Dual-locus, dual-HDR editing permits efficient generation of antigen-specific regulatory T cells with robust suppressive activity.

Adoptive regulatory T (Treg) cell therapy is predicted to modulate immune tolerance in autoimmune diseases, including type 1 diabetes (T1D). However, the requirement for antigen (ag) specificity to optimally orchestrate tissue-specific, Treg cell-mediated tolerance limits effective clinical application. To address this challenge, we present a single-step, combinatorial gene editing strategy utilizing dual-locus, dual-homology-directed repair (HDR) to generate and specifically expand ag-specific engineered Treg (EngTreg) cells derived from donor CD4+ T cells. Concurrent delivery of CRISPR nucleases and recombinant (r)AAV homology donor templates targeting FOXP3 and TRAC was used to achieve three parallel goals: enforced, stable expression of FOXP3; replacement of the endogenous T cell receptor (TCR) with an islet-specific TCR; and selective enrichment of dual-edited cells. Each HDR donor template contained an alternative component of a heterodimeric chemically inducible signaling complex (CISC), designed to activate interleukin-2 (IL-2) signaling in response to rapamycin, promoting expansion of only dual-edited EngTreg cells. Using this approach, we generated purified, islet-specific EngTreg cells that mediated robust direct and bystander suppression of effector T (Teff) cells recognizing the same or a different islet antigen peptide, respectively. This platform is broadly adaptable for use with alternative TCRs or other targeting moieties for application in tissue-specific autoimmune or inflammatory diseases.

Antigen Processing in the Endoplasmic Reticulum Is Monitored by Semi-Invariant αß TCRs Specific for a Conserved Peptide-Qa-1b MHC Class Ib Ligand.

Ag processing in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with Ag processing (ERAAP) is central to presentation of a normal peptide-MHC class I (MHC I) repertoire. Alternations in ERAAP function cause dramatic changes in the MHC I-presented peptides, which elicit potent immune responses. An unusual subset of CD8+ T cells monitor normal Ag processing by responding to a highly conserved FL9 peptide that is presented by Qa-1b, a nonclassical MHC Ib molecule (QFL) in ERAAP-deficient cells. To understand the structural basis for recognition of the conserved ligand, we analyzed the αß TCRs of QFL-specific T cells. Individual cells in normal wild-type and TCRß-transgenic mice were assessed for QFL-specific TCR α- and ß-chains. The QFL-specific cells expressed a predominant semi-invariant TCR generated by DNA rearrangement of TRAV9d-3-TRAJ21 α-chain and TRBV5-TRBD1-TRBJ2-7 ß-chain gene segments. Furthermore, the CDR3 regions of the α- as well as ß-chains were required for QFL ligand recognition. Thus, the αß TCRs used to recognize the peptide-Qa-1 ligand presented by ERAAP-deficient cells are semi-invariant and likely reflect a conserved mechanism for monitoring the fidelity of Ag processing in the ER.

GNTI-122: an autologous antigen-specific engineered Treg cell therapy for type 1 diabetes.

Tregs have the potential to establish long-term immune tolerance in patients recently diagnosed with type 1 diabetes (T1D) by preserving ß cell function. Adoptive transfer of autologous thymic Tregs, although safe, exhibited limited efficacy in previous T1D clinical trials, likely reflecting a lack of tissue specificity, limited IL-2 signaling support, and in vivo plasticity of Tregs. Here, we report a cell engineering strategy using bulk CD4+ T cells to generate a Treg cell therapy (GNTI-122) that stably expresses FOXP3, targets the pancreas and draining lymph nodes, and incorporates a chemically inducible signaling complex (CISC). GNTI-122 cells maintained an expression profile consistent with Treg phenotype and function. Activation of CISC using rapamycin mediated concentration-dependent STAT5 phosphorylation and, in concert with T cell receptor engagement, promoted cell proliferation. In response to the cognate antigen, GNTI-122 exhibited direct and bystander suppression of polyclonal, islet-specific effector T cells from patients with T1D. In an adoptive transfer mouse model of T1D, a mouse engineered-Treg analog of GNTI-122 trafficked to the pancreas, decreased the severity of insulitis, and prevented progression to diabetes. Taken together, these findings demonstrate in vitro and in vivo activity and support further development of GNTI-122 as a potential treatment for T1D.

