Gene expression profiles during human CD4+ T cell differentiation
Myeong Sup Lee1,2,3,
Kristina Hanspers4,
Christopher S. Barker4,5,
Abner P. Korn6 and
Joseph M. McCune1,2,3
1 Gladstone Institute of Virology and Immunology, University of California at San Francisco, San Francisco, CA 94141, USA
2 Department of Medicine and 3 Department of Microbiology and Immunology, University of California at San Francisco, San Francisco, CA 94143, USA
4 Genomics Core Laboratory, Gladstone Institute of Cardiovascular Disease, University of California at San Francisco, San Francisco, CA 94141, USA
5 General Clinical Research Center, Core Genomics Laboratory, San Francisco General Hospital, San Francisco, CA 94110, USA
6 Department of Obstetrics, Gynecology, and Reproductive Biology, San Francisco General Hospital, San Francisco, CA 94110, USA
Correspondence to: J. M. McCune; E-mail: mmccune{at}gladstone.ucsf.edu
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Abstract
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To develop a comprehensive catalogue of phenotypic and functional parameters of human CD4+ T cell differentiation stages, we have performed microarray gene expression profiling on subpopulations of human thymocytes and circulating naive CD4+ T cells, including CD3CD4+CD8 intrathymic T progenitor cells, CD3intCD4+CD8+ double positive thymocytes, CD3highCD4+CD8 single positive thymocytes, CD3+CD4+CD8 CD45RA+CD62L+ naive T cells from cord blood and CD3+CD4+CD8 CD45RA+CD62L+ naive T cells from adult blood. These subpopulations were sort-purified to >98% purity and their expressed RNAs were analyzed on Affymetrix Human Genome U133 arrays. Comparison of gene expression signals between these subpopulations and with early passage fetal thymic stromal cultures identify: (i) transcripts that are preferentially expressed in human CD4+ T cell subpopulations and not in thymic stromal cells; (ii) major shifts in gene expression as progenitor T cells mature into progeny; (iii) preferential expression of transcripts at the progenitor cell stage with plausible relevance to the regulation of expansion and differentiation of these cells; and (iv) preferential expression of potential markers of recent thymic emigrants in naive-phenotype CD4+ T cells from cord blood. Further evaluation of these findings may lead to a better definition of human thymopoiesis as well as to improved approaches to monitor and to augment the function of this important organ of T cell production.
Keywords: gene expression, microarray, naive CD4+ T cell, recent thymic emigrants, thymocytes
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Introduction
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The analysis of T cell differentiation in the mouse has been enhanced by the availability of mouse stocks with defined mutations that are either inborn or introduced and by the ability to examine the fate of adoptively transferred subpopulations of cells. In humans, neither experimental manipulation is readily achieved and observations are instead relegated to in vitro and in vivo model systems that are likely less relevant. These studies have revealed patterns of differentiation that are in many, but not all, aspects similar to those found in the mouse.
CD34+ bone marrow-derived common lymphoid progenitor cells enter the thymus and migrate to the subcapsular region, where they proliferate, differentiate, and rearrange T cell receptor (TCR) gene loci (1). The earliest progenitors that are clearly T-lineage committed are CD34lowCD3CD4+CD8 intrathymic T progenitor (ITTP) cells (2), which respond to interleukin 7 by increasing their rate of proliferation and decreasing their rate of apoptosis (3). Upon maturation to CD3intCD4+CD8+ double positive (DP) thymocytes, a maximally-diverse TCR repertoire is expressed and subjected to positive and negative selection, yielding CD3highCD4+CD8 single positive 4 (SP4) and CD3highCD4CD8+ single positive 8 (SP8) thymocytes [for review, see (4)]. These cells, in turn, migrate into the peripheral circulation as naive phenotype CD4+ and CD8+ T cells, expressing markers such as CD45RA, CD62L, CD27, CD28, CCR9, CD31 and/or CD103 (5,6,7,8). Early in life, when the thymus is active, the naive T cell compartment likely contains a relatively high fraction of recent thymic emigrants (RTEs), analogous to those expressing the ChT1 marker in the chicken (9) and bearing a high frequency of TCR excision circles (10,11). Particularly in adults, in whom the thymus is less active, this phenotypic compartment may also include antigen-naive T cells that have divided homeostatically (12) as well as antigen-experienced cells that have reverted to a naive phenotype (13).
In addition to the similarities between murine and human thymopoiesis, points of variance clearly also exist. For example, early intrathymic progenitors in the human express CD4 whereas those in the mouse can express CD8 instead (14); signaling through Fas induces apoptosis in murine but not in human thymocytes (15), and growth hormone appears to exert differential effects on human and murine thymopoiesis (16).
A more complete understanding of human thymopoiesis could potentially impact on several applied areas of diagnosis and treatment of human immunodeficiency diseases. Thus, identification of genes expressed in early stages of intrathymic T cell proliferation and maturation might yield insights into ways in which these events are regulated. Such information could, in turn, lead to the development of therapeutic maneuvers designed to augment thymic function in times of need. Also, the definition of specific markers for human RTEs would facilitate the measurement of these cells in peripheral blood so that an indirect but minimally invasive estimate of thymic function could be obtained. In the setting of T cell-depleted states (e.g. after bone marrow transplantation or HIV infection), such information could then be used to monitor T cell reconstitution derived from thymus.
