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Pre-T cell receptor self-MHC sampling restricts thymocyte dedifferentiation

Nature volume 613, pages 565–574 (2023)Cite this article

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Abstract

Programming T cells to distinguish self from non-self is a vital, multi-step process that occurs in the thymus1,2,3,4. Signalling through the pre-T cell receptor (preTCR), a CD3-associated heterodimer comprising an invariant pTα chain and a clone-specific β chain, is a critical early checkpoint in thymocyte development within the αβ T cell lineage5,6. PreTCRs arrayed on CD4−CD8− double-negative thymocytes ligate peptides bound to major histocompatibility complex molecules (pMHC) on thymic stroma, similar to αβ T cell receptors that appear on CD4+CD8+ double-positive thymocytes, but via a different molecular docking strategy7,8,9,10. Here we show the consequences of these distinct interactions for thymocyte progression using synchronized fetal thymic progenitor cultures that differ in the presence or absence of pMHC on support stroma, and single-cell transcriptomes at key thymocyte developmental transitions. Although major histocompatibility complex (MHC)-negative stroma fosters αβ T cell differentiation, the absence of preTCR–pMHC interactions leads to deviant thymocyte transcriptional programming associated with dedifferentiation. Highly proliferative double-negative and double-positive thymocyte subsets emerge, with antecedent characteristics of T cell lymphoblastic and myeloid malignancies. Compensatory upregulation of diverse MHC class Ib proteins in B2m/H2-Ab1 MHC-knockout mice partially safeguards in vivo thymocyte progression, although disseminated double-positive thymic tumours may develop with ageing. Thus, as well as promoting β chain repertoire broadening for subsequent αβ T cell receptor utilization, preTCR–pMHC interactions limit cellular plasticity to facilitate normal thymocyte differentiation and proliferation that, if absent, introduce developmental vulnerabilities.

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Fig. 1: Developmental trajectories for thymocyte-like development on MHC+ or MHC− supporting stroma.
Fig. 2: Uncoupling of the transcriptome and TCR β chain repertoire from phenotype in thymocyte-like cells developing on MHC− stroma.
Fig. 3: Single-cell transcriptomics of the MHC− DN4 unusual cluster reveal complex proliferative and lineage abnormalities.
Fig. 4: Single-cell transcriptomics of the MHC− DP abnormal cluster reveal dedifferentiation and reprogramming to include a myeloid programme.

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Data availability

All sequence files have been deposited at NCBI Gene Expression Omnibus (GEO) under accession GSE186049.  Source data are provided with this paper.

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Acknowledgements

This research was supported by NIH NIAID grant AI136301. C.M.M. and P.H.L. were supported additionally by the Expect Miracles Foundation and the Robert and Renée Belfer Foundation. We thank J.-h. Wang for scientific discussion and insight, D. A. Barbie and C. P. Paweletz for facilitating the scRNA-seq analyses, the Dana-Farber/Harvard Cancer Center Specialized Histopathology Core (NIH NCI grant P30 CA006516-57) and S. Moskovitz for graphic design of figures.

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Authors and Affiliations

  1. Laboratory of Immunobiology, Dana-Farber Cancer Institute, Boston, MA, USA

    Jonathan S. Duke-Cohan, Aoi Akitsu, Robert J. Mallis & Ellis L. Reinherz

  2. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    Jonathan S. Duke-Cohan, Aoi Akitsu, Robert J. Mallis & Ellis L. Reinherz

  3. Department of Medicine, Harvard Medical School, Boston, MA, USA

    Jonathan S. Duke-Cohan, Aoi Akitsu & Ellis L. Reinherz

  4. Department of Dermatology, Harvard Medical School, Boston, MA, USA

    Robert J. Mallis

  5. Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, MA, USA

    Cameron M. Messier & Patrick H. Lizotte

  6. Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA

    Jon C. Aster

  7. Department of Biomedical Engineering, Texas A&M University, College Station, TX, USA

    Wonmuk Hwang

  8. Department of Materials Science and Engineering, Texas A&M University, College Station, TX, USA

    Wonmuk Hwang

  9. Department of Physics and Astronomy, Texas A&M University, College Station, TX, USA

    Wonmuk Hwang

  10. Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA

    Matthew J. Lang

  11. Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA

    Matthew J. Lang

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  1. Jonathan S. Duke-Cohan
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Contributions

Conceptualization: J.S.D.-C., A.A., R.J.M., W.H., M.J.L. and E.L.R. Methodology: J.S.D.-C., A.A., R.J.M. and E.L.R. Investigation: J.S.D.-C, A.A., C.M.M., P.H.L. and J.C.A. Writing, original draft: J.S.D.-C. and E.L.R. Writing, review and editing: J.S.D.-C., R.J.M., A.A., W.H., M.J.L. and E.L.R. Funding acquisition: M.J.L., E.L.R. and J.C.A. Supervision: J.S.D.-C. and E.L.R.

