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 [9
], 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+
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 [38
]. 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+
]. 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 [9
]. The overlap between CD4+
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
]. 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+
]. Klrc2 (NKG2C) and Klrk1 (NKG2D) were previously reported in CD8+
]. In addition, we found Klra4 (Ly49D) and Klra19 (Ly49S), not previously described in this context. In unstimulated Itk-/-
T-cells eleven Klr members were found (shown in Table ). Klra3, a7, a8 and b1b were shown to be common to the CD4+
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+
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+
]. 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. ). 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
], 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.