0023-6837/03/8306-765$03.00/0
`LABORATORY INVESTIGATION
`Copyright © 2003 by The United States and Canadian Academy of Pathology, Inc.
`
`Vol. 83, No. 6, p. 765, 2003
`Printed in U.S.A.
`
`Comparative Microarray Analysis of Gene Expression
`During Activation of Human Peripheral Blood T Cells
`and Leukemic Jurkat T Cells
`Zhaosheng Lin, G. Chris Fillmore, Tae-Hyun Um, Kojo S. J. Elenitoba-Johnson, and
`Megan S. Lim
`Department of Pathology (GCF, T-HU, KSJE-J, MSL) and the ARUP Institute for Clinical and Experimental
`Pathology (ZL, KSJE-J, MSL), University of Utah, Salt Lake City, Utah
`
`SUMMARY: Activation of T cells involves a complex cascade of signal
`transduction pathways linking T-cell receptor
`engagement at the cell membrane to the transcription of multiple genes within the nucleus. The T-cell leukemia– derived cell line
`Jurkat has generally been used as a model system for the activation of T cells. However, genome-wide comprehensive studies
`investigating the activation status, and thus the appropriateness, of this cell line for this purpose have not been performed. We
`sought to compare the transcriptional profiles of phenotypically purified human CD2⫹ T cells with those of Jurkat T cells during
`T-cell activation, using cDNA microarrays containing 6912 genes. About 300 genes were up-regulated by more than 2-fold during
`activation of both peripheral blood (PB) T cells and Jurkat T cells. The number of down-regulated genes was significantly lower
`than that of up-regulated genes. Only 79 genes in PB T cells and 37 genes in Jurkat T cells were down-regulated by more than
`2-fold during activation. Comparison of gene expression during activation of Jurkat and PB T cells revealed a common set of
`genes that were up-regulated, such as Rho GTPase-activating protein 1, SKP2, CDC25A, T-cell specific transcription factor 7,
`cytoskeletal proteins, and signaling molecules. Genes that were commonly down-regulated in both PB T cells and Jurkat T cells
`included CDK inhibitors (p16, p19, p27), proapoptotic caspases, and the transcription factors c-fos and jun-B. After activation,
`71 genes in PB T cells and only 3 genes in Jurkat T cells were up-regulated 4-fold or more. Of these up-regulated genes and
`expressed sequence tags, 44 were constitutively expressed at high levels in nonactivated Jurkat cells. Quantitative real-time
`RT-PCR analysis confirmed our microarray data. Our findings indicate that although there is significant overlap in the
`activation-associated transcriptional profiles in PB T cells compared with Jurkat T cells, there is a subset of genes showing
`differential expression patterns during the activation of the two cell types. (Lab Invest 2003, 83:765–776).
`
`M any studies of T-cell activation have been per-
`
`formed using either the human leukemic Jurkat
`cell line (Black et al, 1997; Ghaffari-Tabrizi et al, 1999)
`or primary T cells (Cooper and Pellis, 1998; Ellisen et
`al, 2001). T-cell activation is associated with regulation
`of multiple intracellular signaling events, mediated by
`either protein tyrosine kinases and serine/threonine
`kinases (Rudd, 1999; Tsuchida et al, 1999). Several
`key processes have been well documented during
`T-cell activation, including the activation of phospho-
`lipase C-␥, which is required for TCR– dependent
`activation of IL-2 (Lindholm et al, 1999). Up-regulation
`of protein kinase C, a downstream target of phospho-
`lipase C-␥(Cooper and Pellis, 1998; Ghaffari-Tabrizi et
`al, 1999), results in the stimulation of AP-1 transcrip-
`tional activity, which activates the transcription of
`
`DOI: 10.1097/01.LAB.0000073130.58435.E5
`
`Received March 13, 2003.
`This work was supported by the ARUP Institute for Clinical and Experi-
`mental Pathology and National Institutes of Health grant CA 83984-01
`(to KSJ-EJ).
`Address reprint requests to: Dr. M. S. Lim, Department of Pathology,
`University of Utah, 50 North Medical Drive, Room A565, Salt Lake City,
`Utah 84132. E-mail: Megan.lim@path.utah.edu
`
`c-Jun-N-terminal kinase/stress-activated protein ki-
`nase (Ghaffari-Tabrizi et al, 1999). Other signaling
`molecules such as mitogen-activated/extracellular
`signal-regulated kinase and calcium ions are also
`involved in T-cell activation (Tsuchida et al, 1999).
`Both the leukemic Jurkat T-cell
`line (Black et al,
`1997; Ghaffari-Tabrizi et al, 1999; Tanaka et al, 1997;
`Whisler et al, 1994) and primary PB T cells (Cooper
`and Pellis, 1998; Ellisen et al, 2001; Whisler et al, 1993)
`are commonly used as a model for human T-cell
`activation. Genomic expression programs in primary
`PB T-cell activation have been reported (Diehn et al,
`2002; Ellisen et al, 2001; Feske et al, 2001); however,
`no genomic-scale comparison has been performed to
`analyze the activation-induced gene expression pro-
`files of these two T cell types.
