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Research Article
Constitutive CD8 expression drives innate CD8
+
T-cell
differentiation via induction of iNKT2 cells
Satoshi Kojo* , Michiko Ohno-Oishi*, Hisashi Wada, Sebastian Nieke, Wooseok Seo , Sawako Muroi, Ichiro Taniuchi
Temporal down-regulation of the CD8 co-receptor after receiving
positive-selection signals has been proposed to serve as an
important determinant to segregate helper versus cytotoxic
lineages by generating differences in the duration of TCR sig-
naling between MHC-I and MHC-II selected thymocytes. By con-
trast, little is known about whether CD8 also modulates TCR
signaling engaged by the non-classical MHC-I–like molecule,
CD1d, during development of invariant natural killer T (iNKT) cells.
Here, we show that constitutive transgenic CD8 expression
resulted in enhanced differentiation of innate memory-like CD8
+
thymocytes in both a cell-intrinsic and cell-extrinsic manner, the
latter being accomplished by an increase in the IL-4–producing
iNKT2 subset. Skewed iNKT2 differentiation requires cysteine
residues in the intracellular domain of CD8αthat are essential for
transmitting cellular signaling. Collectively, these findings shed a
new light on the relevance of CD8 down-regulation in shaping the
balance of iNKT-cell subsets by modulating TCR signaling.
DOI 10.26508/lsa.202000642 | Received 8 January 2020 | Revised 16 January
2020 | Accepted 17 January 2020 | Published online 24 January 2020
Introduction
The thymus provides a specific microenvironment that supports
development of several types of T cells, including innate-like T cells,
such as invariant natural killer T (iNKT) cells and mucosal-associated
invariant T (MAIT) cells, and conventional T cells. Signaling via the TCR
plays a central role in driving differentiation of both innate-like and
conventional T cells (Hogquist & Jameson, 2014), although the TCR
diversity and the selecting MHC–presenting antigens are quite dif-
ferent between these two types of T cells; innate-like T cells such as
iNKT cells and MAIT cells express invariant TCRs that recognize non-
peptide antigens presented on non-classical MHC, CD1d and MR1,
respectively (Godfrey et al, 2015), whereas conventional T cells ex-
press diverse TCRs recognizing peptide antigens presented on
classical MHC (Hogquist & Jameson, 2014).
Essential roles of CD4/CD8 co-receptors in TCR/MHC interaction
during differentiation of the two major conventional T-cell subsets,
CD4
+
helper and CD8
+
cytotoxic cells, from common precursors,
CD4
+
CD8
+
double-positive (DP) thymocytes, are well characterized.
Thymocytes positively selected by class II MHC molecules (MHC-II
selected thymocytes) develop into CD4
+
CD8
−
single-positive (SP)
thymocytes that are committed to the helper lineage, whereas
MHC-I–selected thymocytes are directed to become CD4
−
CD8
+
SP
thymocytes committed to the cytotoxic lineage (Ellmeier et al, 1999).
It has been proposed that differences in the duration of the
positive-selection signal instruct distinct fates in post-selection
thymocytes (Singer et al, 2008). Thus, briefer TCR signals in MHC-
I–selected thymocytes caused by temporal down-regulation of the
CD8 co-receptor guide post-selection thymocytes to differentiate
into CD4
−
CD8
+
SP thymocytes. On the other hand, persistent TCR
signals in MHC-II–selected thymocytes supported by constitutive
CD4 expression activate a developmental program toward the
helper-lineage T cells via induction of the zing-finger transcription
factor ThPOK (He et al, 2005;Sun et al, 2005) through antagonizing
a transcriptional silencer in the Zbtb7b gene encoding ThPOK (He
et al, 2008;Setoguchi et al, 2008). Therefore, in what is called the
kinetic signaling model, distinct expression kinetics between CD4
and CD8 co-receptors have been proposed to play a key role in
segregating helper and cytotoxic lineages (Singer et al, 2008). In line
with this model, perturbation of positive-selection signaling du-
ration in MHC-II–selected thymocytes re-directs them to become
CD8
+
cytotoxic-lineage cells (Sarafova et al, 2005;Singer et al, 2008;
Adoro et al, 2012). On the other hand, constitutive transgenic CD8
expression guides about 30% of MHC-I–selected thymocytes to
differentiate into CD4
+
cells (Bosselut et al, 2001). One proposed
explanation for the low efficiency of such redirected differentiation
was down-regulation of the transgenic CD8αchain that hetero-
dimerized with endogenous CD8βchain.
In addition to TCR signals, cytokines play important roles in
controlling T-cell differentiation in the thymus. Signals by IL-7 are
crucial for the differentiation of CD8 SP thymocytes (McCaughtry
et al, 2012). Recently, IL-4 has been shown to support differentiation
of another type of CD8 SP thymocyte with the characteristics of
both the memory and innate cells, which is referred to as innate
memory-like CD8 T cells (Weinreich et al, 2010). The iNKT2 subset of
iNKT cells produces IL-4 and has been shown to be a major source
Laboratory for Transcriptional Regulation, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan
Correspondence: ichiro.taniuchi@riken.jp
*Satoshi Kojo and Michiko Ohno-Oishi contributed equally to this work
©2020Kojo et al. https://doi.org/10.26508/lsa.202000642 vol 3 | no 2 | e202000642 1of13
on 24 January, 2020life-science-alliance.org Downloaded from http://doi.org/10.26508/lsa.202000642Published Online: 24 January, 2020 | Supp Info:
of IL-4 in the thymic environment. Accordingly, an increase in the
numbers of iNKT2 cells, although they represent only a tiny sub-
population of total thymocytes, has a significant impact on the
generation of innate memory-like CD8 T cells (Lee et al, 2013). In
addition to iNKT2 cells, iNKT1 cells expressing IFN-γand iNKT17 cells
expressing IL-17 are also differentiated from iNKT precursors
(Constantinides & Bendelac, 2013). However, little is known about
how balanced differentiation of such iNKT-cell subsets is regulated.
In this study, we generated a novel transgenic mouse model
expressing the CD8αβ heterodimer or the CD8αα homodimer in the
absence of endogenous CD8α/CD8βchains and MHC-II molecules
and observed that two-thirds of MHC-I–selected thymocytes dif-
ferentiated into CD4
−
CD8
+
SP thymocytes, most of which acquired
signatures of innate memory-like CD8 T cells in both cell-intrinsic
and cell-extrinsic manner. The cell-extrinsic mechanism was linked
to results from enhanced differentiation of the iNKT2-cell subset.
Thus, our study sheds new light on the physiological relevance of
down-regulation of the Cd8 gene to fine-tune the balance of iNKT-
cell subsets.
