case_images: PMC9880439_01
case_images: BMA of case 1~3 at diagnosis of MDS (representative images of BM cytomorphology and karyotype analysis). For case 1 (A-C): binuclear myelocyte (A), teardrops (B) and karyotype analysis depicting 45, XX, -5, -7, +add (11) (p15), -17, +mar (C). For case 2 (D~F): binuclear myelocyte (D), giant neutrophils and hyposegmented neutrophils (E), and karyotype analysis depicting 46, XY (F). For case 3 (G-I): basophilic stippling of red cells (G), binuclear myelocyte (H), and karyotype analysis depicting 42~46, XY, del (3) (p24), -5, +add (8) (q24.1), -17, add (18) (q23), +mar, inc[cp9]/46, XY [11].
case_images: fimmu-13-1063986-g001.jpg
case_images: PMC9880439_03_fimmu-13-1063986-g001.jpg
case_images: f1
case_images: As shown in  Figures 1G-I , the morphological assessment exhibited increased cellularity and dysplasia of erythroid and myeloid cells (hypogranular neutrophils, abnormal red blood cells (RBC) of mild anisocytosis and basophilic stippling); FCM showed 11.5% lymphoid cells in the BM; cytogenetics ascertained the abnormal karyotype of 42~46, XY, del (3) (p24), -5, +add (8) (q24.1), -17, add (18) (q23), +mar, inc[cp9]/46, XY [11]; MDS-associated genes revealed the TP53 mutation (c.818G>A, VAF of 45.3%, c.659A>G, VAF of 17%, c.636del, VAF of 11.7%) and DNMT3A mutation (c.939G>A, VAF of 13.3%).
case_images: PMC9880439_02
case_images: BMA of case 1~3 at diagnosis of MDS (representative images of BM cytomorphology and karyotype analysis). For case 1 (A-C): binuclear myelocyte (A), teardrops (B) and karyotype analysis depicting 45, XX, -5, -7, +add (11) (p15), -17, +mar (C). For case 2 (D~F): binuclear myelocyte (D), giant neutrophils and hyposegmented neutrophils (E), and karyotype analysis depicting 46, XY (F). For case 3 (G-I): basophilic stippling of red cells (G), binuclear myelocyte (H), and karyotype analysis depicting 42~46, XY, del (3) (p24), -5, +add (8) (q24.1), -17, add (18) (q23), +mar, inc[cp9]/46, XY [11].
case_images: fimmu-13-1063986-g001.jpg
case_images: PMC9880439_03_fimmu-13-1063986-g001.jpg
case_images: f1
case_images: As shown in  Figures 1G-I , the morphological assessment exhibited increased cellularity and dysplasia of erythroid and myeloid cells (hypogranular neutrophils, abnormal red blood cells (RBC) of mild anisocytosis and basophilic stippling); FCM showed 11.5% lymphoid cells in the BM; cytogenetics ascertained the abnormal karyotype of 42~46, XY, del (3) (p24), -5, +add (8) (q24.1), -17, add (18) (q23), +mar, inc[cp9]/46, XY [11]; MDS-associated genes revealed the TP53 mutation (c.818G>A, VAF of 45.3%, c.659A>G, VAF of 17%, c.636del, VAF of 11.7%) and DNMT3A mutation (c.939G>A, VAF of 13.3%).
case_images: PMC9880439_03
case_images: BMA of case 1~3 at diagnosis of MDS (representative images of BM cytomorphology and karyotype analysis). For case 1 (A-C): binuclear myelocyte (A), teardrops (B) and karyotype analysis depicting 45, XX, -5, -7, +add (11) (p15), -17, +mar (C). For case 2 (D~F): binuclear myelocyte (D), giant neutrophils and hyposegmented neutrophils (E), and karyotype analysis depicting 46, XY (F). For case 3 (G-I): basophilic stippling of red cells (G), binuclear myelocyte (H), and karyotype analysis depicting 42~46, XY, del (3) (p24), -5, +add (8) (q24.1), -17, add (18) (q23), +mar, inc[cp9]/46, XY [11].