`The use of cDNA microarray technology is an effec-
`tive approach for analysis of gene expression profiles
`in T-cell activation (Ellisen et al, 2001; Teague et al,
`1999). The technology is based on two-color fluores-
`cence hybridization in which two cDNA populations
`(from test and reference samples) are labeled sepa-
`rately with red or green fluorochrome and are subse-
`quently hybridized to a microarray containing thou-
`sands of predeposited cDNAs (Schena et al, 1995).
`The ratio of fluorescence intensity (red/green) reflects
`
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`Lin et al
`
`the relative expression level between the tested cDNA
`populations (such as the test sample and the control
`sample). The technology has been used in the analysis
`of gene expression patterns, signaling pathways, and
`identification of new functional genes on a genomic
`scale (Chtanova et al, 2001; Ellisen et al, 2001; Teague
`et al, 1999).
`In this study we sought to compare the gene ex-
`pression profiles of phenotypically purified human
`CD2⫹ T cells with those of Jurkat T cells during
`PHA-induced activation. The results show that general
`patterns of gene expression profiles were similar dur-
`ing activation of Jurkat T cells and peripheral blood
`(PB) T cells. However, a subset of genes showed
`differential expression with varying degrees of gene
`regulation between the Jurkat and PB T cells. This is
`the first comparative analysis of transcription profiles
`on a genomic scale between primary T cells and
`leukemic Jurkat T cells during T-cell activation
`(Gonzales and Bowden, 2002; Rowan et al, 1995).
`
`Results and Discussion
`Overall Gene Expression Patterns Between Jurkat and PB
`T Cells During Activation
`
`The baseline similarity of gene expression between
`Jurkat and PB T cells was evaluated by hybridizing
`cDNA from resting CD2⫹ PB T cells with cDNA from
`nonactivated Jurkat T cells. The correlation coefficient
`between gene expression profiles of the two T cell
`types was 0.674 (Fig. 1), indicating that the two T cell
`types were significantly different. By contrast, cohy-
`bridization of PB T-cell mRNA with themselves re-
`vealed a correlation coefficient of 0.99 (data not
`shown).
`To compare the transcriptional profiles of PB T cells
`and Jurkat T cells during activation, Cy-5-cDNAs from
`either activated Jurkat or PB T cells were hybridized
`with Cy-3-cDNAs from their nonactivated counter-
`parts. Genes that were expressed greater than 2-fold
`(2-fold higher than control) after activation were con-
`
`sidered overexpressed, whereas genes that were ex-
`pressed less than 0.5 fold (2-fold lower than control)
`were considered underexpressed. After 24 hours of
`activation, approximately 300 genes (4.3% of 6912
`genes tested) were overexpressed in each T cell type
`(Table 1). The number of underexpressed genes was
`much lower than that of the overexpressed genes in
`both T cell types, with only 79 genes (1.1% of 6912
`genes tested) in PB T cells and 37 genes (0.5% of
`6912 genes tested)
`in Jurkat T cells. The ratio of
`up-regulated genes, expressed sequence tags (ESTs),
`and unknown genes are also shown in Table 1; the
`accession numbers of
`these ESTs and unknown
`genes are listed in Tables 2 and 3. A significant portion
`of the up-regulated genes represents ESTs and un-
`known genes, highlighting the limited knowledge we
`have regarding the genes involved in T-cell activation.
`In PB T cells, the majority of the 6912 tested genes
`were expressed at similar levels (between the upper
`and lower blue lines in Fig. 1) during activation. This
`result is consistent with other reports that resting T
`cells are not in a quiescent stage; instead, there are
`many cellular transcriptional activities that are needed
`to maintain viability and mobility of unstimulated T
`cells (Boise et al, 1995; Teague et al, 1999; Vella et al,
`1998). Jurkat T cells are derived from a T-precursor
`lymphoblastic lymphoma (Black et al, 1997). As a
`result, many of the genes were expected to be con-
`stitutively expressed before activation treatment; how-
`ever, Jurkat T cells responded to activation treatment
`similar to resting T cells, as reflected by the similar
`numbers of up-regulated genes in both T cell types
`(Table 1).
`
`Functional Groups of Similarly Expressed Genes in Both
`Jurkat and PB T Cells During Activation
`
`Analysis of the 300 genes that were overexpressed in
`each cell type revealed that many were unknown
`genes and ESTs. This suggests the existence of many
`novel, as yet uncharacterized, genes that are involved
`
`Figure 1.
