Downloaded from http://journals.aai.org/jimmunol/article-pdf/203/3/627/1450476/ji1900055.

pdf by guest on 08 November 2024

RESEARCH ARTICLE | AUGUST 01 2019
Coexpression of YY1 Is Required to Elaborate the E ector Functions
Controlled by PLZF in NKT Cells 
Patrick W. Darcy; ... et. al
J Immunol (2019) 203 (3): 627–638.
https://doi.org/10.4049/jimmunol.1900055

Related Content
Distinct PLZF+CD8αα+ Unconventional T Cells Enriched in Liver Use a Cytotoxic Mechanism to Limit Autoimmunity
J Immunol (October,2019)

The Transcription Factor PLZF Is Necessary for the Development and Function of Mouse Basophils
J Immunol (September,2019)

A Transgenic TCR Directs the Development of IL-4+ and PLZF+ Innate CD4 T Cells
J Immunol (July,2013)
 The Journal of Immunology

Coexpression of YY1 Is Required to Elaborate the Effector

Functions Controlled by PLZF in NKT Cells

Patrick W. Darcy,*,1 Kangxin Jin,*,1,2 Louis Osorio,* Lisa K. Denzin,*,†,‡ and

Derek B. Sant’Angelo*,†,‡
The promyelocytic leukemia zinc-finger transcription factor (PLZF) is essential for nearly all of the unique, innate-like functions
and characteristics of NKT cells. It is not known, however, if the activity of PLZF is regulated by other factors. In this article, we
show that the function of PLZF is completely dependent on the transcription factor Yin Yang 1 (YY1). Mouse NKT cells expressing
wild-type levels of PLZF, but deficient for YY1, had developmental defects, lost their characteristic “preformed” mRNA for

Downloaded from http://journals.aai.org/jimmunol/article-pdf/203/3/627/1450476/ji1900055.pdf by guest on 08 November 2024

cytokines, and failed to produce cytokine protein upon primary activation. Immunoprecipitation experiments showed that YY1
and PLZF were coassociated. Taken together, these biochemical and genetic data show that the broadly expressed transcription
factor, YY1, is required for the cell-specific “master regulator” functions of PLZF. The Journal of Immunology, 2019, 203: 627–638.

C
lassic adaptive immunity requires Ag-specific activation The BTB-ZF transcription factor PLZF (Zbtb16) is the signature
of T or B cells, followed by cell differentiation into regulator of the functional and phenotypic dimorphism between
discrete effector subtypes. Only after secondary stimu- NKT cells and conventional T cells. PLZF-deficient NKT cells
lation are the effector functions and cytokines elaborated, enabling lose their normal activated phenotype and do not produce cyto-
these lymphocytes to fully participate in the immune response. kines upon primary activation (3, 4). Ectopic expression of PLZF
Therefore, adaptive immune responses are rather slow to de- in conventional T cells, in contrast, results in the acquisition of
velop and somewhat limited in terms of function. NKT cells, a innate T cell–like characteristics, such as an activated phenotype
unique subset of ab T cells, are, in contrast, poised for im- and the rapid secretion of Th1 and Th2 cytokines in response to
mediate response to activation (1). Minutes after activation, primary activation (5). Interestingly, although PLZF is broadly
NKT cells produce large amounts of a wide array of cytokines. required for all innate-like effector functions, NKT cells are
The breadth of the response allows NKT cells to participate in stratified into distinct effector subgroups. PLZF activity, therefore,
diverse responses, including immunity to pathogens, protection must be modified to direct its subgroup-specific effects.
from autoimmunity, tumor immunity, and regulation of obesity- Activation of T cells via the TCR, TCR plus SLAM signaling, or
induced inflammation, among others (2). Indeed, triggering even by Type 1 inflammation induced by bacterial infection does
NKT cells impacts the activation status of nearly every other cell not induce detectable levels of PLZF expression in noninnate
type of the immune system. The control of NKT cells clearly T cells (6). Suppression of PLZF expression in conventional
straddles a very fine line between tolerance and fulminant T cells occurs early in T cell development and appears to be stably
response. maintained (6). Although initially thought to be expressed con-
tinuously in all NKT cells, it recently was reported that adipose
tissue–resident NKT cells express little or no PLZF (7). Adipose
*Child Health Institute of New Jersey, Rutgers Robert Wood Johnson Medical tissue–resident NKT cells appear to be a distinct lineage that is
School, New Brunswick, NJ 08901; †Graduate School of Biomedical Sciences,
Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ 08901; and selected in the thymus (8). Interestingly, the lack of PLZF ex-
‡
Department of Pediatrics, Rutgers Robert Wood Johnson Medical School, New pression correlates with the acquisition of regulatory functions,
Brunswick, NJ 08901 including an increased capacity to produce IL-10 (7).
1
P.W.D. and K.J. contributed equally to this work. Interest in PLZF has continued to increase because it is now
2
Current address: State Key Laboratory of Ophthalmology, Guangdong Key Lab- known that it also controls the development of innate lymphoid
oratory of Ophthalmology and Visual Science, Zhongshan Ophthalmic Center, Sun
Yat-sen University, Guangzhou, China.
cells (9), MAIT cells (10), and human NK cells (11). Indeed, the
interest in the BTB-ZF family of transcription factors, in general,
ORCIDs: 0000-0002-0108-6948 (K.J.); 0000-0002-5510-9367 (D.B.S.).
has grown enormously over the last few years, as it has been
Received for publication January 17, 2019. Accepted for publication June 4, 2019.
shown that they are often the “toggle switch” that controls key
This work was supported by National Institutes of Health/National Institute of Al-
lergy and Infectious Diseases Grants R01 AI083988 and AI059739 (to D.B.S.),
inflection points during development, such as the commitment of
Robert Wood Johnson Foundation Grant 67038 (to the Child Health Institute of T versus B cells or CD4 versus CD8 T cells (12).
New Jersey), National Cancer Institute Comprehensive Core Grant P30CA072720, In this study, we show that major functions of PLZF are com-
Aresty Research Center undergraduate research fellowships (to P.W.D.), and a
New Jersey Health Foundation Excellence in Research award (to D.B.S.). pletely dependent upon the expression of the transcription factor
Address correspondence and reprint requests to Dr. Derek B. Sant’Angelo, The Child
Yin Yang 1 (YY1) in NKT cells. YY1 belongs to the polycomb
Health Institute of New Jersey, Rutgers Robert Wood Johnson Medical School, 89 protein family, which is a subfamily of zinc-finger transcription
French Street, New Brunswick, NJ 08901. E-mail address: [email protected] factors with four typical zinc-finger DNA binding motifs (13). YY1
The online version of this article contains supplemental material. has been shown to be essential in diverse biological processes,
Abbreviations used in this article: a-GalCer, a-galactosylceramide; IP, immunoprecipitation/ such as hematopoiesis (14), cancer development and progression
immunoprecipitated; KO, knock-out; MFI, mean fluorescent intensity; qPCR, (15, 16), and heart development (17). YY1 modulates chromatin
quantitative PCR; Tg, transgenic; WT, wild type; YY1, Yin Yang 1.
structure and thus gene expression by mediating the formation of
Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 enhancer-promoter loops and by recruiting HAT and HDAC

