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Fli-1 overexpression in hematopoietic progenitors deregulates T cell development and induces pre-T cell lymphoblastic leukaemia/lymphoma.

Smeets MF, Chan AC, Dagger S, Bradley CK, Wei A, Izon DJ - PLoS ONE (2013)

Bottom Line: To address this we retrovirally overexpressed Fli-1 in various in vitro and in vivo settings and analysed its effect on T cell development.We found that Fli-1 overexpression perturbed the DN to DP transition and inhibited CD4 development whilst enhancing CD8 development both in vitro and in vivo.In contrast, we found increased NOTCH1 expression in all Fli-1 T cells and detected Notch1 mutations in all tumours.

View Article: PubMed Central - PubMed

Affiliation: Haematology and Leukaemia Unit, St. Vincent's Institute, University of Melbourne, Fitzroy, Victoria, Australia.

ABSTRACT
The Ets transcription factor Fli-1 is preferentially expressed in hematopoietic tissues and cells, including immature T cells, but the role of Fli-1 in T cell development has not been closely examined. To address this we retrovirally overexpressed Fli-1 in various in vitro and in vivo settings and analysed its effect on T cell development. We found that Fli-1 overexpression perturbed the DN to DP transition and inhibited CD4 development whilst enhancing CD8 development both in vitro and in vivo. Surprisingly, Fli-1 overexpression in vivo eventuated in development of pre-T cell lymphoblastic leukaemia/lymphoma (pre-T LBL). Known Fli-1 target genes such as the pro-survival Bcl-2 family members were not found to be upregulated. In contrast, we found increased NOTCH1 expression in all Fli-1 T cells and detected Notch1 mutations in all tumours. These data show a novel function for Fli-1 in T cell development and leukaemogenesis and provide a new mouse model of pre-T LBL to identify treatment options that target the Fli-1 and Notch1 signalling pathways.

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Fli-1 overexpression perturbs T cell development in vitro and in vivo.A. FL cells were transduced with MigR1 or Fli-1 and used to reconstitute foetal thymic lobes. Cultures were analysed 14 days later with anti-CD4 and CD8 by flow cytometry. Representative plot of at least 4 independent experiments. B. Graph of GFP+ FTOC MigR1 and Fli-1 thymocyte populations. Data are represented as mean ± SEM; n = 4. C. MigR1 and Fli-1 transduced FL cells grown on OP9-DL1 cells for 8 days were analysed for presence of DN1–4 by flow cytometry using Lin−, CD25 and CD44. Plot representative of 5 independent experiments. D. Graph of GFP+ DN cells of MigR1 and Fli-1 transduced FL grown on OP9-DL1 cells. Data are represented as mean ± SEM; n = 5. E. BM cells were transduced with MigR1 or Fli-1 and used to transplant lethally irradiated mice. Pre-leukaemic thymocytes were analysed 3 months later with anti-CD4 and CD8 by flow cytometry. F. Graph of GFP+in vivo MigR1 versus Fli-1 pre-leukaemic thymocyte populations. Data are represented mean ± SEM; n = 4.G. Expression of Fli-1 mRNA in thymocyte subsets (mean ± SEM). Sorted DN1, DN2, DN3, DN4, ISP CD8, DP, CD4 SP and CD8 SP thymocytes were subjected to QRT-PCR for Fli-1. DN data: n = 3;. DP and SP data: n = 2.
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pone-0062346-g001: Fli-1 overexpression perturbs T cell development in vitro and in vivo.A. FL cells were transduced with MigR1 or Fli-1 and used to reconstitute foetal thymic lobes. Cultures were analysed 14 days later with anti-CD4 and CD8 by flow cytometry. Representative plot of at least 4 independent experiments. B. Graph of GFP+ FTOC MigR1 and Fli-1 thymocyte populations. Data are represented as mean ± SEM; n = 4. C. MigR1 and Fli-1 transduced FL cells grown on OP9-DL1 cells for 8 days were analysed for presence of DN1–4 by flow cytometry using Lin−, CD25 and CD44. Plot representative of 5 independent experiments. D. Graph of GFP+ DN cells of MigR1 and Fli-1 transduced FL grown on OP9-DL1 cells. Data are represented as mean ± SEM; n = 5. E. BM cells were transduced with MigR1 or Fli-1 and used to transplant lethally irradiated mice. Pre-leukaemic thymocytes were analysed 3 months later with anti-CD4 and CD8 by flow cytometry. F. Graph of GFP+in vivo MigR1 versus Fli-1 pre-leukaemic thymocyte populations. Data are represented mean ± SEM; n = 4.G. Expression of Fli-1 mRNA in thymocyte subsets (mean ± SEM). Sorted DN1, DN2, DN3, DN4, ISP CD8, DP, CD4 SP and CD8 SP thymocytes were subjected to QRT-PCR for Fli-1. DN data: n = 3;. DP and SP data: n = 2.

