Programmed Death-Ligand 2 Deficiency Exacerbates

International Journal of Molecular Sciences

Article Programmed Death-Ligand 2 Deﬁciency Exacerbates Experimental Autoimmune Myocarditis in Mice

Siqi Li 1, Kazuko Tajiri 1,* , Nobuyuki Murakoshi 1, DongZhu Xu 1, Saori Yonebayashi 1, Yuta Okabe 1, Zixun Yuan 1, Duo Feng 1, Keiko Inoue 1, Kazuhiro Aonuma 1, Yuzuno Shimoda 1, Zoughu Song 1, Haruka Mori 1, Honglan Huang 2, Kazutaka Aonuma 1 and Masaki Ieda 1

1 Department of Cardiology, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575, Japan; [email protected] (S.L.); [email protected] (N.M.); [email protected] (D.X.); [email protected] (S.Y.); [email protected] (Y.O.); [email protected] (Z.Y.); [email protected] (D.F.); [email protected] (K.I.); [email protected] (K.A.); [email protected] (Y.S.); [email protected] (Z.S.); [email protected] (H.M.); [email protected] (K.A.); [email protected] (M.I.) 2 Department of Pathogenobioligy, College of Basic Medical Sciences, Jilin University, Changchun 130012, China; [email protected] * Correspondence: [email protected]; Tel.: +81-29-853-3143

Abstract: Programmed death ligand 2 (PD-L2) is the second ligand of programmed death 1 (PD-1) protein. In autoimmune myocarditis, the protective roles of PD-1 and its first ligand programmed death ligand 1 (PD-L1) have been well documented; however, the role of PD-L2 remains unknown. In this study, we report that PD-L2 deficiency exacerbates myocardial inflammation in mice with

experimental autoimmune myocarditis (EAM). EAM was established in wild-type (WT) and PD-L2- deﬁcient mice by immunization with murine cardiac myosin peptide. We found that PD-L2-deﬁcient

Citation: Li, S.; Tajiri, K.; Murakoshi, mice had more serious inflammatory infiltration in the heart and a significantly higher myocarditis + N.; Xu, D.; Yonebayashi, S.; Okabe, Y.; severity score than WT mice. PD-L2-deficient dendritic cells (DCs) enhanced CD4 T cell proliferation Yuan, Z.; Feng, D.; Inoue, K.; Aonuma, in the presence of T cell receptor and CD28 signaling. These data suggest that PD-L2 on DCs protects + K.; et al. Programmed Death-Ligand against autoreactive CD4 T cell expansion and severe inflammation in mice with EAM. 2 Deficiency Exacerbates Experimental Autoimmune Keywords: cardio-oncology; EAM; myocarditis; immune checkpoint; PD-L2; autoimmunity Myocarditis in Mice. Int. J. Mol. Sci. 2021, 22, 1426. https://doi.org/ 10.3390/ijms22031426 1. Introduction Academic Editor: Guido Iaccarino The programmed cell death protein 1 (PD-1) pathway has been shown to be an Received: 31 December 2020 attractive target for cancer immunotherapy. Immune checkpoint inhibitors targeting PD- Accepted: 28 January 2021 Published: 31 January 2021 1 or one of its ligands such as the programmed cell death ligand 1 (PD-L1) improves the prognosis of various malignancies [1,2]. However, the clinical benefit provided by

Publisher’s Note: MDPI stays neutral these treatments can be accompanied by a unique and distinct spectrum of immune- with regard to jurisdictional claims in related adverse events. Among them, myocarditis is a relatively rare, but potentially life- published maps and institutional affil- threatening cardiovascular side effect; therefore, it has become an emergent problem for iations. clinicians in their daily routines [2,3]. Given these clinical aspects, a better understanding of how disruption of this pathway influences the immune system in the heart is needed. The protective roles of PD-1 and PD-L1 in cardiac immune homeostasis have been well established [4–8]. PD-1−/− mice on a BALB/c background developed autoimmune dilated cardiomyopathy, with the production of autoantibodies against cardiac troponin Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. I[4,5]. PD-1 or PD-L1 deficiency in the autoimmune-prone MRL mice induced lymphocytic + + This article is an open access article myocarditis with massive infiltration of CD4 and CD8 T cells [6,7]. Moreover, adminis- distributed under the terms and tration of anti-PD-1 antibodies exacerbated cardiac myosin peptide-induced experimental conditions of the Creative Commons autoimmune myocarditis (EAM) [9]. However, the role of PD-L2, the second ligand of Attribution (CC BY) license (https:// PD-1, in cardiac autoimmunity remains unknown. creativecommons.org/licenses/by/ In this study, we investigated the effect of PD-L2 deficiency on the development of 4.0/). myocarditis in the EAM model. We found that PD-L2 deficiency caused severe inflamma-