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900055
628 YY1 CONTROLS PLZF IN NKT CELLS

proteins to specific loci (18). Conditional deletion of YY1 at the forward scatter width to forward scatter height and side scatter width to
double-negative stage results in a major block in thymocyte de- side scatter height. Events were acquired on an LSRII cytometer (BD
Biosciences, San Jose, CA), and the data were analyzed with FlowJo
velopment (19). Interestingly, however, the loss of YY1 in thy- software (TreeStar, Ashland, OR).
mocytes is compensated for by simultaneous deletion of p53 (19). NKT cell enrichment was performed using an autoMACS (Miltenyi
Therefore, despite the roles reported for YY1 in some mature Biotec). Cells were stained with the PE-labeled CD1d tetramer, incubated
T cell lineages (20, 21), the function of YY1 during the complex with anti-PE microbeads (Miltenyi Biotec), and separated on autoMACS
process of T cell development appears only to be the control of (Miltenyi Biotec). Samples were at least 80% pure following enrichment.
Cell sorting was performed at the Flow Cytometry/Cell Sorting and
p53’s apoptotic activity. Confocal Microscopy Core Facility at Rutgers Environmental and Occu-
We show that in the absence of YY1, NKT cells acquired the pational Health Sciences Institute or at the Cancer Institute of New Jersey.
typical activated/memory phenotype associated with this T cell
lineage but failed to undergo normal expansion. Furthermore, dis- Coimmunoprecipitation and Western blot: Lck.PLZF Tg mice
tinct from conventional thymocytes, development of YY1-deficient Thirty million thymocytes were lysed in 1 ml of hypotonic lysis buffer
NKT cells was not rescued by deletion of p53. YY1-deficient (0.05% Nonidet P-40, 10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 5 mM
NKT cells, however, had a near-complete defect in their ability to EDTA) and complete protease inhibitor mixture (Roche) and incubated on
ice for 30 min. The cytosolic fraction was discarded. Cell nuclei were
produce cytokines. Remarkably, the profound failure of development incubated on ice for 1 h in a nuclear digestion buffer (20 mM HEPES,
and acquisition of effector functions occurred despite normal PLZF 300 mM NaCl, 20 mM KCl), supplemented with an EDTA-free complete

Downloaded from http://journals.aai.org/jimmunol/article-pdf/203/3/627/1450476/ji1900055.pdf by guest on 08 November 2024

expression levels. Interestingly, we show the two proteins are in protease inhibitor mixture (Roche). Following lysis, nuclear lysates were
complex, raising the possibility that YY1 may be a cofactor sonicated for 3 s at 30% amplitude using a Branson Digital Sonifier. Nuclear
necessary for the function of PLZF. lysates were combined with 1 mg of Ab, either anti-PLZF(D9) or anti-
YY1(sc-7341), and 50 ml of Protein G Agarose Beads (Sigma-Aldrich).
Overall, our data show that the broadly expressed, multifaceted Samples were incubated for 2 h with rotation at 4˚C. Beads were washed
YY1 transcription factor plays a specific role in NKT cells, which is (5 times) with ice-cold PBS. Bound proteins were eluted by boiling for
to enable the function of the “master regulator” functions of PLZF 5 min in reducing sample buffer. The eluted samples were loaded on
for the control of the development and effector functions of Criterion precast TGX gels (Criterion) and separated. Proteins were
transferred to PVDF Transfer Membranes (Immobilon). Primary Abs
NKT cells. used for the Western blot were anti-YY1 (sc1703) and anti-PLZF (D9).

Materials and Methods Coimmunoprecipitation and Western blot: in vivo expanded

Animals NKT cells

YY1 flx.flx mice were obtained from The Jackson Laboratory (Bar Harbor, Mice were injected i.v. via the retro orbital route with 60 mg of
ME) (22) and a colleague (Dr. M. Verzi) at Rutgers University. CD4-Cre a-galactosylceramide (a-GalCer). Seventy-two hours after injection,
mice and p53 flx.flx mice were obtained from The Jackson Laboratory mice were sacrificed, and single-cell suspensions were prepared from
(23, 24). PLZF-Cre mice were developed in our laboratory, as reported (7). spleens. Thirty million splenocytes were lysed in 1 ml of hypotonic
PLZF knockout mice were reported previously and were fully backcrossed lysis buffer (0.05% Nonidet P-40, 10 mM HEPES, 1.5 mM MgCl2,
to C57BL/6 (4). Lck.PLZF transgenic (Tg) mice were reported previously 10 mM KCl, 5 mM EDTA) and complete protease inhibitor mixture
(5). All mouse strains were bred and maintained in the animal facility of (Roche) and incubated on ice for 30 min. The cytosolic fraction was
the Child Health Institute of New Jersey. All experimental protocols and discarded. Cell nuclei were incubated on ice for 1 h in a nuclear di-
procedures were approved by the Institutional Animal Care and Use gestion buffer (20 mM HEPES, 300 mM NaCl, 20 mM KCl), supple-
Committee of the Child Health Institute of New Jersey. Animal Care and mented with an EDTA-free complete protease inhibitor mixture
experimental procedures were carried out in accordance with the guide- (Roche). Following lysis, nuclear lysates were sonicated for 3 s at 30%
lines of the Institutional Animal Care and Use Committee of the Child amplitude using a Branson Digital Sonifier. Nuclear lysates were pre-
Health Institute of New Jersey and the National Institutes of Health Guide cleared by incubation with Protein G Agarose Beads (Sigma-Aldrich)
for the Care and Use of Laboratory Animals. for 30 min at 4˚C. Beads were discarded, and the precleared lysates
were combined with Protein G Agarose Beads and 5 mg of Ab, either
Preparation of single-cell suspensions from lymphoid and anti-PLZF(D9), anti-YY1(sc-7341), or nonspecific Ig. Mock immu-
nonlymphoid tissues noprecipitations (IPs) were also conducted, in which anti-PLZF(D9) or
anti-YY1(sc-7341) Abs were incubated with Protein G Agarose Beds
Whole tissues were dissociated between glass slides and filtered through a in the absence of lysate. Samples were incubated for 2 h with rotation
40-mm mesh. Spleen, lymph node, and thymus samples were treated with at 4˚C. Beads were washed (5 times) with ice-cold PBS. Bound pro-
RBC Lysing Buffer (Sigma-Aldrich). Cells were then counted on a he- teins were eluted by boiling for 5 min in reducing sample buffer. The
macytometer. Liver homogenate was suspended in Percoll (Sigma-Aldrich) eluted samples were loaded on Criterion precast TGX gels (Criterion)
to a final concentration of 30% Percoll. Liver homogenates were then and separated. Proteins were transferred to P Transfer Membranes
centrifuged for 20 min at 2000 rpm in a Sorvall RTH-750 rotor. Lym- (Immobilon). Primary Abs used for the Western blot were anti-YY1
phocytes were then collected from the buffy layer, washed once with FACS (sc1703) and anti-PLZF (D9).
buffer, treated with RBC Lysing Buffer (Sigma-Aldrich), and counted on a
hemacytometer. In vivo activation
Flow cytometry, Abs, autoMACS enrichment, and cell sorting Mice were injected i.v. via the retro orbital route with 10 mg of the NKT cell–
specific agonist a-GalCer. Ninety minutes after injection, splenocytes were
Single-cell suspensions were blocked in FACS buffer with 2% normal harvested.
mouse serum, 0.1% anti-Fcg Ab, and 0.1 mg/ml streptavidin for 15 min at
4˚C, followed by staining with primary Abs for 20 min at 4˚C. Intracellular In vitro activation
staining for transcription factors and cytokines was accomplished using the
Foxp3/Transcription Factor Staining Buffer Set (eBioscience). All intra- NKT cells were incubated in vitro with 50 ng/ml PMA (Sigma-Aldrich) and
cellular staining was performed at room temperature. The following Abs 500 ng/ml ionomycin (Sigma-Aldrich) for 5 h. After 1 h in culture, brefeldin
were used in this study: anti-CD3 (500A2), anti-CD4 (RM4-5), anti-CD8a A (BioLegend) was added to the media at a final concentration of 5 mg/ml.
(53-6.7), anti–IL-17A (Ebio17B7), anti-CD19 (1D3), anti-CD24 (M1/69), RNA isolation and quantitative PCR analysis
anti-CD44 (IM7), CD45.2 (104), anti-CD69 (H1.2F3), anti–IFN-g
(XMG1.2), anti-IL4 (11B11), anti-MHCII (212.A1), anti-NK1.1 (PK136), Sorted NKT cells were lysed in TRIzol solution at a concentration of 1 3
anti-PLZF (Mags.21F7), anti-RORgT (B2D), anti–T-bet (eBio4B10), anti- 106 cells per milliliter. RNA was isolated from the TRIzol solution using
YY1 (sc-7341), anti-TCRvB7 (TR310), and anti-TCRvB8.1/8.2 (MR5-2). the Direct-zol RNA MicroPrep Kit (Zymo Research). Reverse transcrip-
The PBS57-loaded CD1d-tetramer was provided by the National Institutes tion was performed using the GoScript Reverse Transcription System
of Health tetramer core facility. Dead cells were excluded when possible, (Promega). The resultant cDNA was used to perform TaqMan (Life
by DAPI staining, and doublet events were eliminated by comparing Technologies)-based quantitative PCR (qPCR). TaqMan Universal
The Journal of Immunology 629