Results
Developmental pathway to CD4
+
T cells from the CD8 SP stage in
Zbtb7b
ΔTE/ΔTE
mice
The initial activation of the Zbtb7b gene upon receiving positive-
selection signals is achieved mainly by a thymic enhancer (TE) (He
et al, 2008;Muroi et al, 2013). Therefore, removal of the TE from the
Zbtb7b locus results in delayed and low-level expression of ThPOK
in newly selected thymocytes (Muroi et al, 2013). However, se-
quential activation of a proximal enhancer restores ThPOK ex-
pression in a later developmental stage (Muroi et al, 2008). The
impaired ThPOK expression kinetics due to loss of the TE in
Zbtb7b
ΔTE/ΔTE
mice results in redirected differentiation of a small
proportion of MHC-II–selected thymocytes into CD4
−
CD8
+
SP thy-
mocytes (Muroi et al, 2013). On the other hand, enforced ThPOK
expression in CD8
+
T cells was shown to activate some helper-
lineage signature genes (Jenkinson et al, 2007). Given the restored
ThPOK expression in the later developmental stage of MHC-II–
selected thymocytes in Zbtb7b
ΔTE/ΔTE
mice, we examined whether
there are MHC-II–selected thymocytes from some developmental
stages toward the CD8-lineage that acquire a CD4
+
CD8
−
phenotype.
We accomplished this by a fate-mapping approach using an E8I-Cre
transgene, whose expression is driven by the E8I enhancer known
to be activated specifically in mature CD4
−
CD8
+
SP thymocytes
(Ellmeier et al, 1997) and can be monitored by GFP expression from
ires-gfp sequences present in the transgene (Seo et al, 2017). By
crossing the E8I-Cre transgenic mice to a Rosa26
YFP
reporter strain,
about 70% of CD8
+
splenic T cells were marked by YFP expression,
which could be distinguished from the weaker GFP signal derived
from the E8I-Cre transgene (Fig 1A), whereas only 0.02% of CD4
+
splenic T cells were marked by YFP. On the other hand, the per-
centage of YFP-positive CD4
+
T cells was significantly increased, up
to 0.07% on average, in Zbtb7b
ΔTE/ΔTE
mice (Fig 1A and B). This result
indicates that there exists a developmental pathway toward CD4
+
T
cells in the Zbtb7b
ΔTE/ΔTE
mice from a developmental stage where
the E8I-Cre transgene is activated, which occurs after CD4 down-
regulation in control mice (Seo et al, 2017). Such reversible dif-
ferentiation to CD4
+
T cells after possible loss of CD4 expression was
not compatible with the kinetic signaling model, prompting us to
revisit the relevance of distinct co-receptor expression kinetics in
the CD4
+
helper versus CD8
+
cytotoxic lineage choice.
Differentiation of innate memory-like CD8 T cells under
constitutive CD8αβ expression conditions
The effect of constitutive CD8αβ co-receptor expression in vivo was
examined previously in the presence of the endogenous CD8β
chain. Possible down-regulation of the transgenic CD8αchain,
which could dimerize with the endogenous CD8βchain, was dis-
cussed as a possible reason why most of the MHC-I–restricted cells
were differentiated into CD4-negative cytotoxic T cells in this
transgenic model (Bosselut et al, 2001). To analyze the effect of
constitutive CD8αβ expression in the absence of both endogenous
CD8αand CD8βchains, we generated a mutant Cd8 locus, by se-
quential targeting in ES cells, referred to as Cd8
Δab
, that lacks coding
regions for both CD8αand CD8βchains in the Cd8a and Cd8b1
genes, respectively (Figs 2A–Cand S1). For transgenic expression of
CD8αand CD8βchains, a cDNA encoding either chain was inserted
into the Rosa26 locus, generating a Rosa26
8a
and Rosa26
8b
locus,
respectively (Fig 2A). Induction of CD8αand CD8βchains from the
Rosa26
8a
and Rosa26
8b
loci upon Cre-mediated excision of the stop
cassette was confirmed by flow cytometry analyses (Fig 2D).
However, the expression level of transgenic CD8α, which is rep-
resented in CD4
+
population at the lower left panel in Fig 2D was
lower than that of endogenous CD8α(CD8 SP population at the
upper left panel in Fig 2D), presumably because of the weak pro-
moter activity in the Rosa26 locus.
We next examined expression kinetics of transgenic CD8αex-
pression in Rosa26
8a/8a
:Cd8
Δab/Δab
:Cd4-Cre mice. As expected,
expression level of transgenic CD8αfrom the Rosa26 locus was
stable and did not shown down-regulation after positive selection
(Fig S2), confirming constitutive expression of CD8 co-receptor. To
examine differentiation of MHC-I selected–thymocytes, we then
generated I-Ab
−/−
:Rosa26
8a/8b
:Cd8
Δab/Δab
:Cd4-Cre mice, hereafter
referred to as MHC-II
o
:CD8ab-Tg mice. Despite lower transgenic
CD8αβ expression levels, the percentage of mature thymocytes,
defined as the CD24
lo
TCRβ
hi
population, was equivalent between Wt
and MHC-II
o
:CD8ab-Tg mice (Fig 3A). In the mature thymocytes
population of MHC-II
o
:CD8ab-Tg mice, although around 30% cells
acquired CD4 expression, about 70% cells emerged as CD4-negative
cells (Fig 3A). The balance of CD4
+
and CD4
−
cells was maintained
after their egress from the thymus. In the peripheral lymphoid
tissues, such as spleen, of MHC-II
o
:CD8ab-Tg mice, the αβT-cell
population consisted of about 65% CD4
−
cells and 35% CD4
+
cells
(Fig S3A). This proportion of CD4
+
to CD4
−
cells in MHC-II
o
:CD8ab-Tg
mice was similar to that reported in the previous transgenic model
expressing CD8αand CD8βfrom mini-transgenes at equivalent
levels to endogenous CD8αβ (Bosselut et al, 2001), indicating that, in
spite of the lower level of transgenic CD8αβ expression, our CD8αβ
transgenic model is likely to mimic developmental processes in the
previous model.
