Combined Adaptive Immune Mechanisms Mediate Cardiac Injury After COVID-19 Vaccination

Abstract

Background: The COVID-19 pandemic, caused by SARS-CoV-2, has led to the first approval of mRNA vaccines in humans. By producing the full-length SARS-CoV-2 Spike protein, they induce protective antiviral immunity. Acute myopericarditis (AMP) development after vaccination has repeatedly been reported; however, the pathogenesis of this complication remains elusive.

Methods: In-depth phenotyping of peripheral blood T cells was undertaken in cohorts of patients who developed AMP after mRNA vaccination, patients hospitalized for severe COVID-19, and healthy subjects with no cardiac side effects after mRNA vaccine. Validation studies were carried out using an experimental model of cardiac inflammation, in which a shared epitope elicits functional responses in patients and mice and induces AMP.

Results: We show that T cells from patients with AMP recognize vaccine-encoded Spike epitopes homologous to those of cardiac self-proteins. One of these epitopes, mimicking an amino acid sequence from a cardiomyocyte-expressed K⁺ channel, induced AMP in mice. When functional responses to the Kv2 were analyzed, patients with AMP after mRNA vaccination, but not patients with COVID-19, displayed an expanded pattern of cytokine production similar to that observed in AMP mice and in autoimmune myocarditis. Crucially, T-cell autoimmunity segregates to cardiotropic cMet (c-mesenchymal epithelial transition factor)-expressing T cells and is prevented by cMet inhibition, suggesting that heart homing imprinting, permitted by the unique mRNA vaccine biodistribution, is required for AMP development.

Conclusions: AMP development after mRNA vaccines is mediated by distinct immune components, including molecular mimicry, T-cell receptor affinity, and, importantly, homing imprinting.

Keywords: COVID-19; T-lymphocytes; autoimmunity; vaccination.

Figure 1.

Immunization with the mRNA-1273 vaccine induces acute myopericarditis in susceptible mice. Five-week-old male Balb/cAnN mice received 0.25 ng/g of body weight mRNA-1273 vaccine in saline solution intramuscularly in the forelimb (COVID vaccine intramuscular) and were boosted with the same dose 14 days later. Control mice (Con) received saline solution. The development of inflammatory infiltrates and collagen deposition were assessed by hematoxylin-eosin (A) and Masson trichrome staining (B) of the heart 21 days after the first immunization. Column graphs show lymphocyte infiltration and disease scores (A) obtained as described in the Methods. Representative images at 10× and 40× magnification are shown on the right (scale bars=2.5 mm and 50 µm) Statistical analysis was performed with unpaired Student t test. C, Right ventricle (RV) wall thickness, RV diameter, left ventricle ejection fraction (EF), fractional shortening (FS), and diameter at systole and diastole were measured by echocardiography at baseline (day 0) and 21 days after the first immunization. Statistical analysis was performed with 2-way mixed-effects ANOVA followed by Tukey multiple comparison test. D, Tail vein blood was sampled on the same mouse at the indicated time points. The percentage of the indicated T-cell populations was determined by flow cytometry. Statistical analysis was performed with 2-way mixed-effects ANOVA followed by Tukey multiple comparison test. E, Immunofluorescence staining with the indicated markers was performed on heart tissue harvested on day 21. The bar graph shows the mean percentage of cMet (c-mesenchymal epithelial transition factor)⁺ and cMet⁻ T cells measured by QuPath. Statistical analysis was performed with 2-way ANOVA followed by Tukey multiple comparison test. F and G, percentage of cMet⁺ T cells measured in single-cell suspensions from whole hearts and heart-draining lymph nodes. Statistical analysis was performed with unpaired Student t test. All data are shown as median±interquartile range. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001 (N=3, control n=3, mRNA-1273 vaccine intramuscularly n=10).

Figure 2.

Response of T cells from mRNA-1273-immunized mice to Spike epitopes. T cells from mRNA-1273-immunized mice (day 21) were labeled with CFSE and rechallenged with the indicated peptides. Cells were harvested and analyzed 5 days later for CFSE dilution and cMet c-mesenchymal epithelial transition factor expression. The mean division index is shown in A. B through E, Production of the indicated cytokines by cMet⁺ and cMet⁻ T cells was measured 6 hours after restimulation with the indicated antigens by intracellular staining and flow cytometry. Statistical analysis was performed with 2-way ANOVA, followed by Tukey multiple comparison test. All data are shown as median±interquartile range. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001 (N=3, control n=3, PS5 n=10, P87 n=10, P177 n=10).

Figure 3.

