Article

Transcription factor Hoxb5 reprograms B cells into functional T lymphocytes

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Abstract

Deletion of master regulators of the B cell lineage reprograms B cells into T cells. Here we found that the transcription factor Hoxb5, which is expressed in uncommitted hematopoietic progenitor cells but is not present in cells committed to the B cell or T cell lineage, was able to reprogram pro-pre-B cells into functional early T cell lineage progenitors. This reprogramming started in the bone marrow and was completed in the thymus and gave rise to T lymphocytes with transcriptomes, hierarchical differentiation, tissue distribution and immunological functions that closely resembled those of their natural counterparts. Hoxb5 repressed B cell ‘master genes’, activated regulators of T cells and regulated crucial chromatin modifiers in pro-pre-B cells and ultimately drove the B cell fate–to–T cell fate conversion. Our results provide a de novo paradigm for the generation of functional T cells through reprogramming in vivo.

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References

  1. 1.

    Heyworth, C., Pearson, S., May, G. & Enver, T. Transcription factor-mediated lineage switching reveals plasticity in primary committed progenitor cells. EMBO J. 21, 3770–3781 (2002).

  2. 2.

    Kulessa, H., Frampton, J. & Graf, T. GATA-1 reprograms avian myelomonocytic cell lines into eosinophils, thromboblasts, and erythroblasts. Genes Dev. 9, 1250–1262 (1995).

  3. 3.

    Visvader, J. E., Elefanty, A. G., Strasser, A. & Adams, J. M. GATA-1 but not SCL induces megakaryocytic differentiation in an early myeloid line. EMBO J. 11, 4557–4564 (1992).

  4. 4.

    Xie, H., Ye, M., Feng, R. & Graf, T. Stepwise reprogramming of B cells into macrophages. Cell 117, 663–676 (2004).

  5. 5.

    Nutt, S. L., Heavey, B., Rolink, A. G. & Busslinger, M. Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature 401, 556–562 (1999).

  6. 6.

    Rolink, A. G., Nutt, S. L., Melchers, F. & Busslinger, M. Long-term in vivo reconstitution of T-cell development by Pax5-deficient B-cell progenitors. Nature 401, 603–606 (1999).

  7. 7.

    Taghon, T., Yui, M. A. & Rothenberg, E. V. Mast cell lineage diversion of T lineage precursors by the essential T cell transcription factor GATA-3. Nat. Immunol. 8, 845–855 (2007).

  8. 8.

    Laiosa, C. V., Stadtfeld, M., Xie, H., de Andres-Aguayo, L. & Graf, T. Reprogramming of committed T cell progenitors to macrophages and dendritic cells by C/EBPα and PU.1 transcription factors. Immunity 25, 731–744 (2006).

  9. 9.

    Li, P. et al. Reprogramming of T cells to natural killer-like cells upon Bcl11b deletion. Science 329, 85–89 (2010).

  10. 10.

    Cobaleda, C., Jochum, W. & Busslinger, M. Conversion of mature B cells into T cells by dedifferentiation to uncommitted progenitors. Nature 449, 473–477 (2007).

  11. 11.

    Ungerbäck, J., Åhsberg, J., Strid, T., Somasundaram, R. & Sigvardsson, M. Combined heterozygous loss of Ebf1 and Pax5 allows for T-lineage conversion of B cell progenitors. J. Exp. Med. 212, 1109–1123 (2015).

  12. 12.

    Orkin, S. H. & Zon, L. I. Hematopoiesis: an evolving paradigm for stem cell biology. Cell 132, 631–644 (2008).

  13. 13.

    Månsson, R. et al. Molecular evidence for hierarchical transcriptional lineage priming in fetal and adult stem cells and multipotent progenitors. Immunity 26, 407–419 (2007).

  14. 14.

    Chen, J. Y. et al. Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche. Nature 530, 223–227 (2016).

  15. 15.

    Bijl, J. et al. Analysis of HSC activity and compensatory Hox gene expression profile in Hoxb cluster mutant fetal liver cells. Blood 108, 116–122 (2006).

  16. 16.