To develop a more comprehensive catalogue of phenotypic and functional parameters of human T cell differentiation stages, we performed microarray gene expression profiling on CD4+ subpopulations of human thymocytes and circulating naive-phenotype CD4+ T cells. Our choice of subpopulations was based on two major criteria: (i) that they express an unambiguous surface phenotype, permitting sort purification, and (ii) that they represent a discrete stage of human T cell maturation on the basis of previously documented findings. Our decision to focus on CD4+ T cells was based primarily on the important role of this lineage in HIV disease. Five subpopulations meeting these criteria were selected for analysis: CD3CD4+CD8 ITTPs, CD3intCD4+CD8+ DP thymocytes, CD3highCD4+CD8 SP4 thymocytes, CD3+CD4+CD8CD45RA+CD62L+ naive T cells from cord blood (CB4) and CD3+CD4+CD8CD45RA+CD62L+ naive T cells from adult blood (AB4). The less mature ITTP and DP thymocytes might be expected to preferentially express genes important for proliferation, TCR rearrangement and selection in the thymus. Given the expected age-dependent decrease in thymic function, the subpopulation of naive-phenotype CD4+ T cells from cord blood should preferentially express genes specific for recent thymic emigrants compared to the naive-phenotype CD4+ T cells from adult blood. We address major elements of these and other descriptors of each subpopulation below. We also provide a series of detailed supplementary tables for more focused review (see International Immunology Online).
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Methods
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Tissue collection
Human fetal thymus (18 weeks old) was obtained from Advanced Biosciences Resources (Alameda, CA) or removed from SCID-hu Thy/Liv mice previously implanted with human fetal thymus and fetal liver for 68 months (17). All isolation steps up to the point of cell lysis were performed at 4°C. Thymocytes were dissociated by passing the organ through nylon mesh and then resuspended in phosphate-buffered saline (PBS) plus 2% fetal bovine serum (PBSF). For the isolation of naive-phenotype CD4+ T cells, 30 ml of cord blood and adult blood were drawn into tubes containing sodium heparin, thoroughly mixed, transported at 4°C, and processed within 3 h. After ficoll hypaque density centrifugation for 30 min, the peripheral blood mononuclear cell (PBMC) fraction was collected in PBSF. To prepare early passage fetal thymic stromal cultures (TSC), fetal thymus (18 weeks old) was digested in collagenase D in RPMI at 37°C for 30 min; the resultant cells and tissue fragments were cultured for 2 weeks (with periodic media changes and one passage) in modified DMEM containing D-valine (18), penicillin/streptomycin (100 U/100 µg per ml), 2 mM L-glutamine, 10 mM HEPES, and 10% fetal bovine serum. At the end of this time period, the adherent, early passage fetal TSC were monomorphic in appearance. For analysis (e.g. as in Fig. 1D), they were washed once with PBS, dissociated with 0.05% trypsin/0.53 mM EDTA for 5 min at 37°C, and collected by centrifugation for 10 min at 1500 r.p.m.

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Fig. 1. Sort purification of CD4+ subpopulations and purity analysis of thymic stromal cells. (A) Thymocytes were stained with CD8FITC, CD3PE, CD4APC and 7-AAD in PBS containing 2% FBS for 15 min at 4°C. After gating on mononuclear cells using forward scatter (FSC) and side scatter (SSC) (left panel), three subpopulations of viable (7-AAD-negative) CD4+ thymocytes were sort-purified using the regions (R) shown in the middle and right panels: ITTP (CD3CD4+CD8) (R2 and R5); DP (CD3intCD4+CD8+) (R1 and R4); and SP4 (CD3highCD4+CD8) (R2 and R3). (B) Cord blood was stained with CD8FITC, GlyAPE, CD4PECy7, CD45RAECD and CD62LAPC. Mononuclear cells were gated by FSC and SSC (left panel), and divided into populations that were positive or negative for glycophorin A (GlyA) (data not shown). GlyA cells that were CD4+CD8 (middle panel) were sorted for co-expression of CD45RA and CD62L (right panel), to isolate CD4+CD8CD45RA+CD62L+ naive CB4 cells. (C) Adult blood was stained with CD8FITC, CD4PE, CD45RAECD and CD62LAPC. Mononuclear cells were gated by FSC and SSC (left panel), and those that were CD4+CD8 (middle panel) were sorted for co-expression of CD45RA and CD62L (right panel), to isolate CD4+CD8CD45RA+CD62L+ naive AB4 cells. (D) Early passage fetal thymic stromal cultures (TSC), grown for 2 weeks in D-val medium (see Methods), were trypsin-dissociated and stained with 7-AAD and CD45APC. Fewer than 1% of the cells fell into a FSC/SSC gate characteristic of thymocytes (left panel) or expressed CD45 (middle panel), and >90% of the cells were viable, i.e., 7-AAD (right panel).
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Antibody staining and sorting
Thymocytes and PBMCs were filtered through a 70 µm Falcon cell strainer (Becton Dickinson Labware, Franklin Lakes, NJ) and stained in PBSF with the following combinations of antibodies according to the manufacturer's recommendation (for more details, see Fig. 1): for thymocytes, CD8FITC (BD Biosciences, clone SK1) (San Jose, CA), CD3PE (BD Biosciences, clone SK7), CD4APC (CALTAG, clone S3.5) (Burlingame, CA), and 7-AAD (Sigma, St Louis, MO); for cord blood, CD8FITC (BD Biosciences, clone SK1), GlyAPE (BD PharMingen, clone GA-R2), CD4PECy7 (CALTAG, clone S3.5), CD45RAECD (Coulter-Immunotech, clone 2H4) (Miami, Florida), and CD62LAPC (BD PharMingen, Dreg 56); for adult PBMC, CD8FITC, CD4PE (BD Biosciences, clone SK3), CD45RAECD, and CD62LAPC. In the case of cord blood and adult PBMC, cells were also stained with CD8FTIC, CD3PE, 7-AAD and CD4APC in parallel and >99% of CD4+CD8 cells were found to be CD3high (i.e. T cells). The stained cells were then sort-purified on a BD FACS Vantage (BD Biosciences, San Jose, CA) and collected in tubes containing 2 ml of 100% fetal bovine serum. The purity of each subpopulation was confirmed on re-sort analysis directly thereafter and cells were lysed for preparation of RNA. Cells from early passage fetal TSC were analyzed using APC-conjugated antibodies against CD45 (BD Biosciences, clone 2D1) and their viability was assessed using 7-AAD.
