Phosphotyrosine phosphatases (PTPs) constitute a complex family of enzymes that control the balance of intracellular phosphorylation levels to allow cell responses while avoiding the development of diseases. Despite the relevance of CD4 T cell polarisation and effector function in human autoimmune diseases, the expression profile of PTPs during T helper polarisation and restimulation at inflammatory sites has not been assessed. Here, a systematic analysis of the expression profile of PTPs has been carried out during Th1-polarising conditions and upon PKC activation and intracellular raise of Ca2+ in effector cells. Changes in gene expression levels suggest a previously nonnoted regulatory role of several PTPs in Th1 polarisation and effector function. A substantial change in the spatial compartmentalisation of ERK during T cell responses is proposed based on changes in the dose of cytoplasmic and nuclear MAPK phosphatases. Our study also suggests a regulatory role of autoimmune-related PTPs in controlling T helper polarisation in humans. We expect that those PTPs that regulate T helper polarisation will constitute potential targets for intervening CD4 T cell immune responses in order to generate new therapies for the treatment of autoimmune diseases.
CD4 T cells are important components of adaptive immune responses. During antigen stimulation, T cells polarise towards a type of effector cell specialised in controlling different sorts of infections by secreting different cytokines: Effector T helper 1 (Th1) secretes IFN
In humans, protein tyrosine phosphatases (PTPs) constitute a family of more than 100 enzymes that regulate the phosphorylation state of molecular components of signalling networks. The folding of the PTP domain classifies PTPs in four classes: class I, containing the classical nonreceptor and receptor PTPs (NRPTPs and RPTPs, respectively) and the dual specific phosphatases (DSPs) [
Lymphocytes express around 60 to 70 genes coding for PTPs [
Blood cells of healthy adult donors (<65 year old) where obtained from buffy coats processed at the transfusion centre of the “Comunidad de Madrid,” Spain. Peripheral blood mononuclear cells (PBMCs) were obtained by Lymphoprep™ (Rafer, Spain) density gradient centrifugation. Naïve CD4 T cells were isolated from PBMCs using the Naïve CD4+ T cell Isolation Kit II (Miltenyi Biotec, Germany). Purities over 95% were typically obtained as assessed by flow cytometry. For Th1 polarising conditions, the obtained naïve CD4 T cells were cultured for 12 days in RPMI 1640 (Lonza Group, Switzerland) supplemented with 10% FCS (Gibco, USA), Penicillin-Streptomycin 100 U/mL and 100
Th1 polarisation was assessed by analysing the production of IFN
RNA was extracted from naïve, Th1, and Th1-PI cells using the Absolutely RNA Microprep Kit (Agilent Technologies, USA) and the RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). 2
An agglomerative hierarchical tree was implemented in MATLAB (The Mathworks, Inc, USA) by using Euclidean metrics and the ward method. Statistical analysis was implemented in GraphPad Prism version 5.04 (GraphPad Software, USA). Changes in mRNA levels between different conditions (Naïve, Th1, and Th1-PI) obtained for each donor were analysed with a paired
Human naïve CD4 T cells were isolated and polarised to Th1 conditions as detailed in materials and methods. The polarisation was confirmed by the production of IFN
Assessment of Th1 polarisation. (a) Flow cytometry dot plots represent the IFN
Agglomerative hierarchical tree of the gene expression patterns in naïve and Th1 cells. Numbers below the tree indicate the distance among gene patterns. Hitmap represents the average DCT obtained for each gene in both conditions and 3 donors. The calibration bar is shown between 5 and 20 DCTs. Green and red squares point to clusters of upregulated and downregulated genes, respectively. Asterisks indicate those genes whose expression levels were considered to significantly change, as detailed in Table
Expression change of NCs phosphatases induced by the PI treatment. The graph represents the average of the change in DCT between Th1 and Th1-PI cells. Genes upregulated and downregulated are labelled in green and red, respectively. Assessment of regulated genes is explained in materials and methods.
Expression of regulators of the MAPK signalling module in naïve and Th1 cells. (a) Agglomerative hierarchical tree of the expression profile of MAPKs phosphatases in naïve, Th1, and Th1-PI samples. Numbers under the tree indicate the distance among the expression profile. Hitmap represents the average DCT obtained for each gene in all samples and 2 donors. Calibration bar is shown between 5 and 20 DCTs. (b) The average DCT obtained for classical MPKs and PTPN7 in naïve (N) and PI treated Th1 cells (Th1-PI) is plotted. The diagonal line labels the position of genes with equal expression levels in both samples. Labelled genes are those with different expression level assessed as explained in material and methods.
