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Research Article
Lsd1 safeguards T-cell development via suppressing
endogenous retroelements and interferon responses
Miaoran Xia
1,
* , Bingbing Wang
1,2,3,4,5,
*, Wujianan Sun
6,
*, Dengyu Ji
1
, Hang Zhou
1
, Xuefeng Huang
1,2,3,4,5
,
Minghang Yu
1,2,3,4,5
, Ziyang Su
1,2,3,4,5
, Ping Chen
1
,KunQu
6,7
, Xi Wang
1,2,3,4,5
The histone demethylase Lsd1 has been shown to play multiple
essential roles in mammalian biology. However, its physiological
functions in thymocyte development remain elusive. We observed
that the specific deletion of Lsd1 in thymocytes caused significant
thymic atrophy and reduced peripheral T cell populations with
impaired proliferation capacity. Single-cell RNA sequencing com-
bined with strand-specific total RNA-seq and ChIP-seq analysis
revealed that ablation of Lsd1 led to the aberrant derepression of
endogenous retroelements, which resulted in a viral mimicry state
and activated the interferon pathway. Furthermore, the deletion of
Lsd1 blocked the programmed sequential down-regulation of CD8
expression at the DP→CD4
+
CD8
lo
stage and induced an innate
memory phenotype in both thymic and peripheral T cells. Single-
cell TCR sequencing revealed the kinetics of TCR recombination
in the mouse thymus. However, the preactivation state after
Lsd1 deletion neither disturbed the timeline of TCR rearrangement
nor reshaped the TCR repertoire of SP cells. Overall, our study
provides new insight into the function of Lsd1 as an important
maintainer of endogenous retroelement homeostasis in early
T-cell development.
DOI 10.26508/lsa.202302042 | Received 15 March 2023 | Revised 27 June
2023 | Accepted 29 June 2023 | Published online 10 July 2023
Introduction
T-cell development in the thymus is a precise and orderly regulated
multistep process. Briefly, CD4
−
CD8
−
double-negative (DN) thymo-
cytes can be further subdivided into sequential stages, including
the DN1 (CD44
+
CD25
−
), DN2 (CD44
+
CD25
+
), DN3 (CD44
−
CD25
+
), and DN4
(CD44
−
CD25
−
) stages. The cells then differentiate into the CD8
+
TCR
lo
immature single-positive (ISP) stage and the CD4
+
CD8
+
double-
positive (DP) stage and finally develop into mature CD4
+
or CD8
+
single-positive (SP) T cells that migrate to the periphery (1). The
chromatin state (the packaging of DNA with histone proteins) and
its epigenetic regulation modulators play critical roles in the de-
velopment of thymocytes. Histone posttranslational modifications
include phosphorylation, acetylation, ubiquitinylation, methylation,
and others (2). It is critically involved in almost all aspects of T-cell
biology, including lineage commitment, development, activation,
differentiation, and memory formation (3). For example, the loss of
histone methyltransferase Ezh2 has been found to impede T-cell
differentiation in the DN phase (4,5) but not change the production
of mature peripheral T cells (6). However, Vasanthakumar et al
reported that conditional KO of Ezh2 and other polycomb repressive
complex 2 (PRC2) components (Suz12 and Eed) at the DP stage did
not alter the subsequent development of αβ or γδ T-cell devel-
opment in the thymus and spleen (7). Tamoxifen-induced deletion
of the histone deubiquitinase Bap1 in adult mice resulted in severe
thymic atrophy with a block at the DN3 stage and complete loss of
the T-cell lineage (8). It has also been reported that the PRC1, PRC2,
histone methyltransferase G9a, and a variety of lncRNAs influence
the differentiation and maintenance of T helper cells by epige-
netically regulating transcriptional programs associated with dif-
ferent T-cell subsets (9). Understanding the mechanisms of
epigenetic regulation of T-cell development will have important
implications for T-cell biology and translational therapy.
Histone methylation has been believed for a long time to be
irreversible until Lsd1 (Lysine-specific demethylase 1A, encoded by
Kdm1a in mice) was identified to be a bonafide histone demeth-
ylase in 2004 (10). The enzyme can demethylate histone H3 on Lys4
as a transcription corepressor or on Lys9 as a transcription coactivator.
It is essential for a wide range of biological events. Deletion of the
gene results in developmental arrest and the death of mouse
embryos (11,12,13). It is widely reported to be an oncogene that
promotes cancer cell proliferation, migration, and invasion (14,15),
and has been found to repress antitumor T-cell immunity (16).
1
Department of Immunology, School of Basic Medical Sciences, Beijing Key Laboratory for Tumor Invasion and Metastasis, Capital Medical University, Beijing, China
2
Institute of Infectious Diseases, Beijing Key Laboratory of Emerging Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, China
3
Beijing Institute
of Infectious Diseases, Beijing, China
4
National Center for Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, China
5
Department of
Oncology, Capital Medical University, Beijing, China
6
Department of Oncology, The First Affiliated Hospital of USTC, School of Basic Medical Sciences, Division of Life
Sciences and Medicine, University of Science and Technology of China, Hefei, China
7
Institute of Artificial Intelligence, Hefei Comprehensive National Science Center,
Hefei, China
Correspondence: xiwang@ccmu.edu.cn; qukun@ustc.edu.cn; chenping@ccmu.edu.cn
*Miaoran Xia, Bingbing Wang, and Wujianan Sun contributed equally to this work
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Kerenyi et al reported that inducible deletion of Lsd1 in early
hematopoietic stem cells not only compromised early hemato-
poietic differentiation but also strongly interfered with terminal
granulocytic and erythroid differentiation (17). Subsequently, sev-
eral groups have reported that Lsd1 is required for the germinal
center formation and the humoral immune response (18,19,20).
Recently, Lsd1 was found to regulate multiple repressive gene
programs during T-cell development (21). The study showed that
Lsd1 represses genes that are normally down-regulated during the
DN-to-DP transition, such as stem cell-related genes and check-
point molecule genes. However, only a small fraction of H3K4me3
sites, rather than H3K4me1/2, were found to increase, which was
not sufficient for the up-regulation of the observed genes, as
concluded by the authors. The physiological role of Lsd1 in early
T-cell development remains unclear.
