Shuntaro (2025): cGVHD analysis: scRNA and scTCR Sequencing Analysis

Introduction

This is a book that contains all code to reproduce figures related to scRNA/scTCR sequencing analysis of LD IL-2 therapy treated PBMCs, enriched for CD4Treg by flow cytometry cell sorting (7AAD⁻CD3⁺CD4⁺CD25⁺CD127low).

Dysfunctional PD-1 Positive Regulatory T cells Impair Treatment Response after Low-Dose IL-2 Therapy

Shuntaro Ikegawa, Takeru Asano, Bohoon Shim, Jennifer S. Whangbo, Xia Bu, Haesook T. Kim, Shuqiang Li, Kenneth J. Livak, Mehdi Borji, Roger Belizaire, Roman M. Shapiro, Mahasweta Gooptu1,2, Rizwan Romee, Sarah Nikiforow, Vincent T. Ho, Corey Cutler, Joseph H. Antin, Robert J. Soiffer, John Koreth, Gordon J. Freeman, Jeremy M. Simon, Catherine J. Wu, Jerome Ritz

Abstract

CD4 regulatory T cells (Tregs) are indispensable for maintaining immune tolerance, and disruption of Treg homeostasis contributes to various autoimmune diseases. Low-dose interleukin-2 (LD IL-2) therapy preferentially expands Tregs in all patients with chronic graft-versus-host disease (cGVHD); however, clinical response rates remain suboptimal. Here, we identified a dysfunctional subset of PD-1+Tregs that was selectively expanded in non-responders with cGVHD who received LD IL-2. Single-cell transcriptomic, proteomic, and functional profiling revealed that these cells produce IFN-γ, have poor immune suppressive capacity, and are prone to apoptosis. Tregs from non-responders showed upregulation of Th1 signaling, implicating chronic inflammatory cytokines in therapeutic resistance. In this context, IL-12 was found to promote dysfunctional Treg in vitro, and JAK1/2 inhibitor mitigated IFN-γ production by Tregs after LD IL-2 stimulation. These findings uncover the heterogeneity of Tregs, underscore the importance of understanding Treg plasticity, and identify a dysfunctional proinflammatory subset as a potential barrier to LD IL-2 efficacy in cGVHD.