RNA preparation, hybridization and gene expression data collection
Cells were pelleted at 1500 r.p.m. for 5 min and lysed immediately in Buffer RLT (Qiagen RNeasy Mini kit, Valencia, CA). Total cellular RNA was purified following the manufacturer's recommendation and analyzed using the RNA 6000 NanoKit on an Agilent BioAnalyzer 2100 system (Agilent Technologies, Palo Alto, CA); as expected, discrete 28 S rRNA and 18 S rRNA peaks were observed. RNA concentrations were determined spectrophotometrically and all samples had an OD260/OD280 ratio of
1.82.1. Starting with at least 50 ng of total RNA, two rounds of RNA amplification with T7 RNA polymerase were performed using MessageAmp aRNA kit (Ambion, Austin, TX); the final labeling of the cRNA with biotin was performed with Enzo HighYield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY), following the recommendations from the manufacturers. Hybridization, washing, staining and scanning were performed as described in the Affymetrix GeneChip Expression Analysis Technical Manual. The cRNA samples were first hybridized to Affymetrix Test 3 arrays for quality control and subsequently to Affymetrix Human Genome U133 A and B arrays. Hybridizations were carried out at 45°C for 16 h in a rotisserie oven set at 60 r.p.m. Following hybridization, the arrays were washed and stained with streptavidinphycoerythrin in the Affymetrix Fluidics Station 400, using the standard antibody amplification protocol. Arrays were scanned with the Affymetrix GeneArray Scanner at 488 nm and 3 microns per pixel. Expression values were calculated using the Robust Multichip Average (RMA) method. RMA signal value estimates are based on a robust average of background corrected PM intensities and normalization was done using quantile normalization (19,20). All of the signal values in the text are expressed in normal space. In contrast to earlier methods, mismatch oligonucleotide signals are not considered using this method. As shown by Rosati et al. (21), RMA has a lower rate of false positive and false negative calls than earlier methods such as MAS 5.0 (Affymetrix) or dChip (22), thus making it a superior method for detecting differentially expressed transcripts.
Semi-quantitative RTPCR
Five nanograms of total RNA prepared from each cell type was reverse transcribed and amplified sequentially, using the SuperScript One-Step RTPCR kit (Invitrogen, Carlsbad, CA), following the protocol provided by manufacturer, with specific reverse and forward primers. The following primer pairs were used for each of the following gene transcripts: GMFG, [GMFGQF (5'-TGGTGTGCGAGGTAGACCCA-3') and GMFGQR (5'-CCACAGGGCTGGAGAAGATG-3'): amplicon, 267 bp]; TLR7, [TLR7QF (5'-GTATCTGCACACTTGATACAGCAAC-3') and TLR7QR (5'-GGCTTGAACACATGCACGCTG-3'): amplicon, 393 bp]; RASD1, [RASD1QF (5'-GATCAAGAAGATGTGCCCGAG-3') and RASD1QR (5'-GGTCCAGGCTGCTGTTCTTCT-3'): amplicon, 539 bp]; HTR2B, [HTR2BQF (5'-CAACTCAGGTGATGAAACACTTATGC-3') and HTR2BQR (5'-GGAGAAGCGTATCTAGTAGAATGAT-3'): amplicon, 515 bp]; PACAP, [ PACAPQF (5'-CTGGCAAAGGCAGAGACCAAACT-3') and PACAPQR (5'-AGGACTAGAGCTCTTCTCTTGTG-3'): amplicon, 382 bp]; FOXO1A, [FOXO1AQF (5'-CACGCTGTCGCAGATCTACG-3') and FOXO1AQR (5'-TGGATTGAGCATCCACCAAG3'): amplicon, 181 bp]; TSHR, [TSHRQF (5'-CAGTCCAAGGATATGCTTTCAATGG-3') and TSHRQR (5'-CCTTTGGATGGAAGGGCAGT-3'): amplicon, 163 bp]; ARH, [ARHQF (5'-GCATCCTGAAAGCCAGCAAA-3') and ARHQR (5'-TGACAAATAGAGGCCGGTGC-3'): amplicon, 260 bp]; CCR9, [CCR9QF (5'-CCAGCCTTGGCCCTGTT-3') and CCR9QR (5'-CAGACAGACGGTGGACCCA-3'): amplicon, 67 bp]; CCR7, [CCR7QF (5'-CCTCCATCGTTTTCTTCACTGTC-3') and CCR7QR (5'-TTAGGGCGGCGTGGC-3'): amplicon, 63 bp]; P2RX5, [P2RX5QF (5'-CAGCAGTCAGAAGGGGAACGGAT-3') and P2RX5QR (5'-CTCTGTGATGGCTGGTCCCTGT-3'): amplicon, 216 bp]; C1QR1, [C1QR1QF (5'-CTGGAATTACAAGATTTCTATGCAGG-3') and C1QR1QR (5'-TGAGCCTTCCCTCCTCACAG-3'): amplicon, 77 bp]; and GAPDH, [GAPDHQF (5'-ATCTCTGCCCCCTCTGCTG-3') and GAPDHQR (5'-GGGTGTCGCTGTTGAAGTCA-3'): amplicon, 511 bp]. Reverse transcription reactions were carried out at 50°C for 25 min with the reverse primers, followed by PCR reactions with the primer pairs: samples were incubated at 94°C for 2 min initially, then at 94°C for 30 sec, 55°C for 30 sec and 72°C for 1 min, sequentially for 3035 cycles. Finally the samples were incubated at 72°C for 10 min. Amplification of GAPDH was carried out in a linear range (2530 cycles) to evaluate the efficiency of the RTPCR reactions in different cell subpopulations.
