# In Vivo Mechanism of Immune Response to Factor VIII: Project 2

> **NIH NIH U54** · CHILDREN'S HOSP OF PHILADELPHIA · 2022 · $274,989

## Abstract

Project 2: Abstract
Antibody formation against coagulation factor VIII (FVIII) is a major and serious complication in current protein
replacement therapy for the X-linked bleeding disorder hemophilia A. Approximately 20-30% of patients
develop neutralizing antibodies (“inhibitors”) that inhibit coagulation activity, thereby complicating treatment,
increasing risks of morbidity and mortality, and raising treatment costs. FVIII can elicit very high-titer antibody
formation despite being given intravenously at low antigen doses. FVIII-specific B cell responses are
dependent on CD4+ T helper cells and require co-stimulation. However, surprisingly little is known about the in
vivo mechanism of FVIII antigen presentation to T cells or B cell activation. In this proposal, we seek to answer
which antigen presenting cells (APCs) are required for MHC II presentation to CD4+ T cells (leading to FVIII-
specific B cell activation), how these APCs interact to prime FVIII-specific CD4+ T cells, which subsets of CD4+
T cells are induced to promote B cell activation (including T follicular helper, Tfh, cells), and how innate
immune signaling may alter these events. Working with the other projects, we will use these newly established
experimental models to determine the impact of signals derived from the microbiome and the effect of altered
molecular structure of FVIII. We propose three specific aims. Aim 1 is to define the mechanism of in vivo MHC
II presentation of FVIII antigen to CD4+ T cells. We will utilize fluorescently labeled FVIII to study in vivo
antigen uptake, and establish an in vivo MHC II presentation assay based on epitope-tagged FVIII. We will use
a combination of depletion of specific APCs, confocal microscopy, and intravital 2-photon microscopy to
determine the requirements and roles of the critical APCs, their location in lymphoid organs, and their
interactions. We will visualize dendritic cell and T cell migration and clustering of specific T cells around APCs,
and interrogate innate and cytokine responses that increase B cell activation. Aim 2 is to define the mechanism
of in vivo activation of FVIII-specific CD4+ T cells and B cells. Using a combination of depletion
experiments/knock-out strains, adoptive T cell transfer studies, flow cytometry, and cytokine assays, we will
determine the requirements for T and B cell activation, and the subsets of CD4+ T cells that activate B cells
and their requirements for co-stimulation (with particular emphasis on the role of Tfh cells in primary and
memory B cell activation). Aim 3 is to determine the effects of signals derived from innate immune
sensing and the microbiome on B and T cell activation against FVIII. Here, we will study the effects of TLR9
agonists and anti-inflammatory drugs on dendritic cell subsets as an example of a how inflammatory signals
alter inhibitor formation. In collaboration with Project 3, we will determine how an immune stimulatory or
suppressive microbiome alters FVIII ant...

## Key facts

- **NIH application ID:** 10406334
- **Project number:** 5U54HL142012-05
- **Recipient organization:** CHILDREN'S HOSP OF PHILADELPHIA
- **Principal Investigator:** Roland W. Herzog
- **Activity code:** U54 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $274,989
- **Award type:** 5
- **Project period:** 2018-05-01 → 2024-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10406334

## Citation

> US National Institutes of Health, RePORTER application 10406334, In Vivo Mechanism of Immune Response to Factor VIII: Project 2 (5U54HL142012-05). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10406334. Licensed CC0.

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