^{the Exploration of the Tumor Microenvironment in Angioimmunoblastic T-Cell Lymphoma By Single-Cell Sequencing Technology}

Objective:Exploration of the Tumor Microenvironment inAngioimmunoblastic T-cell Lymphoma (AITL) Patients at the Single-cell Level Using Single-cell Sequencing Technology to Guide Understanding of Disease Progression and Treatment.

Methods: 1. Clinical data including age, gender, extranodal infiltration status, Epstein-Barr virus infection status, Eastern Cooperative Oncology Group (ECOG) performance status, International Prognostic Index (IPI) score, disease stage, and treatment regimens were collected.

1. A Fresh lymph node specimens from one of the patients were subjected to single-cell RNA sequencing analysis. Additionally, peripheral blood single-cell RNA sequencing data from one case were downloaded from the 10X Genomics database to serve as a control group.

2. Principal component analysis (PCA) was performed using the RunPCA function. Differential expression genes for various cell types were identified using the FinAllMarkers function with reference to typical cell type marker genes from databases such as CellMarker and PanglaoDB, with highly expressed genes serving as marker genes. Enrichment analysis was conducted using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome databases. Cell communication activities were predicted using CellChat.

Results:1. By annotating cell types, we could see that there was a significant difference between the control group and the case group in the number of B cells, monocytes, and T-helper cells, especially in the number of Naive-CD4+Tcells,Cycling-CD4+T cells,Naive-CD8+ Tcells were significantly different. There were significantly more B cells in the case group than in the control group, and the depletion of monocytes,Naive-CD4+T cells,Cycling-CD4+T cells,Naive-CD8+T cells was significantly more in the case group than in the control group.

1. 2. In the B cells, there were a total of 12 up-regulated genes in the case group compared with the control group, mainly RGS1, EPST1L, TNFAIP3, VMP1, SLC9A9, etc.and there were 33 down-regulated genes, mainly IGHA2, PPP1R14A, LINC01781, IGHG2, IGLC1, COCH and CD27. In monocytes, compared with the control group, there were 164 up-regulated genes in the case group, mainly MB21D2, MPO, PTGDS, DAMM1, ATP8B4, etc.and 29 down-regulated genes, mainly ID1, AC239799.2, CXCL10, etc. In cycling-CD4+T cells, compared with the control group, there were 69 up-regulated genes in the case group, mainly IGHA2, PPP1R14A, LINC01781, IGHG2, IGLC1, COCH and CD27. There were 69 genes in total, mainly CD14, MPEG1, S100A12, CD36, VCAN, GAPDH, etc. and 213 down-regulated genes, mainly STMN1, MHGN2, H2AFZ, etc. In Naive-CD4+T cells, compared with the control group, there were a total of 8 up-regulated genes in the case group, mainly LINC01871, PDE4D, TSHZ2, PHACTR2, etc., and 6 down-regulated genes, mainly IL32, HIST1H1D, TRBC2, etc.

2. 3. Our differential genes in B cells were mainly enriched in the NOD-like receptor signaling pathway, and we also showed a significant increase in the number of differentially regulated genes in Naive-CD4+T cells and Naive-CD8+T cells, respectively, and we also enriched in the NOD1/2 Signaling Pathway, NOD-like receptor signaling pathway in monocytes, Cycling-CD4+T cells and Naive-CD4+T cells, and NOD-like receptor signaling pathway in Bcells, Cycling-CD4+T cells and Naive-CD4+ T cells, pathways associated with cellular inflammatory responses were identified in the enrichment analysis of up-regulated genes, such as RHO GTPases Activate NADPH Oxidases, NF-kappa B signaling pathway, NF-kappa B signaling pathway, NOD1/2 Signaling Pathway. And in the enrichment analysis of Naive-CD8+ T cells and up-regulated genes, pathways such as NF-kappa B signaling pathway (nuclear factor κB signaling pathway), IL-17 signaling pathway (IL-17 and other signaling factors) were also found.

Conclusion：1.The results showed that there were significantly more B cells in the case group than in the control group, while T cells and monocytes were significantly more depleted in the case group than in the control group.

1. In the tumor microenvironment of AITL patients, GAPDH acting on the NF-κB pathway may be related to Cycling-CD4+ T cells, and the NOD-like signaling pathway may play a role in the generation and development of AITL.

Key words: Angioimmunoblastic T-cell Lymphoma; Tumor Microenvironment; single-cell sequencing technology

No relevant conflicts of interest to declare.