In this study, we found that conditional deletion of Lsd1 at the DN
stage led to thymic atrophy and decreased peripheral T-cell
populations with impaired proliferation capacity. We further veri-
fied that the “IFN response”pathway was dramatically up-regulated
throughout all developmental stages by single-cell RNA sequencing
(scRNA-seq) analysis. Moreover, we identified endogenous retro-
elements (EREs) as direct targets of Lsd1, whose H3K4me1 and
H3K4me2 modifications were increased in KO mice rather than IFN-
stimulated genes (ISGs). Notably, after Lsd1 depletion, CD8 ex-
pression failed to be down-regulated in time at the DP→CD4
+
CD8
lo
stage, which could have an impact on T-cell function. Single-cell
TCR sequencing revealed the kinetics of TCR recombination in the
mouse thymus. However, the preactivation state after Lsd1 deletion
neither disturbed the timeline of TCR rearrangement nor reshaped
the TCR repertoire of SP cells. Together, the results indicate that
Lsd1 functions to prevent aberrant stimulation of the IFN pathway
by repressing endogenous EREs and plays a critical role in main-
taining normal T-cell development.
Results
Ablation of Lsd1 disrupts the proliferation capacity of T cells
To elucidate the function of Lsd1 in thymocyte development, we
crossed Lsd1
fl/fl
mice with Lck-Cre mice (Fig S1A), which drives Cre
recombinase expression via the proximal lymphocyte-specific
protein tyrosine kinase (Lck) promoter and effectively deletes the
floxed gene fragment at the DN stage (Fig S1B). To exclude the
possible effect of Lck-driven Cre expression on T-cell development
as reported (22), we used heterozygotes (Lsd1
wt/fl
Lck-Cre) as lit-
termate controls (referred to hereafter as controls). The expression
of Lsd1 in thymocytes was greatly lower in Lsd1
fl/fl
Lck-Cre mice
(referred to hereafter as KO) than in the controls, showing the high
efficiency of Lsd1 deletion (Fig S1C). By performing Western blots on
nuclear extracts, we observed increased H3K4me2 modification in
KO thymocytes, whereas the H3K4me1/3, H3K9me1/2/3, and H3K27ac
modifications were not changed (Fig S1C). This was further confirmed
by flow cytometry. The results showed that H3K4me2 was increased
explicitly in Lsd1-deleted T lineage cells beginning in the DN stage,
whereas the H3K4me1 level was not changed (Fig S1D).
We observed that at 6~10 wk of age, KO mice had a markedly
smaller thymus (Fig 1A) with a 35% reduction in weight (Fig 1B), a
50% reduction in absolute cell numbers (Fig 1C), and an abnormal
architecture with remarkably atrophic cortex and expanded medullary
regions (Fig 1D). Increased cell apoptosis in the Lsd1-KO thymus had
also been detected (Fig S1E). Flow cytometry analysis showed that,
compared with control mice, KO mice exhibited no changes in the
percentages of CD4
−
CD8
−
cells and CD4
+
CD8
+
cells but exhibited a
considerable decrease in the percentages of CD4
+
CD8
−
cells and a
relative increase in the percentages of CD4
−
CD8
+
cells in thy-
mocytes (Fig 1E–G).
We further investigated whether the loss of Lsd1 affects mi-
gration of mature T cells to the periphery. As shown in Fig S2, the
total cell numbers of spleens and lymph nodes were not affected
(Fig S2A). However, the frequencies of CD3
+
T cells in the spleens (Fig
2A) and lymph nodes (Fig S2B) from KO mice were significantly
decreased compared with those of control mice. Moreover, we
found that the expression levels of CD3 on peripheral T cells were
down-regulated (Fig 2B). The percentages of both CD4
+
and CD8
+
T cells were greatly reduced in the spleen (Fig 2C) and lymph nodes
(Fig S2C). This was consistent with a previous report in which Lsd1
was deleted in immature DN thymocytes using Cd2-Cre mice (21).
The authors attributed this finding to the reduced S1pr1 required for
thymocyte emigration on mature SP cells. However, it was inter-
esting that although the CD4/CD8 ratio was altered inside the
thymus in KO mice (Fig 1G), it was not affected in spleens (Fig 2D)
and lymph nodes (Fig S2D). Notably, the proliferation capacities of
both peripheral CD4
+
and CD8
+
T cells under anti-CD3 and anti-CD28
stimulation were impaired after Lsd1 deletion (Fig 2E), which may
have also contributed to the significant decrease in the number of
peripheral T cells. In summary, the loss of Lsd1 disrupted the
numbers and proliferation of mature T cells in the periphery.
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 the 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
compared with control mice (Fig S3A and B). We examined whether
the accumulation of B or NK cells resulted from transdifferentiation
of T-precursors or reactive hyperplasia. YFP was used as an indi-
cator 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 emit fluorescence. Our
results showed that no YFP
+
B cells were observed in the thymus,
bone marrow, spleen, and lymph nodes from KO mice, which in-
dicated that no transdifferentiation from T to B cells occurred after
Lsd1 deletion (Fig 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 (Fig S3C). Considering the similar
frequencies of YFP
+
thymic NK cells in KO mice compared with
control mice, we speculated that it resulted from reactive hyper-
plasia but not transdifferentiation. Consistently, a previous in-
vestigation also observed increased numbers of B cells and NK cells
in the thymus in Lsd1
fl/fl
CD2-Cre mice but not in Lsd1
fl/fl
CD2-Cre DN
thymocyte cultures on OP9-DL1 cells (21). In summary, the observed
increased numbers of other lineage cells were not caused by
transdifferentiation from T cells. This may be caused by the change
Ablation of Lsd1 induces viral mimicry in thymocytes Xia et al. https://doi.org/10.26508/lsa.202302042 vol 6 | no 10 | e202302042 2of16
in the thymic microenvironment after the specific deletion of Lsd1
in thymocytes.
Single-cell sequencing reveals that ablation of Lsd1 disturbs the
programmed down-regulation of CD8 expression at the
DP→CD4
+
CD8
lo
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 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 bio-
informatically 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 of 11 cell
types at different developmental stages of T thymocytes using
SingleR with the ImmGen reference dataset (23). The cell types were
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, γδT and natural killer T (NKT) cells; other lineages,
namely, B cells and dendritic cells (DCs), were also observed (Fig 3B
and C).
Accordingly, we observed similar subpopulation distributions in
the thymi from control and KO mice (Fig 3D). Furthermore, we
calculated the proportions of the different subgroups. Consistent
with our flow cytometry analysis, we observed increased fre-
quencies of other lineages (e.g., B cells and DCs) in the scRNA-seq
data (Fig 3E). Considering the T-cell lineage, the proportions of
Tregs and NKTs were increased, which was confirmed by flow
cytometry (Fig S3D). However, the proportions of other T-cell
subgroups showed no significant differences (Fig 3E). This result
was striking because significant changes were observed in the
percentages of CD4
+
cells and CD8
+
cells in flow cytometry analysis
(Fig 1F). We then tried to determine what made the difference.
