T cell activation and programming from their naïve/resting state, characterized by widespread modifications in chromatin accessibility triggering extensive changes in transcriptional programs, is orchestrated by several cytokines and transcription regulators. PRDM1 and PRDM2 encode for proteins with PR/SET and zinc finger domains that control several biological processes, including cell differentiation, through epigenetic regulation of gene expression. Different transcripts leading to main protein isoforms with (PR +) or without (PR-) the PR/SET domain have been described. Although many studies have established the critical PRDM1 role in hematopoietic cell differentiation, maintenance and/or function, the single transcript contribution has not been investigated before. Otherwise, very few evidence is currently available on PRDM2. Here, we aimed to analyze the role of PRDM1 and PRDM2 different transcripts as mediators of T lymphocyte activation.
Methods
We analyzed the transcription signature of the main variants from PRDM1 (BLIMP1a and BLIMP1b) and PRDM2 (RIZ1 and RIZ2) genes, in human T lymphocytes and Jurkat cells overexpressing PRDM2 cDNAs following activation through different signals.
Results
T lymphocyte activation induced an early increase of RIZ2 and RIZ1 followed by BLIMP1b increase and finally by BLIMP1a increase. The “first” and the “second” signals shifted the balance towards the PR- forms for both genes. Interestingly, the PI3K signaling pathway modulated the RIZ1/RIZ2 ratio in favor of RIZ1 while the balance versus RIZ2 was promoted by MAPK pathway. Cytokines mediating different Jak/Stat signaling pathways (third signal) early modulated the expression of PRDM1 and PRDM2 and the relationship of their different transcripts confirming the early increase of the PR- transcripts. Different responses of T cell subpopulations were also observed. Jurkat cells showed that the acute transient RIZ2 increase promoted the balancing of PRDM1 forms towards BLIMP1b. The stable forced expression of RIZ1 or RIZ2 induced a significant variation in the expression of key transcription factors involved in T lymphocyte differentiation. The BLIMP1a/b balance shifted in favor of BLIMP1a in RIZ1-overexpressing cells and of BLIMP1b in RIZ2-overexpressing cells.
Conclusions
This study provides the first characterization of PRDM2 in T-lymphocyte activation/differentiation and novel insights on PRDM1 and PRDM2 transcription regulation during initial activation phases.
Erika Di Zazzo and Monica Rienzo share first authorship
Maurizio Bifulco and Ciro Abbondanza share last authorship
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Abkürzungen
AKT
Protein kinase B
APC
Antigen presenting cell
BACH2
BTB Domain and CNC Homolog 2
BCL6
B cell lymphoma 6
BLIMP1
B lymphocyte-induced maturation protein 1
CCR C–C
Motif Chemokine Receptor
CD40LG
CD40 Ligand
CIITA MHC
Class II trans-activator
CML
Chronic myelogenous leukemia
CTLA-4
Cytotoxic T-Lymphocyte Antigen 4
CXCR
C-X-C Motif Chemokine Receptor
DLBCL
Diffuse Large B-Cell Lymphoma
FBS
Fetal bovine serum
FOXP3
Forkhead box P3
GAPDH
Glyceraldehydes-3-phophate dehydrogenase
HMTase
Histone methyltransferases
IFN
Interferon
IL
Interleukin
IL2RA
Interleukin 2 receptor subunit alpha
Ion
Ionomycin A
IRF
Interferon regulatory factor
IRS
Insulin receptor substrate
KLF2
KLF Transcription Factor 2
MAPK
Mitogen-activated protein kinase
MEK
Mitogen-activated protein kinase kinase)
PAX5
Paired box 5
PBMCs
SPeripheral blood mononuclear cells
PI3K
Phosphatidylinositol 3-kinase
PMA
Phorbol 12-myristate 13-acetate
PRDMs
PRDF1 and RIZ1 homology domain containing proteins
RIZ
Retinoblastoma protein interacting zinc finger gene
RORC
RAR Related Orphan Receptor C
RPS18
Ribosomal protein S18
STAT
Signal transducer and activator of transcription
TBX21
T-Box Transcription Factor 21
TCR
T cell receptor
Th
T helper
TNFR
Tumor necrosis factor receptor
Background
The activation and programming of T cells from their naïve/resting state is a critical point for the functions of the adaptive immune response. This process, characterized by widespread changes in chromatin accessibility triggering extensive changes in transcriptional programs, is orchestrated by a plethora of signals including cytokines, inflammatory and immune-modulatory products, tissue-specific and transcription regulators that ultimately influence the developmental choices of the T cell [1].
PRDF1 and RIZ1 homology domain containing proteins (PRDMs) are a family of structurally related transcriptional regulators, controlling several biological processes, including differentiation of a variety of cell types [2]. Their protein structure exhibits several zinc fingers preceded by the PR/SET domain, which is a SET-like domain typical of methyltransferases [3, 4]. PRDMs function in epigenetic regulation of gene expression through their intrinsic histone methyltransferases (HMTase) and/or via interactions with chromatin modifying enzymes. For instance, no methyltransferase activity has been demonstrated for PRDM1, which achieves its repressor activity through other proteins, including histone deacetylases [5‐9] whereas the conserved N-terminal PR domain of PRDM2/RIZ1 has an intrinsic H3K9 methyltransferase activity [10, 11]. PRDM genes display a characteristic yin-yang expression pattern where alternative splicing or different promoter use rises to a full-length protein (PR +) and a shorter isoform lacking the PR domain (PR −) [12‐14]. An imbalance of these isoforms is often observed in several malignancies, including leukemias and lymphomas, with the PR + product commonly lost or downregulated both by genetic inactivation or epigenetic silencing and the PR − isoform always expressed at high concentration levels [12‐14].
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PRDM1 gene, widely known as BLIMP1 (B lymphocyte-induced maturation protein 1), encodes for two PR + and PR − major isoforms designated as BLIMP1a and BLIMP1b respectively, which arise from alternative promoter usage. Indeed, BLIMP1b is transcribed from a promoter and exon positioned upstream of exon 4 of the PRDM1 gene. The BLIMP1b protein lacks the first 101 amino acids of BLIMP1a N-terminal region and instead contains three amino acids fused to residues 102–789 of BLIMP1a (see scheme in Fig. 1) [15]. Currently, a lot of studies support its critical role in differentiation, maintenance and/or function of multiple hematopoietic cells of the myeloid and lymphoid lineages [15]. BLIMP1a orchestrates plasma cell differentiation by repressing genetic programs associated with the germinal center stages, while at the same time activating those programs associated with plasma cell functions. Specifically, PRDM1/BLIMP1 drives plasma cell differentiation by repressing several transcription factors, such as Myc, CIITA (MHC class II trans-activator), PAX5 (paired box 5), which is required for B cell fate specification, and BCL6 (B cell lymphoma 6), which promotes germinal centre B cell identity [16‐18]. Notably, BCL6 also acts as a transcriptional repressor and forms a negative loop with PRDM1/BLIMP1 [17]. Besides, PRDM1 expression is induced in activated CD4+ and CD8+ T lymphocytes where it regulates many aspects of their differentiation in effector and memory cells [19]. Indeed, PRDM1/BLIMP1 also acts as a repressor in subsets of effector CD4+ and CD8+ T cells [20‐26]. As for B cells, a reciprocal repression of BCL6 and PRDM1 is crucial for T lymphocytes differentiation. In this cell context, PRDM1/BLIMP1 represses IL-2 (interleukin 2). IL2 regulates the initial expansion of naïve T cells then, acting through STAT5 (signal transducer and activator of transcription 5), it induces PRDM1 expression creating a feedback loop that downregulates its own expression during the later stages of T-cell differentiation [27, 28]. PRDM1/BLIMP1 can act also as a transcriptional activator in regulatory T cells by promoting H3K4 methylation at the IL10 (interleukin 10) locus [20]. A BLIMP1a/BLIMP1b imbalance has been detected in several malignancies derived from B, T, and NK cells [reviewed in 12, 29]. Different regulatory mechanisms, both transcriptional and posttranslational, have been reported to control PRDM1/BLIMP1 expression. Numerous transcription factors, including BCL-6, PAX5, and BACH2, as well as TNFR and cytokine signaling pathways, are known to directly affect PRDM1 transcription [30‐34].
