342 GENE EXPRESSION IN MEMORY AND NAIVE CD4ϩ T CELLS

the difference in gene expression between freshly isolated memory and naive CD4ϩ T cells is small. There are several possible ex- planations that are not mutually exclusive. First, the difference between memory and naive CD4ϩ T cells is small at the gene transcript level but is significant at the protein (functional) level. Second, the current microarray method is not sensitive enough to detect subtle changes of the transcripts between memory and naive CD4ϩ T cells. It is particularly difficult to detect differences in genes that are expressed at low copy number, such as IL-4 and IL-5 genes. Third, recent studies suggest that memory and naive CD4ϩ T cells isolated based on CD45 isoforms can be further divided into functional subsets (64, 65). Analysis of memory cell subsets isolated based on additional cell surface markers, such as the chemokine receptor CCR7, might reveal a greater difference in gene expression between subsets of memory and naive CD4ϩ T cells (28). Further studies with improved cDNA microarray meth- ods are needed to determine the subtle differences in gene expres- sion profiles using subsets of memory CD4ϩ T cells. ϩ Memory and naive CD4 T cells also share significant similar- Downloaded from ities in gene expression. A number of genes that function in main- tenance of the “resting” status of T cells, such as genes involved in antiproliferation and antiapoptosis (53, 66), are expressed in freshly isolated memory and naive CD4ϩ T cells. These genes participate in transcription, signaling, antiproliferation, and cell

survival. The expression of these genes was significantly down- http://www.jimmunol.org/ regulated after activation, suggesting that the “resting” status of memory and naive CD4ϩ T cells is likely to be actively maintained through regulated expression of these antiproliferation and surviv- al-related genes. Among the cDNA clones that are highly expressed in memory CD4ϩ T cells vs naive CD4ϩ T cells, four were significantly up- regulated after stimulation with anti-CD3/CD28, suggesting that

ϩ the higher expression levels of these activation-related genes/ESTs

FIGURE 6. Cytokine expression profiles of memory and naive CD4 T ϩ by guest on September 25, 2021 may contribute to the fast and strong response of memory CD4 cells after stimulation with anti-CD3 or with anti-CD3/CD28. A, Northern T cells upon stimulation. The expression kinetics of CD69 in rest- blot analysis of cytokine genes. Messenger RNA was isolated from resting ϩ memory (M0) and naive (N0) CD4ϩ T cells, stimulated with anti-CD3 (M1 ing and activated CD4 T cells is quite interesting. Freshly iso- and N1) and anti-CD3/CD28 (M2 and N2) for 16 h, are shown. S28 ho- lated memory cells express CD69 mRNA yet have low to unde- mology cDNA used as normalization standard. B and C, Cytokine expres- tectable surface expression of CD69 protein. However, 16 h after sions measured from cell supernatants stimulated with anti-CD3 (B)or stimulation, memory cells expressed high levels of CD69 protein, with anti-CD3/CD28 (C). Culture supernatants of stimulated cells were although the mRNA levels did not change. This suggests that collected at different time intervals and measured for the indicated cytokine CD69 mRNA has a high turnover rate (38) and that there are dif- concentrations by ELISA. Three independent donors were measured and ferent mechanisms regulating CD69 mRNA transcription and pro- the results from a representative donor are shown. tein translation. The consequences of stimulating T cells through the TCR alone or through both TCR and costimulatory receptors have been well the expression levels of those up-regulated genes are higher in documented (3, 67). Here we compared quantitative and qualita- memory cells than in naive CD4ϩ T cells after stimulation, sug- tive changes in gene expression in memory and naive CD4ϩ T gesting that