Interestingly, we observed greatly increased CD8 expression and
decreased CD4 expression in the T.SPinter cells (representing the
intermediate CD4
+
CD8
lo
stage) and CD4Th cells in the Lsd1-deleted
mice (Fig 4A). CD4
+
CD8
+
DP thymocytes differentiate into CD4
+
CD8
lo
cells and then make a lineage choice to become either CD4
+
or CD8
+
SP T cells (Fig 4B)(24). The increased CD8 expression and decreased
CD4 expression in the T.SPinter cells could have led them to be
counted in the CD8
+
SP gate in flow cytometry analysis (Fig 4C).
However, in the scRNA-seq map, they were still defined as T.SPinter
cells based on their general transcriptome. Consistently, the
Figure 1. Ablation of Lsd1 causes thymic atrophy.
(A) Images of thymi from control (n = 3) and KO (n = 3) mice. (B) Weight of the thymi from control (n = 6) and KO (n = 5) mice. (C) Total cell numbers of thymocytes from
control (n = 6) and KO (n = 6) mice. (D) Representative hematoxylin and eosin (H&E) staining of thymi from control and KO mice. Original magnification: ×4 (up), ×10 (down).
Scale bars: 2,000 μm. (E) Representative CD4 versus CD8 staining of total live thymocytes from control and KO mice. (F) Percentages of the indicated populations in total
thymocytes from control (n = 6) and KO (n = 5) mice. DN: CD4
−
CD8
−
double-negative cells, DP: CD4
+
CD8
+
double-positive cells, CD4SP: CD4
+
CD8
−
single-positive cells,
CD8SP: CD4
−
CD8
+
single-positive cells. (G) Ratios of CD4SP versus CD8SP subsets in the thymi from control (n = 6) and KO (n = 5) mice. Control: Lsd1
wt/fl
Lck-Cre, KO: Lsd1
fl/
fl
Lck-Cre. Cumulative data are means ± SEMs. *P< 0.05, **P< 0.01, ***P< 0.001, ns, no significance, as determined by unpaired ttest.
Ablation of Lsd1 induces viral mimicry in thymocytes Xia et al. https://doi.org/10.26508/lsa.202302042 vol 6 | no 10 | e202302042 3of16
proportions of CD4
+
/CD8
+
cells in flow cytometry could be mimicked
by adding T.SPinter cells to the CD8
+
group for abundance analysis
in the scRNA-seq data (Fig 4D). Furthermore, CCR9 was identified as
a surface marker distinguishing T.SPinter cells from CD4Th cells (Fig
4E), consistent with that in the human thymus (25). To confirm this
hypothesis, we stained thymocytes with anti-CCR9 antibodies. As
expected, CCR9
+
and CCR9
−
subgroups were observed under the
CD4
+
CD8
−
gate by flow cytometry (Fig 4F). The CD4
+
CCR9
+
subgroup
size was significantly decreased in KO mice, whereas the number of
CD8
+
CCR9
+
cells was increased.
Furthermore, we investigated the effect of CD8 up-regulation on
T-cell development. It has been reported that constitutively
expressed CD8 can promote most of the MHC-I-restricted thymo-
cytes to develop into innate memory-like CD8
+
T cells rather than
redirecting them to the CD4 helper T-cell lineage (26). Consisten-
tly, we observed increased CD8
+
T cells with an innate-memory
Figure 2. Decreased peripheral T cells with impaired proliferation capacity in Lsd1-deleted mice.
(A) Representative histograms show the percentages of CD3
+
cells in the spleens of control and KO mice. Frequencies of CD3
+
T cells in the thymi from control (n = 6) and
KO (n = 5) mice are statistically shown on the right. (B) The mean fluorescence intensity (MFI) of CD3 expression on thymocytes from control (n = 6) and KO (n = 5) mice.
(C) Representative CD4 versus CD8 staining of splenocytes from control and KO mice. The cumulative data on the frequencies of CD4
+
or CD8
+
cells in the spleen from
control (n = 5) and KO (n = 4) mice are shown on the right. (D) Ratio of CD4
+
versus CD8
+
subsets in the spleen. (E) Analysis of the proliferation capacities of splenic T cells.
Splenocytes from control (n = 3) and KO (n = 3) mice were labeled with CFSE and stimulated with anti-CD3 and anti-CD28 in vitro. After 3 d of culture, the proliferation of
CD4
+
T cells and CD8
+
T cells was examined by flow cytometry for the dilution of CFSE. Control: Lsd1
wt/fl
Lck-Cre,KO:Lsd1
fl/fl
Lck-Cre. Cumulative data are means ± SEMs. *P<
0.05, **P< 0.01, ***P< 0.001, as determined by unpaired ttest.
Ablation of Lsd1 induces viral mimicry in thymocytes Xia et al. https://doi.org/10.26508/lsa.202302042 vol 6 | no 10 | e202302042 4of16
Figure 3. scRNA-seq of thymocytes.
(A) Schematic of procedures for the sorting and scRNA-seq of thymocytes. (B) UMAP visualization of scRNA-seq data for single cells derived from control (n = 3) and KO
(n = 3) mice. Cells were color coded by cell type; each dot represents one cell. ETP, early T-cell progenitor; DN, double-negative cell; ISP, immature single-positive cell;
T.DPbl, blasting double-positive T cell; T.DPsm, small resting double-positive T cell; T.DP69
+
, CD69
+
double-positive T cell, early positive selection; T.SPinter, intermediate
single positive T cell; T.CD4Th, CD4
+
helper T cell; T.CD8, CD8
+
T cell; T.CD4Treg, CD4
+
regulatory T cell; NKT, natural killer T cell; DC, dendritic cell. (C) Expression patterns of
marker genes for each cell subtype. The fraction of cells that expressed the marker genes is indicated by the size of the circle, as shown in the scale on the right. The
means of the expression levels of marker genes are indicated by the color. (D) UMAP visualization of scRNA-seq data derived from control (up) and KO (down) mice. The
cells are color coded by cell type; each dot represents one cell. (E) Cell proportions are shown by cell type in control (n = 3) and KO (n = 3) mice. Control: Lsd1
wt/fl
Lck-Cre,
KO: Lsd1
fl/fl
Lck-Cre. Cumulative data are means ± SEMs. *P< 0.05, as determined by unpaired ttest.
