DOI: 10.5061/dryad.wh70rxx2g
title: Data from: Allospecific splenic Tr1 cells drive effector T cell exhaustion through upregulated Areg-EGFR signaling to promote transplant tolerance

description: Inducing stable tolerance to transplants remains a challenge in
 immunology. Previously, we induced tolerance to allogeneic islets in
 nonhuman primates by preemptive alloantigen delivery to antigen-presenting
 cells in situ. Here, mass cytometry phenotyping with incorporated
 donor-derived MHC-I peptide-loaded MHC-II tetramers revealed accumulation
 of allospecific CD4+ T cell clusters in the spleen of tolerant recipients.
 Areg+Tr1 regulatory and terminally exhausted EGFRhi T (Tex) cells
 represented the predominant allospecific subsets. Trajectory analysis
 showed that antigen-experienced effector memory T cells differentiated
 into suppressive Areg+Tr1 and EGFR+TOX+Nur77+TCF-1- Tex subsets. Cell-cell
 communication mapping showed that exhausted and effector memory T cells
 engaged with allospecific Tr1 cells via the Areg-EGFR axis. Gene silencing
 studies confirmed that Tr1 cells utilize Areg-EGFR signaling to drive the
 metabolic suppression and epigenetic reprogramming of CD4⁺ T cells through
 a Nur77-dependent pathway. These findings point to the splenic Areg⁺Tr1
 cell-EGFR⁺Teff cell axis as a critical immunoregulatory pathway in
 peripheral transplant tolerance. Here we provide the raw and processed
 CyTOF (mass cytometry by time of flight) data and R analysis scripts used
 to arrive at these conclusions for each cell type mentioned above. CyTOF
 data is provided as FCS-format files representing counts for thousands of
 cells and up to 36 mass-tagged protein markers.
description: Raw FCS files were transformed and converted to Seurat objects
 with custom R scripts (process_raw_X.txt and make_seurat_X.txt files,
 respectively) using information contained in sample key files
 (sample_ref_X.txt files). These data are separated into folders comprising
 the data files and analysis files for each respective cell type
 group.
description: # Data from: Allospecific splenic Tr1 cells drive effector T cell
 exhaustion through upregulated Areg-EGFR signaling to promote transplant
 tolerance Dataset DOI:
 [10.5061/dryad.wh70rxx2g](https://doi.org/10.5061/dryad.wh70rxx2g) ##
 Description of the data and file structure There are four separate
 directories representing four main cell groupings presented in the paper.
 Each directory comprises raw and transformed FCS files for the CyTOF data,
 a sample metadata file, R scripts for the transformation of fcs files and
 conversion to Seurat R objects, and the R object itself. ### File:
 scripts_and_data_for_dryad.tar.gz * **Directories** * `Total_cells` *
 `MHCI+_CD4_T_cells` * `MHCI+_PD-1hi` * `MHCI+_Tr1` ## **Contents of each
 [cell_type] directory:**  **`raw`:** directory * untransformed FCS files
 for each tissue in each individual **`transformed`**: directory * arcsinh
 transformed FCS files for each tissue in each individual
 **`process_raw_[cell_type].txt`**: flat text file (R script) * reads in
 raw FCS files, arcsinh transform them, perform centroid correction, and
 write transformed files. **`make_seurat_[cell_type].txt`**: flat text file
 (R script) * reads in transformed FCS files and metadata, creating a
 combined Seurat objects containing each tissue for each animal
 **`sample_ref_[cell_type].txt`** - flat text file (TSV) containing sample
 metadata used in make_seurat_[cell_type].txt script. Columns explained
 below * *file* - name of the .fcs CyTOF file * *tissue* - tissue of origin
 (spleen (spl) or peripheral blood (pbl)) * *cohort* - experimental group
 of animal * *animal* - individual animal ID * *batch* - batch that CyTOF
 data was collected in **`all_data_[cell_type].rds`**: RData file * Seurat
 object containing transformed marker counts for each tissue in each
 individual ## Code/software and versions R version 4+ Seurat version 4.3+
 Necessary packages are shown in the R script files ##