Open Access

Transcriptional signatures of Itk-deficient CD3+, CD4+ and CD8+ T-cells

  • K Emelie M Blomberg1Email author,
  • Nicole Boucheron2,
  • Jessica M Lindvall3,
  • Liang Yu1,
  • Julia Raberger2,
  • Anna Berglöf1,
  • Wilfried Ellmeier2 and
  • CI Edvard Smith1Email author
BMC Genomics200910:233

DOI: 10.1186/1471-2164-10-233

Received: 06 November 2008

Accepted: 18 May 2009

Published: 18 May 2009

Abstract

Background

The Tec-family kinase Itk plays an important role during T-cell activation and function, and controls also conventional versus innate-like T-cell development. We have characterized the transcriptome of Itk-deficient CD3+ T-cells, including CD4+ and CD8+ subsets, using Affymetrix microarrays.

Results

The largest difference between Itk-/- and Wt CD3+ T-cells was found in unstimulated cells, e.g. for killer cell lectin-like receptors. Compared to anti-CD3-stimulation, anti-CD3/CD28 significantly decreased the number of transcripts suggesting that the CD28 co-stimulatory pathway is mainly independent of Itk. The signatures of CD4+ and CD8+ T-cell subsets identified a greater differential expression than in total CD3+ cells. Cyclosporin A (CsA)-treatment had a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Bioinformatic analysis of NFAT-sites of the group of transcripts similarly regulated by Itk-deficiency and CsA-treatment, followed by chromatin-immunoprecipitation, revealed NFATc1-binding to the Bub1, IL7R, Ctla2a, Ctla2b, and Schlafen1 genes. Finally, to identify transcripts that are regulated by Tec-family kinases in general, we compared the expression profile of Itk-deficient T-cells with that of Btk-deficient B-cells and a common set of transcripts was found.

Conclusion

Taken together, our study provides a general overview about the global transcriptional changes in the absence of Itk.

Background

The Tec family of non-receptor protein-tyrosine kinases consists of five members (Bmx, Btk, Itk, Rlk/Txk and Tec); T-cells express Itk, Rlk and Tec, and T-cell receptor (TCR) stimulation leads to the activation of Tec family kinases [1, 2]. A large number of biochemical studies and the generation of mice that are single- or double-deficient for Itk, Tec or Rlk have identified important roles, in particular for Itk, during T-cell development and activation, and in Th2 effector differentiation. Itk-/- mice show impaired positive selection of CD4+ T-cells and it was suggested that Itk modulates signaling thresholds during T-cell development [35]. TCR signaling in naïve T-cells, and therefore activation and proliferation, is impaired in the absence of Itk, and Itk-/- T-cells show defective Th2 polarization [6]. Further, Itk regulates the actin cytoskeleton and is therefore necessary for proper synapse formation and for efficient T-cell activation [7, 8]. More recent data indicate that Itk is involved in signaling pathways that regulate conventional versus innate-like T-cell development. The majority of CD8+ T-cells from Itk-/- as well as from Itk-/-Rlk-/- mice show a more "innate-like" T-cell phenotype, sharing characteristics with conventional memory T-cells, i.e. CD44hi, CD62L- and CD122hi [911]. These cells depend on IL-15, express TCRs specific for non-classical MHC class Ib molecules, and exhibit direct effector functions such as rapid IFNγ production upon PMA/ionomycin stimulation [912]. A significant fraction of innate-like CD44hiCD62L- T-cells has also been described for the CD4+ T-cell lineage in Itk-/- mice [13].

Biochemically, the defects in T-cell activation were linked to an impaired phospholipase C-γ (PLCγ) phosphorylation and activation [5]. PLCγ hydrolyzes phosphatidylinositol-4,5-biphosphate to produce inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 induces the release of intracellular calcium (Ca2+) thereby activating the serine/threonine phosphatase calcineurin. Itk activation results in high levels of IP3, which is required for Ca2+ entry via store-operated channels leading to increased Ca2+ in cells stimulated via the TCR [5]. Both Ca2+ and calmodulin will bind and activate calcineurin, which in turn dephosphorylates serines in the regulatory domain of cytosolic NFAT. This induces a conformational change in NFAT exposing nuclear localization signals allowing its transport into the nucleus [14]. In Itk-deficient T-cells the Ca2+-levels are reduced resulting in impaired NFAT translocation [6]. Mice deficient in NFAT family members share phenotypes with Tec kinase family-deficient mice, as described by Lucas et al. [15]. The NFAT family was first described as binding to and controlling the interleukin 2 (IL-2) promoter and other lymphokine promoters in T-cells [14]. The family consists of five members; NFATc1–4 and NFAT5 [16, 17]. Efficient inhibitors for the activation of NFAT proteins have been developed. Two of these, Cyclosporin A (CsA) and FK506, indirectly inhibit NFAT by blocking the enzymatic activity of calcineurin.

In order to further decipher the role of Itk we have investigated changes in gene expression of CD3+ as well as CD4+ and CD8+ T-lymphocytes in normal and Itk-defective mice. The aim of the study was to (1) define the transcriptome in unstimulated cells, (2) elucidate the influence of anti-CD3 and anti-CD3/CD28-stimulation and (3) to dissect which part of the observed alterations in Itk-deficiency is dependent on the calcineurin/NFAT pathway.

Methods

Mice and generation of T-cells

CD3+ as well as CD4+ and CD8+ T-cells from pooled suspensions of spleen and lymph nodes of Wt and Itk-/- mice on C57BL/6 background were isolated by negative depletion; antibodies used are listed in Additional file 1. The cell suspensions were incubated with the antibodies in PBS supplemented with 2% FCS. Streptavidin beads (BD Pharmingen) were used for negative depletion according to manufacturer's instructions. The purity of the cells was assessed by flow cytometry and was routinely >90% CD3+, >96% CD4+ and >90% CD8+ T-cells. All animal experiments were approved by the Federal Ministry for Science and Research.

T-cell stimulations and Cyclosporin A treatment

Unstimulated as well as stimulated T-cells were studied. Stimulations were performed in 48-well plates, coated with anti-CD3 (1 μg/ml) with or without anti-CD28 (3 μg/ml) in the presence or absence of CsA (1 μg/ml) for 24 hrs. For each stimulus, at least duplicate samples were used in all but one experiment. For the CD4+ T-cells we collected triplicates from the Itk-/- mice and duplicates from the Wt group. For the CD8+ T-cells, we got duplicates from Itk-/-, while we obtained a single sample from Wt owing to the low cell yield for resting Wt CD8+ T-cells. After anti-CD3-stimulation we got a single sample from the CD8+ subset of both Wt and Itk-/-, while for the CD4+ subsets we collected duplicates. To control if the number of differentially expressed probe-sets was truthful in the CD8+ subset, and not due to the lack of replicates, we analyzed the CD4+ in the same way as the CD8+ T-cells. The results were consistent and we found the same number of differentially expressed transcripts when single CD4+ samples were studied separately.

RNA isolation and microarray processing

RNA isolations were done according to RNeasy Mini protocol (Qiagen, Valencia, CA, USA) and microarray processing as previously described [18]. The Affymetrix MOE430 2.0 chips were used. In total 37 arrays were analyzed. The microarray data are accessible through the Gene Expression Omnibus (GEO; GSE12466) [19, 20].

Data and statistical analysis

The processing and primary data analysis was performed in DNA-Chip Analyzer (dChip) [21]. In short, the invariant set normalization method was used [22]. Thereafter, model-based expression values were calculated according to the perfect match (PM)-only model. The criterion for fold-change analysis was set to ≥ 2-fold between groups. Signal values were then used in further statistical analysis steps such as paired and unpaired Student t-test in Excel. Some comparisons were performed using the chi square (χ2) test. Immunoglobulin and histocompatibility transcripts were excluded from the tables, since changes in their expression may be secondary to events unrelated to Itk-deficiency. Also the Xist and Tsix transcripts, X-chromosome encoded and unique to females, as well as Y-specific mRNAs were removed owing to that we used mixed sexes of mice in the experiments. One probe set that corresponded to Itk (1456836_at) was found to also be complementary to an EST gene (recognized at Ensembl) [23] and was therefore only included in the Additional material. We manually annotated a group of genes on the basis of prior knowledge about their role in the immune system. The 900 probe-sets list of differentially expressed transcripts between unstimulated Itk-/- and Wt T-cells was used for this purpose. The classification resulted in 14 subgroups.