Fig. 1
PRDM1 and PRDM2 expression in CD4 + T lymphocyte activation. Bar graphs represent data from qRT-PCR analysis of PRDM1 and PRDM2 main transcripts (BLIMP1a, BLIMP1b, RIZ1, RIZ2) in CD4 + T cells stimulated with anti-CD3/CD28 antibodies. PRDM1 and PRDM2 expression levels in CD4+T cells were calculated using the ΔΔCt method with the indicated control gene. A schematic illustration of human PRDM1 and PRDM2 main products and used amplicons is also reported. To amplify PRDM1 two sets of primers, recognizing specific sequences of BLIMP1a and BLIMP1b transcripts, were used. PRDM2 gene expression was verified using two sets of primers recognizing sequences exclusive of RIZ1 or a common region to both RIZ1 and RIZ2 (and indicated as RIZex8). RIZ2 transcript was measured by subtraction as previously described [46]. The ratio between the PR- and PR + transcripts for each gene was also calculated. The relative expression of activation marker genes CD40LG and IL2RA was also measured. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
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As PRDM1, also PRDM2 gene expresses two main proteins, PRDM2/RIZ1 (PR +) and PRDM2/RIZ2 (PR −), starting from transcripts initiated by two alternative promoters (see scheme in Fig. 1). PRDM2 is expressed in immature progenitor cells and in mature myeloid cells [35]. A selective PRDM2/RIZ1 expression was correlated with myeloid cell differentiation, suggesting a pivotal role for PRDM2 gene products in the proliferation/differentiation transition [36]. Loss of heterozygosity, promoter hypermethylation or PRDM2 gene mutations have been observed in several human cancer types, including Diffuse Large B-Cell Lymphoma (DLBCL) [10, 37]. For example, Riz1 KO mice, which retain Riz2 expression, show a high incidence of DLBCLs and other rare non-hematopoietic cancers [37]. Furthermore, PRDM2 loss is associated with the chronic myelogenous leukemia (CML) collapse to blast crisis [35]. Noteworthy, several reports indicated that PRDM2/RIZ1 is endowed with tumor suppressor activities, whereas PRDM2/RIZ2 acts as an oncogene with putative intrinsic growth-promoting properties [10, 38].
Although previous findings suggest that PRDM2 proteins could have a role in the regulation of the proliferation/differentiation transition in myeloid cells, more extensive investigations in other models (e.g. lymphocytes, hematopoietic stem cells) are advised to confirm a general role of PRDM2 products in the transition from proliferative activity to a quiescent/differentiate state and vice versa [35, 36].
The aim of this study was to evaluate the mechanisms that regulate the expression of the main molecular variants encoded by PRDM1 (BLIMP1a and BLIMP1b) and PRDM2 (RIZ1 and RIZ2) genes, both in lymphocytes from healthy donors and in the Jurkat cell line. Specifically, we aimed to investigate the possible action of RIZ1 and RIZ2 on the gene expression regulation of BLIMP1a and BLIMP1b transcripts and their role in lymphocytes maturation as effectors of the immune response.
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Methods
Isolation, purification, and stimulation of human CD4+and CD8+cells
Human peripheral T lymphocytes were obtained from total peripheral blood mononuclear cells (PBMCs) collected from 10 human samples (60–200 ml, depending on the experimental needs) of 4 healthy donors (authors who contributed to the present manuscript). PBMCs were isolated by density gradient centrifugation (400xg for 30’ at room temperature) on FICOLL-Paque PLUS (GE healthcare Amersham) in 50 mL tubes. After centrifugation the layer of PBMCs was carefully removed and transferred to a new conical tube. Cells were diluted with RPMI-1640 and centrifuged at 200xg for 5’ to remove platelets.
To obtain T naïve cells, PBMCs were resuspended in RPMI-1640 and then transferred to PERCOLL (GE healthcare) 40% and 35%. After centrifugation at 700xg for 25 min at room temperature, cells were washed, suspended in an appropriate volume of RPMI-1640 and counted. T naïve cell concentration was about 6–7 × 106/mL.
To obtain CD4+ and CD8+ T lymphocytes, T naïve cells were incubated for 20’ with specific RosetteSep™ (STEMCELL Technologies), diluted with RPMI-1640 (1:2) and then transferred to FICOLL-Paque PLUS (GE healthcare Amersham) in 50 mL tubes. Isolated cells were analyzed by 6 Color TBNK + Truc assay with BD FACSLyric™ flow cytometer (Becton Dickinson) (Additional file 1: Fig S1).
Isolated cells were maintained in RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS) at 37 °C with 5% CO2 in a humidified atmosphere. For kinetic analysis, purified CD4+ T cells from PBMC were grown in RPMI-1640 25% homolog serum.
T lymphocyte activation and treatments
To induce T lymphocyte activation, 1 × 106 cells were grown in RPMI-1640 5% FBS at 37 °C with 5% CO2 in a humidified atmosphere supplemented with polyclonal activators 50 ng/mL phorbol 12-myristate 13-acetate (PMA) plus 1 μM Ionomycin A (Ion A) (Sigma Aldrich) for 2 h and 6 h. Activation of T cells receptor (TCR) was carried out with antibodies anti-CD3 and anti-CD28 bound to magnetic beads (Dynabeads™, Thermofisher). The percentage of activated cells was measured at 48 h by BD FACSLyric™ flow cytometer (Becton Dickinson) with BD Multitest™ CD8/CD38/CD3/HLA-DR and anti-CD4 PECy7 and anti-CD45 V500C (Becton Dickinson) (Additional file 1: Fig S1).
The mean percentage of activated cells was 33% (SD = ± 8,2%).