    Riddell, J. et al. Reprogramming committed murine blood cells to induced hematopoietic stem cells with defined factors. Cell 157, 549–564 (2014).

  17. 17.

    Ramasamy, I., Brisco, M. & Morley, A. Improved PCR method for detecting monoclonal immunoglobulin heavy chain rearrangement in B cell neoplasms. J. Clin. Pathol. 45, 770–775 (1992).

  18. 18.

    Ehlich, A., Martin, V., Müller, W. & Rajewsky, K. Analysis of the B-cell progenitor compartment at the level of single cells. Curr. Biol. 4, 573–583 (1994).

  19. 19.

    Souabni, A., Cobaleda, C., Schebesta, M. & Busslinger, M. Pax5 promotes B lymphopoiesis and blocks T cell development by repressing Notch1. Immunity 17, 781–793 (2002).

  20. 20.

    Nechanitzky, R. et al. Transcription factor EBF1 is essential for the maintenance of B cell identity and prevention of alternative fates in committed cells. Nat. Immunol. 14, 867–875 (2013).

  21. 21.

    Lin, H. & Grosschedl, R. Failure of B-cell differentiation in mice lacking the transcription factor EBF. Nature 376, 263–267 (1995).

  22. 22.

    Liu, P. et al. Bcl11a is essential for normal lymphoid development. Nat. Immunol. 4, 525–532 (2003).

  23. 23.

    Hu, H. et al. Foxp1 is an essential transcriptional regulator of B cell development. Nat. Immunol. 7, 819–826 (2006).

  24. 24.

    Lin, Y. C. et al. A global network of transcription factors, involving E2A, EBF1 and Foxo1, that orchestrates B cell fate. Nat. Immunol. 11, 635–643 (2010).

  25. 25.

    Müller, M. R. et al. Requirement for balanced Ca/NFAT signaling in hematopoietic and embryonic development. . Proc. Natl. Acad. Sci. USA 106, 7034–7039 (2009).

  26. 26.

    Braunstein, M. & Anderson, M. K. HEB in the spotlight: Transcriptional regulation of T-cell specification, commitment, and developmental plasticity. Clin. Dev. Immunol. 2012, 678705 (2012).

  27. 27.

    Martins, G. & Calame, K. Regulation and functions of Blimp-1 in T and B lymphocytes. Annu. Rev. Immunol. 26, 133–169 (2008).

  28. 28.

    Ferrando, A. A. et al. Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia. Cancer Cell 1, 75–87 (2002).

  29. 29.

    Schwickert, T. A. et al. Stage-specific control of early B cell development by the transcription factor Ikaros. Nat. Immunol. 15, 283–293 (2014).

  30. 30.

    Schebesta, A. et al. Transcription factor Pax5 activates the chromatin of key genes involved in B cell signaling, adhesion, migration, and immune function. Immunity 27, 49–63 (2007).

  31. 31.

    McManus, S. et al. The transcription factor Pax5 regulates its target genes by recruiting chromatin-modifying proteins in committed B cells. EMBO J. 30, 2388–2404 (2011).

  32. 32.

    McMahon, K. A. et al. Mll has a critical role in fetal and adult hematopoietic stem cell self-renewal. Cell Stem Cell 1, 338–345 (2007).

  33. 33.

    Artinger, E. L. et al. An MLL-dependent network sustains hematopoiesis. Proc. Natl. Acad. Sci. USA 110, 12000–12005 (2013).

  34. 34.

    Cobaleda, C., Schebesta, A., Delogu, A. & Busslinger, M. Pax5: the guardian of B cell identity and function. Nat. Immunol. 8, 463–470 (2007).

  35. 35.

    Vaillant, F., Blyth, K., Andrew, L., Neil, J. C. & Cameron, E. R. Enforced expression of Runx2 perturbs T cell development at a stage coincident with β-selection. J. Immunol. 169, 2866–2874 (2002).

  36. 36.

    Zhang, H. et al. MLL1 inhibition reprograms epiblast stem cells to naive pluripotency. Cell Stem Cell 18, 481–494 (2016).

  37. 37.

