PMC

Identification of Mo/MΦ and B cell subpopulations. (A) Bubble plot refers to molecular expression marker in Mo/MΦ clusters. X-axis is the name of the marker gene and Y-axis the name of the Mo/MΦ subpopulation; the size of the bubble represents the ratio of the sum of the marker gene expression in a certain subpopulation to the sum of its expression in all Mo/MΦ cells; the color of the bubble represents the average expression abundance of the marker gene in the Mo/MΦ subpopulation; the more red the bubble, the higher the average expression level of the marker gene in the Mo/MΦ subpopulation. (B) Identification of the 2 Mo/MΦ subpopulations based on marker molecules. Cell subpopulations are represented by different colors (M1 type Mo/MΦ: blue, M2 type Mo/MΦ: red). (C) Bubble plot refers to molecular expression marker in all B cell clusters. X-axis is the name of the marker gene and Y-axis the name of the B cell subpopulation; the size of the bubble represents the ratio of the sum of the marker gene expression in a certain B cell subpopulation to the sum of its expression in all B cells; the color of the bubble represents the average expression abundance of the marker gene in the B cell subpopulations; the more red the bubble, the higher the average expression level of the marker gene in the B cell subpopulation. (D) Identification of 4 B cell subpopulations based on the identified marker genes. The 4 cell subpopulations are represented by different colors (Mature-B cell: blue, pre B cell: orange, Immature-B cell: red, early-pre B cell: green). (E) Pseudo-time analysis of B cell subpopulations. Each subpanel corresponds to previously identified subpopulations as shown in (D). (F) Bubble plot refers to molecular expression marker in mature B cell clusters. X-axis is the name of the marker gene and Y-axis the name of the mature B cell subpopulation; the size of the bubble represents the ratio of the sum of the marker gene expression in a certain mature B cell subpopulation to the sum of its expression in all mature B cells; the color of the bubble represents the average expression abundance of the marker gene in the mature B cell subpopulations; the more red the bubble, the higher the average expression level of the marker gene in the mature B cell subpopulation. (G) Identification of 3 mature B cell subpopulations based on the identified marker genes. The 3 cell subpopulations are represented by different colors (IgM⁺: blue, IgD⁺: red, IgZ⁺: green).

A. Percentage of pro-B/pre-B lymphomas, immature B-cell lymphomas, mature B-cell lymphomas and plasmablastic B-cell lymphomas in Igλ-Myc (wt), heterozygous 3′RR-deficient/Igλ-Myc (∆/wt) and homozygous 3′RR-deficient/Igλ-Myc mice (∆/∆). B. Percentage of total (mature B-cell lymphomas plus plasmablastic B-cell lymphomas) mature B-cell lymphomas in Igλ-Myc (wt), heterozygous 3′RR-deficient/Igλ-Myc (∆/wt) and homozygous 3′RR-deficient/Igλ-Myc mice (∆/∆). ^*p < 0.05, Z test for two population proportions. C. Percentage of total (mature B-cell lymphomas plus plasmabslastic B-cell lymphomas) CD43⁺ mature B-cell lymphomas in Igλ-Myc (wt), heterozygous 3′RR-deficient/Igλ-Myc (∆/wt) and homozygous 3′RR-deficient/Igλ-Myc mice (∆/∆). ^*p < 0.05, ^**p < 0.005, Z test for two population proportions. D. Percentage of total CD5⁺ B-cell lymphomas in Igλ-Myc (wt), heterozygous 3′RR-deficient/Igλ-Myc (∆/wt) and homozygous 3′RR-deficient/Igλ-Myc mice (∆/∆). ^*p < 0.05, Z test for two population proportions. E. Lymphoma clonality. Southern blot analysis was used to examine lymphoma clonality with a J_H4 probe. Genomic DNA was prepared and digested with EcoRI from lymph node cells of Igλ-Myc mice (wt) and homozygous 3′RR-deficient/Igλ-Myc mice (∆/∆). The arrow located the germinal band.

B-cell lymphoma phenotypes in IgH-c-myc transgenic mice. (A) A typical flow cytometry analysis for B-cell lymphoma phenotyping. One representative experiment for a B220⁺CD19⁺IgM⁺IgD⁺CD43⁻CD117⁻CD138⁻ B-cell lymphoma is shown. (B) Percentage of mature (IgM⁺IgD⁺) and immature (IgM^+/−IgD⁻) B-cell lymphomas (24 c-myc-KIE_μ, 19 c-myc-KIC_μ, and 37 c-myc-KIC_α mice). (C) Percentages of CD138⁺ mature B-cell lymphomas in c-myc-KIE_μ, c-myc-KIC_μ, and c-myc-KIC_α mice. The number (n) of mice for each group is indicated. (D) Percentages of CD19⁻ mature B-cell lymphomas in c-myc-KIE_μ, c-myc-KIC_μ, and c-myc-KIC_α mice. Same mice as in panel C.

PD-1⁺ Tim-3⁺ and PD-1⁺ Tim-3⁻ exhausted T cells significantly increase in mice with de novo mature B cell neoplasms. Our mouse model with conditional Utx loss and Braf^V600E expression only in mature B cells develops mature B cell neoplasms de novo with time. Sick mice were euthanized, and BM, the spleen, and LN cells were harvested and analyzed using flow cytometry. Cre-negative mice were also analyzed as controls. a Representative flow plots of PD-1 and Tim-3 expression on CD8⁺ T cells in BM, the spleen, and LNs of control or sick mice. b, c The proportion of PD-1⁺ Tim-3⁺ T cells (b) and PD-1⁺ Tim-3⁻ T cells (c) in BM, the spleen, and LNs of control or sick mice, expressed as a percentage of total CD8⁺ T cells (BM: control n = 26, mature B cell neoplasms n = 17 [plasma cell neoplasms 7, B cell lymphoma 8, LPD 2]; spleen: control n = 28, mature B cell neoplasms n = 20 [plasma cell neoplasms 10, B cell lymphoma 8, LPD 2]; LN: control n = 18, mature B cell neoplasms n = 17 [plasma cell neoplasms 8, B cell lymphoma 7, LPD 2]). d, e The proportion of PD-1⁺ Tim-3⁺ T cells (d) and PD-1⁺ Tim-3⁻ T cells (e) to CD8⁺ T cells in invasion-negative or -positive organs (BM: invasion-negative n = 6 [plasma cell neoplasms 2, B cell lymphoma 4], -positive n = 11 [plasma cell neoplasms 5, B cell lymphoma 4, LPD 2]; spleen: invasion-negative n = 4 [plasma cell neoplasms 2, B cell lymphoma 2], -positive n = 16 [plasma cell neoplasms 8, B cell lymphoma 6, LPD 2]; LN: invasion-negative n = 4 [plasma cell neoplasms 1, B cell lymphoma 2, LPD 1], -positive n = 13 [plasma cell neoplasms 7, B cell lymphoma 5, LPD 1]). The significance of differences was assessed by an unpaired t-test. Horizontal bars indicate means, and error bars represent SD