Int. J. Mol. Sci. 2021, 22, 1426. https://doi.org/10.3390/ijms22031426 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 12

istration of anti-PD-1 antibodies exacerbated cardiac myosin peptide-induced experimental autoimmune myocarditis (EAM) [9]. However, the role of PD-L2, the second lig- and of PD-1, in cardiac autoimmunity remains unknown. Int. J. Mol. Sci. 2021, 22, 1426 2 of 12 In this study, we investigated the effect of PD-L2 deficiency on the development of myocarditis in the EAM model. We found that PD-L2 deficiency caused severe inflammatory infiltration in the heart. PD-L2-deficient dendritic cells (DCs) enhanced CD4+ T cell proliferationtory infiltration in the in thepresence heart. of PD-L2-deficient the T cell receptor dendritic (TCR) cells and (DCs)CD28 enhancedsignaling, CD4suggesting+ T cell thatproliferation PD-L2 on in DCs the presenceprotects agai of thenst T cardiac cell receptor antigen-reactive (TCR) and CD28CD4+ T signaling, cell expansion suggesting and severethat PD-L2 inflammation on DCs protectsin mice with against EAM. cardiac antigen-reactive CD4+ T cell expansion and severe inflammation in mice with EAM. 2. Results 2. Results 2.1. Expression Levels of PD-1 and PD-Ls in the EAM Hearts 2.1. Expression Levels of PD-1 and PD-Ls in the EAM Hearts We first examined the dynamics of inflammatory cells in EAM hearts. The heart in- We first examined the dynamics of inflammatory cells in EAM hearts. The heart flammation peaked at 14 days after the first immunization with cardiac myosin peptide inflammation peaked at 14 days after the first immunization with cardiac myosin peptide as evidenced by an increase in the number of infiltrating CD45+ leukocytes, CD11c+ den- as evidenced by an increase in the number of infiltrating CD45+ leukocytes, CD11c+ dritic cells (DCs), CD4+ T cells, CD11b+Ly6G− monocytes/macrophages, and CD11b+ Ly6G+ dendritic cells (DCs), CD4+ T cells, CD11b+Ly6G− monocytes/macrophages, and CD11b+ + neutrophilsLy6G+ neutrophils (Figure 1). (Figure The 1number). The number of infiltrating of infiltrating CD8 T CD8cells +peakedT cells on peaked day 21 on (Figure day 21 1).(Figure 1).

FigureFigure 1. 1. DynamicsDynamics of of inflammatory inflammatory cells cells in in cardiac cardiac myosin myosin peptid peptide-inducede-induced autoimmune autoimmune myocard myocarditis.itis. Quantification Quantification of of inflammatoryinflammatory cells inin thethe hearts hearts obtained obtained from from mice mice with with experimental experimental autoimmune autoimmune myocarditis, myocarditis, represented represented as a percentage as a per- centageof live cells of live at thecells indicated at the indicated time points time (npoints = 10 (n at = each 10 at time each point). time point). The values The values are expressed are expressed as the asmean the mean± standard ± standarderror errorof the of mean. the mean. * p < * 0.05P < 0.05 vs. dayvs. day 0. 0.

Next,Next, we we examined examined the the expression expression levels levels of of PD-1, PD-1, PD-L1, PD-L1, and and PD-L2 PD-L2 in in the the hearts hearts of of micemice with with EAM. EAM. The The mRNA mRNA expression expression levels levels of of PD-1, PD-1, PD-L1, PD-L1, and and PD-L2 PD-L2 were were markedly markedly upregulatedupregulated on on day day 14 14 in in the the EAM EAM hearts hearts (Figure (Figure 2A).2A). Particularly, Particularly, PD-L2 gene expression increasedincreased approximately approximately 4000-fold 4000-fold compared compared to to the the control control (Figure (Figure 22A).A). Flow cytometric + + analysesanalyses revealed revealed that that the the expression expression of PD-1, PD-L1, and PD-L2 on CD4+ TT cells, cells, CD8 + TT + cells,cells, and and CD11c CD11c+ DCsDCs peaked peaked around around 14–21 14–21 days days after after the the first ﬁrst immunization immunization (Figure (Figure 2B).2B). Thus,Thus, the the expression expression levels levels of of PD-L2, PD-L2, as as well well as as PD-1 PD-1 and and PD-L1, PD-L1, were were upregulated upregulated in in the the heartheart during during cardiac cardiac inflammation. inﬂammation.