RPMI-1640 was supplemented with IL-2 (30U/mL)(Merck), IL-4 (200 ng/mL), IL-6 (200 ng/mL), IFN-α (180 ng/mL) and IFN-γ (200 ng/mL)(GIBCO™, Thermofisher) where described.
PI3K inhibitor, LY294002 (Ly) or Wortmannin (Calbiochem), were used at final concentration of 10 μM and 1 µM, respectively. The MEK1/2 inhibitor PD98059 (PD) and the Src kinase inhibitor PP1 (Alexis) were used at a final concentration of 5 µM.
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Jurkat cell culture and transfection
Jurkat E6.1 (CLS Cell Lines Service GmbH) cells were grown at 37 °C with 5% CO2 in humidified atmosphere in RPMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, and antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin).
The plasmid expressing RIZ1 and RIZ2, were indicated as pSG5_hRIZ1 and pSG5_hRIZ2, respectively. pSG5_hRIZ1 was obtained by cloning in BamH1 site the RIZ1 cDNA from pGEX-RIZ [39] in the FLAG-pSG5 vector. The RIZ2 insert was obtained by PCR amplification of the RIZ1 cDNA sequence with the enzyme PfuTurbo DNA Polymerase (Stragene) using a forward primer containing the BamH1 site and the RIZ2 codon start site and a reverse primer, RIZ2-R, with the stop coding sequence and the BamH1 site (see primer table). To select stable clones of Jurkat cells transfected with pSG5_hRIZ1 and pSG5_hRIZ2, the plasmid pEGFP-C1 (CLONTECH-TaKaRa) was added to the transfection mixture (1:30 compared to FLAG-pSG5, pSG5_hRIZ1 and pSG5_hRIZ2). The EGFP expression was used to evaluate the transfection efficiency and for the resistance to the antibiotic Geniticin G418 (Sigma-Aldrich).
Plasmids for transfection were prepared with Plasmid Midi Kit (Qiagen), according to manufacturer’s instructions.
Cells were transfected using Lipofectamine™ 2000 Reagent in OptiMem I Reduced Serum Medium (Invitrogen™, Thermofisher) for 6 h, following the manufacturer’s instructions. Reaction mixtures were removed, and cells were cultured for an additional 24 h in RPMI1640 with 15% FBS. For Jurkat E6.1 stable clones, selection was performed by addition of 0.8 mg/ml Geneticin G418 (Sigma-Aldrich). pSG5_hRIZ2 shows an epitope tagged by replacing the terminal stop codon with nucleotides encoding FLAG residues (MDYKDDDDK) by PCR. FLAG-pSG5 vector was used as a control together with pEGFP-C1, used as a transfection marker.
Total RNA was extracted from cells using Trizol solution (Invitrogen™, Thermofisher), according to the manufacturer's instructions. The quality and quantity of RNA was assessed by denaturing agarose gel electrophoresis and by spectrophotometry analysis (NanoDrop Technologies), after RNAse-free DNAse-I treatments (Invitrogen™, Thermofisher). RNA was reverse transcribed with SuperScript III (Invitrogen™, Thermofisher) using 500 ng of total RNA; 1 μl of cDNA (60 ng) was used as a template in a PCR reaction with 2.5 U AmpliTaq-Gold (Applied Biosystem™ Termofisher) as previously described [40]. The amplification products were also analyzed by agarose gel electrophoresis. Glyceraldehydes-3-phophate dehydrogenase (GAPDH) was used as housekeeping control gene. Densitometry analysis was performed by ImageJ software and differences were assessed by Student’s t-test with a P < 0.05 considered as significant [40].
Quantitative RT-PCR analysis was performed using the SYBR Green PCR Master Mix (Applied Biosystem™ Termofisher),160 nM of each primer and about 50 ng of cDNA (RNA equivalent) as template in an iCycler thermocycler (Bio-Rad Laboratories). PCR condition were 95 °C for 2 min followed by 45 cycles of 15 s at 95 °C, 60 s at annealing temperature of each primer set and 30 s at 72 °C. All reactions were carried out at least in triplicate for every cDNA template and the melting curves were analyzed to verify the specificity of reaction. The relative gene expression was calculated using the 2−ΔΔCt Pfaffl method [41]. GAPDH or RPS18 were used as housekeeping genes for normalization as indicated [40].
Total cell extracts were separated onto SDS–polyacrylamide gels and blotted onto polyvinylidene difluoride membranes [42]. Western blot analysis was performed as described elsewhere [42] with mouse monoclonal antibody FLAG™ clone M2 (Sigma-Aldrich) and rabbit polyclonal antibody RIZ1 (Abcam Ltd.).
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Statistical analysis
Results are reported as mean ± SD. Three independent experiments in triplicates were performed. GraphPad Prism 9,5 software was utilized to perform Brown-Forsythe and Welch ANOVA tests. Significances were indicated as: *p < 0.05 and particularly significant at **p < 0.01, ***p < 0.001, ****p < 0.0001. Expression correlation analysis was evaluated on the GEPIA2 database (http://gepia.cancer-pku.cn/) using the Spearman correlation coefficient R.
Results
Polyclonal activation of T Lymphocytes increases PRDM1 and PRDM2 expression levels
To investigate the role of the main molecular variants encoded by PRDM1 (BLIMP1a and BLIMP1b) and PRDM2 (RIZ1 and RIZ2) (see scheme in Fig. 1) in the lymphocyte activation we firstly used human peripheral blood lymphocytes and activated them with T cell polyclonal activator phorbol 12-myristate 13-acetate and ionomycin (PMA/Ion) or with anti-CD3/CD28 antibodies at 2 h and 6 h.
Activation of T lymphocytes involves the expression changes of several molecules, both on their surface and within the cell, which clearly characterize them from naïve T cells and can be evaluated by several methods, including gene transcription analysis [43‐45].
In our pilot experiment of RT-PCR analysis, we observed changes of PRDM1 and PRDM2 transcription together with the expression modifications of genes encoding for transcription factors and cytokines involved in lymphocyte activation and differentiation (CD40LG, KLF2, IL2RA, IL2 and MYC) (Additional file 1: Fig. S2).
PRDM2 gene expression was verified using two sets of primers recognizing sequences exclusive of RIZ1 or a common region to both RIZ1 and RIZ2 (and indicated as RIZex8). Because of the extensive similarity between the two gene products, RIZ2 transcript was measured by subtraction as previously described [46]. Similarly, for PRDM1 two sets of primers, recognizing specific sequences of BLIMP1a and BLIMP1b transcripts, were used (see scheme in Fig. 1).
To further detail the early events and the kinetics of PRDM1 and PRDM2 main transcripts we evaluated their expression by qRT-PCR analysis in human CD4+T lymphocytes (greater than 92,5% of total T cells) activated with anti-CD3/CD28 antibodies at different time points (0, 30’, 1 h, 2 h, 4 h, 6 h and 24 h) (Fig. 1). The expression of CD40LG and IL2RA was also analyzed to monitor lymphocyte activation (Fig. 1).