We included in this study all NCs given the unknown function of the majority of them in T cells and some classical RPTPs and NRPTPs, due to their regulatory role in the signalling downstream the TCR and cytokine receptors and in the dynamics of the cellular machinery or due to their association with autoimmune diseases [
Class-I classical RPTP and NRPTP included in this study.
Phosphatase | Substrate | Regulation of T cell activation | Involved in autoimmunity | References |
---|---|---|---|---|
|
SFK | Regulation of TCR signalling: | Not reported | [ |
|
SFK and JAK family kinases | Regulation of TCR and cytokine signalling | MS, AH | [ |
|
SFK | Regulation of TCR signalling | Not reported | [ |
|
STAT3 | Regulation of CD4 T cell development | Not reported | [ |
|
STAT6 | Not reported | Not reported | [ |
|
SFK, JAK1, JAK3 | Regulation of TCR and cytokine signalling | T1D, CD, S | [ |
|
CD247 | Regulation of TCR signalling | Not reported | [ |
|
SFK, ITAMs, ZAP70, SLP-76 | Regulation of TCR signalling | PS | [ |
|
ERK1/2, p38 | Regulation of TCR signalling | Not reported | [ |
|
NSF | Regulation of cytokine secretion | Not reported | [ |
|
Pyk2 | Positive regulator of secondary T cell responses | Not reported | [ |
|
STAT4, STAT6 | Regulation of cytokine signalling | Not reported | [ |
|
HER2 | Not reported | Not reported | [ |
|
ZAP70, LCK, FYN | Regulation of TCR signalling | T1D, RA, SLE | [ |
SFK: Src family kinases, MS: multiple sclerosis, AH: autoimmune hepatitis, T1D: type 1 diabetes, CD: Crohn’s disease, S: synovitis, PS: psoriasis, RA: rheumatoid arthritis, and SLE: systemic lupus erythematosus.
PTP regulated during Th1 polarization. PTPs whose expression levels were regulated during Th1 polarization are shown. Changes in expression were determined as explained in materials and methods. Asterisks represent the result of the paired
Group | Phosphatase | Regulation during Th1 polarization |
|
Substrate | Regulation of T cell activation or polarisation/Involvement in autoimmunity |
---|---|---|---|---|---|
Classical |
|
Upregulation | 2.02/ |
SFK | Regulation of TCR signalling/Not reported [ |
|
Upregulation | 2.08/ |
SFK, ITAMs, ZAP70, SLP-76, Vav1 | Regulation of TCR signalling/PS [ | |
|
Upregulation | 1.54/ |
ERK1/2, p38 | Regulation of TCR signalling/Not reported [ | |
|
Upregulation | 2.45/ |
NSF | Regulation of cytokine secretion/Not reported [ | |
|
Upregulation | 2.40/ |
STAT4, STAT6 | Regulation of cytokine signalling/Not reported [ | |
|
Upregulation | 1.9/ |
HER2 | Not reported/Not reported | |
|
|||||
MKPs and Atypical DSPs | DUSP1 | Downregulation | 3.07/ |
p38, JNK, ERK | T cell activation/Not reported [ |
DUSP7 | Upregulation | 2.10/ |
ERK | Not reported/Not reported | |
DUSP8 | Downregulation | 4.55/ |
JNK, p38 | Not reported/Not reported | |
DUSP13 | Repression | JNK, p38 | Not reported/Not reported | ||
DUSP16 | Downregulation | 1.20/ |
JNK, p38 | Th1/Th2 balance/Not reported [ | |
STYXL1 | Upregulation | 1.19/ |
Catalytically inactive | Not reported/Not reported | |
DUSP21 | Repression | Unknown | Not reported/Not reported | ||
DUSP22 | Upregulation | 2.10/ |
JNK, ERK2, Lck | Regulation of TCR signaling/EAE, SLE [ | |
DUSP23 | Upregulation | 1.57/ |
p38, JNK | Not reported/SLE [ | |
|
|||||
Myotubularins | MTMR1 | Upregulation | 1.43/ |
PI(3)P, PI(3,5)P2 | Not reported/Not reported |
MTMR2 | Upregulation | 2.61/ |
PI(3)P, PI(3,5)P2 | Not reported/Not reported | |
MTMR11 | Induction | Catalytically inactive | Not reported/Not reported | ||
|
|||||
SSHs, CDC14s and PTEN DSPs | SSH2 | Downregulation | 1.68/ |
Cofilin | Not reported/Not reported |
SSH3 | Upregulation | 2.63/ |
Cofilin | Not reported/Not reported | |
TPTE2 | Downregulation | 1.96/ |
PIP | Not reported/Not reported | |
CDKN3 | Upregulation | 5.81/ |
CDK2 | Inhibition of cell cycle/Not reported [ | |
|
|||||
Class III Cys-based PTPs | CDC25A | Upregulation | 6.80/ |
CDKs | Promotion of cell cycle/Not reported [ |
CDC25B | Upregulation | 2.45/ |
CDKs | Not reported/Not reported | |
CDC25C | Induction | CDKs | Not reported/Not reported |
PS: psoriasis. EAE: experimental autoimmune encephalomyelitis. SLE: systemic lupus erythematosus.