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Figure 2. Expression levels of programmed cell death protein 1 (P (PD-1)D-1) and programmed cell de deathath ligands (PD-Ls) in the experimental autoimmune myocarditis myocarditis (EAM) (EAM) hearts. hearts. ( (AA)) mRNA mRNA expression expression of of PD-1, PD-1, PD PD-L1,-L1, and and PD-L2 PD-L2 in in the hearts obtained from mice withwith EAMEAM (days(days 1414 and and 28) 28) and and control control mice mice (n (n = = 6–7). 6–7). (B (B) Flow) Flow cytometric cytometric analysis analysis of of the the expression expression of of PD-1, PD-L1, or PD-L2 on infiltrating CD4+ T cells, CD8+ T cells, and CD11c+MHC II+ dendritic cells in the hearts obtained PD-1, PD-L1, or PD-L2 on infiltrating CD4+ T cells, CD8+ T cells, and CD11c+MHC II+ dendritic cells in the hearts obtained from mice with EAM on days 0, 7, 14, 21, and 35 after first immunization with cardiac myosin peptide (n = 6 at each time from mice with EAM on days 0, 7, 14, 21, and 35 after first immunization with cardiac myosin peptide (n = 6 at each time point). Results are presented as the mean ± standard error of the mean. *P < 0.05 vs. day 0. point). Results are presented as the mean ± standard error of the mean. * p < 0.05 vs. day 0. 2.2. PD-L2 Deficiency Exacerbates Cardiac Inflammation in EAM 2.2. PD-L2 Deficiency Exacerbates Cardiac Inflammation in EAM We then examined the functional significance of PD-L2 in EAM. BALB/c mice are highlyWe susceptible then examined to myocarditis the functional [10], and significance PD-1-deficient of PD-L2 BALB/c in EAM.mice spontaneously BALB/c mice de- are velophighly autoimmune susceptible to myocarditisdilated cardiomyopathy [10], and PD-1-deficient with the BALB/cproduction mice of spontaneously autoantibodies de- againstvelop autoimmune cardiac troponin dilated I [4,5]. cardiomyopathy On the other withhand, the C57BL/6J production mice of are autoantibodies resistant to myocar- against ditiscardiac [11], troponin and PD-1 I [4−,/5−,]. PD-L1 On the−/−, other or PD-L2 hand,−/−C57BL/6J mice on the mice C57BL/6J are resistant background to myocarditis do not [de-11], and PD-1−/−, PD-L1−/−, or PD-L2−/− mice on the C57BL/6J background do not develop spontaneous myocarditis and dilated cardiomyopathy [12]. Therefore, to investigate the ef-

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velopfect spontaneous of PD-L2 on themyocarditis development and of dilated cardiac ca myosinrdiomyopathy peptide-induced [12]. Therefore, myocarditis, to investi- we used − − − gateF1 the hybrids effect ofof PD-L2PD-L2 on/ C57BL/6Jthe developmen mice andt of BALB/ccardiac myosin mice (PD-L2 peptide-induced+/ ). myocar- − − ditis, weAs used shown F1 hybrids in Figure of PD-L23A, PD-L2−/−C57BL/6J+/ mice, mice as and well BALB/c as PD-1 mice+/ (PD-L2mice,+/ developed−). se- vereAs myocarditisshown in Figure with 3A, intense PD-L2 inflammatory+/− mice, as well infiltrates. as PD-1 In+/− mice, contrast, developed wild-type severe (WT) my- mice ocarditis(F1 hybrids with intense of WT C57BL/6Jinflammatory mice infiltrates. and BALB/c In contrast, mice) developed wild-type weak (WT) myocarditis mice (F1 hy- with bridslower of WT myocarditis C57BL/6J severity mice and scores BALB/c (Figure mice)3A,B). developed Heart-to-body weak myocarditis weight ratios with (HW/BW) lower +/− +/− myocarditisin the PD-1 severityand scores PD-L2 (Figuremice 3A,B). were Heart-to-body significantly increasedweight ratios compared (HW/BW) to WTin the mice PD-1(Figure+/−and3 C).PD-L2+/− mice were significantly increased compared to WT mice (Figure 3C).

A WT PD-1+/- PD-L2+/-

1 mm

50 μm

B 5 C 10 * * 4 8 * * 3 6

2 4 HW/BW (mg/g) HW/BW

Myocarditis Score Myocarditis 1 2

0 0 WT PD-1+/- PD-L2+/- WT PD-1+/- PD-L2+/-

Figure 3. PD-L2 deﬁciency exacerbates myocardial inﬂammation in experimental autoimmune my- Figureocarditis. 3. PD-L2 F1 hybridsdeficiency of PD-1 exacerbates−/−, PD-L2 myocardial−/−, and in wild-typeflammation (WT) in miceexperimental on the C57BL/6J autoimmune background myocarditis. F1 hybrids of PD-1−/−, PD-L2−/−, and wild-type (WT) mice on the C57BL/6J background and BALB/c mice (PD-1+/−, PD-L2+/−, and WT, respectively) were immunized twice, on days 0 and and BALB/c mice (PD-1+/−, PD-L2+/−, and WT, respectively) were immunized twice, on days 0 and 7, with cardiac myosin epitope peptide. (A) Representative H&E-stained sections of the hearts on 7, with cardiac myosin epitope peptide. (A) Representative H&E-stained sections of the hearts on µ dayday 14. 14.Scale Scale bars, bars, 1 mm 1 mmor 50 or μ 50m. (Bm.) Myocarditis (B) Myocarditis severity severity in heart in heart sections sections (n = 6–7 (n =per 6–7 group). per group). (C)( Heart-to-bodyC) Heart-to-body weight weight ratios ratios (HW/BW) (HW/BW) of the of mice the mice (n = (n10 = per 10 group). per group). Results Results are presented are presented as as the themean mean ± standard± standard error error of the of themean. mean. *P < *0.05p < 0.05vs. WT. vs. WT.

