Gene expression profile of human T cells following a single stimulation of peripheral blood mononuclear cells with anti-CD3 antibodies

Background Anti-CD3 immunotherapy was initially approved for clinical use for renal transplantation rejection prevention. Subsequently, new generations of anti-CD3 antibodies have entered clinical trials for a broader spectrum of therapeutic applications, including cancer and autoimmune diseases. Despite their extensive use, little is known about the exact mechanism of these molecules, except that they are able to activate T cells, inducing an overall immunoregulatory and tolerogenic behavior. To better understand the effects of anti-CD3 antibodies on human T cells, PBMCs were stimulated, and then, we performed RNA-seq assays of enriched T cells to assess changes in their gene expression profiles. In this study, three different anti-CD3 antibodies were used for the stimulation: two recombinant antibody fragments, namely, a humanized and a chimeric FvFc molecule, and the prototype mouse mAb OKT3. Results Gene Ontology categories and individual immunoregulatory markers were compared, suggesting a similarity in modulated gene sets, mainly those for immunoregulatory and inflammatory terms. Upregulation of interleukin receptors, such as IL2RA, IL1R, IL12RB2, IL18R1, IL21R and IL23R, and of inhibitory molecules, such as FOXP3, CTLA4, TNFRSF18, LAG3 and PDCD1, were also observed, suggesting an inhibitory and exhausted phenotype. Conclusions We used a deep transcriptome sequencing method for comparing three anti-CD3 antibodies in terms of Gene Ontology enrichment and immunological marker expression. The present data showed that both recombinant antibodies induced a compatible expression profile, suggesting that they might be candidates for a closer evaluation with respect to their therapeutic value. Moreover, the proposed methodology is amenable to be more generally applied for molecular comparison of cell receptor dependent antibody therapy. Electronic supplementary material The online version of this article (10.1186/s12864-019-5967-8) contains supplementary material, which is available to authorized users.


Background
Immunosuppressive therapies based on monoclonal antibodies (mAbs) started in the 1980s, with the use of Muromonab-CD3 (OKT3), an antihuman CD3 antibody, for attaining long-term graft survival after organ transplantation 1 . After decades of use, this biopharmaceutical was withdrawn from clinics due to its toxic side effects 2 . However, the emergence of a new generation of (re)engineered recombinant antibodies has sparked hopes that anti-CD3 antibodies may again be used to induce peripheral tolerance 3 , renewing the enthusiasm for CD3-targeted therapies. Hence, anti-CD3 therapy is now being tested for several autoimmune and inflammatory diseases 4,5 . Furthermore, recent clinical data on the use of Teplizumab in type I diabetes 6 contribute to this optimism that new anti-CD3 therapies for autoimmunity and transplantation will become available in a foreseeable time. The administration of anti-CD3 antibodies induces the general activation of T cells, which may lead to a state of tolerance not yet fully understood 3,7 . The proposed mechanism of a peripheral tolerance induction rests upon a potential modulation of regulatory lineages of the CD4 phenotype 8,9 , even though CD8 regulatory cells were also shown to be affected 10,11 . Peripherally induced regulatory cells control the activation of T cells, promoting negative feedback in the inflammatory response. The induction of a more regulatory environment by anti-CD3 antibodies could produce antigen-specific tolerance and alleviate the immune response. More recent data on human clinical data suggest that other mechanisms such as T cell exhaustion 11,12 or the induction of inhibitory receptors on T cells 13,14 could also contribute to the suppression of the immune response.
The effect of anti-CD3 therapy has been addressed in different studies trying to elucidate its mechanism by assessing the genetic profile of T cells induced by those antibodies.
These studies have been performed by microarray analysis 15,16,17 or, more recently, by Next Generation Sequencing (NGS) 18,19 . Nevertheless, in the majority of the investigations, anti-CD3 is not the unique stimulus but is combined with anti-CD28 antibody and/or interleukins, such as IL2. More importantly, these studies are often performed using isolated T cells and thus are in a very different context from the PBMC environment. In the present work, we compared two recombinant antibody fragments, a chimeric fragment and a humanized fragment in an FvFc format, with their prototypic antibody OKT3. The treatment was performed using healthy human donor PBMCs in vitro.
The global changes in the transcriptome profile were assessed using RNA-seq.
Subsequently, their T cell differentiation markers and immunoregulatory signatures were compared. Our data showed that, despite the antibody format, the three anti-CD3 antibodies induced a common pattern of gene expression strongly enriching regulatory genes as well as genes involved in inhibitory signaling. We propose that these comparative analyses could be exploited as a validation tool in designing new and more effective CD3-binding molecules.