Mentions: As Fli-1 is expressed throughout T cell development it was hypothesized that retroviral Fli-1 overexpression could potentially perturb T cell differentiation. In order to test this, we transduced foetal liver (FL) with control MigR1-GFP retrovirus or Fli-1-GFP retrovirus and reconstituted foetal thymic lobes. Fourteen days later, thymocytes were analysed by flow cytometry using antibodies to CD4 and CD8. It can be clearly seen that the Fli-1 foetal thymic organ cultures (FTOC) demonstrated a significant inhibition of the DN to DP transition as evidenced by an increased percentage of DN and a decreased percentage of DP cells (Figure 1A–B). There was also a significant reduction in the percentage of the CD4+ SP population in Fli-1 FTOCs. Finally, an expansion of the percentage of immature (TCRβ−/lo) CD8+ SP (ISP) cells was found in Fli-1 FTOCs.


Fli-1 overexpression in hematopoietic progenitors deregulates T cell development and induces pre-T cell lymphoblastic leukaemia/lymphoma.

Smeets MF, Chan AC, Dagger S, Bradley CK, Wei A, Izon DJ - PLoS ONE (2013)

Fli-1 overexpression perturbs T cell development in vitro and in vivo.A. FL cells were transduced with MigR1 or Fli-1 and used to reconstitute foetal thymic lobes. Cultures were analysed 14 days later with anti-CD4 and CD8 by flow cytometry. Representative plot of at least 4 independent experiments. B. Graph of GFP+ FTOC MigR1 and Fli-1 thymocyte populations. Data are represented as mean ± SEM; n = 4. C. MigR1 and Fli-1 transduced FL cells grown on OP9-DL1 cells for 8 days were analysed for presence of DN1–4 by flow cytometry using Lin−, CD25 and CD44. Plot representative of 5 independent experiments. D. Graph of GFP+ DN cells of MigR1 and Fli-1 transduced FL grown on OP9-DL1 cells. Data are represented as mean ± SEM; n = 5. E. BM cells were transduced with MigR1 or Fli-1 and used to transplant lethally irradiated mice. Pre-leukaemic thymocytes were analysed 3 months later with anti-CD4 and CD8 by flow cytometry. F. Graph of GFP+in vivo MigR1 versus Fli-1 pre-leukaemic thymocyte populations. Data are represented mean ± SEM; n = 4.G. Expression of Fli-1 mRNA in thymocyte subsets (mean ± SEM). Sorted DN1, DN2, DN3, DN4, ISP CD8, DP, CD4 SP and CD8 SP thymocytes were subjected to QRT-PCR for Fli-1. DN data: n = 3;. DP and SP data: n = 2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646842&req=5

