IL13Rα2 expression identifies tissue-resident IL-22-producing PLZF+ innate Tcells in the human liver.

Natural killer T cells are important in the pathogenesis of asthma: The many pathways to asthma

PLZF Outreach: A Finger in Interferon's Pie

The transcription factor promyelocytic leukemia zinc finger (PLZF) is encoded by the BTB domain-containing 16 (Zbtb16) gene. Its repressor function regulates specific transcriptional programs. During the development of invariant NKT cells, PLZF is expressed and directs their effector program, but the detailed mechanisms underlying PLZF regulation of multistage NKT cell developmental program are not well understood. This study investigated the role of acetylation-induced PLZF activation on NKT cell development by analyzing mice expressing a mutant form of PLZF mimicking constitutive acetylation (PLZFON) mice. NKT populations in PLZFON mice were reduced in proportion and numbers of cells, and the cells present were blocked at the transition from developmental stage 1 to stage 2. NKT cell subset differentiation was also altered, with T-bet+ NKT1 and RORγt+ NKT17 subsets dramatically reduced and the emergence of a T-bet-RORγt- NKT cell subset with features of cells in early developmental stages rather than mature NKT2 cells. Preliminary analysis of DNA methylation patterns suggested that activated PLZF acts on the DNA methylation signature to regulate NKT cells' entry into the early stages of development while repressing maturation. In wild-type NKT cells, deacetylation of PLZF is possible, allowing subsequent NKT cell differentiation. Interestingly, development of other innate lymphoid and myeloid cells that are dependent on PLZF for their generation is not altered in PLZFON mice, highlighting lineage-specific regulation. Overall, we propose that specific epigenetic control of PLZF through acetylation levels is required to regulate normal NKT cell differentiation.

Establishment of immune tolerance is crucial to protect humans against asthma. Promyelocytic leukemia zinc finger (PLZF) is an emerging suppressor of inflammatory responses. CCL21-CCR7 signaling mediates tolerance development. However, whether PLZF and CCL21-CCR7 are required for the development of asthma tolerance is unknown. Here, we found that Zbtb16 (coding PLZF) and Ccl21 were upregulated in OVA-induced asthma tolerance (OT) lungs by RNA-seq. PLZF physically interacted with GATA3 and its expression was higher in GATA3+ Th2cells and ILC2s in OT lungs. Zbtb16-knockdown in lymphocytes promoted the differentiation of CD3e+ CD4+ Tcells, particularly those producing IL-4 and IL-5. Moreover, iNKTcells with high expression of PLZF were recruited into the lungs via draining lymph nodes during tolerance. Blockade of CCL21-CCR7 signaling in OT mice decreased the PLZF+ cell population, abolished CCR7-induced PLZF+ iNKT recruitment to the lungs, enhanced Th2responses and exacerbated lung pathology. In OT mice, respiratory syncytial virus (RSV) infection impeded PLZF+ cell and CCR7+ PLZF+ iNKTcellrecruitment to the lungs and increased airway resistance. Collectively, these results indicate that PLZF could interact with GATA3 and restrain differentiation of IL-4- and IL-5-producing Tcells, iNKTcells with high PLZF expression are recruited to the lungs via CCL21-CCR7 signaling to facilitate the development of asthma tolerance.

The transcription factor PLZF (promyelocytic leukemia zinc finger) is required for the development of NKT cell effector functions. Studies described here investigate the gene targets of PLZF in NKT cells. The aims are: 1) to determine the gene expression in wild type and PLZF deficient NKT cells, and 2) to define direct genetic targets of PLZF. A hurdle to carrying out gene expression analysis has been the need to use CD1d tetramers to identify invariant NKT cells. Crosslinking of the TCR, as a result of binding the tetramer, can cause changes in signaling and gene expression. Therefore, for aim 1, we used a BAC eGFP/PLZF reporter mouse system. In this mouse, PLZF expressing cells are labeled with eGFP, which obviates the need for CD1d tetramer. Importantly, GFP expression is retained in NKT cells that develop in PLZF deficient mice. Therefore, "untouched" NKT cells can be collected from PLZF expressing and deficient mice. RNA was isolated from these cells and subjected to microarray analysis. Microarray analysis indicated many gene changes, of which only some will be direct targets of PLZF. Aim 2 will be to carry out Chromatin Immunoprecipitation assays followed by high-throughput sequencing. We will utilize an NKT cell hybridoma cell line that expresses PLZF and has retained the capacity to produce IL-4 and IFN-γ. By combining the data, we expect to define genes that are regulated by PLZF and are necessary for the development of NKT cells.

