Search

The only current curative treatment for chronic lymphocytic leukemia (CLL) is allogenic hematopoietic stem cell transplantation. Chimeric antigen receptor treatment targeting CD19 for CLL achieved some complete responses, suggesting the need for alternative or combinational therapies to achieve a more robust response. In this work, we evaluated CAR-T cells specific for Siglec-6, an antigen expressed in CLL, as a novel CAR-T cell treatment for CLL. We found that detection of SIGLEC6 mRNA and Siglec-6 protein is highly restricted to placenta and immune cells in other tissues and it is not expressed in hematopoietic stem cells. We generated CAR-T cells specific for Siglec-6 based on the sequence of the fully human anti-Siglec-6 antibody (JML1), which was identified in a CLL patient that was cured after allo-hematopoietic stem cell transplantation (alloHSCT), and observed that it specifically targeted CLL cells in vitro and in a xenograft mouse model. Interestingly, a short hinge region increased the activity of CAR-T cells to target cells expressing higher Siglec-6 levels but similarly targeted CLL cells expressing lower Siglec-6 levels in vitro and in vivo. Our results identify a novel CAR-T cell therapy for CLL and establish Siglec-6 as a possible target for immunotherapy.

In Drosophila ovaries, germline stem cells (GSCs) divide asymmetrically in the germaria to produce daughter GSCs and cystoblasts. Single cystoblasts differentiate to form germline cysts with 16 germline cells, all of which are connected by the fusome, a vesiculated structure critical for oocyte specification. We here show that histone H3K9 methyltransferase dg9a is associated with spectrosome/fusome formation in the germarium; dG9a(13414) mutant ovaries have disorganized spectrosome/fusome in about half the germaria, with reduced levels of hu-li tai shao and alpha-SPECTRIN proteins. We found that the amount of germline cells within cysts was reduced and that oocyte determination often failed in egg chambers of the dG9a(13414) mutant ovaries. These results suggest that a mutation in dG9a gene gives rise to anomalous spectrosome/fusome structures, which in turn lead to faulty germ-cell development in Drosophila ovaries.

Up to a third of hemophilia A (HA) patients receiving therapeutic FVIII develop neutralizing antibodies termed "inhibitors". Once inhibitors develop, clinical management of HA patients becomes extremely difficult. Thus, a rational solution would be to establish FVIII-specific immune tolerance to FVIII in high risk patients. To address this issue in a mouse model of human HA, we elected to use an antigen-specific regulatory T cell (Treg) approach. Analogous to the chimeric antigen receptor (CAR) strategy successfully used in cancer immunotherapy, we have created a chimeric receptor comprising a protein antigen or its domain, linked with the transmembrane and signal transduction domains, CD28-CD3ζ. We termed this receptor "BAR" for B-cell-targeting antibody receptor. Human Tregs (CD4+CD25hiCD127low) were retrovirally transduced with a BAR containing FVIII C2 domain (C2-BAR) or FVIII A2 domain (A2-BAR) and expanded successfully in vitro. These cells stained positively with anti-C2 and anti-A2 monoclonal antibodies, respectively, and maintained Treg phenotypic markers in terms of co-expression of Foxp3 and Helios. Control human Tregs were transduced with a BAR containing chicken ovalbumin (OVA-BAR). To test the hypothesis that BAR-transduced Tregs could directly and effectively suppress the activity of specific B cells, a xenogeneic model was employed. On day 0, FVIII-/- HA mice were injected intravenously with 106 transduced human Tregs. The mice were then immunized subcutaneously on day 1 with FVIII in incomplete Freund's adjuvant, and anti-FVIII antibody development was followed. By two weeks after immunization, anti-FVIII antibodies could be detected in the control mice (n = 4). However, in the experimental group (n = 5) that received a mixture of equal number of C2-and A2-BAR Tregs, anti-FVIII antibody development was reproducibly completely blocked for at least 8 weeks. To examine the possible mechanism of BAR Treg suppression, purified B cells and T cells from "tolerized" (A2+C2-BAR) or "control" (OVA-BAR) recipients were mixed and tested for recall responses to FVIII in vitro. The results suggested that the FVIII-specific B cells were directly tolerized while the T-cell response remained intact. Taken together, we report here a successful approach utilizing FVIII-specific BAR-Tregs to directly target FVIII-specific B cells, an approach which could be adapted to address other adverse immune response as well. (Supported in part by a NIH grant HL127495) Disclosures No relevant conflicts of interest to declare.

