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

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 mice had more serious inflammatory infiltration in the heart and a significantly higher myocarditis severity score than WT mice. PD-L2-deficient dendritic cells (DCs) enhanced CD4⁺ T cell proliferation in the presence of T cell receptor and CD28 signaling. These data suggest that PD-L2 on DCs protects against autoreactive CD4⁺ T cell expansion and severe inflammation in mice with EAM.

Keywords: EAM; PD-L2; autoimmunity; cardio-oncology; immune checkpoint; myocarditis.

Figure 1

Dynamics of inflammatory cells in cardiac myosin peptide-induced autoimmune myocarditis. Quantification of inflammatory cells in the hearts obtained from mice with experimental autoimmune myocarditis, represented as a percentage of live cells at the indicated time points (n = 10 at each time point). The values are expressed as the mean ± standard error of the mean. * p < 0.05 vs. day 0.

Figure 2

Expression levels of programmed cell death protein 1 (PD-1) and programmed cell death ligands (PD-Ls) in the experimental autoimmune myocarditis (EAM) hearts. (A) mRNA expression of PD-1, PD-L1, and PD-L2 in the hearts obtained from mice with EAM (days 14 and 28) and control mice (n = 6–7). (B) Flow cytometric analysis of the expression 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 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.

Figure 3

PD-L2 deficiency exacerbates myocardial inflammation in experimental autoimmune 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 7, with cardiac myosin epitope peptide. (A) Representative H&E-stained sections of the hearts on day 14. Scale bars, 1 mm or 50 μm. (B) Myocarditis severity in heart sections (n = 6–7 per group). (C) Heart-to-body weight ratios (HW/BW) of the mice (n = 10 per group). Results are presented as the mean ± standard error of the mean. * p < 0.05 vs. WT.

Figure 4

PD-L2 deficiency increases inflammatory cell infiltration in the experimental autoimmune myocarditis (EAM) heart. (A) Representative flow cytometric plots showing CD45⁺ leukocytes, CD3e⁺ lymphocytes, CD8⁺ T cells, CD4⁺ T cells, CD11b⁺Ly6G⁺ neutrophils, and CD11b⁺CD11c⁺ DCs from WT, PD-1^+/−, and PD-L2^+/− hearts on day 14 after EAM induction. The orange squares indicate CD3e⁺CD45⁺ lymphocytes and the green squares indicate CD11b⁺Ly6G^dull cells. (B) Quantification of inflammatory cells in the hearts obtained from WT mice as well as PD-1^+/− and PD-L2^+/− mice with EAM. Results are presented as the mean ± SEM, n = 7–9 per group. * p < 0.05 vs. WT.

Figure 5

PD-L2 deficiency promotes proinflammatory cytokine expression in the experimental autoimmune myocarditis heart. mRNA expression of inflammatory markers in the hearts of wild-type (WT), PD-1^+/−, and PD-L2^+/- mice at 14 days after immunization. Results are presented as the mean ± standard error of the mean. n = 3–6, * p < 0.05 vs. control, # p < 0.05 vs. WT, and † p < 0.05 vs. PD-1^+/−.

Figure 6

PD-L2 deficiency in dendritic cells (DCs) promoted CD4⁺ T cell proliferation. (A,B) Carboxyfluorescein succinimidyl ester (CFSE)-labeled WT CD4⁺ T cells were co-cultured with bone marrow-derived dendritic cells (BMDCs) generated from wild-type (WT) or PD-L2^−/− mice and stimulated with anti-CD3 and anti-CD28 for 72 h. Proliferation of CD4⁺ T cells was visualized by observing the incremental loss of CFSE fluorescence. Representative histograms (gated on CD4⁺ T cells) and the frequency of the proliferated CD4⁺ T cells among all CD4⁺ cells are shown. n = 3 per group. IL-2 concentrations in the culture supernatants were assessed by ELISA. n = 6 per group. Results are presented as the mean ± SEM. * p < 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-cultured with WT BMDCs and stimulated with anti-CD3 and anti-CD28 for 72 h. Proliferation of CD4⁺ T cells was visualized by observing the incremental loss of CFSE fluorescence. Representative histograms and the frequency of the proliferated CD4⁺ T cells among all CD4⁺ cells are shown. n = 3 per group. IL-2 concentrations in the culture supernatants were assessed by enzyme-linked immunosorbent assay. n = 6 per group. Results are presented as the mean ± standard error of the mean.