Ablation of Lsd1 induces viral mimicry in thymocytes Xia et al. https://doi.org/10.26508/lsa.202302042 vol 6 | no 10 | e202302042 5of16
phenotype expressing CD44 and CXCR3 in KO thymocytes (Fig S4A),
and significantly elevated innate-memory scores of the T.CD8
subgroup in scRNA-seq data (Fig S4B). Similarly, in a previous study
on Lsd1
fl/fl
Cd2-Cre mice, the expression of innate memory T-cell-
associated genes was increased in CD69
−
thymocytes, CD8 SP
thymocytes, and peripheral CD8
+
T cells (21). The authors concluded
that this resulted from the influence of Lsd1 on Bcl11b-mediated
genes because the phenotype was similar to that observed in
Bcl11b-deficient mice, and most of the Bcl11b-repressed genes were
up-regulated in Lsd1
fl/fl
Cd2-Cre CD69
−
thymocytes (21). However, the
interaction of Lsd1 and Bcl11b could not explain the change in CD8
expression. It appears that the innate memory phenotype may be
generated through multiple mechanisms. In summary, the loss of
Lsd1 disturbs the programmed down-regulation of CD8 expression
Figure 4. Ablation of Lsd1 disturbs the programmed down-regulation of CD8 expression at the DP→CD4
+
CD8
lo
stage.
(A) CD4 and CD8a expression in T-cell subgroups. (B, C) A model for the differentiation from DP to SP cells before (B) and after (C) the deletion of Lsd1. (D) CD4SP and
CD8SP abundance in scRNA-seq (right) mimicking that in flow cytometry (left) by adding SPinter cells to the CD8
+
group. (E) DEGs identified as marker genes distinguishing
the T.SPinter and T.CD4Th cells. (F) CCR9
+
T.SPinter cells in CD4
+
(left) and CD8
+
(right) thymocytes identified by flow cytometry. Statistical data are shown below. Control:
Lsd1
wt/fl
Lck-Cre, KO: Lsd1
fl/fl
Lck-Cre. Cumulative data are means ± SEMs. *P< 0.05, **P< 0.01, as determined by unpaired ttest.
Ablation of Lsd1 induces viral mimicry in thymocytes Xia et al. https://doi.org/10.26508/lsa.202302042 vol 6 | no 10 | e202302042 6of16
Figure 5. Deletion of Lsd1 activates the IFN response in thymocytes.
(A) Venn diagrams of the up-regulated genes after Lsd1 deletion in different T-cell subgroups. (B) Violin plots showing the expression of the 10 shared up-regulated
genes in different T subgroups from control and KO mice. (C) Heatmap of the significantly enriched GO terms for the genes overexpressed in different T-cell subgroups
from control and KO mice. (D) Violin plots show the ISG scores of each cell across different T-cell subgroup s from control and KO mice. P< 0.00001 in all compared groups.
Ablation of Lsd1 induces viral mimicry in thymocytes Xia et al. https://doi.org/10.26508/lsa.202302042 vol 6 | no 10 | e202302042 7of16
at the DP→CD4
+
CD8
lo
stage and promotes an innate memory
phenotype in CD8
+
T cells.
Lsd1 regulates activation of the IFN signaling in thymocytes
To determine how Lsd1 regulates thymic T-cell development, we
performed differentially expressed gene (DEG) and pathway en-
richment analyses of the scRNA-seq transcriptome data. As shown
in Fig 5A and B, we found 10 up-regulated 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 down-regulated genes was much lower, and
almost no shared genes were found. Surprisingly, among the
shared up-regulated genes, six genes belonged to the MHC I
complex, and the others were ISGs. Consistently, we found high
enrichment of IFN-response pathways and antigen-processing
pathways across all the T-cell subgroups (Fig 5C). Because the
IFN pathway is a positive regulator of the MHC antigen-processing
machinery (27), we can conclude that the DEGs and their related
pathways are associated with “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 down-regulated at the DN3b
stage and gradually increased during the subsequent develop-
mental stages. After the loss of Lsd1, ISG scores were significantly
elevated in all T-cell stages (Fig 5D), indicating excessive pre-
activation of the IFN pathway during the whole developmental
process. Quantitative polymerase chain reaction (qPCR) analysis
further confirmed the overexpression of MHC I molecules (H2-k1,
H2-d1) and ISGs (Irf7,Irf9,Oas3,Ifit1,Stat1,Nfkb1)(Fig 5E). As known,
T cells mainly secret IFNγunder stimulus (29). Increased expression
of IFN-II (IFN-γ) was determined at both the mRNA and protein
levels in KO mice compared with control mice (Fig S5A and B),
whereas the IFN-I gene (Ifna1,Ifnb) and IFN-III gene (Il28b) were
expressed at much lower levels and even showed some decrease in
expression (Fig S5A) in KO thymocytes. In addition, there is a
possibility that the abnormal up-regulation of IFNγin the thymic
environment also activated the related immune cells and caused
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 Lsd1 ablation was
causing aberrant activation of IFNγsignaling. This was consistent
with a previous report demonstrating that knockdown of Lsd1 in
immature T cells results in the overexpression of genes involved in
IFN/viral response-related functions, according to bulk RNA-seq of
CD69
−
cells and CD69
+
DP cells (21). Our data suggested that IFNγ
signaling was overactivated in all stages of T cells.
We then investigated whether Lsd1directly targets IFN-responsive
genes to repress their expression. Lsd1 is known as an H3K4me1/2
demethylase that acts as a transcription corepressor. We evaluated
whether Lsd1 directly regulates the modification of H3K4me1/2 at
IFN-responsive genes by analyzing 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 (21). They found few changed H3K4me1/2 marks, and
only a small fraction of H3K4me3 marks were increased. To be
more specialized, we analyzed the H3K4me1/2/3 modifications
of ISGs. As shown in Fig 5F, the H3K4me1 and H3K4me2 modifi-
cations of ISGs and the H3K4me2 marks of their enhancers were
decreased after Lsd1 depletion. They exhibited only enhanced
decoration with H3K4me3 marks at their enhancers. This result
indicated that the up-regulation of ISGs could result from other
epigenetic modifications. We conclude that the ISGs are not
directly regulated by the H3K4me1/2 demethylase activity of
Lsd1. In summary, the loss of Lsd1 activates the IFNγresponse in
thymocytes, whereas the overexpression of ISGs is not directly
regulated by Lsd1.
Deletion of Lsd1 derepresses EREs
We then attempted to determine what triggers the IFN response
in Lsd1-deleted thymocytes. Other than the IFNγresponse genes,
the pathway enrichment analysis of scRNA-seq data showed up-
regulation of the “response to the virus”pathway after Lsd1 loss
(Fig 5C), suggesting that the activation of an upstream event,
such as an RNA-sensing pathway, may play an important role.