PCR Master Mix No AmpErase UNG (Life Technologies) was used. The might also occur in undefined hematopoietic stem cells (25–27);
following TaqMan probes were used: B2M (Mm00437762_m1), IL-4 however, if this occurred for YY1, these cells did not contribute to
(Mm00445259_m1), and IFN-g (Mm01168134_m1). Samples were run
on a QuantStudio6 Flex Real-Time PCR System (Life Technology). Rel- the general T cell population in a measurable way. A small subset of
ative expression of IL-4 and IFN-g was calculated by using the DD cycle NKT cells in PLZF-Cre YY1 flx.flx mice continued to express YY1,
threshold method, using B2M as the internal housekeeping control gene. indicating that the gene had not been deleted in those cells (Fig. 1A).
The loss of YY1 expression in NKT cells resulted in an ∼85%
General experiment design and statistical analysis
reduction in both the percentage and absolute numbers of the cells
Data from at least three samples in three or more independent tests were in the thymus (Fig. 1B, 1C). A similar reduction in the number of
collected as detailed in the figure legends. Statistical analysis was performed
using Microsoft Excel or GraphPad Prism. All data were subjected to
NKT cells was also found in peripheral tissues, including the
analysis with Mann–Whitney U test or one-way ANOVA. spleen, lymph nodes, and liver (Fig. 1B, 1C, Supplemental Fig.
1A, 1B). In a previous study, we showed that deletion of YY1 with
Results CD2-Cre reduced total thymic cellularity to ∼1% of wild type
YY1 is required for normal development of NKT cells (WT), whereas deletion at the early double-positive stage with
Deletion of YY1 in NKT cells was induced by crossing mice with Lck-Cre reduced cellularity to ∼30% of WT (19). p53, a gene
an allele of YY1 flanked by flox recombination sites (YY1 flx.flx) known to be inhibited by YY1 (29), was substantially upregulated
(19). Normal thymocyte development was restored by a secondary

Downloaded from http://journals.aai.org/jimmunol/article-pdf/203/3/627/1450476/ji1900055.pdf by guest on 08 November 2024

(22) with a mouse line that expresses the Cre recombinase in
PLZF-expressing cells (PLZF-Cre) (7, 25–27). PLZF expression deletion of p53, which showed that YY1 was necessary for pro-
is induced very early during NKT cell development but is not tection against apoptosis (19). To test if the same mechanism was
expressed in conventional T cells (4, 6, 28). Consistent with this, involved in NKT cell development, we generated mice with
we found that YY1 expression was ablated in NKT cells but conditional “floxed” alleles of both YY1 and TP53 (p53). We then
expressed in conventional, non–PLZF-expressing thymocytes and introduced the PLZF-Cre BAC transgene (PLZF-Cre YY1/p53 flx.
T cells (Fig. 1A). Deletion of the target gene in PLZF-Cre mice flx). This approach induced simultaneous deletion of both genes

FIGURE 1. Cell-specific deletion of YY1 causes a

decrease in the number of NKT cells. The indicated
tissues were collected from C57BL/6 (WT) mice,
PLZF-Cre YY1 flx.flx mice, or PLZF-Cre YY1/p53
flx.flx mice and analyzed by FACS with the indicated
Abs. (A) YY1 expression was compared between WT
NKT cells (MHCII2, CD3+, CD1d tet+, CD242),
PLZF-Cre YY1 flx.flx NKT cells, and PLZF-Cre YY1/
p53 flx.flx NKT cells (left) isolated from the thymus
(top) or spleen (bottom). YY1 expression was also
compared between conventional T cells (MHCII2,
CD3+, CD1dtet2, PLZF2) (right) isolated from WT
mice, PLZF-Cre YY1 flx.flx mice, and PLZF-Cre YY1/
p53 flx.flx mice. Data are representative of five or more
independent experiments. (B and C) The frequency and
absolute numbers of NKT cells (MHCII2, CD3+,
CD1dtet+) in the thymus and spleen of WT mice,
PLZF-Cre YY1 flx.flx mice, and PLZF-Cre YY1/p53
flx.flx mice were determined by FACS. Representative
plots are shown in (B), and pooled data concerning the
frequency (C, top) and absolute number (C, bottom) are
in (C). FACS plots show representative results from at
least five mice analyzed in at least five independent
experiments (A and B). Graphs (C) show compiled data
from six mice, examined in five independent experi-
ments. The horizontal lines indicate the mean (6SEM).
***p , 0.001, determined by one-way ANOVA.
630 YY1 CONTROLS PLZF IN NKT CELLS

soon after positive selection of NKT cells. In contrast to what was based on a failure to upregulate NK1.1, was, however, dis-
reported for conventional T cell development, deletion of p53 in rupted (Fig. 2C, 2D).
YY1-deficient NKT cells did not restore NKT cell development One potential explanation for the failure to progress to the
(Fig. 1B). Both the percentage and total number of NKT cells NK1.1+ stage of development would be the inability to upregulate
were not restored in the thymus, as compared with PLZF-Cre Tbet (31). Although fewer YY12cKO NKT cells express Tbet, the
YY1flx.flx mice or in the spleen (Fig. 1C). Therefore, the transcription factor is clearly induced in the absence of YY1 both
need for YY1 during NKT cell development is distinct from its in thymus and spleen NKT cells (Fig. 2E). Comparison of only the
control of apoptosis mediated by p53 during conventional Tbet-expressing NKT cells shows that the level of expression is
T cell development. very similar (Fig. 2F), with no statistically significant difference
Similar results were obtained with conditional YY1 and p53 between the mean fluorescent intensities (MFI) (Fig. 2G). Overall,
alleles crossed to CD4-Cre (CD4-Cre YY1/p53 flx.flx), which these data indicate YY1 is necessary for some, but not all, aspects
induces deletion early during the double-positive stage of devel- of NKT cell development.
opment (Supplemental Fig. 2). Once again, loss of p53 expression
did not restore the frequency or number of NKT cells in the YY1-deficient NKT cells express WT levels of PLZF
thymus, as compared with CD4-Cre YY1flx.flx mice (Supplemental The failure of YY1-deficient NKT cells to develop normally could
Fig. 2B). Distinct from previous results with CD2-Cre– and Lck- be due to a concurrent loss of PLZF expression (32). PLZF ex-

Downloaded from http://journals.aai.org/jimmunol/article-pdf/203/3/627/1450476/ji1900055.pdf by guest on 08 November 2024