the level of expression of these genes is a molecular cells stimulated with anti-CD3 alone or with anti-CD3/CD28. Af- mechanism that differentiates the response of memory from naive ter anti-CD3 stimulation, both memory and naive CD4ϩ T cells CD4ϩ T cells. down-regulated several groups of genes including antiproliferative Despite recent advances in the phenotypical and functional char- genes and up-regulated many genes functioning in activation and acterizations of memory CD4ϩ T cells, information regarding the proliferation. Although memory CD4ϩ T cells responded to anti- molecular features of these cells is limited. The comparative anal- CD3 stimulation by up-regulating some activation-related genes ysis in this report has revealed some interesting differences as well including cytokine genes, these changes are not sufficient to induce as similarities between freshly isolated memory and naive CD4ϩ T complete activation and proliferation of memory cells. cells. Memory cells express several genes including CD69, inte- In contrast, anti-CD3/CD28 stimulation induced significant ␤ ϩ grin 5, proteoglycan 1, annexin A1, and NK cell protein 4, that changes of gene expression in both memory and naive CD4 T are involved in activation, cell adhesion, and migration (38, 40– cells. Further comparative analysis indicated that memory and na- 43). These processes are critically important for memory cell func- ive CD4ϩ T cells exhibited a similar pattern of gene expression for tions. In addition, memory cells also express higher levels of CDK both up- and down-regulated genes, although expression of the inhibitor 1B that prevent cells from entering the cell cycle (39). up-regulated genes was significantly higher in memory cells after Thus, these small sets of genes provide a glimpse of the delicate anti-CD3/CD28 stimulation (Fig. 4D). Thus, the function of co- balance between expression of genes in activation/migration and in stimulation via CD28 can be viewed as an amplifier of activation maintenance of the “resting” status of memory cells. Nevertheless, signals that enhance the expression of those up-regulated genes. The Journal of Immunology 7343

The precise mechanisms of CD28 signaling in mediating T cell difference in expression levels of those activation up-regulated activation, particularly in up-regulation of activation-induced genes is a molecular basis for the qualitative difference between genes, will require further studies. Taken together, these results memory and naive CD4ϩ T cell response (Fig. 7). indicate that the induction of higher levels of activation-related This expression dosage gradient model for memory CD4ϩ T genes in memory CD4ϩ T cells after activation is a molecular basis cell response was built on the basis of gene expression profiles of for the enhanced cellular response of memory cells. memory and naive CD4ϩ T cells after 16 h of stimulation. Will the Based on results from gene expression and cellular changes in gene expression dosage gradient apply at other time points during memory and naive CD4ϩ T cells at rest and after in vitro stimu- activation as well? What are the functional relationships of those lation, we propose a gene expression dosage gradient model of genes whose expression changes after activation? Further studies memory and naive CD4ϩ T cell response (Fig. 7). In this model, of kinetic changes in gene expression as well as of memory cell memory and naive CD4ϩ T cells express antiproliferation, cell subsets will be able to test our model of the memory cell response survival, and other genes to maintain the “resting” status. Com- and lead to a better understanding of memory CD4ϩ T cells and pared with naive cells, memory T cells may express fewer or lower ultimately the mechanisms of immunological memory. levels of these antiproliferation-related genes and higher levels of activation-related genes. Stimulation through the TCR alone (anti- Acknowledgments