Proliferation assays and cMet⁺ and cMet⁻ T-cell cytokine profiles after peptide stimulation. PBMCs isolated from human blood were labeled with Tag-it-Violet and stimulated with 20 µg/mL of scrambled peptide; MHCα6 partial protein; P87h, PS5h, and P177h peptides; and tetanus toxoid (TT; 10 μg/mL) for 7 days. Proliferation assays show the proliferation index values of CD4⁺ cMet (c-mesenchymal epithelial transition factor)+, and cMet⁻ cells from peripheral blood mononuclear cells of vaccinated healthy controls (Vacc-HCs; A, n=21), patients with post-mRNA vaccination AMP (Vacc-AMP; B, n=11), and patients with COVID-19 (C, n=14). Statistical analysis was performed with Kruskal-Wallis test, followed by Dunn multiple comparisons test. The frequency of peripheral blood cMet⁺ and cMet− T cells producing TNFα (tumor necrosis factor α)⁻ (D), IFN-γ (interferon γ)⁻ (E), IL-4 (interleukin-4; F), IL-17⁻ (G), and IL-22⁻ (H) from the cohorts were analyzed after stimulation with the indicated peptides by intracellular staining and flow cytometry. Statistical analysis was performed with repeated-measures 1-way ANOVA with Dunnett multiple comparisons. All data are shown as median±interquartile range. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 4.

Immunization with the spike peptide PS5 induces acute myopericarditis in susceptible mice. Five-week-old male Balb/cAnN male mice were immunized with PS5 (0.6 mg/kg) in AS03 plus MPLA adjuvant intranasally and boosted with the same dose 14 days later. Control mice received adjuvant alone. The development of inflammatory infiltrates and collagen deposition were assessed by hematoxylin-eosin (A) and Masson trichrome staining (B) of the heart 21 days after the first immunization. Column graphs show lymphocyte infiltration and disease scores (A) obtained as described in Methods. Representative images at 10× and 40× magnification are shown on the right (scale bar=2.5 mm and 50 µm, respectively). Statistical analysis was performed with unpaired Student t test. C, Immunofluorescence staining with the indicated markers was performed on heart tissue harvested on day 21. The bar graph shows the mean percentage of cMet (c-mesenchymal epithelial transition factor)⁺ and cMet⁻ T cells measured by QuPath. Statistical analysis was performed with 2-way ANOVA, followed by Tukey multiple comparisons test. D, Right ventricle (RV) wall thickness, RV diameter, left ventricle ejection fraction (EF), fractional shortening (FS), and diameter at systole and diastole were measured by echocardiography at baseline (day 0) and 21 days after the first immunization. Statistical analysis was performed with 2-way mixed-effects ANOVA, followed by Tukey multiple comparisons test. E, Tail vein blood was sampled on the same mouse at the indicated time points. The percentage of the indicated T-cell populations was determined by flow cytometry. Statistical analysis was performed with 2-way mixed-effects ANOVA, followed by Tukey multiple comparison test. F through I, Percentage of cMet⁺ T cells measured in single-cell suspensions from whole hearts and heart-draining and nondraining lymph nodes. Statistical analysis was performed with unpaired Student t test. J, T cells from PS5-immunized mice (day 21) were labeled with CFSE and rechallenged with the indicated peptides. Cells were harvested and analyzed 5 days later for CFSE dilution and cMet expression. The mean division index is shown. Statistical analysis was performed with 2-way ANOVA, followed by Tukey multiple comparisons test. K through M, Production of the indicated cytokines by cMet⁺ and cMet⁻ T-cells was measured 6 hours after restimulation with the indicated antigens by intracellular staining and flow cytometry. Statistical analysis was performed with 2-way ANOVA, followed by Tukey multiple comparison test. All data are represented as median±interquartile range. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001 (N=3, control n=3, PS5 n=10, P87 n=10, P177 n=10).

Figure 5.