Cre–mediated deletion of YY1, deletion with CD4-Cre did not re- pression levels were compared by flow cytometry between WT
sult in a consistent reduction in the cellularity or development NKT cells and YY12 cKO NKT cells from PLZF-Cre YY1 flx.flx
of thymocytes (Supplemental Fig. 2C). Simultaneous deletion of mice. Because PLZF expression levels change as NKT cells de-
p53 and YY1, therefore, did not impact development of conven- velop (4), we compared expression within the discrete stages
tional thymocytes when deleted later in development (Supplemental defined by CD44 and NK1.1 expression. The level of PLZF ex-
Fig. 2D). pression was found to be statistically indistinguishable between
Overall, these data show that deletion of YY1 either early in WT NKT cells and YY12 cKO NKT cells at all stages of de-
thymocyte development (with CD4-Cre) or post–positive selection velopment (Fig. 3A–C). These data showed that YY1 was not
(with PLZF-Cre) resulted in a similar disruption of NKT cell required for PLZF expression and, more importantly, that devel-
development. Most importantly, the PLZF-Cre data show that opment of NKT cells was disrupted despite normal PLZF
the impact of YY1 on NKT cell development is cell intrinsic and, expression. PLZF expression in YY1-deficient NKT cells from
because NKT cells can be detected with the CD1d-tetramer, the CD4-Cre YY1 flx/flx mice was also found to be similar to WT
recombination event required for the production of the invariant (Supplemental Fig. 3A). YY1 deficiency induced by CD4-Cre did,
TCR is intact in the absence of YY1. Finally, the mechanism for however, result in an increase in CD24 + stage 0 cells
reduced NKT cell development is not apoptosis as a consequence (Supplemental Fig. 3B), which, as expected, have not yet upreg-
of upregulation of p53. ulated expression of PLZF. A previous report suggested that PLZF
was not expressed in YY1-deficient NKT cells (32); however, the
NKT cell maturation is only partially compromised by loss accumulation of PLZF-negative stage 0 cells was not appreciated.
of YY1 Finally, to determine if PLZF impacted the expression of YY1,
After positive selection, NKT cells progress through a develop- WT NKT cells and PLZF knock-out (KO) NKT cells were
mental program during which they acquire an activated/memory compared by flow cytometry. WT and PLZF-deficient NKT
phenotype. Following downregulation of CD24, discrete devel- cells isolated from the thymus (Fig. 3D) and the spleen
opment steps are typically demarked first by expression of CD44 (Fig. 3E) expressed statistically indistinguishable levels of YY1
and then NK1.1. These steps include stage 1 (CD442, NK1.12) (Fig. 3F, 3G).
and upregulation of CD44 (stage 2: CD44+, NK1.12), followed Collectively, these data indicate that these two transcription
by expression of NK1.1 (stage 3: CD44+, NK1.1+) (Fig. 2A). In factors do not directly regulate each other’s protein expression
the absence of PLZF, NKT cells fail to acquire the characteristic levels. Furthermore, because PLZF is expressed at WT levels, yet
activated phenotype (3, 4). Most notably, PLZF-deficient NKT cells NKT cells fail to fully develop, these data raise the possibility that
do not fully upregulate CD44 or NK1.1, consistent with a stage 1 YY1 is either directly or indirectly required for the function of
phenotype (Fig. 2A). PLZF.
In contrast (to PLZF-deficient NKT cells), the majority of YY1-
deficient NKT cells from PLZF-Cre YY1 flx.flx (denoted as YY1-deficient NKT cells do not produce cytokines
YY12cKO) acquired the activated CD44 high phenotype WT and PLZF-Cre YY1 flx.flx mice were i.v. injected with 10 mg
(Fig. 2A). NKT cells from PLZF-Cre YY1 flx.flx that continued to of the NKT cell–specific agonist ligand, a-GalCer. Ninety minutes
express YY1 because of failed deletion of the gene (denoted as after injection, mice were sacrificed, spleens were removed, and
YY1+cKO) similarly upregulated CD44, as did NKT cells from NKT cells were enriched by sorting with magnetic beads. The
WT mice. NK1.1 expression was, however, reduced in the cells were made permeable and stained for IL-4 and IFN-g. YY1-
YY12cKO NKT cells. Therefore, based on upregulation of CD44, expressing NKT cells from the PLZF-Cre YY1 flx.flx (YY1+cKO)
YY12 cKO NKT cell development progressed to a stage 2–like mice produced both cytokines at levels that were indistinguishable
phenotype, but continued development to stage 3 was impeded from NKT cells from WT mice (Fig. 4A, 4B). In sharp contrast,
(Fig. 2B). The YY1-sufficient NKT cells (YY1+ cKO NKT cells) almost none of the YY12 cKO NKT cells produced cytokines
from the PLZF-Cre YY1 flx.flx mice displayed no developmental (Fig. 4A, 4B).
defects as compared with WT NKT cells, indicating the YY12 YY1-deficient NKT cells may have a deficiency in migration or
cKO NKT cell developmental defect was cell intrinsic. NKT cells localization that could impact their in vivo response to injected
leave the thymus at stage 2 and upregulate NK1.1 expression ligands. Alternatively, the loss of YY1 might impede TCR sig-
in peripheral tissues, such as the spleen (30). In the spleen, naling. To bypass these concerns, we isolated NKT cells from WT
too, YY1 2 cKO NKT cells were found to upregulate CD44 and PLZF-Cre YY1 flx.flx mice and activated them in vitro by use
similarly to YY1-sufficient NKT cells. Further differentiation, of PMA/ionomycin. Similar to in vivo activation, YY12cKO
The Journal of Immunology 631

Downloaded from http://journals.aai.org/jimmunol/article-pdf/203/3/627/1450476/ji1900055.pdf by guest on 08 November 2024

FIGURE 2. YY1-deficient NKT cells acquire an effector/memory phenotype but fail to reach stage 3 of development. The indicated tissues were
collected from C57BL/6 (WT) mice, PLZF KO mice, or PLZF-Cre YY1 flx.flx mice and analyzed by FACS with the indicated Abs. NKT cells (MHCII2,
CD3+, CD1dtet+, CD242) isolated from PLZF-Cre YY1 flx.flx mice were stained for YY1. The NKT cells from the PLZF-Cre YY1 flx.flx mice that stained
YY12 are denoted as YY12 cKO NKT cells. The NKT cells from the PLZF-Cre YY1 flx.flx mice that stained YY1+ are denoted as YY1+ cKO NKT cells.
(A and B) NKT cells isolated from the thymus of 4-wk-old WT mice, PLZF KO mice, and PLZF-Cre YY1 flx.flx mice were stained for CD44 and NK1.1.
Representative FACs plots are shown in (A). Pooled data concerning the distribution of NKT cells in each stage are (Figure legend continues)
632 YY1 CONTROLS PLZF IN NKT CELLS

FIGURE 3. PLZF and YY1 expression are in-

dependent. The indicated tissues were collected
from C57BL/6 (WT) mice, PLZF-Cre YY1 flx.flx
mice, or PLZF KO mice and analyzed by FACS
with the indicated Abs. (A) PLZF expression was
determined by flow cytometry and compared be-
tween WT stage 0 NKT cells (MHCII2, CD3+,
CD1dtet+, CD24+) and stage 0 NKT cells isolated
from PLZF-Cre YY1 flx.flx mice. (B and C) PLZF
expression was determined by flow cytometry and
compared between WT NKT cells and YY12 cKO
at the same developmental stage. Representative

Downloaded from http://journals.aai.org/jimmunol/article-pdf/203/3/627/1450476/ji1900055.pdf by guest on 08 November 2024

histograms are shown in (B). Cumulative MFI data
for PLZF are plotted in (C). NKT cell stages were
defined as follows: stage 1 (CD242, CD442,
NK1.12) (left), stage 2 (CD242, CD44+, NK1.12)
(middle), and stage 3 (CD242, CD44+, NK1.1+)
(right). NKT cells isolated from the thymus (D and
F) and spleen (E and G) of WT mice and PLZF KO
mice were stained for YY1. Representative histo-
grams of YY1 expression are shown in (D) and (E).
Pooled data concerning the MFI of YY1 are shown
in (F) and (G). Each symbol (C, F, and G) represents
an individual mouse. Data are from three or more
independent experiments representing (C) seven, (F)
four, or (G) three biological replicates. The hori-
zontal lines indicate the mean (6SEM). The p
values were determined by Mann–Whitney U test
(C, F, and G).

NKT cells did not produce either IFN-g or IL-4 (Fig. 4C, 4D). NKT cells but were absent from PLZF-deficient NKT cells
Both WT NKT cells and YY1+ cKO NKT cells produced both (Fig. 4D). Despite the presence of some YY1-sufficient
cytokines under the same activation conditions. NKT cells, the NKT cells from the PLZF-Cre YY1 flx.flx mice
Unlike conventional T cells, the mRNA for IL-4 and other showed a substantial reduction in the level of these cytokine
cytokines is present in NKT cells, even prior to activation (Fig. 4E). mRNAs. Therefore, despite WT expression levels, in the absence
This “preformed” mRNA likely plays a direct role in the rapid of YY1, PLZF was unable to induce an open configuration at these
production of cytokines by NKT cells. PLZF-deficient NKT cells gene loci, thereby preventing continuous mRNA transcription,
do not have preformed cytokine mRNA, however, which pre- which prevents the rapid release of cytokines.
sumably is a major reason for why PLZF-deficient NKT cells Overall, these experiments show that despite expressing WT
cannot produce cytokines upon primary activation (4). The fail- levels of PLZF, YY1-deficient NKT cells could not produce their
ure of YY1-deficient NKT cells to produce cytokines suggested characteristic burst of cytokines in response to activation. These
that a similar mechanism might be involved. Total NKT cells data imply that as in development of NKT cells, YY1 expression is
(both YY12 cKO and YY1+ cKO) were sorted from WT, PLZF- necessary for the function of PLZF. Therefore, the lineage-specific
deficient, and PLZF-Cre YY1 flx.flx mice. RNA was isolated, and master regulator of NKT cell effector functions, PLZF, requires
levels of specific cytokine message were measured by qPCR. As coexpression of the ubiquitously expressed YY1 transcription
expected, mRNA for IL-4 and IFN-g were readily detected in WT factor for its activity.