Epithin/PRSS14 proteolytically regulates angiopoietin receptor Tie2 during transendothelial migration.

Epithin/PRSS14, a type II transmembrane serine protease, is involved in normal epithelial development and tumor progression. Here we report, as an interacting substrate of epithin, a receptor tyrosine kinase Tie2 that is well known for important roles in the vessel stability. Epithin interacts with and degrades the Tie2 extracellular portion that contains the ligand-binding domain. Epithin is located in the neighbor of Tie2-expressing vessels in normal tissue. Furthermore, epithin can cleave and degrade Tie2 not only in the same cell but also from neighboring cells nearby, resulting in the degradation of the Tie2 ectodomain. The remaining Tie2 fragment was highly phosphorylated and was able to recruit a downstream effector, phosphatidylinositol 3-kinase. Knocking down epithin expression using short hairpin RNA in thymoma cell severely impaired the migration through endothelial cells that show the actin rearrangement during the process. The diminution of epithin protein expression in 4T1 breast cancer cells caused the significant decrease in the number of transendothelial migrating cells in vitro as well as in those of metastasizing tumor nodules in vivo, Therefore, we propose that epithin, which regulates endothelial Tie2 functions, plays a critical role in the fine tuning of transendothelial migration for normal and cancer cells.

Novel NF-κB reporter mouse for the non-invasive monitoring of inflammatory diseases.

Bioluminescence imaging is useful for non-invasively monitoring inflammatory reactions associated with disease progression, and since NF-κB is a pivotal transcription factor that alters expressions of inflammatory genes, we generated novel NF-κB luciferase reporter (NF-κB-Luc) mice to understand the dynamics of inflammatory responses in whole body, and also in various type of cells by crossing NF-κB-Luc mice with cell-type specific Cre expressing mice (NF-κB-Luc:[Cre]). Bioluminescence intensity was significantly increased in NF-κB-Luc (NKL) mice exposed to inflammatory stimuli (PMA or LPS). Crossing NF-κB-Luc mice with Alb-cre mice or Lyz-cre mice generated NF-κB-Luc:Alb (NKLA) and NF-κB-Luc:Lyz2 (NKLL) mice, respectively. NKLA and NKLL mice showed enhanced bioluminescence in liver and macrophages, respectively. To confirm that our reporter mice could be utilized for the non-invasive monitoring of inflammation in preclinical models, we conducted a DSS-induced colitis model and a CDAHFD-induced NASH model in our reporter mice. In both models, our reporter mice reflected the development of these diseases over time. In conclusion, we believe that our novel reporter mouse can be utilized as a non-invasive monitoring platform for inflammatory diseases.

Pancreatic islet-specific engineered Tregs exhibit robust antigen-specific and bystander immune suppression in type 1 diabetes models.

Adoptive transfer of regulatory T cells (Tregs) is therapeutic in type 1 diabetes (T1D) mouse models. Tregs that are specific for pancreatic islets are more potent than polyclonal Tregs in preventing disease. However, the frequency of antigen-specific natural Tregs is extremely low, and ex vivo expansion may destabilize Tregs, leading to an effector phenotype. Here, we generated durable, antigen-specific engineered Tregs (EngTregs) from primary human CD4+ T cells by combining FOXP3 homology-directed repair editing and lentiviral T cell receptor (TCR) delivery. Using TCRs derived from clonally expanded CD4+ T cells isolated from patients with T1D, we generated islet-specific EngTregs that suppressed effector T cell (Teff) proliferation and cytokine production. EngTregs suppressed Teffs recognizing the same islet antigen in addition to bystander Teffs recognizing other islet antigens through production of soluble mediators and both direct and indirect mechanisms. Adoptively transferred murine islet-specific EngTregs homed to the pancreas and blocked diabetes triggered by islet-specific Teffs or diabetogenic polyclonal Teffs in recipient mice. These data demonstrate the potential of antigen-specific EngTregs as a targeted therapy for preventing T1D.

PRSS14/Epithin is induced in macrophages by the IFN-γ/JAK/STAT pathway and mediates transendothelial migration.

