Integrative Transcriptomic Analysis Identifies CXCL12 as a Candidate Hub Gene Associated with CD4⁺ T-Cell Immune Network Remodeling in Secondary Lymphedema

Introduction: Secondary lymphedema is accompanied by a persistent chronic inflammatory response and remodeling of the immune microenvironment. Among them, CD4⁺ T cells and their functional subgroups play a crucial role. This study identified potential candidate molecules in the CD4⁺ T cell-related immune microenvironment, providing new research clues for subsequent mechanism-oriented intervention studies. Methods: In this study, we used the hdWGCNA method to establish a co-expression network of CD4⁺ T cells in secondary lymphedema, enabling the identification of key immune regulatory modules. We performed differential expression analysis using bulk transcriptomics and systematically analyzed the immune microenvironment of secondary lymphedema using CIBERSORT, Hallmark ssGSEA, and CellChat. This study primarily relies on computational analysis, employing numerous bioinformatics tools and algorithms for data integration and interpretation, aiming to explore the potential regulatory role of CD4⁺ T cells in the immune microenvironment from a holistic perspective. To further verify the results of the computational analysis, the selected key genes were experimentally validated using qPCR in a mouse model. Furthermore, computational virtual cell knockout analysis was applied for the first time in single-cell analysis of secondary lymphedema to assess the potential regulatory roles of key candidate genes in the immune regulatory network. This method provides more computational predictions and hypotheses for the study, and further experimental validation is needed to establish the actual functions and mechanisms of action of these genes. Results: This study identified 11 cell types using single-cell sequencing and found that CD4⁺ T cells transitioned from a resting to an activated state in SL tissue (resting CD4⁺ T cells decreased from 43.9% to 22.8%, while activated CD4⁺ T cells increased from 17.7% to 41.9%). Cell communication analysis showed that CD4⁺ T cells are key signal receivers in the microenvironment, with the CXCL12–CXCR4 axis significantly enriched in communication between fibroblasts and CD4⁺ T cells. Six co-expressed modules were identified using hdWGCNA, with CXCL12 associated with both the immune regulation-related M1 module and the T cell signaling-related M4 module. CXCL12 was significantly upregulated in SL tissue, exhibiting good diagnostic efficacy (AUC = 0.844), and was positively correlated with follicular helper T cells and negatively correlated with resting CD4⁺ memory T cells. In addition, this study validated the upregulation of Cxcl12 expression in a mouse model; virtual knockout analysis showed that only about 2% of gene expression was significantly altered after CXCL12 deletion, involving immune regulation and matrix remodeling-related genes such as AXL, IFITM3, and IGFBP7. Conclusion: This study reveals CXCL12 as a potential candidate molecule for the immune regulation of secondary lymphedema, which may mediate the interaction between fibroblasts and CD4⁺ T cells through the CXCL12–CXCR4 axis. However, the causal relationship still needs functional verification. Keywords: CXCL12, CD4⁺ T cells, immune remodeling, immune network, secondary lymphedema