# Project 3

> **NIH NIH P01** · UNIVERSITY OF FLORIDA · 2023 · $300,058

## Abstract

The adaptive arm of the human immune system provides exquisite protection from pathogens, but the highly
variable receptors can turn on self-tissues when immunoregulatory checkpoints are broken due to genetic risk
and inflammatory events. A breakdown in tolerance impacting T cells is thought to be a critical immune
checkpoint during the natural history of type 1 diabetes (T1D). Of the approximately 150 independent loci
identified by genome-wide association studies as contributing to polygenic T1D risk, the major histocompatibility
complex (MHC) remains the largest factor conferring risk due to its influence over thymic selection of the T cell
receptor (TCR) repertoire and impact on peripheral T cell activation. Despite this critical link, little is known about
how high-risk alleles like HLA-DR4 impact TCR selection, activation thresholds, and distribution across tissues.
Our compelling preliminary data suggest that the risk-associated DR4/DQ8 haplotype, which has been linked
with insulin autoreactivity, is hyper-expressed on the surface of antigen presenting cells (APCs) and associated
with a unique TCR signature. Additional gene variants impacting T cell co-stimulation and differentiation are also
enriched in subjects with T1D, yet their mechanistic contributions toward T1D autoimmunity remain poorly
characterized. Project 3 proposes to address knowledge gaps governing these key aspects influencing the TCR
repertoire and activation requirements in health and disease. We hypothesize that MHC risk, and additional
non-MHC protein-coding risk variants, lead to aberrant T cell activation and differentiation thresholds and result
in the loss of T cell tolerance in T1D. Specifically, T1D-associated risk variants alter the functional avidity of the
MHC:peptide:TCR complex, and risk variants in the molecules SIRPG and CD226 alter T cell signaling, resulting
in activation of autoreactive effector T cells as well as defective immunoregulation by regulatory T cells (Treg).
These studies aim to investigate the mechanisms by which MHC class II (Aim 1) and non-MHC risk variants
(Aim 2) control autoreactive T cells through the analysis of pancreatic draining lymph nodes (pLN), spleen, and
peripheral blood from subjects with T1D and those at-risk for disease development (collected through Core A).
We will leverage polygenic risk scoring and broad immunophenotyping data generated by Core B to direct case
selection and analysis with state-of-the-art single-cell profiling and focused functional assays utilizing both
genotype-selected and gene-edited samples. To investigate individual clones and risk alleles, we will modify
primary human T cell specificity and function through lentiviral gene expression systems and CRISPR/Cas9
genome engineering for use in ex vivo pancreas slice culture and in vitro isogenic cellular modeling systems, in
collaboration with Projects 1 & 2, respectively. Data from this Project are expected to inform on the T cell
activation checkpoint...

## Key facts

- **NIH application ID:** 10549504
- **Project number:** 2P01AI042288-25
- **Recipient organization:** UNIVERSITY OF FLORIDA
- **Principal Investigator:** Todd Michael Brusko
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $300,058
- **Award type:** 2
- **Project period:** 1997-09-30 → 2028-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10549504

## Citation

> US National Institutes of Health, RePORTER application 10549504, Project 3 (2P01AI042288-25). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10549504. Licensed CC0.

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