GVHD_Reproducibility

GVHD Data Analysis

Overview

This repository contains the code and data analysis pipeline for our study on acute Graft-versus-Host Disease (GVHD) following allogeneic hematopoietic cell transplantation (allo-HCT) in collaboration with Reshef Lab. Acute GVHD is a potentially fatal complication where donor T cells attack recipient tissues. Our study aims to identify reliable biomarkers and uncover the mechanisms of T cell-mediated damage using a combination of in vitro assays, high-throughput sequencing, and spatial transcriptomics.

Study Design

To understand the interaction between donor T cells and host tissues:

We performed mixed lymphocyte reactions (MLRs) combined with high-throughput TCR sequencing to identify alloreactive T cell fingerprints across 20 patients, with 5–12 timepoints per patient. A subset of patients received Post-Transplant Cyclophosphamide (PTCy). We profiled immune cells in gut biopsies from 14 GVHD patients and two normal donors using paired single-cell transcriptomics and TCR sequencing. We further examined spatial transcriptomics data to elucidate the tissue architecture and cell-cell interactions underlying GvHD.

Data Access

• Raw FASTQ files: GEO GSE307215
• TCR raw data: https://drive.google.com/drive/folders/1bum2Z2LbJ6CuspDRHoZm_UG5jxtA4tZ7?usp=share_link
• Visium raw data and intermediate files: https://drive.google.com/drive/folders/1N2SJfrH7kDa3tjfcgwfZ4RbEJegAvXhT?usp=share_link (Starfysh deconvolution signature: GVHD_spatial_signature_v8_major_curated_unique_epi_tcells_subset_Transition_EPI_REFINE.csv)
• scRNA raw data and intermediate files: https://drive.google.com/drive/folders/1yV3ByTQL-CTCRNtnZf6tnRKFycLZseWp?usp=share_link (meta file: scRNAseq_TCR_samples_meta_LS-15_f.xlsx)
• MLR raw data and meta file: https://drive.google.com/drive/folders/1oyI_0J4Lk4-Sx1CYpHzdhNx-I0KqO4Bf?usp=sharing
• MLR PTcy analysis (5 additional patients) raw data and meta files: https://drive.google.com/drive/folders/1ftcSLg4Bx-BiXCFiQpUNSSJ-2spo8bAa?usp=drive_link

Figures

The table below maps each published figure to the notebook(s)/script(s) that generate it, following the layout used in decipher_reproducibility. Paths are relative to this repository's root. MLR_Pipelines/README.md and DecipherTCR/README.md contain additional per-panel detail for their pipelines.

Note on figure numbering: a few notebooks contain leftover code comments referencing figure numbers that no longer match the published manuscript (e.g. # Figure 4a/4B/4C/4F/4G in Sc&TCR_Pipelines/4., 6., 7.; # Figure 6A/Figure 7A in the two StarfyshHD Starfysh notebooks; "supplementary figure 22" in StarfyshHD/CellphoneDB_Analysis.ipynb), which is one ahead of, or otherwise diverged from, the final published numbering — most likely from a mid-revision figure reorder. The mapping below follows the published figure numbers, verified against the specific genes, sample sizes, and statistical tests named in each caption rather than the in-code labels.

Figure 1

• MLR_Pipelines/notebooks/01_patient_figures_and_diversity.Rmd (b)
• MLR_Pipelines/notebooks/02_clone_analysis_corrected_fig1C.Rmd (c)
• MLR_Pipelines/notebooks/04_cumulative_figures_corrected.Rmd (d)
• MLR_Pipelines/notebooks/03_grade_ptcy_analysis_corrected.Rmd (e–g)

Figure 2

• DecompTCR/pipelines/basis_decomposition_generate.ipynb (a)
• DecompTCR/pipelines/basis_decomposition_final.ipynb (b)
• MLR_Pipelines/MLR_rcode_work_for_all_sample_combined_final_ptcy_github.Rmd (c–e)
• MLR_Pipelines/notebooks/05_figure3_annotation_variants.Rmd (f–h)