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Correspondence to Jonathan S. Duke-Cohan or Ellis L. Reinherz.

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Extended data figures and tables

Extended Data Fig. 1 Schematic for FACS isolation of thymocyte subsets (DN3a, DN3b, DN4, DP) for 10X scRNA-Seq and single cell TCR α and β chain clonotype sequencing.

Sorted cells were isolated as DN3a cells (CD25+CD44−CD28−), DN3b cells (CD25+CD44−CD28+), DN4 (CD25−CD44−CD28+) cells, and DP (CD4+CD8+) cells.

Extended Data Fig. 2 Cluster delineation of DN3a to DPsm cell transitions.

For each transition, data from the Immune Genome Project (IGP) microarray and RNA-Seq data was used to construct a panel representing genes with the highest fold-change between phenotypically defined stages of thymocyte differentiation. The gene panel was then used to query the MHC+ thymocyte clusters identified by UMAP projection. Combination of library phenotype together with good fit to the interrogating gene panel permitted identification of cluster relationships and developmental trajectories. a. Delineation of early post-β selection checkpoint DN3a/3b thymocytes from pre-β selection checkpoint DN3a thymocytes by differential gene expression. The left-hand heatmap depicts a panel selected by comparison of DN3b thymocyte gene expression from the IGP with DN3a cell expression. The same genes were examined for expression in the clusters defined as DN3a and DN3a/3b in Fig. 1B (right-hand heatmap). The volcano plot depicts the log2-fold increase of expression in the DN3a/3b population over DN3a for the expected normal developmental trajectory (x-axis). Note that for all volcano plots reported here, only the significantly changed transcripts are depicted (Padj < 0.05; y-axis). b. Delineation of late post-β selection checkpoint DN3b/4 thymocytes from early pre-β selection checkpoint DN3a/3b thymocytes by differential gene expression. The heatmap on the far left depicts a panel selected by comparison of DN4 thymocyte gene expression from the IGP with DN3b cell expression (neither DN3a/3b nor DN3b/4 transitional states are explicitly defined in the IGP database). Transcripts in red were predicted from IGP data to be upregulated in the DN3b to DN4 transition but are downregulated for the conditions reported here. c. Delineation of late post-β selection checkpoint DN3b/4 thymocytes from DPbl thymocytes by differential gene expression. The DPbl cluster was extracted from the DP library and delineated from the more mature DPsm population by transcriptome signature as described below. d. Delineation of mature DPsm thymocytes from cycling DPbl thymocytes by differential gene expression. The heatmap on the far left depicts a panel selected by comparison of DPsm thymocyte gene expression from the IGP with DPbl cell. Note that during the DPbl to DPsm transition, significant cell cycling transcripts were downregulated thus significantly upregulated transcripts in the volcano plot represent the DPbl cells.

Extended Data Fig. 3 Delineating the ILC-γ/δ TCR thymocyte cluster and pro-apoptotic cluster from the main α/β TCR lineage pathway.

a. Distinguishing ILC-γ/δ-like cells from DN3b/4 in the DN4 libraries by gene expression. The heatmap on the left shows a manually curated panel of gene transcripts selected by likely high representation in either DN3b/4 or ILC-γ/δ-like cells. Log2 Fold-change (L2FC) and Padj in the DN4 libraries for differential expression between the DN3b/4 clusters and ILC-γ/δ-like clusters are shown in the volcano plot to the right with transcripts associated with ILC development are highlighted in light purple (Id2, Zbtb16, Gata3, Rora). TCR γ and δ transcripts are highlighted in green, and Trbv transcripts highlighted in blue. b. Gene expression profile of the pro-apoptotic cluster. The dominant pro-apoptotic cluster upregulated gene expression changes are similar between all the MHC+ libraries on comparison with the 2 dominant clusters within each of these libraries. All log2-fold changes (L2FC) are relative only to the 3 clusters listed in each heatmap (i.e. local) and not to the average across all clusters in that library.

