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

Abstract

Programming T cells to distinguish self from non-self is a vital, multi-step process that occurs in the thymus^1-4. 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 lineage^5,6. 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 strategy^7-10. 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.

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 (P_adj < 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 P_adj 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-2K^b 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-2K^b (n = 3)). For all box plots, the box bounds the 1^st to 3^rd 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-2K^b 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-2K^b 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-2K^b 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-2K^b 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.

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).

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 x 10⁻⁵), H2-T3 (TL) (p = 3 x 10⁻⁷), H2-T22 (p = 1 x 10⁻⁷) and H2-T-ps (p = 4 x 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.

Fig. 1.. Developmental trajectories for thymocyte-like development on MHC⁺ or MHC^– supporting stroma.

a. Schematic depicting representative gene transcript levels during key thymocyte developmental transitions (based on array data from the Immune Genome Project). Upper panel: Early DN1-DN3 proliferation is driven by thymocyte Notch signalling, represented here by the Erg and Hes1 transcripts. Myeloid development is suppressed by downregulation of Spi1 (coding for PU.1) during the DN2a to DN2b transition and T lineage commitment following the Bcl11a to Bcl11b switch. Lower panel: following entry in to the DN3 stage, the preTCR with invariant pTα (pTCRα) is expressed. PreTCR signalling downregulates Notch-driven proliferation, inhibits TCR β locus recombination, downregulates Ptcra and upregulates the indicated transcripts. b. UMAP projection of k-means clustering (k = 10) for DN3a, DN3b, DN4, and DP libraries for cells developing on either MHC⁺ or MHC⁻ stroma. All libraries are projected into the same space to permit direct comparison. Process for assignment of labels to each cluster is defined in the text. c. Cluster developmental trajectories of individual libraries and relationship of individual clusters to phenotypically characterized thymocyte subsets. Projection of the individual FACS-sorted libraries (labeled in large font) into the primary space allows initial assignment of clusters expressing distinct transcriptomes (labelled in small font). d. MHC⁻ thymocyte-like cells progress developmentally by phenotype from DN3a to the DP stage but with altered distribution on comparison with MHC⁺ cells. To focus only on the αβTCR lineage, ILC-γ/δ-like T cells and pre-apoptotic/apoptotic cells are excluded. For each library, the proportion of each defined developmental cluster is depicted. P < 2.5 x 10⁻⁷ for difference between MHC⁺ and MHC⁻ cells/stage distributions (Chi-square statistic).

Fig. 2.. Uncoupling of the transcriptome and repertoire from phenotype in thymocyte-like cells developing on MHC⁻ stroma.

a. The DN3b/4 cluster in the MHC⁻ DN4 library harbours a population with characteristics of cells not having passed through the preTCR signalling checkpoint. The MHC⁻ DN3b/4 cluster in the DN4 library is split into two subclusters, one corresponding to the DN3b/4 cluster in the MHC⁺ DN4 library (“main”, brown) and one corresponding to a set poorly represented in the MHC⁺ DN4 library (“tail”, orange). b. Transcript expression in MHC⁺ and MHC⁻ DN3b/4 cells. Transcripts well-expressed and marking the transition to DN3b/4 cells (Ikzf3, Rorc, Cd2; Suppl. Fig. 3) remain low in the MHC⁻ “tail” subcluster, transcripts expected to be downregulated remain high (Fig. 1a), and robust TCR β chain upregulation is not observed (Extended Data. Fig. 2b). c-f. Stage-specific analysis of β chain clonotype representation/ 10,000 cells in d9 MHC⁺, MHC⁻, and scH-2K^b OP9-DL4 thymocyte-like development cultures. Representative of 6 experiments examining MHC⁺ (n = 5), MHC⁻ (n = 6), scH-2K^b (n = 3).