case_images: fimmu-13-1063986-g001.jpg
case_images: PMC9880439_03_fimmu-13-1063986-g001.jpg
case_images: f1
case_images: As shown in  Figures 1G-I , the morphological assessment exhibited increased cellularity and dysplasia of erythroid and myeloid cells (hypogranular neutrophils, abnormal red blood cells (RBC) of mild anisocytosis and basophilic stippling); FCM showed 11.5% lymphoid cells in the BM; cytogenetics ascertained the abnormal karyotype of 42~46, XY, del (3) (p24), -5, +add (8) (q24.1), -17, add (18) (q23), +mar, inc[cp9]/46, XY [11]; MDS-associated genes revealed the TP53 mutation (c.818G>A, VAF of 45.3%, c.659A>G, VAF of 17%, c.636del, VAF of 11.7%) and DNMT3A mutation (c.939G>A, VAF of 13.3%).
case_images: PMC9880439_04
case_images: Identification of cell populations in BM samples from patient 1 developing tMDS after CAR T therapy and an age-matched HD using scRNA-seq. UMAP projection of scRNA-seq data for 24,919 hematopoietic cells from patient 1 and HD based on 10 principal component analysis consolidations and the 1,000 most variable genes in the data set. (A) t-SNE plot of all the 24,919 single cells from tMDS patient and HD. Each dot represents a single cell; the dot colors indicate source of donors. (B) t-SNE plot of all the 24,919 single cells colored by predicted hematopoietic populations in tMDS patient and HD. Cells were annotated based on known linease-specific marker genes. (C) Dot plot of differentially key cell-type marker genes. (D) Stacked barplots show the frequencies of 14 cell types in bone marrow from tMDS patient and HD, colored based on cell type.
case_images: fimmu-13-1063986-g002.jpg
case_images: PMC9880439_04_fimmu-13-1063986-g002.jpg
case_images: f2
case_images: In total, after quality control, 13,939 and 10,980 single cell libraries from a tMDS patient and HD, respectively, were retained for further analysis, as shown in  Figure 2A . These 24,919 BM-derived cells were segregated into 14 populations ( Figure 2B ) based on expression of established marker genes ( Figure 2C ). The frequencies of different cell types in tMDS and normal BM were also shown in  Figure 2D .
case_images: Transcriptional changes of the HSPC/CMP population in the tMDS patient identified with scRNA-seq. (A) Heatmap of the top 50 significantly differentially expressed genes between t-MDS versus HD HSPC/CMP populations, highlighting the distribution of transcript expression within the clusters. (B) Volcano plot revealing significantly differentially expressed genes between t-MDS versus HD HSPC/CMP populations (FDR <= 0.05 and log2 fold change>=1.0, <=-1.0).
case_images: fimmu-13-1063986-g003.jpg
case_images: PMC9880439_04_fimmu-13-1063986-g003.jpg
case_images: f3
case_images: We identified 80 genes that were significantly differentially expressed (log2 fold change >=1.0, <=-1.0, false discovery rate [FDR] <= 0.05) ( Figures 3A, B ;  Supplementary Table 1 ), with genes upregulated (n=17) and downregulated (n=63).
case_images: Dissection and clustering of T/NK subpopulations in the tMDS patient and HD. (A) t-SNE plot shows all identified T/NK subpopulations in tMDS and normal BM samples. The dot colored by source of donors. (B) UMAP plot of T/NK subpopulations from Figure 2B-represented CTL, Th and NK/NKT subsets. These T/NK cells can be divided into 13 subsets. (C) Dot plot of differently key cell-type marker genes. (D) Pie charts show relative ratio of cells annotated as CTL or Th cells in BM samples by scRNA-seq. (E) FCM analysis for T cells (CD3, CD4 and CD8) in the tMDS BM sample.
case_images: fimmu-13-1063986-g004.jpg
case_images: PMC9880439_04_fimmu-13-1063986-g004.jpg
case_images: f4
case_images: T/NK heterogeneity and states, we utilized UMAP to re-cluster 1,585 T/NK-like cells ( Figure 4A ) with more details. Thus, the T/NK lineage was divided into 13 clusters ( Figure 4B, BM environment of MDS. Besides, the tMDS aspirate tended to have proportionally more CTL and less Th than normal BM ( Figure 4D ). In accordance, the FCM analysis of this tMDS BM also showed an elevated CTL/T cell ratio of 65.66% ( Figure 4E ).