`Overall expression profile of 6912 genes between resting peripheral blood (PB) T cells and Jurkat T cells. Red and green data points indicate overexpressed and
`underexpressed genes, respectively. Genes above the upper blue lines represent those that were overexpressed by greater than 2-fold, whereas genes below the lower
`blue line represent those that were underexpressed by more than 2-fold.
`
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`

`Gene Expression in T-Cell Activation
`
`Table 1. Numbers of Differentially Expressed Genes During Activation of Jurkat and PB T Cellsa
`
`Experiments (Cy5 vs Cy3)
`
`ⱖ2-fold
`ⱖ2-fold
`ⱖ4-fold
`down-regulated
`up-regulated
`up-regulated
`(total/known genes:EST:unknown genes)
`
`Activated PB T cells vs Resting PB T cells
`Activated Jurkat vs Jurkat T cells
`
`71/10:4:57
`3/0:0:3
`
`300/76:30:194
`301/62:97:142
`
`79/28:34:17
`37/19:8:10
`
`a Fold changes shown represent the ratio of gene expression in activated vs nonactivated cells.
`
`Table 2. GenBank Accession Numbers of Overexpressed ESTs and Unknown Genes (>2-fold) During PB
`T-Cell Activationa
`
`19676
`13016
`13011
`20025
`21901
`18363
`23175
`20032
`12742
`300237
`23911
`23405
`12554
`11690
`23377
`23419
`
`150221
`249856
`135527
`233365
`134753
`135094
`123065
`123817
`154789
`295866
`197637
`194704
`193617
`246449
`711857
`141627
`198694
`121275
`110582
`110987
`303048
`206816
`23616
`23533
`23114
`22355
`23398
`21634
`23121
`23454
`20394
`19745
`19739
`19776
`21269
`13083
`22388
`21228
`19734
`21232
`20070
`
`23496
`23492
`9101
`7863
`23436
`23477
`23453
`23691
`11984
`21464
`21462
`23635
`20280
`18764
`15458
`23618
`11054
`20373
`20384
`20382
`8290
`19958
`19957
`18832
`19969
`7053
`23592
`23590
`9252
`23598
`23580
`23536
`23535
`22419
`23563
`19873
`12704
`22162
`20892
`20891
`21003
`
`23157
`20784
`23162
`23159
`23245
`23216
`23256
`23250
`23207
`21943
`12602
`20826
`21978
`23132
`15871
`10260
`21845
`17148
`16911
`23049
`23031
`23096
`23059
`7536
`16099
`20766
`23122
`23105
`20716
`12530
`21893
`23280
`20981
`23349
`23358
`10512
`20952
`20900
`168
`20916
`66686
`
`17979
`23964
`15787
`20563
`22782
`13404
`20538
`20512
`9683
`12406
`20618
`21755
`22859
`22815
`23967
`18197
`17017
`14557
`15578
`23714
`21540
`22688
`23713
`15521
`16657
`23707
`23703
`7275
`20492
`22701
`1588
`7268
`21589
`20419
`18925
`21597
`23010
`21790
`20620
`20640
`23007
`
`23384
`23380
`23375
`23364
`23363
`23366
`23365
`22265
`6660
`21150
`10586
`22246
`23388
`23385
`20236
`7049
`7039
`21388
`22495
`21332
`20228
`19836
`21173
`23188
`23181
`23396
`23393
`23362
`18493
`18457
`17373
`23520
`2355
`23524
`23523
`17573
`20150
`19787
`2317
`20178
`22342
`
`a This list was generated using GeneSpring software and the database available when this manuscript was prepared. Some of the genes listed are now identified
`known genes.