Fig. 3.. Single cell transcriptomics of the MHC⁻ DN4 unusual cluster reveal complex proliferative and lineage abnormalities.

a. Volcano plot of significant transcript differences (P<0.05) between the DN4 unusual population and the DN3b/4 cluster in the same MHC⁻ DN4 library. Upregulated DN4 unusual transcripts are shown to the right of zero on the x-axis, downregulated to the left. Functional significance of highlighted transcripts is listed in the inset. 14 other Trbv transcripts were downregulated but did not meet the P<0.05 threshold. b. Violin plots depicting selected log-normalised transcript levels for the MHC⁺ DN4 library DN3b/4 cluster (2021 cells), MHC⁻ DN4 library DN3b/4 cluster (2493 cells), and MHC⁻ DN4 unusual cluster (1776 cells). For box plots, the box bounds the 1^st to 3^rd 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. Cluster colours are as depicted in Figs. 1 and 2. P values as in panel a. c. Cluster distribution of top 20 DN4 clonotypes developing on MHC⁺ stroma (left panel) with expected developmental trajectory into DN3b/4 cluster (right panel). Each colour represents a unique clonotype and circle diameter is proportional to the cell number expressing that specific β chain (see cell count scale common to Fig. 3c and Fig. 3d). Colour/clonotype specification is unique to the panel and bears no relation to colour use in Fig. 3d or Fig. 4c, d. Right panel depicts clonotype tracking from cluster to cluster with cluster/track colouring concordant with left panel. d. Distribution of top 20 DN4 clonotypes developing on MHC⁻ stroma similar to depiction in Fig. 3c with right panel depicting clonotype tracking from cluster to cluster. e. ssGSEA scores for MHC⁺ DN3b/4 (green), MHC⁻ DN3b/4 (light orange), and MHC⁻ DN4 unusual (red) clusters compared with gene subset modules exhibiting co-ordinated up- or down-regulation in the indicated cancers (MSigDb C4). f. Comparison of the DN4 unusual cluster developing on MHC⁻ with the DN3b/4 cluster developing on DN4 MHC⁺ stroma (positive control) for transcripts reported as dysregulated in human T-ALL. Shaded boxes identify regulatory transcripts over-expressed in the indicated forms of human T-ALL (columns 1-3). Heatmap in column 4 depicts log2-fold difference for the DN4 unusual cluster compared with the MHC⁻ DN3b/4 cluster (scale to right) and significance (P value).

Fig. 4.. Single cell transcriptomics of the MHC⁻ DP abnormal cluster disclose dedifferentiation and reprogramming to include a myeloid programme.

a. Volcano plot of significant transcript differences (P<0.05) between the DP abnormal population and the DPbl cluster in the same MHC⁻ DP library. b. Violin plots depicting indicated log-normalised transcript levels for selected transcripts well-expressed in the MHC⁺ DP library DPbl cluster (1212 cells), MHC⁻ DP library DPbl cluster (3044 cells), and MHC⁻ DP abnormal cluster (2512 cells). Cluster colours are as depicted in Figs. 1 and 2. P values as in panel a. c. Cluster distribution of top 20 DP clonotypes developing on MHC⁺ stroma (left panel) together with clonotype tracking (right panel). Colour/ clonotype specification is unique to the panel and bears no relation to colour use in Fig. 4d or Fig. 3c, d. Right panel maintains colour concordance with left panel. d. DP cluster distribution of top 20 clonotypes developing on MHC⁻ stroma with right panel depicting clonotype tracking. e. ssGSEA scores for MHC⁺ DPbl (green), MHC⁻ DPbl (light orange), and MHC⁻ DP abnormal (red) cells analysing correlation with module gene sets with co-ordinated gene regulation in the indicated tumours (MSigDb C4). f. Comparison of MHC⁻ DP abnormal cluster with the DP cluster developing on MHC⁺ stroma for transcripts reported as dysregulated in human AML (T-ALL/AML common) or as overexpressed in CD34⁺ leukaemic stem cells (LSC) from AML patients (LSC17). g. Co-expression of Spi1 and Trbv transcripts in Spi1⁺ MHC⁻ DP abnormal cells. Cell numbers for MHC⁺ DPbl, MHC⁻ DPbl, and MHC⁻ DP abnormal as in panel b, and 221 DP abnormal Spi1⁺ cells. h. Schematic of proposed dedifferentiation for thymocytes developing in the MHC⁻ condition (pink area). For box plots, the box bounds the 1^st to 3^rd 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.