iNKT2-cell development by CD8 expression Kojo et al. https://doi.org/10.26508/lsa.202000642 vol 3 | no 2 | e202000642 2of13
We next examined expression of Runx3, a central transcription
factor for cytotoxic T-cell development, by using a Runx3
tdTomato
reporter allele (Kojo et al, 2017). CD4
−
mature thymocytes emerged in
MHC-II
o
:CD8ab-Tg mice expressed Runx3-tdTomato, albeit at a lower
level than that in control CD8
+
T cells in control mice (Fig 3A), in-
dicating that these cells are related to the cytotoxic lineage. To
further characterize the CD4
−
mature thymocytes in MHC-II
o
:CD8ab-
Tg mice, we performed transcriptome analyses by RNA-seq. Principal
component analyses indicated that CD4
−
mature thymocytes that
had differentiated in MHC-II
o
:CD8ab-Tg mice were different from
control wild-type CD8 SP thymocytes (Fig S3B). Analyses of differ-
entially expressed genes revealed that Gzmk,Zbtb16,andTbx21
genes were up-regulated in the CD4
−
mature thymocytes (Fig 3B). We
also confirmed higher expression of Eomes, one of the signature
genes for innate memory-like CD8 T cells (Weinreich et al, 2010), in
CD8 SP thymocyte population of MHC-II
o
:CD8ab-Tg mice in subse-
quent qRT-PCR and flow cytometry analyses (Figs 3B and S3C). CD44
and CD122 are known surface markers for innate memory-like CD8 T
cells. Higher CD44 expression was detected in about half of the CD4
−
mature thymocytes in MHC-II
o
:CD8ab-Tg mice and CD122 expression
was detected in a half of the CD44
hi
cells (Figs 3C and S3D). An in-
crease in CD44
hi
CD122
hi
cells was also observed in spleen CD4
−
αβT-
cell population (Fig S3E). These observations demonstrate that half of
the CD4
−
mature thymocytes that emerge in MHC-II
o
:CD8ab-Tg mice
are innate memory-like CD8 T cells. An increase in the CD44
hi
CD122
hi
subset was also induced by transgenic CD8αβ expression in an MHC-
sufficient background (Fig 3C). We also found that expression of just
the CD8αchain that could function as a CD8αα homodimer resulted
in enhanced differentiation of CD44
hi
CD122
hi
innate memory-like CD8
T cells (Fig 3C). Thus, under lower but constitutive CD8αβ or CD8αα
expression, more than half of the MHC-I–selected cells remain to be
differentiated into CD4
−
cytotoxic lineage–related cells with a skewed
differentiation toward innate memory-like CD8
+
cells.
Increase in the iNKT2 subset under constitutive CD8αβ expression
During the analysis of promyelocytic leukaemia zinc finger (PLZF)
expression, we noticed that around 40% of CD4
+
mature thymocytes
expressed a higher level of PLZF than that in innate memory-like CD8 T
cells (Fig 4A). Innate-like CD4
+
T cells, including CD1d-restricted
iNKT cells (Kovalovsky et al, 2008) and MR1-restricted MAIT cells
(Rahimpour et al, 2015), have been shown to express PLZF at high
levels. Interestingly, differentiation of innate memory-like CD8 T cells
in the thymus has been shown to be enhanced by IL-4 secreted by
iNKT2 cells (Verykokakis et al, 2010;Weinreich et al, 2010;Lee et al,
2013). We, therefore, examined CD4
+
PLZF
hi
cells in MHC-II
o
:CD8ab-Tg
mice in terms of iNKT characteristics, such as expression of the in-
variant TCR, which can be specifically detected by CD1d dimers loaded
with αGalCer (αGC) (Matsuda et al, 2000). About 30% of CD4
+
mature
thymocyte of MHC-II
o
:CD8ab-Tg micewerestainedwithCD1d-αGC and
were positive for transgenic CD8 expression, whereas only 5% of those
cells were stained in control mice (Figs 4B and S4A). Such iNKT cells
werepresentatasimilarratioinCD4
+
T-cell population in MHC-II
o
mice expressing only endogenous CD8αβ (Fig 4B). On the contrary, in
MHC-II–sufficient background, both frequency and numbers of iNKT
cells were reduced in our CD8ab-Tg mice as was previously reported
(Lantz & Bendelac, 1994), whereas these are restored in MHC-II–
deficient background (Fig S4A). These observations indicate that iNKT
cells are one of the major CD4
+
subsets arising after elimination of
MHC-II–selected thymocytes. Whereas PLZF expression was not de-
tected in the CD4
+
CD1d-αGC
−
population in wild-type control mice,
there remained a small proportion of CD1d-αGC
−
PLZF
+
CD4
+
cells in the
thymus of MHC-II
o
:CD8ab-Tg mice as well as MHC-II
o
mice (Fig 4B).
Although the frequency of iNKT cells was similar between MHC-
II
o
and MHC-II
o
:CD8ab-Tg mice, an increase in innate memory-like
CD8
+
T cells occurred only in MHC-II
o
:CD8ab-Tg mice (Fig 3C). Given
the important role of IL-4 to support innate memory-like CD8
+
T-cell
Figure 1. Differentiation pathway to CD4
+
T cells after E8I activation in Thpok
DTE/DTE
mice.
(A) Histograms showing GFP expression from the E8I-Cre transgene and YFP expression from the Rosa26-STOP-YFP allele of splenic CD4
+
CD8
−
and CD4
−
CD8
+
T cells of
mice with indicated genotypes. One representative of at least three experiments. (B) Graph showing summary of the percentage of YFP
+
CD4
+
CD8
−
splenic T cells of Zbtb7
+/+
,
Zbtb7b
+/ΔTE
,andZbtb7b
ΔTE/ΔTE
mice that are hemizygous for E8I-Cre and Rosa26-STOP-YFP transgene. Mean ± SD. ***P< 0.001 (Kruskal–Wallis test with Dunn’smultiple
comparisons test).
iNKT2-cell development by CD8 expression Kojo et al. https://doi.org/10.26508/lsa.202000642 vol 3 | no 2 | e202000642 3of13
differentiation (Verykokakis et al, 2010;Lee et al, 2013), we asked
whether differentiation of iNKT-cell subsets is different between
MHC-II
o
and MHC-II
o
:CD8ab-Tg mice. After CD24 down-regulation, the
developmental stages of iNKT cells are divided into the CD44
−
NK1.1
−
stage 1, the CD44
+
NK1.1
−
stage2,andtheCD44
+
NK1.1
+
stage 3 (Benlagha
et al, 2002). In the thymus of MHC-II
o
:CD8ab-Tg mice, proportions of
CD44
−
NK1.1
−
stage 1 and CD44
+
NK1.1
−
stage 2 cells were significantly
increasedcomparedwithcontrolandMHC-II
o
mice and, this was
accompanied by a relative decrease in CD44
+
NK1.1
+
stage 3 cells (Fig
4C). The T-bet
lo
PLZF
hi
iNKT2-cell subset is included in the stage 1 and
stage 2 iNKT-cell population (Constantinides & Bendelac, 2013;Lee
et al, 2013). As expected, the proportion of the T-bet
lo
PLZF
hi
iNKT2-cell
subset was significantly higher in MHC-II
o
:CD8ab-Tg mice than in wild-
type and MHC-II
o
mice (Fig 4C), which was consistent with an increase
in the number of iNKT2 cells in MHC-II
o
:CD8ab-Tg mice (Fig S4B). We
further confirmed an increase in the iNKT2 subset by using other
Figure 2. Transgenic mouse line expressing CD8αand CD8βchain in the absence of endogenous CD8αand CD8βchains.