To Toanalyze analyze the the impact impact of ofPD-L2 PD-L2 deficiency deficiency on oninflammatory inflammatory cellular cellular infiltrate infiltrate in the in the EAMEAM hearts, hearts, flow flow cytometric cytometric analyses analyses were were performed performed on on day day 14 following immunization.immuniza- +/− tion.The The hearts hearts of of PD-L2 PD-L2+/− micemice with with EAM EAM showed showed significantly significantly more more inflammatory inflammatory cell cell + + + infiltration, such as an increased number of CD45+ leukocytes, CD3+ T cells, CD4+ T cells, infiltration,+ such as an increased+ + number of CD45 leukocytes,+ CD3+ T cells, CD4 T cells, CD8CD8+ T cells,T cells, CD11b CD11b+Ly6GLy6G+ neutrophils,neutrophils, and CD11b and CD11b+CD11cCD11c+ DCs, thanDCs, in than the hearts in the heartsof WT of WT mice with EAM (Figure4A,B). PD-1 +/− mice with EAM also had a significantly greater mice with EAM (Figure 4A,B). PD-1+/− mice with EAM also had a significantly greater number of infiltrating cells in the heart than WT mice with EAM (Figure4A,B). number of infiltrating cells in the heart than WT mice with EAM (Figure 4A,B).

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A 1.83 3.65 82.9

3.05 WT

15.3

9.88 1.19 68.9

+/- 3.82 PD-1

16.9

8.95 1.08 68.8

+/- 5.00 PD-L2

10.2 CD3e CD8 CD11b CD11c CD45 CD4 Ly6G CD11b B 25 * 15 2.5 * 20 * 2.0 * 10 * 15 1.5 cell (%) cell cell(%) + + T cell (%) T cell

10 + 1.0 5 CD3e CD45 CD4 5 0.5

0 0 0.0 WT PD-1+/- PD-L2+/- WT PD-1+/- PD-L2+/- WT PD-1+/- PD-L2+/-

0.3 * 10 6 * * 8 * * (%)cell 0.2 * + 4

cell (%) 6 + CD11c - T cell (%) cell T + Ly6G 4 0.1 + 2 Ly6G CD8 2 + CD11b

0.0 0 CD11b 0 WT PD-1+/- PD-L2+/- WT PD-1+/- PD-L2+/- WT PD-1+/- PD-L2+/-

Figure 4. PD-L2 deficiency increases inflammatory cell infiltration in the experimental autoimmune myocarditis (EAM) Figure 4. PD-L2 deficiency increases inflammatory cell infiltrati+on in the experimental+ autoimmune myocarditis+ (EAM)+ heart. (A) Representative flow cytometric plots showing CD45+ leukocytes, CD3e+ lymphocytes, CD8+ T cells,+ CD4 T heart. (A) Representative+ + flow cytometric plots+ showing+ CD45 leukocytes,+/ CD3e− lymphocytes,+/− CD8 T cells, CD4 T cells, CD11bcells, CD11b+Ly6G+Ly6G neutrophils,neutrophils, and CD11b and CD11b+CD11c+CD11c DCs fromDCs WT, from PD-1 WT,+/−, PD-1 and PD-L2, and+/− hearts PD-L2 on dayhearts 14 after on day EAM 14 afterinduction. EAM + + + dull Theinduction. orange Thesquares orange indicate squares CD3e indicate+CD45 CD3e+ lymphocytesCD45 lymphocytes and the green and squares the green indicate squares CD11b indicate+Ly6G CD11bdull cells.Ly6G (B) Quantifi-cells. +/− +/− cation(B) Quantification of inflammatory of inflammatory cells in the hearts cells in obtained the hearts from obtained WT mice from as WT well mice as PD-1 as well+/− and as PD-1 PD-L2+/− andmice PD-L2 with EAM.mice Results with areEAM. presented Results as are the presented mean ± SEM, as the n mean = 7–9± perSEM, group. n = * 7–9P < per0.05 group. vs. WT. * p < 0.05 vs. WT.

2.3.2.3. PD-L2 PD-L2 Deficiency Deficiency Promotes Promotes Proinflammatory Proinflammatory Cytokine Expression in the EAM Heart ToTo further evaluateevaluate the the effects effects of of PD-L2 PD-L2 deficiency deficiency on on EAM, EAM, we we examined examined cytokine cytokine and andchemokine chemokine milieu milieu in the in heart. the heart. The heartThe heart homogenates homogenates from PD-L2from PD-L2 mice showedmice showed increased in- creasedgene expression gene expression levels of levels cytokines of cytokines such as interleukinsuch as interleukin (IL)-1β, IL-6,(IL)-1 interferonβ, IL-6, interferon (IFN)-γ, (IFN)-transformingγ, transforming growth growth factor (TGF)- factorβ (TGF)-1, tumorβ1, tumor necrosis necrosis factor factor (TNF)- (TNF)-α, chemokineα, chemokine (C-C (C-Cmotif) motif) ligand ligand (CCL)1–3, (CCL)1–3, chemokine chemokine (C-X-C (C-X-C motif) motif) ligand ligand (CXCL) (CXCL) 1, 2, and1, 2, 10, and and 10, atrial and

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atrial natriuretic peptide (ANP) (Figure 5). PD-1+/− mice with EAM showed similar trends natriuretic peptide (ANP) (Figure5). PD-1 +/− mice with EAM showed similar trends as as PD-L2+/− mice, but the expression levels were relatively weak (Figure 5). PD-L2+/− mice, but the expression levels were relatively weak (Figure5).