Cellular metabolism controlled by the signaling pathways has a crucial role in dictating the outcome of T cell activation and effector function. Reactive oxygen species (ROS) are byproducts of aerobic metabolism and contribute to both physiological and pathological conditions as a second messenger. In T cells, ROS levels increase upon activation and antigen specific responses of T cells are compromised in the absence of ROS production by mitochondria. However, it is not well understood how NKT cells’ functions are controlled by cell metabolism. In the current study, we investigated the role of ROS in NKT cell function. We found that ROS levels were similar among CD4, CD8 and NKT cell subsets in the thymus but NKT cells showed dramatically increased ROS in the spleen and liver but not in adipose tissues. ROS in the peripheral NKT cells were primarily produced by NADPH oxidases not mitochondria. Accordingly, ROS high NKT cells were susceptible to oxidative stress and underwent apoptotic cell death. Furthermore, ROS play an important role regulating the inflammatory function of NKT cells because antioxidant treatment of NKT cells showed reduced frequencies of IFN-γ+ and IL-17+ cells. In line with this, freshly isolated ROS-high NKT cells had more NKT1 and NKT17 cells but less NKT2 than ROS-low cells. Importantly, these characteristics of NKT cells are regulated by PLZF (Promyelocytic Leukaemia Zinc Finger) protein as evidenced by reduced ROS in NKT cells from adipose tissues that do not express PLZF and also those from PLZF haplodeficient mice. Conversely, ROS were highly elevated in CD4 T cells from mice ectopically expressing PLZF. Together, our study revealed for the first time that ROS regulate NKT cell functions through PLZF.

The promyelocytic leukaemia zinc finger (PLZF) protein is a member of the BTB/POZ zinc finger family of transcription factors. This family of transcription factors also include Th-Pok, BCL-6 and MAZR which are known to play important roles in the immune system. Here we demonstrate that PLZF deficient mice have a severe impairment in iNKT cell lineage development leading to a lack of iNKT cells in the thymus and periphery. Real-time PCR showed that PLZF mRNA is highly expressed in iNKT cells. This correlates with flow cytometry analysis which revealed exclusive expression of PLZF protein in iNKT cells within the immune system. Radiation chimeras showed that PLZF is required for iNKT cell development in a cell intrinsic manner. These results identify PLZF as a key factor in iNKT development. They also underline the BTB/POZ family of transcription factors as important factors in the immune system.

Development of invariant natural killer T (iNKT) cells in the thymus requires cell-cell interaction through invariant TCR (iTCR) and CD1d, which induces expression of the transcription factor, promyelocytic leukemia zinc finger (PLZF). However, the signaling pathway linking iTCR and PLZF remains unclear. Here, we report that a serine/threonine kinase, protein kinase D (PKD), plays a pivotal role in iNKT cell development. In T cell-specific PKD-deficient (Prkd2/3∆CD4) mice, PLZF induction and iNKT cell generation were severely impaired, which were rescued by introduction of a PLZF transgene. We identified the transcription factor Ikaros as a substrate of PKD upon iTCR stimulation. Knock-in mice carrying a phosphorylation-defective mutant Ikaros (Ikzf1S267/275A) exhibited an impairment of iNKT cell development, whereas conventional T cells were normal. In iNKT cells, Ikaros binds to the upstream region of the PLZF gene to induce its transcription. Mutant mice lacking the Ikaros-binding site (Zbtb16∆IBS) generated fewer iNKT cells than WT mice. These results suggest that PKD links iTCRs to PLZF induction through Ikaros, thereby mediating iNKT cell development.

The broad complex, tramtrack, bric-a-brac-zinc finger (BTB-ZF) transcription factor promyelocytic leukemia zinc finger (PLZF) is required for development of the characteristic innate/effector functions of NKT cells. In this study, we report the characterization and functional analysis of transgenic mouse T cells with forced expression of PLZF. PLZF expression was sufficient to provide some memory/effector functions to T cells without the need for Ag stimulation or proliferation. The acquisition of this phenotype did not require the proliferation typically associated with T cell activation. Furthermore, PLZF transgenic cells maintained a diverse TCR repertoire, indicating that there was no preferential expansion of specific clones. Functionally, PLZF transgenic CD4 and CD8 lymphocytes were similar to wild type memory cells, in that they had similar requirements for costimulation and exhibited a similar pattern of cytokine secretion, with the notable exception that transgenic T cells produced significantly increased levels of IL-17. Whereas transgene-mediated PLZF expression was not sufficient to rescue NKT cell development in Fyn- or signaling lymphocytic activation-associated protein (SAP)-deficient mice, the acquisition of memory/effector functions induced by PLZF in conventional T cells was independent of Fyn and SAP. These data show that PLZF is sufficient to promote T cell effector functions and that PLZF acts independently of SAP- and Fyn-mediated signaling pathways.

