Towards antigen-specific Tregs for type 1 diabetes: Construction and functional assessment of pancreatic endocrine marker, HPi2-based chimeric antigen receptor.

Type 1 Diabetes (T1D) is an autoimmune disease marked by direct elimination of insulin-producing ß cells by autoreactive T effectors. Recent T1D clinical trials utilizing autologous Tregs transfers to restore immune balance and improve disease has prompted us to design a novel Tregs-based antigen-specific T1D immunotherapy. We engineered a Chimeric Antigen Receptor (CAR) expressing a single-chain Fv recognizing the human pancreatic endocrine marker, HPi2. Human T cells, transduced with the resultant HPi2-CAR, proliferated and amplified Granzyme B accumulation when co-cultured with human, but not mouse ß cells. Furthermore, following exposure of HPi2-CAR transduced cells to islets, CD8+ lymphocytes demonstrated enhanced CD107a (LAMP-1) expression, while CD4+ cells produced increased levels of IL-2. HPi2-CAR Tregs failed to maintain expansion due to a persistent tonic signaling from the CAR engagement to unexpectantly HPi2 antigen present on Tregs. Overall, we show lack of functionality of HPi2-CAR and highlight the importance of careful selection of CAR recognition driver for the sustainable activity and expandability of engineered T cells.

SAMHD1 Posttranscriptionally Controls the Expression of Foxp3 and Helios in Human T Regulatory Cells.

Clinical application of Ag-specific T regulatory cells (Tregs) offers promise for the treatment of undesirable immune diseases. To achieve this goal, long-term expansion of Tregs is required to obtain sufficient numbers of cells. However, human Tregs are not stable ex vivo. Therefore, we previously developed an innovative Treg expansion protocol using 25mer-phosphorothioated random oligonucleotides (ODNps25). The addition of ODNps25 successfully resulted in the stabilization of engineered Ag-specific Tregs; however, the mechanism is not fully characterized. We first identified sterile α motif histidine-aspartate-domain containing protein 1 (SAMHD1) as an ODNps25-binding protein using a UV-cross-linking pull-down strategy. SAMHD1 physically interacted with the 3' untranslated region of Foxp3 mRNA and was translocated from nucleus to cytoplasm after ODNps25 treatment. Importantly, addition of ODNps25 enhanced the interaction of SAMHD1 and Foxp3 mRNA significantly, and this interaction was increased by TCR stimulation. Because ODNps25 binds to the nuclease (HD) domain of SAMHD1, we then established that overexpression of a dNTPase-deficient mutant (D137N) in Tregs significantly stabilized the expression level of the Foxp3 protein. Furthermore, we found that TCR stimulation upregulates phosphorylation of the threonine residue (Thr592), which is a regulatory site to control SAMHD1 activity, and phosphorylation of Thr592 is critical to control SAMHD1 activity to stabilize the expression of Foxp3 and Helios in Tregs. Taken together, we suggest that the interaction of ODNPs25 in HD or phosphorylation of Thr592 by TCR stimulation interferes with nuclease activity of SAMHD1, thereby stabilizing 3' untranslated region of Foxp3 and Helios mRNAs in long-term culture.

Targeting Antigen-Specific B Cells Using Antigen-Expressing Transduced Regulatory T Cells.

Controlling immune responses in autoimmunity and to biotherapeutics is an unmet need. In hemophilia, for example, up to one third of patients receiving therapeutic factor VIII (FVIII) infusions develop neutralizing Abs termed "inhibitors." To address this problem in a mouse model of hemophilia A, we used an Ag-specific regulatory T cell (Treg) approach in which we created a novel B cell-targeting chimeric receptor composed of an FVIII Ag domain linked with the CD28-CD3ζ transmembrane and signaling domains. We termed these "BAR" for B cell-targeting Ab receptors. CD4+CD25hiCD127low human Tregs were retrovirally transduced to express a BAR containing the immunodominant FVIII C2 or A2 domains (C2- and A2-BAR). Such BAR-Tregs specifically suppressed the recall Ab response of spleen cultures from FVIII-immunized mice in vitro and completely prevented anti-FVIII Ab development in response to FVIII immunization. Mechanistic studies with purified B cells and T cells from tolerized or control recipients demonstrated that the FVIII-specific B cells were directly suppressed or anergized, whereas the T cell response remained intact. Taken together, we report in this study a successful proof-of-principle strategy using Ag-expressing Tregs to directly target specific B cells, an approach which could be adapted to address other adverse immune responses as well.