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Results
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RNA expression profiles from subpopulations of human T cells
To define the mRNA expression profile of cells at various stages of human T cell differentiation, the following subpopulations were sort-purified using the gates shown in Fig. 1: CD3CD4+CD8 ITTPs (R2 and R5 in Fig. 1A), CD3intCD4+CD8+ DP thymocytes (R1 and R4 in Fig. 1A), CD3highCD4+CD8 SP4 thymocytes (R2 and R3 in Fig. 1A), CD3+CD4+CD8CD45RA+CD62L+ naive T cells from cord blood (CB4) (Fig. 1B) and CD3+CD4+CD8CD45RA+CD62L+ naive T cells from adult blood (AB4) (Fig. 1C). The purity of each subpopulation on re-sort analysis was >98% (data not shown). To better discriminate the expression of CD4+ T cell lineage-specific genes from more ubiquitous housekeeping genes, early passage fetal thymic stromal cultures (TSC) with <1% thymocyte contamination were also prepared (Fig. 1D). Total cellular RNA was extracted from at least 1.5 x 105 cells of each purified subpopulation and was amplified by two cycles of cDNA synthesis and in vitro transcription. Amplified RNA samples were then hybridized to Affymetrix microarrays, with 45 000 probe sets displaying
33 000 genes corresponding to 39 000 transcripts (see Methods). For each subpopulation, replicate samples were obtained from different donors, including two for DP thymocytes, CB4 and early passage fetal TSC, and three for ITTPs, SP4 and AB4.
All samples were normalized using RMA, the method of choice for detection of differentially expressed transcripts. The average background level for arrays was 32 as determined by hybridization to negative controls. We established 3x the background (or 100) as a cut-off value for determining signal to noise. About 30% of transcripts (13 573/44 928) were called positive by this method. By comparison, we generally expect to see
50% of probe sets to be positive using the Affymetrix present/absent call system. Thus we believe this to be a very conservative method for determining present transcripts.
The data have been deposited in their entirety in the Gene Expression Omnibus website at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/geo) (GEO accession no. GSE1460). To determine the quality of the sample preparation and the relationship of expressed genes between different cell subpopulations, hierarchical clustering analysis was performed using the data from the entire gene set (see Fig. 2). In each case, replicate samples for a given subpopulation clustered together more closely than samples from any other subpopulation. ITTPs and DP thymocytes had expression profiles that were more closely related to each other than to those of SP4 thymocytes. SP4 thymocytes, in turn, demonstrated an expression profile that was not identical to those of naive CB4 and AB4 T cells. The naive CB4 and AB4 subpopulations were most closely related. Contrariwise, the expression profile of the non-hematopoietic, early passage fetal TSC population was not related to those of any of the CD4+ thymocyte or T cell subpopulations. In general, these results are consistent with the expected biological properties of each subpopulation, providing confidence in the methods used to isolate cells and to prepare RNA for analysis.

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Fig. 2. Hierarchical clustering of gene expression data for all subpopulations. Hierarchical clustering of all samples using all genes was performed using Pearson centered distance metric and complete linkage rule in Soochika version 1.0 (Strand Genomics, India). Replicates of each subpopulation are shown and are found to cluster more closely to each other than to those of other subpopulations.
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Two approaches were taken to determine in detail whether the arrays detected anticipated patterns of gene expression (Table 1). First, the T-lineage expression profiles were inspected for the presence of contaminating cell populations (Table 1, top). Marker genes of the monocyte, macrophage, dendritic cell, B cell, NK cell and erythroid lineages were not observed. Additionally, differential expression of IL3R
(CD123) was not observed in the ITTPs compared to all other subpopulations, making it unlikely that this subpopulation contained substantial numbers of phenotypically similar plasmacytoid dendritic cells (23). The expression profiles were then inspected for genes known to be important for T cell differentiation (Table 1, bottom). In most cases, there was a close match between expected and actual results: (i) differentiation markers found on all hematopoietic cells (e.g. CD45) were expressed at high levels in each of the T cell subpopulations, but not in early passage fetal TSC; (ii) those markers known to be present at early stages of T cell differentiation (e.g. CD1A and preTCR
) were down-regulated after the DP thymocyte stage; (iii) genes required for TCR rearrangement (e.g. RAG1, RAG2, TdT) were differentially expressed in ITTPs and in DP thymocytes compared to SP4 thymocytes and circulating naive CB4 and AB4 T cells; and (iv) genes required for TCR signaling (e.g. CD3
, CD3
and CD3
) were highly expressed in all of the T lineage subpopulations. In several cases (e.g. RAG2 and CD4), although genes were differentially expressed in a predictable pattern, their signal values were relatively low. In aggregate, these data suggest that the array profiles should reveal known as well as previously unappreciated changes in mRNA expression during various stages of T cell differentiation, but may underestimate the biological relevance of some transcripts.