1500 4 5000 *#† 15 *#† 4000 3 1000 10 3000 2 2000 500 5 expression (fold) expression (fold) expression expression (fold)

expression (fold) expression 1 1000 Il6 Il4 Il17 Il1b NDND ND 0 0 0 0 Ctrl EAM Ctrl EAM Ctrl EAM Ctrl EAM 1500 *#† 200 8 *# 4000 *#† *# *#† 150 6 1000 3000

100 4 2000 500 expression (fold) expression (fold) expression (fold) expression

expression (fold) 50 2 1000 Ifng Tnf Ccl1 Tgfb1 0 0 0 0 Ctrl EAM Ctrl EAM Ctrl EAM Ctrl EAM 250 *#† *#† *# 200 400 100 *# 200 *#† 80 150 300 150 60 100 200 100 40 expression (fold) expression (fold) expression (fold) expression (fold) expression 50 100 50 20 Ccl2 Cxcl2 Ccl3 Cxcl1 0 0 0 0 Ctrl EAM Ctrl EAM Ctrl EAM Ctrl EAM 2000 20 *# 1500 15 *# WT 1000 10 PD-1+/-

+/- expression (fold) 500 PD-L2 expression (fold) 5 Nppa Cxcl10 0 0 Ctrl EAM Ctrl EAM Figure 5. PD-L2PD-L2 deficiency deficiency promotes promotes proinflamma proinflammatorytory cytokine expression in the experimental autoimmune myocarditis − heart. mRNA expression ofof inflammatoryinflammatory markersmarkers in in the the hearts hearts of of wild-type wild-type (WT), (WT), PD-1 PD-1+/+/−,, andand PD-L2PD-L2+/-+/- micemice at at 14 14 days days after immunization. Results areare presentedpresented asas the the mean mean± ± standardstandard errorerror ofof the the mean. mean. n n = = 3–6, 3–6, * *pP << 0.05 vs.vs. control, ##Pp < 0.05 vs. WT, WT, and † Pp << 0.05 0.05 vs. vs. PD-1 PD-1+/+/−. −.

2.4.2.4. PD-L2 PD-L2 Deficiency Deﬁciency in in De Dendriticndritic Cells Cells Promotes Promotes CD4 CD4++ TT Cell Cell Proliferation Proliferation EAMEAM is a CD4 is a+CD4 T cell-mediated+ T cell-mediated disease, disease, and DCs and are DCs the are major the major antigen-presenting antigen-presenting cells andcells key and players key players in the in thepriming priming of appropriate of appropriate CD4 CD4+ T+ cellT cell responses responses [3]. [3]. To To assess assess the the effecteffect of of PD-L2 PD-L2 deficiency deficiency on CD4 + TT cell cell function, function, we we perfor performedmed a a co-culture co-culture experiment experiment inin vitro. vitro. First, we assessedassessedwhether whether PD-L2 PD-L2 deficiency deficiency in in DCs DCs affects affects CD4 CD4+ T+ cellT cell proliferation. proliferation.As shown As shown in Figure in Figure6A, 6A, WT WT CD4 CD4+ T+ T cells cells co-cultured co-cultured with withbone bone marrow-derivedmarrow-derived DCs (BMDCs)(BMDCs) from from PD-L2 PD-L2−/−− /mice− mice showed showed significantly significantly more more proliferation proliferation than than those those co-cul- co- turedcultured with with WT WT BMDCs. BMDCs. In addition, In addition, CD4 CD4+ T +cellsT cells co-cultured co-cultured with with PD-L2 PD-L2−/− BMDCs−/− BMDCs pro- ducedproduced significantly significantly more more IL-2 IL-2 than than WT WT BMDCs BMDCs (Figure (Figure 66B).B). OnOn the the other other hand, hand, PD-L2 PD-L2−/−−/− CD4CD4++ TT cells cells co-cultured co-cultured with WT BMDCs did notnot affectaffect CD4CD4++ T cellcell proliferationproliferationand and IL-2 IL- 2production production (Figure (Figure6C,D). 6C,D). These These results results suggest suggest that that PD-L2 PD-L2 expressed expressed in DCs in mayDCs inhibitmay in- T hibitcell proliferation, T cell proliferation, thereby thereby suppressing suppressing the development the development of autoimmune of autoimmune myocarditis. myocarditis.