Validation of differentially expressed genes using quantitative RT-PCR

Total RNA (100 ng) was reversed-transcribed into cDNA with AMV reverse transcriptase using random hexamer primers (Roche Applied Science, IN, USA). TaqMan Gene Expression Assays from Applied Biosystems were used to confirm the microarray data and it was done as previously described [24]. The validated genes were Klrg1 (Mm00516879_m1), Klra3 (Mm01702813_m1) and Klra7 (Mm01183384_m1). 18S rRNA was used as endogenous control.

Chromatin-immunoprecipitation for detection of NFATc1-binding

Whole splenic and thymic cells were used for chromatin-immunoprecipitation (ChIP) assay. The cells were lysed by ammoniumchloride solution to remove erythrocytes, counted and divided into three groups each. One group was untreated, while the other two were treated with anti-CD3ε(1 μg/ml) with or without pre-treatment of CsA (1 μg/ml) for 1 hour. The stimulated cells were incubated for 24 hrs in 37°C with 5% CO2. The protocol for ChIP was described by Yu et al. [25] with the following modifications. After sonication and centrifugation, lysates were incubated with 1 μg of polyclonal anti-NFATc1 (K-18) antibody (sc-1149-R, Santa Cruz Biotechnology, Inc.) or rabbit normal Ig overnight at 4°C with rotation. Identification of targets was done by PCR using primers for the genes corresponding to IL2, IL7R, Schlafen1, Bub1, Ctla2a and Ctla2b (Additional file 2).

Results

Transcriptional changes in the absence of Itk in unstimulated CD3+ T-cells

In order to survey Itk-dependent transcriptional signatures we initially conducted microarray analysis on MACS-sorted CD3+ primary, unstimulated T-cells from Itk-/- and Wt mice. The number of probe-sets changing ≥ 2-fold in Itk-/- compared to Wt samples was 900 (2% of the total number of probe-sets on the MOE 430 2.0 chips), which is equivalent to 56% up- and 44% down-regulated probe-sets (33% with p < 0.05). From the 900-list we show the 60 most significantly up- and down-regulated transcripts in Itk-deficiency (Table 1). Most up-regulated were the killer cell lectin-like receptors Klra3 and Klra8, followed by granzyme M. Oligoadenylate synthetase-like 2 (Oasl2) was the most down-regulated transcript, next was actinin alpha 2 (Actn2). Furthermore, from the 900 probe-sets we have manually extracted 106 immune response-related genes and divided them into 14 different subgroups (Table 2). Of the 106 genes 10% were Klrs and 8.5% encoded transcription factors (Table 3). Additional file 3 shows the individual genes in each category.
Table 1

The 60 most up- and down-regulated transcripts in Itk-deficiency (unstimulated cells)

Probe set

Gene Symbol, Gene Title

Itk-/- vs Wt

t-test

1453196_a_at

Oasl2, 2'-5' oligoadenylate synthetase-like 2

-7.91

0.008

1448327_at

Actn2, actinin alpha 2

-3.97

0.016

1437445_at

Trpm1, transient receptor potential cation channel, subfamily M, member 1

-3.72

0.051

1445450_x_at

A530021J07Rik

-3.71

0.012

1421234_at

Hnf1a, HNF1 homeobox A

-3.45

0.044

1448485_at

Ggt1, gamma-glutamyltransferase 1

-3.39

0.005

1445194_at

Cnksr2, connector enhancer of kinase suppressor of Ras 2

-3.31

0.034

1418545_at

Wasf1, WASP family 1

-3.01

0.016

1451548_at

Upp2, uridine phosphorylase 2

-2.91

0.049

1434722_at

Ampd1, adenosine monophosphate deaminase 1

-2.85

0.004

1436836_x_at

Cnn3, calponin 3, acidic

-2.79

0.005

1429274_at

2310010M24Rik

-2.46

0.001

1455442_at

Slc6a19, solute carrier family 6 member 19

-2.34

0.005

1432383_a_at

Armc9, armadillo repeat containing 9

-2.29

0.020

1417928_at

Pdlim4, PDZ and LIM domain 4

-2.28

0.055

1444801_at

2900041M22Rik

-2.27

0.029

1443570_at

Cops3, COP9 (constitutive photomorphogenic) homolog, subunit 3

-2.25

0.005

1421895_at

Eif2s3x, eukaryotic translation initiation factor 2, subunit 3

-2.25

0.013

1418055_at

Neurod4, neurogenic differentiation 4

-2.23

0.020

1453009_at

Gene name not assigned for this probe set

-2.22

0.014

1429350_at

Eid3, EP300 interacting inhibitor of differentiation 3

-2.21

0.039

1420877_at

Sept6, septin 6

-2.17

0.001

1438825_at

Calm3, Calmodulin 3

-2.16

0.022

1434915_s_at

Lrrc19, leucine rich repeat containing 19

-2.12

0.041

1436103_at

Rab3ip, RAB3A interacting protein

-2.12

0.006

1456751_x_at

A530021J07Rik

-2.12

0.000

1439254_at

Gene name not assigned for this probe set

-2.11

0.037

1418003_at

1190002H23Rik

-2.11

0.037

1449634_a_at

Anks1b, ankyrin repeat and sterile alpha motif domain containing 1B

-2.09

0.047

1418990_at

Ms4a4d, membrane-spanning 4-domains, subfamily A, member 4D

-2.09

0.031

1421182_at

Clec1b, C-type lectin domain family 1, member b

3.7

0.038

1424842_a_at

Arhgap24, Rho GTPase activating protein 24

3.74

0.014

1418340_at

Fcer1g, Fc receptor, IgE, high affinity I, gamma polypeptide

3.76

0.016

1444214_at

Tubb1, tubulin, beta 1

3.79

0.044

1452666_a_at

Tmcc2, transmembrane and coiled-coil domains 2

3.88

0.033

1457001_at

Cenpk, centromere protein K

3.9

0.004

1449340_at

Sostdc1, sclerostin domain containing 1

3.91

0.014

1434115_at

Cdh13, cadherin 13

3.95

0.054

1434955_at

March1, membrane-associated ring finger (C3HC4) 1

4.04

0.000

1439397_at

Fmn1, formin 1

4.06

0.026

1448749_at

Plek, pleckstrin

4.13

0.010

1426171_x_at

Klra7, killer cell lectin-like receptor, subfamily A, member 7

4.14

0.003

1436778_at

Cybb, cytochrome b-245, beta polypeptide

4.15

0.012

1448025_at

Sirpb1, signal-regulatory protein beta 1

4.2

0.046

1420789_at

Klra5, killer cell lectin-like receptor, subfamily A, member 5

4.22

0.018

1441887_x_at

EG622976

4.26

0.017

1438553_x_at

Gene name not assigned for this probe set

4.28

0.011

1417765_a_at

Amy1, amylase 1, salivary

4.29

0.016

1451263_a_at

Fabp4, fatty acid binding protein 4

4.32

0.020

1427866_x_at

Gene name not assigned for this probe set

4.43

0.037

1454200_at

Zeb2, zinc finger E-box binding homeobox 2

4.57

0.022

1420492_s_at

Smr3a, submaxillary gland androgen regulated protein 3A

4.79

0.006

1427503_at

AI324046

4.85

0.024

1437463_x_at

Tgfbi, transforming growth factor, beta induced

5.04

0.005

1419348_at

Psp, parotid secretory protein

5.11

0.019

1419874_x_at

Zbtb16, zinc finger and BTB domain containing 16

5.46

0.005

1442025_a_at

Gene name not assigned for this probe set

5.48

0.002

1449501_a_at

Gzmm, granzyme M

6.39

0.013

1425436_x_at

Klra3, killer cell lectin-like receptor, subfamily A, member 3

9.92

0.000

1425417_x_at

Klra8, killer cell lectin-like receptor, subfamily A, member 8

35.69

0.000

The down-regulated transcripts are shown with "-"

Table 2

Groups of genes expressed in the immune response group

Immune response groups

Number of genes involved

Immune response groups

Number of genes involved

Chemokine receptors

5

Interleukins

4

Chemokines

8

Intracellular signaling components

7

Colony stimulating factor receptors

4

Killer cell lectin-like receptors

11

Fc receptors

5

Miscellaneous

24

Granzymes

4

Other surface antigens with CD-designation

17

Interferon-related genes

3

Toll-like receptors

2

Interleukin receptors

4

Transcription factors

9

Table 3

The genes found in Killer cell lectin-like receptor and transcription factor groups from Table 2

