1. Ablation of lsd1 in early T cells leads to thymic atrophy and decreases peripheral T cells.
To elucidate the function of Lsd1 in thymocyte development, we crossed Lsd1fl/fl mice with Lck-Cre mice (Figure S1A), which drives the Cre recombinase expression by proximal Lck (lymphocyte protein tyrosine kinase) promoter and effectively deletes the floxed gene fragment at the DN stage (Figure S1B). To exclude the possible effect of Lck-driven Cre expression on T cell development as reported [24], we used heterozygotes (Lsd1wt/flLck-Cre) as littermate controls (referred to hereafter as control). The expression of Lsd1 in Lsd1fl/flLck-Cre mice (referred to hereafter as KO) is significantly reduced in thymocytes compared to the control, showing a high-efficiency Lsd1 deletion (Figure S1C). And the modification of H3K4me2 was increased explicitly in Lsd1-deleted T lineage cells, while the H3K4me1 level was not changed (Figure S1D).
We observed that at 6 ~ 10 weeks of age, KO mice had a markedly smaller thymus (Fig. 1A) with a 35% reduction in thymocyte weight (Fig. 1B), a 50% reduction in absolute cell numbers (Fig. 1C), and an abnormal architecture with medullary regions remarkably expanded (Fig. 1D). Flow cytometry analysis showed that, as compared to control mice, no change in the percentages of CD4−CD8− cells and CD4+CD8+ cells, but a considerable decrease in the percentages of CD4+CD8− cells and a relative increase in the percentages of CD4−CD8+ cells were observed in the thymocytes from KO mice (Fig. 1E-G).
We further investigated whether the loss of Lsd1 affects the mature T cells migrating to the peripheral. As shown in Figure S2, the total cell numbers of spleens and lymph nodes were not affected (Figure S2A). However, the frequencies of CD3+ T cells in spleens (Fig. 2A) and lymph nodes (Figure S2B) from KO mice were significantly decreased, as compared to control mice. Moreover, the expression level of CD3 was downregulated (Fig. 2B). The percentages of CD4+ and CD8+ T cells were greatly reduced in the spleen (Figs. 2C) and lymph nodes (Figure S2C). Interestingly, although the CD4/CD8 ratio altered inside the thymus in KO mice (Fig. 1G), it was not affected in spleens (Fig. 2D) and lymph nodes (Figure S2D). The proliferation capacities of both the mature peripheral CD4+ and CD8+ T cells under anti-CD3 and anti-CD28 stimulation were disrupted in KO mice (Fig. 2F). In summary, the loss of Lsd1 disrupted the numbers and proliferation of mature T cells in the peripheral.
More interestingly, we observed that the specific deletion of Lsd1 in thymocytes at the DN stage also affected the development of other immune cells in thymus. The percentages and absolute numbers of thymic B (CD4−CD8−B220+) cells and thymic NK cells (CD4−CD8−CD122+NK1.1+) were increased significantly in KO mice, as compared to control mice (Figure S3A & B). We examined whether the accumulation of B or NK cells results from trans-differentiation of T-precursors or reactive hyperplasia. The yellow fluorescence protein (YFP) was used as an indicator of the activation of Lck-Cre recombinase, which is believed to be specifically expressed in T-lineage cells. If a cell is derived from the T cell precursor, it will express YFP and obtain fluorescence. Our results showed that no YPF+ B cells were observed in the thymus, bone marrow, spleen, and lymph nodes from KO mice, which indicated that no trans-differentiation from T to B cells occurred after Lsd1-deletion (Figure S3C). Of note, it was unexpected that thymic NK cells could also activate the Lck-Cre recombinase as there were YFP+ NK cells in the control thymus (Figure S3C). Considering the similar frequencies of YFP+ thymic NK cells in KO mice compared with control mice, we speculated it to be resulted from reactive hyperplasia but not trans-differentiation. Consistently, previous investigation also observed the increased B cells and NK cells in the thymus in Lsd1fl/flCD2-Cre mice, but not in Lsd1fl/flCD2-Cre DN thymocyte cultures on OP9-DL1 cells [22]. In summary, the observed increased number of other lineage cells is not caused by the trans-differentiation from T cells. It may be caused by the change in the thymic microenvironment after the specific deletion of Lsd1 in thymocytes.
2. Ablation of lsd1 disturbs the programmed downregulation of CD8 at the DP→CD4+CD8lo stage
To better understand the effect of Lsd1 on T cell development in the thymus, we profiled thymocytes from three control mice and three KO mice with single-cell RNA sequencing (scRNA-seq) based on a 10× genomics platform and obtained a total of 39,857 cells (Fig. 3A). After filtering low-quality cells with abnormal numbers of expressed genes, high mitochondrial gene expression, and doublets, the remaining bioinformatically identified cells from all six thymi (38,966 cells) with an average of 1,583 genes per cell were combined for downstream analysis.