(A) A scheme showing structures of Rosa26
8a
, Rosa26
8b
,Cd8, and Cd8
Δab
loci on the mouse chromosome 6. Orange triangles and green ovals represent loxP and FRT
sequences, respectively. hCD2 was inserted to replace the Cd8a locus. Arrows indicate direction of transcription at the Cd8 and Rosa26 loci. PA, polyA adenylation signals;
Neo,neomycin-resistance gene; ires, internal ribosomal entry sites. (B) Gel images of genotyping PCR showing deletion of the second exon at the Cd8b1 locus and insertion
of the hCD2 cDNA at the Cd8a locus. (C, D) Flow cytometry analyses for CD4 and CD8a expression of total thymocytes of Cd8
+/+
and Cd8
Δab/Δab
mice (C) and TCRβ
+
spleen T
cells of Rosa26
8a/+
and Rosa26
8a/+
: CD4-Cre mice (D). (C, D) Histogram showing intracellular staining of CD8βchain in total thymocytes of Cd8
+/+
and Cd8
Δab/Δab
mice (C)
and surface CD8βexpression of spleen CD4
+
T cells of Rosa26
8b/+
:CD4-Cre and Rosa26
8a/8b
: CD4-Cre mice (D). CD8βexpression by wild-type CD4
+
T cells is shown as an open
histogram as a negative control. One representative of two independent experiments.
iNKT2-cell development by CD8 expression Kojo et al. https://doi.org/10.26508/lsa.202000642 vol 3 | no 2 | e202000642 4of13
markers, Plzf and RORγt, that define iNKT2 subset as Plzf
hi
RORγt
−
population (Fig S4C). In addition, after ex vivo PMA/ionomycin stim-
ulation, thymic iNKT cells of MHC-II
o
:CD8ab-Tg mice produced more
IL-4 than wild-type control mice (Fig 4D), confirming the increase in
the iNKT-cell subset that functionally produces IL-4. These observa-
tions indicate that constitutive expression of the CD8αβ co-receptor
results in a skewed differentiation toward the iNKT2-cell subset.
Cell-intrinsic and cell-extrinsic mechanisms for innate-like CD8
T-cell development
Our results showed that differentiation of both innate memory-like
CD8 thymocytes and the iNKT2-cell subset is enhanced in MHC-II
o
:
CD8ab-Tg mice. Given the known role of IL-4 secreted by iNKT2 cells
in supporting innate memory-like CD8 thymocyte differentiation
(Verykokakis et al, 2010;Lee et al, 2013), we next tested to what
extents the increase in the iNKT2-cell subset has impacts on innate
memory-like CD8 T-cell differentiation in MHC-II
o
:CD8ab-Tg mice.
To accomplish this aim, we set up mixed bone marrow chimera
experiments in which equal numbers of bone marrow progeni-
tors from CD45.1 wild-type mice and CD45.2 wild-type or MHC-II
o
:
CD8ab-Tg mice were co-injected together into sublethal irradiated
MHC-II
o
host mice. In the host mice that received CD45.1 wild-
type and CD45.2 wild-type bone marrow cells, the percentages
of CD44
+
CD122
+
innate memory-like cells in mature CD4
−
CD8
+
SP
thymocytes were similar between CD45.1
+
and CD45.2
+
populations
Figure 3. Differentiation of innate-like CD8
+
Tcell
with under constitutive CD8αβ expression.
(A) Flow cytometry analyses for CD4, CD8, TCRβ, CD24,
and Runx3-tdTomato expression by various thymocyte
subsets of mice with the indicated genotypes.
Representative results of more than three
independent analyses. Numbers in the plot indicate the
percentage of cells in each quadrant. Graphs
showing summary of cell numbers of total and mature
thymocytes and the percentage of CD4
+
and CD4
−
subset
in mature thymocytes. Mean ± SD. ***P< 0.001
(unpaired ttest, two-sided). (B) RNA-seq analyses of
CD8 SP cells from indicated genotypes (left). The top 40
differentially expressed genes observed between Wt
and Tg are shown. Histograms showing protein
expression level of selected genes in indicated
genotypes (right). (C) Dot plots showing CD44 and
CD122 expression in mature CD8 SP thymocyte of the
indicated genotypes (left). Representative results of
more than three independent analyses. Graph
showing summary of percentage of CD44
hi
CD122
+
population in mature thymocytes of indicated
genotypes (right). Mean ± SD. **P< 0.01, ***P< 0.001,
****P< 0.0001 (one-way ANOVA with Tukey’s multiple
comparison).
iNKT2-cell development by CD8 expression Kojo et al. https://doi.org/10.26508/lsa.202000642 vol 3 | no 2 | e202000642 5of13
Figure 4. Skewed differentiation of the iNKT2 subset by constitutive CD8αβ expression.
(A) Dot plots and histograms showing CD4, CD8, and PLZF expression in mature thymocytes of the indicated genotypes (left two panels). Representative results of at
least three experiments. Graph showing a summary of five independent experiments (right). Mean ± SD. ***P< 0.001 (unpaired ttest with Welch’s correction, two-sided).
(B) Histograms showing CD1d-αGC anti-PLZF staining of the indicated thymocyte subsets and genotypes (left). Graph showing summary of four independent experiments
(right). Mean ± SD. ***P< 0.001 (one-way ANOVA with Tukey’s multiple comparison). (C) Contour and dot plots showing frequencies in various stages and subsets of
thymic iNKT cells from the indicated genotypes (upper). CD44
−
NK1.1
−
CD24
−
stage 1 cells, CD44
+
NK1.1
−
stage 2 cells, and CD44
+
NK1.1
+
stage 3 cells. T-bet
lo
PLZF
hi
is the iNKT2
iNKT2-cell development by CD8 expression Kojo et al. https://doi.org/10.26508/lsa.202000642 vol 3 | no 2 | e202000642 6of13
andwerelessthan1%inbothpopulation(Fig 5A). In striking contrast,
the percentage of CD45.1
+
CD44
+
CD122
+
CD8α
+
cells were increased to
around 5% when progenitors from CD45.2 MHC-II
o
:CD8ab-Tg mice
were included (Fig 5A). The degree of CD45.1
+
CD44
+
CD122
+
CD8 SP
thymocyte differentiation tended to be correlated with the degree
of chimerism of CD45.2
+
cells to CD45.1
+
cells. These results are
consistent with previous studies showing that iNKT2-mediated
cell-extrinsic mechanisms, such as an IL-4–enriched thymic envi-
ronment, support innate memory-like CD8 T-cell differentiation
(Verykokakis et al, 2010;Lee et al, 2013). Although actual involve-
ment of IL-4–producing iNKT2-cell subsets in enhanced differen-
tiation of innate memory-like CD8 T cells in MHC-II
o
:CD8ab-Tg needs
to get confirmed by genetics using mice deficient for IL-4 or CD1d,
it is likely that skewed differentiation of iNKT2 cells could serve
as a cell-extrinsic mechanism driving innate memory-like CD8 T
differentiation.