An expression level increase in RIZ1 and RIZ2 transcripts (at major extend) was observed already at 30’ upon lymphocytes activation. At subsequent time points, the RIZ1 expression is constant over the time, while RIZ2 fluctuates until 6 h where no RIZ2 expression was detected (Fig. 1). BLIMP1a is not expressed in basal conditions and its expression grows progressively to reach the maximum expression level at 4 h. After this time point, the BLIMP1a slows down and maintains a high expression level than the starting point (Fig. 1). An increase in BLIMP1b expression level was detected already 30’ after T cell activation, and gained the maximum expression level at 2 h, which is sustained at 4 h. Thereafter, as BLIMP1a, also BLIMP1b slows down and maintains a higher expression level than the starting point (Fig. 1). An increase in the expression levels of both PRDM1 and PRDM2 has been observed upon 24 h activation (Fig. 1). The CD40LG increases rapidly upon activation reaching its maximum expression level at 2 h after the CD3/CD28 stimulation. Subsequently, its expression level progressively decreases maintaining constant level greater than time 0 (Fig. 1). The IL2RA expression slowly increases during T cell activation, and it peaks upon 4 h CD3/CD28 treatment. At the following time points IL2RA expression remains unchanged and higher than control cells (Fig. 1). In addition, we assessed the ratio among BLIMP1b/BLIMP1a and RIZ2/RIZ1. In basal condition CD4+T lymphocytes express prevalently BLIMP1b; during CD4+T lymphocyte activation BLIMP1b/BLIMP1a decreases progressively in favor of BLIMP1a. In basal conditions, CD4+T lymphocytes express predominantly RIZ2; during CD4+T lymphocyte activation RIZ2/RIZ1increases until 1 h where it begins to slow down reaching the lower level at 6 h.
PRDM2 encodes for several transcripts, which can be distinguished also for the presence of different 3’-ends produced by alternative splicing, although they still need to be experimentally validated (see scheme in Additional file 1: Fig S3) [10]. Thus, we also analyzed these slightly different C- terminal tails during T lymphocyte activation utilizing specific oligonucleotides. Interestingly, the amplicon representing the long tail (RIZ ex10) displays the same lower trend as RIZ1 transcript expression levels during T cell activation whereas the higher expression of short tail amplicon (RIZ ex9a) prevalently correlates with RIZ2 (Additional file 1: Fig S3).
Overall, these data suggested the participation of PRDM2 transcripts other than PRDM1 ones in the early stages of lymphocyte activation.
PI3K and Src modulate PRDM1 and PRDM2 gene expression during T lymphocytes activation
Anti-CD3/CD28 or PMA/Ion treatment prompt the activation of various downstream signaling pathways [47‐49]. To figure out the cell signaling pathways activated at cell membrane, transduced within the cell, and targeting PRDM1 and PRDM2 gene expression, we employed Ly294002 (Ly) or Wortmannin PI3K inhibitors and the PP1 Src kinase inhibitor to treat T lymphocytes activated with anti-CD3/CD28 antibodies for 2 h and 6 h.
The qRT-PCR analysis showed that Ly and Wortmannin treatment reduced the RIZ1 expression levels and increased the total RIZ products expression levels, probably due to a RIZ2 increase, thus reverting the effect induced by T cell activation on RIZ1 and potentiating the effect on total RIZ (RIZex8) at 2 h. In addition, Ly and Wortmannin increased both RIZ1 and RIZex8 at 6 h. Otherwise, PP1 treatment augmented both RIZ1 and RIZex8 expression levels observed upon T cell activation already at 2 h (Fig. 2).
Fig. 2
PRDM1 and PRDM2 relative gene expression by qRT-PCR analysis in activated T cell treated with Ly, PP1 Src kinase inhibitor and Wortamannin PI3K inhibitor for 2 h and 6 h. PRDM1 and PRDM2 transcript levels were compared to their expression in naïve T cells (with expression value equal to 1). RPS18 was used as control gene. Three independent experiments in triplicates were performed and data expressed as mean ± SD. ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001
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Wortmannin hindered the BLIMP1a increase induced at 2 h and 6 h without affecting the BLIMP1b expression at 2 h, whereas increased BLIMP1b expression levels at 6 h (Fig. 2). Ly prevented the expression level increase of BLIMP1a at 2 h and 6 h, potentiated the increase of BLIMP1b at 2 h and has not produced any effect on BLIMP1b at 6 h. PP1 prevented and potentiated the BLIMP1a and BLIMP1b increase respectively at 2 h, and has not modified BLIMP1a and BLIMP1b expression at 6 h (Fig. 2). Altogether, these results suggested that the signaling pathways involving PI3K and Src kinase modulates PRDM1 and PRDM2 gene expression during T lymphocyte activation.
IL2, IL4 and IFNA modify the PRDM1 and PRDM2 gene expression levels in activated T lymphocytes
Cytokines trigger intracellular signaling cascades regulating gene expression in target cells, which lead to their activation, proliferation, and differentiation [1, 43]. IL2 provides an additional signal to the antigen recognition that triggers cyclin-dependent kinases and promotes the S phase cell cycle entry through STAT1/STAT3/STAT5 activation [43]. Therefore, IL2 promotes T cell proliferation and differentiation. IL4, by inducing the cell signaling transduction through STAT5/STAT6 and IRS-2 promotes naïve T lymphocyte differentiation in Th2 cells. In addition, IL4 represents the typical growth factor for Th2 cells. IFN-α modulates STAT1/STAT2 activation and prompts Th1 polarization. Thus, we aimed to assess the effect of IL2, IL4 and IFNA on PRDM1 and PRDM2 transcriptional levels in T lymphocytes isolated from healthy donor PBMCs. As shown in Fig. 3, IL2 modified PRDM2 gene expression at 2 h and 6 h, by increasing RIZ1 expression levels without significantly altering PRDM2/RIZex8 amount; however, IL2 induced a RIZ2 downregulation at 2 and 6 h. IL4 increased both RIZ1 and PRDM2/RIZex8 expression levels at 2 h and 6 h; IFNA upregulated RIZ1at 2 and 6 h and particularly influenced PRDM2/RIZex8 expression at 2 h revealing a RIZ2 up-regulation (Fig. 3). As regards to PRDM1, IL2 downregulated both transcripts at 2 h and BLIMP1a also at 6 h. IL4 modulated only BLIMP1b by increasing its expression at 2 h and 6 h. IFN-α down-regulated BLIMP1a and up-regulated BLIMP1b at 2 and 6 h (Fig. 3).