`
`in T-cell activation. Activation of T cells leads to
`expression of multiple genes that are involved in
`cellular events such as proliferation, cytoskeletal con-
`struction, apoptosis, and secretion of cytokines (El-
`lisen et al, 2001; Teague et al, 1999). To determine the
`
`functional groups of genes that were commonly up- or
`down-regulated in T-cell activation, we examined the
`identities of 93 overexpressed and 15 underexpressed
`genes in both Jurkat and PB T cells. Of the genes that
`were up- and down-regulated by 1.5 fold or more, the
`
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`

`Lin et al
`
`Table 3. GenBank Accession Numbers of Overexpressed ESTs and Unknown Genes (>2-fold) During Jurkat T-Cell
`Activationa
`
`346134
`233365
`127408
`294916
`211202
`346299
`247901
`245426
`128735
`246073
`134753
`154789
`156023
`193617
`155128
`340840
`67037
`207379
`21634
`23121
`144951
`292452
`203772
`196125
`209583
`199327
`196350
`122161
`23454
`292207
`198694
`135527
`276286
`711857
`128775
`292679
`196148
`201207
`66377
`135212
`135450
`
`293683
`129567
`129922
`292171
`144802
`132323
`128627
`130801
`138837
`194656
`234380
`194401
`194704
`193533
`197637
`195358
`295866
`142397
`137760
`137797
`160730
`244299
`139189
`248535
`297102
`247710
`121521
`38465
`293417
`110904
`243770
`110582
`66390
`245485
`294133
`206816
`110987
`122913
`123196
`122126
`123065
`
`127185
`120631
`246143
`196109
`295492
`295594
`128118
`20308
`22594
`21462
`19945
`21434
`6957
`23675
`19908
`21408
`18880
`8292
`16657
`17740
`8290
`19958
`19957
`18832
`21495
`11984
`21388
`19836
`19894
`8173
`9275
`9255
`9252
`19824
`9259
`23641
`15458
`23660
`23647
`23618
`18764
`
`23177
`8556
`21751
`22843
`21755
`9683
`510760
`241874
`17017
`17803
`7248
`7298
`17842
`21505
`23713
`23700
`15578
`23714
`18132
`23911
`22806
`15787
`18109
`18009
`10043
`17979
`20584
`23256
`23246
`22115
`20892
`15069
`23222
`8789
`15068
`23224
`12742
`20906
`10512
`20952
`9984
`
`8745
`20716
`17148
`16099
`21887
`18239
`23096
`23049
`15871
`10260
`23188
`23181
`17265
`972
`23175
`23137
`23132
`23162
`23155
`23023
`21279
`20116
`20178
`20156
`21266
`19734
`19721
`21253
`19737
`23562
`23542
`15521
`23580
`23536
`11813
`2317
`23535
`23523
`21232
`20007
`21150
`
`23380
`10586
`22265
`23477
`23471
`21228
`9101
`23465
`19696
`20025
`7863
`13963
`66864
`23362
`136801
`138168
`198961
`127542
`20280
`23592
`17373
`18457
`5546
`23399
`135094
`247082
`123817
`127710
`7053
`23606
`3210
`23297
`21173
`20014
`
`a This list was generated using GeneSpring software and the database available when this manuscript was prepared. Some of the genes listed are now identified
`known genes.
`
`genes with known functions were grouped together
`(Tables 4 and 5). Among the known up-regulated
`genes (Table 4), several genes involved in cellular
`proliferation, cell adhesion, and cell cycle progres-
`sion were identified, including CDC kinases, inte-
`grin, and Rho-activating protein-encoding genes
`(Klekotka et al, 2001; Lew and Reed, 1993; Welsh
`and Assoian, 2000). Genes encoding for protein
`synthesis machinery (eukaryotic translational termi-
`nation factor 1, ribosomal proteins L7a, L31, L21),
`cytoskeletal elements (myosin, actin, tubulin, calpo-
`nin), and cell surface antigens (CD68, CD36, CD47)
`were also commonly up-regulated in both cell types
`during activation.
`Interestingly, a group of KIAA
`genes encoding proteins with potential signaling
`
`function, involved in cell migration (Chen et al, 2002)
`and metastasis (Lee et al, 2002), were also up-
`regulated upon activation.
`The down-regulated genes in both activated Jurkat
`and PB T cells consisted mostly of genes encoding
`CDK inhibitors, proapoptotic caspases, and transcrip-
`tion factors C-Fos and Jun-B (Table 5). Down-
`regulation of CDK inhibitors is critical for inhibition of
`cell cycle progression. The stimulation of cell prolifer-
`ation via PHA activation has been shown to induce the
`down-regulation of p27Kip and p21Waf CDK inhibitors,
`resulting in the release of cells into G2/S phase of the
`cell cycle (Polyak et al, 1994; Sawada et al, 1996). The
