Table 3.

Challenges impeding progress toward the development of effective T-cell biomarkers in T1D

Challenges	Potential solutions and technological advances needed
Biological
High repertoire diversity and low precursor frequency of autoreactive T cells in peripheral blood	Develop or improve assays capable of measuring the complex mixture of autoreactive T cells
	Implement new technologies and approaches for identifying pathogenic signatures, including high-dimensional flow cytometry, mass cytometry, and barcoded antibodies or pMHC multimers for use in scRNA-Seq approaches
	Develop sensitive molecular biomarkers capable of detecting signatures of autoreactive T cells, including TCR immunosequencing
Large numbers of genetic risk variants impacting cellular function	Create isogenic cellular systems to identify causative SNPs and elucidate their impact on T-cell function
	Employ well-characterized biobanks with genotype-selectable donor samples
High degree of heterogeneity in T-cell phenotypes among subjects with T1D	Conduct functional testing on subjects with defined phenotypic profiles
	Define and control for covariates leading to heterogeneity in T-cell responses
	Design and conduct interventional trials using targeted populations with mechanistic outcomes
Transient or variable autoreactivity over the natural history of the disease	Build robust longitudinal and interventional cohorts with sufficient clinical samples
Process
Low sample volumes in peripheral blood of pediatric samples	Work toward miniaturizing functional assays
	Develop surrogate markers of autoreactivity that do not require large sample volumes
Need for measures that correlate T-cell autoreactivity with endogenous β-cell mass and/or function	Develop assays capable of detecting signals from autoreactive T cells in circulation reflective of ongoing pathology within T1D islets
	Characterize the degree of overlap between tissues and peripheral blood signatures
Need to understand the pathogenic potential of T-cell subsets or reactivities	Create biomimetic devices to model the islet:immune microenvironment
	Employ new technologies to test the function of antigen-specific T cells in viable pancreatic tissue sections
Need for assay reproducibility and interoperability	Employ independent validation cores and sample resources capable of repeating assays to test reproducibility and robustness
	Incentivize replication testing
Paradigms
Focus on limited epitopes from known autoantigens	Consider nonnative peptides, hybrid peptides, posttranslationally modified peptides
	Implement novel high-throughput unbiased peptide screens
	Implement novel computational approaches to model peptides capable of activating T cells through the TCR:MHC complex
Consider alternate concepts to explain origins of autoreactivity	Improve understanding of endogenous stress response and host response to commensal bacteria and viral agents, for example
Focus on classical T1D pathogenesis	Broaden studies to include longitudinal studies of T cells in cancer subjects receiving immune checkpoint inhibitors
	Understand autoreactivity emanating from rare genetic variants with high penetrance of T1D
T-cell–centric approaches	Broaden studies to better understand T cell:B cell and T cell:APC interactions
	Understand exogenous signals that can break T-cell tolerance
Heavy focus on the pathogenic features of T-cell autoreactivity in subjects with known genetic risk	Better understand the principles related to the mechanisms by which the MHC class II haplotype of DR15-DQ6 influences the T-cell repertoire and leads to dominant protection from disease

APC, antigen-presenting cell; pMHC, peptide MHC; scRNA-Seq, single-cell RNA sequencing; SNPs, single nucleotide polymorphisms.