Fig. 3
Cytokine treatments regulate PRDM1 and PRDM2 gene expression levels in activated T cells. BLIMP1a, BLIMP1b, RIZ1, RIZex8 and RIZ2 gene expression analyses by qRT-PCR in activated T cells were compared to naïve T cells after treatment with IL2, IL4 and IFNA at 2 h and at 6 h. Three independent experiments in triplicates were performed and data expressed as mean ± SD. *P < 0.05 vs control cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
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Analysis of PRDM1 and PRDM2 transcripts in different T cell subpopulations
To explore the T cell subset in which polarizing cytokines target PRDM1 and PRDM2 genes expression during differentiation induction, lymphocytes were previously separated into naïve CD4+ and CD8+ T positive cells, activated with anti-CD3/CD28 antibodies and with IL2 supplemented with different further cytokines, as IL4, IL6, IFNA and IFNG for 48 h (Fig. 4A). We also measured the levels of GATA3, which is critical for the development, differentiation and function of CD4+ T cell subsets, as well as CD8+ T cells [50].
Fig. 4
Analysis of different T cell subpopulations. (A) PRDM2/RIZ, PRDM1/BLIMP1, GATA3 and (B) CD40LG, IL2RA, MYC and KLF2 genes were assayed in CD4+ and CD8+ positive cells activated with anti-CD3/CD28 antibodies and treated with IL-2 supplemented with IL4, IFNG, IFNA and IL6 for 48 h. As in the previous experiments, PR + and PR- transcripts of RIZ and BLIMP1 were tested (see also Fig. 1 for transcript details). Three independent experiments in triplicates were performed and data expressed as mean ± SD. ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
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Anti-CD3/CD28 and IL2 treatment of CD4+ T lymphocytes increased PRDM2 expression levels. However, IL4, IL6 or INFG cytokines, but not IFNA, were able to dramatically revert the effect of IL2 on PRDM2 expression level increase (Fig. 4A). Similarly, IL2 treatment of CD4+ T lymphocytes produced a consistent increase of both PRDM1 molecular forms, particularly of BLIMP1a. All the cytokines supplemented to IL2, excluding IFNA, resulted in a reduction of both BLIMP1a and BLIMP1b expression level (Fig. 4A).
In CD8+ T activated lymphocytes, treatment with IL2 reduced RIZ1 expression levels and increased RIZ2 expression level. The other cytokines supplemented to IL2 induced a downregulation of RIZ2 and an upregulation of RIZ1. In CD8+ activated T lymphocytes, IL2 treatment upregulated both PRDM1 transcripts. However, no significant effect was found for BLIMP1b expression with the addition of a second cytokine, except for IL4 that reduced its expression. Besides, BLIMP1a ever decreased in the presence of a second cytokine.
Interestingly, in the same experiment shown in Fig. 4A, our gene expression analysis in CD4+, and CD8+ T subpopulations during T cell activation revealed an upregulation of CD40LG, IL2RA, MYC and KLF2, paralleling previous literature data [51‐54] (Fig. 4B).
Collectively, T cell subpopulations revealed a different response to the “third signal” provided by the several cytokines supplemented to the activated T cells treated with IL2 when we observed the PRDM gene expression levels. Indeed, while BLIMP1a and BLIMP1b variants exhibited a substantial similarity in their trend, PRDM2 products showed a great variation.
PRDM1 and PRDM2 expression level analysis during Jurkat cell activation
The Jurkat cell line is an immortalized T lymphocyte cell line originally obtained from the peripheral blood sample of a young patient with T cell leukemia [55]. The Jurkat cell line has often been used as a prototypical T cell line to study multiple events in T cell biology, including signal transduction pathways involved in lymphocyte activation [56]. Moreover, considering the high difficulties to transfect normal lymphocytes, this cell line is suitable for evaluating the overexpression of genes by transfection. Firstly, we assessed the PRDM1 and PRDM2 expression levels in Jurkat cell line by qRT-PCR analysis. In standard culture conditions (see Methods), Jurkat cells expressed prevalently PRDM2; besides, they showed similar expression levels of PRDM1 transcripts whereas RIZ2 was expressed at higher levels than RIZ1 (about six folds) (Fig. 5). Then, we assessed the expression levels of PRDM1 and PRDM2 transcripts at early time points after activation with PMA/Ion (45’, 80’, 2 h, 4 h and 8 h) in Jurkat cells (Fig. 5). As observed in T cell lymphocytes, both PRDM1 and PRDM2 transcripts showed a cyclic expression variation even though with different magnitudes and kinetics. Specifically, in basal conditions and at 45’ the predominant PRDM2 isoform is RIZ2 that is downregulated at 2 h and 4 h. At 4 h a decrease of both isoforms was observed. Upon 8 h activation, RIZ1 and RIZ2 expression level is equal to the level observed at 45’ (Fig. 5).
Fig. 5
Gene expression analysis of PRDM1 and PRDM2 in Jurkat cells. On the left upper side, the basal levels of BLIMP1a, BLIMP1b, RIZ1, RIZex8 and RIZ2 in Jurkat cells are represented as fold on RPS18 control gene. The other bar graphs represent data from qRT-PCR analysis of the same transcripts in Jurkat cells following activation with PMA/Ion. Expression levels were calculated using the ΔΔCt method with the indicated control gene. Three independent experiments in triplicates were performed and data expressed as mean ± SD. ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
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A cyclic variation in BLIMP1a and BLIMP1b expression level has been observed during lymphocyte activation: both BLIMP1a and BLIMP1b are upregulated at 45'. BLIMP1a increased at 2 h and 8 h while at 80’ and 4 h BLIMP1a showed an expression level similar to that observed in basal conditions. An opposite trend has been revealed for BLIMP1b at the same time (Fig. 5).
PRDM2 transcript overexpression in Jurkat cells
Hypothesizing a role of master regulator gene for PRDM2 in T lymphocyte activation and a hierarchical organization in the expression level regulation of PRDM genes, Jurkat cell line was transiently transfected with plasmids encoding for RIZ1 (pSG5_rRIZ1), RIZ2 (pSG5_hRIZ2) and with the empty vector (pSG5) to investigate the ability of RIZ1 and RIZ2 to modulate the gene expression of pivotal factors involved in lymphocyte activation and differentiation. The RIZ1 and RIZ2 overexpression was evaluated after transfection by qRT-PCR of reverse-transcribed total cellular RNA and Western blot analysis. qRT-PCR analysis revealed a significant increase of RIZ1 and RIZ2 transcripts compared to control cells (pSG5) (Additional file 1: Fig S4). RIZ1 protein level was evaluated by Western blot analysis with the monoclonal antibody recognizing the PR domain exclusive of RIZ1. RIZ2 protein overexpression was revealed using a monoclonal antibody recognizing the Flag epitope. Altogether, both qRT-PCR and Western blot analyses confirmed RIZ1 and RIZ2 overexpression (Additional file 1: Fig S4).
Then, we analyzed if the overexpression of RIZ1 and RIZ2 was able to modulate PRDM1 expression level and modify the ratio among the BLIMP1a and BLIMP1b forms.
As depicted in Fig. 6, RIZ1 transient overexpression in Jurkat cell line has not significantly modified BLIMP1a and BLIMP1b gene expression levels, while RIZ2 overexpression increased BLIMP1b without affecting BLIMP1a expression levels. When we co-transfected both RIZ1 and RIZ2, we observed a reduction of both BLIMP1a and BLIMP1b transcripts. Besides, RIZ1 overexpression increased the BLIMP1a/BLIMP1b ratio also in the presence of RIZ2 (> 2 folds). RIZ2 alone, instead, reduced the ratio in favor of BLIMP1b (Fig. 6).