Killer cell lectin-like receptors

Probe set

Gene title

Itk -/- vs Wt

1458642_at

killer cell lectin-like receptor family E member 1 (NKG2I)

2.6

1451664_x_at

killer cell lectin-like receptor subfamily A, member 12 (Ly49L)

2.13

1422065_at

killer cell lectin-like receptor subfamily B member 1B (Ly55B/Ly55D)

3.16

1425005_at

killer cell lectin-like receptor subfamily C, member 1 (NKG2A/2B)

2.13

1420790_x_at

killer cell lectin-like receptor, subfamily A, member 16 (Ly49P)

-2.62

1426127_x_at

killer cell lectin-like receptor, subfamily A, member 18 (Ly49R)

2.99

1426140_x_at

killer cell lectin-like receptor, subfamily A, member 19 (Ly49S)

2.73

1425436_x_at

killer cell lectin-like receptor, subfamily A, member 3 (Ly49C)

9.92

1420789_at

killer cell lectin-like receptor, subfamily A, member 5 (Ly49E)

4.22

1426171_x_at

killer cell lectin-like receptor, subfamily A, member 7 (Ly49G)

4.14

1425417_x_at

killer cell lectin-like receptor, subfamily A, member 8 (Ly49H)

35.69

Transcription factors

Probe set

Gene title

Itk -/- vs Wt

1416916_at

E74-like factor 3

2.97

1457441_at

early B-cell factor 1

*

1416301_a_at

early B-cell factor 1

*

1435172_at

eomesodermin homolog (Xenopus laevis)

2.56

1426001_at

eomesodermin homolog (Xenopus laevis)

3.07

1421303_at

IKAROS family zinc finger 1

-2.2

1422537_a_at

inhibitor of DNA binding 2

2.03

1447640_s_at

pre B-cell leukemia transcription factor 3

2.05

1460407_at

Spi-B transcription factor (Spi-1/PU.1 related)

2.38

1429427_s_at

transcription factor 7-like 2, T-cell specific, HMG-box

2.41

1419874_x_at

zinc finger and BTB domain containing 16

5.46

* Early B-cell factor 1 showed variable expression changes for different probe sets

The down-regulated transcripts are shown with "-"

Transcriptional changes in the absence of Itk in stimulated CD3+ T-cells

Stimulating the Itk-/- and Wt T-cells with anti-CD3 resulted in 804 differentially expressed probe-sets in Itk-deficiency (74% up- and 26% down-regulated, 68% with p < 0.05), while after anti-CD3/CD28-stimulation the number was reduced to 409 (78% up- and 22% down-regulated; 58% with p < 0.05) as depicted in Figure 1a. Between CD3- and CD3/CD28-stimulations, the overlap was 252 probe-sets (see Table 4 for a list of the 60 most up- and down-regulated transcripts). We show there that Itk was the most down-regulated transcript in Itk-deficiency, followed by Crabp2, which encodes cellular retinoic acid binding protein 2. This is a 15 kD regulator of retinoic acid signaling recently reported to be differentially expressed in acute lymphoblastic leukaemia [26]. Other down-regulated transcripts were IL-2 and IL-3.
Table 4

The 60 most up- and down-regulated transcripts in Itk-deficiency after anti-CD3- (1) and anti-CD3/CD28-stimulation (2)

Probe set

Gene Symbol, Gene Title

Itk-/- vs Wt (1)

t-test

Itk-/- vs Wt (2)

t-test

1457120_at

Itk, IL2-inducible T-cell kinase

-6.96

0.007

-6.63

0.009

1451191_at

Crabp2, cellular retinoic acid binding protein II

-5.31

0.013

-2.79

0.054

1449990_at

Il2///LOC630222, interleukin 2

-3.89

0.002

-5.17

0.030

1436194_at

Prelid2, PRELI domain containing 2

-3.54

0.036

-2.42

0.077

1437935_at

4930486G11Rik, RIKEN cDNA

-3.44

0.032

-3.16

0.079

1439995_at

Nhedc2, Na+/H+ exchanger domain containing 2

-2.92

0.021

-2.71

0.028

1441971_at

Gene name not assigned for this probe set

-2.91

0.050

-2.88

0.082

1426243_at

Cth, cystathionase

-2.9

0.000

-2.8

0.072

1438380_at

Ddx47, DEAD box polypeptide 47

-2.69

0.002

2.03

0.300

1450566_at

Il3, interleukin 3

-2.68

0.031

-2.81

0.005

1420843_at

Ptprf, protein tyrosine phosphatase, receptor type,

-2.49

0.027

-2.07

0.222

1448788_at

Cd200, Cd200 antigen

-2.47

0.014

-2.43

0.011

1427049_s_at

Smo, smoothened homolog (Drosophila)

-2.46

0.000

-2.45

0.015

1422070_at

Adh4, alcohol dehydrogenase 4 (class II)

-2.33

0.015

-2.26

0.157

1456226_x_at

Ddr1, discoidin domain receptor family, member 1

-2.29

0.014

-2.91

0.038

1419136_at

Akr1c18, aldo-keto reductase family 1, member C18

-2.09

0.052

2.41

0.008

1433571_at

Serinc5, serine incorporator 5

-2

0.011

-2.05

0.125

1425832_a_at

Cxcr6, chemokine (C-X-C motif) receptor 6

5.34

0.000

3.27

0.085

1437463_x_at

Tgfbi, transforming growth factor, beta induced

5.36

0.045

2.4

0.012

1421802_at

Ear1, eosinophil-associated, ribonuclease A family, member 1

5.38

0.017

2.8

0.000

1448620_at

Fcgr3, Fc receptor, IgG, low affinity III

5.57

0.036

3.72

0.004

1438855_x_at

Tnfaip2, tumor necrosis factor, alpha-induced protein 2

5.6

0.0034

3.12

0.000

1450009_at

Ltf, lactotransferrin

5.68

0.080

2.03

0.034

1416514_a_at

Fscn1, fascin homolog 1

5.7

0.000

3.69

0.096

1451948_at

Gm1409, gene model 1409

5.81

0.002

3.45

0.136

1451675_a_at

Alas2, aminolevulinic acid synthase 2, erythroid

5.84

0.003

2.92

0.096

1420330_at

Clec4e, C-type lectin domain family 4, member e

5.89

0.014

3.15

0.091

1420699_at

Clec7a, C-type lectin domain family 7, member a

5.98

0.014

4.05

0.004

1427747_a_at

Lcn2, lipocalin 2

5.98

0.056

2.23

0.011

1427503_at

AI324046, expressed sequence AI324046

6.33

0.005

3.48

0.055

1419082_at

Serpinb2, serine (or cysteine) peptidase inhibitor, clade B, member 2

6.34

0.001

2.51

0.125

1419627_s_at

Clec4n, C-type lectin domain family 4, member n

6.36

0.021

2.6

0.0129

1448213_at

Anxa1, annexin A1

6.43

0.069

3.07

0.029

1419874_x_at

Zbtb16, zinc finger and BTB domain containing 16

6.62

0.032

5.3

0.001

1417898_a_at

Gzma, granzyme A

6.71

0.009

5.59

0.001

1419598_at

Ms4a6d, membrane-spanning 4-domains, subfamily A, member 6D

6.73

0.005

3.27

0.083

1429889_at

Faim3, Fas apoptotic inhibitory molecule 3

6.73

0.047

3.01

0.041

1415904_at

Lpl, lipoprotein lipase

6.76

0.054

4.1

0.026

1449254_at

Spp1, secreted phosphoprotein 1

6.8

0.028

3.79

0.020

1427910_at

Cst6, cystatin E/M

7.1

0.023

4.17

7.43E-06

1438553_x_at

Gene name not assigned for this probe set

7.11

0.011

4.63

0.055

1434150_a_at

Mettl7a///Ubie, methyltransferase like 7A

7.14

0.000

4.04

0.185

1442025_a_at

AI467657, expressed sequence AI467657

7.48

0.041

6.52

0.000

1436778_at

Cybb, cytochrome b-245, beta polypeptide

7.77

0.077

4.03

0.003

1439426_x_at

Lyz, lysozyme

7.78

0.006

2.98

0.011

1449846_at

Ear2///Ear3, eosinophil-associated, ribonuclease A family, member 2

8.62

0.008

3.54

0.031

1434194_at

Mtap2, microtubule-associated protein 2

8.73

0.035

5.78

0.000

1427866_x_at

Beta globin

9.29

0.001

5.44

0.074

1450912_at

Ms4a1, membrane-spanning 4-domains, subfamily A, member 1

9.4

0.087

4.31

0.036

1422873_at

Prg2, proteoglycan 2, bone marrow

9.81

0.028

4.19

0.026

1419764_at

Chi3l3, chitinase 3-like 3

10.51

0.070

4.1

0.004

1422411_s_at

Ear1///Ear12///Ear2///Ear3, eosinophil-associated, ribonuclease A family, member 1

