Itm2a, a Target Gene of GATA-3, Plays a Minimal Role in Regulating the Development and Function of T Cells

The integral membrane protein 2a (Itm2a) is one of the BRICHOS domain-containing proteins and is structurally related to Itm2b and Itm2c. It is expressed preferentially in the T lineage among hematopoietic cells and is induced by MHC-mediated positive selection. However, its transcriptional regulation and function are poorly understood. Here we showed Itm2a to be a target gene of GATA-3, a T cell-specific transcription factor. Deficiency of Itm2a had little impact on the development and function of polyclonal T cells but resulted in a partial defect in the development of thymocytes bearing a MHC class I-restricted TCR, OT-I. In addition, Itm2a-deficient mice displayed an attenuated T helper cell-dependent immune response in vivo. We further demonstrated that Itm2b but not Itm2c was also expressed in T cells, and was induced upon activation, albeit following a kinetic different from that of Itm2a. Thus, functional redundancy between Itm2a and Itm2b may explain the minimal phenotype of Itm2a deficiency

PTPN22.6, a Dominant Negative Isoform of PTPN22 and Potential Biomarker of Rheumatoid Arthritis

PTPN22 is a tyrosine phosphatase and functions as a damper of TCR signals. A C-to-T single nucleotide polymorphism (SNP) located at position 1858 of human PTPN22 cDNA and converting an arginine (R620) to tryptophan (W620) confers the highest risk of rheumatoid arthritis among non-HLA genetic variations that are known to be associated with this disease. The effect of the R-to-W conversion on the phosphatase activity of PTPN22 protein and the impact of the minor T allele of the C1858T SNP on the activation of T cells has remained controversial. In addition, how the overall activity of PTPN22 is regulated and how the R-to-W conversion contributes to rheumatoid arthritis is still poorly understood. Here we report the identification of an alternative splice form of human PTPN22, namely PTPN22.6. It lacks the nearly entire phosphatase domain and can function as a dominant negative isoform of the full length PTPN22. Although conversion of R620 to W620 in the context of PTPN22.1 attenuated T cell activation, expression of the tryptophan variant of PTPN22.6 reciprocally led to hyperactivation of human T cells. More importantly, the level of PTPN22.6 in peripheral blood correlates with disease activity of rheumatoid arthritis. Our data depict a model that can reconcile the conflicting observations on the functional impact of the C1858T SNP and also suggest that PTPN22.6 is a novel biomarker of rheumatoid arthritis

Deficiency of Itm2a does not affect the selection and activation of OT-II T cells.

<p><b>A</b>. Thymocytes of WT/OT-II and KO/OT-II mice were stained with anti-CD4, anti-CD8, and anti-Vβ5. Representative FACS plots are shown. The means and standard deviations of total thymocytes and CD4⁺Vβ5⁺ thymocytes from three experiments are shown in the left panels. <b>B</b>. Splenocytes of WT/OT-II and KO/OT-II mice were stimulated with ovalbumin peptides at indicated concentration for 3 days. The percentages of live cells were determined based on the FSC/SSC gate and are shown. The cells were also stained with anti-CD25 and anti-CD69. Representative FACS plots are shown.</p

Impaired immune response to Th cell-dependent antigens in the absence of Itm2a.

<p>WT (N = 8) and Itm2aKO (N = 7) mice were immunized with TNP-KLH in CFA at tail base. Two weeks after the immunization, the level of TNP-specific IgG was quantified with ELISA (<b>A</b>). The percentage of plasma cells (CD138⁺B220^dull) among splenocytes was calculated (<b>B</b>). T cells of draining (inguinal) and non-draining (axillary) lymph nodes were further stained with anti-ICOS/anti-PD-1. ICOS⁺PD-1⁺ Tfh cells were gated (<b>C</b>). The means and standard deviations of the percentage of Tfh among lymph node Th cells are shown in the right panel of <b>C</b>. Draining LN cells were restimulated with TNP-KLH for 24 hours. The concentration of IFN-γ and IL-2 in the supernatant of the stimulated cells was quantified with ELISA (D). Statistical analyses were performed with unpaired Student's t tests.</p

<i>Itm2a</i> is a target gene of GATA-3.

<p><b>A</b>. Total RNA was prepared from DP thymocytes of WT and G3KO mice. The transcript level of <i>Itm2a</i> was quantified with real time PCR. The transcript level was then normalized against that of <i>Actb</i>. The data shown are means and standard deviation of three experiments. <b>B</b>. A schematic diagram of the 2 kb promoter of <i>Itm2a</i>. The location and sequence of the conserved and mutated GATA sites are shown. The black boxes represent exon of <i>Itm2a</i>. The thick arrow marks the transcription start site. <b>C</b>. M12 cells were transfected with the indicated luciferase reporter constructs along with a GATA-3 expression vector or an empty vector. The relative luciferase activity was calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096535#s2" target="_blank">Materials and Methods</a>. The data shown are means and standard deviations of three independent experiments.</p

Deficiency of Itm2a has no impact on the function of peripheral T cells.