We identified 28 clusters with distinct transcriptomic signatures using unbiased clustering and uniform manifold approximation and projection (UMAP) analysis. We identified the clusters to twelve cell types at different developmental stages of T thymocytes using SingleR with the ImmGen reference dataset [25]. The cell types are ETP-DN3a, DN3b-ISP, T.DPbl (blasts), T.DPsm (small resting), T.DP69+ (early positive selection), T.SPinter (intermediate), T.CD4Th, T.CD8, T.CD4Treg, NKT (natural killer T cells), as well as other lineages: B cells, DCs (dendritic cells) (Fig. 3B & C).
Accordingly, we observed similar subpopulation distribution in the thymi from control and KO mice (Fig. 3D). TCRβ transcripts were noticed as early as in DN3b-ISP subgroup, and TCRα transcripts appeared at the CD69+ DP stage in both control and KO thymi (Fig. 3D), indicating that Lsd1-deletion didn’t disturb the timeline of the TCR V(D)J gene recombination events. Furthermore, we calculated the proportions of the different subgroups. Consistent with our flow cytometry analysis, we observed the increased frequencies of other lineages (e.g., B cells and DC cells) in the scRNA-seq data (Fig. 3E). However, although the percentages of CD4+ cells and CD8+ cells altered in the flow cytometry analysis (Fig. 1F), no significant differences were found in the most T cell subgroups from control and KO mice by scRNA-seq (Fig. 3E). We then tried to figure out what made the difference. Interestingly, in the scRNA-seq analysis, the greatly increased CD8 expression and decreased CD4 expression in the T.SPinter cells (representing the intermediate CD4+CD8lo stage) and CD4Th cells were observed from Lsd1-deleted mice (Fig. 3F). It is known that CD4+CD8+ DP thymocytes will differentiate into CD4+CD8lo cells and then make lineage choice to become either CD4+ or CD8+ SP T cells (Fig. 3G) [26]. The increased CD8 expression and decreased CD4 expression in CD4+CD8lo cells led them to be counted in the CD8+ SP gate in flow cytometry analysis (Fig. 3H). However, in the scRNA-seq map, they were still defined as CD4+CD8lo cells based on their general transcriptome. In summary, the loss of Lsd1 disturbed the programmed downregulation of CD8 at the DP→CD4+CD8lo stage.
3. Deletion of Lsd1 activates the interferon (IFN) response in thymocytes
To determine how Lsd1 regulates the thymic T cell development, we performed differentially expressed genes (DEGs) and pathway enrichment analysis of the scRNA-seq transcriptome data. As shown in Fig. 4A & B, we found ten upregulated genes (H2-Q4, H2-T22, H2-D1, H2-K1, H2-Q7, B2m, Ifi27, Usp18, Osa1a, and ly6a) shared by T cell subgroups at different stages (ETP-DN3a, DN3b-ISP, T.DP, T.CD8, T.CD4). The number of downregulated genes was much fewer, and almost no shared genes were found. Surprisingly, among the shared upregulated genes, six genes belong to the MHC I complex, and the others are interferon-stimulating genes (ISGs). Consistently, we found high enrichment of IFN response pathways and antigen processing pathways across all the T cell subgroups (Fig. 4C). Since the IFN pathway is a positive regulator of the MHC antigen-processing machinery [27], we can conclude that the DEGs and their related pathways focus on ‘the activation of IFN response’. We further evaluated the IFN response using an ISG module score [28]. Interestingly, in the control thymocytes, the ISG score was high at the early ETP-DN3a stage. Then it was downregulated at the DN3b stage and gradually increased during the subsequent developmental stages. After the loss of Lsd1, ISG scores were significantly elevated in all T cell stages (Fig. 4D), indicating an excessive pre-activation of IFN pathway during the whole developmental process. By quantitative polymerase chain reaction (qPCR) analysis, the overexpression of MHC I molecules (H2-k1, H2-d1) and interferon-stimulating genes (Irf7, Irf9, Oas3, Ifit1,Stat1, Nfkb1) were further confirmed (Fig. 4E). However, the IFN-I gene (Ifna, Ifnb1) and IFN-III gene (Il28b) were expressed at low levels and even showed some decrease (Figure S4A) in KO thymocytes. Instead, increased expression of IFN-II (IFN-γ) was determined at both mRNA and protein levels in KO mice, as compared to control mice (Figure S4A and Figure S4B). In addition, the abnormal upregulation of IFN-γ in the thymic environment could also activate the related immune cells, and cause the increased numbers of other lineage cells (B cells, NK cells, DCs) in the Lsd1-deleted thymus, as we observed in flow cytometry and scRNA-seq analysis. These data indicated that the Lsd1 ablation is strongly related to the aberrant activation of IFN signaling.