Reactive oxygen species (ROS) are byproducts of aerobic metabolism and contribute to both physiological and pathological conditions as second messengers. ROS are essential for activation of T cells, but how ROS influence NKT cells is unknown. In the present study, we investigated the role of ROS in NKT cell function. We found that NKT cells, but not CD4 or CD8 T cells, have dramatically high ROS in the spleen and liver of mice but not in the thymus or adipose tissues. Accordingly, ROS-high NKT cells exhibited increased susceptibility and apoptotic cell death with oxidative stress. High ROS in the peripheral NKT cells were primarily produced by NADPH oxidases and not mitochondria. We observed that sorted ROS-high NKT cells were enriched in NKT1 and NKT17 cells, whereas NKT2 cells were dominant in ROS-low cells. Furthermore, treatment of NKT cells with antioxidants led to reduced frequencies of IFN-γ- and IL-17-expressing cells, indicating that ROS play a role in regulating the inflammatory function of NKT cells. The transcription factor promyelocytic leukemia zinc finger (PLZF) seemed to control the ROS levels. NKT cells from adipose tissues that do not express PLZF and those from PLZF haplodeficient mice have low ROS. Conversely, ROS were highly elevated in CD4 T cells from mice ectopically expressing PLZF. Thus, our findings demonstrate that PLZF controls ROS levels, which in turn governs the inflammatory function of NKT cells.

Expression of promyelocytic leukemia zinc finger (PLZF) protein directs the effector differentiation of invariant NKT (iNKT) cells and IL-4(+) γδ NKT cells. In this study, we show that PLZF is also required for the development and function of IL-17(+) γδ T cells. We observed that PLZF is expressed in fetal-derived invariant Vγ5(+) and Vγ6(+) γδ T cells, which secrete IFN-γ and IL-17, respectively. PLZF deficiency specifically affected the effector differentiation of Vγ6(+) cells, leading to reduced numbers of mature CD27(-)CD44(+) phenotype capable of secreting IL-17. Although PLZF was not required for Vγ5(+) γδ T cells to develop, when these cells were reprogrammed into IL-17-secreting cells in Skint-1 mutant mice, they required PLZF for their effector maturation, similarly to Vγ6(+) γδ T cells. The impaired effector differentiation of PLZF-deficient Vγ6(+) γδ T cells was not due to increased apoptosis and it was related to reduced proliferation of immature CD27(+)CD44(-) Vγ6(+) γδ T cells, which was required for their differentiation into mature CD27(-)CD44(+) IL-17-secreting cells. Thus, the present study identifies that PLZF function is not restricted to NKT or IL-4(+) T cells, but it also controls the development of IL-17(+) γδ T cells.

Spermatogonial stem cells (SSCs) are an important tool for fertility preservation and species conservation. The ability to expand SSCs by in vitro culture is a crucial premise for their use in assisted reproduction. Because SSCs represent a small proportion of the germ cells in the adult testis, culture success is aided by pre-enrichment through sorting techniques based on cell surface-specific markers. Given the importance of the domestic cat as a model for conservation of endangered wild felids, herein we sought to examine culture conditions as well as molecular markers for cat SSCs. Using a cell culture medium for mouse SSCs supplemented with glial cell-derived neurotrophic factor (GDNF), germ cells from prepuberal cat testes remained viable in culture for up to 43days. Immunohistochemistry for promyelocytic leukaemia zinc finger (PLZF) protein on foetal, prepuberal and adult testis sections revealed a pattern of expression consistent with the labelling of undifferentiated spermatogonia. Fluorescence-activated cell sorting (FACS) with an antibody against epithelial cell adhesion molecule (EPCAM) was used to sort live cells. Then, the gene expression profile of EPCAM-sorted cells was investigated through RT-qPCR. Notably, EPCAM (+) cells expressed relatively high levels of CKIT (CD117), a surface protein typically expressed in differentiating germ cells but not SSCs. Conversely, EPCAM (-) cells expressed relatively high levels of POU domain class 5 transcription factor 1 (POU1F5 or OCT4), clearly a germ line stem cell marker. These results suggest that cat SSCs would probably be found within the population of EPCAM (-) cells. Future studies should identify additional surface markers that alone or in combination can be used to further enrich SSCs from cat germ cells.