Global change in the gene expression profile in human T cells induced by anti-CD3 treatments
To compare the effects of each of the three anti-CD3 antibodies on human T cells, the gene expression profiles were analyzed. T cells were obtained from 72-hour untreated or treated PBMCs with one of the three anti-CD3 antibodies: OKT3, FvFc M (OKT3 scFv fused to human IgG1 Fc), and a humanized version of this FvFc (FvFc R). Anti-CD3 was used as the sole stimulus. To avoid any further stimulation, T cells were obtained by negative selection, using magnetic beads for cell surface markers. The purity of the T cell population was assessed by flow cytometry and was above 96% (Supplementary Figure   S1). The transcriptomes of stimulated and unstimulated T cells from a single individual were obtained by performing sequencing in two replicates. More than 55 million pairedend reads of 150 bp length were obtained. The reads were mapped to the human reference genome (hg19); of the total reads, 84% to 94% were mapped (Table 1).
Subsequently, we assessed differentially expressed genes (DEGs) by comparing each anti-CD3 antibody-treated sample with the control of unstimulated T cells. The gene sets found to be differentially expressed in the different treatments are shown as a MA plot in Figure   1A and as a Venn diagram in Figure 1B. OKT3 treatment resulted in a larger set of differentially expressed genes (7089) with a fold change of less than -0.8 or above 0.8, followed by FvFc R treatment with 2425 DEG and FvFc M treatment with 1406 DEG. We found 860 genes that were equally regulated among the treatments, considering a padj ≤ 0.05. Except for FvFc R treatment, DEGs were mostly downregulated. FvFc R induced the most unbalanced DEG dataset, with 58% (1,419) upregulated over 41% (1,006) downregulated DEGs. The gene regulation profile promoted by FvFc R was more similar to OKT3 than FvFc M, even though the cluster analysis suggested a similar DEG profile for each treatment ( Figure 1C).

Associations of DEGs with Gene Ontology categories
Anti-CD3 stimulation was shown to affect different set of genes 18,19 . Therefore, functional characterization of the differentially expressed genes was performed using GO term enrichment analysis. Anti-CD3 activated and repressed DEGs were separately classified for the GO category "biological process". Upregulated genes were dominated by terms associated with cell proliferation (Figure 2), reflecting the anti-CD3 associated activation of T cells. To visualize changes in GO term enrichment and coverage (completeness), immune-associated terms were selected among up-and downregulated DEGs for each antibody treatment, focusing on those associated with the immune response and inflammation typically associated with anti-CD3 therapy ( Figure 3).
All antibodies induced a similar profile of GO term enrichment, coverage and FDR adjusted p-value, shown by radar plots (Figure 3). Among the upregulated genes, the predominance of OKT3-induced GO term coverage was less obvious. Between selected terms, the most enriched GO term among the upregulated genes was the Regulation of Regulatory T Cell Differentiation (GO:0045589), but terms for the regulation of IFNγ (GO:0032729), IL-10 (GO:0032653) and IL-12 production (GO:0032655) were also highlighted.
The downregulated DEG set enriched terms reflected categories that fade after antibody treatment. It is notable that, among the GO terms enriched by genes repressed after treatment, the term "regulation of inflammatory response" (GO:0050727), was the most conspicuous. Furthermore, the terms "immune response-regulating signaling pathway" (GO:0002433) and "activation of immune response" (GO:0002253) were also evident ( Figure 3).

