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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.

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.