Fig. 6
Gene expression analysis of lymphocyte activation related transcription factors in Jurkat transiently RIZ overexpressing cells. PRDM1 transcript levels were compared in Jurkat cells transiently transfected with the indicated encoding plasmids (with the expression value of the empty vector equal to 1). The ratio between the PR- and PR + transcripts for each gene was also calculated. RIZ2 overexpression reduced the ratio between BLIMP1a and BLIMP1b in favor of BLIMP1b. The expression of GATA3, TBX21, FOXP3, RORC, CD40LG was also measured in Jurkat cells in both basal conditions (ΔCt method as fold on RPS18 control gene) and after 36 h transient transfection with the plasmid encoding for RIZ1, RIZ2 or with pSG5 control (ΔΔCt method with the indicated control gene). Three independent experiments in triplicates were performed and data expressed as mean ± SD. ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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In standard culture conditions, Jurkat cells exhibited different expression levels of transcription factors involved in lymphocyte differentiation (Fig. 6). Specifically, they expressed high levels of GATA3, alike CD40LG, moderate levels of FOXP3 and RORC (about 10 folds lower than CD40LG) and very low levels of TBX21 (about 100 folds lower than CD40LG). Interestingly, the overexpression of RIZ1 or RIZ2 in Jurkat cells altered the expression of these factors; RIZ2 overexpressing cells showed a significant reduction in GATA3 expression. In addition, RIZ2 overexpression reduced RORC expression level while RIZ1 overexpression has not significantly affected its transcription. In both RIZ1 and RIZ2 overexpressing cells we observed a reduction in CD40LG expression, while FOXP3 was upregulated only in RIZ1 overexpressing cells (Fig. 6). Collectively, RIZ2 forced expression through an imbalance of RIZ1/RIZ2 ratio in favor of RIZ2, induced a BLIMP1b increase. Consequently, the balance among BLIMP1a and BLIMP1b has shifted in disfavor of the first form. Otherwise, a balance in favor of BLIMP1a was promoted by RIZ1 overexpression through BLIMP1b repression. In conclusion, we speculate that PRDM2 may act upstream PRDM1 by regulating its gene expression. In addition, upon PRDM2 overexpression the main transcription factors involved in lymphocyte differentiation showed a down-regulation except for FOXP3 that is up-regulated by RIZ1 suggesting a putative PRDM2 involvement in the regulation of lymphocyte differentiation. Meanwhile, RIZ1 and/or RIZ2 could act on CD40LG in T lymphocytes activation with a negative feedback mechanism of regulation. TBX21 was expressed at very low levels; thus, we were not able to appreciate statistically significant variations.
Polyclonal activation of Jurkat cells stably transfected with RIZ1 and RIZ2
To investigate the possible effect of stable overexpression of PRDM2 molecular variants on target genes, qRT-PCR analysis was performed in Jurkat cells stably transfected with the plasmid encoding for RIZ1 (pSG5_rRIZ1), RIZ2 (pSG5_hRIZ2) or with pSG5 control vector (see Additional file 1: Fig S4 for details). In non-activated cells, only RIZ2 stable overexpression increased BLIMP1a level, as shown in Fig. 7. Instead, both RIZ1 and RIZ2 increased BLIMP1b expression levels. In addition, RIZ2 significantly increased the expression level of the main transcription factor of lymphocyte differentiation, GATA3, a similar effect as observed for BLIMP1b, while RIZ1 increased it to a lesser extent. Besides, a reduction of BCL6 expression was detected in RIZ2 overexpressing cells. No significant changes were observed for FOXP3, RORC and TBX21. CD40LG was noticeably increased by RIZ1, and to a less extent by RIZ2.
Fig. 7
A Gene expression analysis of the main transcription factors related to lymphocyte activation in Jurkat stably transfected cells. qRT-PCR analysis of BLIMP1a, BLIMP1b, GATA3, FOXP3, RORC, TBX21, BCL6, CD40LG, KLF2, CTLA4, IL2, IL2RA, CCR4, CCR6, and CXCR3 on Jurkat cells transfected with the plasmid encoding for RIZ1, RIZ2 or with pSG5 control vector and treated with or without treatment with polyclonal activators for 6 h. Three independent experiments in triplicates were performed and data expressed as mean ± SD. ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
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Activation of Jurkat cells stably transfected with pSG5_rRIZ1 or pSG5_hRIZ2 increased the level of both PRDM1 transcripts compared to control cells. RIZ1 overexpression reduced the BLIMP1a and BLIMP1b expression increase induced by cell activation, particularly for BLIMP1b. RIZ2 overexpression did not affect the BLIMP1a expression level whereas significantly amplified BLIMP1b expression increase observed after activation (of about 2.5 folds) (Fig. 7).
The RIZ1 overexpression prevented the FOXP3 expression level increase observed upon activation. No significant variations were detected for TBX21. Only the RIZ1 overexpression augmented GATA3 expression level increase observed in Jurkat cell activation. Despite its elevated basal level, CD40LG showed a conserved inducibility with a markedly higher increase of total levels in RIZ1 overexpressing cells. By observing the expression of genes encoding chemokine receptors related to the effector functions of different lymphocyte subsets, the CCR4 gene was significantly increased in activated RIZ1 and RIZ2 overexpressing cells. Instead, CCR6 was repressed by both RIZ1 and RIZ2 while CXCR3 was repressed only by RIZ2 (Fig. 7). Moreover, RIZ1 and RIZ2 overexpression did not modify the IL2 levels in basal conditions but halved the IL2 upregulation observed in activated lymphocytes. RIZ1 overexpression increased IL2RA levels in basal conditions. Both RIZ1 and RIZ2 overexpression did not significantly modify IL2RA upon lymphocyte activation. RIZ1 and RIZ2 overexpression increased the basal level of CTLA4 that was not modulated during lymphocyte activation (Fig. 7).
PRDM1 and PRDM2 transcripts positively correlate with expression of T cell activation genes
To further support the relationship between PRDM1 and PRDM2 and lymphocyte activation we explored the expression of their transcripts by in silico analysis of GEPIA2 database [57]. Specifically, we measured the Spearman correlation coefficient R to evaluate the expression correlation of PRDM1, PRDM2 and their main transcripts on three datasets from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx): whole blood, thymoma normal and EBV-transformed lymphocytes. The mRNA levels of the analyzed genes were normalized by RPS18 expression. As depicted in Fig. 8A, a high positive correlation was found between PRDM1 and PRDM2 (R = 0.76) when their whole transcription was considered. When we analyzed the PR + (RIZ1 and BLIMP1a) and PR- (RIZ2 and BLIMP1b) transcripts, we found a stronger correlation in both cases (R = 0.93 and R = 0.88 respectively). Interestingly, high correlations were also observed between PRDM1 and PRDM2 transcripts with GATA3 (R > 0.7) (Fig. 8B). Again, when we matched the whole PRDM1 or PRDM2 transcripts with gene signatures of immune response, a moderate correlation was found for PRDM1 (R ≥ 0.57) whereas a high correlation resulted for PRDM2 (R ≥ 0.85) (Fig. 8C).