The majority of classical PTPs analysed (10 out of 14) were found inside the group of high expression, including
Our analysis uncovered a relation between the upregulated expression and the function of some PTPs. For example,
The genes
We did not detect mRNA of the atypical MKPs
22 NCs were found in the group of highly expressed PTPs (Figure
MTMs dephosphorylate the position 3 of phosphatidylinositol phosphate (PIP) molecules PI(3)P and PI(3,5)P2, making them important regulators of the endosomal compartment, the cytoskeleton, and ion channels [
We also found phosphatase death (PD) MTMs expressed in CD4 T cells, including the highly expressed SBF1 (MTMR5), MTMR10, and MTMR12, the middle expressed MTMR9, and the inducible with Th1 polarisation MTMR11 (Figure
Inside the group of middle expressed PTPs, MTMR2 was found upregulated by Th1-polarising conditions and PI treatment (Table
The cytoskeleton regulators SSH2 and SSH3 were found downregulated and upregulated, respectively, by Th1-polarising conditions (Table
Among the 9 out of 10 classical MKPs [
The group of MKPs are characterised by sharing MAPK substrates. For example, ERK is dephosphorylated by 13 different MKPs. Interestingly, it has been proposed that the spatial distribution of dephosphorylated MAPKs is regulated by the binding of MKPs, such as the nucleocytoplasmic location of ERK by nuclear DUSP5 and cytoplasmic DUSP6, and the accumulation of ERK in the nucleus by DUSP2, DUSP4, and DUSP5 [
Consistent with this complex scenario, Th1 polarisation and PI treatment of Th1 cells induced dramatic changes in the expression profile of ERK-directed MKPs and, consequently, remarkable differences were found between naïve and Th1 restimulated cells (Th1-PI) (Figures
Some atypical MKPs were found regulated by Th1-polarising conditions (Table
Among cell cycle regulators, CDC14A and CDC25B were found in the group of highly expressed PTPs. Interestingly, CDC25B, a negative regulator of the cell cycle was upregulated by Th1-polarising conditions (Table
The systematic analysis performed in this study reveals several genes coding for PTPs that are regulated during Th1 polarisation and restimulation of effector cells. Interestingly, the mRNA level of the majority of the regulated genes coding for NCs was increased during Th1 polarisation, which suggests a regulatory role of these PTPs in Th1 polarisation or effector function. By contrast downmodulated genes during polarising conditions might be involved in initial immune responses by naïve T cells. In general, changes in expression levels found during polarisation might indicate a role in achieving a healthy balance of T helper polarisation. Our data also suggest an important compartmentalisation of dephosphorylated ERK functions during the T cell responses at inflammatory sites. Finally, the obtained results also suggest the existence of PTPs that might regulate components of the cellular machinery (including the endosomal compartment, the cytoskeleton, and the activity of ion channels) during T cell immune responses. The regulatory role of these PTPs should be investigated.
Regarding autoimmunity, the expression of
In the mouse model the atypical DUSP22 has been proposed to inhibit TCR signalling and to control the development of experimental autoimmune encephalomyelitis [
The authors declare that there are no conflicts of interest regarding the publication of this paper.
This work was supported by funds of the Spanish Ministry of Economy and Competitiveness (SAF2012-33218) and the European Union (FP7-PEOPLE-2012-CIG) to Pedro Roda-Navarro. These projects supported Patricia Castro-Sánchez. Rocio Ramirez-Munoz was supported by a fellowship for educating researchers (FPI Fellowship) assigned to the SAF2012-33218 Project.