    Zhou, X., Marks, P. A., Rifkind, R. A. & Richon, V. M. Cloning and characterization of a histone deacetylase, HDAC9. Proc. Natl. Acad. Sci. USA 98, 10572–10577 (2001).

  38. 38.

    Krivega, I., Dale, R. K. & Dean, A. Role of LDB1 in the transition from chromatin looping to transcription activation. Genes Dev. 28, 1278–1290 (2014).

  39. 39.

    Kokavec, J. et al. The ISWI ATPase Smarca5 (Snf2h) is required for proliferation and differentiation of hematopoietic stem and progenitor cells. Stem Cells 35, 1614–1623 (2017).

  40. 40.

    Starr, T. K., Jameson, S. C. & Hogquist, K. A. Positive and negative selection of T cells. Annu. Rev. Immunol. 21, 139–176 (2003).

  41. 41.

    Limón, A. et al. High-titer retroviral vectors containing the enhanced green fluorescent protein gene for efficient expression in hematopoietic cells. Blood 90, 3316–3321 (1997).

  42. 42.

    Goldschneider, I., Komschlies, K. L. & Greiner, D. L. Studies of thymocytopoiesis in rats and mice. I. Kinetics of appearance of thymocytes using a direct intrathymic adoptive transfer assay for thymocyte precursors. J. Exp. Med. 163, 1–17 (1986).

  43. 43.

    Wolfer, A., Wilson, A., Nemir, M., MacDonald, H. R. & Radtke, F. Inactivation of Notch1 impairs VDJβ rearrangement and allows pre-TCR-independent survival of early αβ lineage thymocytes. Immunity 16, 869–879 (2002).

  44. 44.

    Ye, J., Ma, N., Madden, T. L. & Ostell, J. M. IgBLAST: an immunoglobulin variable domain sequence analysis tool. Nucleic Acids Res. 41, W34–40 (2013).

  45. 45.

    Tang, F. et al. RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nat. Protoc. 5, 516–535 (2010).

  46. 46.

    Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).

  47. 47.

    Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

  48. 48.

    Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).

  49. 49.

    Yu, G., Wang, L. G., Han, Y. & He, Q. Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16, 284–287 (2012).

  50. 50.

    Trickett, A. & Kwan, Y. L. T cell stimulation and expansion using anti-CD3/CD28 beads. J. Immunol. Methods 275, 251–255 (2003).

  51. 51.

    Lan, P., Tonomura, N., Shimizu, A., Wang, S. & Yang, Y. G. Reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and CD34+ cell transplantation. Blood 108, 487–492 (2006).

  52. 52.

    Jiang, X. et al. Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity. Nature 483, 227–231 (2012).

  53. 53.

    Ai, S. et al. EED orchestration of heart maturation through interaction with HDACs is H3K27me3-independent. eLife 6, e24570 (2017).

  54. 54.

    He, A. et al. Dynamic GATA4 enhancers shape the chromatin landscape central to heart development and disease. Nat. Commun. 5, 4907 (2014).

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Acknowledgements

We thank T. Cheng, D. Pei and E. H. Bresnick for comments on the manuscript; Z. Liu (Institute of Biophysics, CAS, China) for Rag1−/− mice; and the animal center and instrument center of Guangzhou Institutes of Biomedicine and Health for the animal care, cell sorting and skin imaging. Supported by the Major National Research Project of China (2015CB964401 to J.W.; 2015CB964404 to Y.-G.Y. and Z.H.; 2015CB964902 to J.D.; and 2015CB964901 to H.W., CAS Key Research Program of Frontier Sciences (QYZDB-SSW-SMC057), the Major Scientific and Technological Project of Guangdong Province (2014B020225005), the Strategic Priority Research Program of the Chinese Academic of Sciences (XDA01020311), the co-operation program of the Guangdong Natural Science Foundation (2014A030312012), the National Natural Science Foundation of China (31471117 and 81470281 to J.W.; 31600948 to D.Y.; 91642208 to Y.-G.Y.; and 81770222 to D.W.), the National Key Research and Development Program of China (2017YFA0103401 to B.L.; and 2017YFA0103402 to A. H.) and the US National Institutes of Health (AI079087 and HL130724 to D.W.).