PRSS14/Epithin (also known as matriptase and ST14), a member of the type II transmembrane serine proteases, is primarily found in a subpopulation of normal epithelial cells and in epithelial cancers. Its known functions include maintaining the epithelial barrier, thymic development, and cancer progression. In this study, we show that several macrophage cell lines and activated bone marrow-derived macrophages also express PRSS14/Epithin. Surface expression, as well as cytoplasmic expression, was detectable upon activation by IFN-γ, but not TNF-α or TGF-ß. Induction of the protein appeared to be restricted to macrophages. IFN-γ showed a biphasic regulation in RAW264.7 cells, and upregulated expression was sustained for several days. This induction by IFN-γ was partially through the increase of PRSS14/Epithin mRNA production, which is downstream of the JAK pathway, shown by the inhibition by tyrphostin AG490. Using chromatin immunoprecipitation, we verified that two sites among six putative STAT1 binding sites in the PRSS14/Epithin promoter were occupied by STAT1 upon activation. Treatment with IFN-γ enhanced the serum-triggered transendothelial migration of RAW264.7 cells, but not that of PRSS14/Epithin knock-down RAW264.7 cells, although they express multiple markers such as ICAM1, CD80, and CD40 at normal levels. These data strongly suggest that PRSS14/Epithin plays an important role in the transendothelial migration of activated macrophages in the inflammatory microenvironment, and the mode of action is similar to the events in cancer metastasis.

TSCOT+ thymic epithelial cell-mediated sensitive CD4 tolerance by direct presentation.

Although much effort has been directed at dissecting the mechanisms of central tolerance, the role of thymic stromal cells remains elusive. In order to further characterize this event, we developed a mouse model restricting LacZ to thymic stromal cotransporter (TSCOT)-expressing thymic stromal cells (TDLacZ). The thymus of this mouse contains approximately 4,300 TSCOT+ cells, each expressing several thousand molecules of the LacZ antigen. TSCOT+ cells express the cortical marker CDR1, CD40, CD80, CD54, and major histocompatibility complex class II (MHCII). When examining endogenous responses directed against LacZ, we observed significant tolerance. This was evidenced in a diverse T cell repertoire as measured by both a CD4 T cell proliferation assay and an antigen-specific antibody isotype analysis. This tolerance process was at least partially independent of Autoimmune Regulatory Element gene expression. When TDLacZ mice were crossed to a novel CD4 T cell receptor (TCR) transgenic reactive against LacZ (BgII), there was a complete deletion of double-positive thymocytes. Fetal thymic reaggregate culture of CD45- and UEA-depleted thymic stromal cells from TDLacZ and sorted TCR-bearing thymocytes excluded the possibility of cross presentation by thymic dendritic cells and medullary epithelial cells for the deletion. Overall, these results demonstrate that the introduction of a neoantigen into TSCOT-expressing cells can efficiently establish complete tolerance and suggest a possible application for the deletion of antigen-specific T cells by antigen introduction into TSCOT+ cells.

Interaction of microtubules and actin with the N-terminus of ßPix-b(L) directs cellular pinocytosis.

ßPix is a Rac/Cdc42 guanine nucleotide exchange factor (GEF) that is known to be a regulator of actin cytoskeleton remodeling. Recently, a novel splicing isoform, ßPix-b(L), was identified as an alternative translational product of the ßPix-b mRNA with an extended N-terminus comprising a partial calponin homology (CH) domain and a serine-rich (SR) domain. However, the cellular function of ßPix-b(L) is largely unknown. In the current study, we analyzed the genomic DNA structure and cellular functions of ßPix-b(L). The results of this study demonstrate that ßPix is composed of 24 exons and 21 introns spanning around 100 kb. RT-PCR experiments revealed that there are two forms of ßPix mRNA with distinct 5' UTRs that are the result of alternative splicing of exon 1 and 2 from ßPix genomic DNA. In addition, affinity chromatography analysis and a pull-down assay with the N-terminal region of ßPix-b(L) revealed that ßPix-b(L) interacts with tubulin and actin via its N-terminal CH and SR domains, respectively. Interaction with tubulin enabled ßPix-b(L) to bundle the microtubule and form membrane protrusions. Furthermore, the N-terminus of ßPix-b(L) was also critical for its localization to cellular vesicles. Functionally, ßPix-b(L) induced pinocytosis through cooperative action of the CH and Dbl homology (DH) domains, demonstrating the role of ßPix-b(L) in the regulation of membrane dynamics.

