Session: 321. Coagulation and Fibrinolysis: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Bleeding and Clotting, Bleeding disorders, Translational Research, Hemophilia, Assays, Genetic Disorders, Diseases, Immune mechanism, Immunology, Biological Processes, Molecular biology, Technology and Procedures, Gene editing, Multi-systemic interactions, Pathogenesis
Aims: We aim to establish an ITI testing platform using PWHA’s peripheral blood nuclear cells (PBMCs) to humanizing a novel immunodeficient mouse model that minimizes GvHD and enhances B cell survival. We also investigate PD-1/PD-L1 expression in Treg/plasma cells and TFH cell activation to elucidate the mechanisms of ITI therapy.
Methods:
Using CRISPR/Cas9 technology, we generated NSG-KDIA-HA mice lacking mouse major histocompatibility (H2) genes and the F8 gene, which were further transduced with AAV-hBLyS for long-term expression of human B lymphocyte stimulator (hBLyS). Genotyping and phenotyping were by DNA sequencing, flow cytometry and ELISA and the one-stage clotting assays. The hBLyS-NSG-KDIA-HA mice were engrafted with peripheral blood mononuclear cells (PBMCs) from 5 FVIII inhibitor-positive PWHA (FVIII-PWHA), and from hemophilic C57BL/6 mice challenged to carry high titer FVIII inhibitors (FVIII-B6-HA). Mice underwent either an ITI protocol (FVIII 200 IU/kg, BIW) mimicking patient conditions or received regular FVIII infusions (maintain groups, FVIII 50 IU/kg, BIW) as controls. Samples were collected during and at experimental endpoints for analysis.
Results: We generated the hBLyS-NSG-KDIA-HA mice and confirmed their loss of MHC molecules, their residual FVIII activity of 3.0 ± 1.7 %, and their expressing hBLyS in plasma (35~110 ng/mL). The historic FVIII inhibitor titers in the 5 FVIII-PWHA were 18.3~147.2 BU, and 215.1 ± 53.0 BU in FVIII-B6-HA mice (n=32). The FVIII-B6-HA’s PBMCs-engrafted BLyS-NSG-KDIA-HA mice (n=12) exhibited 3.2 % B cells (H2Kb+mCD19+) at endpoint. Their FVIII inhibitors were 2.7 ± 2.3 BU on week 8 and decreased to < 0.6 BU (0.5 ± 0.3 BU) in the ITI groups (n=4), in contrast to the 1.2 ± 0.7 BU in the maintain groups (n=4). In the FVIII-PWHA humanized hBLyS-NSG-KDIA-HA mice, we detected 28.0 ± 20.3% patients’ cells (hCD45+, n=20) at week 6 and gradually decreased to 9.0 ± 6.6% at endpoint (n=10). The FVIII inhibitor titers peaked to 1.4 ± 0.9 BU at week 8 (n=18). Of the 5 patients, successful ITI were confirmed in 4, showing titer < 0.6 BU at endpoint, whereas 1 failed in ITI, exhibiting 1.1 ± 0.3 BU/1.8 ± 0.2 BU at week 8/week 20 (endpoint). At endpoint analysis, the FVIII-PWHA-humanized mice carried different B cell subsets, as 56.7% of B cells were mature B cells (hCD27-hCD38+) in the spleen and 51.8% of B cells were plasma cells (hCD27+hCD38+) in the bone marrow.
To investigate the mechanism for ITI, we engrafted the hBLyS-NSG-KDIA-HA mice with FVIII-B6-HA splenocytes. The engraftment rate was 24.9 ± 13.0 % donor cells (H2Kb+) in circulation at week 2 which expanded to 66.6 ± 5.0 % at week 20 (n=7). In the ITI groups, the FVIII inhibitor titers were 16.0 ± 6.2 BU (week 8) decreased to 6.5 ± 2.0 BU (n=3) at endpoint, while in the maintain group, the titers were consistently > 20 BU (n=4). Germinal center-like structure was identified by H&E staining and by immunofluorescence staining of recipient’s spleen, revealing a “white pulp-like” structure with donor T cells recruited at the central arteriole surrounded by B cells. Analysis of the splenocytes showed upregulated PD-L1 in the Treg cells and upregulated PD-1 in the B cells (mCD19+mCD138+) in the ITI group as compared to respective counterparts in the maintain group (n=7, p<0.05).
Conclusion: We have developed a novel humanized ITI testing mouse model that significantly shortens testing duration for FVIII-PWHA. Our findings highlight the potential involvement of the PD-L1/PD-1 axis in ITI mechanisms.
Disclosures: Chou: Sanofi: Other: travel support.
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