CD100 modulates cytotoxicity of CD8+ T cells in patients with acute myocardial infarction

Abstract

Background: CD100 is an immune semaphorin family member that highly expressed on T cells, which take part in the development of acute myocardial infarction (AMI). Matrix metalloproteinases (MMPs) are important mediators for membrane-bound CD100 (mCD100) shedding from T cells to generate soluble CD100 (sCD100), which has immunoregulatory effect on T cells. The aim of this study was to investigate modulatory role of CD100 on CD8⁺ T cell activity in AMI patients.

Methods: Peripheral sCD100 and MMP-2 level, as well as mCD100 level on T cells was assessed in patients with stable angina pectoris (SAP), unstable angina pectoris (UAP), and AMI. The regulatory function of MMP-2 on mCD100 shedding, sCD100 formation, and cytotoxicity of CD8⁺ T cells was analyzed in direct and indirect contact co-culture system.

Results: AMI patients had higher peripheral sCD100 and lower mCD100 expression on CD8⁺ T cells in comparison with SAP, UAP, and controls. CD8⁺ T cells in AMI patients showed elevated direct cytotoxicity, enhanced cytokine production, and increased perforin/granzyme B secretion. Recombinant sCD100 stimulation promoted cytolytic function of CD8⁺ T cells in controls and AMI patients. Furthermore, AMI patients also had elevated circulating MMP-2 level. Recombinant MMP-2 stimulation induced mCD100 shedding from CD8⁺ T cells and sCD100 generation, resulting in enhancement of CD8⁺ T cell cytotoxicity in AMI patients.

Conclusion: Up-regulation of MMP-2 might contribute to elevation of mCD100 shedding and sCD100 formation, leading to increased cytotoxicity CD8⁺ T cells in AMI patients.

Keywords: Acute myocardial infarction; CD100; Immunoregulation; T lymphocytes.

Fig. 1

Comparison of soluble CD100 (sCD100) and matrix metalloproteinase-2 (MMP-2) level in the plasma among control group (n = 20), stable angina pectoris (SAP) group (n = 22), unstable angina pectoris (UAP) group (n = 20), and acute myocardial infarction (AMI) group (n = 23). Plasma sCD100 and MMP-2 was measured by ELISA. a Plasma sCD100 level was increased in AMI group than in control, SAP, and UAP group. b Plasma MMP-2 level was increased in AMI group than in control, SAP, and UAP group. Individual level for each subject was shown. The horizon lines indicated means, while the error bars indicated standard deviations. One-way ANOVA and SNK-q test was used for statistical analysis

Fig. 2

Comparison of membrane bound CD100 (mCD100) expression in CD4⁺ T cells and CD8⁺ T cells in the plasma among control group (n = 20), stable angina pectoris (SAP) group (n = 22), unstable angina pectoris (UAP) group (n = 20), and acute myocardial infarction (AMI) group (n = 23). a PBMCs were stained by anti-CD3, CD4, CD8, and CD100, and the representative flow dots and histograms of CD100 expression in CD4⁺ T cells and in CD8⁺ T cells in control, SAP, UAP, and AMI group were shown. The isotype control for CD100 positive and negative cells was also shown. b Comparison of CD100⁺CD4⁺ cells percentage among control, SAP, UAP, and AMI group. There was no significant difference of CD100⁺CD4⁺ cells percentage among control, SAP, UAP, and AMI group. c Comparison of CD100 mean fluorescence intensity (MFI) in CD4⁺ T cells among control, SAP, UAP, and AMI group. There was no significant difference of CD100 MFI in CD4⁺ T cells among control, SAP, UAP, and AMI group. d Comparison of CD100⁺CD8⁺ cells percentage among control, SAP, UAP, and AMI group. CD100⁺CD8⁺ cells percentage was reduced in AMI group compared with control, SAP, and UAP group. e Comparison of CD100 MFI in CD8⁺ T cells among control, SAP, UAP, and AMI group. CD100 MFI in CD8⁺ T cells was down-regulated in AMI group compared with control, SAP, and UAP group. Individual level for each subject was shown. The horizon lines indicated means, while the error bars indicated standard deviations. One-way ANOVA and SNK-q test was used for statistical analysis

Fig. 3

Influence of recombinant human CD100 stimulation to cytokine production and perforin/granzyme B secretion by CD8⁺ T cells from controls (n = 10) and acute myocardial infarction (AMI) patients (n = 10). CD8⁺ T cells were stimulated with recombinant human CD100 for 24 h. Cells and supernatants were then harvested. a IFN-γ and b TNF-α level in the supernatants was measured by ELISA, and was compared between controls and AMI patients, and between presence and absence of CD100 stimulation. c Perforin and d granzyme B production by CD8⁺ T cells was measured by ELISPOT. The number of spot-forming cells (SFC) was compared between controls and AMI patients, and between presence and absence of CD100 stimulation. Individual level for each subject was shown. The horizon lines indicated means, while the error bars indicated standard deviations. One-way ANOVA and SNK-q test was used for statistical analysis

Fig. 4

Influence of recombinant human CD100 stimulation to CD8⁺ T cells cytotoxicity from HLA-A02 restricted controls (n = 8) and acute myocardial infarction (AMI) patients (n = 10). CD8⁺ T cells were stimulated with recombinant human sCD100 for 24 h. Cells were washed twice, and 10⁴ of stimulated CD8⁺ T cells were co-cultured with 5 × 10⁴ of HUVECs in direct contact and indirect contact co-culture system. Supernatants were harvested 48 h post co-culture. a LDH level in the cultured supernatants were measured using LDH Cytotoxicity Assay Kit. The percentage of HUVECs death was calculated, and was compared between controls and AMI patients, and between presence and absence of CD100 stimulation. b IFN-γ and c TNF-α level in the supernatants was assessed by ELISA, and was compared between controls and AMI patients, and between presence and absence of CD100 stimulation. Individual level for each subject was shown. The horizon lines indicated means, while the error bars indicated standard deviations. One-way ANOVA and SNK-q test was used for statistical analysis

Fig. 5

Influence of recombinant human matrix metalloproteinase-2 (MMP-2) stimulation to CD100 expression in acute myocardial infarction (AMI) patients (n = 10). CD8⁺ T cells were stimulated with recombinant human MMP-2 for 24 h. sCD100 level in the supernatants was measured by ELISA, and mCD100 expression in CD8⁺ T cells was investigated by flow cytometry. a sCD100 level in the supernatants was increased in response to MMP-2 stimulation. b The percentage of CD100⁺ within CD8⁺ T cells was reduced in response to MMP-2 stimulation. c CD100 mean fluorescence intensity (MFI) in CD8⁺ T cells was down-regulated in response to MMP-2 stimulation. Individual level for each subject was shown. The horizon lines indicated means, while the error bars indicated standard deviations. Student t test was used for statistical analysis

Fig. 6

Influence of recombinant human matrix metalloproteinase-2 (MMP-2) stimulation to CD8⁺ T cells cytotoxicity from HLA-A02 restricted acute myocardial infarction (AMI) patients (n = 9). 10⁴ of CD8⁺ T cells were stimulated with recombinant human MMP-2 in the presence or absence of anti-CD100 neutralization antibody, and were co-cultured with 5 × 10⁴ HUVEC in a direct contact manner. LDH level in the cultured supernatants were measured using LDH Cytotoxicity Assay Kit. The percentage of HUVECs death was calculated, and was compared among groups. Individual level for each subject was shown. The horizon lines indicated means, while the error bars indicated standard deviations. One-way ANOVA and SNK-q test was used for statistical analysis