Objective To investigate the effects of glial cell line-derived neurotrophic factor(GDNF)on the gene transcriptions and expressions of promyelocytic leukaemia zinc finger(PLZF)and c-Kit in spermatogonial stem cells(SSC).Methods SSC was cultured in DMEM plus different concentrations of GDNF(0,10,50,100 ng/ml).Quantitative reverse transcription PCR was used to detect the mRNA of PLZF and c-Kit.The expressions of PLZF and c-Kit protein were detected by Western blot assay.Results With the control group and 10ng/ml of GDNF group cell growth has no obvious influence,but with 50 ng/ml of GDNF group and 100 ng/ml of GDNF group cell growth was enhanced significantly.In the control group,the PLZF mRNA was 0.28±0.13 and c-Kit mRNA was 0.65±0.21.In the 10 ng/ml of GDNF group,PLZF mRNA was 0.27±0.14 and c-Kit tuRNA was 0.62±0.19.Compared with the control group,10 ng/ml of GDNF group had not influence on PLZF and c-Kit mRNA.In the 50 ng/ml of GDNF group PLZF mRNA was 0.64±0.28 and c-Kit mRNA was 0.34±0.15.Compared with the control group,50 ng/ml of GDNF enhanced the transcription of PLZF mRNA(P＜0.05),decreased the transeription of c-Kit mRNA (P＜0.05).In the 100 ng/ml of GDNF gmup,PLZF mRNA was 0.68±0.27 and c-Kit mRNA was 0.28±0.18.In the 100 ng/ml of GDNF group,there was no difference compared with 50 ng/ml of GDNF.In the control group,PLZF protein was 0.34±0.13 and c-Kit protein was 0.72±0.27.In 10 ng/ml of GDNF group,PLZF protein was 0.38±0.18 and c-Kit protein was 0.69±0.26.Compared with the control group,10 ng/ml of GDNF had no influence on PLZF and c-Kit protein.In 50 ng/ml of GDNF group,PLZF protein was 0.68±0.26 and c-Kit protein was 0.35±0.15.50 ng/ml of GDNF enhanced the expression of PLZF protein(P＜0.05),decreased the expression of c-Kit protein(P＜0.05).In 100 ng/ml of GDNF group PLZF protein was 0.70±0.27 and c-Kit protein Was 0.32±0.11,100 ng/ml of GDNF had no influence on PLZF and c-Kit protein compared with 50 ng/ml of GDNF.Conclusions Higher concentration of GDNF could enhance the transcription and expression of PLZF,and decrease the transcription and expression of c-Kit in SSC.GDNF has possible role to regulate and control proliferation and differentiation of SSC. Key words: Spermatogonia; Glial cell line-derived neurotrophic factor; Promyelocytic leukaemia zinc finger; Proto-oncogene proteins; c-kit

NKT cells constitute a small population of T cells developed in the thymus that produce large amounts of cytokines and chemokines in response to lipid Ags. Signaling through the Vα14-Jα18 TCR instructs commitment to the NKT cell lineage, but the precise signaling mechanisms that instruct their lineage choice are unclear. In this article, we report that the cytoskeletal remodeling protein, p21-activated kinase 2 (Pak2), was essential for NKT cell development. Loss of Pak2 in T cells reduced stage III NKT cells in the thymus and periphery. Among different NKT cell subsets, Pak2 was necessary for the generation and function of NKT1 and NKT2 cells, but not NKT17 cells. Mechanistically, expression of Egr2 and promyelocytic leukemia zinc finger (PLZF), two key transcription factors for acquiring the NKT cell fate, were markedly diminished in the absence of Pak2. Diminished expression of Egr2 and PLZF were not caused by aberrant TCR signaling, as determined using a Nur77-GFP reporter, but were likely due to impaired induction and maintenance of signaling lymphocyte activation molecule 6 expression, a TCR costimulatory receptor required for NKT cell development. These data suggest that Pak2 controls thymic NKT cell development by providing a signal that links Egr2 to induce PLZF, in part by regulating signaling lymphocyte activation molecule 6 expression.

Jarid2 is a reported component of three lysine methyltransferase complexes, polycomb repressive complex 2 (PRC2) that methylates histone 3 lysine 27 (H3K27), and GLP-G9a and SETDB1 complexes that methylate H3K9. Here we show that Jarid2 is upregulated upon TCR stimulation and during positive selection in the thymus. Mice lacking Jarid2 in T cells display an increase in the frequency of IL-4-producing promyelocytic leukemia zinc finger (PLZF)(hi) immature invariant natural killer T (iNKT) cells and innate-like CD8(+) cells; Itk-deficient mice, which have a similar increase of innate-like CD8(+) cells, show blunted upregulation of Jarid2 during positive selection. Jarid2 binds to the Zbtb16 locus, which encodes PLZF, and thymocytes lacking Jarid2 show increased PLZF and decreased H3K9me3 levels. Jarid2-deficient iNKT cells perturb Th17 differentiation, leading to reduced Th17-driven autoimmune pathology. Our results establish Jarid2 as a novel player in iNKT cell maturation that regulates PLZF expression by modulating H3K9 methylation.