`fact that some cytokines such as IL-4 (Boise et al,
`1995; Vella et al, 1998) can only induce activated T
`
`768 Laboratory Investigation • June 2003 • Volume 83 • Number 6
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`

`Table 4. Functional Groups of Genes That Were Up-Regulated in Both Jurkat and PB T Cells During Activationa
`
`Gene Expression in T-Cell Activation
`
`Genban
`
`Common
`name
`
`Description
`
`Fold change in expression
`
`Activated PB T
`cells vs resting
`PB T cells
`
`Activated Jurkat vs
`nontreated Jurkat T
`cells
`
`Proliferation/cell cycles
`AA443506
`ARHGAP1
`AA487634
`ARHGDIB
`R22239
`SKP2
`H15504
`ANXA7
`R48796
`ITGAL
`W44701
`IL3RA
`R09063
`CDC25A
`H75547
`CLK1
`AA459292
`CKS1
`Antiapoptosis
`H21071
`Transcription
`factors
`AA480071
`
`NAIP
`
`TCF7
`
`TFAP4
`
`RPL5
`RPL10A
`RPL21
`RPS14
`
`CNN2
`MYL6
`KRT8
`ACTG2
`TUBA1
`
`MERTK
`BRCA1
`
`CD36
`CD47
`CD68
`CD81
`
`AA284693
`Protein synthesis
`AA496838
`R01139
`AA464034
`H73727
`Cytoskeleton
`AA284568
`AA488346
`W32281
`T60048
`AA180912
`Proto-oncogenes
`AA436591
`H90415
`Surface antigens
`H69048
`AA455448
`AA421296
`AA486653
`Potential signaling
`molecules
`H05563
`AA456352
`N22435
`T90374
`R91264
`N79669
`AA486524
`T74606
`
`Rho GTPase-activating protein 1
`Rho GDP dissociation inhibitor (GDI) beta
`S-phase kinase-associated protein 2 (p45)
`Annexin A7
`Integrin, alpha L [antigen CD11A (p180)]
`Interleukin 3 receptor, alpha
`Cell division cycle 25A
`CDC-like kinase 1
`CDC28 protein kinase 1
`
`Neuronal apoptosis inhibitory protein
`
`Transcription factor 7 (T-cell specific,
`HMG-box)
`Transcription factor AP-4
`
`Ribosomal protein L5
`Ribosomal protein L10a
`Ribosomal protein L21
`Ribosomal protein S14
`
`Calponin 2
`Myosin, light polypeptide 6
`Keratin 8
`Actin, gamma 2
`Tubulin, alpha 1 (testis specific)
`
`c-mer proto-oncogene tyrosine kinase
`Breast cancer 1, early onset
`
`CD36 antigen (collagen type I receptor)
`CD47 antigen (Rh-related antigen)
`CD68 antigen
`CD81 antigen
`
`2.1
`1.5
`1.5
`1.5
`1.5
`1.5
`3.6
`1.5
`1.5
`
`1.9
`
`1.5
`
`1.5
`
`1.9
`2.0
`3.2
`3.6
`
`1.7
`2.1
`1.5
`1.7
`1.5
`
`1.5
`1.5
`
`1.5
`2.1
`2.3
`1.9
`
`1.6
`1.6
`1.7
`1.7
`1.7
`1.7
`2.1
`1.8
`
`2.3
`1.5
`1.8
`1.9
`1.8
`1.6
`1.6
`1.9
`1.5
`
`2.7
`
`2.0
`
`1.7
`
`1.6
`1.5
`1.7
`1.8
`
`2.1
`1.7
`1.9
`1.6
`1.8
`
`1.5
`2.0
`
`1.6
`2.1
`2.6
`1.6
`
`2.3
`1.6
`1.6
`2.5
`2.0
`2.0
`2.4
`2.3
`
`KIAA0182
`KIAA0224
`KIAA0229
`KIAA0798
`KIAA0205
`KIAA0231
`KIAA0264
`KIAA0057
`
`KIAA0182 protein
`KIAA0224 gene product
`KIAA0229 protein
`KIAA0798 gene product
`KIAA0205 gene product
`KIAA0231 protein
`KIAA0264 protein
`KIAA0057 gene product
`
`a Fold changes shown represent the ratio of gene expression in activated vs nonactivated cells.
`
`cells (but not resting T cells) to proliferate also sup-
`ports these data.
`Caspases are a group of proteases that are gener-
`ally associated with apoptosis. Activation of several
`caspases including caspase-1, -3, -8, and -9 has been
`reported to induce T-cell apoptosis (MacFarlane et al,
`
`2000; Morley et al, 2000; Ruiz et al, 2001; Takahashi et
`al, 2001; Varghese et al, 2001). Our data showed that
`several caspases (caspase-1, -3, -4, -7, -8, -9) were
`down-regulated in both Jurkat and PB T cells after 24
`hours of activation (Table 5). This occurred during the
`period of T-cell proliferation in which cell numbers
`
`Laboratory Investigation • June 2003 • Volume 83 • Number 6 769
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`Lin et al
`
`Table 5. Functional Groups of Genes That Were Down-Regulated During Activation of Both Jurkat and PB T Cellsa
`
`Gene name
`
`Cell growth and/or maintenance
`p16
`p19
`p27
`CDKI 3
`Death
`caspase 1
`caspase 3
`caspase 4
`caspase 7
`caspase 8
`caspase 9
`Transcription regulator
`C-fos
`jun-B
`
`Fold change in expression
`
`Activated PB T cells
`vs resting PB T cells
`
`Activated Jurkat vs
`nontreated Jurkat T cells
`
`⫺2.2
`⫺2.1
`⫺1.8
`⫺2.2
`
`⫺1.7
`⫺1.5
`⫺1.9
`⫺1.5
`⫺1.5
`⫺1.9
`
`⫺1.8
`⫺1.5
`
`⫺3.0
`⫺2.5
`⫺2.1
`⫺2.4
`
`⫺2.6
`⫺1.9
`⫺1.9
`⫺2.4
`⫺1.6
`⫺2.5
`
`⫺2.1
`⫺1.9
`
`a Fold changes shown represent the ratio of gene expression in activated vs nonactivated cells.
