RNA and TCR Sequencing Shed Light on Mechanisms of Treg Suppression in a Murine Model of Acute GvHD
Authors: Lohmeyer Juliane K. , Hirai Toshihito , Turkoz Mustafa , Koehler Natalie , Baker Jeanette , Buhler Stephane , Villard Jean , Ji Xuhuai , Simonetta Federico , Negrin Robert S. 
Published: November 2020

Introduction
Regulatory T cell (Treg) based therapies are a promising approach for graft-versus-host disease (GvHD) prevention and treatment. However, mechanisms of Treg suppression of alloreactive conventional T cells (Tcon) in GvHD are incompletely understood. In this study, we performed paired RNA and T cell receptor (TCR) sequencing analysis on Tcon and Treg before and after transplantation to further elucidate Treg suppressive function during in vivo suppression of acute GvHD in an MHC major-mismatch mouse model.
Methods
CD45.2 Thy1.2 BALB/c mice were lethally irradiated (8.8 Gy) and transplanted with 5x106 T cell- depleted bone marrow cells (TCD-BM) from CD45.1 Thy1.2 C57Bl/6 mice alone or together with CD45.2 Thy1.2 C57Bl/6 FoxP3/GFP+ Treg (1x106) on day 0. On day 2, CD45.1 Thy1.1 C57Bl/6 Tcon (1x106; CD4:CD8 ratio = 2:1) were injected to induce GvHD. Irradiated (11 Gy) syngeneic C57Bl/6 recipients receiving C57Bl/6 TCD-BM and CD45.1 Thy1.1 Tcon alone were used as controls. On day 8, donor Thy1.1+ CD45.1+ CD4 and CD8 Tcon and Thy1.2+CD45.2+ FoxP3/GFP+ Treg were isolated by FACS from spleens and lymph nodes. T cell subsets before injection and recovered at day 8 were analyzed by paired RNA and TCR sequencing.
Results
The transcriptomic analysis revealed a dominant effect of the allogeneic transplant procedure on the clustering of the different T cell populations. Principal component analysis (PCA) of the top 1000 most differentially expressed genes revealed that 68% of the variance was explained by PC1, which clearly segregated CD4and CD8Tcon recovered at day 8 from allogeneic recipients from cells before injection or recovered from syngeneic recipients (Fig. 1). PC1 was mainly driven by naïve T cell genes (Ccr7, Sell, Il6ra, Il6st, Foxo1) that were progressively downregulated along PC1. Analysis of the TCR repertoire based on sequencing of the TCR alpha chain revealed a progressive clonal restriction along PC1 in CD4 and CD8 T cells (Fig. 1). Accordingly, clonal overlap between cells collected at day 8 and cells analyzed before injection were reduced in allogeneic recipients (Morisita Index [MI], CD4: 0.06±0.01; CD8: 0.02±0.01) compared to syngeneic controls (MI, CD4: 0.23±0.26; CD8: 0.57±0.05). Treg administration did not affect CD4 or CD8 T cell segregation along PC1, suggesting that they minimally interfered with cell activation and differentiation during GvHD. Accordingly, Treg did not inhibit clonal restriction (Fig.1) nor the reduction in clonal overlap (MI, CD4: 0.04±0.01; CD8: 0.01±0.01), indicating that Treg did not inhibit the initial activation of alloreactive T cells clones. Treg impact on CD4 but not CD8 Tcon transcriptome was revealed by PC2 (Fig.1). Treg induced the downregulation of TH1-signature genes (Tbx21, Il12rb1, Il12rb2, Stat4) and proinflammatory genes (Il18rap) while promoting up-regulation of anti-inflammatory genes (Il18bp), TH2 signature genes (Ccr4, Il4) and Il2 in CD4Tcon. Moreover, gene set enrichment analysis (GSEA) revealed that Treg treatment significantly impacted gene sets involved in metabolic processes in CD4and CD8Tcon, leading to a global up-regulation of genes encoding for enzymes involved in oxidative phosphorylation (OXPHOS) and downregulation of genes encoding for enzymes contributing to glycolysis (Slc2a1, Hk1, Pfkl, Pfkp, Pkm).
Treg recovered at day 8 preserved a distinct transcriptomic signature observed before injection and further enhanced by the up-regulation of genes involved in Treg activation and suppressive function (Gata3, Tnfrsf18, Tnfrsf4, Icos, Ccr1, Ccr4, Il9r). GSEA in Treg revealed significant up-regulation of genes in the OXPHOS signature. TCR repertoire analysis showed clonal restriction of Treg during GvHD. Direct comparison of clone frequencies in Treg and CD4Tcon showed smaller clonal overlap on day 8 (MI=0.005±0.006) compared to day 0 (MI= 0.11±0.004) suggesting that Treg and CD4Tcon responses during GvHD are engaging different cell clonotypes triggered by different epitopes or antigens.
Conclusion
Our results indicate that Treg treatment did not interfere with Tcon activation and differentiation of alloreactive Tcon clones in a model of acute GvHD. Treg predominantly affected CD4Tcon and to a lesser extent CD8Tcon transcriptome, modulating the transcription of genes encoding pro- and anti-inflammatory molecules as well as enzymes involved in metabolic processes.

Disclosures
Turkoz: Adicet Bio: Current Employment, Current equity holder in private company. Negrin:Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company; BioEclipse Therapeutics: Current equity holder in private company; Amgen: Consultancy; KUUR Therapeutics: Consultancy; Biosource: Current equity holder in private company; UpToDate: Honoraria.