Extended Data Fig. 4 Development and TCR repertoire analyses for cells growing on MHC+, MHC− and scH-2Kb stromal support cells.

a. Total cell recoveries after 9d development from 2,000 seeded HSC (Representative of 6 experiments examining MHC+ (n = 5), MHC− (n = 6), and scH-2Kb (n = 3)). For all box plots, the box bounds the 1st to 3rd quartiles; where visible, the dotted line within represents mean, and the solid line represents median. Whiskers above and below (maximum and minimum) are defined as (quartile 3 + 1.5 * interquartile range) and (quartile 1 – 1.5 * interquartile range), respectively. P (= 0.0204) determined by two-tailed t test. b. Apparent thymocyte developmental stage representation as fraction of total cells for cultures represented in panel a. c. Stage-specific analysis of β chain clonotype representation/10,000 cells in d9 MHC+, MHC−, and scH-2Kb OP9-DL4 development cultures. Representation of data from replicate experiments of data in Fig. 2c–f. d. TCR β chain clonotype diversity at DN4 on MHC+, MHC−, and scH-2Kb stroma. The total number of TCR β chain clonotypes (black) recovered from 104 cells of each DN4 population isolated after growth for 9d on the varying OP9-DL4 stroma is represented by an ellipse of area in direct proportion to unique clonotype count (5 independent experiments). Percentage shared clonotypes of the total for each condition (MHC+ in blue, MHC− in pink, and scH-2Kb in green) is depicted. Note that the area of overlap only approximates degree of sharing to maintain consistent orientation of the ellipses for presentation. The overlap of MHC+ and scH-2Kb for experiments 4 and 5 is <1% and too small to represent in this format. Statistics and P calculated from two-tailed t test presented on left.

Source data

Extended Data Fig. 5 Transcriptome and selected phenotype comparison of MHC+ and MHC− OP9-DL4 cells and select gene expression profiles for the DN4 unusual and DPbl abnormal populations.

a. Comparison of MHC+ and MHC− OP9-DL4 stromal cells for transcriptome and phenotypic differences. 93.6% of transcripts detected shared by MHC+ and MHC− stroma. b. Correlation between cell transcriptomes. Square of two-tailed Pearson correlation coefficient (R2 = 0.958) ideally greater than 0.92 under optimal experimental conditions. c. Differential gene expression is <4% of all transcripts detected. d. Loss of CD1d surface expression in B2m/Tap2 KO MHC− OP9-DL4 and confirmation of lack of MHC Class II expression in MHC+ and MHC− OP9-DL4. e. Raet expression in MHC+ and MHC− OP9-DL4. f. Select transcripts significantly differentially expressed between the MHC− DN3b/4 cluster and the DN4 “unusual” cluster. Heatmap depicts log2-fold change (L2FC) of the DN4 “unusual” cluster relative to the DN3b/4 cluster. Actual L2FC values are listed within the heatmap. g. Co-expression of Cd4 transcript with Cd8a and/or Cd8b1 transcripts in an overlay of the MHC− libraries focussed on the DN4 unusual, DPbl, and DP abnormal clusters. h. Characteristic myeloid gene transcript expression maps to the MHC− DP abnormal cluster. i. Full-length clonotypic TCR β chain transcript expression in 82 of 221 Spi1+ cells (37.1%) in the MHC− DP abnormal cluster. j. Mpo-expressing cells in the DP abnormal cluster and the Mpo+Spi1+ subset co-express T lineage Lck and/or Cd3e. k. DP cell yields after 12 d for DN3a and DN4 cells seeded onto MHC+ or MHC- stromal cells. l. Relative expression by qRT-PCR (normalised to Actb = 1000) of Mpo and Spi1 in DP cells developing from DN3a cells seeded 12 d earlier onto MHC+ or MHC− stromal cells (Cells pooled from 3 separate cultures; n = 7 qRT-PCR replicates; Mpo: P < 0.00001, Spi1: P = 0.000655). m. Relative expression (normalised to Actb = 1000) of Mpo and Spi1 in DP cells developing from DN4 cells seeded 12 d earlier onto MHC+ or MHC− stromal cells (Cells pooled from 3 separate cultures; Mpo: n = 8 qRT-PCR replicates; Spi1: n = 4 qRT-PCR replicates); for l, m: mean ± s.d.; P from two-tailed t-test; representative of 2 independent experiments; Mpo: P = 0.000013, Spi1: P = 0.000233).

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Extended Data Fig. 6 Highly proliferating clonotypic progeny cluster together by transcriptional signature.

a. MHC+ DN4 20 most highly represented clonotypes by cell number. b. MHC− DN4 20 most highly represented clonotypes by cell number. The identical MHC+ and MHC− DN4 clonotypic cells to those presented in Fig. 3d and Extended Data Table 3a are shown in their mapped positions in the UMAP projection. Each clonotype is represented for each panel in a unique colour with cell number indicated in key. Note that colours are not directly related to those used in Fig. 3d.