EREs account for ~40% of mammalian genomes, and the si-
lencing of EREs is controlled by the state of histone methylation
(30). 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 to detect noncoding
RNAs. Increased numbers of transcripts in both sense and an-
tisense directions from all ERE subfamilies were detected in the
Lsd1-deleted thymocytes, including LTR-containing endogenous
retroviruses (ERV1s, ERVKs, and ERVLs) and non-LTR elements
(LINEs and SINEs) (Fig 6A and B). ChIP-seq analysis showed that
the loci of up-regulated EREs had a great increase in H3K4me1
and H3K4me2 levels, and a mild increase in H3K4me3 levels in KO
mice, which indicated that Lsd1 directly targets these EREs to
regulate their expression (Fig 6C). The overexpression of EREs
can contribute to the generation of dsRNAs, which trigger IFN
signaling activation. In addition, we detected the expression levels
of the dsRNA sensors Tlr3, Mda5 (encoded by Ifih1), and Rig-I
(encoded by Ddx58) and the DNA sensors Sting (encoded by
Sting1) and Cgas in Lsd1-deleted thymocytes and found that they
were all increased as expected (Fig 6D). These data suggested that
ablation of Lsd1 derepresses the transcription of a group of EREs,
resulting in aberrant activation of the IFNγresponse in mouse
thymocytes. Consistently, Lsd1 has been reported to be an ERV
suppressor in embryonic stem (ES) cells (30) and regulates the ERV-
IFN pathway in melanoma cells (16).
(E) The expression of MHC I molecules (H2-k1,H2-d1) and ISGs (Irf7,Irf9,Oas3,Ifit1,Stat1,Nfkb1) in control and KO thymocytes analysed by qPCR. The error bars represent
the SD between triplicates in one of three experiments. (F) H3K4me1, H3K4me2, and H3K4me3 ChIP-seq signals at ISG loci (up) and their enhancer regions (down) in control
and KO thymocytes. Control: Lsd1
wt/fl
Lck-Cre, KO: Lsd1
fl/fl
Lck-Cre. Cumulative data are means ± SEMs. *P< 0.05, **P< 0.01, ***P< 0.001, ns, no significance, as determined by
unpaired ttest.
Ablation of Lsd1 induces viral mimicry in thymocytes Xia et al. https://doi.org/10.26508/lsa.202302042 vol 6 | no 10 | e202302042 8of16
We next investigated the biological effects of the Lsd1 deletion-
induced viral mimicry state and IFNγsignaling activation.
We observed that compared with those in control mice, the per-
centages of TCR
hi
CD69
+
CD24
+
immature T cells in KO mice were
decreased, whereas those of TCR
hi
CD69
−
CD24
−
mature T cells were
increased, indicating a premature state of thymic T cells (Fig S6).
Moreover, in addition to the innate memory phenotype of CD8
thymocytes mentioned before, we found elevated innate memory
scores in all Lsd1-deleted T subgroups by analyzing the scRNA
transcriptome (Fig S4B). In addition, the mature T cells in the pe-
riphery of KO mice also showed an effector/memory phenotype
in the absence of antigen stimulation (Fig S4C). It has been
indicated that up-regulation of CD8 expression is associated
with the innate memory phenotype in thymic CD8 SP cells. We
believe that continuous viral mimicry stimulation and activated
IFNγsignaling could be another way to contribute to this phe-
notype (31).
TCR formation and selection in Lsd1-deleted thymocytes
TCR signaling is essential for T-cell activation. Notably, we observed
decreased expression of TCR receptors on Lsd1-deleted SP cells
(Fig S6), suggesting that dysplasia of T-cell function resulted in a
premature activation state. Moreover, it is known that TCR re-
combination events control T-cell development as major check-
points. Thus, we also investigated the kinetics of TCR recombination
by single-cell TCR sequencing. TCRβtranscripts were noticed as
early as in the DN3b-ISP subgroup, and TCRαtranscripts appeared
in large numbers at the CD69
+
DP stage in both control and KO thymi
(Fig 7A), indicating that Lsd1 deletion did not disturb the timeline of
TCR V(D)J gene recombination events. Furthermore, we analyzed the
patterns of the TCR repertoire in different cell types associated with
our annotation.
For TCRβ, we observed recombination of the D1 gene with J1 and
J2 segments and the D2 gene with J2 segments, whereas nearly no
Figure 6. Deletion of Lsd1 derepresses EREs.
(A) A volcano plot showing differentially expressed EREs (both forward and reverse strands) in total thymocytes from control and KO mice. Increased loci are shown in
red and decreased loci are shown in blue. The top 10 significantly expressed loci are labeled. (B) The differential expression of ERE classes comparing thymocytes from
control and KO mice. (C) H3K4me1, H3K4me2, and H3K4me3 ChIP-seq signals at genomic loci of the up-regulated EREs in thymocytes from KO mice compared with those
from control mice. (D) The expression of the RNA and DNA sensors in control and KO thymocytes were analyzed by qPCR. Control: Lsd1
wt/fl
Lck-Cre, KO: Lsd1
fl/fl
Lck-Cre.
The error bars represent the SD between triplicates in one of three experiments. *P< 0.05, **P< 0.01, ***P< 0.001, as determined by unpaired ttest.
Ablation of Lsd1 induces viral mimicry in thymocytes Xia et al. https://doi.org/10.26508/lsa.202302042 vol 6 | no 10 | e202302042 9of16
use of D2-J1 (Fig 7B and C) was observed in either control or KO
thymocytes. This should be associated with their position in the
genome, where D2 is located after J1 (32)(Fig 7D). The Vβ-D-Jβ
diversity increased after the development process in both control
and KO thymocytes (Fig 7B). For TCRα, we found that the proximal V
segments and J segments recombined first during development;
Figure 7. TCRβrepertoire revealed by scRNA-seq.
(A) UMAP visualization of scRNA-seq data derived from control and KO mice. Cells were color coded by TCR receptors; each dot represents one cell. TRA, TCR αchain; TRB,
TCR βchain. (B, C) Heatmap showing the proportion of each TCRβV-D-J pattern present at progressive stages during T-cell development. The red asterisk indicates
significantly higher usage compared with the other group. (D) Schematics illustrating the genomic location of the Vβ, D, and Jβgene segments.
Ablation of Lsd1 induces viral mimicry in thymocytes Xia et al. https://doi.org/10.26508/lsa.202302042 vol 6 | no 10 | e202302042 10 of 16
this was followed by the recombination of the distal segments (Fig
S7), as previously described in humans (25). For both TCRβand
TCRα, contractions of their repertoire during the transition from DP
to SP were observed. In addition, different V(D)J patterns were
shown in the developed CD4.Th, Treg, and CD8 cells (Figs 7C and S8).
These changes suggest differences between Vβand Vαgenes’
affinity to MHC molecules and self-antigen peptides during positive
and negative selection. It has been shown that the loss of Lsd1
causes the preactivation of T cells by derepressing ERE expression.
However, generally, we observed similar diversity and TCR reper-
toires in different cell types of control and KO mice. Several V-D-J
patterns were found to be used differently in DP thymocytes.
However, the differences were diminished in SP thymocytes after
the “selection”process. That is, unlike the conventional clonal
expansion of TCRs after foreign antigen stimulation, endogenous
ERE stimulation did not cause a bias in the selection of TCR
clones.