Author information

Author notes

  1. These authors contributed equally: Mengyun Zhang, Yong Dong and Fangxiao Hu.

Affiliations

  1. CAS Key Laboratory of Regenerative Biology and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China

    • Mengyun Zhang
    • , Yong Dong
    • , Fangxiao Hu
    • , Dan Yang
    • , Qianhao Zhao
    • , Cui Lv
    • , Ying Wang
    • , Chengxiang Xia
    • , Qitong Weng
    • , Xiaofei Liu
    • , Peiqing Zhou
    • , Tongjie Wang
    • , Yuxian Guan
    • , Rongqun Guo
    • , Lijuan Liu
    • , Yang Geng
    • , Hongling Wu
    • , Juan Du
    • , Jiekai Chen
    •  & Jinyong Wang
  2. University of Chinese Academy of Sciences, Beijing, China

    • Mengyun Zhang
    • , Yong Dong
    • , Ying Wang
    • , Chengxiang Xia
    • , Qitong Weng
    • , Peiqing Zhou
    • , Rongqun Guo
    • , Jiekai Chen
    •  & Jinyong Wang
  3. Institute of Molecular Medicine, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Peking University, Beijing, China

    • Chen Li
    •  & Aibin He
  4. The First Hospital and Institute of Immunology, Jilin University, Changchun, China

    • Zheng Hu
    •  & Yong-Guang Yang
  5. National Key Laboratory of Medical Immunology & Institute of Immunology, Second Military Medical University, Shanghai, China

    • Sheng Xu
  6. State Key Laboratory of Proteomics, Translational Medicine Center of Stem Cells, 307-Ivy Translational Medicine Center, Laboratory of Oncology, Affiliated Hospital, Academy of Military Medical Sciences, Beijing, China

    • Bing Liu
  7. Biomedical Research Center of South China, College of Life Sciences, Fujian Normal University, Fuzhou, Fujian, China

    • Demin Wang
  8. Blood Research Institute, Blood Center of Wisconsin, Milwaukee, WI, USA

    • Demin Wang
  9. Columbia Center for Translational Immunology, Columbia University College of Physicians and Surgeons, New York, NY, USA

    • Yong-Guang Yang

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Contributions

M.Z., Y.D. and F.H. performed the core experiments and contributed equally to this work; D.Y., Q.Z., C. Lv, Y.W., C.X., Q.W., X.L. and C. Li performed multiple experiments; P.Z., T.W., Y.G., R.G., L.L., Y.G. and H.W. performed certain in vitro experiments; J.D., Z.H., S.X., J.C., A.H., B.L., D.W., Y.-G.Y. and J.W. discussed the data and edited the manuscript; B.L., D.W. and J.W. wrote the manuscript; and J.W. designed the project and provided final approval of the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jinyong Wang.

Integrated supplementary information

  1. Supplementary Figure 1 Flow cytometry analysis of B cell development in recipients transplanted with 15-TF virus mixture transduced pro-pre-B cells

    (a) Flow cytometry analysis of the purity of sorted Ter119-Mac1-CD3-CD4-CD8-CD19+B220+CD93+IgM- pro-pre-B cells. (b) Schematic diagram of 15-TF virus transduced pro-pre-B cells transplantation strategy. (c) Flow cytometry analysis of donor derived CD19+B220+IgMhiIgDlo immature B and CD19+B220+IgMloIgDhi mature B cells in bone marrow (BM), spleen and peripheral blood (PB) of 15-TF pro-pre-B cell recipient mice (CD45.1+ C57BL/6) four weeks post-transplantation. (d) Statistical analysis of immature and mature B cells in BM, Spleen, and PB. Each symbol represents an individual mouse, and small horizontal lines indicate the mean (± s.d.). *P < 0.05, **P < 0.01, ***P < 0.001 (two-sided-independent Student’s t-test), n = 3 biological replicates. (e) Flow cytometry analysis of donor derived CD19+B220+CD93+IgM- pro-pre-B cells in the bone marrow of 15-TF mice. (f) Statistical analysis of donor derived pro-pre-B cells in BM of 15-TF pro-pre-B cell recipient mice (CD45.1+ C57BL/6). Each symbol represents an individual mouse, and small horizontal lines indicate the mean (± s.d.). ***P < 0.001 (two-sided-independent Student’s t-test, n = 3 biological replicates). Data are representative of two independent experiments (c, e).

