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Blood, Vol. 109, Issue 1, 281-289, January 1, 2007
Oncogenic role of Pax5 in the T-lymphoid lineage upon ectopic expression from the immunoglobulin heavy-chain locus
Blood Souabni et al.
109: 281
Supplemental materials for: Souabni et al, Vol 109, Issue 1, 281-289
Files in this Data Supplement:
- Document S1. Supplemental materials and methods (PDF, 31.6 KB).
- Figure S1. Partial block of B-cell development at the pro–B- to pre–B-cell transition in IgHP5ki/+ Pax5–/– mice (PDF, 174 KB) -
Bone marrow (A) and spleen (B) of 2-month-old wild-type, Pax5–/–, and IgHP5ki/+ Pax5–/–- mice were analyzed by flow cytometry for expression of the indicated B-cell markers. The percentage of cells is shown in the quadrants. Similar results were obtained with 3 additional IgHP5ki/+ Pax5–/–- mice.
- Figure S2. Failure of IgHP5ki/+ progenitors to contribute to T lymphopoiesis in mixed bone marrow chimeras (PDF, 45.4 KB) -
(A) Hematopoiesis. Bone marrow cells from IgHP5ki/+ mice (Ly5.2/Ly5.2) and IgHµMT/+ mice (Ly5.1/Ly5.2) were used at a ratio of 1:1 to reconstitute lethally irradiated wild-type C57BL/6 mice (Ly5.1/Ly5.1). The IgHµMT/+ mouse was used as a reference because it also carries 1 nonfunctional IgH allele due to deletion of the µ membrane exon (µMT).1 Nine months after transplantation, the donor bone marrow cells were identified as Ly5.2+ cells, and the different cell types were defined by the second gate indicated. The Ly5.1 expression profile of the gated cells is shown to the right together with the relative percentage of Ly5.1– (IgHP5ki/+) and Ly5.1+ (IgHµMT/+) cells. This analysis revealed a chimerism of 28:72 (P5ki/µMT) for granulocytes (Gr1+Mac1+) and 49:51 for B cells (CD19+B220+), indicating that B cells preferentially developed from the IgHP5ki/+ HSCs. (B) T lymphopoiesis. IgHP5ki/+ progenitors (Ly5.2/Ly5.2) failed to contribute to T-cell development because the earliest CD44+CD25– (DN1) thymocyte precursors and all subsequent developmental stages (DN, DP, and SP cells) expressed the Ly5.1 marker characteristic of the IgHµMT/+ donor cells.
- Figure S3. Increased pro-B cell compartment in IgHP5ki mutant mice (PDF, 62.7 KB) -
(A) Expansion of the pro–B-cell compartment in IgHP5ki mutant mice. Bone marrow cells from 3-week-old mice of the indicated genotypes were analyzed by flow cytometry, and the relative percentages of B220+ B lymphocytes and Mac1+ myeloid cells are shown in the respective quadrants. (B) A 2-fold increase of pro-B cells. The percentage of c-Kit+B220+ pro-B cells and Mac1+ myeloid cells of the total bone marrow are shown together with the standard deviation for each genotype (n ≥ 4). (C) Normal B-cell development past the pro–B-cell stage in IgHP5ki/+ mice (ki/+). The percentages of pre-B cells (B220+IgM–), B cells (B220+IgM+), and total B lymphocytes (B220+CD19+) are shown for the bone marrow of wild-type and IgHP5ki/+ mice at the age of 3 weeks (n=4/genotype).
- Figure S4. Infiltration of adjacent tissues and different organs by IgHP5ki/P5ki lymphoma cells (PDF, 794 KB) -
(A,B) Infiltration of lymphoma cells from the lymph node into the adjacent tissue in tumor mice 11 (A) and 81 (B). Histologic sections of the lymph nodes were stained with eosin and hematoxylin. A black line indicates the limit of the lymph node capsule. (C,D) Infiltration of secondary lymphoma cells in the spleen (D), liver (E), and kidney (F) of recipient mouse 4 that was intravenously injected with lymphoma cells from the thymus of the IgHP5ki/P5ki tumor mouse 14. Histologic analysis of the different organs was performed 7 weeks after cell transplantation. The spleen architecture of the recipient mouse 4-14 (D) is completely disrupted in comparison to a control wild-type spleen (C) with its typical B-cell follicles (FO).
- Figure S5. Transplantation and in vitro culturing of IgHP5ki/P5ki tumor cells (PDF, 72.7 KB) -
Thymocytes of the IgHP5ki/P5ki tumor mouse 15 were analyzed by flow cytometry as well as by injection into the tail vein of syngeneic wild-type mice. The thymocytes of a mouse given a transplant were analyzed 44 days after injection, whereas the remaining cells were cultured in vitro on stromal ST2 cells for 10 days prior to flow cytometric analysis.
- Figure S6. TCRb rearrangements in Pax5-induced T-cell lymphomas (PDF, 79.2 KB) -
(A) The presence of different Dβ2-Jβ2.4 and Dβ2-Jβ2.3 rearrangements in lymphoma cells of the spleen (S) and thymus (T) of the IgHP5ki/P5ki tumor mouse 19 and IgHP5ki/+ tumor mouse 34, respectively. Open and closed symbols denote the PCR fragments for which the DNA sequences are shown below. The Dβ2 and Jβ2 sequences are indicated in boldface and the nucleotides at their junction in small letters. C indicates control tail DNA; GL, germline PCR product. (B) PCR analysis of Dβ1-Jβ1 and Dβ2-Jβ2 rearrangements in lymphomas from the spleen and thymus of tumor mouse 34.
- Figure S7. GFP expression of DN and DP thymocytes in IkPax5/+ mice (PDF, 51.4 KB) -
GFP expression (gray surface) is shown for IkPax5/+ DN and DP thymocytes, whereas the black line indicates the absence of GFP expression in the corresponding wild-type thymocytes.
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