In transplantation, the indirect pathway of allorecognition — recipient antigen-presenting cells processing donor alloantigen into self-MHC-restricted peptides for CD4 T cells — persists long after direct-pathway responses wane and is thought to drive chronic rejection. Murine work (Ali et al., Lancet 2015 conference abstract; Cell Reports 2016) showed indirect CD4 responses are heterogeneous: responses against donor MHC class I allopeptide are long-lasting, whereas class II responses decay, and class-I-allopeptide-specific regulatory T cells (Tregs) transferred weeks after transplantation prevented alloantibody and allograft vasculopathy while class-II-specific Tregs did not. This raised a two-part clinical question that remains unresolved: which indirect-pathway alloantigen specificities actually dominate clinical rejection in human transplant recipients, and whether antigen-specific Treg therapy can be targeted, dosed, and scheduled safely against those specificities in humans. Recent progress — human HLA-DQ-derived epitope mapping in donor-specific-antibody-positive recipients (Immunity 2025) and phase 1 safety trials of polyclonal, donor-antigen-reactive, and CAR-engineered Tregs (ONE Study and successors) — has advanced both components but no human study has established an indirect-pathway dominance map or delivered epitope-specific Tregs with validated dose and schedule.

Background

A transplanted organ is recognized by recipient T cells through distinct allorecognition pathways. In the direct pathway, T cells recognize intact donor MHC (major histocompatibility complex) molecules on donor antigen-presenting cells (APCs); this response is strong but short-lived because donor APCs are rapidly eliminated. In the indirect pathway, recipient APCs phagocytose donor material and present processed donor-derived peptides (allopeptides) on self MHC class II to CD4 T cells; this pathway persists for the life of the graft and is considered a principal driver of chronic rejection — the slow destruction of grafts by immune injury, evident as allograft vasculopathy (progressive intimal thickening of graft vessels) in hearts and as interstitial fibrosis and inflammation in kidneys — largely by providing help to B cells producing donor-specific alloantibody (DSA). Regulatory T cells (Tregs) are CD4+CD25+FOXP3+ suppressor T cells that can be expanded ex vivo; 'antigen-specific' Tregs are those whose suppressive activity is directed at a defined antigen, e.g. by sorting with peptide-MHC multimers (fluorescent reagents made of peptide bound to MHC that stain only T cells whose receptor matches that specificity) or by engineering a T-cell receptor (TCR) or chimeric antigen receptor (CAR) of defined specificity.

Indirect CD4 responses were first dissected in murine cardiac-transplantation studies. A Lancet 2015 conference abstract and its full-length paper (Ali et al., Cell Reports 2016, cardiac transplantation from the bm12.Kd.IE donor strain, which carries defined MHC class I and class II mismatches, into C57BL/6 recipients) tracked indirect CD4 responses against donor MHC class I, class II, and H-Y (male-specific) minor histocompatibility antigens using adoptively transferred monoclonal TCR-transgenic reporter T cells (TCR75 for class I allopeptide, Tea and Mar for other specificities). Indirect responses proved heterogeneous: the class I allopeptide-specific response persisted and kept dividing (continual processing of graft parenchymal cells), while the class II allopeptide response decayed within about two weeks, reflecting rapid destruction of donor hematopoietic APCs. Class I allopeptide-specific Tregs transferred at transplantation or three weeks later abrogated germinal-center (the lymphoid structures where B cells mature antibody responses) alloantibody responses and allograft vasculopathy, whereas class II-specific Tregs failed at the late time point — suggesting that targeting dominant, long-lasting indirect specificities, including with late dosing, might prevent chronic rejection. The full paper added a caution: the persistent class-I-specific population showed suboptimal recall responses and could not provide co-stimulation-independent B-cell help, so murine immunodominance is itself not fully settled. Standard reviews (Carnel et al., Transplantation 2022) frame indirect allorecognition as central to chronic rejection but note the clinical parameters remain poorly defined.

Human data relevant to the specificity component: HLA-DQ DSA carries the highest rejection risk; the Immunity 2025 study mapped indirect CD4 epitopes in HLA-DQ DSA-positive kidney and heart recipients to two polymorphic hotspots of donor HLA-DQ, and a parallel murine epitope (H2-Kd residues 287-301) proved sufficient to drive DSA in mice; the computational PIRCHE framework (Predicted Indirectly ReCognizable HLA Epitopes) predicts such epitopes from donor-recipient HLA mismatches, with experimental validation emerging. Human data relevant to the therapy component: the ONE Study (Lancet 2020) established that regulatory cell products, including polyclonal Tregs, can be given safely to living-donor kidney recipients; a ONE Study pilot (Guinan et al., Am J Transplant 2023) administered donor-antigen-reactive Tregs to three recipients days after transplantation with encouraging safety; a liver-transplantation pilot (Todo et al., Hepatology 2016) used donor-stimulated Treg-enriched products; and HLA-A*02-directed CAR-Tregs entered first-in-human testing (STEADFAST study design, 2022), though these recognize intact donor HLA class I rather than indirect allopeptides. None of these delivers Tregs specific for defined indirect-pathway allopeptides, and none tests late post-transplant dosing against chronic rejection endpoints.

Problem Statement

Determine, in human transplant recipients, (1) which indirect-pathway alloantigen specificities — donor-derived peptides presented on recipient MHC class II — dominate clinically evident rejection, particularly chronic rejection, across the major rejection phenotypes (acute and chronic, cellular and antibody-mediated) and at least the principal solid-organ settings for which human data exist (e.g. kidney and heart), where dominance denotes the specificities whose indirect CD4 responses are most frequent and persistent and most strongly associated with donor-specific alloantibody and chronic rejection in human recipients; and (2) whether antigen-specific regulatory T cell therapy (Tregs are suppressive CD4 T cells; antigen-specific Tregs are directed at a defined antigen) directed against those dominant specificities can be delivered to humans with defined antigen targeting, dosing, and scheduling, including late post-transplant administration aimed at preventing chronic rejection, without unacceptable safety consequences — resolved, in the absence of any human epitope-specific Treg delivery to date, by an evidence-grounded regimen design that specifies antigen targeting, dose, and schedule with a supporting safety rationale, rather than by new clinical-trial data. The two components form one experimental-medicine objective: the specificity map supplies the therapeutic target, and the therapy component tests whether targeting it is safe and pharmacodynamically effective (suppression of the targeted indirect alloresponse).

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