`
`increased by 3-fold (data not shown). This is consis-
`tent with a previous report demonstrating that the
`activation of caspase 1 and 3 in CD4⫹ and CD8⫹ T
`cells was time dependent. The maximum activities of
`caspase-1 and -3 were observed after the proliferative
`period, at Days 7 and 10 (Ruiz et al, 2001).
`The proto-oncogenes jun-B and c-fos are generally
`involved in proliferation-associated transcriptional ac-
`tivation (Sylvester et al, 1998; Wang et al, 1997); thus,
`our results of their down-regulation were unexpected.
`Our results are, however, in keeping with those of
`Ellisen et al (2001) who observed down-regulation of
`jun-B and c-fos in ConA-treated PB T cells. In our
`experiments, jun-B and c-fos were down-regulated in
`both Jurkat and PB T cells during activation; this is
`consistent with other reports (Kreuzer et al, 1997;
`Passegue and Wagner, 2000) that C-Fos and Jun-B
`may be involved in the transcriptional activation of
`antiproliferative genes such as p16INK4a.
`Diehn et al (2002) performed a study of gene ex-
`pression profiles during PB T-cell activation. To com-
`pare our results with theirs, we used their web-based
`GeneXplorer software and Figure 1 (available at http://
`genome-www.stanford.edu/costimulation)
`to search
`for the expression levels of 22 randomly selected
`genes from our list of genes that were up- or down-
`regulated during T-cell activation. The expression lev-
`els of these genes showed a high level of concordance
`between the two studies (18 of 22). Among the con-
`sistently expressed genes, there were proliferation-
`related genes (four), CDK inhibitors (four), caspases
`(three), and transcription factors (two).
`
`Differences in Gene Expression Between Jurkat and PB T
`Cells During Activation
`
`Interestingly, many activation-induced genes in PB T
`cells were constitutively overexpressed to similar lev-
`
`els in the non-activated Jurkat T cells (Fig. 2, A and C).
`Of the 300 overexpressed (by 2-fold) genes and ESTs,
`more than one-third (118 genes and ESTs) were
`expressed at similar levels in nonactivated Jurkat cells
`(Fig. 2A, Fig. 3). Although the vast majority of genes
`were similarly expressed between Jurkat and PB T
`cells during activation, one of the most significant
`differences between the expression profiles of the two
`cell types was the total number of genes that were
`expressed greater than 4-fold after activation (Table
`1). Although 71 genes and ESTs were expressed
`greater than 4-fold in PB T cells, only three genes were
`overexpressed to this degree in Jurkat T cells. Among
`the ⵑ300 2-fold up-regulated genes during T-cell
`activation, there were only 93 genes (31%) that were
`common between the two T cell types (Fig. 3).
`Among the 71 genes and ESTs overexpressed by
`4-fold or higher in activated PB T cells, more than half
`were constitutively overexpressed in nontreated Jur-
`kat cells (Fig. 2C). These data suggest that when using
`Jurkat cells as model of T-cell activation, the number
`of activated genes may be underrepresented because
`many are constitutively overexpressed. The constitu-
`tively overexpressed genes (greater than 2-fold)
`in
`Jurkat cells (Table 6) most likely reflect the activated
`status of these leukemic T cells and probably are
`important in the pathogenesis of the neoplasm. Similar
`observations have been made when expression of
`phosphoinositide (PI)3-kinase was compared between
`PB T cells and Jurkat cells (Astoul et al, 2001). The
`finding that many of the constitutively overexpressed
`genes in Jurkat cells were also overexpressed in
`activated PB T cells suggests that genes induced
`during activation of T cells may be involved in leuke-
`mogenesis. Indeed, growth factors such as transform-
`ing growth factor-␤and TNF activate several signaling
`pathways including the PI3-kinase pathway (Kim et al,
`2002) and the nuclear factor-␬B pathway (Natoli et al,
`
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`Gene Expression in T-Cell Activation
`
`Figure 2.
`Distribution of genes overexpressed greater than 2-fold during activation. A, Of the 6912 genes tested, 300 were overexpressed greater than 2-fold in PB T cells (a).
`Among these 300 overexpressed genes, 93 genes were also overexpressed in Jurkat T cells during activation (b), and 118 genes were expressed more than 2-fold
`higher in nonactivated Jurkat than in resting PB T cells (c). B, Profile of 301 genes overexpressed more than 2-fold in Jurkat T cells (a) during activation, in PB T
`cells (b) during activation, and in resting PB T cells compared with nonactivated Jurkat T cells (c). C, Profile of 71 genes up-regulated by 4-fold in PB T cells (a) during
`activation, in Jurkat T cells (b) during activation, and in resting PB T cells compared with nonactivated Jurkat T cells (c). Red points indicate overexpressed genes
`and green points represent underexpressed genes. Genes above the upper blue line were overexpressed by more than 2-fold, whereas genes below the lower blue
`line were underexpressed by more than 2-fold.