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ABWT DCs + PD-L2-/- DCs + WT CD4 T cells WT CD4 T cells 100 * 200

80 * 150 T cell (%) cell T

+ 60 77.4 89.0 100 Count 40 IL-2 (pg/ml) 20 50

Proliferated CD4 0 0 CFSE WT PD-L2-/- WT PD-L2-/-

CDWT DCs + WT DCs + WT CD4 T cells PD-L2-/- CD4 T cells 100 160

80 120 T cell (%) cell T

+ 60 75.1 69.9 80

Count 40 IL-2 (pg/ml) IL-2 40 20

Proliferated CD4 0 0 CFSE WT PD-L2-/- WT PD-L2-/-

+ FigureFigure 6. PD-L2 deficiency deficiency in dendritic cells (DCs) promoted CD4 + TT cell cell proliferation. proliferation. ( (AA,B,B)) Carboxyfluorescein Carboxyfluorescein succin- succin- imidylimidyl ester (CFSE)-labeled(CFSE)-labeled WTWT CD4 CD4++ TT cellscellswere were co-cultured co-cultured with with bone bone marrow-derived marrow-derived dendritic dendritic cells cells (BMDCs) (BMDCs) generated gener- atedfrom from wild-type wild-type (WT) (WT) or PD-L2 or PD-L2−/− mice−/− mice and and stimulated stimulated with with anti-CD3 anti-CD3 and and anti-CD28 anti-CD28 for 72 for h. 72 Proliferation h. Proliferation of CD4 of +CD4T cells+ T cellswas visualizedwas visualized by observing by observing the incremental the incremental lossof loss CFSE of CF fluorescence.SE fluorescence. Representative Representative histograms histograms (gated (gated on CD4 on+ CD4T cells)+ T cells)and the and frequency the frequency of the of proliferated the proliferated CD4+ TCD4 cells+ T among cells among all CD4 all+ cellsCD4+ are cells shown. are shown. n = 3 pern = group.3 per group. IL-2 concentrations IL-2 concentra- in tionsthe culture in the supernatantsculture supernatants were assessed were assessed by ELISA. by nELISA. = 6 per n group.= 6 per Resultsgroup. areResults presented are pr asesented the mean as the± meanSEM. *± pSEM.< 0.05 *P vs. < 0.05 vs. WT. (C,D) CD4+ T cells were isolated from WT or PD-L2−/− mice and labeled with CFSE. Then, they were co- WT. (C,D) CD4+ T cells were isolated from WT or PD-L2−/− mice and labeled with CFSE. Then, they were co-cultured cultured with WT BMDCs and stimulated with anti-CD3 and anti-CD28 for 72 h. Proliferation of CD4+ T cells was visual- with WT BMDCs and stimulated with anti-CD3 and anti-CD28 for 72 h. Proliferation of CD4+ T cells was visualized by ized by observing the incremental loss of CFSE fluorescence. Representative histograms and the frequency of the prolifer- + atedobserving CD4+ theT cells incremental among all loss CD4 of+ CFSE cells fluorescence.are shown. n Representative= 3 per group. histogramsIL-2 concentrations and the frequency in the culture of the supernatants proliferated CD4were + assessedT cells among by enzyme-linked all CD4 cells immunosorbent are shown. n = 3assay. per group. n = 6 per IL-2 grou concentrationsp. Results are in presented the culture as supernatants the mean ± standard were assessed error byof theenzyme-linked mean. immunosorbent assay. n = 6 per group. Results are presented as the mean ± standard error of the mean.

3.3. Discussion Discussion ThisThis is thethe firstfirst study study to to show show that that PD-L2 PD-L2 suppresses suppresses cardiac cardiac autoimmunity. autoimmunity. Our study Our studyrevealed revealed a critical a critical role of role PD-L2 of PD-L2 in controlling in controlling autoimmune autoimmune heart heart disease. disease. PD-L2 PD-L2 was wassparsely sparsely detected detected in normal in normal hearts hearts but was bu upregulatedt was upregulated under inflammatory under inflammatory conditions. condi- PD- tions.L2 deficiency PD-L2 deficiency accelerated accelerated myocardial myocardial inflammation inflammation in cardiac myosin in cardiac peptide-induced myosin peptide- EAM. inducedPD-L2-deficient EAM. PD-L2-deficient DCs enhanced CD4DCs +enhancedT cell proliferation CD4+ T cell in proliferation the presence in of the TCR presence and CD28 of TCRsignaling. and CD28 These signaling. findings suggestThese findings that PD-L2 suggest in DCs that may PD-L2 protect in DCs against may cardiacprotect antigen-against cardiacreactive antigen-reactive CD4+ T cell expansion CD4+ T and cell severe expansion inflammation and severe in autoimmuneinflammation myocarditis. in autoimmune myocarditis.PD-L1 and PD-L2 are the two known ligands of PD-1 and they exhibit different ex- pressionPD-L1 patterns. and PD-L2 Unlike are thethe widelytwo known expressed ligands PD-L1 of PD-1 on theand surface they exhibit of many different cell types, ex- pressionPD-L2 expression patterns. Unlike was originally the widely reported expressed to be PD-L1 restricted on tothe activated surface of DCs, many macrophages, cell types, PD-L2and bone expression marrow-derived was originally mast cells reported [13]. In to the be presentrestricted study, to activated we found DCs, that macrophages, PD-L2 was ex- andpressed bone on marrow-derived heart-infiltrating mast DCs cells and its[13]. expression In the present increased study, with we inflammation found that PD-L2(Figure was2). expressedMore recently, on heart-infiltrating PD-L2 expression DCs has and been its detected expression in various increasedcancers with [14inflammation–16], resting (Fig- peri- uretoneal 2). More B1 cells recently, [17], and PD-L2 activated expression T cells has [18 ].been In our detected study, in PD-L2 various was cancers transiently [14–16], detected rest- + + ingon theperitoneal surface B1 of heart-infiltratingcells [17], and activated CD4 and T cells CD8 [18].T cells In our during study, myocardial PD-L2 was inflammation transiently detected(Figure2 ).on The the peaksurface of PD-L2of heart-infiltrating expression on CD4 inflammatory+ and CD8+ T cells cells was during delayed myocardial compared in- flammationto the peak of(Figure myocardial 2). The inflammation peak of PD-L2 (Figure expression1,2). PD-1/PD-Ls on inflammatory as immune cells was checkpoint delayed comparedmolecules downregulateto the peak of Tmyocardial cell activity infla duringmmation immune (Figure responses 1,2). [PD19].-1/PD-Ls Due to the as natureimmune of checkpointimmune checkpoints, molecules downregulate PD-L2 may play T cell an importantactivity during role in immune regulating responses immune [19]. responses Due to to prevent autoimmune heart damage.