Beta-Pix-dynamin 2 complex promotes colorectal cancer progression by facilitating membrane dynamics.

PURPOSE: Spatiotemporal regulation of cell membrane dynamics is a major process that promotes cancer cell invasion by acting as a driving force for cell migration. Beta-Pix (ßPix), a guanine nucleotide exchange factor for Rac1, has been reported to be involved in actin-mediated cellular processes, such as cell migration, by interacting with various proteins. As yet, however, the molecular mechanisms underlying ßPix-mediated cancer cell invasion remain unclear. METHODS: The clinical significance of ßPix was analyzed in patients with colorectal cancer (CRC) using public clinical databases. Pull-down and immunoprecipitation assays were employed to identify novel binding partners for ßPix. Additionally, various cell biological assays including immunocytochemistry and time-lapse video microscopy were performed to assess the effects of ßPix on CRC progression. A ßPix-SH3 antibody delivery system was used to determine the effects of the ßPix-Dyn2 complex in CRC cells. RESULTS: We found that the Src homology 3 (SH3) domain of ßPix interacts with the proline-rich domain of Dynamin 2 (Dyn2), a large GTPase. The ßPix-Dyn2 interaction promoted lamellipodia formation, along with plasma membrane localization of membrane-type 1 matrix metalloproteinase (MT1-MMP). Furthermore, we found that Src kinase-mediated phosphorylation of the tyrosine residue at position 442 of ßPix enhanced ßPix-Dyn2 complex formation. Disruption of the ßPix-Dyn2 complex by ßPix-SH3 antibodies targeting intracellular ßPix inhibited CRC cell invasion. CONCLUSIONS: Our data indicate that spatiotemporal regulation of the Src-ßPix-Dyn2 axis is crucial for CRC cell invasion by promoting membrane dynamics and MT1-MMP recruitment into the leading edge. The development of inhibitors that disrupt the ßPix-Dyn2 complex may be a useful therapeutic strategy for CRC.

Gene editing to induce FOXP3 expression in human CD4+ T cells leads to a stable regulatory phenotype and function.

Thymic regulatory T cells (tTregs) are potent inhibitors of autoreactive immune responses, and loss of tTreg function results in fatal autoimmune disease. Defects in tTreg number or function are also implicated in multiple autoimmune diseases, leading to growing interest in use of Treg as cell therapies to establish immune tolerance. Because tTregs are present at low numbers in circulating blood and may be challenging to purify and expand and also inherently defective in some subjects, we designed an alternative strategy to create autologous Treg-like cells from bulk CD4+ T cells. We used homology-directed repair (HDR)-based gene editing to enforce expression of FOXP3, the master transcription factor for tTreg Targeted insertion of a robust enhancer/promoter proximal to the first coding exon bypassed epigenetic silencing, permitting stable and robust expression of endogenous FOXP3. HDR-edited T cells, edTregs, manifested a transcriptional program leading to sustained expression of canonical markers and suppressive activity of tTreg Both human and murine edTregs mediated immunosuppression in vivo in models of inflammatory disease. Further, this engineering strategy permitted generation of antigen-specific edTreg with robust in vitro and in vivo functional activity. Last, edTreg could be enriched and expanded at scale using clinically relevant methods. Together, these findings suggest that edTreg production may permit broad future clinical application.

A peptide-based fluorescent probe images ERAAP activity in cells and in high throughput assays.

ERAAP is an intracellular amino-peptidase that plays a central role in determining the repertoire of peptides displayed by cells by MHC class I molecules, and dysfunctions in ERAAP are linked to a variety of diseases. There is therefore great interest in developing probes that can image ERAAP in cells. In this report we present a fluorescent probe, termed Ep, that can image ERAAP activity in live cells. Ep is composed of a 10 amino acid ERAAP substrate that has a donor quencher pair conjugated to it, composed of BODIPY and dinitro-toluene. Ep undergoes a 20-fold increase in fluorescence after ERAAP cleavage, and was able to image ERAAP activity in cell culture via fluorescence microscopy. In addition, we used Ep to develop a high throughput screen for ERAAP inhibitors, and screened an electrophile library containing 1460 compounds. From this Ep based screen we identified aromatic alkyne-ketone as a lead fragment that can irreversibly inhibit ERAAP activity. We anticipate numerous applications of Ep given its unique ability to image ERAAP within cells.