Fig. 8
Expression correlation analysis of PRDM1, PRDM2 and the main transcripts with the indicated gene/transcript was performed on GEPIA2 platform using the Spearman correlation coefficient R. Datasets evaluated: whole blood, thymoma normal and EBV-transformed lymphocytes. PRDM2-001 from GEPIA2 database represents RIZ1 transcript, PRDM2-003 represents RIZ2 transcript, PRDM1-001 represents BLIMP1a transcript and PRDM1-002 represents BLIMP1b transcript. Scatter plots of mRNA levels were normalized by RPS18 expression. A Correlation analysis between PRDM1 and PRDM2 gene expression; B Correlation between PRDM1, PRDM2 and their main transcripts with GATA3; C Correlation between PRDM1, PRDM2 and their main transcripts with the indicated signatures
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Discussion
PRDM family genes play a pivotal role in the control of several aspects of cell behavior, such as cell cycle progression, cell development and differentiation as well as the homeostasis maintenance of immune system cells [2, 3, 13]. The lymphocyte differentiation is mainly due to histone modifications caused by methyltransferase proteins [58, 59]. A very close relationship between PRDM gene expression and lymphocyte function regulation could exist [60]. To date, several studies have discussed the PRDM1 pivotal role in lymphocyte activation and differentiation. Indeed, BLIMP1 protein is considered an essential regulator of terminal B cell differentiation into antibody-secreting plasma cells and controls the differentiation of Th1/Th2 cells [15, 61]. PRDM2 products are expressed in T lymphocytes where they interact with GATA-3, an essential transcription factor for T cell development and the Th2 lymphocyte differentiation, thereby suggesting that PRDM2 regulates GATA-3 function [62]. The role of PRDM2/RIZ in lymphocytes has not been elucidated so far. Several findings highlighted the relationship between PRDM2 and proliferation control, represented by the Yin-Yang paradigmatic model. Here, we have investigated the role of PRDM2 gene and its molecular variants using the human peripheral blood lymphocytes. Indeed, normal lymphocytes represent a good model to evaluate events induced by mitogenic signals, understand their control mechanisms than tumor cell lines or cells transformed with oncogenes. We have analyzed the transcription of PRDM1 and PRDM2 variants through qRT-PCR because of the high specificity and sensitivity of this method. Besides, it represents a proper tool to study the early events of T cell activation. Indeed, it is well established that distinct transcriptional profiles and chromatin modifications are observed during T cell response [63].
Our findings represent the first evidence on the putative role of PRDM2 proteins (RIZ1 and RIZ2) in the commitment of T lymphocytes to the functions subsequent to their activation. Moreover, as regard to PRDM1, we have also characterized the expression of the different PRDM1/BLIMP1 (a and b) transcripts during activation. Specifically, we have analyzed the transcription signature of these genes in both human peripheral blood T lymphocytes and Jurkat cells after activation operating through the “first” signal mediated by TCR, and the “second” signal triggered by CD28, or through the cell signaling transduction pathways downstream the receptors. Noteworthy, our findings are also supported by in silico data on GEPIA2; indeed, gene expression correlation analyses clearly indicated a high correlation between PRDM1 and PRDM2, and between their transcripts and other recognized genes involved in immune response (Fig. 8).
We observed that the activation of PBMC or naïve CD4+ T lymphocytes induced an increase in the expression levels of the PRDM2/RIZ and PRDM1/BLIMP1 genes, with a correlated increase of MYC gene expression, a target of both PRDM1 and PRDM2 proteins [64, 65] (Fig. 1). The expression profile of PRDM1 and PRDM2 variants over the time showed the early increase of RIZ2 and RIZ1 followed by BLIMP1b increase and finally by BLIMP1a increase. The “first” and the “second” signals shifted the balance between the PR + and the PR- forms in favor of the PR- ones, for both studied genes. Furthermore, we have also evidenced that the short tail amplicon (RIZ ex9a) prevalently correlates with RIZ2 transcription (Additional file 1: Fig S3); however, further studies are still needed to fully elucidate the functional role of these PRDM2 transcription variants [10].
These genes showed a trend similar to that of the Immediate Early Growth Response Genes, which determine important changes in the cellular state of Jurkat T cells [66]. Specifically, they regulate the switch of resting lymphocytes from quiescent to activated state, followed by cell proliferation essential for clonal expansion and subsequent differentiation. Furthermore, to determine the role of the first and second signal transduction pathways, TCR and CD3/CD28 respectively, in the gene expression regulation of PRDMs, the effects of several inhibitors of key enzymes of transduction pathways were evaluated, such as PI3 and MEK1/2 kinases. The obtained results suggested that the signaling pathway involving PI3K modulates the RIZ1/RIZ2 ratio in favor of RIZ1 gene during T lymphocyte activation while the balance versus RIZ2 was promoted by the MAPK pathway. Similarly, in the CML model, RIZ1 was induced from myeloid cells, where PI3K/AKT upregulates RIZ1 [67].
The presence of cytokines that mediate different Jak/Stat signaling pathways are observed as agents capable of modulating the expression of PRDM1/PRDM2 genes and the relationship of their forms (Fig. 2). IL-2/STAT5 promotes the increase in the RIZ1 amount compared to RIZ2; instead, IL-4/STAT6 induces an increase of PRDM2/RIZ, most likely of the RIZ2 form. Besides, IFN-∝/STAT1/2/IRF9 produces the most marked increase in RIZ2. All these effects seem to be very quick, already at 2 hrs whereas at 6 hrs we observed non-significant variation in expression. In the same T cells, stimulated as above, we observed an early increase in the BLIMP1b form with IFN-∝ and late as with IL-2 or IL-4, while the increase in BLIMP1a is only late. The relationship between the PRDM1 forms towards the form a is evident in the presence of IL-2 and IL-4 while with IFN-∝ the increase in the form a balances the form b. Overall, this analysis would confirm what we observed with the activation of the 1st and 2nd signals, i.e. the early increase of the PR-minus transcripts compared to the PR-plus ones. The physiological activation by the antigen presenting cell (APC) generates clonal expansion by means of 1st and 2nd signals and the 3rd signal, which is prompted by the presence of “inducing” cytokines produced by different innate cells present in the microenvironment or by the APCs themselves, promotes the differentiation of T cell subtypes. The presence of all the three signals, simulated in the CD4+ T cells and CD8+ T cells populations, involves a highly different expression trend between PRDM2 and PRDM1 for many of the cytokines added, such as IL-4, IFN-γ, IFN-∝ and IL-6, to the main stimulus CD3/CD28 + IL-2 (Fig. 4). In summary, it is likely that in CD4+ cells IL-4 produces a reduction in the induction of PRDM1 and a repression of PRDM2. The latter effect is also observed with other pro-differentiating cytokines, such as IFN-γ and IL-6. In CD8+ T cells, the RIZ2 and BLIMP1b increase occurs through the activation with IL-2 and CD3/CD28 alone, while the presence of cytokines pushes the prevalence of RIZ1 (IL-4) or the repression of RIZ2 (IFN-∝ and IL-6). On the other hand, BLIMP1b increases, and in the presence of IL-4, a balance is produced towards a while b form is repressed. In the presence of the other cytokines, IFN-γ, IFN-∝ and IL-6, the negative regulation of a form promotes a greater balance towards the b form. From these findings, we deduce that the response of the CD4+ or CD8+ effector T cells is different regarding the expression of the PRDM1 and PRDM2 genes and their variants, if we compare the trend of T cell activation markers (KLF2, CD40LG, IL2RA, MYC). The used model of enriched polyclonal human T cells, which represent the sum of different T subsets (Th1, Th2, TH17, TFh etc.), does not allow a precise assessment of the associations between PRDM1 and PRDM2 transcripts and the different cytokine stimuli, but these data solicit further correlation and functional studies to better understand the cause/effect role of PRDM1 and PRDM2 and their variants in the mechanism of action of the cytokines themselves.