Clinical application of antigen-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 phosphorothioated random oligonucleotides (ODNps25). The addition of ODNps25 successfully resulted in the stabilization of engineered antigen-specific Tregs, however, the mechanism is not fully characterized. We first identified SAMHD1 (sterile alpha motif and HD domain 1) as an ODNps25 binding protein using a UV-crosslinking pull-down strategy. SAMHD1 physically interacted with the 3′ untranslated region (UTR) 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. Since ODNps25 binds to the nuclease (HD) domain of SAMHD1, we then established that overexpression of a nuclease-deficient mutant (D137N) in Tregs significantly stabilized the expression level of Foxp3 protein. This mutant also enhanced stability of a luciferase reporter, followed by the 3′ UTR of Foxp3 mRNA. Additionally, we also found that phosphorylation of the threonine residue (Thr592), which is a regulatory site to control SAMHD1 activity, was clearly downregulated in Tregs. Taken together, we suggest that ODNs interact with the HD of SAMHD1 to interfere with its nuclease activity, thereby stabilizing 3′ UTR of FoxP3 mRNA in long-term culture.

Expanded antigen-specific engineered regulatory T cells (Tregs) have been proposed for potential clinical application for the treatment of undesirable immune responses, such as inhibitor responses in hemophilia A patients and autoimmune diseases. By providing an antigen-specific T-cell receptor (TCR) to polyclonal natural Tregs, we suggested that antigen-specific engineered Tregs would migrate specifically to particular target tissues and induce antigen-specific immune tolerance in the local milieu. Previously, we developed FVIII C2-specific Tregs using a long-term stabilization protocol in vitro and demonstrated that these stabilized engineered Tregs successfully modulated FVIII-specific T-cell- and B-cell immune responses. Herein, we examined the mechanism of suppression by antigen-specific engineered Tregs compared to polyclonal normal natural Tregs. Initially, we tested whether these FVIII-specific engineered Tregs were able to suppress neighboring activated T-cell effectors locally. We found that FVIII C2-specific Tregs strongly suppressed myelin basic protein (MBP)-specific T effectors by presentation of both specific antigens in same APC population. However, we also observed that C2-specific Tregs could suppress MBP-specific T effectors presented on different APCs. These results imply contactless suppressive function of C2-specific engineered Tregs. Using a modified trans-well suppression assay, in which physical distance and clear separation between Tregs and a set of T effectors was created, we found that C2-specific activated Tregs showed significant contactless suppression only when T effectors were also present. In addition, and confirming previous studies with polyclonal Tregs, suppression by FVIII-specific engineered Tregs could be overcome by increasing the dose of IL-2 in co-culture media. This suggests that Tregs act, in part, by usurping IL-2 needed by T effectors to proliferate. Surprisingly, neutralization of CTLA-4 did not interfere with FVIII C2-specific suppression of engineered Tregs in contrast to the reversal seen with anti-CD3e-driven non-specific immunosuppression. Our data strongly suggest that suppressive function of FVIII-specific engineered Tregs is not restricted to cell-to-cell contact. Rather cross-talk of engineered Tregs and T effectors potentially generate a contactless suppressive mechanism to suppress other FVIII-specific multiple effector cells in the local milieu for effective immune tolerance. Understanding the mechanism of contactless suppression mechanism should provide critical clues to develop more effective engineered Tregs as a therapeutic tool in hemophilia A. (Supported by NIH grants HL061883 and HL126727) Disclosures Kim: Henry Jackson Foundation: Other: patent filed. Zhang:Henry Jackson Foundation: Other: patent filed. Scott:Henry Jackson Foundation: Other: patent filed.