  2. Supplementary Figure 2 Flow cytometry analysis of B and T cell development in Hoxb5-overepressing transgenic mice

    (a) Schematic diagram of Hoxb5 knock in mouse model. The Hoxb5-EGFP expression cassette was inserted into ROSA26 (Hoxb5LSL/+ mice). (b) Flow cytometry analysis of CD19+B220+IgMhiIgDlo immature B and CD19+B220+IgMloIgDhi mature B cells in BM, Spleen and PB of Hoxb5LSL/+ CD19-Cre and littermate Hoxb5LSL/+ control mice (8-week-old). Representative plots were shown. (c) Flow cytometry analysis of CD19+B220+CD93+IgM- pro-pre-B cells in the bone marrow of Hoxb5LSL/+ CD19-Cre and littermate Hoxb5LSL/+ control mice (8-week-old). Representative plots were shown. (d-e) Flow cytometry analysis of DN cells in thymus (d) gated from Ter119-Mac1-CD19- population, T cells in PB, Spleen, and LN (e) gated from Ter119-Mac1- population of Hoxb5LSL/+Vav-Cre mice and littermate control (8-week-old mice). Representative plots of DN cells in thymus and T cells in PB, Spleen, LN and BM were shown. (f) Flow cytometry analysis of Lin-CD44+c-kithiCD25- early T cell lineage progenitors (ETP) in thymus and BM from the Hoxb5LSL/+Vav-Cre mice and littermate control (8-week-old mice). Representative plots were shown. Data are representative of two independent experiments (b-f).

  3. Supplementary Figure 3 Competitive bone marrow transplantation using Hoxb5-overepressing transgenic mice or Hoxb5-deficent transgenic mice as donors

    (a-c) Flow cytometry analysis of hematopoietic lineages in PB (a), BM (b), and spleen (c) of representative Hoxb5LSL/+Vav-Cre and Hoxb5LSL/+ mice (12-week-old). (d) Flow cytometry analysis of Lin-CD127+c-kitintSca-1int common lymphoid progenitors (CLP) in bone marrow of representative Hoxb5LSL/+Vav-Cre and Hoxb5LSL/+ mice. (e) Flow cytometry analysis of double negative (DN) cells in thymus gated from Ter119-Mac1-CD19-CD45.2+ population. Hoxb5LSL/+Vav-Cre and Hoxb5LSL/+ mice were analyzed. (f) Competitive transplantation analysis of Hoxb5LSL/+Vav-Cre group and Hoxb5LSL/+ (Ctr group). Half million total bone marrow cells from either Hoxb5LSL/+Vav-Cre or Hoxb5LSL/+ mice (CD45.2+) with equivalent number of competitor cells (CD45.1+) were retro-orbitally transplanted into lethally irradiated (9 Gy) individual CD45.1+ recipients. Donor contributions in PB of recipient mice were shown. Control group (n = 5 mice), and Hoxb5LSL/+Vav-Cre group (n = 11 mice). Donor chimerism for total cells, Mac1+ myeloid cells,,CD19+ B cells and CD3+ T cells were shown respectively. Each symbol represents an individual mouse, and small horizontal lines indicate the mean (± s.d.). (g-i) Flow cytometry analysis of hematopoietic lineages in PB (g), BM (h), and spleen (i) of representative Hoxb5-/- and wild type littermate mice (12-week-old). (j) Flow cytometry analysis of Lin-CD127+c-kitintSca-1int CLP in bone marrow of representative Hoxb5-/- and wild type littermate mice (12-week-old). (k) Flow cytometry analysis of DN cells in thymus gated from Ter119-Mac1-CD19-CD45.2+ population of representative Hoxb5-/- and wild type littermate mice (12-week-old). (l) For competitive transplantation, half million total bone marrow cells from either Hoxb5-/- or WT mice (CD45.2+) with equivalent number of competitor cells (CD45.1+) were retro-orbitally transplanted into lethally irradiated (9 Gy) individual CD45.1+ recipients. Donor contributions in peripheral blood of recipient mice were shown. Control group (n = 5 mice), and Hoxb5-/- group (n = 7 mice). Donor chimerism for total cells, Mac1+ myeloid cells, CD19+ B cells and CD3+ T cells were shown respectively. Each symbol represents an individual mouse, and small horizontal lines indicate the mean (± s.d.). Data are representative of three independent experiments (a-e, g-k).