<p><b>A & B</b>. Splenocytes of WT and Itm2aKO mice were stimulated with anti-CD28 (2 µg/ml) and indicated amount of anti-CD3 for three days. The expression of CD25 by CD4⁺ and CD8⁺ T cells was examined with FACS and is shown in <b>A</b>. The concentration of IL-2 and IFN-γ in the supernatant of stimulated cells was quantified with ELISA. The means and standard deviations of three independent experiments are shown in <b>B. C</b>. Naive Th cells of WT and Itm2aKO mice were differentiated in vitro under Th1, Th2, and Th17 polarization conditions, and re-stimulated with PMA/ionomycin. The production of indicated cytokines was examined with intracellular cytokine staining. Representative FACS plots are shown. <b>D</b>. CTL activity of WT and Itm2aKO CD8⁺ T cells was examined according to the protocol described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096535#s2" target="_blank"><i>Material and Methods</i></a>. The data shown are means and standard deviations of three independent experiments.</p

Potential functional redundancy between Itm2a and Itm2b.

<p><b>A</b>. The transcript level of <i>Itm2a</i>, <i>Itm2b</i>, and <i>Itm2c</i> in WT thymocytes and WT Th1 cells were quantified with real time PCR, and normalized against that of <i>Actb</i>. The means and standard deviations of three experiments are shown. <b>B</b>. WT naive Th cells were stimulated in vitro with anti-CD3 (1 µg/ml)/anti-CD28 (2 µg/ml). Whole cell extract was harvested at indicated time points and probed with indicated antibodies. The data shown are representative of two independent experiments.</p

Itm2a regulates the thymic development but not the activation of OT-I cells.

<p><b>A</b>. Thymocytes of WT/OT-I and KO/OT-I mice were stained with anti-Vβ5, anti-α/β TCR, anti-CD4, and anti-CD8, and. representative TCR/Vβ5 FACS plots are shown in the left column. The CD4/CD8 FACS plots of TCR⁺Vβ5⁺ cells are shown in the right column. The DP, post-selected CD4+CD8dull (PS), and CD8SP populations of TCR⁺Vβ5⁺ cells are gated. B. The numbers of DP, PS, and CD8SP Vβ5⁺ cells were enumerated from three pairs of mice. The means and standard deviations are shown. Statistical analysis was performed with Student's t tests. <b>C</b>. The histograms of CD69 and CD5 of indicated Vβ5⁺ thymocytes of WT/OT-I and KO/OT-I mice are overlaid and shown. D. Splenocytes and lymph nodes cells of WT/OT-I and KO/OT-I mice were stained with anti-Vβ5 and anti-CD8. Representative CD8/Vβ5 FACS plots and the absolute numbers of CD8⁺Vβ5⁺ cells are shown. <b>E</b>. Splenocytes of WT/OT-I and KO/OT-I mice were stimulated with ovalbumin peptides at the indicated concentrations for 3 days. The percentages of live cells were determined based on the FSC/SSC gate and are shown. The cells were also stained with anti-CD25 and anti-CD69. Representative FACS plots are shown.</p

Deficiency of Itm2a has little impact on the differentiation of immune cells.

<p><b>A</b>. Generation of Itm2a-deficient mice. Schematic diagrams of <i>Itm2a</i> gene (top), the targeting construct (middle), and deleted allele (bottom) are shown. Exons are represented with gray boxes and numbered. Regions for homologous recombination are marked with dashed lines. Neo and TK stand for neomycin cassette and thymidine kinase. <b>B</b>. T cells were purified from control (WT) and two Itm2aKO mice and stimulated with anti-CD3 for 72 hours. Cell extract from the stimulated cells was probed with anti-Itm2a and anti-Hsp90. <b>C</b>. Thymocytes of WT and Itm2aKO mice were enumerated, and the means and standard deviations are shown in the left panel. <b>D & E</b>. The thymocytes were also stained with anti-CD4/anti-CD8 (<b>D</b>) and anti- α/β TCR, and TCR^high cells were further stained with antibodies against various Vβ chains indicated in <b>E</b>. <b>F</b>. The thymocytes were stained with anti-CD4/anti-Foxp3 and anti-TCR/NK1.1. <b>G</b>. Splenocytes and lymph node cells of WT and Itm2aKO mice were stained with anti-CD4 and anti-CD8. <b>H</b>. The splenocytes were also stained with anti-TCR/anti-B220. B220⁺ cells were then further separated by anti-IgM/anti-IgD staining. The data shown in <b>D–H</b> are representative FACS plots of at least three independent experiments.</p