-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

720 Immune Mechanisms and Cytokine Profiles in Hemophilia a Patients Undergoing Low-Dose ITI and Emicizumab Prophylaxis: Implications for ITI Success and Failure

Program: Oral and Poster Abstracts
Type: Oral
Session: 322. Hemophilia A and B: Clinical and Epidemiological: Impact of Novel Therapies on Outcomes, Including Long-Term
Hematology Disease Topics & Pathways:
Research, Clinical Research, Immunology, Biological Processes
Monday, December 9, 2024: 11:45 AM

Jessica O. Frade-Guanaes, MSc1*, Carolina Costa-Lima, MD1,2*, Vanessa B. Faiotto, MSc1*, Ana P. Francisco1*, Nívia M. Foschi, MD1*, Andrea L. A. Sambo, RN1*, Mariana M. T. Hosokawa, RN1*, Valdirene Stahl, RN1*, Luiz G. R. Lima1*, Bianca Stefanello1*, Lucas W. Weiss Santos, MD1*, Samuel S. Medina, MD1*, Marina P. Colella, MD, PhD1*, Silmara A. De Lima Montalvão, PhD1*, Gabriela G. Yamaguti-Hayakawa1* and Margareth C. Ozelo, MD, PhD1,2

1Hemocentro UNICAMP, University of Campinas, Campinas, SP, Brazil
2Department of Internal Medicine, School of Medical Sciences, University of Campinas, UNICAMP, Campinas, SP, Brazil

Hemophilia A (HA) treatment has relied on factor VIII (FVIII) replacement therapy, using either plasma-derived or recombinant FVIII (rFVIII) products. However, the development of inhibitors poses a significant challenge to this approach. To counter this, immune tolerance induction (ITI) is employed to restore in ~60% the effectiveness of FVIII therapy. With the advent of emicizumab, the benefits of ITI are being re-evaluated, given its associated treatment burden and high cost. A deeper understanding of the immune mechanisms underlying persistent inhibitors could provide insights into the inhibitor’s persistence and the predictors of ITI success.

This longitudinal study aims to evaluate the cells and cytokines associated with immune response during low-dose ITI along with emicizumab prophylaxis.

The study included ten severe HA patients with high-responding inhibitors attempting ITI for the first time. Patients initially received 4-week emicizumab loading dose (3mg/kg), followed by low-dose ITI (rFVIII 50IU/kg, 3x/wk) combined with emi maintenance dose (1.5mg/kg/wk). After achieving tolerance, patients continued emi prophylaxis for an additional year, with a reduced FVIII dose (25 IU/kg 1x/wk). Peripheral blood mononuclear cells (PBMCs) were collected during the screening period, before ITI (baseline), during ITI, and in the final year of additional emi prophylaxis. The cryopreserved PBMCs were thawed and cultured in 96-well plates with RPMI-1640 medium for six days. Antigen-presenting cells were initially stimulated with IL4 (10ng/mL) and GM-CSF (20ng/mL), followed by IL2 (10IU/mL) to promote proliferation, and challenged with rFVIII (1IU/well) on day 2. On day 5, cells were further challenged with rFVIII, anti-CD3, and anti-CD28. After 18h, cytokine intracellular transport was halted using GolgiPlug and GolgiStop, maintaining this culture condition for 6h. The effector responses of CD4+T cells (Th1, Th2, and Th17), along with memory and regulatory T and B cells, were assessed using a CytoFlex flow cytometer. This included analysis of cytokine markers for CD4, CD19, and CD3. The cytokines and transcription factors evaluated were IL2, IL4, IL6, IL17A, IL21, IL10, TGFβ, IFNγ, T-bet, GATA-3, FoxP3, and Bcl6. Cells were labeled with CTLA-4, PD-1, CD40, and CD40L to determine the immune checkpoint response. To evaluate the frequency of memory and regulatory B cells, cells were labeled with CD38, CD27, IL10, PD-L2, IgG, IgM, and IgD.

Ten participants, including nine children with a median age of 2.3 years at the start of ITI (range 1-30 years), were enrolled between July 2021 and January 2023. Six patients (60%) met the criteria for complete ITI success, three (30%) failed ITI, and one remained on ongoing ITI with a low-titer inhibitor. During follow-up, immune response screening showed that patients who did not achieve complete ITI success had a higher frequency of IL21 (p=0.01) and IL4 (p=0.03) in CD4+T cells. This suggests a possible involvement of T follicular helper (Tfh) cells, as indicated by a non-significant increase in Bcl6 frequency. These findings correlated with inhibitor levels (r=0.6, p=0.002 for IL21 and r=0.6, p=0.04 for IL4). Although IL17A frequency did not show a statistically significant difference, it was also correlated with inhibitor levels (r=0.5, p=0.008). Additionally, Th1 cells showed increased activity in patients who failed ITI, with elevated levels of T-bet (p=0.03) and TNFα (p=0.03). The failure group also had a higher frequency of Th2 cells, which was associated with increased GATA-3 frequency (p=0.01). No differences were observed in the frequency of Tregs (CD4+CD25+FoxP3+) or CTLA-4. However, in patients who achieved tolerance, there was a significant increase in PD-1 frequency (p=0.03), supporting the role of Tregs. Inhibitory regulatory B cells did not show differences in this cohort, likely due to the limited number of B cells available for evaluation, requiring further data. Analysis of one patient with a high inhibitor titer strongly suggests that, despite the immune system's effort to produce a regulatory response by increasing Tregs over time, this response was insufficient, as the increase in Tregs did not effectively enhance IL10 production, maintaining a high frequency of IL21.

Our data suggest that IL21 is a key cytokine involved in the pathophysiology of inhibitor maintenance and may be associated with ITI failure.

Disclosures: Costa-Lima: Roche: Speakers Bureau. Faiotto: BioMarin: Speakers Bureau. Hosokawa: Roche: Speakers Bureau. Colella: Eurofarma: Honoraria; Novartis: Speakers Bureau. Montalvão: Roche: Speakers Bureau; Takeda: Speakers Bureau. Yamaguti-Hayakawa: BioMarin: Honoraria, Speakers Bureau. Ozelo: BioMarin: Honoraria, Research Funding, Speakers Bureau; Novo Nordisk: Honoraria, Other: grant review, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Other: grant review, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding, Speakers Bureau; Sanofi: Honoraria, Research Funding, Speakers Bureau; Takeda: Honoraria, Research Funding, Speakers Bureau; CSL Behring: Other: grant review, Speakers Bureau.

<< Previous Abstract | Next Abstract
*signifies non-member of ASH