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In the co-culture experiments, PD-L2 deficiency in DCs increased TCR- and CD28- mediated CD4+ T cell proliferation (Figure6A), which was consistent with a published report that showed PD-L2-deficient splenic antigen-presenting cells (APCs) enhanced CD4+ T cell activation [20]. In the presence of anti-PD-1 antibody, CD4+ T cells exhib- ited similar proliferation when activated by WT or PD-L2-deficient APCs [20]. Together, these results indicate that PD-L2 may negatively regulate CD4+ T cell proliferation in a PD-1-dependent manner. We observed that PD-L2 expression on CD4+ T cells was markedly upregulated on day 21 after EAM induction (Figure2). However, PD-L2 deficiency in CD4 + T cells did not affect T cell proliferation (Figure6B). In this study, we could not elucidate the role of PD-L2, expressed on CD4+ T cells, in the pathophysiology of EAM. Further investigation is needed to determine whether PD-L2 in CD4+ T cells is involved in the development and progression of autoimmune myocarditis. Myocarditis is an inflammatory disease of the myocardium that is generally self- limited. However, in several cases, prolonged inflammation eventually results in cardiomyopathy, which is a potentially lethal disorder characterized by progressively impaired cardiac function [21,22]. Myocarditis can be triggered by many different environmental agents, including viral and bacterial infections, toxins, and drugs [23,24], and subsequent autoimmune response is thought to contribute to the disease progression to cardiomyopathy [25,26]. In this study, we clearly showed the protective roles of PD-L2 in cardiac autoimmune responses. Our findings suggest that the PD-L2 pathway might serve as a novel therapeutic target in the treatment of myocarditis.

4. Materials and Methods 4.1. Mice PD-1−/− mice were kindly provided by Dr. Honjo [4], and PD-L2−/− mice were generated at the Laboratory Animal Resource Center, University of Tsukuba. BALB/c mice were purchased from CLEA, Japan. F1 hybrids of PD-1−/−or PD-L2−/− mice on the C57BL/6J background and BALB/c mice were generated (PD-1+/− and PD-L2+/− mice, respectively). Male PD-1+/− and PD-L2+/− mice and WT littermates (6–8 weeks of age) were used in the in vivo experiments. All animal experiments were approved by the Institutional Animal Experiment Committee of the University of Tsukuba (approved number: 20037, approved date: 1 June 2020) and conformed to the NIH Guide for the Care and Use of Laboratory Animals.

4.2. EAM Induction Mice were subcutaneously immunized with 100 µg of myosin heavy chain-α (MyHC- α) (MyHC-α614–629 [Ac-RSLKLMATLFSTYASADR-OH]; Toray Research Center) emulsiﬁed 1:1 in PBS/complete Freund’s adjuvant (1 mg/mL; H37Ra; Sigma–Aldrich) on days 0 and 7, as described previously [27–29]. On day 14, mice were euthanized, and their hearts were removed for further analysis.

4.3. Histopathological Examination The hearts were fixed in 4% paraformaldehyde in PBS, embedded in paraffin wax, sectioned into 3-µm thick sections, and stained with hematoxylin and eosin for histological analysis. Myocarditis severity was scored in hematoxylin and eosin-stained sections using grades from 0–4: 0, no inflammation; 1, >25% of the heart section involved; 2, 25–50%; 3, >50–75%; and 4, >75%, as described previously [27]. The analysis was performed in a blinded manner.

4.4. Flow Cytometric Analyses Heart inflammatory cells were isolated and processed as previously described [30,31]. For the flow cytometric analysis of the surface markers and cytoplasmic cytokines, the cells were stained directly using fluorochrome-conjugated mouse-specific antibodies and ana- Int. J. Mol. Sci. 2021, 22, 1426 9 of 12

lyzed using the FACSVerse instrument (BD Biosciences, San Jose, CA, USA). The following monoclonal antibodies were used for ﬂow cytometric analysis: Anti-CD279 (PD-1, TONBO Biosciences, San Diego, CA, USA), anti-CD274 (PD-L1, TONBO Biosciences, San Diego, CA, USA), anti-CD273 (PD-L2, Invitrogen, Waltham, MA, USA), anti-CD45 (TONBO Biosciences, San Diego, CA, USA), anti-CD4 (TONBO Biosciences, San Diego, CA, USA), anti-CD8 (eBioscience, San Diego, CA, USA), anti-CD11b (BioLegend, San Diego, CA, USA), anti-CD11c (TONBO Biosciences, San Diego, CA, USA), and anti-Ly6G (BD Pharmingen, San Jose, CA, USA).