Figure 3

• Sc&TCR_Pipelines/4. after annotation analysis.ipynb (a)
• Sc&TCR_Pipelines/6. T_cell_pipline_T_cell_subsets_signatures.ipynb (b, c, f)
• Sc&TCR_Pipelines/7. T_cell_pipline_proportion_analysis.ipynb (d, g, h)
• Sc&TCR_Pipelines/10.Donor_recipient_by_cell_type_freemuxlet.ipynb (e)

Figure 4

• Sc&TCR_Pipelines/11.Migration_analysis.ipynb (a–f)
• DecipherTCR/2. decipher_ananlysis_GVHD_main_Tcells_CD8_conv_deg_4cluster_clean_with_figures_mobile_balanced.ipynb (g–i)

DecipherTCR/1. decipher_ananlysis_GVHD_all_Tcells_all_genes_final.ipynb is a prerequisite step (loads the raw AnnData and runs Decipher training) for notebook 2 above; it contains no savefig calls itself and does not directly produce any panel.

Figure 5

• StarfyshHD/starfysh_ST_celltype_proportion_with_figures_clean.ipynb (a–c)
• StarfyshHD/starfysh_ST_distance_analysis.ipynb (a, d–f)

Figure 6

• StarfyshHD/starfysh_ST_distance_analysis.ipynb (a, b)

Panel c is a non-code schematic (credit: SciStories).

Supplementary Figure 1

• MLR_Pipelines/notebooks/01_patient_figures_and_diversity.Rmd

Supplementary Figure 2

• MLR_Pipelines/notebooks/04_cumulative_figures_corrected.Rmd

Supplementary Figure 3

• MLR_Pipelines/notebooks/02_clone_analysis_corrected_fig1C.Rmd

Supplementary Figure 4

• MLR_Pipelines/notebooks/02_clone_analysis_corrected_fig1C.Rmd
• MLR_Pipelines/notebooks/03_grade_ptcy_analysis_corrected.Rmd

Supplementary Figure 5

• DecompTCR/pipelines/basis_decomposition_generate.ipynb

Supplementary Figure 6

• DecompTCR/pipelines/basis_decomposition_final.ipynb (a, b)
• DecompTCR/pipelines/tcrdist.ipynb (c)
• MLR_Pipelines/Basis_Hydrophobicity_CDR3Length_Figures.Rmd (d, e)

Supplementary Figure 7

• DecompTCR/pipelines/basis_decomposition_final.ipynb

Supplementary Figure 8

• MLR_Pipelines/MLR_rcode_work_for_all_sample_combined_final_ptcy_github.Rmd (a)
• MLR_Pipelines/notebooks/03_grade_ptcy_analysis_corrected.Rmd (b–f)

Supplementary Figure 9

• MLR_Pipelines/notebooks/02_clone_analysis_corrected_fig1C.Rmd
• MLR_Pipelines/notebooks/03_grade_ptcy_analysis_corrected.Rmd

Supplementary Figure 10

• Sc&TCR_Pipelines/4. after annotation analysis.ipynb

Supplementary Figure 11

• Sc&TCR_Pipelines/7. T_cell_pipline_proportion_analysis.ipynb

Supplementary Figure 12

• Sc&TCR_Pipelines/7. T_cell_pipline_proportion_analysis.ipynb

Supplementary Figure 13

• Sc&TCR_Pipelines/7. T_cell_pipline_proportion_analysis.ipynb (a, c)
• Sc&TCR_Pipelines/9. Clonotype_T_cell_pipline_Migration.ipynb (b, e)
• Sc&TCR_Pipelines/11.Migration_analysis.ipynb (d)

Supplementary Figure 14

• Sc&TCR_Pipelines/10.Donor_recipient_by_cell_type_freemuxlet.ipynb

Supplementary Figure 15

• Sc&TCR_Pipelines/6. T_cell_pipline_T_cell_subsets_signatures.ipynb (b)
• Sc&TCR_Pipelines/7. T_cell_pipline_proportion_analysis.ipynb (a, d–g)