Engineered MBP-specific human Tregs ameliorate MOG-induced EAE through IL-2-triggered inhibition of effector T cells.

Expanded polyclonal T regulatory cells (Tregs) offer great promise for the treatment of immune-mediated diseases. Inhibition by Tregs is under the control of the T-cell receptor (TCR). Therefore, we created Tregs with defined antigen specificity, using a recombinant T-cell receptor isolated from a myelin-basic protein specific T-cell clone of a multiple sclerosis (MS) patient (Ob2F3). We expressed this TCR using a retroviral expression vector in human Tregs from peripheral blood. We observed that transduced Tregs were activated in vitro in response to myelin basic protein (MBP) peptide on DR15 antigen-presenting cells (APC) and upregulated Treg markers, Foxp3, LAP and Helios. These engineered MBP-specific Tregs could suppress MBP-specific T effector cells, and were also able to suppress T cells with other specificities after Tregs had been activated through the TCR. Importantly, we showed that these engineered Tregs were able to function effectively in the presence of strong TLR-induced inflammatory signals, and that MBP-specific Tregs ameliorated EAE in myelin oligodendrocyte glycoprotein (MOG)-immunized DR15 transgenic mice. We further demonstrated in vitro that IL-2 produced by neighboring effector T cells activated MBP-specific Tregs, initiating contact-independent suppression to T effectors in local milieu. Mechanistic studies demonstrated that bystander suppression in vivo may involve transfer of soluble mediators, enhanced by cell contact between Tregs and effectors. Taken together, we show that engineered clonal MBP-specific Tregs are able to suppress autoimmune pathology in EAE. This approach may serve as a cellular therapy for MS patients with the common DR15 haplotype that is associated with disease susceptibility.

ATP-degrading ENPP1 is required for survival (or persistence) of long-lived plasma cells.

Survival of antibody-secreting plasma cells (PCs) is vital for sustained antibody production. However, it remains poorly understood how long-lived PCs (LLPCs) are generated and maintained. Here we report that ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is preferentially upregulated in bone marrow LLPCs compared with their splenic short-lived counterparts (SLPCs). We studied ENPP1-deficient mice (Enpp1 -/- ) to determine how the enzyme affects PC biology. Although Enpp1 -/- mice generated normal levels of germinal center B cells and plasmablasts in periphery, they produced significantly reduced numbers of LLPCs following immunization with T-dependent antigens or infection with plasmodium C. chabaudi. Bone marrow chimeric mice showed B cell intrinsic effect of ENPP1 selectively on generation of bone marrow as well as splenic LLPCs. Moreover, Enpp1 -/- PCs took up less glucose and had lower levels of glycolysis than those of wild-type controls. Thus, ENPP1 deficiency confers an energetic disadvantage to PCs for long-term survival and antibody production.

Human Tregs Made Antigen Specific by Gene Modification: The Power to Treat Autoimmunity and Antidrug Antibodies with Precision.

Human regulatory CD4+ T cells (Tregs) are potent immunosuppressive lymphocytes responsible for immune tolerance and homeostasis. Since the seminal reports identifying Tregs, vast research has been channeled into understanding their genesis, signature molecular markers, mechanisms of suppression, and role in disease. This research has opened the doors for Tregs as a potential therapeutic for diseases and disorders such as multiple sclerosis, type I diabetes, transplantation, and immune responses to protein therapeutics, like factor VIII. Seminal clinical trials have used polyclonal Tregs, but the frequency of antigen-specific Tregs among polyclonal populations is low, and polyclonal Tregs may risk non-specific immunosuppression. Antigen-specific Treg therapy, which uses genetically modified Tregs expressing receptors specific for target antigens, greatly mitigates this risk. Building on the principles of T-cell receptor cloning, chimeric antigen receptors (CARs), and a novel CAR derivative, called B-cell antibody receptors, our lab has developed different types of antigen-specific Tregs. This review discusses the current research and optimization of gene-modified antigen-specific human Tregs in our lab in several disease models. The preparations and considerations for clinical use of such Tregs also are discussed.