Interestingly, in this setting, the percentage of CD44
+
CD122
+
cells
differentiated from CD45.2 MHC-II
o
:CD8ab-Tg progenitors was always
higher (threefold) than that from CD45.1 wild-type cells (Fig 5A). This
higher percentage of CD44
+
CD122
+
cells in the CD45.2
+
population
suggests that besides cell-extrinsic mechanisms such as higher level
of IL-4 produced by iNKT2 cells, constitutive CD8αβ expression ac-
tivates uncharacterized cell-intrinsic mechanisms that makes MHC-
I–restricted cells more prone to activate a developmental program
toward the innate memory-like CD8 thymocyte.
We next examined development of the iNKT-cell subset in the
bone marrow chimera host mice by assessing the ratio of CD45.2
+
cells to CD45.1
+
cells at the three stages of iNKT-cell development.
This was performed individually for each host mouse because iNKT-
cell reconstitution efficiency varied in each host. In the mixed
chimera setting of CD45.1 wild-type with CD45.2 wild-type progen-
itors, the ratio of CD45.2
+
cells to CD45.1
+
cells in each of the three
iNKT-cell stages was around one in all host mice (Fig 5B). On the
contrary, the proportion of CD45.2
+
cells relative to CD45.1
+
cells at
the CD44
−
NK1.1
−
stage 1 and the CD44
+
NK1.1
−
stage 2 was significantly
increased in host mice injected with CD45.1 wild-type and CD45.2
MHC-II
o
:CD8ab-Tg progenitors (Fig 5B). We also confirmed an in-
creased proportion of the T-bet
lo
PLZF
hi
iNKT2-cell subset in this
setting (Fig 5B). These results clearly demonstrate that cell-intrinsic
mechanisms are involved in the skewed differentiation toward
iNKT1 and iNKT2-cell subsets in MHC-II
o
:CD8ab-Tg mice, although
involvement of cell-extrinsic mechanisms is not formally excluded.
Intracellular signaling from the CD8αchain is required for iNKT2
skewing
Our results revealed that constitutive CD8αβ expression enhanced
iNKT2-cell differentiation in at least a cell-intrinsic manner. Recently,
TCR signal strength was shown to be involved in regulating the dif-
ferentiation of iNKT-cell subsets. Thus,strongTCRsignalingpromotes
iNKT2 and iNKT17 development (Tuttle et al, 2018;Zhao et al, 2018),
suggesting that constitutive CD8αβ expression might enhance TCR
signaling during iNKT-cell development, either by aiding in antigen
recognition and/or modulating intracellular TCR signals. Consistent
with this notion, expression level of CD5, an indicator of TCR signal
strength, was higher on thymic iNKT cells that were developed under
constitutive CD8 expression (Fig S4D). iNKT cells are selected by CD1d
molecules on DP thymocytes (Constantinides & Bendelac, 2013).
Contrary to the previous study showing CD1d down-regulation by
transgenic CD8 expression (Engel et al, 2010), there was no significant
change in CD1d expression levels on DP thymocytes upon transgenic
CD8αα or CD8αβ expression (Fig S4E), suggesting that CD1d expression
level is unlikely to be involved in enhancement of TCR signaling by
transgenic CD8 expression. Two cysteine residues in the CD8αcyto-
plasmic tail are essential for co-receptor function in TCR stimulation
by classical MHC-I via interacting with Lck kinase and recruitment of
LAT (Turner et al, 1990;Hoeveler & Malissen, 1993;Bosselutetal,1999).
We, therefore, tested whether these cysteine residues are necessary
to enhance iNKT2-cell differentiation by generating a Rosa26
8aCA
allele that expresses a mutant CD8αchaininwhichtwocysteine
residues are replaced with alanine (Fig 6A). Unlike in the MHC-II
o
:
CD8ab-Tg mice, the frequency and numbers of mature CD8 SP thy-
mocytes were significantly decreased in the MHC-II
o
mice expressing
CD8α
CA
mutant protein (MHC-II
o
:CD8a
CA
b−Tg mice) (Fig 6B), indicating
that co-receptor function supporting conventional MHC-I–restricted
cell development was abrogated by these amino acid replacements.
When we examined the iNKT-cell population, defined as
CD24
lo
TCRβ
+
CD1d-αGC
+
,inMHC-II
o
:CD8a
CA
b−Tg mice, there was no
increase in the percentage of the iNKT2-cell subset (Fig 6C). These
observations indicate that intracellular signaling through the CD8α
chain is necessary to drive enhanced iNKT2-cell development.
It remains unclear whether CD8αβ or CD8αα interacts directly with
the non-classical MHC-I molecule CD1d. Nevertheless, our results
clearly demonstrated that constitutive CD8αexpression alone is
sufficient to affect iNKT-cell subset differentiation. We then exam-
ined expression kinetics of the endogenous CD8αchain during iNKT-
cell development. At the CD24
hi
CD44
−
NK1.1
−
stage 0, most of iNKT-cell
precursors express CD8αand CD4 and more than half of these cells
down-regulate CD8αexpression (Fig S5). During the transition from
CD24
hi
stage 0 to the CD24
lo
CD44
−
NK1.1
−
stage 1, CD8αexpression was
efficiently down-regulated. The ThPOK transcription factor is es-
sential to terminate Cd8 gene expression in conventional MHC-I–
restricted cytotoxic T cells (He et al, 2005;Rui et al, 2012)aswellasin
iNKT cells (Engel et al, 2010). Interestingly, CD8αdown-regulation at
stage 0 seemed to precede ThPOK induction (Fig S5). Thus, similar to
conventional cytotoxic T cells (Muroi et al, 2008), the initial CD8 down-
regulation during iNKT-cell development is independent of ThPOK.
