Hodaka Fujii

Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells

The molecules that define human regulatory T cells (Tregs) phenotypically and functionally remain to be fully characterized. We recently showed that activated human Tregs express mRNA for a transmembrane protein called glycoprotein A repetitions predominant (GARP, or LRRC32). Here, using a GARP-specific mAb, we demonstrate that expression of GARP on activated Tregs correlates with their suppressive capacity. However, GARP was not induced on T cells activated in the presence of TGFβ, which expressed high levels of FOXP3 and lacked suppressive function. Ectopic expression of FOXP3 in conventional T cells was also insufficient for induction of GARP expression in most donors. Functionally, silencing GARP in Tregs only moderately attenuated their suppressive activity. CD25+ T cells sorted for high GARP expression displayed more potent suppressive activity compared with CD25+GARP− cells. Remarkably, CD25+GARP− T cells expanded in culture contained 3–5 fold higher IL-17-secreting cells compared with either CD25+GARP+ or CD25−GARP− cells, suggesting that high GARP expression can potentially discriminate Tregs from those that have switched to Th17 lineage. We also determined whether GARP expression correlates with FOXP3-expressing T cells in human immunodeficiency virus (HIV) −infected subjects. A subset of HIV+ individuals with high percentages of FOXP3+ T cells did not show proportionate increase in GARP+ T cells. This finding suggests that higher FOXP3 levels observed in these HIV+ individuals is possibly due to immune activation rather than to an increase in Tregs. Our findings highlight the significance of GARP both in dissecting duality of Treg/Th17 cell differentiation and as a marker to identify bona fide Tregs during diseases with chronic immune activation.

Plasmodium Circumsporozoite Protein Promotes the Development of the Liver Stages of the Parasite

The liver stages of malaria are clinically silent but have a central role in the Plasmodium life cycle. Liver stages of the parasite containing thousands of merozoites grow inside hepatocytes for several days without triggering an inflammatory response. We show here that Plasmodium uses a PEXEL/VTS motif to introduce the circumsporozoite (CS) protein into the hepatocyte cytoplasm and a nuclear localization signal (NLS) to enter its nucleus. CS outcompetes NFkappaB nuclear import, thus downregulating the expression of many genes controlled by NFkappaB, including those involved in inflammation. CS also influences the expression of over one thousand host genes involved in diverse metabolic processes to create a favorable niche for the parasite growth. The presence of CS in the hepatocyte enhances parasite growth of the liver stages in vitro and in vivo. These findings have far reaching implications for drug and vaccine development against the liver stages of the malaria parasite.

Protein tyrosine kinase Pyk2 mediates the Jak‐dependent activation of MAPK and Stat1 in IFN‐γ, but not IFN‐α, signaling

Two distinct types of interferon, IFN‐α/β and IFN‐γ, commonly exhibit antiviral activities by transmitting signals to the interior of the cell via their homologous receptors. Receptor stimulation results in the activation of distinct combinations of Janus family protein tyrosine kinases (Jak PTKs); Jak1/Tyk2 and Jak1/Jak2 for IFN‐α/β and IFN‐γ, respectively. Jak PTK activation by these IFNs is commonly followed by tyrosine phosphorylation of the transcription factor Stat1 at Y701, which is essential for dimerization, translocation to the nucleus and DNA‐binding activity. To gain full transcriptional activity, Stat1 also requires serine phosphorylation at S727. In this paper we demonstrate that Pyk2, which belongs to another PTK family, is critical for the Jak‐mediated MAPK and Stat1 activation by IFN‐γ, but not IFN‐α. Pyk2 is selectively associated with Jak2 and activated by IFN‐γ. Overexpression of PKM, a dominant interfering form of Pyk2, in NIH 3T3 cells results in a strong inhibition of the IFN‐γ‐induced activation of Erk2, serine phosphorylation of Stat1 and Stat1‐dependent gene transcription. Finally, the antiviral action of IFN‐γ, but not IFN‐α, is severely impaired by PKM overexpression. Thus, the two types of IFN may utilize distinct Jak‐mediated Erk2, and possibly other MAPK activation pathways for their antiviral action.