FVIII-specific human chimeric antigen receptor T-regulatory cells suppress T- and B-cell responses to FVIII.

Replacement therapy with factor VIII (FVIII) is used in patients with hemophilia A for treatment of bleeding episodes or for prophylaxis. A common and serious problem with this therapy is the patient's immune response to FVIII, because of a lack of tolerance, leading to the formation of inhibitory antibodies. Development of tolerogenic therapies, other than standard immune tolerance induction (ITI), is an unmet goal. We previously generated engineered antigen-specific regulatory T cells (Tregs), created by transduction of a recombinant T-cell receptor (TCR) isolated from a hemophilia A subject's T-cell clone. The resulting engineered T cells suppressed both T- and B-cell effector responses to FVIII. In this study, we have engineered an FVIII-specific chimeric antigen receptor (ANS8 CAR) using a FVIII-specific scFv derived from a synthetic phage display library. Transduced ANS8 CAR T cells specific for the A2 domain proliferated in response to FVIII and ANS8 CAR Tregs were able to suppress the proliferation of FVIII-specific T-effector cells with specificity for a different FVIII domain in vitro. These data suggest that engineered cells are able to promote bystander suppression. Importantly, ANS8 CAR-transduced Tregs also were able to suppress the recall antibody response of murine splenocytes from FVIII knockout mice to FVIII in vitro and in vivo. In conclusion, CAR-transduced Tregs are a promising approach for future tolerogenic treatment of hemophilia A patients with inhibitors.

Plasma cell alloantigen ENPP1 is expressed by a subset of human B cells with potential regulatory functions.

Plasma cell alloantigen 1 (PC1), also known as ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), is an enzyme involved primarily in hydrolysis of adenosine triphosphate at the cell surface. Although the expression pattern of PC1 is relatively broad, its expression in B cells is found at significant levels only in terminally differentiated germinal center B cells, plasma cells and a subset of B-1a cells in mice. Here we describe studies designed to determine whether expression of PC1 might define novel populations of human B cells with similarities to mouse B cells. We found that PC1 is expressed in small populations of human B lineage cells in peripheral blood, cord blood, tonsils, bone marrow and pediatric peritoneal fluid, with the highest levels in plasma cells. The characteristics of human PC1(+) B cells differ from mouse peritoneal B-1a subsets and from features of the human CD20(+)CD27(+)CD43(+)CD70(-) B-cell subset proposed to be human B-1 cells. Expression of PC1 was greatly increased in B cells stimulated with the combination of CD40 ligand, interleukin (IL)-4 and IL-21. In addition, PC1(+) B cells activated CD4(+) T regulatory cells. ENPP1 thus defines a subset of human B cells that differs significantly from mouse peritoneal B-1a and proposed human B-1 cells.

Tolerogenic nanoparticles to induce immunologic tolerance: Prevention and reversal of FVIII inhibitor formation.

The immune response of hemophilia A patients to administered FVIII is a major complication that obviates this very therapy. We have recently described the use of synthetic, biodegradable nanoparticles carrying rapamycin and FVIII peptide antigens, to induce antigen-specific tolerance. Herein we test the tolerogenicity of nanoparticles that contains full length FVIII protein in hemophilia A mice, focusing on anti-FVIII humoral immune response. As expected, recipients of tolerogenic nanoparticles remained unresponsive to FVIII despite multiple challenges for up to 6 months. Furthermore, therapeutic treatments in FVIII-immunized mice with pre-existing anti-FVIII antibodies resulted in diminished antibody titers, albeit efficacy required longer therapy with the tolerogenic nanoparticles. Interestingly, durable FVIII-specific tolerance was also achieved in animals co-administered with FVIII admixed with nanoparticles encapsulating rapamycin alone. These results suggest that nanoparticles carrying rapamycin and FVIII can be employed to induce specific tolerance to prevent and even reverse inhibitor formation.

