Study on Organ-Specific T Cell Differentiation in Acute Graft-Versus-Host Disease (AGVHD)

Academic Background

Acute Graft-Versus-Host Disease (AGVHD) is a common and severe complication following Allogeneic Hematopoietic Stem Cell Transplantation (HCT), caused by the immune attack of donor T cells on host tissues. Although the pathological mechanisms of AGVHD have been studied to some extent, the differentiation mechanisms of T cells in specific organs remain unclear. The specific responses of T cells in different organs may be regulated by the local microenvironment, but the specific mechanisms of this regulation have not been fully elucidated. AGVHD affects multiple organs, including the gastrointestinal tract, skin, liver, and lungs, but there are significant differences in the pathological features and immune responses in different organs. Understanding these differences is crucial for developing treatment strategies targeting specific organs.

To investigate this issue, the research team used a Nonhuman Primate (NHP) model to conduct an in-depth study on the differential differentiation of T cells in the liver and lungs during AGVHD through various systems immunology methods, including multiparameter flow cytometry, population-based transcriptomic analysis, Single-Cell RNA Sequencing (scRNA-seq), and T Cell Receptor Sequencing (TCR-seq).

Source of the Paper

The study was conducted by Kayleigh Ingersoll Omdahl, Rene S. Bermea, Victor Tkachev, Leslie S. Kean, and other researchers from multiple renowned institutions, including Boston Children’s Hospital, Harvard Medical School, and Massachusetts General Hospital. The paper was published on January 29, 2025, in the journal Science Translational Medicine, titled “Organ-specific microenvironments drive divergent T cell evolution in acute graft-versus-host disease.”

Research Process

1. Research Model and Samples

The study used rhesus macaques as the AGVHD model, with analysis conducted 7-8 days post-transplantation. The study was divided into three groups: AGVHD group (allogeneic HCT, n=12), autologous HCT control group (auto-HCT, n=4), and healthy control group (Healthy Controls, HCs, n=26). The extent of inflammation in the liver and lungs was assessed through histopathological analysis.

2. Flow Cytometry Analysis

The researchers used multiparameter flow cytometry to analyze the phenotypic characteristics of T cells in the AGVHD group, auto-HCT group, and HCs. The focus was on the memory phenotypes of CD8+ and CD4+ T cells, tissue-resident markers (e.g., CD69 and CD103), proliferation markers (Ki67), and cytotoxicity markers (Granzyme B). Additionally, unsupervised clustering of T cells was performed using the FlowSOM algorithm to identify phenotypic differences in T cells across different organs.

3. Transcriptomic Analysis

The researchers used NHP-specific gene arrays to conduct transcriptomic analysis of CD3+ T cells in the AGVHD group, auto-HCT group, and HCs. The gene expression differences among different organs and groups were assessed through UMAP dimensionality reduction and hierarchical clustering. Further comparison of the specific gene expression pathways in liver and lung T cells revealed that lung T cells were associated with extracellular matrix remodeling and chemotaxis, while liver T cells were associated with nucleic acid metabolism and proliferation.

4. Single-Cell RNA Sequencing and TCR Sequencing

To investigate the differentiation process of T cells in more detail, the researchers isolated CD45+CD3+ T cells from the liver and lungs of AGVHD animals and performed single-cell RNA sequencing and TCR sequencing. Data were batch-corrected using the Python scVI package, and pseudotime analysis was conducted using the Monocle3 software to trace the differentiation trajectories of T cells. The study found that as clonal expansion increased, lung T cells gradually enriched CX3CR1+ CD8+ effector T cells, while liver T cells enriched Eomes+ CD8+ effector memory T cells.

5. Shared Clonotype Analysis

The researchers also analyzed T cell receptor clonotypes (Shared Clonotypes, SCs) that existed in both the liver and lungs, finding that even with the same TCR, the transcriptomes of T cells in the liver and lungs exhibited significant differences. This indicates that T cell differentiation depends on signals from the local microenvironment rather than antigen specificity.

Research Results

1. Histopathological Analysis: The liver and lungs in the AGVHD group showed significant inflammatory responses, with the liver exhibiting periportal inflammation and the lungs showing perivascular inflammation and interstitial pneumonia.

2. Flow Cytometry Analysis: The liver and lungs in the AGVHD group were infiltrated by CD8+ effector memory T cells (TEM) and CD4+ central memory T cells (TCM), with these cells highly expressing the proliferation marker Ki67 and the cytotoxicity marker Granzyme B.

3. Transcriptomic Analysis: Lung T cells significantly upregulated genes related to chemotaxis and extracellular matrix remodeling, while liver T cells upregulated genes related to nucleic acid metabolism and proliferation. These results suggest that the differentiation of T cells in specific organs is regulated by the local microenvironment.

4. Single-Cell RNA Sequencing and TCR Sequencing: As the degree of clonal expansion increased, lung T cells gradually differentiated into CX3CR1+ CD8+ effector T cells, while liver T cells differentiated into Eomes+ CD8+ effector memory T cells. Even with the same TCR, the transcriptomes of T cells in the liver and lungs exhibited significant differences.

Conclusion

This study revealed the differentiation mechanisms of T cells in specific organs during AGVHD, emphasizing the significant impact of the local microenvironment on T cell transcriptomic programming. The study found that the differentiation and functional states of T cells in the liver and lungs are influenced by organ-specific signals, and this differentiation is closely related to clonal expansion. This discovery provides new insights for developing immune therapies targeting specific organs, particularly laying the foundation for targeted treatments for lung and liver AGVHD.

Highlights of the Study

1. Organ-Specific T Cell Differentiation: For the first time, the differential differentiation mechanisms of T cells in the liver and lungs were revealed in an AGVHD model.
2. Application of Single-Cell Technology: The clonal expansion and differentiation trajectories of T cells were investigated in depth through single-cell RNA sequencing and TCR sequencing.
3. Clinical Significance: The study results provide new targets and strategies for developing immune therapies targeting specific organs, especially offering hope for the treatment of lung and liver AGVHD.

Other Valuable Information

The study also found that the differentiation of lung and liver T cells is closely related to clonal expansion, suggesting that inhibiting T cell expansion may be key to controlling organ-specific immune responses. Additionally, the study found that lung and liver T cells enriched different chemokines and metabolic pathways, providing potential targets for developing organ-specific immune-modulating drugs.