CD3 alone) induces down-regulation of antiproliferation and other We thank Drs. Dan Longo, Stephen Shaw, Patricia Gearhart, and Ron genes that are required for maintaining the resting condition and Wange for critical reading of the manuscript. We thank the National In- up-regulation of genes that are required for activation. However, stitutes of Health Blood Bank for providing blood samples, Drs. Carl June the expression level of those up-regulated genes is not high enough and Bruce Levine for providing anti-CD3 and anti-CD3/CD28-conjugated Downloaded from to lead to cytokine production and proliferation in naive cells and magnetic beads, Shannon Murray for technical assistance, and William Wood for printing the cDNA microarray filters. can only produce low levels of a limited number of cytokines and undergo partial proliferation in memory cells. In contrast, stimu- References lation through the TCR and costimulatory receptor CD28 (anti- 1. Ahmed, R., and D. Gray. 1996. Immunological memory and protective immunity: CD3/CD28) induces a similar down-regulation of antiproliferation understanding their relation. Science 272:54. 2. Sprent, J. 1997. Immunological memory. Curr. Opin. Immunol. 9:371. genes but higher expression levels of up-regulated genes that are http://www.jimmunol.org/ ϩ 3. Glimcher, L. H., and K. M. Murphy. 2000. Lineage commitment in the immune sufficient to completely activate both memory and naive CD4 T system: the T helper lymphocyte grows up. Genes Dev. 14:1693. cells. However, even in the presence of costimulatory stimulation, 4. McHeyzer-Williams, M. G., and R. Ahmed. 1999. B cell memory and the long- memory cells express higher levels of those activation up-regu- lived . Curr. Opin. Immunol. 11:172. 5. Doherty, P. C., and J. P. Christensen. 2000. Accessing complexity: the dynamics lated genes and secrete more cytokines than naive cells. Thus, our of virus-specific T cell responses. Annu. Rev. Immunol. 18:561. model proposes that activation of CD4ϩ T cells requires two se- 6. Dutton, R. W., L. M. Bradley, and S. L. Swain. 1998. T cell memory. Annu. Rev. Immunol. 16:201. quential molecular events: down-regulation of genes required for 7. Morimoto, C., N. L. Letvin, J. A. Distaso, W. R. Aldrich, and S. F. Schlossman. maintenance of the resting status and up-regulation of genes re- 1985. The isolation and characterization of the human suppressor inducer T cell quired for activation and proliferation, and that the quantitative subset. J. Immunol. 134:1508. 8. Rose, L. M., A. H. Ginsberg, T. L. Rothstein, J. A. Ledbetter, and E. A. Clark. by guest on September 25, 2021 1985. Selective loss of a subset of T helper cells in active multiple sclerosis. Proc. Natl. Acad. Sci. USA 82:7389. 9. Tedder, T. F., M. D. Cooper, and L. T. Clement. 1985. Human lymphocyte differentiation HB-10 and HB-11. II. Differential production of B cell growth and differentiation factors by distinct helper T cell subpopulations. J. Im- munol. 134:2989. 10. Sanders, M. E., M. W. Makgoba, and S. Shaw. 1988. Human naive and memory T cells: reinterpretation of helper-inducer and suppressor-inducer subsets. Immu- nol. Today 9:195. 11. Young, J. L., J. M. Ramage, J. S. Gaston, and P. C. Beverley. 1997. In vitro responses of human CD45RObrightRAϪ and CD45ROϪRAbright T cell subsets and their relationship to memory and naive T cells. Eur. J. Immunol. 27:2383. 12. Weng, N. P., B. L. Levine, C. H. June, and R. J. Hodes. 1995. Human naive and memory T lymphocytes differ in telomeric length and replicative potential. Proc. Natl. Acad. Sci. USA 92:11091. 13. Byrne, J. A., J. L. Butler, and M. D. Cooper. 1988. Differential activation re- quirements for virgin and memory T cells. J. Immunol. 141:3249. 14. Sanders, M. E., M. W. Makgoba, C. H. June, H. A. Young, and S. Shaw. 1989. Enhanced responsiveness of human memory T cells to CD2 and CD3 receptor- ϩ mediated activation. Eur. J. Immunol. 