`
`1998) to ultimately induce cell survival and inhibit
`apoptosis. The expression of
`transcription factors
`such as jun and fos are also activated by PI3-kinase
`and AKT (Duan et al, 2002; Walsh et al, 2002) and
`contribute to T-cell survival. Cross-linking of cell sur-
`face proteins such as CDW52 (CAMPATH-1 antigen),
`a glycopeptide antigen expressed on T and B lympho-
`cytes, by anti-CD52 antibody mediates effective
`growth inhibition, supporting its important role in
`T-cell transformation (Rowan et al, 1998). Another cell
`
`surface antigen, endoglin (a recently recognized com-
`ponent of the transforming growth factor-␤ receptor
`complex), is a strong marker of neovascularization and
`may thus play a role in Jurkat T-cell leukemogenesis
`(Calabro et al, 2003). Furthermore, several other genes
`in this list (Table 6) may be influenced by the persistent
`activation of the PI3-kinase pathway. For example,
`matrix metalloproteinase secretion in some cell types
`occurs via the PI3 kinase-AKT pathway (Ellerbroek et
`al, 2001). Matrix metalloproteinases are critical for cell
`
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`Lin et al
`
`Figure 3.
`A Venn diagram showing the overlaps of up-regulated genes during T-cell
`activation. Among the 300 genes that were up-regulated more than 2-fold (left
`circle) during PB T-cell activation, 93 of them were also overexpressed more
`than 2-fold during activation of Jurkat T cells (right circle). Approximately 118
`genes (bottom circle) up-regulated during PB T cell activation were constitu-
`tively up-regulated in nonstimulated Jurkat T cells compared with resting PB
`T cells.
`
`adhesive and invasive properties of malignant cells
`including leukemic T cells (Stetler-Stevenson et al,
`1997) and may contribute to the neoplastic phenotype
`of Jurkat T cells. Further functional studies of these
`differentially expressed genes may lead to identifica-
`tion of novel leukemia-specific genes.
`
`Validation of Gene Expression by Real-Time RT-PCR
`
`To validate the gene expression patterns observed in
`our microarray experiments, we used the same source
`of mRNAs to perform quantitative fluorescent real-
`time RT-PCR (Elenitoba-Johnson et al, 2002). The
`RT-PCR quantitation of nine selected gene transcripts
`(Table 7) showed that the trend of gene expression
`during activation of both Jurkat and PB T cells was
`consistent between the microarray and RT-PCR data.
`The overall correlation coefficient between relative
`expression levels from the microarray analysis and
`from RT-PCR was 0.81. Using this methodology, our
`previous validation of microarray data has shown
`approximately 70% concordance (Fillmore et al, 2002;
`Robetorye et al, 2002).
`In summary, comparative microarray studies of
`PHA-induced activation of PB T cells and Jurkat cells
`reveal that genes associated with proliferation, protein
`synthesis, cytoskeletal construction, and signaling
`were similarly up-regulated, whereas genes associ-
`ated with cell cycle inhibition and apoptosis were
`down-regulated at similar levels. A subset of genes
`induced during PB T-cell activation, however, were
`constitutively expressed at high levels in nonactivated
`
`772 Laboratory Investigation • June 2003 • Volume 83 • Number 6
`
`Jurkat cells, suggesting a role for these genes in the
`neoplastic transformation and/or biology of T-ALL.
`Finally, with the exception of a small subset of genes,
`the high degree of overlap between the gene expres-
`sion profiles of the two cell types suggests that Jurkat
`T cells remain a suitable model for T-cell activation.
`
`Materials and Methods
`Isolation of Human PB T Cells
`
`Fresh PB was obtained from normal donors. To isolate
`T cells, buffy coat samples were collected from blood
`using Ficoll-Paque (Amersham Pharmacia, Piscat-
`away, New Jersey), following the manufacturer’s in-
`structions. CD2⫹ T cells were then isolated by Dynal
`beads coated with CD2-specific mAbs (Dynal CD2
`CELLection Kit; DYNAL Biotech, Oslo, Norway), fol-
`lowing the manufacturer’s protocol.
`
`T-Cell Activation
`
`Both CD2⫹ T cells and leukemic Jurkat T cells were
`cultured in RPMI-1640 (Life Technologies, Rockville,
`Maryland) containing 10% fetal bovine serum and
`activated in the same medium supplemented with 1%
`(v/v) PHA (Life Technologies, Carlsbad, California) for
`24 hours at 37° C in 5% CO2.