pone-0062346-g001: Fli-1 overexpression perturbs T cell development in vitro and in vivo.A. FL cells were transduced with MigR1 or Fli-1 and used to reconstitute foetal thymic lobes. Cultures were analysed 14 days later with anti-CD4 and CD8 by flow cytometry. Representative plot of at least 4 independent experiments. B. Graph of GFP+ FTOC MigR1 and Fli-1 thymocyte populations. Data are represented as mean ± SEM; n = 4. C. MigR1 and Fli-1 transduced FL cells grown on OP9-DL1 cells for 8 days were analysed for presence of DN1–4 by flow cytometry using Lin−, CD25 and CD44. Plot representative of 5 independent experiments. D. Graph of GFP+ DN cells of MigR1 and Fli-1 transduced FL grown on OP9-DL1 cells. Data are represented as mean ± SEM; n = 5. E. BM cells were transduced with MigR1 or Fli-1 and used to transplant lethally irradiated mice. Pre-leukaemic thymocytes were analysed 3 months later with anti-CD4 and CD8 by flow cytometry. F. Graph of GFP+in vivo MigR1 versus Fli-1 pre-leukaemic thymocyte populations. Data are represented mean ± SEM; n = 4.G. Expression of Fli-1 mRNA in thymocyte subsets (mean ± SEM). Sorted DN1, DN2, DN3, DN4, ISP CD8, DP, CD4 SP and CD8 SP thymocytes were subjected to QRT-PCR for Fli-1. DN data: n = 3;. DP and SP data: n = 2.
Mentions: As Fli-1 is expressed throughout T cell development it was hypothesized that retroviral Fli-1 overexpression could potentially perturb T cell differentiation. In order to test this, we transduced foetal liver (FL) with control MigR1-GFP retrovirus or Fli-1-GFP retrovirus and reconstituted foetal thymic lobes. Fourteen days later, thymocytes were analysed by flow cytometry using antibodies to CD4 and CD8. It can be clearly seen that the Fli-1 foetal thymic organ cultures (FTOC) demonstrated a significant inhibition of the DN to DP transition as evidenced by an increased percentage of DN and a decreased percentage of DP cells (Figure 1A–B). There was also a significant reduction in the percentage of the CD4+ SP population in Fli-1 FTOCs. Finally, an expansion of the percentage of immature (TCRβ−/lo) CD8+ SP (ISP) cells was found in Fli-1 FTOCs.

Bottom Line: To address this we retrovirally overexpressed Fli-1 in various in vitro and in vivo settings and analysed its effect on T cell development.We found that Fli-1 overexpression perturbed the DN to DP transition and inhibited CD4 development whilst enhancing CD8 development both in vitro and in vivo.In contrast, we found increased NOTCH1 expression in all Fli-1 T cells and detected Notch1 mutations in all tumours.

View Article: PubMed Central - PubMed

Affiliation: Haematology and Leukaemia Unit, St. Vincent's Institute, University of Melbourne, Fitzroy, Victoria, Australia.

ABSTRACT
The Ets transcription factor Fli-1 is preferentially expressed in hematopoietic tissues and cells, including immature T cells, but the role of Fli-1 in T cell development has not been closely examined. To address this we retrovirally overexpressed Fli-1 in various in vitro and in vivo settings and analysed its effect on T cell development. We found that Fli-1 overexpression perturbed the DN to DP transition and inhibited CD4 development whilst enhancing CD8 development both in vitro and in vivo. Surprisingly, Fli-1 overexpression in vivo eventuated in development of pre-T cell lymphoblastic leukaemia/lymphoma (pre-T LBL). Known Fli-1 target genes such as the pro-survival Bcl-2 family members were not found to be upregulated. In contrast, we found increased NOTCH1 expression in all Fli-1 T cells and detected Notch1 mutations in all tumours. These data show a novel function for Fli-1 in T cell development and leukaemogenesis and provide a new mouse model of pre-T LBL to identify treatment options that target the Fli-1 and Notch1 signalling pathways.

Show MeSH
Related in: MedlinePlus