19:803. FIGURE 7. Gene expression dosage gradient model of memory CD4 15. Horgan, K. J., G. A. van Seventer, Y. Shimizu, and S. Shaw. 1990. Hyporespon- ϩ T cells response. Freshly isolated memory and naive CD4 T cells pre- siveness of “naive” (CD45RAϩ) human T cells to multiple receptor-mediated dominantly express genes (green) that are required for maintaining the stimuli but augmentation of responses by co-stimuli. Eur. J. Immunol. 20:1111. “resting” statue. Compared with naive cells, memory CD4ϩ T cells express 16. Farber, D. L., M. Luqman, O. Acuto, and K. Bottomly. 1995. Control of memory CD4 T cell activation: MHC class II molecules on APCs and CD4 ligation inhibit fewer or lower levels of these “resting” maintenance genes. These resting memory but not naive CD4 T cells. Immunity 2:249. ϩ cell expressed genes are down-regulated once memory and naive CD4 T 17. Babbitt, B. P., P. M. Allen, G. Matsueda, E. Haber, and E. R. Unanue. 1985. cells receive an activation signal from the engagement of TCR or TCR and Binding of immunogenic peptides to Ia histocompatibility molecules. Nature costimulatory receptor CD28. Activation also induces an up-regulation of 317:359. 18. Harding, F. A., J. G. McArthur, J. A. Gross, D. H. Raulet, and J. P. Allison. 1992. genes (red) that function in proliferation and differentiation. Dependent CD28-mediated signalling co-stimulates murine T cells and prevents induction of upon the stimulation conditions, the expression levels of these activation anergy in T-cell clones. Nature 356:607. up-regulated genes were only slightly increased in memory and naive cells 19. Dubey, C., M. Croft, and S. L. Swain. 1996. Naive and effector CD4 T cells differ after TCR engagement alone and were significantly increased after the in their requirements for T cell receptor versus costimulatory signals. J. Immunol. 157:3280. engagement of both TCR and costimulatory receptors (CD28). The degree 20. Schwartz, R. H. 1996. Models of T cell anergy: is there a common molecular of cellular response assessed by cytokine production in memory and naive mechanism? J. Exp. Med. 184:1. CD4ϩ T cells after in vitro stimulation can be directly attributed to the 21. Weaver, C. T., and E. R. Unanue. 1990. The costimulatory function of - ϩ presenting cells. Immunol. Today 11:49. expression levels of activation up-regulated genes. Memory CD4 T cells ϩ 22. Luqman, M., and K. Bottomly. 1992. Activation requirements for CD4 T cells express higher levels of activation up-regulated genes and secrete more differing in CD45R expression. J. Immunol. 149:2300. ϩ cytokines than naive CD4 T cells. 23. Gray, D. 2000. Thanks to the memory. Nat. Immunol. 1:11. 7344 GENE EXPRESSION IN MEMORY AND NAIVE CD4ϩ T CELLS

24. Ehlers, S., and K. A. Smith. 1991. Differentiation of T cell lymphokine gene 45. Agarwal, S., and A. Rao. 1998. Modulation of chromatin structure regulates expression: the in vitro acquisition of T cell memory. J. Exp. Med. 173:25. cytokine gene expression during T cell differentiation. Immunity 9:765. 25. Kristensson, K., C. A. Borrebaeck, and R. Carlsson. 1992. Human CD4ϩ T cells 46. Ginsberg, D., G. Vairo, T. Chittenden, Z. X. Xiao, G. Xu, K. L. Wydner, expressing CD45RA acquire the lymphokine gene expression of CD45ROϩ T- J. A. DeCaprio, J. B. Lawrence, and D. M. Livingston. 1994. E2F-4, a new helper cells after activation in vitro. Immunology 76:103. member of the E2F transcription factor family, interacts with p107. Genes Dev. 26. Conlon, K., J. Osborne, C. Morimoto, J. R. Ortaldo, and H. A. Young. 1995. 8:2665. Comparison of lymphokine secretion and mRNA expression in the CD45RAϩ 47. Parra, E., K. McGuire, G. Hedlund, and M. Dohlsten. 1998. Overexpression of and CD45ROϩ subsets of human peripheral blood CD4ϩ and CD8ϩ lympho- p65 and c-Jun substitutes for -1 costimulation by targeting the CD28RE within cytes. Eur. J. Immunol. 25:644. the IL-2 promoter. J. Immunol. 160:5374. 