Interferon regulatory factor 8 (IRF8) interacts with the B cell lymphoma 6 (BCL6) corepressor BCOR.

B cell lymphoma 6 (BCL6) corepressor (BCOR) was discovered as a BCL6-interacting corepressor, but little is known about its other biological activities in normal B cell development and function. Previously, we found that interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein, directly targets a large number of genes in germinal center B cells including BCL6. In this study, we screened potential binding partners of IRF8 using a retrovirus-based protein complementation assay screen in a mouse pre-B cell line. We found that IRF8 interacts directly with BCOR and that the α-helical region of IRF8 and the BCL6 binding domain of BCOR are required for this interaction. In addition, IRF8 protein interacts directly with BCL6. Using an siRNA-mediated IRF8 knockdown mouse B cell lymphoma cell line, we showed that IRF8 represses Bcor and enhances Bcl6 transcription. Taken together, these data suggest that a complex comprising BCOR-BCL6-IRF8 modulates BCL6-associated transcriptional regulation of germinal center B cell function.

The 3'-5' DNA exonuclease TREX1 directly interacts with poly(ADP-ribose) polymerase-1 (PARP1) during the DNA damage response.

The main function of the 3'-5' DNA exonuclease TREX1 is to digest cytosolic single-stranded DNA to prevent activation of cell-intrinsic responses to immunostimulatory DNA. TREX1 translocates to the nucleus following DNA damage with its nuclear activities being less well defined. Although mutations in human TREX1 have been linked to autoimmune/inflammatory diseases, the mechanisms contributing to the pathogenesis of these diseases remain incompletely understood. Here, using mass spectrometry and co-immunoprecipitation assays and in vivo overexpression models, we show that TREX1 interacts with poly(ADP-ribose) polymerase-1 (PARP1), a nuclear enzyme involved in the DNA damage response. Two zinc finger domains at the amino terminus of PARP1 were required for the interaction with TREX1 that occurs after nuclear translocation of TREX1 in response to DNA damage. Functional studies suggested that TREX1 may contribute to stabilization of PARP1 levels in the DNA damage response and its activity. These results provide new insights into the mechanisms of single-stranded DNA repair following DNA damage and alterations induced by gene mutations.

Oligodeoxynucleotides stabilize Helios-expressing Foxp3+ human T regulatory cells during in vitro expansion.

Foxp3(+) regulatory T cells (Tregs) maintain self-tolerance and adoptive therapy, and using Foxp3(+) Tregs has been proposed as treatment for autoimmune diseases. The clinical use of Tregs will require large numbers of cells and methods for in vitro expansion of Tregs are being developed. Foxp3(+) Tregs can be divided into 2 subpopulations based on expression of the transcription factor, Helios. Foxp3(+)Helios(+) Tregs (70%) are thymic-derived, whereas Foxp3(+)Helios(-) Tregs (30%) are induced in the periphery. Foxp3(+)Helios(+) Tregs differ from Foxp3(+)Helios(-) Tregs in terms of epigenetic changes at the Foxp3 locus, their capacity to produce effector cytokines, and their stability of Foxp3 expression on days to weeks of expansion in vitro. Addition of a 25 mer DNA oligonucleotide of random composition for a short period during the expansion of Foxp3(+) Tregs in vitro results in prolonged stabilization of the Foxp3(+)Helios(+) subpopulation and yields an optimal population for use in cellular biotherapy.

The structural complexity of the human BORIS gene in gametogenesis and cancer.