CD3 stimulation
Anti-CD3 antibody therapy is strongly associated with an over secretion of cytokines, also known as a "Cytokine Storm" 4 . The deleterious consequences of the cytokine production are assumed to be promoted by the Fc part of the molecule, and novel humanized antibodies can circumvent these consequences by inducing a nonmitogenic effect. Our data suggest that the in vitro administration of all three anti-CD3 antibodies induce the upregulation of several cytokine genes, including INFG, IL17A, IL17F, LIF and TNF ( Figure   4). However, when we analyzed the expression of IL17 in human donors by RT-qPCR, we noticed that even though the IL17A gene expression was consistently expressed along all treatments in the NGS panel, its induction was variable among antibody-treated donor T cells ( Figure 5A). The FvFc R and OKT3 treatment also induced upregulation of IL6 and IL32. OKT3 treatment induced additional interleukins such as IL1B, IL2, IL3, IL9, IL13, IL12B, IL21 and IL22 ( Figure 4).
Cytokine receptors were also induced after antibody treatment, including strong upregulation of the IL2 receptor subunit genes, IL2RA and IL2RB ( Figure 4). IL2RA expression was also tested in the qPCR panel of treated donor T cells, suggesting that any form of anti-CD3 induces the expression of the IL2 receptor α-chain, also known as CD25 Anti-CD3 antibody treatment induced the upregulation of several interleukin and interleukin receptors genes, but only a few interleukins and receptors were downregulated due to antibody treatment. IL10 and IL24 expression was significantly repressed after OKT3 and FvFc R treatment, while IL18BP was repressed by OKT3 and FvFc M. In addition, OKT3 treatment also reduced the expression of IL18 ( Figure 4). IL10 was further investigated by qPCR. Notwithstanding, the qPCR panel suggested that OKT3 treatment had a variable effect on IL10 expression among treated donor cells, and the FvFc-based antibody had no significant effect ( Figure 5D).

Downregulation of interleukin receptors makes T cells less sensitive to their cognate
cytokine. The NGS panel suggested that OKT3 treatment might interfere with signaling of interleukins IL10, IL11 and IL13, due to the downregulation of IL10RA, IL10RB, IL11RA and IL13RA1 ( Figure 4). IL6R was downregulated after treatment with OKT3 and FvFc R. The IL17RA codes for IL17A specific receptor and was found to be downregulated after OKT3 treatment, with a barely significant q-value (0.0069); nevertheless, the qPCR panel confirmed this tendency for downregulation after treatment with any of the antibodies ( Figure 5E). The IL17RC gene, which codes for a receptor for both IL17A and IL17F, was found to be downregulated after both OKT3 and FvFc M treatment. The receptor for IL7, IL7R, was shown to be downregulated with both FvFc R and OKT3 treatment. The qPCR panel corroborated these results, suggesting that most donor T cells respond to any anti-CD3 antibody format, reducing the IL7R expression levels ( Figure 5F).
Anti-CD3 stimulation regulates phenotypic marker genes Activation of resting T cells by anti-CD3 antibodies can induce cell differentiation, and indeed, several phenotypic markers are modulated after antibody treatment. Resting T cells can differentiate in several lineages of effector and regulatory phenotypes, and specific genetic markers can characterize these T cell phenotypes. We compared several markers for CD4 and CD8 subpopulations depicted as panels to visualize their possible differentiation ( Figure 6). To confirm prototype marker expression levels found in the NGS panel, qPCR analyses were performed using anti-CD3 treated T cells ( Figure 7). Some expression markers are key for charactering T cell subpopulations. The Th1 marker TBX21, which codes for the TBET transcription factor, was shown to be significantly induced only with OKT3 treatment in the NGS panel ( Figure 4). The qPCR panel corroborated the NGS data ( Figure 7A), suggesting a minimal effect of FvFc antibodies on TBX21 expression. STAT4, another Th1 marker, was also only induced by OKT3 in the NGS experiment, but qPCR data suggests that FvFc R could also affect the expression levels of STAT4 in stimulated cells 20,21 (Figures 4 and 7B). GATA3, a Th2 phenotypic marker, was not significantly induced in NGS or qPCR data (Figures 4 and 7C). However, other characteristic markers of this subtype were induced 21,22 (Figure 6).
In addition, we also analyzed markers for the Th17 subpopulation 23,24 (Figures 4, 5, 6 and 7). The gene that codes for ROR ϒ t, RORC was found to be slightly upregulated after treatment with both OKT3 and FvFc M antibodies ( Figure 4), but without significance (padj > 0.01). In the qPCR panel, RORC was shown to be barely activated in all three treatments ( Figure 7D). IL17A, known to be produced by Th17 cells, was upregulated in the NGS panel, but these data were not supported by qPCR, which suggests a variable and mild regulation of this gene ( Figure 5A). The third marker, STAT3, was found to be induced by OKT3 in the NGS data and was induced by OKT3 and FvFc R treatments, as measured by qPCR ( Figure 7E). Interestingly, the FvFc M antibody induced a very contrasting effect on different donors. Half of the donors showed an upregulated profile, while the other half showed a downregulated profile.
T cells can assume a regulatory phenotype, and many regulatory markers were found in this analysis 25,26 (Figure 6). FOXP3, a major transcription factor that is associated with the human T regulatory phenotype, was upregulated in the NGS data for all antibody treatments. These data were corroborated by qPCR ( Figure 7F Modulations of CD8 T cell markers were also observed after anti-CD3 treatment, suggesting changes in the CD8 T cell population 27,28 (Figure 6). Among these markers, EOMES and KLRG1 were repressed after all the anti-CD3 treatments, but GMZB was strongly induced by anti-CD3. These three markers were also tested by qPCR, which confirmed the tendency of the NGS data ( Figure  Anti-CD3 stimulation modulates genes that encode nuclear receptor transcription factors Nuclear receptors integrate a family of transcription factors that respond to hormones and hydrophobic molecules that have been associated with the control of the immune response 30 . Thus, the PFAM family for Nuclear Receptor (PF00104), was used to probe antibody-induced DEGs. Anti-CD3 treatment induced the expression of PF00104-associated genes. OKT3 induced 7 genes, while FvFc R induced 3 and FvFc M induced 2 genes. The orphan nuclear receptor gene NR4A1 was activated in all treatments at a padj < 10 -5 .
Three other PF00104 annotated genes were found in two of three treatments: NR4A3, RORC, and VDR ( Figure 4). NR4A3 codes for a mitogen-associated nuclear receptor (http://www.uniprot.org/uniprot/Q92570). RORC is mentioned above as a marker for lymphocyte lineages. VDR codes for the vitamin D3 receptor, and its overexpression was detected in all antibody treatments by qPCR ( Figure 7P).
Among the downregulated DEGs, peroxisome proliferator-activated receptor gamma (PPARG), a gene associated with the development of Tregs, was found to be 4-to 9-fold less expressed than that in the unstimulated T cells (Figure 4). Moreover, the THRA gene that codes for thyroid hormone receptor alpha was also repressed in all treatments. therapy was used as an adjuvant for acute episodes of graft rejection, but its use was discontinued due to pronounced side effects 2 . However, despite the prolonged clinical use, the mechanism of action of OKT3 is still uncertain. In this study, human T cells were treated with anti-CD3 antibodies in vitro, within the complexity of the PBMC milieu, in an attempt to simulate the natural ambiance that occurs in the intravenous administration of therapeutic anti-CD3. This in vitro experimental model was used to compare the mouse mAb OKT3 with two recombinant antibody fragments inspired by the mAb: a humanized and a chimeric human IgG1 in the FvFc format (scFv-hinge-CH2-CH3).