shown in (B). The stages were defined as follows: stage 1 (CD242, CD442, NK1.12), stage 2 (CD242, CD44+, NK1.12), and stage 3 (CD242, CD44+,
NK1.1+). (C and D) Splenic NKT cells isolated from WT mice, PLZF-Cre YY1 flx.flx mice, and PLZF KO mice were stained for CD44 and NK1.1. A
representative FACs plot is shown in (C). Pooled data concerning the frequency of splenic NK1.1+ NKT cells are depicted in (D). (E) Tbet expression was
compared between WT NKT cells and YY12 cKO NKT cells from the thymus (left) and spleen (right). (F and G) Tbet expression levels were compared
between Tbet+ WT NKT cells and Tbet+ YY12 cKO NKT cells. A representative histogram is shown in (F), and cumulative data from four mice are shown
in (G). FACS plots show typical results from indicated tissues, and graphs show compiled data from four mice, examined in three or more independent
experiments. The horizontal lines indicate the mean (6SEM). **p , 0.01, ***p , 0.001, determined by one-way ANOVA (B and D) or Mann–Whitney
U test (G).
The Journal of Immunology 633

FIGURE 4. YY1-deficient NKT cells do

not produce cytokines upon primary acti-
vation. (A and B) C57BL/6 (WT) and
PLZF-Cre YY1 flx.flx (YY1 cKO) mice
were injected i.v. with a-GalCer (10 mg)
suspended in saline. Ninety minutes after

Downloaded from http://journals.aai.org/jimmunol/article-pdf/203/3/627/1450476/ji1900055.pdf by guest on 08 November 2024

injection, splenic NKT were harvested and
stained for IL-4 and IFN-g. Representa-
tive FACs plots depicting IL-4 and IFN-g
expression in WT NKT cells (MHCII2,
CD3+, CD1dtet+, CD242), YY12 cKO
NKT cells, and YY1+ cKO NKT cells are
depicted in (A), and cumulative data from
four experiments are shown in (B). (C and
D) NKT cells isolated from the spleens of
WT and YY1 cKO mice were activated
in vitro for 5 h by culture with PMA (50 ng/ml)
and ionomycin (500 n/ml). Activated NKT cells
were stained for IL-4, IFN-g, and YY1.
Representative histograms (C) and aggre-
gate data (D) show the production of IFN-g
(left) and IL-4 (right) by WT NKT cells,
YY1+ cKO NKT cells, and YY12 cKO
NKT cells, following in vitro activation. (E)
NKT cells were FACs sorted from WT,
PLZF KO, and YY1 cKO mice. RNA was
prepared, and cDNA was generated. qPCR
was used to measure the presence of IL-4
and IFN-g mRNA in the sorted NKT cell
populations. Data are representative of four
(B and E) or three (D) independent experi-
ments and biological replicates. The hori-
zontal lines indicate the mean (6SEM).
*p , 0.05, **p , 0.01, ***p , 0.001, de-
termined by one-way ANOVA (B, D, and E).

Subset development RORgt. Consistent with the known functions of these transcription
The propensity of NKT cell subsets to have particular effector factors, NKT1, NKT2, and NKT17 cells are skewed toward pro-
functions can be defined by the expression of Tbet, PLZF, and ducing INFg, IL-4, or IL-17, respectively (34).
RORgt (33). Therefore, to further explore the impact of the loss of Most WT thymic NKT cells belong to the NKT1 subpopulation
YY1, we determined if expression of these transcription factors (Fig. 5A). In contrast, the majority of thymic YY12 cKO NKT cells
was altered in YY1-deficient NKT cells. NKT1 cells express had a NKT2-like profile (Fig. 5A). The NKT17 subpopulation was
PLZF and Tbet, NKT2 cells express high levels of PLZF, but no substantially diminished in the YY12 cKO NKT cell population
Tbet and NKT17 cells express intermediate levels of PLZF and compared with WT (Fig. 5B). Although the percentage of NKT2
634 YY1 CONTROLS PLZF IN NKT CELLS

Downloaded from http://journals.aai.org/jimmunol/article-pdf/203/3/627/1450476/ji1900055.pdf by guest on 08 November 2024

FIGURE 5. NKT cell subset development in the absence of YY1. FACs analysis was used to determine the expression of key transcription factors
associated with NKT cell effector functions in YY1-deficient thymus NKT cells. NKT cells isolated from the thymuses of C57BL/6 (WT) mice and PLZF-
Cre YY1 flx.flx mice were stained for the transcription factors PLZF, Tbet, and ROR gT. Gating schemes and representative FACS data for the (A) NKT1,
NKT2, and (B) NKT17 populations are shown. (C) The frequency and absolute number of thymic WT NKT cells and thymic YY12 cKO NKT cells in these
subpopulations are summarized. Data are from four or more independent experiments representing four biological replicates. The horizontal lines indicate
the mean (6SEM). *p , 0.05, determined by Mann–Whitney U test (C).

cells was increased, the absolute number of NKT2 cells was YY1+cKO versus 20% YY12cKO). Vb2, which typically rep-
statistically unchanged (Fig. 5C). Reflecting the thymus, spleen resents ,5% of total NKT cells, was not changed (Supplemental
YY12cKO cells also had an increased NKT2 population but Fig. 3C). Therefore, TCR usage was similar, suggesting that the
decreased frequencies of NKT1 and NKT17 cells (Fig. 6A–C). strength of signaling during development was not substantially
The increased percentage of NKT2 cells did not, however, reflect altered. Overall, although substantially altered in frequency, based
increased numbers of the cells as compared with WT (Fig. 6C). on transcription factor expression, all three NKT cell effector sub-
Finally, we tested the possibility that changes in subset distri- types develop in the absence of YY1.
bution might reflect altered TCR usage, which can impact the
strength of signal received during selection (35–37). Approxi- Interaction of YY1 with PLZF
mately 70% of YY1+cKO and YY12cKO NKT cells expressed To determine if YY1 might be directly influencing the function of
Vb8 (Supplemental Fig. 3C). The frequency of the less-common PLZF, we tested if the two transcription factors were complexed by
Vb7-expressing NKT cells was, however, altered ∼2-fold (10% carrying out co-IP experiments (Fig. 7). Reported mass spectrometry
The Journal of Immunology 635

Downloaded from http://journals.aai.org/jimmunol/article-pdf/203/3/627/1450476/ji1900055.pdf by guest on 08 November 2024

FIGURE 6. NKT cell subsets in the spleen in the absence of YY1. FACs analysis was used to determine the expression of key transcription factors
associated with NKT cell effector functions in YY1-deficient spleen NKT cells. Splenic NKT cells isolated from WT and PLZF-Cre YY1 flx.flx mice were
stained for PLZF, Tbet, and ROR gT (A and B). The frequency and absolute number of splenic WT NKT cells and YY1-deficient NKT cells present in the
NKT1, NKT2, and NKT17 subpopulations are summarized in (C). Data are from four or more independent experiments representing four biological
replicates. The horizontal lines indicate the mean (6SEM). *p , 0.05, **p , 0.01, determined by Mann–Whitney U test (C).