Histogen Layers Contributing to Adventitious Bud Formation Are Determined by their Cell Division Activities.

Saintpaulia ionantha is propagated by adventitious buds in horticulture, and periclinal chimeral cultivars are usually difficult to propagate. However, some periclinal chimeral cultivars can be propagated with adventitious buds, and the mechanism of which has been unknown. Striped flower cultivars "Kaname," "Concord," and "Monique" were used to investigate what causes flower color separation in adventitious shoot-derived plants by tissue culture. These cultivars were revealed to have mutated flavonoid 3', 5' hydroxylase (SiF3'5'H), WDR1 (SiWDR1), or flavonoid 3 hydroxylase (SiF3H), respectively, in their L1 layer. From our previous study using "Kaname," all flowers from adventitious shoots were colored pink, which was the epidermal color of mother plants' flowers. We used "Concrd" and "Monique" from which we obtained not only monochromatic-colored plants the same as the epidermal color of mother plants, but also plants with a monochromatic colored plants, same as the subepidermal color, and a striped flower color the same as mother plants. Histological observations revealed that epidermal cells divided actively at 14 d after culture and they were involved in the formation of adventitious shoots in the cultured leaf segments of "Kaname." On the other hand, in "Concord" and "Monique," the number of divided cells in the subepidermis was rather higher than that of epidermal cells, and subepidermal cells were sometimes involved in shoot formation. In addition, the plant and leaf size of L1-derived plants from "Concord" and "Monique" were non-vigorous and smaller than those derived from the subepidermal layer. In conclusion, periclinal chimeral cultivars of Saintpaulia can be divided into two types. One type has a high cell division activity in the L1 layer, from which only single flower-colored plants derived from L1 can be obtained as adventitious shoots. Another type has a low cell division activity in the L1 layer, from which striped flower-colored plants the same as mother plants derived from several layers including L1 can be obtained as adventitious shoots. In the periclinal chimeral cultivar capable of propagation with adventitious shoots, the possibility was shown that cells in the L2 layer could form shoots by involving cells of the L1 layer with a low division activity.

Assembly of the draft genome of buckwheat and its applications in identifying agronomically useful genes.

Buckwheat (Fagopyrum esculentum Moench; 2n = 2x = 16) is a nutritionally dense annual crop widely grown in temperate zones. To accelerate molecular breeding programmes of this important crop, we generated a draft assembly of the buckwheat genome using short reads obtained by next-generation sequencing (NGS), and constructed the Buckwheat Genome DataBase. After assembling short reads, we determined 387,594 scaffolds as the draft genome sequence (FES_r1.0). The total length of FES_r1.0 was 1,177,687,305 bp, and the N50 of the scaffolds was 25,109 bp. Gene prediction analysis revealed 286,768 coding sequences (CDSs; FES_r1.0_cds) including those related to transposable elements. The total length of FES_r1.0_cds was 212,917,911 bp, and the N50 was 1,101 bp. Of these, the functions of 35,816 CDSs excluding those for transposable elements were annotated by BLAST analysis. To demonstrate the utility of the database, we conducted several test analyses using BLAST and keyword searches. Furthermore, we used the draft genome as a reference sequence for NGS-based markers, and successfully identified novel candidate genes controlling heteromorphic self-incompatibility of buckwheat. The database and draft genome sequence provide a valuable resource that can be used in efforts to develop buckwheat cultivars with superior agronomic traits.

Identifying subpopulations of thymic epithelial cells by flow cytometry using a new specific thymic epithelial marker, Ly110.