Effect of an exclusive anti-CD3 stimulation
Currently, most antibody therapies rely on full-sized mAbs, derived from chimeric, humanized or fully human sequences, but new molecular formats may represent technological and economical alternatives. The FvFc format used here represents a novel solution as a single-chained homodimeric molecule that mimics heteromultimeric mAbs 31,32,33,34,35,36 . The DEG profiles induced by each antibody format were very similar as judged from the enrichment analysis, despite the larger DEG set induced by OKT3, especially for the repressed DEG set. The ontology-based classification for up-and downregulated DEGs suggests that all antibody formats induce a very similar profile, marked by a sharp mitotic response (with a low p-value), and a higher, even significant, pvalue for "Immune"-related GO. It is noteworthy that FvFc compares positively for several terms, such as regulation of "regulatory T cell" and "interleukin-10 production" and "inflammatory response." Overall, despite the larger set of OKT3 DEGs, FvFc molecules could enrich GO terms at least similarly.
The broader coverage of GO terms of OKT3 DEGs may reflect their greater mitogenic stimulus 31 , while the humanized FvFc displayed a skewed DEG profile, yet preserving its function. Further analyses suggest that the chimeric molecule FvFc R reproduces the OKT3 DEG profile more accurately than FvFc M, despite the better binding proprieties of the latter molecule. Therefore, the humanization process seems to have preserved the original OKT3 paratope in the recombinant molecules, suggesting them as alternative CD3 binders for clinical anti-CD3 therapy.
The mitogenic activity of OKT3 and other anti-CD3 antibodies renders them especially investigated for therapeutics 37,38,39,40 . The analysis of differentially expressed gene ontology classification suggests that all three anti-CD3 antibodies modulate a distinct number of genes related to cell proliferation and mitosis. This supports a significant impact of anti-CD3 therapy on T cell proliferation as observed for the proliferation marker