Identification of a regulatory T cell specific cell surface molecule that mediates suppressive signals and induces Foxp3 expression.

Regulatory T (Treg) cells control immune activation and maintain tolerance. How Tregs mediate their suppressive function is unclear. Here we identified a cell surface molecule, called GARP, (or LRRC32), which within T cells is specifically expressed in Tregs activated through the T cell receptor (TCR). Ectopic expression of GARP in human naïve T (TN) cells inhibited their proliferation and cytokine secretion upon TCR activation. Remarkably, GARP over-expression in TN cells induced expression of Treg master transcription factor Foxp3 and endowed them with a partial suppressive function. The extracellular but not the cytoplasmic region of GARP, was necessary for these functions. Silencing Foxp3 in human Treg cells reduced expression of GARP and attenuated their suppressive function. However, GARP function was not affected when Foxp3 was downregulated in GARP-overexpressing cells, while silencing GARP in Foxp3-overexpressing cells reduced their suppressive activity. These findings reveal a novel cell surface molecule-mediated regulatory mechanism, with implications for modulating aberrant immune responses.

Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR.

Isolation of specific genomic regions retaining molecular interactions is necessary for their biochemical analysis. Here, we established a novel method, engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP), for purification of specific genomic regions retaining molecular interactions. We showed that enChIP using the CRISPR system efficiently isolates specific genomic regions. In this form of enChIP, specific genomic regions are immunoprecipitated with antibody against a tag(s), which is fused to a catalytically inactive form of Cas9 (dCas9), which is co-expressed with a guide RNA (gRNA) and recognizes endogenous DNA sequence in the genomic regions of interest. enChIP-mass spectrometry (enChIP-MS) targeting endogenous loci identified associated proteins. enChIP using the CRISPR system would be a convenient and useful tool for dissecting chromatin structure of genomic regions of interest.

Regulation of protein kinase C isoform proteins in phorbol ester-stimulated Jurkat T lymphoma cells.

Activation of protein kinase C (PKC) in T cells leads to a variety of responses including IL-2 production and IL-2 receptor expression. PKC consists of several isoforms that exhibit some different in vitro properties. We have set up a Western blotting system to explore the regulation of PKC isoforms during T cell activation. In Jurkat T lymphoma cells, PKC alpha, beta, delta, epsilon, and zeta were detected. PKC alpha and beta existed primarily in the cytosol, translocated to the membrane fraction after 10 minutes of treatment with PMA, and almost completely disappeared within 16 h. A larger fraction of PKC delta and epsilon existed in the membrane fraction compared to PKC alpha or beta, and PKC epsilon translocated to the membrane fraction rapidly. Translocation of PKC delta was not apparent after 1 h treatment with PMA, but total PKC delta protein was reduced within 4 to 6 h of treatment. Consistent with this, overnight treatment with PMA caused down-regulation of both PKC delta and epsilon, but to a lesser degree than was observed with PKC beta. Anti-PKC zeta antibody detected two bands at 82 and 75 kDa. The 75-kDa band existed mostly in the cytosol fraction and showed no translocation or down regulation after PMA. We present evidence that this 75-kDa band represents PKC zeta. Similar PMA-induced translocation responses were observed in murine thymocytes showing that the responses are not unique to PKC isoforms in Jurkat. These results demonstrate that it is possible for the PKC isoforms to be differentially regulated during T cell activation.

Regulation of Cell Proliferation by Interleukin-3-induced Nuclear Translocation of Pyruvate Kinase

Extracellular signaling molecules bound to cell surface receptors can regulate nuclear function with consequences for cell proliferation, differentiation, and function. To regulate nuclear function, signals must be transduced across the nuclear envelope to propagate the signal from the cytoplasm to the nucleus. Therefore, many signaling responses induce the nuclear translocation of transcription factors, kinases, and others. By using inducible translocation trap, a reporter gene-based system to detect inducible nuclear translocation, we found that the M2 isoform of pyruvate kinase, a key enzyme in glycolysis, translocates into the nucleus by interleukin-3, but not by epidermal growth factor, stimulation. The C domain of the M2 isoform of pyruvate kinase was sufficient for interleukin-3-induced nuclear translocation. Interleukin-3-induced nuclear translocation of the M2 isoform of pyruvate kinase was dependent on the activation of Jak2. Overexpression of the M2 isoform of pyruvate kinase protein fused with a nuclear localization signal enhanced cell proliferation in the absence of interleukin-3, suggesting that the nuclear pyruvate kinase plays an important role in cell proliferation.