Discussion
Results shown in this study clearly demonstrate that constitutive
expression of CD8αβ or CD8αα influences the proportion of iNKT-
cell subsets, with skewing to the iNKT2 subset in a cell-intrinsic
subpopulation. One representative result of at least four experiments. Graphs showing summary of at least four experiments (lower). Mean ± SD. *P<0.05,**P< 0.01, ***P<
0.001 (one-way ANOVA with Tukey’s multiple comparison). (D) Histogram showing intracellular IL-4 staining in the thymic iNKT population after stimulation with PMA and
ionomycin for 4 h. One representative of two experiments.
iNKT2-cell development by CD8 expression Kojo et al. https://doi.org/10.26508/lsa.202000642 vol 3 | no 2 | e202000642 7of13
Figure 5. Cell-intrinsic and cell-extrinsic mechanisms for differentiation of innate-like CD8
+
T cells.
(A) Gating strategy of the flow cytometry analyses for the frequency of the innate memory-like CD8
+
T-cell population in mixed bone marrow chimera experiments. One
representative result from three independent mice (left). Graphs showing summary of innate CD8
+
T-cell frequencyin the indicated genotypes (right). Mean ± SD. Probability is
calculated using two-tailed unpaired ttest with Welch’s correction (right upper panel). The scatter plot indicates the relationships between CD45.2 chimerism and frequency of
innate CD8
+
T cells. r indicates Pearson’s correlation coefficient. (B) Dot plots showing iNKT-cell stages and iNKT2 subpopulations in mixed bone marrow chimera experiments
(top). One representative result from three independent mice. Graph showing summary of frequencies of iNKT-cell developmental stages and iNKT2 subpopulation (bottom).
Mean ± SD. Indicated probabilities are calculated using two-tailed unpaired ttest with or without Welch’s correction (right upper panel).
iNKT2-cell development by CD8 expression Kojo et al. https://doi.org/10.26508/lsa.202000642 vol 3 | no 2 | e202000642 8of13
manner, generating an IL-4–rich thymic microenvironment that
fosters differentiation of innate memory-like CD8
+
T cells. In ad-
dition to the cell-extrinsic mechanism, constitutive CD8αβ ex-
pression somehow conditions MHC-I–restricted cells to become
more prone to acquire innate memory-like signatures in a cell-
intrinsic manner.
Recent studies reported that TCR signal strength influences
iNKT-cell subset differentiation (Tuttle et al, 2018;Zhao et al, 2018).
Attenuated function of ZAP70, which plays an essential role in early
TCR signaling events via phosphorylation of LAT, impaired differ-
entiation of iNKT2 and iNKT17, but not iNKT1 cells (Tuttle et al, 2018;
Zhao et al, 2018). The expression level of CD5, which parallels
TCR signaling intensity, and induction of Nur77, another marker
reflecting TCR strength, is higher in iNKT2 cells (Moran et al, 2011;Lee
et al, 2013;Tuttle et al, 2018). These findings highlight that strong TCR
signaling promotes iNKT2-cell development, at least in part by
sustaining Egr2 expression, which results in increases of chromatin
accessibility of iNKT2-specific regulatory elements harboring Egr2-
and NFAT-binding motifs (Tuttle et al, 2018). Therefore, it is con-
ceivable that constitutive CD8αβ expression enhances TCR sig-
naling in iNKT precursors. However, a previous study failed to detect
direct binding of CD8αβ to the non-classical MHC-I molecule, CD1d
(Engel et al, 2010), leaving open the question of how CD8αβ en-
hances TCR signaling engaged by CD1d. Our results showed that the
CD8αα homodimer, which has a distinct affinity for conventional
MHC-I and a thymus leukemia antigen (TL), another non-classical
MHC-I molecule, from CD8αβ co-receptor (Leishman et al, 2001;
Gangadharan & Cheroutre, 2004), also functions to enhance iNKT2
development. This suggests that CD8αβ is unlikely to help iNKT
precursors recognize antigen on CD1d as it does for antigens on
classical MHC-I as a co-receptor, although the possibility that
CD8αβ increases binding affinity of invariant TCR with antigen/CD1d
molecules is not formally excluded. Using a retrogenic transgenic
mouse system, Cruz Tleugabulova et al showed that TCR half-life is
more important to modulate iNKT subset differentiation than
avidity (Cruz Tleugabulova et al, 2016). This finding is not only in line
Figure 6. Signals from CD8αchain are essential for iNKT2 skewing.
(A) Schematic structure of the C227/229A mutant CD8a chain inserted in the Rosa2 6 locus. Two cysteine residues at positions 227 and 229 in the CD8αchain cytoplasmic
tail were changed to alanine. (B) Dot plots showing CD24 and TCRβexpression to define mature thymocytes and CD4 and CD8 expression in mature thym ocytes. Numbers
in the dot plots indicate percentage of cells in the indicated gates (left). Graph showing summary of absolute number of mature CD8 SP thymocytes of mice with indicated
genotype (right). Mean ± SD. ***P< 0.001, ****P< 0.0001 (one-way ANOVA with Tukey’s multiple comparison). (C) Dot plot showing T-bet and PLZF expression in thymic
iNKT cells of mice of the indicated genotype (top). Numbers indicate percentage of cells in the indicated gate that defines iNKT2 subset. Graph showing a summary of
iNKT2 frequency among iNKT cells of mice with the indicated genotype (bottom). Mean ± SD. **P< 0.01, ***P< 0.0001 (one-way ANOVA wit h Tukey’s multiple comparison). TM,
transmembrane domain.
iNKT2-cell development by CD8 expression Kojo et al. https://doi.org/10.26508/lsa.202000642 vol 3 | no 2 | e202000642 9of13
with the idea that CD8αβ does not increase affinity for antigen/
CD1d complexes but also raise the possibility that CD8αβ stabilizes
the interaction between TCR and antigen/CD1d complexes. On the
other hands, our results show that two cysteine residues within the
intracellular domain of CD8αchain are required for skewed dif-
ferentiation toward the iNKT2 subset. These cysteine residues
function as a docking module for Lck and LAT (Turner et al, 1990;
Hoeveler & Malissen, 1993). Such an intracellular association of
CD8αchain with TCR/CD3 complexes and signaling molecules in the
absence of antigen recognition suggests a possible role of CD8α
chain to strengthen TCR signaling independently of extracellular
binding of CD8αto CD1d. Given the involvement of ZAP70 in shaping
the development of iNKT subsets, the capacity of the CD8αchain to
interact with the molecular machinery in early TCR signaling events
is likely to be essential to strengthen or prolong TCR signaling. At
any rate, given that CD4 is constitutively expressed during iNKT-cell
development and interacts with Lck with higher affinity than CD8α,
it is possible that CD8αmodulates intracellular signaling from
invariant TCR engaged by antigen/CD1d complexes differently from
CD4.