These observations highlight the relationship between stimulus and response downstream of the PRDM1 and PRDM2 genes and of the different expressivity of their variants. Thus, the question is how and in what way the different pathways are concatenated and if there could be a cause-effect hierarchy between the early activation of RIZ1 and RIZ2 and the late activation of BLIMP1a and BLIMP1b. To this purpose, we performed overexpression experiments of PRDM2/RIZ isoforms in Jurkat cell line (although they already overexpress RIZ2 at baseline condition) to reproduce significant variations of RIZ1 and RIZ2 as those induced by the 1st/2nd signals over time and observed in T cells from PBMCs and assess the effects on BLIMP1a and BLIMP1b expression levels. This approach allowed us observing that the acute (transiently) increase of RIZ2 promotes the balancing of the PRDM1 forms towards the b form (Fig. 5). Noteworthy, the generation of stable Jurkat clones overexpressing RIZ1 or RIZ2 allowed us to evidence a different equilibrium of the BLIMP1a/b molecules in basal conditions beyond the modification of the response to the polyclonal activators (Fig. 6). The RIZ2-mediated adaptation of the cells led to a basal increase in BLIMP1b and preserved its induction by the 1st/2nd signals. Obviously, through an autocrine mechanism, cells can promote the expression of IL2, which is not expressed in basal conditions in the different stable RIZ1 or RIZ2 overexpressing cell lines. Stable RIZ1 cell line promotes a lower increase in the baseline BLIMP1b level and, on the other hand, a strong repression effect of the 1st/2nd signal induction, thus highlighting the importance of RIZ1 and RIZ2. GATA3 gene appears strongly modulated under basal conditions in RIZ2 overexpressing cells in a similar manner as BLIMP1b. Otherwise, in RIZ1 overexpressing cells, GATA3 increases little in basal conditions but responds to activation stimuli. This evidence confirms the hypothesis of a close relationship between PRDM2 and GATA3, and it indicates significant differences between the action of the PRDM2 forms on the GATA3 expression regulation. Different changes of other analyzed genes (FOXP3, RORC, BCL6, etc.), compared to BLIMP1 and GATA3, seem to confirm the different activity of the PRDM2 forms, RIZ1 and RIZ2, both in the increase or decrease of the basal amount and in the induction capacity by the activation signals. This finding is in line with the basal GATA3 increase, which predisposes to the cell function increase in the T helper 2 direction. Likely, RIZ2 could work in concert with GATA3 to strengthen its functions, including the modulation of CCR4 expression. Instead, comparing the effects on other key genes, such as those coding for chemokine receptors, CCR6 and CXCR3, we observed a general reduction in the induction of their expression following activation. Therefore, we can assume that RIZ2 strengthens the polarization of T lymphocytes in Th2. RIZ1, as RIZ2, is capable of repressing CCR6 and CXCR3 but not of increasing CCR4 levels. Probably the effect could be due to the concomitant repression of BLIMP1b (Fig. 7).
Interestingly, the CD40LG high expression level in basal condition, which was particularly observed in RIZ1 overexpressing cells than RIZ2 ones, and the CD40LG up-regulation, observed during cell activation, could be related to the variations in the expression levels of positive and negative regulators induced by the adaptation of the cell line to the overexpression of PRDM2 forms that mimic key steps of cell activation or memory cell differentiation.
In summary, the stable forced expression of RIZ1 or RIZ2 in clones of Jurkat T cell line induced a significant variation in the expression level of key transcription factors involved in T lymphocyte differentiation observed already in steady state. Accordingly, these clones showed a different ability to modulate the expression level of these transcription factors upon cell activation. The PRDM1a/b balance has shifted in favor of BLIMP1a in RIZ1-overexpressing cells and in favor of BLIMP1b in RIZ2-overexpressing cells. In addition, GATA3 expression level is noticeably modified by both RIZ1 and RIZ2.
Conclusions
Overall, our observations represent the first step for the characterization of PRDM2 in lymphocyte activation/differentiation. In the future, we point out to clarify deeply the mechanism of action and functions of PRDM2 in lymphocyte activation/differentiation. In our opinion, it could be interesting evaluating the gene expression regulation by performing time-course Next Generation Sequencing (NGS) analysis in single cell. Furthermore, it could be remarkable to study the chromatin architecture of genes regulated by PRDM2 proteins, identifying any PRDM2 responsive element sequence. Considering that literature data shows the involvement of other genes of the family, including PRDM15 in lymphoproliferative diseases [60], the preliminary evidence of our study allows us to hypothesize the use of drugs directed against RIZ or BLIMP1 to prevent or modify the response of cells to activation and cell differentiation and as a useful tool also for cancer therapy.
Consistent with its established role as an essential regulator of immune cell function, numerous PRDM1-associated risk alleles have been linked with autoimmune pathologies [15, 68]. Noteworthy, a recent meta-analysis of multi-trait genome-wide association studies identified PRDM2 as a locus associated with six autoimmune and allergic diseases thus suggesting the implication of this gene [69]. Abnormal gene expression level or expression of genes containing deleterious variants can be at the basis of these genetic diseases. Currently, several molecular tools have been developed and are in clinical trials for therapeutic purposes acting to restore the physiological gene expression [70]. Thus, the whole knowledge of the transcription regulation mechanisms involving PRDM1 and PRDM2 genes in the immune biology can provide the molecular bases for the application of these new strategies to the autoimmune diseases.
Acknowledgements
We thank our colleagues Dr Luciano Pezone, Dr Grazia Maglio and Dr Marina D’Agostino for their technical assistance.
Declarations
Ethics approval and consent to participate
Human peripheral T lymphocytes utilized in this study were obtained from total peripheral blood mononuclear cells (PBMCs) collected from authors who consented to participate.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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