Hemophilia A is an X-linked disorder, in which mutations in the coagulation Factor VIII (FVIII) gene lead to a loss of FVIII function and serious bleeding episodes. These episodes can be treated with recombinant FVIII protein replacement. Unfortunately, ~25% of hemophilia A patients produce inhibitory anti-FVIII antibodies because of lack of tolerance. Thus, it is necessary to develop effective tolerogenic therapies to prevent, as well as reverse, inhibitor formation. Previously, we 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 bind MHC tetramers, proliferate in response to a specific FVIII epitope, and suppress effector responses to FVIII. In this study, we engineered a FVIII-specific chimeric antigen receptor (ANS8CAR) using a FVIII-specific scFv derived from a synthetic phage display library. Following initial experiments in naïve CD4 T cells, this CAR was introduced into human Tregs. Western blot and specific staining with FVIII verified CAR expression. Transduced ANS8CAR Tregs proliferated in response to FVIII and were able to suppress the proliferation of FVIII-specific T effector cells in vitro. Additionally, the proliferation of T effector cells with different FVIII domain specificity was suppressed as well when ANS8CAR-transduced Tregs were activated with FVIII. Thus, engineered cells are able to promote bystander suppression. Cytokine expression of ANS8CAR-transduced Tregs was comparable to expression of untransduced and TCR-transduced Tregs indicating that the regulatory phenotype of Tregs was not negatively influenced by ANS8CAR expression. In conclusion, CAR-transduced Tregs seem to be a promising alternative to TCR-transduced Tregs for a future tolerogenic treatment of hemophilia A patients with inhibitory FVIII-specific antibodies. Supported by NIH grants HL061883 and HL126727 (DWS), the Society of Thrombosis and Hemostasis Research, and the Günter Landbeck Excellence Award (AS). Disclosures Kim: Henry Jackson Foundation: Other: patent filed. Scott:Henry Jackson Foundation: Other: patent filed.

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.

ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), also known as PC1 (plasma cell alloantigen 1), is a homodimeric type II transmembrane glycoprotein that mediates nucleotide recycling by breaking down ATP to AMP. Previous studies of mouse B lineage cells showed that levels of PC1 expression could distinguish two distinct subsets of peritoneal B -1a cells. In addition, PC1 expression increases progressively on germinal center B cells and splenic plasma cells with highest levels on bone marrow plasma cells. To determine if PC1 might also help define human B1-like cells, we have used a mouse mAb to human PC1 to characterize its expression on human cells. The frequency of PC1-positive cells from cord blood (mean 6%, n=10) was higher than that from adult peripheral blood (1.34%, n=15). These PC1-positive cells are mostly derived from CD27- naïve B cells expressing CD38, IgM and IgD, and were detected in plasmablast/plasma cells in peripheral blood. In contrast to B-1a cells in mice, human PC1-positive cells did not secrete natural Abs or produce IL10. Expression of PC1 was greatly increased on purified peripheral blood B cells stimulated with anti-CD40, IL4 and IL21. These activated PC1-positive cells expressed low levels of CD5, HLA-DR, surface IgM, and high levels of B cell activation markers and CD138. Our results indicated that PC1-positive B cell populations in humans are different from either CD20+CD27+CD43+ B1 cells in human, or CD5+ B-1a cells in mice.

BCOR has been discovered as a BCL-6 interacting co-repressor, but little is known about its biological activities in normal B cell development and function. Previously, we found that the interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein, ICSBP, directly targets a large number of genes in germinal center (GC) B cells including BCL6. In this study, we screened potential binding partners of IRF8 using a retrovirus-based protein complementation assay in a mouse pre-B cell line. We found that IRF8 interacts directly with BCOR and the alpha helical region of IRF8 is required for this interaction. Using a 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 comprised of BCOR-IRF8 modulates BCL6 associated transcriptional regulation of B cell function. Additional studies to evaluate the possible role of BCOR-IRF8 complex are in progress.

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.