4.5. RNA Extraction and Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction All hearts of mice, removed for performing reverse transcription polymerase chain reaction, were snap frozen and stored at −80 ◦C. Subsequently, the tissue was homoge- nized using the bead kit (MagNA Lyser Green Beads; Roche Diagnostics, Indianapolis, IN, USA) in accordance with the manufacturer’s instructions. Total RNA was extracted using the RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. cDNA was synthesized from 1 µg of total RNA per sample using the Omniscript RT kit (Qiagen, Hilden, Germany). A quantitative reverse transcription polymerase chain reaction was performed on the LightCycler 480 system (Roche Applied Science, Indianapolis, IN, USA) using a Universal Probe Library (Roche Ap- plied Science, Indianapolis, IN, USA). Hypoxanthine-guanine phosphoribosyltransferase (HPRT) RNA was used as an internal control. Gene expression values were calculated using the 2−∆Ct method.

4.6. T Cell Proliferation Assay Naïve CD4+ T cells were isolated from splenocytes of mice using mouse CD4 (L3T4) MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). BMDCs were generated as previously described [32]. CD4+ T cells were labeled with 32 µL of 5 mM carboxyﬂuorescein succinimidyl ester (CFSE, Dojindo, Kumamoto, Japan) in 8 mL of PBS with 0.1% BSA for 10 min at 37 ◦C. The staining reaction was stopped with 1 mL of FCS (Lonza, Basel, Switzerland). CFSE-labeled 5 × 105 CD4+ T cells were co-cultured with 105 BMDCs and stimulated with 5 µg/mL of anti-CD3 and 3 µg/mL of anti-CD28 antibodies in 96-well round-bottom plates for 72 h. Proliferation of CD4+ T cells was visualized by observing the incremental loss of CFSE ﬂuorescence using the FACSCalibur instrument (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (Tree Star, San Carlos, CA, USA).

4.7. ELISA The concentrations of IL-2 in cell culture supernatants were measured using the DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer’s instructions.

4.8. Statistics All data are expressed as the mean ± standard error of the mean (SEM). Normality was veriﬁed using the Shapiro–Wilk test. Statistical analyses were performed using the unpaired two-tailed t-test or Mann–Whitney U test for comparison between two groups. For multiple comparisons, one-way analysis of variance with the Newman–Keuls post hoc test or Kruskal–Wallis analysis with the post hoc Steel–Dwass or Steel test was performed. Results with P < 0.05 were considered as statistically signiﬁcant. All statistical analyses were performed using JMP software (SAS Institute, Cary, NC, USA).

5. Conclusions PD-L2 plays a pivotal role in suppressing cardiac autoimmunity. PD-L2 on DCs protects against autoreactive CD4+ T cell expansion and severe inﬂammation in mice with EAM. Int. J. Mol. Sci. 2021, 22, 1426 10 of 12

Author Contributions: Each author made substantial contributions to the conception and design of this work, or the acquisition, analysis, and interpretation of the data. All authors have aided in drafting the manuscript, approved to submit this version, and agree to be personally accountable for their contributions to this study. Conceptualization, S.L. and K.T.; methodology, S.L. and K.T.; validation, S.Y., Z.Y., D.F., Y.O., K.I., K.A., Y.S., Z.S., H.M., and H.H.; investigation, S.L. and K.T.; writing—original draft preparation, S.L. and K.T.; writing—review and editing, S.Y. and M.I.; vi- sualization, S.L. and K.T.; supervision, D.X., N.M., and K.A.; project administration, K.T. and M.I.; funding acquisition, K.T. and M.I. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by JSPS KAKENHI, grant number 20K08396, KANAE Foundation for the Promotion of Medical Science, the Bayer Scholarship for Cardiovascular Research, and the Takeda Science Foundation. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of University of Tsukuba (protocol code 20037 and date of approval 1 June 2020). Informed Consent Statement: Not applicable. Data Availability Statement: The data that support the findings of this study are available from the corresponding author (K.T.), upon reasonable request. Acknowledgments: The authors would like to thank Yumi Isaka and Akiko Miyamoto for their technical assistance. Conflicts of Interest: The authors declare no conflict of interests.

Abbreviations

APC antigen-presenting cell BMDC bone marrow-derived dendritic cell CCL chemokine (C-C motif) ligand CFSE carboxyﬂuorescein succinimidyl ester CXCL chemokine (C-X-C motif) ligand DC dendritic cell EAM experimental autoimmune myocarditis H&E hematoxylin and eosin IFN-γ interferon-γ IL interleukin MyHC-α myosin heavy chain-α PD-1 programmed death 1 PD-L1 programmed death ligand 1 PD-L2 programmed death ligand 2 TCR T cell receptor TGF-β transforming growth factor beta TNF tumor necrosis factor WT wild-type

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