We then investigated whether the Lsd1 directly targets at the IFN responsive genes to repress their expressions. Lsd1 is known as an H3K4me1/2 demethylase to act as a transcription co-repressor. We evaluated whether the Lsd1 directly regulate the modification of H3K4me1/2 at the IFN responsive genes,. by analyzing the publicly available chromatin immunoprecipitation sequencing (ChIP-seq) data obtained from thymocytes in which Lsd1 was knocked out at the DN stage by CD2-Cre recombinase [22]. Unexpectedly, as shown in Fig. 4F, the H3K4me1 and H3K4me2 modification of ISGs and their enhancers were decreased after Lsd1 depletion, although there was a slight increase in the H3K4me3 modification. The results indicated that the ISGs are not directly regulated by the H3K4me1/2 demethylase activity of Lsd1. In summary, the loss of Lsd1 activates the interferon (IFN) response in thymocytes, while the over-expression of ISGs is not directly regulated by Lsd1.
4. Deletion of Lsd1 de-represses endogenous retroelements
We then try to figure out what triggers the IFN response in Lsd1-deleted thymocytes. Other than the IFN response genes, the pathway enrichment analysis on scRNA-seq data showed upregulated ‘response to the virus’ after Lsd1 loss (Fig. 4C), suggesting that the activation of an upstream event, such as an RNA-sensing pathway, may play an important role. It is known that endogenous retroelements (EREs) account for about 40% of mammalian genomes, and the silencing of EREs is controlled by the state of histone methylation [29]. We wondered if there was an abnormal transcription of EREs triggering IFN signaling after Lsd1 deletion. As our scRNA-seq focused on poly-A eukaryotic mRNAs, we further performed strand-specific total RNA-seq of thymocytes for detecting the non-coding RNAs. Increased transcripts in both sense and anti-sense directions from all ERE subfamilies were detected in the Lsd1-deleted thymocytes, including long terminal repeat (LTR)-containing endogenous retroviruses (ERV1s, ERVKs and ERVLs) and non-LTR elements (LINEs and SINEs) (Fig. 5A & B). ChIP-seq analysis showed that the loci of upregulated EREs had a great increase in H3K4me1 and H3K4me2 levels, as well as a mild increase in H3K4me3 level in KO mice, which indicated that the Lsd1 directly targets at EREs to regulate their expression (Fig. 5C). The overexpression of EREs can contribute to the generation of dsRNAs, which trigger IFN activation. In addition, we detected the expression levels of double-stranded RNA (dsRNA) sensors, Tlr3, Mda5 (encoded by Ifih1), and Rig-I (encoded by Ddx58), and DNA sensors, Sting (encoded by Sting1) and Cgas in Lsd1-deleted thymocytes, and found they were all increased as expectedly (Fig. 5D). These data suggested that ablation of Lsd1 could de-repress the transcription of a group of EREs, resulting in the aberrant activation of IFN response in mouse thymocytes. Consistently, Lsd1 has been reported to be an ERV suppressor in embryonic stem (ES) cells [29] and regulates the ERV-IFN pathway in melanoma cells [17].
5. Lsd1 deletion promotes an innate-memory phenotype of T cells
We next investigated the biological effects of Lsd1 deletion-induced viral mimicry state and IFN signaling activation. We observed, as compared to control mice, the percentages of TCRhiCD69+CD24+ immature T cells decreased in KO mice, and that of TCRhiCD69−CD24− immature T cells increased, which indicated a pre-mature state of thymic T cells (Figure S5). Moreover, we observed increased CD44+ (Fig. 6A), CXCR3+ (Fig. 6B), and Eomes+ (Fig. 6C) CD8+ T cells in KO thymocytes. Thus there was an elevated fraction of innate-memory CD8+ T cells induced by Lsd1-deletion. Furthermore, we used the innate-memory score, which was evaluated by a set of related genes, to determine the state of thymocytes based on their scRNA transcriptome. The results showed elevated innate-memory scores in all Lsd1-deleted T subgroups (Fig. 6D). Consistently, the mature T cells in the peripheral from KO mice also showed an effector/memory phenotype in the absence of antigen stimulation (Fig. 6E). While the conventional memory is induced by foreign antigens, the continuous viral mimicry stimulation and activated IFN signaling [30] could contribute to the innate-memory phenotype of T cells in KO mice. Together, conditional deletion of Lsd1 in T precursors leads to severe thymic atrophy and impaired periphery T cell pool. It induces the IFN activation by de-repressing the endogenous retroelements and promotes an innate-memory phenotype of both thymic and peripheral T cells (Fig. 7).