Expression profiles reveal genes important for human T cell differentiation and function
To determine which genes might be expressed during human T cell differentiation, signal values from each of the T cell subpopulations were compared in a pairwise fashion to those obtained from early passage fetal TSC. 4084 transcripts were found to be expressed differentially (P < 0.05, two-tailed, unpaired t-test), 639 of which had an average signal value difference of >100 (>3-fold background) for the T cell subpopulations relative to early passage fetal TSC. Among these, 45 were expressed at a >10-fold higher level in every T cell subpopulation, 110 were >5-fold higher, and 269 were >3-fold higher (see Supplementary table 1). The first set of 45 (corresponding to 40 discrete) transcripts fell into several general classes of transcripts (see Table 2 for functional annotations and background references, and Fig. 3 for relative expression level). One class encodes molecules clearly associated with T cell differentiation, including nine known to be important for progression to the DP thymocyte stage (e.g. TRB, CD3
, PTPRC, LAT, BCL11B) or important for progression to the more mature SP state (e.g. CD3
, CD3
, LCK, SATB-1). A second class of genes encodes molecules known to play a significant role in immune function that is not absolutely T-lineage restricted (e.g. ZNFN1A1, LEF-1, IL7R, LTB, CXCR4, FYB, HCLS1, ICOS, SH2D1A and ARHGDIB). A third class of genes encodes molecules with plausible, but heretofore undocumented, roles in T cell differentiation and/or function. For example, overexpression of ITM2A in lymphocytes in transgenic mice results in partial downregulation of CD8 in DP thymocytes, indicating a potential role for it in intrathymic differentiation; CDW52 and MAL are located in lipid rafts and may play a role in TCR signaling; GMFG, a member of the glia maturation factor family, may inhibit the activity of ERK1/ERK2 in the context of TCR signaling since GMF stimulated by phorbol myristate acetate in astrocytes could inhibit ERK1/ERK2 activity; and CD53, a molecule that is upregulated at the late DP thymocyte stage, may play a role in intrathymic T cell selection. A fourth class of genes encodes molecules with an unknown contribution to T cell differentiation and function, including: UCP2 (which impacts on macrophage function by limiting reactive oxygen species), CORO1A (which may be important for phagocytic function), TXNIP (a regulator of cellular redox state), AQP3 (a water transporter), and CUGBP2 (an RNA binding protein). Finally, there are five hypothetical proteins and six ESTs with no known function.
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Table 2. Transcripts showing preferential (>10-fold higher) expression in T-lineage cells compared to early passage fetal TSC
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Fig. 3. Gene expression profile of selected genes. Signal values (in log2 space) from array data for selected transcripts of each category were plotted on the y-axis over cell types on the x-axis.
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Expression profiles distinguish between successive stages of T cell differentiation
To determine which genes might be preferentially expressed during discrete stages of T cell differentiation, the expression profiles were successively filtered to meet the following criteria (see Supplementary table 2 for details): (i) an overall average signal difference among all the T subpopulations that was >100 (to assure that any apparent differences had high signal to background/noise values: >3-fold background signal); (ii) a P-value < 0.05 using ANOVA (analysis of variance: KruskalWallis H test, considering the expression levels of each gene separately over all replicates in all subpopulations) among the signal values from all the T subpopulations; and (iii) a >3-fold average expression change overall among the lymphocyte subpopulations. This filter yielded a total of 1521 transcripts that, in turn, generated a self-organizing map (SOM) with six major expression clusters displayed across the range of T cell differentiation (see Fig. 4). Cluster 1 (C1) encompasses genes expressed at high levels during early stages of thymocyte differentiation (e.g. the ITTP and DP thymocyte stages) while C6 includes those expressed highly at later stages (e.g. SP4, CB4 and AB4). There are four additional clusters that demark intermediate stages: C2 (ITTP-specific), C3 (DP-specific), C4 (SP4-specific) and C5 (naive CB4 and AB4 T cell-specific). The greatest degree of expression change appears to occur at the transition between DP and SP4 thymocytes (see gene numbers within the clusters C1, C3, C4 and C6) while the least amount of change appears to exist between the profiles of naive-phenotype CB4 and AB4 T cells. Genes that were preferentially expressed in each cluster were inspected.

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Fig. 4. Self-organizing map (SOM) analysis of gene expression data for sorted subpopulations. 1521 transcripts were identified as being statistically and biologically significant (see text) and were analyzed using the self-organizing map (SOM) algorithm in Soochika version 1.0. Initial SOM analysis by 4x6 nodes gave 23 minor clusters (not shown). Then, similar nodes were merged by visual inspection into six major clusters. Cluster 1 (C1) (503 transcripts) showing high signal values (in red) in both ITTP and DP thymocytes and low signal values (in green) in SP4 thymocytes and in CB4, and AB4 T cells; C2 (159 transcripts) showing high signal values primarily in ITTP; C3 (135 transcripts) showing high signal values in DP thymocytes; C4 (98 transcripts) showing high signal values in SP4 thymocytes; C5 (352 transcripts) showing high signal values in the CB4 and AB4 naive T cell subpopulations. C6 (174 transcripts) shows the opposite expression pattern of C1, with high signal values in SP4 thymocytes and in CB4, and AB4 T cells.
