Figure 5.
Human IL-7Rα expression leads to the dose-dependent development of T-cell leukemias that are sensitive to Bcl-2 inhibition. (A) Flow cytometry analysis for hIL-7Rα within Linneg thymocytes from 12-week-old animals of the indicated genotypes. FMO, fluorescence minus 1 negative control. (B) Difference from FMO of MFI of hIL-7Rα within each thymocyte subpopulation in heterozygous or homozygous animals. Standard deviation (SD) is indicated. **P < .01; ***P < .001; ****P < .0001; Student t test. (C) Survival corresponding to the indicated genotypes. No leukemias were observed in the CD2neg hIL-7R+/− cohort (not shown). *P < .05; log-rank, Mantel-Cox test . CD2neg hIL-7R+/+; n = 23; CD2pos hIL-7R+/−; n = 28; and CD2pos hIL7R+/+; n = 18. (D-F) Analysis of a representative leukemic animal euthanized when moribund. (D) Dot plots show CD4/CD8 coreceptor and CD8/TCRβ expression within Linneg thymocytes (Thy) and the presence of the same cells in the spleen (Spn) and bone marrow (BM). (E) CD8/Ki67 expression in thymus (Thy), Spn, and BM. (F) Animal that presented with a very large thymus and enlarged spleen vs control. (G) Survival of Rag−/−γc−/− or Rag−/−γc−/−IL-7−/− recipients of leukemic cells (4 × 105; n = 8 per group). ***P < .001, log-rank, Mantel-Cox. (H) Mutational burden map of single-nucleotide and indel variants with predicted high and moderate impact in functionally relevant genes and drivers of T-ALL or pediatric leukemias in CD2pos hIL-7R leukemias. (I) Il7 messenger RNA expression levels relative to Hprt1 in leukemic cells from CD2pos hIL-7R leukemias were quantified by quantitative real-time reverse transcription polymerase chain reaction. Average of triplicate experiments and SD are shown. (J) Bcl-2 flow cytometry analysis of CD4posTCRβpos normal SP thymocytes and CD8posTCRβneg leukemic cells of the same animal as in panels D-F. (K) Cells from 2 different leukemias (12895 and 14941) were cultured in the presence of the indicated doses of the Bcl-2 inhibitor venetoclax and in the absence (red bars) or presence (purple bars) of IL-7. Data show viability at 48 hours. One-way analysis of variance with Tukey’s correction for multiple comparisons. #P < .0001, venetoclax in the presence of IL-7 vs IL-7 alone; §P < .0001, venetoclax in the absence of IL-7 vs medium alone.

Human IL-7Rα expression leads to the dose-dependent development of T-cell leukemias that are sensitive to Bcl-2 inhibition. (A) Flow cytometry analysis for hIL-7Rα within Linneg thymocytes from 12-week-old animals of the indicated genotypes. FMO, fluorescence minus 1 negative control. (B) Difference from FMO of MFI of hIL-7Rα within each thymocyte subpopulation in heterozygous or homozygous animals. Standard deviation (SD) is indicated. **P < .01; ***P < .001; ****P < .0001; Student t test. (C) Survival corresponding to the indicated genotypes. No leukemias were observed in the CD2neg hIL-7R+/− cohort (not shown). *P < .05; log-rank, Mantel-Cox test . CD2neg hIL-7R+/+; n = 23; CD2pos hIL-7R+/−; n = 28; and CD2pos hIL7R+/+; n = 18. (D-F) Analysis of a representative leukemic animal euthanized when moribund. (D) Dot plots show CD4/CD8 coreceptor and CD8/TCRβ expression within Linneg thymocytes (Thy) and the presence of the same cells in the spleen (Spn) and bone marrow (BM). (E) CD8/Ki67 expression in thymus (Thy), Spn, and BM. (F) Animal that presented with a very large thymus and enlarged spleen vs control. (G) Survival of Rag−/−γc−/− or Rag−/−γc−/−IL-7−/− recipients of leukemic cells (4 × 105; n = 8 per group). ***P < .001, log-rank, Mantel-Cox. (H) Mutational burden map of single-nucleotide and indel variants with predicted high and moderate impact in functionally relevant genes and drivers of T-ALL or pediatric leukemias in CD2pos hIL-7R leukemias. (I) Il7 messenger RNA expression levels relative to Hprt1 in leukemic cells from CD2pos hIL-7R leukemias were quantified by quantitative real-time reverse transcription polymerase chain reaction. Average of triplicate experiments and SD are shown. (J) Bcl-2 flow cytometry analysis of CD4posTCRβpos normal SP thymocytes and CD8posTCRβneg leukemic cells of the same animal as in panels D-F. (K) Cells from 2 different leukemias (12895 and 14941) were cultured in the presence of the indicated doses of the Bcl-2 inhibitor venetoclax and in the absence (red bars) or presence (purple bars) of IL-7. Data show viability at 48 hours. One-way analysis of variance with Tukey’s correction for multiple comparisons. #P < .0001, venetoclax in the presence of IL-7 vs IL-7 alone; §P < .0001, venetoclax in the absence of IL-7 vs medium alone.

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