Together, the findings indicated that conditional deletion of Lsd1
in immature T cells led to severe thymic atrophy and disrupted the
numbers and proliferation of mature T cells in the periphery. The
programmed down-regulation of CD8 at the DP→CD4
+
CD8
lo
stage
was disturbed. Moreover, a subgroup of EREs was derepressed
with increased H3K4me1/2 modification, inducing the activa-
tion of IFNγsignaling at all developmental stages. An innate-
memory phenotype of thymic and peripheral T cells was
promoted, but no significant shift in the TCR repertoire was found
(Fig 8).
Discussion
In this study, we found that Lsd1 is critical for normal T-cell de-
velopment and the maintenance of the peripheral T-cell pool. The
cellularity of the KO thymus decreased by half; thus, significant
thymic atrophy was observed, consistent with that observed in
Lsd1
fl/fl
Cd2-Cre mice (21). DN and DP cells are present in the cortex.
Although their proportions were not disturbed, their decreased
Figure 8. Proposed model for the role of
Lsd1 in thymocyte development.
Under normal conditions (left), Lsd1
demethylates H3K4me1 and H3K4me2 at the
gene loci of a group of EREs and represses
their transcription. The thymocytes maintain
tonic IFNγsignaling for normal maturation.
After the deletion of Lsd1 in thymocytes
(right), H3K4me1 and H3K4me2 modifications
at the gene loci of a group of EREs are
increased, which leads to an accumulation
of dsRNAs and aberrantly activates IFNγ
signaling at all developmental stages of
thymocytes. In the tissue level, deletion of
Lsd1 causes severe thymic atrophy with cortex
atrophy and medulla expansion. Decreased
numbers of peripheral T cells were also
observed. Notably, the programmed down-
regulation of CD8 expression at the
DP→CD4
+
CD8
lo
stage is disrupted, and an
innate memory phenotype is induced in both
thymic and peripheral T cells.
Ablation of Lsd1 induces viral mimicry in thymocytes Xia et al. https://doi.org/10.26508/lsa.202302042 vol 6 | no 10 | e202302042 11 of 16
absolute numbers led to a remarkably atrophic cortex in the KO
thymus; and the increased thymic B and DC cells, which are po-
sitioned in the cortico-medullary junction and medullary region,
respectively, may contribute to the medullary expansion. Fur-
thermore, scRNA-seq revealed similar frequencies of different
T-cell subgroups in control and KO mice, except Tregs and NKTs.
Interestingly, CD8 expression could not be down-regulated in a
timely manner at the intermediate SP stage (CD4
+
CD8
lo
CCR9
+
)inKO
mice, whereas CD4 expression unexpectedly decreased. However, it
did not redirect the following development path to CD4 or CD8 SP
cells, as similar CD4/CD8 ratios were observed in the thymus and
periphery. Instead, an innate-memory phenotype of CD8
+
cells was
found. This is consistent with a previous study in which CD8 was
overexpressed in thymocytes (26). However, more investigation into
how Lsd1 regulates CD8 and CD4 expression at the DP to CD4
+
CD8
lo
stage is needed.
After the loss of Lsd1, the results of DEG and pathway en-
richment analyses focused on aberrant activation of IFNγsig-
naling across all the T-cell subgroups, suggesting that this
pathway was closely regulated by Lsd1 during T-cell develop-
ment. Consistently, overexpression of IFN response genes has
been observed after the deletion of Lsd1 via Cd2-Cre (22).
However, in that study, it remained unclear why the overex-
pressed genes in Lsd1-deleted thymocytes were not always
associated with increased H3K4 methylation, and the authors
concluded that Lsd1 indirectly affects their expression. Here, our
study further revealed a subgroup of EREs that exhibited in-
creased H3K4me1/2 modification in the KO thymus and could be
a direct target of Lsd1. Unlike other viruses, EREs are ancient
retroviruses that integrate into host genomic DNA in a germline
cell and are inherited by the host’s offspring. The epigenetic
regulation of ERE transcription in mammalian germ cells and
early embryonic development is well documented (33,34). Re-
cent investigations have also demonstrated that the regulation
of ERE transcription plays an important role in human and
mouse cancer cells (16,35,36). However, little is known about its
function in the development of immune cells. Pathogen-induced
thymus atrophy is a common phenomenon in infectious dis-
eases, featuring contracted thymic parenchyma and reduced cell
numbers (37). Here, we reported that under specificpathogen-
free conditions, the loss of Lsd1 in thymocytes caused a similar
phenotype by derepressing the expression of a subgroup of EREs.
The viral mimicry state induced a general IFN response in thy-
mocytes and increased IFN-γexpression levels. Previous in-
vestigations have shown that up-regulation of IFN signaling and
increased IFN-γsecretion in the thymic microenvironment are
involved in the mechanisms underlying infection-induced thy-
mus atrophy (37). As increased apoptosis has been observed in
Lsd1-deleted thymocytes, there could be IFN-γinduced cell death
causing thymic atrophy in the KO thymus. On the other hand,
T-cell development requires tonic type I IFN signaling. IFN-βis
constitutively expressed in thymic medullary epithelial cells, and
maturing medullary thymocytes respond to constitutively pro-
duced IFN targeting STAT1 and IRF7 (38,39,40,41). Overall,
maintaining IFN signaling at a moderate level is necessary for the
normal development and maturation of thymic T cells. Thus, Lsd1
acts as an important controller of ERE-IFN signaling.
TCR signaling plays a critical role in programmed T-cell de-
velopment and is highly associated with T-cell activation. Com-
bined with the scRNA-seq data, we were able to analyze the TCR
repertoire among different cell types in the mouse thymus. The
initial recombination showed a strong bias, whereas the diversity
increased along with DP blasting. Selection is known to shape the
TCR repertoire, and enrichment and deletions of TCR V(D)J pat-
terns could be observed at the SP stages. However, the pre-
activation state after Lsd1 deletion neither disturbed the timeline
of TCR rearrangement nor reshaped the TCR repertoire of SP cells,
which may have been because of the innate response to ERE
expression rather than acquired immunity against a specific
foreign antigen.