  4. Supplementary Figure 4 PCR bands of B cell-specific BCR IgH V(D)J rearrangements and T cell-specific TCRβ V(D)J rearrangements in sorted single iT cells

    (a-c) PCR of B cell-specific VHJ558 and VHQ52 rearrangements and T cell-specific Vβ2-DJβ2, Vβ4-DJβ2, Vβ5.1-DJβ2 and Vβ8-Jβ2 rearrangements in sorted single iT cells of recipients transplanted with (a) retro-Hoxb5 pro-pre-B cells (Retro-iT), (b) single iT cells of CD19-Hoxb5 pro-pre-B recipients (KI-iT) and (c) single iT cells of Tet-Hoxb5 pro-pre-B recipients (Dox-iT) four weeks after transplantation. Wild-type bulk pro-pre-B cells and T cells were used as positive controls (PC). Water was used as DNA template negative control to exclude DNA contaminants (NC). 200 ng DNA of each amplified single lymphocyte genome was used as template for PCR of BCR IgH V(D)J and TCRβ V(D)J rearrangements.

  5. Supplementary Figure 5 iT cells generated from Rag1-/- mice exhibit immune functions

    (a) Three million sorted GFP+ pro-pre-B cells from CD19-Hoxb5 transgenic mice were transplanted into individual Rag1-/- recipient mice lacking mature T cells. Flow cytomerty analysis of PB were performed three weeks post-transplantation. Plots of three representative recipients from ten animals of three independent experiments were shown. (b) Images of allogeneic skin-grafted Rag1-/- and Hoxb5-Rag1-/- mice. Representative images of rejected allogeneic skin tissues from three mice (day 8) and successfully grafted skin tissue control (day 15) were taken. Three weeks before the skin transplantation, three million sorted GFP+ pro-pre-B cells from Hoxb5LSL/+CD19-Cre mice were transplanted into individual Rag1-/- mice. Donor skin tissues were from BALB/c mice.

  6. Supplementary Figure 6 Analysis of iT cells in Tet-Hoxb5 pro-pre-B recipients

    (a) Schematic diagram of targeting strategy using Tet-Hoxb5 transgenic model for conditional expression of Hoxb5. The indicated expression cassette Tet-Hoxb5-BFP was inserted into ROSA26 locus. The BFP reporting Hoxb5 can be induced by Doxycycline (Dox). (b) Schematic strategy of B to T conversion by transient expression of Hoxb5 using Tet-Hoxb5 model. (c-d) Fow cytometry analysis of the iT cells gated from Ter119-Mac1-CD19-CD45.2+ in the PB and spleen of Tet-Hoxb5 pro-pre-B cell recipients maintained on Dox-water (1 mg/mL) for four weeks (c) and recipients maintained on conditions of Dox withdrawal 2 weeks prior to analysis (d). Wild-type CD45.2 C57BL/6 mice were used as negative control. Recipient mice were analysed four weeks post-transplantation. (e) Q-PCR of ectopic Hoxb5 in BFP- and BFP+ thymic CD3+ T cells from Tet-Hoxb5-NOD-SCID mice. Wild-type CD45.2 C57BL/6 CD3+ thymic T cells were used as negative control (n = 6 biological replicates). Data are representative of three independent experiments (c, d).