Pioneering studies proposed that CD8αβ expression during
iNKT-cell development inhibits this development through a process
of negative selection (Lantz & Bendelac, 1994). However, emergence
of CD8
+
iNKT cells in ThPOK-deficient mice indicated that CD8
expression does not provide full inhibitory signals (Engel et al,
2010). Recently, reduction of iNKT-cell numbers in SLAM family
receptor–deficient mice was shown to stem from increased apo-
ptosis because of strong TCR signals (Lu et al, 2019). Thus, the SLAM
family receptor functions to attenuate TCR signals to foster iNKT-
cell development, implying that differentiation of iNKT cells is
impaired once TCR signal strength exceeds some thresholds.
Therefore, enhanced TCR signaling by constitutive CD8αβ expres-
sion inhibits differentiation of some iNKT precursors that receive
excessive TCR signals, whereas other iNKT precursors that can
survive under constitutive CD8αβ expression are directed toward
the iNKT2-cell developmental pathway. It is possible that such
iNKT2 cells that emerged under constitutive CD8αβ expression are
exposed to prolonged TCR signals and produce an increased
amount of IL-4, generating the IL-4–rich thymic microenvironment
despite a small increase in their numbers.
A previous publication discussed the possibility that down-
regulation of endogenous CD8βchain could be what limits the
redirection of MHC-I selected cells into CD4
+
helper T cells (Bosselut
et al, 2001). However, in the absence of endogenous CD8β, the
proportion of CD4
+
versus CD4
−
cells was about one-third, which is
similar to that observed in the previous study (Bosselut et al, 2001),
implying that this is not the case. Moreover, one-third of the CD4
+
T
cells are CD1d-restricted iNKT cells, and the rest of the CD4
+
T
population contains a significant proportion of PLZF
+
innate-like
cells with uncharacterized MHC-restriction. Therefore, at least in
our experimental setting for transgenic CD8αβ expression, most
MHC-I–selected cells differentiate into CD8
+
cytotoxic-lineage cells.
This finding demonstrates that a persistent positive-selection
signal alone is not sufficient to instruct the helper-lineage fate
to MHC-I restricted cells. Thus, our results challenge the model in
which a distinct duration of positive-selection signals between
MHC-I–and MHC-II–selected thymocytes generated by distinct
kinetics of expressions of the CD4 versus CD8αβ co-receptor is
central to mechanisms that govern the helper versus cytotoxic
lineage choice. Our results also revealed that down-regulation of
CD8 plays an essential role in preventing development of innate
memory-like CD8 T cells, rather than in inducing conventional
cytotoxic fate, at least in part by shaping differentiation of iNKT-cell
subsets. By using several experimental systems, disruption of CD4/
MHC-II–mediated positive-selection signals had been previously
shown to result in a redirected differentiation of MHC-II restricted
cells into CD8
+
cytotoxic-lineage cells (Sarafova et al, 2005;Adoro et
al, 2012). This genetic evidence supports the concept that persis-
tence of positive-selection signals is necessary to guide MHC-II–
selected thymocytes to become the helper-lineage T cells. On the
contrary, whether persistence of MHC-I–mediated TCR signaling is
sufficient to instruct the helper-lineage fate has not yet been well
established. Given the lower expression level of transgenic CD8αβ
in this study, it remains possible that the signal strength was not
sufficient to instruct the helper-lineage fate in this setting. How-
ever, the ratio of CD4
+
versus CD4
−
mature thymocytes in this study
was similar to that observed in mice expressing transgenic CD8αβ
at a level similar to endogenous CD8αβ (Bosselut et al, 2001). In
addition, the lower expression level of transgenic CD8αβ or CD8αα
was sufficient to influence TCR signals from invariant TCR on iNKT
precursors. Therefore, it is conceivable that persistence of MHC-
I–derived positive-selection signals alone is not sufficient to in-
struct the helper-lineage fate, although it is still important to test
thymocyte fate decision by achieving constitutive CD8αβ expres-
sion at the correct level (Littman, 2016). Our results suggest that
there must be uncharacterized differences in positive-selection
signals between MHC-I–and MHC-II–restricted thymocytes beyond
duration of signal length. Understanding of such differences in
positive-selection signals engaged by distinct MHCs is extremely
important and awaits experiments in future studies.
The unique kinetics of CD8 expression, known as co-receptor
reversal, was discovered 20 yr ago (Brugnera et al, 2000;Cibotti et al,
2000). CD8 co-receptor reversal is controlled at the transcriptional
level and is accomplished by temporal termination of Cd8 gene
expression in all post-selection thymocytes and sequential Cd8
reactivation specifically in the cytotoxic-lineage cells. However, the
molecular mechanisms underlying such CD8 expression with its
unique kinetics remains uncharacterized. Our results revealed a
novel relevance of Cd8 down-regulation to shaping iNKT-cell subset
differentiation, raising the possibility that mechanisms causing Cd8
down-regulation might be evolved to fine-tune innate T-cell dif-
ferentiation in species that lack conventional cytotoxic T cells or in
the extant positive-selection process of cytotoxic T cells by classical
MHC-I molecules. However, expression of the CD8αβ co-receptor on
cytotoxic-lineage cells is beneficial to efficiently mount adaptive
immune responses by assisting recognition of peptide antigen
presented on MHC-I molecules in the periphery, leading us to
speculate that mechanism(s) that reactivate Cd8 gene might have
been acquired later. In mice, the E8I enhancer, one of six enhancers
in the Cd8 locus, shows CD8
+
cytotoxic lineage–specific activity in a
reporter transgenic assay (Ellmeier et al, 1997). However, CD8αβ co-
receptor expression by MHC-I–restricted cells is maintained in the
absence of the E8I enhancer (Ellmeier et al, 1998), indicating that
compensatory mechanisms operate to maintain CD8αβ co-receptor
iNKT2-cell development by CD8 expression Kojo et al. https://doi.org/10.26508/lsa.202000642 vol 3 | no 2 | e202000642 10 of 13
expression on MHC-I–restricted cytotoxic T cells (Ellmeier et al, 2002).
Such mechanisms for CD8 expression are likely to have been estab-
lished under evolutional pressures to secure MHC-I–dependent
pathogen clearance.
