# Basic and Translational Mechanisms of Alloimmunization to RBC Transfusion.  Project 1

> **NIH NIH P01** · UNIVERSITY OF VIRGINIA · 2024 · $401,492

## Abstract

Humoral immunization to alloantigens on red blood cells (RBC alloimmunization) remains a major complication,
and in some cases barrier to transfusion therapy for myriad diseases. However, the tendency to become
alloimmunized is not evenly distributed; rather, patients tend to become alloimmunized to multiple alloantigens
(responders) or none at all despite chronic transfusion (nonresponders). However, the factors that regulate
responder vs. nonresponder status remain poorly defined. Rates of RBC alloimmunization is increased in
patients with autoimmune pathologies, such as systemic lupus erythematosus (SLE). We have recently
discovered that mice with SLE-like biology (i.e. develop anti-nuclear antibodies (ANAs)) have an increased rate
of RBC alloimmunization. These mice develop ANAs due to a specific cluster of variant genes (NBA2) that
contains multiple orthologues to human genes associated with SLE. Importantly, the NBA2 mice do not display
organ pathology associated with SLE, leading to the hypothesis that ANA are an isolated component of SLE that
affects RBC alloimmunization. Moreover, in NBA2 mice, toll like receptor 7 (TLR7) and TLR9 are known to
regulate ANA formation through mutual antagonism (TLR7 promotes and TLR9 suppresses). These findings
lead to the central hypothesis of this project: Central Hypothesis: Anti-nuclear antibodies cause increased RBC
alloimmunization through activation of B cells as a result of dysregulated TLR7 and TLR9 signaling. This
hypothesis is tested using a novel B cell receptor transgenic mouse that recognizes an authentic human blood
group antigen (Kpb epitope) on the human KEL glycoprotein. The anti-Kpb mouse is combined with NBA2 mice
that are then transfused with transgenic RBCs expressing the KEL-K2 variant of human KEL, which contains the
Kpb antigen. This approach allows an in vivo mechanistic study of early activation and differentiation of naïve B
cells specific for an RBC antigen on transfused RBCs. Specific aims 1 and 2 test the role of ANAs on RBC
alloimmunization, including the manipulation of TLR7 and TLR9 signaling to rigorously test the central
hypothesis. Specific aim 3 uses the human cohort of SCD patients studied in project 4. Patients with SCD
have high rates of ANAs. The presence or absence of ANAs in patient plasma will be correlated with patterns
of alloimmunization. Patient leukocytes will be stimulated in vitro, in the presence or absence of ANA containing
plasma and/or TLR7 and TLR9 agonism or antagonism, using an assay that measures B cell activation and
differentiation in response to CD4+ follicular T cell help. Synergy between projects comes from crosstalk between
purinergic signaling (project 2) and TLR7 and TLR9, that reticulocytes (project 3) provide agonists for TLR7 and
TLR9, and the prediction that genetic variations known to predispose humans to ANA formation will emerge as
associations in the prospective GWAS study on RBC alloimmunization in patients with SCD (project 4). T...

## Key facts

- **NIH application ID:** 11070284
- **Project number:** 5P01HL169552-02
- **Recipient organization:** UNIVERSITY OF VIRGINIA
- **Principal Investigator:** JAMES C. ZIMRING
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $401,492
- **Award type:** 5
- **Project period:** 2023-09-10 → 2028-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11070284

## Citation

> US National Institutes of Health, RePORTER application 11070284, Basic and Translational Mechanisms of Alloimmunization to RBC Transfusion.  Project 1 (5P01HL169552-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/11070284. Licensed CC0.

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