analysis suggested that such an association might occur (38). We thymocytes, followed by Western blotting for YY1 (Fig. 7A). The
used cells from mice carrying a transgene for PLZF under the results showed that YY1 was specifically detected in association
control of the T cell–specific Lck promoter (PLZF Tg) in which with PLZF only in PLZF Tg thymocytes and not in negative
all T cells express PLZF (5). We have previously shown that control PLZF-deficient or WT thymocytes. Importantly, these re-
conventional T cells from these PLZF Tg mice take on innate-like sults were confirmed by capturing YY1 from the thymocytes,
T cell characteristics, suggesting that PLZF functions similarly in followed by Western blotting for PLZF (Fig. 7B). Western blotting
this system as it does in NKT cells. Importantly, the expression of of the input nuclear lysates showed that YY1 was present in
PLZF in T cells from these mice was shown to be similar to en- thymocytes from all three mice; however, PLZF was only detected
dogenous PLZF expression in NKT cells (5). Therefore, this is in thymocytes isolated from PLZF Tg mice (Fig. 7C). As ex-
ectopic expression but not overexpression. pected, PLZF was not detected in total thymocytes from WT mice
To determine if PLZF and YY1 interact, PLZF complexes were because of the very low frequency of PLZF-expressing T cells.
captured from nuclear lysates isolated from thymocytes from PLZF Next, we confirmed this result in primary NKT cells. The
Tg mice or from control WT or PLZF-deficient (PLZF KO) scarcity of NKT cells in WT mice makes direct biochemical assays
636 YY1 CONTROLS PLZF IN NKT CELLS

proliferation, culling via apoptosis, migration, and interaction with

thymic stromal cells, which ultimately results in a self-referential,
self-tolerant, and functionally heterogeneous T cell repertoire (39–
41). Disruption of any of these steps leads to aberrant or blocked
T cell development. Nearly all the factors that control this process
for conventional T cells are also essential for NKT cell develop-
ment. Furthermore, disruption of positive or negative selection
owing to defects in the TCR due to loss of TCRb and/or TCRa-
chain rearrangement (42) or loss of key proteins required for TCR
signaling pathways, such as Lck, ZAP-70 (43, 44), LAT (45),
SLP-76 (46), PLCg1 (47), or VAV1 (48), also blocks NKT cell
development. Therefore, it is clear that much of NKT cell de-
velopment is similar to the development of conventional T cells.
A number of genes, however, have been shown to be uniquely
essential for NKT development. For example, loss of expression of
either the tyrosine kinase Fyn (49) or its adapter protein SAP (50)

Downloaded from http://journals.aai.org/jimmunol/article-pdf/203/3/627/1450476/ji1900055.pdf by guest on 08 November 2024

cause the specific loss of NKT cells, whereas conventional T cell
development is not perturbed. This is potentially due to a specific
need for SLAM family member signaling (51, 52). Also, because
NKT cells express a canonical TCRa-chain, encoded by the Va14
gene segment joined to the distally located Ja18 gene segment,
disruption of the lifespan of the double-positive thymocytes im-
pacts NKT cell development, as such is the case in the absence of
RORgt (53) or HEB (42).
Although multiple gene deficiencies are now known to impact
NKT cell development, the BTB-ZF transcription factor, PLZF,
appears to be the primary, if not only, known transcription factor
essential for the development of the characteristic NKT innate-like
effector functions and phenotype (3, 5). Importantly, among
T cells, PLZF is only expressed in innate-like T cells (NKT cells,
gd NKT cells, and MAIT cells) and is not induced in conventional
FIGURE 7. Co-IP of PLZF and YY1 demonstrates a molecular inter- T cells (6). The full mechanism by which PLZF endows the innate
action between the two transcription factors. (A–C) Nuclear lysates
phenotype in T cells is, however, unknown.
were generated from PLZF-deficient (PLZF KO), C57BL/6 (WT), and
Lck.PLZF Tg (PLZF Tg) thymocytes. Lysates were IP with (A) PLZF- or
We generated mice with a deletion of YY1 that was largely re-
(B) YY1-specific Abs, followed by Western blot with Abs against YY1 or stricted to NKT cells. Deletion of YY1 resulted in a sharp reduction
PLZF to test for the coassociation of the two transcription factors. (C) in the total number of NKT cells in the thymus, spleen, liver, and,
Western blot of thymocyte lysates (input) from the same lysates as (A) and in contrast to PLZF deficiency, the lymph nodes. A similar, albeit,
(B) were probed with the indicated Ab (anti-PLZF, top; anti-YY1, bottom). moderately more severe phenotype is found using thymocyte-
Data are representative of four independent experiments. (D) Nuclear specific expression of Cre controlled by the CD4 promoter (32),
lysates were generated from the spleens of a-GalCer–treated mice. Total which deletes YY1 earlier in development.
lysate (lane 1), lysate IP with a PLZF-specific Ab (lane 2), YY1-specific Of most interest, however, is that YY1-deficient NKT cells lost
Ab (lane 4), or nonspecific Ig (lane 6) were used. Mock PLZF IP (lane 3) the capacity to produce cytokines following primary activation.
and mock YY1 IP (lane 5) were loaded in the indicated lanes. Western blot
Loss of rapid cytokine production is likely caused by a failure to
of the samples were probed with anti-PLZF Ab. Data are representative of
four independent experiments.
produce preformed mRNA, which also occurs in the absence of
PLZF. These data suggest that YY1 is required to establish the
genetic program that is ultimately controlled by PLZF. However,
such as this difficult. To overcome this, we first expanded NKT cells PLZF clearly can direct some gene expression activity in the ab-
in vivo by injection of a-GalCer. Three days postactivation, ∼20 sence of YY1. For example, upregulation of CD44, which is a
million NKT cells could be obtained from the spleen, representing characteristic of nearly all PLZF-expressing lymphocytes (4, 54),
an ∼20-fold increase (Supplemental Fig. 4A). Importantly, still occurs. The CD44 gene has been shown by chromatin IP to be
NKT cells postactivation continue to express both YY1 and PLZF a direct target of PLZF (55). These data show that YY1 is only
(Supplemental Fig. 4B, 4C). Nuclear isolates from purified necessary for certain aspects of PLZF function, and, therefore, it is
NKT cells were IP with anti-YY1 Ab, followed by Western blot possible that PLZF and YY1 control both complementary and
with anti-PLZF. Associated PLZF protein was specifically de- independent aspects of NKT cell development and function.
tected only in the lane with precipitated YY1 (Fig. 7D). Despite the loss of preformed cytokine mRNA, the complete
Overall, these data showed that YY1 is in complex with PLZF, disruption of cytokine production, and the failure to accumulate in
suggesting that YY1 might directly control the function of the thymus and liver, YY1-deficient NKT cells expressed WT
PLZF in NKT cells. levels of PLZF. These data show that PLZF function, but not
expression, is dependent on coexpression of YY1. YY1, however,
Discussion clearly is not sufficient for NKT cell effector functions because
T cell progenitors, derived from bone marrow hematopoietic stem PLZF-deficient NKT cells express WT levels of the transcription
cells, migrate to the thymus and are signaled to become early factor. This codependency implies there is a direct, functional
thymocyte progenitors. These early thymocytes undergo a well- coordination. Our finding that the two proteins are in a complex
regulated, stepwise development involving TCR rearrangement, suggests that YY1 might directly modulate the function of PLZF in
The Journal of Immunology 637

NKT cells. Understanding this interplay will be an important step 13. Shi, Y., E. Seto, L. S. Chang, and T. Shenk. 1991. Transcriptional repression by
YY1, a human GLI-Krüppel-related protein, and relief of repression by adeno-
toward understanding the transcriptional programming of NKT cells. virus E1A protein. Cell 67: 377–388.
This is, to our knowledge, the first report of the requirement 14. Lu, Z., C. C. Hong, G. Kong, A. L. F. V. Assumpção, I. M. Ong, E. H. Bresnick,
for a coexpression of YY1 and a BTB-ZF transcription factor J. Zhang, and X. Pan. 2018. Polycomb group protein YY1 is an essential reg-
ulator of hematopoietic stem cell quiescence. Cell Rep. 22: 1545–1559.
for cellular function. BTB-ZF transcription factors, for example, 15. Castellano, G., E. Torrisi, G. Ligresti, G. Malaponte, L. Militello, A. E. Russo,
ThPOK, LRF, Bcl6, and MAZR, regulate many of the key in- J. A. McCubrey, S. Canevari, and M. Libra. 2009. The involvement of the
flection points during the development of the immune system as transcription factor Yin Yang 1 in cancer development and progression. Cell
Cycle 8: 1367–1372.
well as basic biological functions, such as male germ cell homeo- 16. Patten, D. K., G. Corleone, B. Gy}orffy, Y. Perone, N. Slaven, I. Barozzi,
stasis, axial-skeletal patterning, liver development, brain function, E. Erd}os, A. Saiakhova, K. Goddard, A. Vingiani, et al. 2018. Enhancer mapping
etc. The molecular mechanisms by which BTB-ZF transcription uncovers phenotypic heterogeneity and evolution in patients with luminal breast
cancer. Nat. Med. 24: 1469–1480.
factors execute their functions remain poorly described. It is pos- 17. Morikawa, Y., J. Leach, and J. F. Martin. 2013. Yin-Yang 1, a new player in early
sible that molecular coordination with YY1 may be necessary for heart development. Circ. Res. 112: 876–877.
18. Weintraub, A. S., C. H. Li, A. V. Zamudio, A. A. Sigova, N. M. Hannett,
the function of BTB-ZF transcription factors in these other settings, D. S. Day, B. J. Abraham, M. A. Cohen, B. Nabet, D. L. Buckley, et al. 2017.
in addition to its function in NKT cells. YY1 itself has been shown YY1 is a structural regulator of enhancer-promoter loops. Cell 171: 1573–
to play a role in the development of the immune system. Perhaps 1588.e28.
19. Chen, L., D. P. Foreman, D. B. Sant’Angelo, and M. S. Krangel. 2016. Yin yang
some functions that are attributed to YY1 are actually a conse- 1 promotes thymocyte survival by downregulating p53. J. Immunol. 196: 2572–