10.54

0.010

4.61

0.000

1418722_at

Ngp, neutrophilic granule protein

11.39

0.031

3.74

0.006

1450989_at

LOC100047300///Tdgf1, teratocarcinoma-derived growth factor

11.64

0.009

2.89

0.076

1419394_s_at

S100a8, S100 calcium binding protein A8

12.08

0.071

4.6

0.034

1425436_x_at

Klra3, killer cell lectin-like receptor, subfamily A, member 3

12.34

0.002

14.75

0.000

1415897_a_at

Mgst1, microsomal glutathione S-transferase 1

12.62

0.082

3.77

0.006

1448756_at

S100a9, S100 calcium binding protein A9

12.97

0.077

5.15

0.010

1426171_x_at

Klra7, killer cell lectin-like receptor, subfamily A, member 7

14.08

0.027

14.02

0.015

1425417_x_at

Klra8, killer cell lectin-like receptor, subfamily A, member 8

21.07

0.026

17.99

0.000

The down-regulated transcripts are shown with "-"

https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-10-233/MediaObjects/12864_2008_Article_2117_Fig1_HTML.jpg
Figure 1

The number of differentially expressed probe-sets in Itk-deficiency. a. Venn diagram showing overlapping probe-sets in CD3+ Itk-defective T-cells, unstimulated (upper), anti-CD3- (left) and anti-CD3/CD28-stimulated (right). All the comparisons were made against Wt and with the criterion ≥ 2-fold. b. Quantitative RT-PCR confirms up-regulated expression of Klra3, Klra7 and Klrg1 in Itk-deficiency. The bar charts show relative amount of Klr mRNA compared to unstimulated Wt CD3+ T-cells (Wt C).

Stimulation affected the majority of the transcripts in the same direction as observed in unstimulated cells (p < 10-6) (only in 5/252 cases the CD3- or CD3/CD28-stimulations showed opposite fold-changes; Additional file 4). We show there that the most induced mRNA was Klra8 (Ly49H) (21-fold-change upon anti-CD3-stimulation). Other up-regulated Klrs were Klra3 (Ly49C), Klra5 (Ly49E), Klra7 (Ly49G), Klra19 (Ly49S), Klrc1 (NKG2A/2B), Klrd1 (CD94), Klre1 (NKG2I) and Klrg1 (2P1-Ag). By quantitative RT-PCR we confirmed the up-regulated expression of Klrg1, Klra3 and Klra7 in Itk-defective samples (Fig. 1b). Two transcription factors, inhibitor of DNA binding 2 (Id2) and eomesodermin, were also found up-regulated. Thus, in CD3+ cells, differential transcriptional signatures between Wt and Itk-deficient cells were more pronounced in unstimulated when compared to activated cells.

We continued to analyze the activation-dependent signatures in Wt and Itk-/- T-cells separately. The number of probe-sets changing ≥ 2-fold after anti-CD3-stimulation (compared to the unstimulated state) in the Wt samples was 4252, and the corresponding number after co-stimulation was 4385 (Figure 2). The overlap between the two stimulations was 3713 (87% and 85%, respectively; Additional file 5). However, the differences were significantly more pronounced in anti-CD3 versus anti-CD3/CD28 activated cells in the Itk-defective group, with only 50% of the transcripts in the co-stimulated group overlapping with the CD3-stimulated (p < 10-6) (Additional file 6). Thus, co-stimulation had much greater effect on Itk-deficient than on Wt cells. We further examined some of the immune response-related genes previously mentioned. Ten members of the Klr family were up-regulated in unstimulated Itk-defective compared to Wt samples (Table 3), while after stimulation the majority of Klrs were down-regulated in both Wt and Itk-defective T-cells. Down-regulation of Klrs were also reported for human cells from healthy individuals in a recent paper by Wang et al., where primary human T-cells were analyzed after anti-CD3/CD28-stimulation [27]. With respect to cytokine expression, IL-2 and IL-6 were found up-regulated upon anti-CD3-stimulation in both Wt and Itk-deficient samples when compared to the corresponding unstimulated cells. In contrast, IL-16 and IL-18 were down-regulated (Additional files 5 and 6). Two cytokines, whose expression was only altered in Itk-deficient cells upon anti-CD3-stimulation, were IL-10 (up-regulated) and IL-33 (down-regulated) (data not shown). IL-33 is a novel IL-1 family cytokine, IL1F11/IL-33, playing an important role in eosinophil-mediated inflammation [28]. Interestingly, Itk-deficient mice have previously been shown to have reduced lung inflammation, eosinophil infiltration and mucous production after induction of allergic asthma [29]. No other cytokines were differentially expressed. In the stimulated Wt samples we also observed altered expression of several transcription factors such as Zbtb16 (encoding the transcriptional regulator PLZF), Id2 and Spi-C, while in Itk-defective cells we found Zbtb16, Spi-C and Id3 to be differentially expressed upon stimulation.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-10-233/MediaObjects/12864_2008_Article_2117_Fig2_HTML.jpg
Figure 2

The amount of differentially expressed probe-sets in Wt and Itk-defective CD3 + T-cells following stimulation. The upper panel represents the Wt T-cells and the lower the Itk-defective T-cells. The left panel symbolizes the anti-CD3-stimulation and the right panel the anti-CD3/CD28-stimulation. All the stimulations were compared to the untreated condition. The arrows denote the number of overlapping probe-sets.

CD4+ and CD8+ T-cell signatures in Itk-deficiency

As T-cells can be divided into CD4+ and CD8+ subsets, and as these subsets have very distinct functions and gene regulations, we examined Itk-deficiency in MACS purified CD4+ and CD8+ Wt and Itk-deficient T-cells. The Itk-deficient CD4+ and CD8+ T-cells are known from previous studies to be of a memory-like phenotype, characterized by the markers CD44 and CD122 [911, 13]. Since CD122 expression is enhanced by the transcription factors eomesodermin and T-box 21 (T-bet), we looked for their expression in our data. Eomesodermin was previously reported to be up-regulated in Itk-deficient T-cells [9] and we found the expression of eomesodermin much higher in the CD8+ T-cell population compared to CD4+ in unstimulated condition. The same was also seen with T-bet. Taken together, the observed expression pattern of eomesodermin and T-bet is in agreement with previously published studies and thus validates our microarray data. Moreover, our analysis also includes new knowledge related to these transcripts, namely how they respond to activation of T-cells as well as the effect of CsA (Additional file 7).

Both CD4+ and CD8+ T-cell subsets in Itk-deficient mice have been shown to differ in phenotype compared to the Wt mice. In the absence of Itk, a higher percentage of each subset expresses surface markers, typical for memory phenotype cells, such as CD44hi and CD122hi [9, 10, 13]. We sought to determine whether this was also reflected by their transcriptomes. The number of transcripts differentially expressed between unstimulated Itk-/- and Wt in the CD4+ population was 2050, while in the CD8+ population the number was higher (n = 6907). The 60 most up- and down-regulated transcripts from each subset are shown in Tables 5 and 6. Among those are genes already mentioned, e.g. eomesodermin, Klra3 and 8, T-bet and Granzyme M. In these groups we also found Zbtb16. Interestingly, Zbtb16 was 12-fold up-regulated in CD4+ cells and 45-fold down-regulated in CD8+ cells, also suggesting a highly efficient separation of the two subsets. Based on these findings PLZF was selected for further studies presented elsewhere [30]. The most pronounced changes were seen in the CD8+ population (Table 6), with 69-fold down-regulation of Clca1, which is a calcium-activated chloride-channel, of importance in airway epithelial cells. Two up-regulated transcripts were the PTB-domain containing MAP-kinase regulator Dok5 (29-fold) and α-tubulin (30-fold), whose expression in T-cells, to our knowledge, was not previously reported. After anti-CD3-stimulation we found the number of differentially expressed transcripts reduced in both subsets, approximately 30% and 47% fewer probe-sets in CD4+ and CD8+, respectively. The overlapping probe-sets between the unstimulated and the anti-CD3-stimulated conditions are shown in Figure 3. 82% of the transcripts in the CD4+ subset were also found in the CD8+ population in unstimulated cells. The percentage of overlapping transcripts decreased with stimulation.
Table 5

