Peanut Allergy Oral Immunotherapy Drives Single-Cell Multi-omic Changes in Peanut-reactive T cells Associated with Sustained Unresponsiveness
Oral immunotherapy (OIT) is the only FDA-approved treatment for peanut allergy (PA). Peanut-reactive (pr) CD4+ T cells are pivotal in PA pathogenesis as well as OIT-induced desensitization (DS). However, the underlying pr CD4+ T cell immune mechanisms leading to sustained unresponsiveness (SU) after OIT discontinuation remain largely unknown. We analyzed single-cell RNA and protein immunophenotypes and TCR repertoires of pr CD4+ T cells from a phase 2 peanut OIT trial cohort. We identify increased cytotoxicity-related phenotypes and Th1 CTL (cytotoxic T lymphocytes)-like cell clonal expansion during OIT in pr T effector memory cells, while Th2-related phenotypes and Th2a-like cell clonal expansion decreased. OIT participants who achieved SU had lower baseline Th2-related phenotypes, elevated cytotoxicity-related gene signatures in pr Teff cells post-OIT and higher CD39 expression in pr Tregs (T regulatory cells) after OIT withdrawal. These findings clarify OIT-induced CD4+ T cell tolerance mechanisms and can guide effective allergen-specific OIT strategies. Overall design: To comprehensively characterize the heterogeneous pr CD4+ T cell, including pr Teff CD4+ T cells (CD4+CD154+) and pr Treg CD4+ T cells (CD4+CD137+CD154-), and their longitudinal responses in OIT, we used peripheral blood mononuclear cells (PBMC) from 31 POISED (Our phase 2 randomized, controlled peanut OIT trial: Peanut Oral Immunotherapy: Safety, Efficacy, Discovery; NCT 02103270) participants: 23 active participants who underwent OIT and passed DBPCFC at week 104 (14 peanut-0 participants, of whom 7 passed (SU) and 7 failed (DS) at week 117, and 9 peanut-300 participants, of whom 5 passed and 4 failed DBPCFC at week 117); 8 placebo participants; and 5 age- and gender-matched non-peanut allergic controls. We stimulated PBMCs with peanut solution for 18 hours, and sorted pr CD4+ T cells (CD4+CD137+ and/or CD4+CD154+) and non-reactive (nr) CD4+ T cells (CD4+CD154-CD137-). Sorted cells were processed using the BD Rhapsody™ platform for single cell RNA-sequencing (scRNA-seq) with a focus on 432 immune-relevant target transcripts (399 commercially pre-defined target panel complemented with 33 targets of interest), and 42 immune-relevant Ab-seq markers for protein level detection as well as single-cell paired TCRaß sequencing.