Extended Data Fig. 7 Transcriptome comparison of DN and ISP thymocyte subsets from MHC+ and MHC− mice.

a. Thymocyte subset cell recoveries from thymi of MHC+ and MHC− mice. Mean ± s.d. shown; 3 mice/group; ** p = 0.0169; *** p < 0.0039; **** p < 0.0003 determined by 2-tailed t-test. b. Log2-fold change in expression from global population mean for the MHC− knocked out genes (B2m, H2-Ab1), classical and minor MHCI genes, and MHCII genes. Note that for each thymocyte subset there are 3 replicates except for the MHC− DN4 cells for which there are duplicates. Asterisks highlight transcripts that are upregulated across all MHC− libraries on comparison with MHC+ Q10 (p = 7 × 10−5), H2-T3 (TL) (p = 3 × 10−7), H2-T22 (p = 1 × 10−7) and H2-T-ps (p = 4 × 10−5). P calculated using two-tailed Chi-square test. c. Log2-fold change in expression of all development stage marker genes depicted in Fig. 1a. d. Log2-fold change in TCR Vβ chain segment (Trbv) expression. Mean depicted of triplicates for all libraries except for duplicates for MHC− DN4 samples. e. Log2-fold change in Bcl2a1 family transcripts (upper panel), canonical Bcl2 transcripts (middle panel), and Pim1 protooncogene (lower panel). Mean values presented. f. Log2-fold change in TCR Vα chain segment (Trav) expression. Mean depicted of triplicates for all libraries except for duplicates for MHC− DN4 samples. g. Display of haematopoietic/immune organs from an MHC− dKO mouse with massive thymic growth at 15 months and from age-matched MHC+ control. h. FACS analysis of single cell thymic and splenocyte suspensions stained for CD4 and CD8. Numbers next to gates indicate % of cells in that gate. i. Haematoxylin and eosin staining of representative organs from an age-matched MHC+ wt B6 mouse and an MHC− dKO mouse with leukaemic growth. Thymic cortex indicated by ‘c’, and thymic medulla by ‘m’. Cancellous bone indicated by ‘ca’. Arrow indicates leukaemic cell accumulation adjacent and around a hepatic vein. j. Immunohistochemistry of tumour cells in dKO thymus for TdT (immature thymocytes), CD8 (T lineage) and neutrophil elastase (myeloid lineage) and in dKO spleen metastatic focus for the intracellular domain of Notch 1 (NICD1). i, j: For each tissue and condition, the complete section was examined down to the cellular level and the image presented (~1% of each total section) is representative of that complete section. White bar in all images represents 100 μm.

Source data

Extended Data Table 1 Top 20 DPsm TCR β clonotypes developing on scH-2Kb stroma at d9
Full size table
Extended Data Table 2 Non-classical MHC class I expression in MHC+ and MHC− OP9-DL4
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Extended Data Table 3 Well-represented clonotypes in MHC+ and MHC− libraries
Full size table

Supplementary information

Reporting Summary

Supplementary File 1.

Gene expression level (UMI/cell), log2fold change (global), and Padj for all clusters in all MHC+ libraries – scRNA-seq. MS Excel file: Supplementary-Information-File1.

Supplementary File 2.

Gene expression level (UMI/cell), log2fold change (global), and Padj for all MHC- clusters – scRNA-seq. MS Excel file: Supplementary-Information-File2.xlsx

Supplementary File 3.

TCR β chain clonotypes for DN3, DN4, DPbl and DPsm thymocytes developing on MHC+, MHC-, and scH-2Kb/VSV8 stroma. MS Excel file: Supplementary-Information-File3.

Supplementary File 4.

Representative FACS separation profiles of developing thymocyte-like subsets in vitro and thymocyte subsets in vivo. PDF file: Supplementary-Information-File4.

Supplementary File 5.

Gene expression for DN3a, DN3b, DN4 and ISP cells isolated from MHC+ and MHC- mice. MS Excel file: Supplementary-Information-File5.

Supplementary File 6.

Gene expression analysis of a chromosome Y transcript panel and expression-matched autosomal panel to address aberrant clonal HSC expansion contributing to the MHC- DN4 unusual and DP abnormal populations. MS Excel file: Supplementary-Information-File6.

Source data

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Duke-Cohan, J.S., Akitsu, A., Mallis, R.J. et al. Pre-T cell receptor self-MHC sampling restricts thymocyte dedifferentiation. Nature 613, 565–574 (2023). https://doi.org/10.1038/s41586-022-05555-7

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