Downloaded from http://journals.aai.org/jimmunol/article-pdf/203/3/627/1450476/ji1900055.pdf by guest on 08 November 2024

quence of the gross dysfunction of BTB-ZF transcription factors in 2582.
the absence of YY1. 20. Hwang, S. S., Y. U. Kim, S. Lee, S. W. Jang, M. K. Kim, B. H. Koh, W. Lee,
J. Kim, A. Souabni, M. Busslinger, and G. R. Lee. 2013. Transcription factor
In summary, we show that complete loss of YY1 in NKT cells YY1 is essential for regulation of the Th2 cytokine locus and for Th2 cell dif-
leads to a loss of the expansion and function of NKT cells. These ferentiation. Proc. Natl. Acad. Sci. USA 110: 276–281.
disrupted functions occur despite WT expression levels of PLZF. 21. Hwang, S. S., S. W. Jang, M. K. Kim, L. K. Kim, B. S. Kim, H. S. Kim, K. Kim,
W. Lee, R. A. Flavell, and G. R. Lee. 2016. YY1 inhibits differentiation and
Combined, these data, to our knowledge, identify a new function function of regulatory T cells by blocking Foxp3 expression and activity. Nat.
for YY1, which is to mediate specific functions of PLZF. Commun. 7: 10789.
22. Affar el, B., F. Gay, Y. Shi, H. Liu, M. Huarte, S. Wu, T. Collins, E. Li, and
Y. Shi. 2006. Essential dosage-dependent functions of the transcription factor
Acknowledgments yin yang 1 in late embryonic development and cell cycle progression. Mol. Cell.
Biol. 26: 3565–3581.
We thank Joshua Vieth from the Child Health Institute of New Jersey for 23. Marino, S., M. Vooijs, H. van Der Gulden, J. Jonkers, and A. Berns. 2000. In-
critical analysis of the data and/or help with experimental procedures and duction of medulloblastomas in p53-null mutant mice by somatic inactivation
Arthur Roberts from the Cancer Institute of New Jersey Flow Cytometry of Rb in the external granular layer cells of the cerebellum. Genes Dev. 14:
Core Facility for cell sorting. 994–1004.
24. Lee, P. P., D. R. Fitzpatrick, C. Beard, H. K. Jessup, S. Lehar, K. W. Makar,
M. Pérez-Melgosa, M. T. Sweetser, M. S. Schlissel, S. Nguyen, et al. 2001. A
Disclosures critical role for Dnmt1 and DNA methylation in T cell development, function,
and survival. Immunity 15: 763–774.
The authors have no financial conflicts of interest. 25. Thapa, P., M. W. Chen, D. C. McWilliams, P. Belmonte, M. Constans,
D. B. Sant’Angelo, and V. S. Shapiro. 2016. NKAP regulates invariant
NKT cell proliferation and differentiation into ROR-gt-Expressing NKT17 cells.
References J. Immunol. 196: 4987–4998.
1. Bendelac, A., P. B. Savage, and L. Teyton. 2007. The biology of NKT cells. 26. Thapa, P., S. Romero Arocha, J. Y. Chung, D. B. Sant’Angelo, and V. S. Shapiro.
Annu. Rev. Immunol. 25: 297–336. 2017. Histone deacetylase 3 is required for iNKT cell development. Sci. Rep. 7:
2. Bandyopadhyay, K., I. Marrero, and V. Kumar. 2016. NKT cell subsets as key 5784.
participants in liver physiology and pathology. Cell. Mol. Immunol. 13: 337–346. 27. Uddin, M. N., D. A. Sultana, K. J. Lorentsen, J. J. Cho, M. E. Kirst,
3. Savage, A. K., M. G. Constantinides, J. Han, D. Picard, E. Martin, B. Li, M. L. Brantly, D. Califano, D. B. Sant’Angelo, and D. Avram. 2016. Tran-
O. Lantz, and A. Bendelac. 2008. The transcription factor PLZF directs the scription factor Bcl11b sustains iNKT1 and iNKT2 cell programs, restricts
effector program of the NKT cell lineage. Immunity 29: 391–403. iNKT17 cell program, and governs iNKT cell survival. Proc. Natl. Acad. Sci.
4. Kovalovsky, D., O. U. Uche, S. Eladad, R. M. Hobbs, W. Yi, E. Alonzo, K. Chua, USA 113: 7608–7613.
M. Eidson, H. J. Kim, J. S. Im, et al. 2008. The BTB-zinc finger transcriptional 28. Hosokawa, H., M. Romero-Wolf, M. A. Yui, J. Ungerbäck, M. L. G. Quiloan,
regulator PLZF controls the development of invariant natural killer T cell ef- M. Matsumoto, K. I. Nakayama, T. Tanaka, and E. V. Rothenberg. 2018. Bcl11b
fector functions. Nat. Immunol. 9: 1055–1064. sets pro-T cell fate by site-specific cofactor recruitment and by repressing Id2
5. Kovalovsky, D., E. S. Alonzo, O. U. Uche, M. Eidson, K. E. Nichols, and and Zbtb16. Nat. Immunol. 19: 1427–1440.
D. B. Sant’Angelo. 2010. PLZF induces the spontaneous acquisition of memory/ 29. Sui, G., B. Affar, Y. Shi, C. Brignone, N. R. Wall, P. Yin, M. Donohoe,
effector functions in T cells independently of NKT cell-related signals. M. P. Luke, D. Calvo, S. R. Grossman, and Y. Shi. 2004. Yin Yang 1 is a negative
J. Immunol. 184: 6746–6755. regulator of p53. Cell 117: 859–872.
6. Zhang, S., A. Laouar, L. K. Denzin, and D. B. Sant’Angelo. 2015. Zbtb16 30. Benlagha, K., T. Kyin, A. Beavis, L. Teyton, and A. Bendelac. 2002. A thymic
(PLZF) is stably suppressed and not inducible in non-innate T cells via T cell precursor to the NK T cell lineage. Science 296: 553–555.
receptor-mediated signaling. Sci. Rep. 5: 12113. 31. Townsend, M. J., A. S. Weinmann, J. L. Matsuda, R. Salomon, P. J. Farnham,
7. Lynch, L., X. Michelet, S. Zhang, P. J. Brennan, A. Moseman, C. Lester, C. A. Biron, L. Gapin, and L. H. Glimcher. 2004. T-bet regulates the terminal
G. Besra, E. E. Vomhof-Dekrey, M. Tighe, H. F. Koay, et al. 2015. Regulatory maturation and homeostasis of NK and Valpha14i NKT cells. Immunity 20: 477–
iNKT cells lack expression of the transcription factor PLZF and control the 494.
homeostasis of T(reg) cells and macrophages in adipose tissue. Nat. Immunol. 32. Ou, X., J. Huo, Y. Huang, Y. F. Li, S. Xu, and K. P. Lam. 2019. Transcription
16: 85–95. factor YY1 is essential for iNKT cell development. Cell. Mol. Immunol. 16: 547–
8. Vieth, J. A., J. Das, F. M. Ranaivoson, D. Comoletti, L. K. Denzin, and 556.
D. B. Sant’Angelo. 2017. TCRa-TCRb pairing controls recognition of CD1d 33. Lee, Y. J., K. L. Holzapfel, J. Zhu, S. C. Jameson, and K. A. Hogquist. 2013.
and directs the development of adipose NKT cells. [Published erratum appears in Steady-state production of IL-4 modulates immunity in mouse strains and is
2017 Nat. Immunol. 18: 951.] Nat. Immunol. 18: 36–44. determined by lineage diversity of iNKT cells. [Published erratum appears in
9. Constantinides, M. G., B. D. McDonald, P. A. Verhoef, and A. Bendelac. 2014. 2014 Nat. Immunol. 15: 305.] Nat. Immunol. 14: 1146–1154.
A committed precursor to innate lymphoid cells. Nature 508: 397–401. 34. Monteiro, M., and L. Graca. 2014. iNKT cells: innate lymphocytes with a di-
10. Rahimpour, A., H. F. Koay, A. Enders, R. Clanchy, S. B. Eckle, B. Meehan, verse response. Crit. Rev. Immunol. 34: 81–90.
Z. Chen, B. Whittle, L. Liu, D. P. Fairlie, et al. 2015. Identification of pheno- 35. Tuttle, K. D., S. H. Krovi, J. Zhang, R. Bedel, L. Harmacek, L. K. Peterson,
typically and functionally heterogeneous mouse mucosal-associated invariant L. L. Dragone, A. Lefferts, C. Halluszczak, K. Riemondy, et al. 2018. TCR
T cells using MR1 tetramers. J. Exp. Med. 212: 1095–1108. signal strength controls thymic differentiation of iNKT cell subsets. Nat. Com-
11. Eidson, M., J. Wahlstrom, A. M. Beaulieu, B. Zaidi, S. E. Carsons, P. K. Crow, mun. 9: 2650.
J. Yuan, J. D. Wolchok, B. Horsthemke, D. Wieczorek, and D. B. Sant’Angelo. 36. Zhao, M., M. N. D. Svensson, K. Venken, A. Chawla, S. Liang, I. Engel,
2011. Altered development of NKT cells, gd T cells, CD8 T cells and NK cells P. Mydel, J. Day, D. Elewaut, N. Bottini, and M. Kronenberg. 2018. Altered
in a PLZF deficient patient. PLoS One 6: e24441. thymic differentiation and modulation of arthritis by invariant NKT cells
12. Beaulieu, A. M., and D. B. Sant’Angelo. 2011. The BTB-ZF family of tran- expressing mutant ZAP70. Nat. Commun. 9: 2627.
scription factors: key regulators of lineage commitment and effector function 37. Moran, A. E., K. L. Holzapfel, Y. Xing, N. R. Cunningham, J. S. Maltzman,
development in the immune system. J. Immunol. 187: 2841–2847. J. Punt, and K. A. Hogquist. 2011. T cell receptor signal strength in Treg and
638 YY1 CONTROLS PLZF IN NKT CELLS