MKI67 and the T-specific activation marker CD25 (IL2RA). The activation of T cells by OKT3
and other anti-CD3 antibodies is usually associated with the clinical efficacy of this antibody 39 . However, upregulation of activation markers does not correlate with antibody mitogenic activity, since non mitogenic anti-CD3 antibodies may also induce activation markers in vivo 40 . Therefore, these data corroborate a previous characterization of the FvFc R antibody, shown to be less mitogenic than OKT3 31 , despite inducing several activation markers, as observed in the present study.
Most models for anti-CD3 therapy rely on CD4 regulatory cells 41,42,43 , but the majority of data supporting it came from mouse models. Recent data on humanized antibodies in clinical trials highlight the role of CD8 cells in tolerance associated with anti-CD3 therapy, suggesting a two-phase model: a short-term depletion of T cells followed by induction of regulatory mechanisms 6 . A burst of cell activation initially induces mitotic mechanisms.
Our data suggest that even after three days of anti-CD3 stimulation, activated T cell DEGs are still dominated by a mitotic signature, as seen by GO term enrichment, but, along with, barely detected emerging immunoregulatory mechanisms.
Several regulatory phenotypes have been proposed, along with genes usually associated with a regulatory function 44 . For CD4 cells, regulatory cells are distinguished from effector cells that are classified as Th1, Th2, and Th17. A TBET signature with high production of IFNγ characterizes Th1 cells, but TBX21, which codes for TBET, is only weakly upregulated by OKT3, in line with previous observations 43 . Th2 cells do not appear to be induced by anti-CD3 since no significant alteration in GATA3 expression was observed. Beyond that, markers for Th17 and T regulatory cells are predominantly found in anti-CD3-treated cells.
Among those with the Th17 phenotype, IL17A, IL17F, and IL16 were upregulated, and FOXP3, GITR, LAG3, and CTLA4 were characteristic of the regulatory phenotype. These markers were all observed to some degree in each of treatments but commonly were weakly expressed among donors stimulated with FvFc M.
The anti-CD3 treatment seemed to bias toward a Th17/Treg polarity, as suggested before 45,46 . However, FOXP3, an important marker of regulatory cells, was only weakly induced after PBMC stimulation. It is possible that by analyzing gene expression after 72 h of anti-CD3 induction, we missed the transient FOXP3 peak kinetics 18,19 . Moreover, the activation of IRF4, a late effect of FOXP3 activation, represses FOXP3 and may negatively affect its expression 47 . IRF4 was upregulated in all the anti-CD3-treated cells.
Interestingly, anti-CD3-treated cells showed an apparent decrease in the mRNA levels of CD127 (IL7R), the IL-7 receptor, for which downregulation is considered to be a hallmark of a bona fide regulatory phenotype in humans 29,48,49 .
IL10 is a marker for regulatory CD8 and the CD4 (Tr1) phenotype 50,51 . We found no significant IL10 regulation except for a slight decline due to treatment with OKT3.
However, we noted an enrichment of the "regulation of Interleukin-10" GO term, suggesting that the machinery for IL10 production was activated in anti-CD3-treated cells.
FvFc R antibody was previously shown to induce a high IL10/IFNγ ratio compared to OKT3 in anti-CD3-stimulated PBMCs 31 , although no significant induction of IL10 was observed in the present study. Nevertheless, the increase in the IL10/IFNγ ratio observed by Silva and colleagues could be explained in part by a more consistent induction of the IFNG gene in OKT3-treated T cells, or likewise due to a non-lymphocytic origin of the produced IL10 probed in the whole PBMCs 31 .
Clinical data on novel humanized antibodies suggest new mechanisms of anti-CD3 action in humans. In mice, studies have suggested that anti-CD3 therapy induces immunosuppression dependent on CD4 T cells, with stimulated helper cells developing a regulatory phenotype. However, in humans, the CD8 lineage also seems to contribute to the tolerogenic effect of anti-CD3, either by inducing differentiation into CD8 regulatory cells 15,29 or by leading CD8 T cells to exhaustion 11,12 . Data from Teplizumab clinical trials suggested that the immunosuppressive effect of the humanized antibody is due to anergic and exhausted CD8 cells 12,52 , along with CD8 and CD4 Tregs 15 . Nonetheless, inhibitory receptors were clearly activated in our model system, including PDCD1, CTLA4, and LAG3, suggesting that inhibition of the immune response and inflammation after 72 h of a proliferative stimulus might have led to an exhausted phenotype 14 . Otherwise, inhibitory receptors may not indicate exhaustion, but a detuning of CD8 T activation. It is possible that these inhibitory receptors signify the transition from a highly activated T cell state toward a differentiation/memory profile 11,53 . The high and consistent induction of the PDCD1 gene (PD-1) observed here suggests a general detuning of anti-CD3-treated cells, that may underlie the basis of anti-CD3 therapeutic effects.
The detuning of T cell following PD-1 expression may contribute to the effect of anti-CD3, but other molecular players may also contribute to immunosuppression. IDO1 is a tryptophan catabolic enzyme known to induce regulatory T cells and immunosuppression 54,55 . Although usually produced by monocytes, a CD4+IDO+ lymphocyte population had been characterized 56 . Anti-CD3 treatment induced IDO1 upregulation in T cells, although not uniformly among donors. In this sense, a putative IDO-producing T cell could trigger a profound regulatory effect by locally restricting available tryptophan. This finding may represent an alternative mechanism of T-cellinduced immunosuppression that could be therapeutically exploited.