27. Shin, H. C., N. Benbernou, S. Esnault, and M. Guenounou. 1999. Expression of 48. Li-Weber, M., M. Giasi, and P. H. Krammer. 1998. Involvement of Jun and Rel IL-17 in human memory CD45ROϩ T lymphocytes and its regulation by protein proteins in up-regulation of -4 gene activity by the T cell accessory kinase A pathway. Cytokine 11:257. molecule CD28. J. Biol. Chem. 273:32460. 28. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two 49. Cantrell, D. A. 1996. T cell antigen receptor signal transduction pathways. Can- subsets of memory T lymphocytes with distinct homing potentials and effector cer Surv. 27:165. functions. Nature 401:708. 50. Suzuki, Y., C. Demoliere, D. Kitamura, H. Takeshita, U. Deuschle, and 29. Ohshima, Y., L. P. Yang, M. N. Avice, M. Kurimoto, T. Nakajima, M. Sergerie, T. Watanabe. 1997. HAX-1, a novel intracellular protein, localized on mitochon- C. E. Demeure, M. Sarfati, and G. Delespesse. 1999. Naive human CD4ϩ T cells dria, directly associates with HS1, a substrate of Src family tyrosine kinases. Are a major source of lymphotoxin ␣. J. Immunol. 162:3790. J. Immunol. 158:2736. 30. Marazzi, S., S. Blum, R. Hartmann, D. Gundersen, M. Schreyer, S. Argraves, 51. Bui, J. D., S. Calbo, K. Hayden-Martinez, L. P. Kane, P. Gardner, and V. von Fliedner, R. Pytela, and C. Ruegg. 1998. Characterization of human fi- S. M. Hedrick. 2000. A role for CaMKII in T cell memory. Cell 100:457. broleukin, a fibrinogen-like protein secreted by T lymphocytes. J. Immunol. 161: 52. Kuo, C. T., and J. M. Leiden. 1999. Transcriptional regulation of T lymphocyte 138. development and function. Annu. Rev. Immunol. 17:149. 31. Brown, P. O., and D. Botstein. 1999. Exploring the new world of the genome 53. Kuo, C. T., M. L. Veselits, and J. M. Leiden. 1997. LKLF: a transcriptional with DNA microarrays. Nat. Genet. 21:33. regulator of single-positive T cell quiescence and survival. Science 277:1986. 32. Alizadeh, A. A., M. B. Eisen, R. E. Davis, C. Ma, I. S. Lossos, A. Rosenwald, 54. Rouault, J. P., R. Rimokh, C. Tessa, G. Paranhos, M. Ffrench, L. Duret, Downloaded from J. C. Boldrick, H. Sabet, T. Tran, X. Yu, et al. 2000. Distinct types of diffuse large M. Garoccio, D. Germain, J. Samarut, and J. P. Magaud. 1992. BTG1, a member B-cell lymphoma identified by gene expression profiling. Nature 403:503. of a new family of antiproliferative genes. EMBO J. 11:1663. 55. Deblandre, G. A., O. P. Marinx, S. S. Evans, S. Majjaj, O. Leo, D. Caput, 33. Teague, T. K., D. Hildeman, R. M. Kedl, T. Mitchell, W. Rees, B. C. Schaefer, G. A. Huez, and M. G. Wathelet. 1995. Expression cloning of an interferon- J. Bender, J. Kappler, and P. Marrack. 1999. Activation changes the spectrum but inducible 17-kDa membrane protein implicated in the control of cell growth. not the diversity of genes expressed by T cells. Proc. Natl. Acad. Sci. USA J. Biol. Chem. 270:23860. 96:12691. 56. Scherle, P., T. Behrens, and L. M. Staudt. 1993. Ly-GDI, a GDP-dissociation 34. Rogge, L., E. Bianchi, M. Biffi, E. Bono, S. Y. Chang, H. Alexander, C. Santini, inhibitor of the RhoA GTP-binding protein, is expressed preferentially in lym- G. Ferrari, L. Sinigaglia, M. Seiler, et al. 2000. Transcript imaging of the devel-

phocytes. Proc. Natl. Acad. Sci. USA 90:7568. http://www.jimmunol.org/ opment of human T helper cells using oligonucleotide arrays. Nat. Genet. 25:96. 57. Kanzaki, T., A. Olofsson, A. Moren, C. Wernstedt, U. Hellman, K. Miyazono, 35. Weng, N., B. L. Levine, C. H. June, and R. J. Hodes. 1996. Regurated expression L. Claesson-Welsh, and C. H. Heldin. 1990. TGF-␤1 binding protein: a compo- of telomerase activity in human T lymphocyte development and activation. nent of the large latent complex of TGF-␤1 with multiple repeat sequences. Cell J. Exp. Med. 183:2471. 61:1051. 