`
`Separation of Total RNA and mRNA
`
`Total RNA was extracted by using the TRIZol reagent
`(Life Technologies, Carlsbad, California). Messenger
`RNA was purified from total RNA using poly(T)-coated
`beads (Oligotex mRNA Isolation Kit; Qiagen, Valencia,
`California), according to the manufacturer’s protocol.
`
`Preparation and Hybridization of Fluorescent-Labeled
`cDNA
`
`For each comparative array hybridization, fluores-
`cent cDNA was synthesized by reverse transcription
`from a test sample (activated T-cell mRNA or acti-
`vated Jurkat mRNA) in the presence of Cy5-dUTP
`and from a reference sample (nonactivated T-cell
`mRNA or Jurkat mRNA) with Cy3-dUTP, using the
`Superscript II RT kit (Life Technologies, Carlsbad,
`California) with several modifications. The RT reac-
`tion mixture contained 0.5 mM dATP, dCTP, and
`dGTP, 0.2 mM dTTP, and 0.1 mM Cy5- or Cy3-dUTP
`(Amersham Pharmacia). The labeling reaction was
`stopped by the addition of 5 ␮l of 500 mM EDTA. The
`mRNA was hydrolyzed by adding 10 ␮l of 1 N NaOH
`and heating to 65° C for 1 hour. The resulting
`alkaline solution was neutralized with 1 M Tris-HCl
`(pH 7.5). Unincorporated nucleotides were removed
`using a Biospin 6 column (Bio-Rad Laboratories,
`Hercules, California). The two fluorescently labeled
`cDNA samples were mixed, concentrated using a
`Microcon 30 microconcentrator (Amicon Inc., Bev-
`erly, Massachusetts), and hybridized to an arrayed
`slide (Research Genetics, Huntsville, Alabama) con-
`taining 6912 clones as described previously (Manos
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`Table 6. Constitutively Overexpressed Genes and ESTs in Nontreated Jurkat T cellsa in Comparison to Nonactivated PB T
`Cells
`
`Gene Expression in T-Cell Activation
`
`GenBank/Unigene
`
`Cell growth and/or maintenance
`Hs.153889
`Hs.2017
`Hs.2206
`Transcription regulator
`Hs.75889
`Hs.83428
`
`Hs.44450
`Hs.169465
`Cellular
`component
`AA278759
`AA410265
`Hs.1908
`Others
`R38270
`AA410517
`N73222
`R38105
`H70017
`H17882
`R47979
`AA446108
`Hs.2248
`Hs.75462
`Hs.100000
`Hs.112405
`Hs.59847
`Hs.29748
`Hs.28102
`Hs.3452
`Hs.151031
`Hs.77961
`Hs.159120
`Hs.3842
`Hs.10590
`Hs.155609
`Hs.15953
`Hs.76807
`Hs.21278
`Hs.180029
`Hs.62954
`Hs.347
`Hs.155485
`Hs.151738
`Hs.8249
`Hs.16291
`Hs.8248
`Hs.13059
`R39288
`
`Description
`
`Transforming growth factor-␤ precursor
`Latent transforming growth factor-␤ binding protein 1
`TNF type 2 receptor-associated protein
`
`V-jun avian sarcoma virus 17 oncogene homolog
`Nuclear factor of kappa light polypeptide gene enhancer in B
`cells
`Sp3 transcription factor
`FOS-related antigen 1
`
`Proteoglycan 1, secretory granule
`Lysosomal-associated multispanning membrane protein-5
`Hematopoetic proteoglycan core protein
`
`Homo sapiens clone 24734 mRNA sequence
`Protease inhibitor 6 (placental thrombin inhibitor)
`ESTs, moderately similar to hypothetical protein (H. sapiens)
`ESTs
`Mannosidase, alpha, class 2A, member 1
`Kallmann syndrome 1 sequence
`Human HLA-DR ␣-chain mRNA
`Endoglin
`Interferon ␥-induced protein 10
`Human BTG2
`S100 calcium-binding protein A8 (calgranulin A)
`S100 calcium-binding protein A9 (calgranulin B)
`ESTs
`ESTs
`ESTs
`ESTs
`Human Aac11
`Major histocompatibility complex, class I, C
`CDW52 antigen (CAMPATH-1 antigen)
`Homo sapiens ATPase homolog
`ESTs
`CAMP-dependent protein kinase regulatory subunit type I
`ESTs
`HLA class II histocompatibility antigen
`ESTs
`Human guanine nucleotide exchange factor
`Enolase 1 (alpha)
`Lactotransferrin
`Huntingtin-interacting protein 2
`Matrix metalloproteinase 2
`ESTs
`ESTs
`Ubiquinone oxidoreductase precursor
`ESTs
`ESTs
`
`a These genes were overexpressed greater than 4-fold during PB T-cell activation.
`
`and Jones, 2001). Hybridization