Functional dissection of the cytoplasmic subregions of the IL-2 receptor βc chain in primary lymphocyte populations

The interleukin 2 (IL‐2) receptor βc chain (IL‐2Rβc) is known to regulate the development and function of distinct lymphocyte populations. Thus far, the functions of the IL‐2Rβc cytoplasmic subregions have been studied extensively by using cultured cell lines; however, this approach has limitations with respect to their functions in distinct primary lymphocyte populations. In the present study, we generated mice each expressing a mutant form of an IL‐2Rβc transgene, lacking the cytoplasmic A‐ or H‐region, on an IL‐2Rβc null background. We show that lack of the H‐region, which mediates activation of the Stat5/Stat3 transcription factors, selectively affects the development of natural killer cells and T cells bearing the γδ T cell receptor. This region is also required for the IL‐2‐induced proliferation of T cells in vitro, by upregulating IL‐2Rα expression. In contrast, the A‐region, which mediates activation of the Src family protein tyrosine kinase (PTK) members, contributes to downregulation of the T cell proliferation function. The IL‐2Rβc null mutant mice develop severe autoimmune symptoms; these are all suppressed following the expression of either of the mutants, suggesting that neither the Stats nor the Src PTK members are required. Thus, our present approach offers new insights into the functions of these cytoplasmic subregions of the IL‐2Rβc chain.

Regulation of the Expression of GARP/Latent TGF-β1 Complexes on Mouse T Cells and Their Role in Regulatory T Cell and Th17 Differentiation

GARP/LRRC32 was defined as a marker of activated human regulatory T cells (Tregs) that is responsible for surface localization of latent TGF-β1. We find that GARP and latent TGF-β1 are also found on mouse Tregs activated via TCR stimulation; however, in contrast to human Tregs, GARP is also expressed at a low level on resting Tregs. The expression of GARP can be upregulated on mouse Tregs by IL-2 or IL-4 exposure in the absence of TCR signaling. GARP is expressed at a low level on Tregs within the thymus, and Treg precursors from the thymus concomitantly express GARP and Foxp3 upon exposure to IL-2. The expression of GARP is independent of TGF-β1 and TGF-β1 loading into GARP and is independent of furin-mediated processing of pro–TGF-β1 to latent TGF-β1. Specific deletion of GARP in CD4+ T cells results in lack of expression of latent TGF-β1 on activated Tregs. GARP-deficient Tregs develop normally, are present in normal numbers in peripheral tissues, and are fully competent suppressors of the activation of conventional T cells in vitro. Activated Tregs expressing GARP/latent TGF-β1 complexes are potent inducers of Th17 differentiation in the presence of exogenous IL-6 and inducers of Treg in the presence of IL-2. Induction of both Th17-producing cells and Tregs is caused preferentially by Tregs expressing the latent TGF-β1/GARP complex on their cell surface rather than by secreted latent TGF-β1.

Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP)

Biochemical analysis of molecular interactions in specific genomic regions requires their isolation while retaining molecular interactions in vivo. Here, we report isolation of telomeres by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using a transcription activator-like (TAL) protein recognizing telomere repeats. Telomeres recognized by the tagged TAL protein were immunoprecipitated with an antibody against the tag and subjected to identification of telomere-binding molecules. enChIP-mass spectrometry (enChIP-MS) targeting telomeres identified known and novel telomere-binding proteins. The data have been deposited to the ProteomeXchange with identifier PXD000461. In addition, we showed that RNA associated with telomeres could be isolated by enChIP. Identified telomere-binding molecules may play important roles in telomere biology. enChIP using TAL proteins would be a useful tool for biochemical analysis of specific genomic regions of interest.