Peripheral T cell numbers were markedly reduced after the
deletion of Lsd1, consistent with the findings of a previous study
on Lsd1
fl/fl
Cd2-Cre mice (21). They reported a defect in mature SP
cell emigration with reduced expression of the emigration
marker S1pr1 on mature SP cells. Moreover, we found reduced
CD3 expression and impaired proliferation capacity of the pe-
ripheral T cells in KO mice, which may have also contributed to
the atrophic peripheral T-cell pool. It would be interesting to
gain a deeper understanding of the impact of Lsd1 on the
function of mature T cells. Recently, a research group knocked
out Lsd1 by crossing Cd4-Cre transgenic mice with Lsd1-floxed
mice. Because Lsd1 significantly decreased beginning at the
mature peripheral T cells in their study, little effect on thymocyte
development was found. They demonstrated that Lsd1 loss in
peripheral CD8
+
T cells resulted in an increased pool of
progenitor-exhausted CD8
+
T cells, providing a sustained source
for more differentiated T cells with a stronger tumor-killing
capacity (42). Similarly, a “memory CD8
+
T-cell signature”and
an enrichment of “positive regulation of IFN-γproduction”were
observed in these Lsd1-deficient CD8
+
tumor-infiltrating cells.
Together, these data suggest that Lsd1 is involved in similar
biological pathways at different developmental stages of T cells.
For CD4
+
T cells, we found no change in the percentage of Th cells
but an increase in the percentage of Tregs in the thymus. The
effect of Lsd1 on the function and differentiation of peripheral
CD4
+
Tcellsisstillunknown.
Lsd1 is up-regulated in many cancers and plays a key role in
carcinogenesis. Numerous Lsd1 inhibitors are undergoing clinical
trialsforcancertherapy(43). Recently, it was shown that targeting
Lsd1 in B16 tumor cells can also regulate host antitumor T-cell
immunity. Lsd1 inhibition reinforces dsRNA stress and IFN re-
sponses, which up-regulates the expression of MHC-I molecules
and the checkpoint receptor PD-L1 in tumor cells (16). Similar
pathways have been found in tumor cells controlled by other
histone modulators, such as Kdm5b (35). Here, our study reveals
that Lsd1 plays an important role in governing the IFN pathway by
controlling ERE transcription for the normal development of
thymic T cells and sustaining the peripheral T-cell pool, which
should be taken into consideration in Lsd1 inhibitor-based cancer
therapy. Our findings highlight the impact of the viral mimicry
state on the early development of T cells and suggest that early
onset infections invading the thymus could have potential ad-
verse effects on T-cell development and the building of the im-
mune system.
Ablation of Lsd1 induces viral mimicry in thymocytes Xia et al. https://doi.org/10.26508/lsa.202302042 vol 6 | no 10 | e202302042 12 of 16
Materials and Methods
Mice
Lsd1
fl/fl
mice (loxP-flanked Lsd1 allele at exons 5 and 6) (17), Lck-
Cre mice (44), and YFP-transgenic mice were kind gifts from
Dana-Farber Cancer Center, Boston, MA USA. The deletion was
reported to be >50% in immature CD44
+
DN cells and almost
100%completedinCD44
−
DN or DP and SP cells (44). 6–10-wk-old
mice were used for the experiments. All mice were bred under
specific pathogen-free conditions. All experiments were ap-
proved by the Capital Medical University Animal Care and Use
Committee.
Flow cytometry
Single-cell suspensions were prepared from the thymus, spleen,
and lymph nodes. Fluorochrome-conjugated antibodies against
CD4 (RM4-5; eBioscience), CD8 (53-6.7; BD Biosciences), CD3
(17A2; BioLegend), CD44 (IM7; BD Biosciences), CD62L (MEL-14;
BioLegend), B220 (RA3-6B2; BioLegend), NK1.1 (PK136; BD Bio-
sciences), CD122 (TM-β1; BD Biosciences), CD69 (H1.2F3; eBio-
science), TCRβ(H57-597; eBioscience), and CD24 (M1/69; eBioscience)
were used for staining. FVS510 (BD Biosciences) was used for
dead/live cell staining. For intracellular staining, cells were
first stained with surface antibodies and then fixed and per-
meabilized with freshly prepared fixation/permeabilization
working solution (BD Biosciences) according to the manufac-
turer’s instructions. Then, the cells were stained with anti-IFNγ
(XMG1.2; eBioscience), anti-H3K4me1 (D1A9; CST) or anti-H3K4me2
(Y47; Abcam) diluted in permeabilization buffer. Data were acquired
on an LSRFortessa (BD Biosciences) and analyzed with FlowJo
Software (version 10.6.2). For cell sorting, single-cell suspen-
sions isolated from the thymus were sorted on a FACSAria II (BD
Biosciences).
Histological analysis
Thymi harvested from control and Lsd1
fl/fl
Lck-Cre mice were fixed in
10% formalin solution and embedded in paraffin. Haematoxylin–
eosin (HE) standard staining was performed by Servicebio Tech-
nology Co., Ltd.
RNA extraction and qPCR
Whole thymi were dissected, and total cellular RNA was
extracted by RNA Isolater Total RNA Extraction Reagent (Vazyme)
following the manufacturer’s instructions. Total RNA (1 μg)
was reverse transcribed using HiScript QRT SuperMix (+gDNA
Wiper) (Vazyme) to generate cDNA. With ChamQ SYBR qPCR Master
Mix (Vazyme), qPCR was performed on the Real-Time PCR detection
system. All data were normalized to β-actin mRNA levels, and the
2
−4CT
method was used to calculate the expression levels of target
mRNAs. The primers used in the qPCR analysis are presented in
Table S1.
T-cell proliferation
T-cell proliferation was assessed by CFSE dilution with FACS (45,46).
Briefly, a single-cell suspension of splenocytes was prepared and
labeled with CFSE (CellTrace CFSE Cell Proliferation Kit; Thermo
Fisher Scientific). The labeled cells were cultured in 96-well plates
precoatedwith5μg/ml anti-CD3 monoclonal antibodies and
5μg/ml anti-CD28 monoclonal antibodies. After 72 h, splenocytes
were stained with APC-Cy7 anti-mouse CD4 (GK1.5), Percp-Cy5.5 anti-
mouse CD8 (53-6.7), and FVS510 to exclude dead cells. Non-CFSE-
labeled splenocytes and unstimulated splenocytes were used as
controls.