BACKGROUND: BORIS/CTCFL is a paralogue of CTCF, the major epigenetic regulator of vertebrate genomes. BORIS is normally expressed only in germ cells but is aberrantly activated in numerous cancers. While recent studies demonstrated that BORIS is a transcriptional activator of testis-specific genes, little is generally known about its biological and molecular functions. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that BORIS is expressed as 23 isoforms in germline and cancer cells. The isoforms are comprised of alternative N- and C-termini combined with varying numbers of zinc fingers (ZF) in the DNA binding domain. The patterns of BORIS isoform expression are distinct in germ and cancer cells. Isoform expression is activated by downregulation of CTCF, upregulated by reduction in CpG methylation caused by inactivation of DNMT1 or DNMT3b, and repressed by activation of p53. Studies of ectopically expressed isoforms showed that all are translated and localized to the nucleus. Using the testis-specific cerebroside sulfotransferase (CST) promoter and the IGF2/H19 imprinting control region (ICR), it was shown that binding of BORIS isoforms to DNA targets in vitro is methylation-sensitive and depends on the number and specific composition of ZF. The ability to bind target DNA and the presence of a specific long amino terminus (N258) in different isoforms are necessary and sufficient to activate CST transcription. Comparative sequence analyses revealed an evolutionary burst in mammals with strong conservation of BORIS isoproteins among primates. CONCLUSIONS: The extensive repertoire of spliced BORIS variants in humans that confer distinct DNA binding and transcriptional activation properties, and their differential patterns of expression among germ cells and neoplastic cells suggest that the gene is involved in a range of functionally important aspects of both normal gametogenesis and cancer development. In addition, a burst in isoform diversification may be evolutionarily tied to unique aspects of primate speciation.

Expression of a testis-specific form of Gal3st1 (CST), a gene essential for spermatogenesis, is regulated by the CTCF paralogous gene BORIS.

Previously, it was shown that the CTCF paralogous gene, BORIS (brother of the regulator of imprinted sites) is expressed in male germ cells, but its function in spermatogenesis has not been defined. To develop an understanding of the functional activities of BORIS, we generated BORIS knockout (KO) mice. Mice homozygous for the null allele had a defect in spermatogenesis that resulted in small testes associated with increased cell death. The defect was evident as early as postnatal day 21 and was manifested by delayed production of haploid cells. By gene expression profiling, we found that transcript levels for Gal3st1 (also known as cerebroside sulfotransferase [CST]), known to play a crucial role in meiosis, were dramatically reduced in BORIS KO testes. We found that CST is expressed in testis as a novel testis-specific isoform, CST form F(TS), that has a short exon 1f. We showed that BORIS bound to and activated the promoter of CST form F(TS). Mutation of the BORIS binding site in the promoter reduced the ability of BORIS to activate the promoter. These findings define transcriptional regulation of CST expression as a critical role for BORIS in spermatogenesis.

dSETDB1 and SU(VAR)3-9 sequentially function during germline-stem cell differentiation in Drosophila melanogaster.

Germline-stem cells (GSCs) produce gametes and are thus true "immortal stem cells". In Drosophila ovaries, GSCs divide asymmetrically to produce daughter GSCs and cystoblasts, and the latter differentiate into germline cysts. Here we show that the histone-lysine methyltransferase dSETDB1, located in pericentric heterochromatin, catalyzes H3-K9 trimethylation in GSCs and their immediate descendants. As germline cysts differentiate into egg chambers, the dSETDB1 function is gradually taken over by another H3-K9-specific methyltransferase, SU(VAR)3-9. Loss-of-function mutations in dsetdb1 or Su(var)3-9 abolish both H3K9me3 and heterochromatin protein-1 (HP1) signals from the anterior germarium and the developing egg chambers, respectively, and cause localization of H3K9me3 away from DNA-dense regions in most posterior germarium cells. These results indicate that dSETDB1 and SU(VAR)3-9 act together with distinct roles during oogenesis, with dsetdb1 being of particular importance due to its GSC-specific function and more severe mutant phenotype.

LPS-induced CD53 expression: a protection mechanism against oxidative and radiation stress.

The CD53 antigen is a member of the tetraspanin membrane protein family that is expressed in the lymphoid-myeloid lineage. Its biological role remains unknown. Using microarrays, we identified CD53 as one of the principal genes up-regulated by exposure of macrophages to LPS. Northern blot analysis confirmed the induction of CD53 in RAW264.7 macrophages treated with LPS or SNAP (a nitric oxide donor). Cells stably transfected with sense CD53 cDNA had increased levels of intracellular GSH and lower levels of peroxide, and were more resistant to H2O2 and to UVB irradiation. Cells harboring antisense CD53 had the opposite properties. We propose that the induction of CD53 is a major mechanism by which macrophages protect themselves against LPS-induced oxidative stress and UVB irradiation.