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2572 Inhibitor-Positive Hemophilia a Patients-Derived Humanized Mouse Models Revealed the Connection of PD-1/PD-L1 Interactions to Successful Immune Tolerance Induction (ITI) Therapy

Program: Oral and Poster Abstracts
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
Sunday, December 8, 2024, 6:00 PM-8:00 PM

Li-Fu Chen1*, Sheng-Chieh Chou2*, Yen-Ting Chang1*, I-Shing Yu3*, Yu-Chun Liao4*, Chien-Hung Liu5*, Shih-Fan Wu3*, Te-Hsien Liu3*, Kuei-Liang Chen3*, Cheng-Pu Sun6*, Ying-Hui Su, M. Sc.7*, Chin-Yu Cheng5*, Bin-Bin Lin5*, Cheng-An Hsu8*, Mi-Hua Tao9*, Wen-Chien Chou, MD, PhD10 and Shu-Wha Lin11*

1National Taiwan University, Taipei, TWN
2Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
3National Taiwan University College of Medicine, Taipei, Taiwan
4Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine,, National Taiwan University, Taipei City, Taiwan
5National Taiwan University Hospital, Taipei, Taiwan
6Academia Sinica, Taipei, Taiwan
7Clinical Laboratory Technician, Childhood Cancer Foundation of R.O.C., Taipei City, Taiwan
8National Taiwan University Hospital, Taipei, TWN
9Academia Sinica, Taipei, TWN
10Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan
11Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, Department of Clinical Laboratory Sciences and Medical Biotechnology, Taipei, Taiwan

Background: Patients with hemophilia A (PWHA) are at risk of developing FVIII inhibitors. To date, immune tolerance induction (ITI) therapy is still the only way to induce tolerance with FVIII. Developing mouse models for ITI testing offers a promising approach to benefit PWHA. However, humanizing immunodeficient mice with patients' immune cells poses challenges such as graft-versus-host disease (GvHD) and poor survival of human B cells, limiting their utility for ITI studies. Furthermore, while studies suggest associations between FVIII immune responses and PD-L1/PD-1 expression in Treg/plasma cells and TFH cell activation, conclusive mechanisms underlying successful ITI induction remain elusive.

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.

*signifies non-member of ASH