Materials and Methods
Mice
Thpok
ΔTE
mice (Muroi et al, 2013), Rosa26-STOP-YFP mice (Srinivas
et al, 2001), and Runx3-tdTomato reporter mice (Kojo et al, 2017)
have been described. I-Aβ–deficient mice were from Taconic. To
generate a Cd8
Δab
allele, we first replaced exon 2 in the Cd8b1 gene
with a neomycin resistant gene (neo
r
)flanked with FRT sites by
homologous recombination with the targeting vector in MI ES cells,
establishing ES clone 67. We next removed the neo
r
gene from the
ES clone 67 by transient transfection of an FLP recombinase ex-
pression vector and isolated an ES clone harboring the Cd8
Δb
allele,
into which the targeting vector used to generate the Cd8a
h2PA
allele
(Wada et al, 2018) was transfected to replace exon 1 of the Cd8a
gene with an hCD2 cDNA and the neo
r
gene. After isolation of ES
clones that had undergone homologous recombination with the
second targeting vector at the Cd8a gene, we screened them to
determine which allele, Cd8 or Cd8
Δb
, was targeted by analyzing the
type of FLP recombinase-mediated recombination occurring after
transduction of a retrovirus vector encoding FLP, and then isolated
ES clones harboring the Cd8
+/ΔbaCD2N
genotype. After removal of the
neo
r
gene from the Cd8
+/ΔbaCD2N
ES clone by transient transfection
of the FLP recombinase expression vector, ES clones were used to
generate chimera mice by aggregation. To generate the Rosa26
8a
or Rosa26
8b
allele, a cDNA fragment encoding CD8αor CD8βwas
PCR-amplified by RT-PCR using thymus mRNA, sequenced, and
cloned into the AscI site of the CTV vector (#159212; Addgene). A
cDNA encoding the mutant CD8α
CA
chain harboring alanine
substitutions at two cysteine residues in the CD8αcytoplasmic tail
was amplified by overlap PCR. All mice were maintained in the
specific pathogen-free animal facility at the RIKEN IMS, and all
animal procedures were in accordance with institutional guideli nes
for animal care and with the protocol (28-017) approved by the
Institutional Animal Care and Use Committee of RIKEN Yoko-
hama Branch.
Flow cytometry and cell sorting
Single-cell suspensions from the thymus, spleen, and lymph nodes
were prepared by mashing tissues through a 70-μm cell strainer (BD
Bioscience). Single-cell suspensions were stained with the fol-
lowing antibodies purchased from BD Bioscience, eBiosciences, or
BioLegend: CD4 (RM4-5), CD8a (53-6.7), CD24 (M1/69), CD44 (IM7),
CD122 (TM-β1), TCRβ(H57-597), NK1.1 (PK136), CD69 (H1.2F3), and CCR7
(4B12). Murine CD1d-dimers X I (557599; BD Biosciences) obtained
from BD Biosciences were loaded with αGalCer (KRN7000; Funa-
koshi) and were labelled with antimouse IgG1 proximal enhancer
(550083; BD Biosciences) or antimouse IgG1 BV510 (740421; BD
Biosciences). For intracellular staining of transcription factors, the
cells were stained with cell surface molecules, and then fixed and
permeabilized using the Transcription Factor Buffer Set (562574; BD
Biosciences). Permeabilized cells were stained with antibodies
from BD Biosciences, eBiosciences, or BioLegend: T-bet (4B10), PLZF
(9E12), Eomes (Dan11mag), and ThPOK (T43-94). To stain intracellular
IL-4, total thymocytes were stimulated with 50 ng/ml in ml of
phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) and 1.5 μMof
ionomycin for 4 h in the presence of BD Golgi Stop (554724; BD
Biosciences). Then, the cells were stained with cell surface mole-
cules and were fixed and permeabilized using BD Cytofix/Cytoperm
Kit (554714; BD Biosciences). Permeabilized cells were stained
with anti-IL-4 antibody (11B11; BD Biosciences). Multi-color flow
cytometry analysis was performed using a BD FACSCanto II (BD
Bioscience), and data were analyzed using FlowJo (BD Bioscience)
software. Cell subsets were sorted using a BD FACSAria II or III (BD
Biosciences).
Mixed bone marrow chimera
Recipient MHC-II-deficient mice were sublethally irradiated at 950
rad and were reconstituted with 5 × 10
6
bone marrow cells from
CD45.2
+
wild-type mice or CD45.2
+
MHC-II
o
:CD8αβ Tg mice with 5 × 10
6
CD45.1
+
bone marrow cells. At 8–12 wk after transfer, the spleen and
thymus were analyzed by flow cytometry.
Quantitative RT-PCR
Total cellular RNA was extracted from purified cell subsets using
Trizol (Thermo Fisher Scientific) and treated with RNase-free DNase
I (Thermo Fisher Scientific). cDNA was synthesized from total RNA
using the SuperScriptII First Strand Synthesis System (Invitrogen).
Quantitative RT-PCR was performed using the ABI/PRISM 7000
sequence detection system with an internal fluorescent TaqMan
probe. Primers for Zbtb7b (He et al, 2005)andEomes (Intlekofer
et al, 2005) were previously described.
RNA-seq
RNA was extracted from sorted CD8
+
and CD4
−
mature thymocytes of
Wt and MHC-II
o
:CD8αβ Tg mice by TRIzol (Thermo Fisher Scientific),
followed by RNeasy Micro Kit (QIAGEN) according to the manu-
facturer’s protocols. Sequencing libraries were prepared using a
NEBNext RNA Library Prep Kit for Illumina (E7530; NEB) with Poly(A)
mRNA Magnetic Isolation Module (E7490; NEB) according to the
manufacturer’s protocol. Single-end 50-bp reads were obtained by
Illumina HiSeq 1500. The reads were mapped by Tophat v2.1.1 onto
the mouse genome mm10 and counts were generated using HTSeq
v.0.6.1. The DESeq2 R package was used to perform principle com-
ponent analysis and to generate the heat map of differentially
expressed gene with normalized counts of wild-type (GSE48138)
and Tg (GSE132059)induplicatesinthisstudy.
Accession codes
GEO: raw sequencing data and processed files, GSE48138 and
GSE132059.
iNKT2-cell development by CD8 expression Kojo et al. https://doi.org/10.26508/lsa.202000642 vol 3 | no 2 | e202000642 11 of 13
Supplementary Information
Supplementary Information is available at https://doi.org/10.26508/lsa.
202000642.
Acknowledgements
We are grateful to T Ishikura for aggregation of ES cells, Y Taniguchi for
mouse genotyping, N Yoza for cell sorting, and Dr Wilfried Ellmeier for critical
reading of the manuscript. This work was supported by the Grants-in-Aid for
Scientific Research (B) (19390118) from JSPS and the Grants-in-Aid for Sci-
entific Research on Innovative Areas (17H05805 and 19H04820) from the
MEXT in Japan (I Taniuchi).
Author Contributions
S Kojo: formal analysis and investigation.
M Ohno-Oishi: formal analysis and investigation.
H Wada: formal analysis and investigation.
S Nieke: methodology.
W Seo: formal analysis.
S Muroi: methodology.
I Taniuchi: formal analysis and writing—original draft.
Conflict of Interest Statement
The authors declare that they have no conflict of interest.
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