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Early (C1) vs Late (C6) stages of T cell differentiation
134 transcripts in C1 showed >3-fold higher expression in the ITTP and DP thymocyte subpopulations than in the subpopulations of SP4 thymocytes and CB4 and AB4 T cells. Reciprocally, 76 transcripts in C6 showed >3-fold higher expression in the SP4 thymocytes and in CB4 and AB4 T cell subpopulations than in both ITTP and DP thymocyte subpopulations (see Supplementary table 3). Using public databases, many of these transcripts could be assigned to categories with distinct biological functions (Table 3). In less mature cells (e.g. ITTPs and DP thymocytes), gene expression was skewed (in 28 out of 57 categorized cases) towards those important in cell cycle regulation, cell cycle progression, mitosis, DNA replication, recombination, or repair. By contrast, genes encoding cell surface receptors dominated the expression profiles of more mature SP4 thymocytes and CB4 and AB4 T cells (in 18 out of 37 categorized cases). These results are reflective of the highly proliferative state of early progenitors and the fact that more differentiated cells often remain quiescent until triggered by external stimuli. It is interesting to note that different molecules were used for similar biological functions at varying stages of T cell maturation: (i) members of the lipid/glycolipid-presenting CD1 family were preferentially expressed in less mature cells while peptide-presenting MHC antigens were preferentially expressed in more mature cells (see Fig. 3 for relative expression level) (71); (ii) transcription factors AEBP1, UHRF1, TFDP2, MYB and GFI1 were represented in less mature cells while FOXO1A, KLF2, ID2, and SCML1 were represented in more mature cells; and (iii) less mature cells may express PACAP, FAIM and BIRC5 for regulation of apoptosis, while more mature cells may use STK17A, TNFRSF25 and TNFSF10.
ITTP-specific gene expression
Twenty-eight transcripts were expressed in ITTPs at levels 3-fold or more higher than in any other isolated T cell subpopulation (see Table 4 for the stage-specific gene list and their relevant references and Supplementary table 3 for the actual data). Some transcripts encode molecules known to be important for immune function (see Fig. 3 for relative expression level), e.g. HOXA9 (a transcription factor necessary for maturation of CD3CD4CD8 triple negative thymocytes), TLR7 (a Toll-like receptor important for antiviral immune response), IGJ (a peptide thought to be important in the stabilization of IgM and IgA) and TNFSF4 (a costimulator of T cell activation). Other expressed genes in this group have known molecular but unclear immunological functions, e.g. FXYD2 (a modulator of the sodium/potassium ATPase channel), GUCY1B3 (the beta component of guanylate cyclase complex), GUCY1A3 (the alpha component of guanylate cyclase complex) and two small monomeric members of Ras GTPase superfamily (RAB13 and RRAS2).
DP thymocyte-specific gene expression
Twenty-eight transcripts were expressed in DP thymocytes at levels 3-fold or more higher than in any other isolated T cell subpopulation (Table 4). Many of these genes have apparent relevance to thymocyte differentiation and/or T cell function (see Fig. 3 for relative expression level). For instance, RORC is necessary for DP thymocyte survival and positive selection; SLAM and SH2D1A (SLAM-associated protein) are important for T cell activation and for modulation of Th1 and Th2 responses; the CD1 family proteins, CD1C and CD1D, are essential for the development of a major subset of NK T cells; the proto-oncogene, BCL6, is necessary for germinal center formation and the establishment of memory T and B cell pools; CHS1 has been implicated in intracellular sorting of peptides and EFNB2 is involved with T cell co-stimulation. There are also several transcripts preferentially expressed in the DP thymocyte subpopulation that have no known relationship to the immune system, including: ELOVL4, an elongase of very long chain fatty acids associated with macular degeneration diseases; ANKRD3, an ankyrin repeat domain-containing kinase involved in PKC-mediated NF-
B activation; RASD1, a small GTPase of the RAS superfamily that inhibits GPCR signaling; CPLX1, a component of the exocytotic core complex involved in calcium-evoked exocytosis; SLC7A3, a cationic amino acid transporter; and BG1, an acyl-CoA synthetase.
SP4 thymocyte-specific gene expression
Sixteen transcripts were expressed in SP thymocytes at levels 3-fold or higher than in any other isolated T cell subpopulation (Table 4). Among these, several are known to be important for thymocyte differentiation and/or T cell function (see Fig. 3 for relative expression level): CTSL, a protein essential for the maturation of NK T cells and a subpopulation of CD4 T cells; IRAK2, which mediates activation of the NF-
B and MAPK pathways; IER3, an inhibitor of T cell apoptosis that helps to regulate T cell homeostasis during immune responses, and EGR3, an early growth response transcription factor contributing to T cell differentiation. There are several highly expressed genes in SP4 thymocytes with known molecular functions and unclear relevance to the immune system, including: two Ras/MAPK signaling pathway regulators (DUSP4 and SPRY2); HTR2B (a receptor for serotonin), important for heart development in vivo; CLDN1, a tight junction component necessary for mammalian epidermal barrier maintenance in vivo; NR4A2, a nuclear receptor necessary for dopamine neurogenesis and associated with Parkinson's disease; SERPINE2, a serine protease inhibitor involved in synaptic transmission in the brain; and PDE4D, a cAMP-specific phosphodiesterase important for normal growth, survival, and fertility.
