No abstract

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.

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.

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.