Supplementary Figure 16

• Sc&TCR_Pipelines/7. T_cell_pipline_proportion_analysis.ipynb

Supplementary Figure 17

• Sc&TCR_Pipelines/15a. Mapping_clones_between_MLR_and_tissue_TCRB_with_new_patients_all_Tcells.ipynb
• Sc&TCR_Pipelines/15b. Mapping_clones_between_MLR_and_tissue_TCRB_with_new_patients_map_to_donors.ipynb

Supplementary Figure 18

• Sc&TCR_Pipelines/11.Migration_analysis.ipynb

Supplementary Figure 19

• Sc&TCR_Pipelines/11.Migration_analysis.ipynb

Supplementary Figure 20

• Sc&TCR_Pipelines/7. T_cell_pipline_proportion_analysis.ipynb

Supplementary Figure 21

• Sc&TCR_Pipelines/13. CellTypist_analysis_for_T_cells_3_resi_cluster.ipynb

Supplementary Figure 22

• Sc&TCR_Pipelines/12.Tcell_clonaltype_analysis_with_refined_cluster.ipynb

Supplementary Figure 23

• Sc&TCR_Pipelines/TRB_clonality_validation.ipynb

Supplementary Figure 24

• DecipherTCR/2. decipher_ananlysis_GVHD_main_Tcells_CD8_conv_deg_4cluster_clean_with_figures_mobile_balanced.ipynb

Supplementary Figure 25

• DecipherTCR/2. decipher_ananlysis_GVHD_main_Tcells_CD8_conv_deg_4cluster_clean_with_figures_mobile_balanced.ipynb

Supplementary Figure 26

• StarfyshHD/starfysh_ST_celltype_proportion_with_figures_clean.ipynb

Supplementary Figure 27

StarfyshHD/CellSAM_Segmentation.ipynb is the notebook this figure should come from, but the file is currently empty (0 bytes) in this repository and will need to be restored.

Supplementary Figure 28

• StarfyshHD/Starfysh_tutorial_integration_all_with_no_arch_with_figures_final_github.ipynb

Supplementary Figure 29

• StarfyshHD/starfysh_ST_celltype_proportion_with_figures_clean.ipynb

Supplementary Figure 30

• StarfyshHD/starfysh_ST_distance_analysis.ipynb (a, b)
• StarfyshHD/CellphoneDB_Analysis.ipynb (c)

Supplementary Figure 31

Gating strategy for cell sorting (flow cytometry); not produced by a notebook in this repository.

Supplementary Figure 32

• Sc&TCR_Pipelines/7. T_cell_pipline_proportion_analysis.ipynb

Supplementary Figure 33

• Sc&TCR_Pipelines/11.Migration_analysis.ipynb

Acknowlegement

This work was made possible by the collaboration between the Azizi Lab and Reshef Lab, as well as our clinical partners at CUIMC and CCTI. We thank all participating patients and acknowledge the funding sources that supported this research.

Citation

Spatiotemporal Single-Cell Analysis Reveals T Cell Clonal Dynamics and Phenotypic Plasticity in Human Graft-versus-Host Disease Lingting Shi, Ajna Uzuni, Ximi K. Wang, Michael Pressler, David W. Harle, Shami Chakrabarti, Rodney Macedo, Kirubel Belay, Christian A. Gordillo, Erik Raps, Jia Yi (Ady) Zhang, Achille Nazaret, Joy L. Fan, Yinuo Jin, Xumin Shen, Joshua S. Fuller, Tamjeed Azad, Jessie Huang, Pranik Chainani, Julian A. Abrams, Armando Del Portillo, Markus Y. Mapara, Mohamed Alhamar, Megan Sykes, José L. McFaline-Figueroa, Elham Azizi, Ran Reshef bioRxiv 2025.05.24.655962; doi: https://doi.org/10.1101/2025.05.24.655962