36. Carlisle, A. J., V. V. Prabhu, A. Elkahloun, J. Hudson, J. M. Trent, 58. Koegl, M., T. Hoppe, S. Schlenker, H. D. Ulrich, T. U. Mayer, and S. Jentsch. W. M. Linehan, E. D. Williams, M. R. Emmert-Buck, L. A. Liotta, P. J. Munson, 1999. A novel ubiquitination factor, E4, is involved in multiubiquitin chain as- and D. B. Krizman. 2000. Development of a prostate cDNA microarray and sembly. Cell 96:635. statistical gene expression analysis package. Mol. Carcinog. 28:12. 59. Roy, N., M. S. Mahadevan, M. McLean, G. Shutler, Z. Yaraghi, R. Farahani, 37. Liu, K., M. M. Schoonmaker, B. L. Levine, C. H. June, R. J. Hodes, and S. Baird, A. Besner-Johnston, C. Lefebvre, and X. Kang. 1995. The gene for N. Weng. 1999. Constitutive and regulated expression of telomerase reverse tran- neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal scriptase (hTERT) in human lymphocytes. Proc. Natl. Acad. Sci. USA 96:5147. muscular atrophy. Cell 80:167. 38. Lopez-Cabrera, M., A. G. Santis, E. Fernandez-Ruiz, R. Blacher, F. Esch, 60. Goodwin, R. G., D. Friend, S. F. Ziegler, R. Jerzy, B. A. Falk, S. Gimpel, by guest on September 25, 2021 P. Sanchez-Mateos, and F. Sanchez-Madrid. 1993. Molecular cloning, expres- D. Cosman, S. K. Dower, C. J. March, A. E. Namen, and. 1990. Cloning of the sion, and chromosomal localization of the human earliest lymphocyte activation human and murine interleukin-7 receptors: demonstration of a soluble form and antigen AIM/CD69, a new member of the C-type animal superfamily of homology to a new receptor superfamily. Cell 60:941. signal-transmitting receptors. J. Exp. Med. 178:537. 61. Kotenko, S. V., C. D. Krause, L. S. Izotova, B. P. Pollack, W. Wu, and S. Pestka. 39. Sherr, C. J., and J. M. Roberts. 1995. Inhibitors of mammalian G1 cyclin-depen- 1997. Identification and functional characterization of a second chain of the in- dent kinases. Genes Dev. 9:1149. terleukin-10 receptor complex. EMBO J. 16:5894. 40. Dahl, C. A., R. P. Schall, H. L. He, and J. S. Cairns. 1992. Identification of a 62. Penninger, J. M., and G. R. Crabtree. 1999. The actin cytoskeleton and lympho- novel gene expressed in activated natural killer cells and T cells. J. Immunol. cyte activation. Cell 96:9. 148:597. 63. Maekawa, M., T. Ishizaki, S. Boku, N. Watanabe, A. Fujita, A. Iwamatsu, 41. Hauzenberger, D., J. Klominek, S. E. Bergstrom, and K. G. Sundqvist. 1995. T T. Obinata, K. Ohashi, K. Mizuno, and S. Narumiya. 1999. Signaling from Rho lymphocyte migration: the influence of interactions via adhesion molecules, the to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. Science T cell receptor, and cytokines. Crit. Rev. Immunol. 15:285. 285:895. 42. Rothenberg, M. E., J. L. Pomerantz, W. F. Owen, S. Avraham, R. J. Soberman, 64. Bell, E. B., S. M. Sparshott, and C. Bunce. 1998. CD4ϩ T-cell memory, CD45R K. F. Austen, and R. L. Stevens. 1988. Characterization of a human subsets and the persistence of antigen: a unifying concept. Immunol. Today 19: proteoglycan, and augmentation of its biosynthesis and size by interleukin 3, 60. interleukin 5, and granulocyte/macrophage colony stimulating factor. J. Biol. 65. Garcia, S., J. DiSanto, and B. Stockinger. 1999. Following the development of a Chem. 263:13901. CD4 T cell response in vivo: from activation to memory formation. Immunity 43. Walther, A., K. Riehemann, and V. Gerke. 2000. A novel ligand of the formyl 11:163. peptide receptor: annexin I regulates neutrophil extravasation by interacting with 66. Marrack, P., T. Mitchell, D. Hildeman, R. Kedl, T. K. Teague, J. Bender, the FPR. Mol. Cell 5:831. W. Rees, B. C. Schaefer, and J. Kappler. 2000. Genomic-scale analysis of gene 44. Louie, D. F., K. K. Gloor, S. C. Galasinski, K. A. Resing, and N. G. Ahn. 2000. expression in resting and activated T cells. Curr. Opin. Immunol. 12:206. Phosphorylation and subcellular redistribution of high mobility group proteins 14 67. Chambers, C. A., and J. P. Allison. 1999. Costimulatory regulation of T cell and 17, analyzed by mass spectrometry. Protein Sci. 9:170. function. Curr. Opin. Cell Biol. 11:203.