PMC10266344_01
Clinical presentations of the studied patient. (A) The first column shows the Wright-staining of the periphery blood and bone marrow aspirate. The staining identifies small abnormal lymphocytes with round or irregular nuclei and coarse chromatin. The second column shows the H&E staining of paraffin sections of abdominal lymph node biopsies. The lymphoid tissues are hyperplasia, with uniformly small cell size, with round or irregular nuclei and coarse chromatin. The third and fourth column were immunohistochemistry of lymph nodes, suggesting CD2+, CD3+, CD5 partial+, PAX-5 Lymphatic follicle+ tumor cells. (B) Immunophenotypes of the malignant T cells by flow cytometry. (C) PET-CT scans of the patient. The red arrows indicate enlarged lymph nodes in bilateral axilla, intraperitoneal cavity, retroperitoneum and bilateral inguinal area, with increased radioactive uptake. Whole body PET images suggest that diffused radioactive uptakes are increased in the four limb bones, trunk bones, and pelvic medullary cavity.
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Figure 1A ). Flow cytometry revealed a group of mature TCRalphabeta+ ( Figure 1B ). TCR gene rearrangement assay suggested TCR rearrangement in TCRB and TCRG but not TCRD. The patient's lab examinations were summarized in  Table S1 . The liver was also enlarged, with increased radioactive uptake (SUV max 2.80) ( Figure 1C ). B ,  Figure S1 ): CD4 and CD8 T cells (CD3E, CD4, CD8A, CD8B); natural killer (NK) cells (GNLY, NKG7); B cells (CD19, CD79A); Hematopoietic stem and progenitor cells (STMN1, CA2, CDK6, MKI67); we also identified two monocyte subtypes: CD14+CD16- classical monocytes (c_Mono) and CD14loCD16+ nonclassical monocytes (nc_Mono). Besides these normal cells, we revealed a cluster of T cell lineage tumor cells, accounting for 52.7% and 44.4% of the cell population in BM and PBMC, respectively ( Figures 2A, C ). Three tumor subsets have been identified ( Figure 2D ): Tumor_1 is the major subset, accounting for 84.8% of the tumor cells, Tumor_2 and Tumor_3 were relative smaller tumor subsets, account for 7.2% and 8.0%, respectively.
Single cell analysis of the unclassified TCL patient samples. (A) Cell types revealed by scRNA-seq and presented by UMAP. (B) Dotplot of the canonical marker genes used to annotate the cell types. Dot size represents % of cells of that cluster expressing the given gene, while color indicates the expression level of that cluster. (C) Cell type count and % in the BM and PBMC samples. (D) UMAP of re-clustered malignant T cells, cells are colored by different tumor subsets. (E) UMAP of malignant T cells, each color represents a distinct TCR clonotype. Tumor_1 and Tumor_2 belong to the same clonotype (blue dots), while Tumor_3 contains diverse clonotypes. (F) Dotplot of the marker genes used in flow cytometry analysis, single cell expression data are consistent with flow cytometry results. (G) Unbiasedly identified signature genes of each tumor subset.
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B ,  Figure S1 ): CD4 and CD8 T cells (CD3E, CD4, CD8A, CD8B); natural killer (NK) cells (GNLY, NKG7); B cells (CD19, CD79A); Hematopoietic stem and progenitor cells (STMN1, CA2, CDK6, MKI67); we also identified two monocyte subtypes: CD14+CD16- classical monocytes (c_Mono) and CD14loCD16+ nonclassical monocytes (nc_Mono). Besides these normal cells, we revealed a cluster of T cell lineage tumor cells, accounting for 52.7% and 44.4% of the cell population in BM and PBMC, respectively ( Figures 2A, C ). Three tumor subsets have been identified ( Figure 2D ): Tumor_1 is the major subset, accounting for 84.8% of the tumor cells, Tumor_2 and Tumor_3 were relative smaller tumor subsets, account for 7.2% and 8.0%, respectively. TCR information, we found that Tumor_1 and Tumor_2 cells shared the same TCR sequences, suggesting that they arose from the same tumor ancestor cell and evolved into two branches ( Figure 2E ). Tumor_3 had a distinct TCR profile compared to Tumor_1 and Tumor_2 ( Figure 2E ). TRAJ47 + CVVIYGNKLVF), one beta chain (TRBV10-2 + TRBJ2-2 + CASSANTGELFF) and one gamma chain (TRGV9 + TRGJ1 + CALVAVGREEFYYKKLF), but no delta chain. The four TCR chains had comparable expression levels according to their TCR V gene expressions ( Figure S2 ). T cells usually express either a pair of alphabeta chains or a pair of gammadelta chains, it is very rare to observe the co-expressing of alphabeta and gammadelta chains in a single T cell. To our knowledge, here, we identified the first case of T cell lymphoma with alpha, beta and gamma chains presenting together in a single tumor cell. The co-expressing of alpha, beta and gamma chains indicated a unique putative cell of origin of these tumor cells, the ancestor cell might be a combined alphabeta and gammadelta T cell. This special TCR presentation can be related to the pathogenesis of this rare tumor subtype, explaining the poor prognosis of the patient. The Tumor_3 cluster were not monoclonal expanded, the biggest clonotype in Tumor_3 contained 48 cells (13.8%). CD2, CD3 positive, CD8A negative but CD8B positive. CD5 and CD7 were only presented at Tumor_3 cells, consistent with the partial positive observation in the flow cytometry data ( Figure 2F ,  Figure S4 ). Figure 2G ). Tumor_1 and Tumor_2 displayed cytotoxic T cell phenotypes by expressing a cytotoxic module, including GZMK and GZMA, PRF1 etc., which supported their putative CD8 T cell and gammadelta T cell origins. Several potential therapeutic target genes were also identified in Tumor_1 and Tumor_2 ( Figure 2G ). For example, CCL5, abnormal expression and activity of CCL5/CCR5 axis have been found in hematological malignancies and solid tumors. Therapeutic strategies targeting CCL5/CCR5 might be effective for these tumor cells. Tumor_1, but it was featured by high expression of proliferation markers such as MKI67, STMN1, suggesting they are the actively expansion tumor cells. Tumor_3 were the pre-malignant T cell cluster with a distinct expression profile ( Figure 2G ,  Table S5 ).