Genes expressed specifically in both naive CB4 and AB4 T cells
Twenty-five transcripts were expressed in both naive CB4 and AB4 T cells at levels 3-fold or higher than in any thymocyte subpopulation (see Fig. 3 for relative expression level) (Table 4). Some of these (e.g. IL6R and IAN4L1) have clear relevance to immune function. Most, however, have known functions of uncertain relevance to the immune system. These highly expressed genes include: ARH, an adaptor molecule important for LDL receptor endocytosis; NOG, a TGF-ß antagonist important for patterning of the neural tube and cartilage morphogenesis; ATP10A, an aminophospholipid-transporting membrane ATPase important for normal behavior; PLEKHA1, a pleckstrin homology domain-containing protein that mediates phosphatidylinositol 3,4-bisphosphate-dependent cytoskeletal reorganization; PASK, a kinase implicated in sugar metabolism and translation; and two enzymes involved in nucleotide metabolism (UP and AK5).
Genes expressed in a pattern consistent with markers of recent thymic emigrants (RTE)
Candidate genes specific for RTEs could conceivably meet several criteria: (i) high expression in SP4 thymocytes, low or intermediate expression in CB4, and negligible expression in AB4 (SP4>CB4>AB4), or (ii) higher expression in naive phenotype CB4 than in naive phenotype AB4 T cells (CB4>AB4). As shown in Supplementary table 4, 32 transcripts satisfied the first criterion, with signal value differences >100 among the three compared subpopulations, expression levels >3-fold higher in SP4 thymocytes than in CB4 or AB4 cells, P-values < 0.05 between SP4 thymocytes and others, and expression levels higher in CB4 than in AB4. 240 transcripts satisfied the second criterion, with signal value differences >100 between CB4 and AB4, and expression levels >2-fold higher in naive CB4 compared to naive AB4 T cells. Within these two groups of transcripts, those that encode known or putative plasma membrane proteins with extracellular domains are amongst the most interesting as they might be used to phenotypically identify RTEs. Among four such genes in the first group (SP4>CB>AB), the expression level of two is only marginally (<1.5-fold; CD1B and CCRL2) higher in CB4 compared to AB4, while one is not expressed in CB4 (HTR2B: see Fig. 5); these are unlikely to represent candidate RTE markers. The other gene, also showing relatively low differences in signal values (1.5-fold), is a G protein-coupled receptor, CCR9. Among many transcripts found in the second group (CB4>AB4), several genes such as P2RX5 (3.5-fold higher expression in CB4 compared to AB4), C1QR1 (2-fold), and CCR7 (2-fold) were confirmed to be expressed differentially by independent RTPCR (see below and Fig. 5) among the cell surface molecules tested.

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Fig. 5. Confirmation of expression patterns of representative genes by semi-quantitative RTPCR. Top panel: Signal values (in log2 space) from array data for representative transcripts of each category were plotted on the y-axis over cell types on the x-axis. Bottom panel: RTPCR amplification products were loaded on a 1.5% agarose gel followed by ethidium bromide staining. The letters at the bottom refer to cell subpopulations from which the products were amplified: I (intrathymic T progenitor cells), D (double positive thymocytes), S (single positive CD4 thymocytes), C (cord blood CD4 cells), A (adult blood CD4 cells), and T (thymic stromal cells). Amplification of GAPDH in the linear range was carried out to evaluate the efficiency of the RTPCR reactions in different cell subpopulations. Two independent samples for each cell type were analyzed, with representative results shown here. The relative amounts of amplified products were measured by densitometry over the lowest, measured sample for each transcript as one: TLR7 (8, 2, 1, 1), RASD1 (1, 15, 1, 1), HTR2B (1,1,10,1), PACAP (15, 20, 4, 1), FOXO1A (1, 3, 9, 15), TSHR (15, 30, 1, 1), ARH (1, 4, 15, 15), CCR9 (4.5, 7.0, 1.5, 1.0), CCR7 (1, 3, 6, 3), P2RX5 (1.5, 3.0, 3.5, 1.0), C1QR1 (3, 6, 2,1), GMFG (20, 30, 20, 10, 1) and GAPDH (1.0, 1.3, 1.3, 1.3, 1.3, 1.3).
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To obtain independent confirmation of the microarray expression patterns, representative genes in each category were evaluated by semi-quantitative RTPCR. As shown in Fig. 5, expression patterns of most, if not all, were consistent with the microarray data: the expression of mRNA for TLR7 was ITTP-specific (>4-fold), RASD1 was DP-specific (>10-fold), HTR2B was SP4-specific (
10-fold), ARH was naive CD4 T-cell specific (>3-fold), PACAP was more highly expressed in immature ITTPs and DPs (>3-fold), FOXO1A was more highly expressed in mature SPs and CB cells (
3-fold) and GMFG expression was restricted to hematopoietic cells and not found in early passage fetal TSC (>10-fold).
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Discussion
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This study has generated a descriptive profile of expressed genes in discrete and successive stages of human T cell differentiation: ITTP, DP thymocyte, SP4 thymocyte and naive-phenotype CD4+ T cells from cord blood (CB4) and adult blood (AB4). The analysis appears to be a reliable reflection of the biological potential of each population: all subpopulations were enriched to >98% purity; replicate samples from different donors showed similar signal values; most genes known to be expressed in each subpopulation had high signal values while those known to be expressed in other, potentially-contaminating cell subpopulations were not observed; and expression patterns of a dozen representative genes, tested by independent semi-quantitative RTPCR, matched the results of the microarray analysis. Some broad and interesting patterns of gene expression were revealed. First, a series of transcripts is preferentially expressed in human T cell subpopulations and not in early passage fetal TSC. Second, SOM analysis indicates that major shifts in gene expression occur as progenitor T cells mature into progeny (e.g. from the DP to the SP4 thymocyte stages). Third, a large proportion of the transcripts that are highly expressed at the progenitor cell stage (e.g. within ITTP and DP thymocyte) are important for cell cycle progression or proliferation whereas those expressed at more mature stages (e.g. within SP4, CB4 and AB4 cells) are cell surface receptors. Fourth, many stage-specific transcripts are noticeable and these may be important for the associated biology of each unique stage of T cell differentiation. Finally, some transcripts are preferentially expressed in CB4 as opposed to AB4 cells and may represent markers of RTEs. To our knowledge, this is the most extensive published catalogue of genes enriched in discrete human T cell stages.