Conclusions
Novel therapeutic anti-CD3 antibodies development could focus on regulatory associated GO term enrichment and specific subpopulation markers. The in vitro assay proposed here, based on a simple and economical procedure, seems to be efficient to compare novel antibody molecules before clinical evaluation. Development of new antibodies or novel pharmaceutical association could benefit from this in vitro methodology, allowing a novel discovery pipeline based on a System Biology approach.
In conclusion, we used a deep transcriptome sequencing method for comparing three anti-CD3 antibodies regarding Gene Ontology enrichment and immunological marker expression. The present data showed that both recombinant antibodies induced a compatible expression profile, suggesting that they might be candidates for closer evaluation concerning their therapeutic value. Moreover, the proposed methodology is amenable to be more generally applied for molecular comparison purposes.

Donors
Peripheral blood was collected from seven healthy individuals enrolled in this study (Supplementary Table S1). For NGS a single donor was analyzed and for qPCR assays, seven healthy individuals were enrolled. All human blood experiments were performed in accordance to the Ethics Committee of the University of Brasilia guidelines, which approved the study protocol (CAAE: 32874614.4.0000.0030). A written informed consent was obtained from all human donors. Antibodies OKT3 was purchased from eBioscience (San Diego, CA, USA). The humanized antibody fragment FvFc R is a single-chain FvFc molecule and was previously described (FvFc version R) 31 Figure S1).

RNA extraction
Total RNA was extracted from T cells isolated after PBMC stimulation using the miRNeasy® Mini Kit (Qiagen, Valencia, CA, USA) as described before 46 . RNA integrity and purity were evaluated using a Bioanalyzer 2100 (Agilent Technologies Genomics, Santa Clara, CA, USA). All RNA samples used in this work showed an RIN > 7. Sample sequencing and differential gene expression

Analysis of gene functions
The enrichment GO terms for biological processes of DEG were also assessed. For this purpose, upregulated (log 2 FC >1.2) and downregulated (log 2 FC <1.2) gene set enrichment analyses were performed using functional categories of the database Gene Ontology (GO).
The Panther software 63,64 was used to calculate enrichment, p-values and FDR adjusted pvalues. The super category "biological process" was used, and within this category, GO terms related to the immune system and inflammatory process were further investigated.
Nuclear receptor analysis was performed exclusively for the Pfam family PF00104 of the Pfam database (http://pfam.xfam.org/). Members of PF00104 were searched in the DEG set using regular expression and analyzed individually.

Gene expression analysis by qPCR assays
Quantitative PCR was performed as previous described 46 . Briefly, total RNA isolated from T cells was utilized for cDNA synthesis using an RT 2 First Strand Kit (Qiagen, Valencia, CA, USA). The expression genes were quantified using RT² qPCR SYBR Green/ROX MasterMix (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. The housekeeping gene B2M was used as the endogenous control. qPCR assays were performed using an ABI Step          Supplementary Files