  7. Supplementary Figure 7 Flow cytometry analysis of LSK and CLP cells in recipient mice, and T development after thymic transplantation

    (a) Flow cytometry analysis of phenotypic CD2-CD3-CD4-CD8-B220-Mac1-Gr1-Ter119- (Lin-)c-kit+Sca-1+ cells (LSK) and Lin-CD127+c-kitintSca-1int (CLP) cells in the bone marrow of 15-TF pro-pre-B cell recipients. (b) Flow cytometry analysis were performed on phenotypic CD2-CD3-CD4-CD8-B220-Mac1-Gr1-Ter119- (Lin-)c-kit+Sca-1+ cells (LSK) and Lin-CD127+c-kitintSca-1int (CLP) cells in the bone marrow of retro-Hoxb5 mice. (c) Flow cytometry analysis of phenotypic CD2-CD3-CD4-CD8-B220-Mac1-Gr1-Ter119- (Lin-) c-kit+Sca-1+ cells (LSK) and Lin-CD127+c-kitintSca-1int (CLP) cells in the bone marrow of CD19-Hoxb5 pro-pre-B cell recipient mice. (d) Flow cytometry analysis of phenotypic CD2-CD3-CD4-CD8-B220-Mac1-Gr1-Ter119- (Lin-)c-kit+Sca-1+ cells (LSK) and Lin-CD127+c-kitintSca-1int (CLP) cells in the bone marrow of recipients 4 weeks post-transplantation with BFP+ Tet-Hoxb5 pro-pre-B cells. The recipient mice were maintained on drinking water containing 1 mg/ml doxycycline one day prior to transplantation and for continuous two weeks. Data are representative of three independent experiments (a-d). (e-g) Flow cytometry analysis of DN cells in thymus (e) gated from Ter119-Mac1-CD19- population, T cells in lymph node (f), spleen and bone marrow (g) gated from Ter119-Mac1-CD19- population of recipients three weeks after thymic transplantation. A quarter of total wild type thymocytes (CD45.1+) were transplanted into sublethally irradiated individual recipients (CD45.2+) by intra-thymus injection. Data are representative of two independent experiments (e-g).

  8. Supplementary Figure 8 Flow cytometry analysis of iT lymphocytes in Bio-Hoxb5 pro-pre-B recipient mice, and Runx2 and Smarca5 CHIP-Seq peaks

    (a) Schematic diagram of Bio-tagged Hoxb5 vector cassettes. Bio-tagged Hoxb5 sequence was constructed into the pMY-IRES-GFP retro-vector. (b) Flow cytometry analysis of intra-cellular biotin in GFP control virus or Bio-Hoxb5 virus transduced pro-pre-B cells isolated from Rosa26BirA/BirA transgenic mice. Cells were analysed 72 hours post-virus transplantation. (c-d) Flow cytometry analysis of T cells in PB, LN and Spleen (c) gated from Ter119-Mac1-GFP+ population and DN cells in thymus (d) gated from Ter119-Mac1-CD19- GFP+ population of Bio-Hoxb5 pro-pre-B recipient mice six weeks post-transplantation. (e) ChIP-Seq profiles Hoxb5 binding tracks in pro-pre-B on Runx2 and Smarca5 gene locus. Data are representative of two independent experiments (b-e).

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1-8

  2. Life Sciences Reporting Summary

  3. Supplementary Table 1

    Primers used to identify the integrated ectopic genes

  4. Supplementary Table 2

    Single cell PCR analysis of integrated ectopic transcription factors in individual GFP+ T lymphocytes

  5. Supplementary Table 3

    Primers used for single cell PCR analysis of IgH V(D)J and Tcrb V(D)J rearrangements

  6. Supplementary Table 4

    Ig Blast results of BCR IgH V(D)J rearrangements and TCRβ V(D)J rearrangements in splenic T lymphocytes

  7. Supplementary Table 5

    Differential expression genes (> 2 fold, p adj < 0.05)

  8. Supplementary Table 6

    Differential expression genes (> 1.2 fold, p adj < 0.01)

  9. Supplementary Table 7

    Differential expression transcription factors (> 1.20 fold, p adj < 0.01)

  10. Supplementary Table 8

    Hoxb5-binding peak-related genes overlapped with DEGs of RNA-Seq in pro-pre-B cells

  11. Source Data, Figure 1

  12. Source Data, Figure 2

  13. Source Data, Figure 4

  14. Source Data, Figure 5

  15. Source Data, Figure 7