iNKT cell development demonstrated by a novel fluorescent reporter mouse. 47. Fu, G., Y. Chen, M. Yu, A. Podd, J. Schuman, Y. He, L. Di, M. Yassai,
J. Exp. Med. 208: 1279–1289. D. Haribhai, P. E. North, et al. 2010. Phospholipase Cgamma1 is essential for
38. Mathew, R., M. P. Seiler, S. T. Scanlon, A. P. Mao, M. G. Constantinides, T cell development, activation, and tolerance. J. Exp. Med. 207: 309–318.
C. Bertozzi-Villa, J. D. Singer, and A. Bendelac. 2012. BTB-ZF factors recruit 48. Turner, M., P. J. Mee, A. E. Walters, M. E. Quinn, A. L. Mellor, R. Zamoyska,
the E3 ligase cullin 3 to regulate lymphoid effector programs. Nature 491: and V. L. Tybulewicz. 1997. A requirement for the Rho-family GTP exchange
618–621. factor Vav in positive and negative selection of thymocytes. Immunity 7:
39. Petrie, H. T., and J. C. Zúñiga-Pflücker. 2007. Zoned out: functional mapping of 451–460.
stromal signaling microenvironments in the thymus. Annu. Rev. Immunol. 25: 49. Eberl, G., B. Lowin-Kropf, and H. R. MacDonald. 1999. Cutting edge: NKT cell
649–679. development is selectively impaired in Fyn- deficient mice. J. Immunol. 163:
40. Sant’Angelo, D. B., B. Lucas, P. G. Waterbury, B. Cohen, T. Brabb, 4091–4094.
J. Goverman, R. N. Germain, and C. A. Janeway, Jr. 1998. A molecular map of 50. Pasquier, B., L. Yin, M. C. Fondanèche, F. Relouzat, C. Bloch-Queyrat,
T cell development. Immunity 9: 179–186. N. Lambert, A. Fischer, G. de Saint-Basile, and S. Latour. 2005. Defective
41. Krangel, M. S. 2007. T cell development: better living through chromatin. Nat. NKT cell development in mice and humans lacking the adapter SAP, the X-
Immunol. 8: 687–694. linked lymphoproliferative syndrome gene product. J. Exp. Med. 201: 695–701.
42. D’Cruz, L. M., J. Knell, J. K. Fujimoto, and A. W. Goldrath. 2010. An essential 51. Borowski, C., and A. Bendelac. 2005. Signaling for NKT cell development: the
role for the transcription factor HEB in thymocyte survival, Tcra rearrangement SAP-FynT connection. J. Exp. Med. 201: 833–836.
and the development of natural killer T cells. Nat. Immunol. 11: 240–249. 52. Lu, Y., M. C. Zhong, J. Qian, V. Calderon, M. Cruz Tleugabulova, T. Mallevaey,
43. Negishi, I., N. Motoyama, K. Nakayama, K. Nakayama, S. Senju, and A. Veillette. 2019. SLAM receptors foster iNKT cell development by re-
S. Hatakeyama, Q. Zhang, A. C. Chan, and D. Y. Loh. 1995. Essential role for ducing TCR signal strength after positive selection. Nat. Immunol. 20: 447–457.
ZAP-70 in both positive and negative selection of thymocytes. Nature 376: 53. Egawa, T., G. Eberl, I. Taniuchi, K. Benlagha, F. Geissmann, L. Hennighausen,
435–438. A. Bendelac, and D. R. Littman. 2005. Genetic evidence supporting selection of
44. Wiest, D. L., J. M. Ashe, T. K. Howcroft, H. M. Lee, D. M. Kemper, I. Negishi, the Valpha14i NKT cell lineage from double-positive thymocyte precursors.

Downloaded from http://journals.aai.org/jimmunol/article-pdf/203/3/627/1450476/ji1900055.pdf by guest on 08 November 2024

D. S. Singer, A. Singer, and R. Abe. 1997. A spontaneously arising mutation in Immunity 22: 705–716.
the DLAARN motif of murine ZAP-70 abrogates kinase activity and arrests 54. Alonzo, E. S., R. A. Gottschalk, J. Das, T. Egawa, R. M. Hobbs, P. P. Pandolfi,
thymocyte development. Immunity 6: 663–671. P. Pereira, K. E. Nichols, G. A. Koretzky, M. S. Jordan, and D. B. Sant’Angelo.
45. Zhang, W., C. L. Sommers, D. N. Burshtyn, C. C. Stebbins, J. B. DeJarnette, 2010. Development of promyelocytic zinc finger and ThPOK-expressing innate
R. P. Trible, A. Grinberg, H. C. Tsay, H. M. Jacobs, C. M. Kessler, et al. 1999. gamma delta T cells is controlled by strength of TCR signaling and Id3.
Essential role of LAT in T cell development. Immunity 10: 323–332. J. Immunol. 184: 1268–1279.
46. Pivniouk, V., E. Tsitsikov, P. Swinton, G. Rathbun, F. W. Alt, and R. S. Geha. 55. Mao, A. P., M. G. Constantinides, R. Mathew, Z. Zuo, X. Chen, M. T. Weirauch,
1998. Impaired viability and profound block in thymocyte development in mice and A. Bendelac. 2016. Multiple layers of transcriptional regulation by PLZF in
lacking the adaptor protein SLP-76. Cell 94: 229–238. NKT-cell development. Proc. Natl. Acad. Sci. USA 113: 7602–7607.