Bulk RNA-seq data processing
The raw reads were aligned to the reference genome mm10 using
STAR aligner (version 2.7.9a) with the parameters “--clip3pNbases=0
–clip5pNbases=10 10 –winAnchorMultimapNmax 200 –out-
FilterMultimapNmax 100 –outSAMstrandField intronMotif”.The
repeatmasker annotation GTF file for mm10 was obtained using the
UCSC table browser (47). NCBI RefSeq mm10 was used for gene
annotation. Reads falling in either annotated repeat regions or
genes were counted using featureCounts (48) (version 2.0.1) with
the parameters“-p -B -M -t exon -s 2”and differential accessibility
analysis was performed with DESeq2 (49) (version 1.34.0) with
default parameters. Repeat regions with the same “gene_id”but
different loci were treated as different repeat regions. Repeat re-
gions with P-values < 0.05 and |log
2
(FC)| values > 0.5 were con-
sidered differentially expressed. In addition, the log
2
(FC) values
were calculated by subtracting the log
2
-transformed mean counts
in each group.
scRNA-seq data processing
The raw sequencing data of the thymus cells from control and Lsd1
KO mice were processed using Cell Ranger software (version 6.0.1)
against the GRCm38 mouse reference genome with the default
parameters. First, we filtered low-quality cells with detected gene
numbers between 200 and 4,000 and less than 10% mitochondrial
unique molecular identifiers using Seurat (version 4.0.6). Subse-
quently, we used Scrublet (50) (version 0.2.3) to eliminate doublets
among control and KO mice. We used the default parameters for
Scrublet (i.e., Eq. min_gene_variability_pctl = 85, n_prin_comps = 30,
threshold = 0.25) and detected 12 doublets in the WT mice and three
doublets in the KO mice. After removing the doublets, we nor-
malized the gene counts for each cell using the NormalizeData
function of Seurat (51) with the default parameters. The top 2,000
highly variable genes were used for principal component analysis.
For downstream data processing, we used the SelectInte-
grationFeatures function in Seurat to select features for inte-
gration and used the top 2,000 features to identify the anchor cells
in control mice and KO mice using the FindIntegrationAnchors
function in Seurat. We then used the IntegrateData function in
Seurat to integrate the cells from control mice and KO mice. We
clustered all the cells based on the integrated gene expression
matrix using Seurat with a parameter resolution=1.5 and generated
28 clusters. To display the cells in a two-dimensional space, we
Ablation of Lsd1 induces viral mimicry in thymocytes Xia et al. https://doi.org/10.26508/lsa.202302042 vol 6 | no 10 | e202302042 13 of 16
performed PCA on the integrated dataset and used the first 15
principal components (PCs) for UMAP analysis.
Differential expression analysis
To identify DEGs between two groups of clusters, we used the
Wilcoxon rank-sum test in Seurat to evaluate the significance of
each gene. log
2
(FC) was calculated by subtracting log
2
-transformed
mean counts in each group. Genes with a P-value < 0.01 and
|log
2
(FC)| > 0.25 were considered differentially expressed.
Gene functional annotation
Gene Ontology, gene set enrichment analysis, and KEGG pathway
analyses for DEGs were performed using the Metascape (52)
webtool (www.metascape.org), which supports statistical analysis
and visualization of functional profiles for genes and gene clusters.
Calculation of gene set scores
The gene sets of the innate memory score and ISG score were
obtained from the original article (21,53) (Table S2). The gene set
scores were calculated with the built-in function scanpy.tl.score
genes in Scanpy.
scTCR-seq data processing
The TCR sequence data from 10× Genomics were processed using
Cell Ranger software (version 6.0.1) with the manufacturer-supplied
mouse VDJ reference genome. For each sample, the output file
filtered_contig_annotations.csv, containing TCR α- and β-chain
CDR3 nucleotide sequences, was used for downstream analysis.
Only those assembled chains that were productive, highly confi-
dent, full length, with a valid cell barcode and an unambiguous
chain type (for example, alpha) assignment were retained. If a cell
had two or more qualified chains of the same type, only the chain
with the highest unique molecular identifier count was qualified
and retained.
ChIP-seq data processing
TheChIP-seqdatawereobtainedfromapublicstudyinwhich
Lsd1 was knocked out by Cd2-Cre recombinase (21). Consistent
with our study, a reduction in Lsd1 was observed at the DN stage.
The raw reads were aligned to the reference genome mm10 using
Bowtie2 (54) aligner (version 2.2.5). The resultant SAM files were
converted to BAM files with samtools (version 1.3.1). Duplicate
reads were filtered using Picard. MACS3 (55) (version 3.0.0a7) was
used to call peaks on the BAM files. The bedGraph files containing
signal per million reads produced from MACS3 were converted to
bigWig files with the UCSC-toolkit. ChIP-seq signals of ISGs and
differentially expressed EREs were extracted and visualized with
the deepTools (56)(version3.5.1)commandcomputeMatrix and
plotProfile from bigWig files.
Statistical analyses
Statistical analyses were performed using GraphPad Prism software
(version 8.0). The statistical significance was determined with ttest.
AP-value of less than 0.05 was considered statistically significant.
For scRNA-seq data, statistical analysis was performed with the
Python (version 3.8.10) package “scipy”(version 1.7.0).
Data Availability
The accession numbers for the raw data of scRNA-seq and
strand-specific total RNA-seq are GSA:CRA007488 and CRA007498,
respectively.
Supplementary Information
Supplementary Information is available at https://doi.org/10.26508/lsa.
202302042.
Acknowledgements
We thank all the faculties at the Flow Cytometry Core of Capital Medical
University for assistance with flow cytometry sorting and analyzing. We
thank Han Yan (Tianjin Medical University) for helping with the
model figure drawing. We thank the bioinformatics support from
Genewiz Company. We thank the USTC supercomputing center and
the School of Life Science Bioinformatics Center for providing com-
putational resources for this project. This work was supported by
the National Natural Science Foundation of China (grant# 32270635,
81972652 and 81171899 to X Wang, grant# 82201918 to M Xia, grant,
#32022014 to P Chen, grant#91940306, T2125012, 31970858, and
31771428 to K Qu), the Ministry of Science and Technology of People’sRe-
public of China (grant# 2014CB910100 to X Wang), ScientificRese-
arc h Common Program of Beijing Municipal Commission of Education
(grant#KM201910025026 to M Xia), CAS Project for Young Scientists in Basic
Research (grant# YSBR-005 to K Qu), and Fundamental Research Funds
for the Central Universities (grant# YD2070002019, WK9110000141, and
WK2070000158 to K Qu).
Author Contributions
M Xia: conceptualization, data curation, formal analysis, funding
acquisition, investigation, visualization, and writing—original draft.
B Wang: conceptualization, data curation, formal analysis, and
visualization.
W Sun: software, formal analysis, and visualization.
D Ji: validation.
H Zhou: investigation.
X Huang: conceptualization and validation.
M Yu: conceptualization and resources.
Z Su: conceptualization.
P Chen: conceptualization, supervision, funding acquisition, method-
ology, project administration, and writing—review and editing.
K Qu: conceptualization, data curation, software, supervision, funding
acquisition, methodology, and writing—review and editing.
Ablation of Lsd1 induces viral mimicry in thymocytes Xia et al. https://doi.org/10.26508/lsa.202302042 vol 6 | no 10 | e202302042 14 of 16
X Wang: conceptualization, resources, supervision, funding acqui-
sition, project administration, and writing—review and editing.
Conflict of Interest Statement
The authors declare that they have no conflict of interest.
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