A large proportion of transcripts (19/29 in Table 2) that are enriched >10-fold in all T cell populations, but not in early passage fetal TSC, are known to have a significant role in various immunological functions, especially in thymopoiesis. Accordingly, exploring the role of other transcripts in this group might reveal important roles for them in T cell differentiation and functions as well. This exercise could be extended to the group of genes enriched >5-fold (110 transcripts) and 3-fold (269 transcripts) in all T cell populations; additional genes known to be important for T cell development and biology belong to this group (e.g. GATA3, TCF7 and VAV1 for the 5-fold group, and DOCK2 for the 3-fold group) (Supplementary table 1) (116,117,118,119).
Conservative criteria were used to select biologically significant genes: transcripts had to have signal values >3-fold above background and with >3-fold differential expression among the compared cell types. Since each transcript is probed with a different probeset, it is not possible to assign a minimum detection threshold across the full range of 44 928 transcripts and absolute transcript levels cannot be assigned on the basis of detected signal values. Possibly, some transcripts that are important for various stages of T cell differentiation may not have met the selection criteria and the biological significance of other transcripts (e.g. those for RAG2 and CD4) might have been underestimated. In the final analysis, our experimental approach assigns greatest confidence to the differential expression of genes that have relatively high signal levels.
Recently, expression profiles were generated for similar and/or overlapping populations of murine thymocytes (but not circulating naive CD4+ T cells) (120). Although this study used an array with a different gene set and studied somewhat different cell subpopulations, one finding is clear in each case: in less mature cell populations [e.g. ITTP and DP in human, and DN4 (CD44CD25CD4CD8)/DPL (CD4+CD8+Large) in mice], a large number of cell cycle-related genes is expressed. Another report has provided the gene expression profile of human hematopoietic stem cells and early lymphoid progenitors (121). In conjunction with this report, we now have a relatively complete view of the expression profiles of T progenitors through to the CD4 naive T cell stage.
Other than the broad patterns of gene expression profile summarized above among the cell types studied, several genes stand out as particularly interesting: PACAP, TSHR, CCR9 and CCR7. Compared to mature peripheral T cells, thymocytes (especially ITTP and DP) are known to be sensitive to various conditions/reagents (122) and may express a series of proapoptotic genes. Among those that are highly expressed in these immature cells (Table 3), PACAP, associated with caspase 2, is known to be proapoptotic in several cell lines and in primary B cells (123) and may drive immature thymocyte apoptosis through the caspase pathway. Among the surface molecules highly expressed in ITTP and DP (see Table 3 and Fig. 4), ligand binding through the TSHR (thyroid stimulating hormone receptor) might result in enhanced thymopoiesis. Indeed, constitutive activation of this receptor by autoantibodies is thought to be responsible for the thymic hyperplasia that is often associated with Graves' disease (124). Among the RTE candidates listed in Table 4, the most interesting candidates for markers include CCR9 (from the first group) and CCR7 (from the second group). CCR9 has been recently reported as a potential marker of RTEs in humans, with the proportion of circulating CCR9+ naive CD4+ T cells declining with age (6). The relatively high expression of CCR7 in CB4 naive T cells is interesting because it, along with CD62L, mediates homing of T cells to secondary lymphoid organs, it is expressed at high levels on resting but not activated T cells (125), and it also has reduced expression on CD4+ T cells as a function of age (126). The combination of these markers may serve to delineate RTEs with greater precision than existing markers. Further evaluation of these findings may lead to a better definition of human thymopoiesis as well as to improved approaches to monitor and to augment the function of this important organ of T cell production.
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Supplementary data
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Supplementary data are available at International Immunology Online.
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Acknowledgements
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This work was carried out in part in the General Clinical Research Center at San Francisco General Hospital and was supported by Grants R01-AI45865 and R37-AI40312 (to J.M.M.) from the NIAID and by Grant 5-MO1-RR00083 from the Division of Research Resources, National Institutes of Health. We are grateful to the members of the Gladstone Flow Cytometry Core (Marty Bigos, Cris Bare and Dax Arguello) for their assistance and advice with high quality sorting of lymphocyte subpopulations. We thank Dr Juan Vargas of the SFGH Department of Obstetrics, Gynecology, and Reproductive Biology for providing cord blood samples and Jose Rivera for providing fetal thymus from SCID-hu Thy/Liv mice. We thank Dr Jean Yang for helpful discussions regarding analysis of the microarray data.
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Abbreviations
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AB4 | naive CD4+ T cells from adult blood |
CB4 | naive CD4+ T cells from cord blood |
DP | double positive thymocyte |
ITTP | intrathymic T progenitor |
RTE | recent thymic emigrant |
SOM | self-organizing map |
SP4 | single positive 4 thymocyte |
SP8 | single positive 8 thymocyte |
TCR | T cell receptor |
TSC | thymic stromal culture |
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Notes
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Transmitting editor: P. W. Kincade
Received 12 February 2004,
accepted 17 May 2004.
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