Patients with VACC-AMP and PS5-immunized mice display functional T-cell responses to the homologous Kv2 peptide. PBMCs isolated from human blood were labeled with Tag-it-Violet and stimulated with 200 ng/mL scrambled and autologous Kv2.1h (human) peptides for 7 days. A, Mean proliferation index of CD4⁺ cMet (c-mesenchymal epithelial transition factor)⁺ T cells from peripheral blood mononuclear cells (PBMCs) of vaccinated healthy controls (Vacc-HC; n=8), patients with post-mRNA vaccination AMP (Vacc-AMP; n=9), and patients with COVID-19 (n=10) patients measured by flow cytometry. Statistical analysis was performed with paired Student t test. B, The frequency of cytokine-producing peripheral blood cMet⁺ T cells among peripheral blood cMet⁺ T cells from the cohorts were analyzed after stimulation with the indicated peptides, using intracellular staining and flow cytometry. Statistical analysis was performed with paired Student t test. B, Frequencies of cytokine-producing cMet⁺ T cells in response to the scrambled peptide were subtracted from those of cMet⁺ T cells stimulated with Kv2.1 peptide in a paired manner. Statistical analysis was performed with paired Student t test. C, To compare the size of cytokine-producing T cell subsets in patients with Vacc-AMP (n=9) and COVID-19 (n=10), frequencies of cytokine producers in response to scrambled peptide were subtracted in a paired manner from those in response to the Kv2.1h peptide. Statistical analysis was performed with unpaired Student t test. D, Five-week-old Balb/cAnN male mice were immunized with PS5 (0.6 mg/kg) in AS03 plus MPLA adjuvant intranasally and boosted with the same dose 14 days later. Control mice received adjuvant alone. T cells from PS5-immunized mice (day 21) were labeled with CFSE and rechallenged with the homologous Kv2.1m (murine) peptide. Cells were harvested and analyzed 5 days later for CFSE dilution and cMet expression by flow cytometry. The mean division index of cMet⁻ and cMet⁺ T cells from controls and immunized mice is shown. Statistical analysis was performed with 2-way ANOVA, followed by Tukey multiple comparison test. E, Production of the indicated cytokines by cMet⁺ and cMet⁻ T-cells was measured 6 hours after restimulation with the Kv2.1m peptide by intracellular staining and flow cytometry. Statistical analysis was performed with 2-way ANOVA, followed by Tukey multiple comparison test. Data are shown as median±interquartile range (A through C) or median±interquartile range (D). *P<0.05, **P<0.01, ***P<0.005, ****P<0.001 (N=3, control n=5, PS5 n=5).

Figure 6.

Pharmacological cMet inhibition substantially reduces the severity of PS5-induced myopericarditis in mice. Five-week-old Balb/cAnN mice were immunized with PS5 (0.6 mg/kg) in AS03 plus MPLA adjuvant intranasally and boosted with the same dose 14 days later. Control mice received adjuvant alone. Some mice received intraperitoneal injections of the cMet (c-mesenchymal epithelial transition factor) inhibitor PHA-665752 (9.6 mg/kg, PS5+INH) from day 8 to day 17 after immunization. The development of inflammatory infiltrates and collagen deposition were assessed by hematoxylin-eosin (A) and Masson trichrome staining (B) of the heart 21 days after the first immunization. Column graphs show lymphocyte infiltration and disease scores (A) obtained as described in Methods. Representative images at 10× and 40× magnification are shown on the right (scale bar=2.5 mm). Statistical analysis was performed with 1-way ANOVA, followed by Dunnett multiple comparison. C, Immunofluorescence staining with the indicated markers was performed on heart tissue harvested on day 21. The bar graph shows the mean percentage of cMet⁺ and cMet⁻ T cells measured by QuPath. Statistical analysis was performed with 2-way ANOVA, followed by Tukey’s multiple comparisons test. D, Right ventricle (RV) wall thickness, RV diameter, left ventricle ejection fraction (EF), fractional shortening (FS), and diameter at systole and diastole were measured by echocardiography at baseline (day 0) and 21 days after the first immunization. Statistical analysis was performed with 2-way mixed-effects ANOVA, followed by Tukey multiple comparison test. E, Tail vein blood was sampled on the same mouse at the indicated time points. The percentage of the indicated T cell populations was determined by flow cytometry. Statistical analysis was performed with 2-way mixed-effects ANOVA, followed by Tukey multiple comparison test. F through I, Percentage of cMet⁺ T cells measured in single-cell suspensions from whole hearts, heart-draining lymph nodes, spleen, and nondraining lymph nodes. Statistical analysis was performed with 1-way ANOVA, followed by Dunnett multiple comparison. All data are shown as median±interquartile range. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001 (N=3, control n=6, PS5 n=5, PS5+INH n=10).

Figure 7.

Pharmacological cMet inhibition reduces functional responses by cMet⁺ T cells in PS5-immunized mice. T cells from PS5-immunized mice (day 21) were labeled with CFSE and rechallenged with the indicated peptides. Cells were harvested and analyzed 5 days later for CFSE dilution and cMet (c-mesenchymal epithelial transition factor) expression. A, Mean division index of cMet⁻ and cMet⁺ T cells from controls and immunized mice with and without cMet inhibitor. Statistical analysis was performed with 2-way ANOVA, followed by Tukey multiple comparison test. B through E, Production of the indicated cytokines by cMet⁺ and cMet⁻ T cells was measured 6 hours after restimulation with the indicated antigens by intracellular staining and flow cytometry. Statistical analysis was performed with 2-way ANOVA, followed by Tukey multiple comparison test. All data are shown as median±interquartile range. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001 (N=3, control n=6, PS5 n=5, PS5+INH n=10).