The 60 most up- and down-regulated transcripts in Itk-defective CD4+ T-cells (unstimulated cells)

Probe set

Gene Symbol, Gene Title

Itk-/- vs Wt

t-test

1436386_x_at

OTTMUSG00000010671

-7.51

0.012

1444708_at

Tmem29, transmembrane protein 29

-5

0.014

1434418_at

Lass6, LAG1 homolog, ceramide synthase 6

-4.93

0.001

1438354_x_at

Cnn3, Calponin 3, acidic

-4.81

0.030

1430988_at

2810407C02Rik

-3.84

0.028

1430827_a_at

Ptk2, PTK2 protein tyrosine kinase 2

-3.68

0.002

1458977_at

A530021J07Rik

-3.36

0.006

1439778_at

Cables1, Cdk5 and Abl enzyme substrate 1

-3.26

0.018

1427675_at

V1ra2, vomeronasal 1 receptor, A2

-3.22

0.041

1448338_at

Pgcp, plasma glutamate carboxypeptidase

-3.16

0.001

1458945_at

AU015148

-3.16

0.038

1421507_at

Olfr78, olfactory receptor 78

-3.07

0.021

1457120_at

Itk, IL2-inducible T-cell kinase

-2.96

0.002

1456178_at

Bambi-ps1, BMP and activin membrane-bound inhibitor, pseudogene (Xenopus laevis)

-2.94

0.000

1455907_x_at

Phox2b, paired-like homeobox 2b

-2.88

0.050

1452474_a_at

Art3, ADP-ribosyltransferase 3

-2.86

0.026

1459508_at

C85600

-2.79

0.007

1440761_at

4833422C13Rik

-2.77

0.052

1446412_at

Gene name not assigned for this probe set

-2.71

0.012

1441221_at

Gene name not assigned for this probe set

-2.7

0.019

1427632_x_at

Cd55, CD55 antigen

-2.65

0.042

1439181_at

BC043301

-2.63

0.032

1434473_at

Slc16a5, solute carrier family 16 (monocarboxylic acid transporters), member 5

-2.59

0.001

1448002_x_at

2610001J05Rik

-2.57

0.018

1455425_at

BB001228

-2.57

0.016

1419620_at

Pttg1, pituitary tumor-transforming 1

-2.53

0.000

1453009_at

Gene name not assigned for this probe set

-2.39

0.000

1455740_at

Hnrnpa1, heterogeneous nuclear ribonucleoprotein A1

-2.31

0.048

1429413_at

Cpm, carboxypeptidase M

-2.28

0.003

1416441_at

Pgcp, plasma glutamate carboxypeptidase

3.12

0.005

1425216_at

Ffar2, free fatty acid receptor 2

3.13

0.036

1448471_a_at

Ctla2a, cytotoxic T lymphocyte-associated protein 2 alpha

3.13

0.006

1450334_at

Il21, interleukin 21

3.13

0.013

1423091_a_at

Gpm6b, glycoprotein m6b

3.21

0.032

1435339_at

Kctd15, potassium channel tetramerisation domain containing 15

3.24

0.044

1428197_at

Tspan9, tetraspanin 9

3.37

0.001

1449036_at

Rnf128, ring finger protein 128

3.44

0.002

1449361_at

Tbx21, T-box 21

3.5

0.012

1447839_x_at

Adm, adrenomedullin

3.62

0.031

1418318_at

Rnf128, ring finger protein 128

3.77

0.021

1419647_a_at

Ier3, immediate early response 3

3.78

0.005

1448961_at

Plscr2, phospholipid scramblase 2

4.06

0.012

1449280_at

Esm1, endothelial cell-specific molecule 1

4.15

0.013

1427445_a_at

Ttn, titin

4.32

0.006

1425471_x_at

Gene name not assigned for this probe set

4.33

0.050

1438553_x_at

Gene name not assigned for this probe set

4.35

0.019

1423231_at

Nrgn, neurogranin

4.66

0.002

1416846_a_at

Pdzrn3, PDZ domain containing RING finger 3

4.69

0.002

1430946_at

2600014E21Rik

4.82

0.031

1426001_at

Eomes, eomesodermin homolog (Xenopus laevis)

5.43

0.024

1422280_at

Gzmk, granzyme K

5.6

0.002

1427608_a_at

Tcrg-V1, T-cell receptor gamma, variable 1

5.62

0.044

1434194_at

Mtap2, microtubule-associated protein 2

5.71

0.040

1434115_at

Cdh13, cadherin 13

6.15

0.047

1455435_s_at

Chdh, choline dehydrogenase

6.54

0.041

1449864_at

Il4, interleukin 4

6.77

0.031

1424011_at

Aqp9, aquaporin 9

7.23

0.011

1420678_a_at

Il17rb, interleukin 17 receptor B

9.84

0.029

1442025_a_at

Gene name not assigned for this probe set

11.69

0.008

1419874_x_at

Zbtb16, zinc finger and BTB domain containing 16

12.07

0.009

The down-regulated transcripts are shown with "-"

Table 6

The 60 most up- and down-regulated transcripts in Itk-defective CD8+ T-cells (unstimulated cells)

Probe set

Gene Symbol, Gene Title

Itk-/- vs Wt

1417852_x_at

Clca1, chloride channel calcium activated 1

-68.71

1419874_x_at

Zbtb16, zinc finger and BTB domain containing 16

-45.13

1436759_x_at

Cnn3, calponin 3, acidic

-39.91

1454869_at

Wdr40b, WD repeat domain 40B

-34.02

1427054_s_at

Abi3bp, ABI gene family, member 3 (NESH) binding protein

-31.51

1437992_x_at

Gja1, gap junction protein, alpha 1

-25.36

1416203_at

Aqp1, aquaporin 1

-24.68

1437279_x_at

Sdc1, syndecan 1

-24.33

1448182_a_at

Cd24a, CD24a antigen

-21.41

1456956_at

Ikzf2, IKAROS family zinc finger 2

-19.8

1442025_a_at

Gene name not assigned for this probe set

-19.78

1439422_a_at

C1qdc2, C1q domain containing 2

-19.69

1454086_a_at

Lmo2, LIM domain only 2

-18.62

1416330_at

Cd81, CD81 antigen

-17.08

1451867_x_at

Arhgap6, Rho GTPase activating protein 6

-17.08

1456060_at

Maf, avian musculoaponeurotic fibrosarcoma (v-maf) AS42 oncogene homolog

-16.84

1419014_at

Rhag, Rhesus blood group-associated A glycoprotein

-16.79

1416193_at

Car1, carbonic anhydrase 1

-16.49

1450744_at

Ell2, elongation factor RNA polymerase II 2

-14.83

1456147_at

St8sia6, ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 6

-14.78

1456475_s_at

Prkar2b, protein kinase, cAMP dependent regulatory, type II beta

-14.57

1460431_at

Gcnt1, glucosaminyl (N-acetyl) transferase 1, core 2

-14.03

1437171_x_at

Gsn, gelsolin

-13.64

1417777_at

Ltb4dh, leukotriene B4 12-hydroxydehydrogenase

-13.54

1437935_at

4930486G11Rik

-13.51

1434499_a_at

Ldhb, lactate dehydrogenase B

-13.44

1450333_a_at

Gata2, GATA binding protein 2

-13.33

1423569_at

Gatm, glycine amidinotransferase (L-arginine:glycine amidinotransferase)

-12.94

1435884_at

Itsn1, intersectin 1 (SH3 domain protein 1A)

-12.72

1425145_at

Il1rl1, interleukin 1 receptor-like 1

-12.6

1449313_at

Klk1b5, kallikrein 1-related peptidase b5

13.38

1454106_a_at

Cxxc1, CXXC finger 1 (PHD domain)

