Transcription factor Hoxb5 reprograms B cells into functional T lymphocytes

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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Fig. 1: Screening for transcription factors involved in the B cell–to–T cell conversion.
Fig. 2: Expression of retro-Hoxb5 in pro-pre-B cells converts B cells into T lymphocytes in vivo.
Fig. 3: Conversion of B lymphocytes into T lymphocytes in the CD19-Hoxb5 model.
Fig. 4: Immunological function of Hoxb5-induced iT lymphocytes.
Fig. 5: Hoxb5-induced iT lymphocytes show normal immunological function in vivo.
Fig. 6: Transient expression of Hoxb5 reprograms B cells into T lymphocytes in the Tet-Hoxb5 model.
Fig. 7: Hoxb5 directly converts B lymphocytes into ETP-like cells in the BM.
Fig. 8: Hoxb5 targets in pro-pre-B cells.

Change history

  • 18 June 2018

    In the version of this article initially published, some identification of the supplementary information was incorrect. The items originally called Supplementary Tables 1, 2, 3, 4 and 5 should be Source Data Figures 1, 2, 4, 5 and 7, respectively; those originally called Supplementary Tables 6, 7 and 8 should be Supplementary Tables 1, 2 and 3, respectively; and those originally called Source Data Figures 1, 2, 4, 5 and 7 should be Supplementary Tables 4, 5, 6, 7 and 8, respectively. The errors have been corrected in the HTML version of the article.

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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.).

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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.

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Correspondence to Jinyong Wang.

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Integrated supplementary information

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).

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).

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).

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.

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.

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).

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).

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

Supplementary Text and Figures

Supplementary Figures 1-8

Life Sciences Reporting Summary

Supplementary Table 1

Primers used to identify the integrated ectopic genes

Supplementary Table 2

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

Supplementary Table 3

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

Supplementary Table 4

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

Supplementary Table 5

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

Supplementary Table 6

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

Supplementary Table 7

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

Supplementary Table 8

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

Source Data, Figure 1

Source Data, Figure 2

Source Data, Figure 4

Source Data, Figure 5

Source Data, Figure 7

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Zhang, M., Dong, Y., Hu, F. et al. Transcription factor Hoxb5 reprograms B cells into functional T lymphocytes. Nat Immunol 19, 279–290 (2018). https://doi.org/10.1038/s41590-018-0046-x

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