We generated monoclonal antibodies reacting to a mouse thymic epithelial cell specific membrane protein, Thymic Stromal Co-transporter (TSCOT)/Ly110. These antibodies showed specificity to the peptide sequences derived from TSCOT/Ly110 determined by specific peptide inhibition in flow cytometric analyses with cells expressing the protein on the surface. TSCOT/Ly110 expressing subpopulation can be identified among the CDR1(+) or 6C3(+) cortical epithelial cells. Furthermore, CDR1 positive cortical thymic epithelial cells can be separated into further distinguishable populations; CDR1(+)6C3(+)Ly110(+), CDR1(+)6C3(-)/(low)Ly110(+), CDR1(+)Ly110(-). Some of TSCOT/Ly110 expressing cells negative for both CDR1 and 6C3 markers were found at the earlier stages of development, while most of the cells are positive for both at 1-week-old stage. After then, downregulation in 6C3 and/or CDR1 expression was noticed until 16 weeks of age. These results suggest that TSCOT/Ly110 is a new marker for the subpopulation of CDR1(+) or 6C3(+) epithelial cells in the neonatal and adult thymus and is useful for the studies on the epithelial cell differentiation process.

Impact of regulated secretion on antiparasitic CD8 T cell responses.

CD8 T cells play a key role in defense against the intracellular parasite Toxoplasma, but why certain CD8 responses are more potent than others is not well understood. Here, we describe a parasite antigen, ROP5, that elicits a CD8 T cell response in genetically susceptible mice. ROP5 is secreted via parasite organelles termed rhoptries that are injected directly into host cells during invasion, whereas the protective, dense-granule antigen GRA6 is constitutively secreted into the parasitophorous vacuole. Transgenic parasites in which the ROP5 antigenic epitope was targeted for secretion through dense granules led to enhanced CD8 T cell responses, whereas targeting the GRA6 epitope to rhoptries led to reduced CD8 responses. CD8 T cell responses to the dense-granule-targeted ROP5 epitope resulted in reduced parasite load in the brain. These data suggest that the mode of secretion affects the efficacy of parasite-specific CD8 T cell responses.

The quantitative assessment of MHC II on thymic epithelium: implications in cortical thymocyte development.

The dynamics of MHC II expression in various thymic stromal compartments was investigated. By including MHC II in flow cytometry in addition to the cortical CDR1, medullary UEA-1 and pan-epithelial G8.8 markers, thymic stromal compartments were subdivided into at least six different populations. The total level of surface and cytoplasmic MHC II from fresh cortical thymic epithelial cells (cTECs) of normal mouse was as high as MHC II levels in medullary thymic epithelial cells (mTECs). MHC II levels as well as the percentages and cycling status of thymic epithelial cell populations expressing MHC II were not static during post-natal development, suggesting quantitative flexibility in presenting signals to the developing thymocytes. Although there was no evidence found for regulation of surface MHC II levels by TCR or by IFN-gamma, the absence of class II transactivator reduced both the level of MHC II expression and the number of MHC II+ cells. Surprisingly, MHC II molecules were found to form distinct focal aggregates on the surface of cTEC but not mTEC using high-resolution analysis by confocal microscopy. Moreover, these aggregates were formed independent of TCR or TCR-bearing cells in the thymus. These aggregates could potentially generate a functional unit containing a much higher local MHC II concentration to yield a higher avidity interaction. We discuss possible mechanisms for positive selection by weak interactions in the presence of such preformed MHC II aggregate units in cTEC.

betaPix-b(L), a novel isoform of betaPix, is generated by alternative translation.

betaPix (Pak-interacting exchange factor) isoforms are recently identified guanine nucleotide exchange factors (GEFs) for Rho family GTPases, Rac/Cdc42, that are key players in the regulation of actin dynamics. Here we show that a novel 105-kDa betaPix isoform, betaPix-bL, is generated by alternative translation of betaPix-b mRNA. Translation of betaPix-bL starts at an atypical initiation site, GTG, that is located 57 nucleotides downstream from the newly identified 5' end of betaPix-b cDNA. The expression of two isoforms, betaPix-b and betaPix-bL, from betaPix-b mRNA is controlled by an internal ribosome entry site (IRES)-driven mechanism. Comparing to betaPix-b, betaPix-bL contains additional 105 amino acids composed of a calponin homology (CH) domain and a serine-rich sequence in the N-terminus. The expression of betaPix-bL in rat brain is developmentally regulated and high in the embryonic stages, suggesting that the function of betaPix-bL is more heavily required during the early stages of brain development.