13.41

1442639_at

Gene name not assigned for this probe set

13.76

1443006_at

Gene name not assigned for this probe set

13.93

1433449_at

Snx32, sorting nexin 32

14.78

1423603_at

Zfpm1, zinc finger protein, multitype 1

14.98

1441770_at

Ppat, phosphoribosyl pyrophosphate amidotransferase

15.29

1420343_at

Gzmd, granzyme D

15.42

1445596_at

Gene name not assigned for this probe set

15.54

1452985_at

Uaca, uveal autoantigen with coiled-coil domains and ankyrin repeats

16.55

1420233_at

Gene name not assigned for this probe set

16.79

1423020_at

Gene name not assigned for this probe set

17.39

1454481_at

Mif, macrophage migration inhibitory factor

17.64

1427426_at

Kcnq5, potassium voltage-gated channel, subfamily Q, member 5

18.06

1424698_s_at

Gca, grancalcin

18.46

1447574_s_at

Slc32a1, Solute carrier family 32 (GABA vesicular transporter), member 1

18.73

1431854_a_at

4930452B06Rik

18.94

1460267_at

Dmrt3, doublesex and mab-3 related transcription factor 3

19.76

1417197_at

Wwc2, WW, C2 and coiled-coil domain containing 2

20.76

1442788_at

Afap1, actin filament associated protein 1

20.91

1447292_at

Actr1b, ARP1 actin-related protein 1 homolog B (yeast)

21.96

1431878_at

Grhl2, grainyhead-like 2 (Drosophila)

22.18

1449501_a_at

Gzmm, granzyme M (lymphocyte met-ase 1)

23.58

1436500_at

Rps24, Ribosomal protein S24

25.96

1425436_x_at

Klra3, killer cell lectin-like receptor, subfamily A, member 3

26.39

1456130_at

LOC553091

27.36

1454240_at

Nfe2l3, nuclear factor, erythroid derived 2, like 3

29.25

1422641_at

Dok5, docking protein 5

29.31

1417375_at

Tuba4a, tubulin, alpha 4A

30.36

1425417_x_at

Klra8, killer cell lectin-like receptor, subfamily A, member 8

131.81

The down-regulated transcripts are shown with "-"

https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-10-233/MediaObjects/12864_2008_Article_2117_Fig3_HTML.jpg
Figure 3

Overlapping probe-sets in unstimulated and anti-CD3-stimulated Itk-deficient CD4 + (left) and CD8 + (right) T-cell populations. Each circle is a comparison between Itk-defective and Wt samples. All the comparisons were made with the criterion ≥ 2-fold.

The largest number of differentially expressed transcripts was observed in the unstimulated groups of Itk-deficient CD4+ and CD8+ T-cell subsets. By subtracting the overlapping 1495 probe-sets (Figure 3) from unstimulated Itk-deficient CD4+ and CD8+ cells, respectively, we characterized separate core groups of transcripts for each subset. The remaining number of probe-sets in the CD4+ population was 324 (Additional file 8), while it was more than 10-fold higher in the CD8+ group (Additional file 9). Interestingly, two members of the Klr family (Klrb1a (Ly55a) and Klrb1c (NK-1.1)) were present in the CD4+population, while four other members were found in the CD8+ subset (Klra4 (Ly49D), Klra19 (Ly49S), Klrc2 (NKG2C) and Klrk1 (NKG2D)). Five Klrs were in common between unstimulated CD4+ and CD8+ groups, they were Klra3, a7, a8, a22 and b1b (by comparing 2050 and 6907 in Fig. 3). The differentially expressed NK/innate cell-related transcripts were not limited to cell surface markers, since RNA for the cytotoxic protein Granzyme M was strongly enriched in the Itk-deficient population, again confirming that NK- and innate cells have overlapping transcriptomes [31].

Itk-deficiency mimics calcineurin inhibition

Tec-family kinases activate PLCγ and are therefore important regulators of Ca2+-mobilization and the calcineurin/NFAT pathway [5, 32]. However, Tec-family kinases regulate also other signaling pathways. To investigate which of the Itk-related changes is the consequence of an impaired calcineurin/NFAT pathway, we compared the expression profiles of anti-CD3 ± CD28 stimulated Itk-/- CD3+ T-cells and of CsA-treated Wt T-cells. CsA specifically inhibits calcineurin and by that affects downstream signaling and the activation of the transcription factors of the NFAT-family. Based on the dependency of Itk and/or calcineurin, three groups of genes could be identified: Itk- and calcineurin-dependent (Itk/CN); Itk-dependent and calcineurin-independent (Itk/non-CN) and Itk-independent and calcineurin-dependent (non-Itk/CN).

Altogether, after anti-CD3-stimulation 4613 probe-sets were differentially expressed in CsA-treated cells compared to untreated, and after co-stimulation the number was reduced by 15% to 3936. The gene numbers observed in Itk-deficient compared to Wt cells were 804 and 409 after anti-CD3- and anti-CD3/CD28-stimulation, respectively (Figure 1a). About 60% of the probe-sets that were changed ≥ 2-fold in Itk-/- compared to Wt after anti-CD3-stimulation were also found in the CsA-treated samples (Figure 4, Itk/CN anti-CD3, showing the 10 most highly-regulated transcripts). In co-stimulated cells 45% of the probe-sets were the same (Figure 4, Itk/CN anti-CD3/CD28). When comparing the Itk-dependent probe-sets being calcineurin-dependent in both stimulations the overlap was 113 (Additional file 10). As expected, IL-2 was found in that group, confirming the biological relevance of our data, since IL-2 is known to be both Itk- and calcineurin-dependent [16, 33]. Among other genes found in this group were Zbtb16 (up-regulated) and Crabp2 (down-regulated). Interestingly, the transcript for chemokine (C-motif) ligand 1 (Xcl1) was down-regulated in the CsA-treated cells while it showed increased expression in Itk-deficient samples. Furthermore, cytotoxic T lymphocyte-associated protein 2 alpha and beta (Ctla2a and Ctla2b) were up-regulated. Interestingly, their altered expression was more pronounced in the CsA-treated (>3 times higher) than in the Itk-deficient samples. Two granzyme-encoding genes, Gzma and Gzmk, were also found among those that were Itk- and calcineurin-dependent.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-10-233/MediaObjects/12864_2008_Article_2117_Fig4_HTML.jpg
Figure 4

Anti-CD3- and anti-CD3/CD28-stimulations leading to Itk- and calcineurin-dependent and -independent transcriptional signatures. Three different groups of genes exist in each stimulation condition. The groups are Itk- and calcineurin-dependent (Itk/CN), Itk-dependent and calcineurin-independent (Itk/non-CN) and Itk-independent and calcineurin-dependent (non-Itk/CN). 113 probe-sets are overlapping between Itk/CN groups in the two stimulations. In each group the 10 most highly regulated transcripts are presented. All the chosen genes passed the t-test criterion of p < 0.05. The down-regulated transcripts are shown with "-". The transcripts in the two Itk/CN groups passed the criterion in at least one of the two comparisons. Genes previously known to be calcineurin-regulated are grey-shaded [16]. Arrows denoting signal transduction from CD28 have been omitted for clarity.

The fractions of Itk/non-CN genes (322 vs 225- for anti-CD3- and anti-CD3/CD28-stimulated T-cells, respectively) shared 95 probe-sets, corresponding to 89 transcripts (Additional file 11). Among them were three up-regulated members of the Klrs; Klra5, Klra8 and Klre1. After co-stimulation, a much smaller number of probe-sets were Itk-dependent compared to anti-CD3-stimulation only (p < 10-6). The transcripts being calcineurin-dependent but Itk-independent (non-Itk/CN group) were 4131 and 3752 in anti-CD3- and co-stimulated cells, respectively. It is interesting to note that CsA-treatment, but not Itk-deficiency (the non-Itk/CN anti-CD3 group), results in severely reduced transcript levels for IFNγ. Previous studies show that an immediate IFNγ release is a hallmark of the innate CD8-population [9, 10].

