Single-Cell Transcriptomics Reveals Immunosuppressive Microenvironment and LAG3-FGL1 Axis Impairs Effector T Lymphocytes in Hepatitis-Associated Aplastic Anemia
Authors: Cheng Xuelian , Chen Ming , Yang Tong , Duan Yongjuan , Zhang Jingliao , Zhang Yingchi , Zhang Li , Zou Yao , Yang Wenyu , Zhu Xiaofan , Chang Lixian 
Published: December 2024

Hepatitis-associated aplastic anemia (HAAA) is a rare illness, characterized by onset of pancytopenia with a hypoplastic bone marrow. The differences in the pathogenesis of HAAA and aplastic anemia (AA) have not been fully elucidated, especially the relationship between hepatitis and AA, resulting in the unclear pathogenesis of HAAA.

Here in this study, single-cell RNA sequencing was used to compare gene expression profiles in bone marrow of 6 HAAA patients versus 3 severe AA (SAA) patients, 2 healthy donors (HD) as the control group. Unbiased bioinformatics analysis revealed that HAAA induced lymphoid-biased differentiation. The proportion of CD8 effector, gamma deta T and MAIT cells were higher than that in SAA and healthy group. We compared the composition of CD8+ effector, CD8+ memory, CD8+ Naive, gamma deta T, MAIT cells in the trajectory of T lymphocytes differentiation by pseudo-time analysis from HAAA, SAA and HD samples. The differentiation trajectories of CD8+ T cells in HAAA resembled those in SAA samples and different those in HD samples. Alignment of BM cellular composition with hematopoietic developmental trajectories revealed potential functional roles for CD8+ effector T cells in HAAA.

Further, we found exhaustion-related markers lymphocyte activation gene 3 (LAG3) and TGFβ1 were highly expressed in HAAA, not programed cell death protein 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4). The gene LAG3 was highly expressed in CD8 effector cells, gamma deta T and MAIT cells. Lymphocyte activation gene 3 (LAG3), an immune checkpoint molecule expressed on activated T cells, functions as a negative regulator of immune responses. We further compared the expression differences of LAG3 in different HAAA samples and found that LAG3 was highly expressed in 3 patients, while the remaining patients had no significant difference in expression levels compared to SAA. A more detailed analysis revealed that in these three patients with CD8+ effector T cell clones proliferation. Further analysis suggests that high expression of LAG3 might be positively correlated with the severity of hepatitis. Fibrinogen-like protein 1 (FGL1) and Galectin-3 (GAL3) have been identified as two major ligands of LAG3. Through in vitro experiments, our data revealed that FGL1 can be captured in the plasma and the content in HAAA was significantly higher than that in AA and normal groups. The addition of FGL1-Fc protein suppressed the response of HAAA, which highly expressed LAG3. FGL1-Fc attenuated CD8+ effector T cells proliferation and secretion of inflammatory factors by binding to LAG3. These observations may provide favorable evidence that the FGL1-LAG3 signaling axis is a potential driver of HAAA development.

In conclusion, this study provided a landscape description of the immunosuppressive features in T lymphocytes in HAAA, and suggested an important role of FGL1-LAG3 axis in T cell function. Immune-checkpoint blockade might be a approach for the treatment of HAAA and the heterogeneity in treatment response to anti-LAG3 in HAAA.