NFAT-binding genes that are Itk- and calcineurin- dependent

The comparison of Itk-deficient and CsA-treated Wt T-cells revealed 482 up- or down-regulated transcripts upon anti-CD3-stimulation. In order to identify putative NFAT-binding sites (GGAAA), we selected 24 genes for bioinformatic analyses. The genes were chosen as being highly regulated in the CsA or Itk-/- comparisons after anti-CD3-stimulation. 19/24 of these genes were also significantly regulated after co-stimulation with anti-CD3/CD28. 15/24 genes had putative NFAT-sites in the 500 bp region upstream of the transcriptional start site (Additional file 12). We identified 1 to 2 binding sites in 4 of those genes: IL7R, Bub1, Ctla2a and Ctla2b, as well as upstream of the translation initiation of Schlafen1 (Slfn1) gene (Figure 5a). To test whether NFAT binds to the promoter region of the genes in vivo, chromatin-immunoprecipitation experiments were performed. As a positive control, we used the IL-2 promoter region known to contain functional NFAT-sites bound by NFATc1 [34, 35]. ChIP assays demonstrated NFATc1 binding to the IL-2 promoter region as expected and revealed anti-CD3 induced binding of NFATc1 to the selected regions of the five genes. They were also shown to be bona fide calcineurin-regulated genes owing to that the induced binding was reversed by CsA-treatment (Fig. 5b). A heat-map presenting the signal intensities of the above mentioned genes is shown in Figure 5c.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-10-233/MediaObjects/12864_2008_Article_2117_Fig5_HTML.jpg
Figure 5

NFAT-binding genes being Itk- and calcineurin-dependent. a. Promoter regions with NFAT-binding sites in IL2, IL7R, Bub1, Slfn1 (Schlafen1), Ctla2a and Ctla2b genes are shown with the binding site(s) represented by black boxes. This identification was done by bioinformatic analyses of the 500 bp region upstream of each gene's transcriptional start site. This approach identified 1–2 NFAT binding sites within the promoter regions of these genes as well as upstream of the translation initiation of Slfn1 gene. The numbers below each box represent the position of the binding site in correlation to the transcription start. The arrows indicate forward and reverse primers. b. NFATc1-binding in IL2, IL7R, Bub1, Slfn1, Ctla2a and Ctla2b genes. CD3+ T-cells were isolated from Wt thymus (thy) and spleen (spl) as described in experimental procedures. The PCR pictures were analyzed with Fluoro-S gel documentation equipment (BioRad Laboratories, CA) with a CCD camera, and further evaluated using the Quantity One software. Input; DNA before IP, NTC; no template control. c. Signal intensities of the six genes in Wt unstimulated, Wt anti-CD3-stimulated, Wt anti-CD3-stimulated + CsA-treated, Itk-defective unstimulated and Itk-defective anti-CD3- stimulated samples. The figure was made in dChip [21]. The color scale in the lower part of the picture corresponds to the mean expression of a gene. The red color represents expression level above mean expression of a gene across all samples, the white color is mean expression and the blue color represents expression lower than the mean.

Transcripts regulated by Tec-family kinases

Itk is crucial for T-cell development and activation. Similarly, Btk is essential for proper differentiation and activation of B-cells [36, 37]. In a previous study, we investigated the genes modulated by Btk [24]. There was a pronounced overlap 18/38 (47%) between differentially expressed genes from the Itk-defective T-cells (900-list) and the previously published list of Btk-deficient genes from splenic B-cells as analyzed by the U74Av2 chip with approximately 12 000 genes [24]. The overlapping transcripts are shown in Table 7. Sixteen of the 18 genes were similarly regulated, which shows a highly significant co-variation (p = 0.01). Among these genes were those for transcription factors (Id2, Ikaros and Spi-C), cell membrane spanning (Csf1r, Mrc1 and Vcam1) and secreted proteins (Aif1, Igf1 and Tgfbi).
Table 7

Overlapping transcripts between Btk-/- B-cells and Itk-/- T-cells

U74Av2

MOE 430 2.0

Gene Symbol, Gene Title

Fold change Btk-/-/Itk-/-

102330_at

1418204_s_at

Aif1, allograft inflammatory factor 1

7.47/2.28

95546_g_at

1419519_at

Igf1, insulin-like growth factor 1

6.5/2.46

103226_at

1450430_at

Mrc1, mannose receptor, C type 1

5.42/6.06

95597_at

1423414_at

Ptgs1, prostaglandin-endoperoxide synthase 1

4.68/2.43 **

95597_at

1436448_a_at

Ptgs1, prostaglandin-endoperoxide synthase 1

4.68/2.19 **

96020_at

1417063_at

C1qb, complement component 1, q subcomponent, beta polypeptide

4.57/6.6 ***

96020_at

1434366_x_at

C1qb, complement component 1, q subcomponent, beta polypeptide

4.57/5.87 ***

96020_at

1437726_x_at

C1qb, complement component 1, q subcomponent, beta polypeptide

4.57/4.46 ***

104354_at

1419873_s_at

Csf1r, colony stimulating factor 1 receptor

4.4/3.96 **

104354_at

1423593_a_at

Csf1r, colony stimulating factor 1 receptor

4.4/2.95 **

103736_at

1448005_at

Sash1, SAM and SH3 domain containing 1

3.97/6.79

103454_at

1418555_x_at

Spic, Spi-C transcription factor (Spi-1/PU.1 related)

3.93/3.75 *

103454_at

1449134_s_at

Spic, Spi-C transcription factor (Spi-1/PU.1 related)

3.93/8.5 *

92877_at

1415871_at

Tgfbi, transforming growth factor, beta induced

3.9/3.46 ***

92877_at

1437463_x_at

Tgfbi, transforming growth factor, beta induced

3.9/5.04 ***

92877_at

1448123_s_at

Tgfbi, transforming growth factor, beta induced

3.9/4.52 ***

92877_at

1456250_x_at

Tgfbi, transforming growth factor, beta induced

3.9/4.6 ***

92223_at

1449401_at

C1qc, complement component 1, q subcomponent, C chain

3.73/5.47

92558_at

1448162_at

Vcam1, vascular cell adhesion molecule 1

3.63/5.62

102065_at

1418243_at

Fcna, ficolin A

3.6/5.27

103070_at

1416985_at

Sirpa, signal-regulatory protein alpha

3.42/3.58

102860_at

1424923_at

Serpina3g, serine (or cysteine) peptidase inhibitor, clade A, member 3G

3.05/2.08 ***

99476_at

1453931_at

Col14a1, collagen, type XIV, alpha 1

2.95/(-)2.51

93013_at

1422537_a_at

Id2, inhibitor of DNA binding 2

2.77/2.03 **

102293_at

1421303_at

Ikzf1, IKAROS family zinc finger 1

(-)2.7/(-)2.2

99413_at

1419610_at

Ccr1, chemokine (C-C motif) receptor 1

(-)4.13/2.62 *

The down-regulated transcripts are shown with "(-)"

* Denotes significant changes in Itk-/- T-cells. * p ≤ 0.05 ** p ≤ 0.01 *** p ≤ 0.001

Discussion

For any analysis of individuals with defective genes there are important considerations related to the choice of accurate controls and the adequate interpretation of the data. This is nicely exemplified in Itk-deficiency. Thus, when mice with Itk-deficiency are immunized and generate impaired responses it is unclear to what extent the impairment is caused by the reduced numbers of mature T-lymphocytes as compared to the increased innate populations versus that the mature as well as the innate populations are deficient because they lack Itk. The net outcome is the sum of these alterations. The same is true in microarray experiments or when phenotypic markers are assayed by other means. In the likely event that the innate populations themselves are further altered owing to lack of Itk, the corresponding population may not even exist in the Wt. The same principle is true for any mutant gene, and it is important to be aware of this fact when interpreting data, including expression profiling, related to such defects. In this report we describe the phenotypic changes in Itk-deficiency and make comparisons to CsA-treatment. Owing to the very large number of genes with altered expression, we here provide an overview of the observed changes. We pinpoint some of the interesting findings obtained from this dataset. However, the original gene profiling data, available to any investigators at GEO, could be analyzed in different ways, depending on the biological question to be answered.

T-cells deficient for the Tec-family kinase Itk have severe impairment during T-cell activation. Furthermore, Itk has also been shown to be involved in signaling pathways that regulate the development decisions of conventional versus innate-like T-cell development [913], since CD8+ T-cells and a certain fraction of CD4+ T-cells have an innate-like T-cell phenotype. Collectively, these studies revealed that Itk has a crucial and important function in T-cells. In this study we performed an Affymetrix microarray expression analysis to investigate how Itk-deficiency affects the expression profile in T-cells. The effect of Itk-deficiency was investigated in CD3+ T-cells, as well as in the CD4+ and CD8+ T-cell subsets. These signatures for the first time reveal the transcriptome of Itk-deficiency.

The most pronounced changes were observed in resting Itk-deficient compared to Wt CD3+ T-cells. This is in agreement with the previous findings that more genes are expressed in untreated cells as compared to stimulated T-lymphocytes [3840]. Thus, after anti-CD3/CD28-stimulation the number of differentially expressed transcripts was dramatically decreased in Itk-defective (down by approximately 50%) compared to Wt cells. This suggests that the CD28 co-stimulatory pathway is less dependent of Itk. It was previously shown that Itk was a negative regulator of CD28-signaling in CD4+ T-cells [41]. However, sorted naïve CD4+ T-cells from Itk-deficient mice had normal CD28 co-stimulatory responses when compared to Wt cells [42], showing that CD28-signaling is not dependent on Itk in these cells. Our result confirms that Itk is not essential for CD28-signaling and suggests that a great deal of the TCR signaling defects in Itk-/- T-cells is rescued by CD28 co-stimulation in vitro. However, expression of genes that is essential for T-cell proliferation like Il2 remain Itk-dependent after co-stimulation.

It was satisfying to observe the most pronounced transcriptional changes in CD8+ cells, since Itk-deficiency is known to predominantly affect this subpopulation [911]. The overlap between CD4+ and CD8+ subsets was highest in untreated cells indicating an innate-like pattern also of the CD4+ population. Moreover, a recent paper showed that CD4+ T-cells in Itk-deficient mice have a memory phenotype with expression of typical surface markers such as CD62Llow and CD44high [13]. When looking at the specific transcripts for each T-cell subset we found differences in expression of Klrs, two members in CD4+ and four in CD8+. Klrb1a (Ly55a) and Klrb1c (NK-1.1) were found in CD4+ T-cells. To our knowledge, only NK-1.1 was previously reported for Itk-deficient CD4+ T-cells [13]. Klrc2 (NKG2C) and Klrk1 (NKG2D) were previously reported in CD8+ T-cells [11]. In addition, we found Klra4 (Ly49D) and Klra19 (Ly49S), not previously described in this context. In unstimulated Itk-/- CD3+ T-cells eleven Klr members were found (shown in Table 3). Klra3, a7, a8 and b1b were shown to be common to the CD4+ and CD8+ subsets. Interestingly, we found Klra3 and Klra7 to also be calcineurin-dependent, while Klra8 was only Itk-dependent. Of note is also that a cytosolic protein known to characterize NK-cells, granzyme M, was present in the data. It has recently been shown to be expressed in NK-cells and cytotoxic T-cells with innate immune function [31]. Here, we show for the first time up-regulation of this transcript in CD8+ Itk-defective T-cells. As expected, more differentially expressed genes were revealed following separation into the CD4+ and CD8+ subsets. In a mixed population changes that affect both subsets in a similar way are preferentially detected.

Itk-deficiency partially mimicked CsA-treatment, since there was a large overlap of affected transcripts. However, we observed that CsA had a much greater effect on transcriptional regulation compared to loss of Itk, especially after co-stimulation. Approximately 4000 genes were affected by CsA following either anti-CD3- or anti-CD3/CD28-stimulation, while the corresponding numbers for those also affected by Itk was 482 and 184, respectively. 113 probe-sets were shared between Itk-defective and CsA-treated T-cells independent of stimulation. Among them we found Zbtb16, encoding the transcriptional regulator PLZF, and Xcl1, which is also called lymphotactin or ATAC, a chemokine mainly produced by activated CD8+ T-cells [43, 44]. Also, Crabp2 was found in this comparison showing its calcineurin-dependency. Crabp2 is involved in regulating access of retinoic acid to its nuclear receptors, is developmentally regulated [45], and has been implicated in various forms of tumors. In addition, our analysis revealed that some of the Itk-induced changes are independent of the Ca2+/calcineurin pathway (322 and 225 transcripts after anti-CD3- and anti-CD3/CD28-stimulation, respectively). In this study we did not treat Itk-deficient cells with CsA. Such experiments could give further insights into the calcineurin-dependent regulation.

One interesting example of how different members of a gene family are differentially affected by Itk-deficiency and CsA-treatment is provided by the Granzyme family. Granzymes are serine proteases expressed in cytotoxic lymphocytes [46]. Interestingly, Granzymes A and K were both Itk- and calcineurin-dependent, while granzymes E and M were found to be Itk-dependent and calcineurin-independent after anti-CD3-stimulation. Both granzymes A and K induce caspase-independent cell death. Not much is known about granzyme E, while granzyme M is known to induce cell death in a caspase- and mitochondria-independent way [46]. Granzyme B was only affected in CsA-treated samples and has been shown to be involved in the induction of caspase-dependent apoptosis. Collectively, this demonstrates that expression of granzymes is differentially controlled.

The comparison of Itk-deficiency and CSA-treated CD3+ T-cells led also to the identification of novel NFAT target genes. The combination of a bioinformatics approach and chromatin-immunoprecipitation assays revealed that IL7R, Schlafen1, Bub1, Ctla2a and Ctla2b are novel Itk- and calcineurin-dependent genes with seemingly functional NFAT-binding sites. However, they can be regulated in different ways, e.g. Ctla2a and Ctla2b, IL7R and Slfn1 were negatively regulated, while IL-2 and Bub1 were positively regulated by CN-dependent pathways. The same regulation pattern was observed in unstimulated Itk-deficient samples, but after anti-CD3-stimulation IL7R and Slfn1 became positively regulated by Itk (Fig. 5c). Members of the Schlafen (Slfn) protein family have been implicated in the regulation of cell growth and T-cell development. Furthermore, similar to the Il2 gene [47], AP-1 and NF-κB are reported to regulate Slfn2 expression [48]. Bub1 (budding uninhibited by benomyl) is a serine/threonine kinase that has a function in the mitotic spindle checkpoint and is mutated in certain types of human cancer [49]. Ctla2a and Ctla2b are cysteine proteinase inhibitors expressed in activated T-cells and mast cells [50]. Both naïve and memory T-cells have high levels of IL7R, and IL7 is required for their homeostasis [51]. Furthermore, recently it was shown that Wt and Itk-deficient CD4+ T-cells express similar levels of IL7R (CD127) [13]. Certain genes in the Itk/CN group did not have bona fide NFAT-sites as determined by our bioinformatic approach. This could be due to that the current data base algorithms are not good enough to predict the putative sites or that the chromosomal stretches harboring NFAT-sites are located outside the 500 bp region that we choose to study. Future studies will aim to reveal a possible link between the altered expression of these genes and the phenotype of Itk-deficiency.

Finally, a comparative analysis of Itk-deficient T-cells and Btk-deficient B-cells revealed a significant overlap of transcripts, indicating that there is a common Tec family gene expression profile in lymphocytes. The fact that 16/18 genes had a similar fold-induction in T- and B-cells (p < 0.05) suggests common regulation of these genes by Itk and Btk. Conversely, the observation that two transcripts (Col14a1 and Ccr1) were differentially expressed may simply reflect that B- and T-cells represent different lineages, each characterized by unique features of their transcriptomes. Of the six most up-regulated genes in Btk-deficiency [24] all of them were >2-fold changed in cells lacking Itk, eight of which were also significantly altered in Itk-deficient T-cells (with p-values ranging from <0.05 to <3 × 10-5). Tgfbi, which was up-regulated in Btk-/- and confirmed as highly increased in Itk-/- (p < 3 × 10-5) samples, encodes an extracellular protein that mediates cell adhesion to collagen, laminin and fibronectin via its interaction with integrins [52]. Since these 16 genes are common to both Btk- and Itk-dependent transcriptomes it seems likely that the corresponding promoters could be activated through signaling components controlled by either pathway. Future identification of regulatory elements targeted by common factors could reveal the underlying mechanism.

Conclusion

This report is the first to define the transcriptional signature of cells from Itk-/- mice. The transcriptome of Itk-deficient cells revealed that there is a large overlap with regular CD4+ and CD8+ cells. Future studies analyzing different stages of innate, memory-like cells from Wt mice will aid in unraveling to what extent the innate population of Itk-deficiency also shows unique features, which differ from normal mice.

Declarations

Acknowledgements

We would like to thank the Affymetrix core facility at Novum, BEA, Bioinformatics and Expression Analysis, which is supported by the board of research at the Karolinska Institutet and the research committee at the Karolinska hospital. This work was supported by the Swedish Science Council, the Swedish Cancer Fund, the European Council FP7 grant EURO-PADnet, the Wallenberg foundation, the Stockholm County Council (research grant ALF-projektmedel medicin), the Special Research Area SFB-F23 (project SFB-F2305) of the Austrian Science Fund (FWF), and by the START program (grant Y-163) of the FWF and the Austrian Ministry of Science and Research (BM:WF).

Authors’ Affiliations

(1)
Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital Huddinge
(2)
Division of Immunobiology, Institute of Immunology, Center for Physiology, Pathophysiology and Immunology, Medical University of Vienna
(3)
Department for Informatics, Center for Bioinformatics

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© Blomberg et al; licensee BioMed Central Ltd. 2009

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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