Treatment with saturating dose of conventional anti-CD28 monoclonal antibody well tolerated in pig acute myocardial infarction model

Figures

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  Figure 1. Anti-pig CD28 mAb 3D11 blocks ligand binding. (A) Representative histograms showing pCD80-hIg binding to wt or pCD2`8-transgenic A20J cells in the absence or presence of 3D11 mAb. (B) Summary graph of MFI of pCD80-hIg under the conditions shown. (C) MFI of mouse CD80-mScarlet on porcine CD4+ T cells after trogocytosis from transgenic MEFs (4 h incubation). (D) MFI of mouse CD86 on porcine CD4+ T cells after trogocytosis from mouse splenocytes (incubation over-night). (E) Set-up as for (D). (C–E) Anti-CD3 mAb: 1 μg/ml mAb PPT-3. 3D11 and MOPC-21 mAb: 10 μg/ml. Concentrations Fab fragments: 20, 5, 1.25 μg/ml. (B–E) Means + SD of duplicates. Data representative of at least two experiments with similar results. One-way ANOVA followed by Tukey’s post-hoc test (Holm-Sidak in E). **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Figure 2. Fab fragments of 3D11 mAb inhibit T cell costimulation. CFSE-labelled porcine splenocytes were stimulated with Con A for four days in the presence or absence of Fab fragments of anti-pCD28 mAb 3D11 or 4D12 (20 μg/ml each). (A) Representative CFSE dilution profiles of gated CD4+ cells. (B) The FlowJo™ proliferation tool was used to determine division indices. Means + SD of duplicate cultures. Concentrations of Fab fragments: 20, 10, 5, 2.5 and 1.25 μg/ml. Concentration of 4D12 mAb: 10 μg/ml. #: 20 μg/ml 3D11-Fab plus 10 μg/ml 4D12 mAb. One-way ANOVA followed by Holm-Sidak test. *p <0.05, **p < 0.01, ***p < 0.001: Comparison with cells cultured with Con A only. The experiment was repeated three times with similar result.

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  Figure 3. Similar cardiac function as determined by echocardiography in pigs on day 7 after MI, irrespective of antibody treatment. mAb 3D11 was infused on day 3 after MI induction. (A) Left ventricular enddiastolic volume (LVEDV) in the apical 4-chamber view (A4C) was similar on the day of MI induction as well as on days 3 and 7 after MI without differences between both groups. (B) Left ventricular internal diastolic diameter (LVIDd) in parasternal long-axis view (PLAX) is stable over time after MI without differences between both groups. (C) No differences in cardiac output over time and between both groups. (D) Left ventricular ejection fraction (EF) by the Teichholz method in PLAX and (E) fractional shortening (FS) in PLAX decreased over time due to MI induction without differences between both groups. (F) Stroke volume (SV) by LVOT diameter and LVOT curve is stable over time after MI without differences between both groups. Repeated measures two-way ANOVA followed by Tukey’s post-hoc test. 0.05 ≤ p < 0.1 are shown as numbers. *p < 0.05.

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  Figure 4. mAb 3D11 infusion decreased CD28 expression on CD4+ T cells of blood, spleen and mediastinal lymph nodes. mAb 3D11 was infused on day 3 after MI induction. (A) Detection of mAb 3D11 on CD4+ T cells of peripheral blood on the day of MI induction as well as on days 3 and 7 post MI induction. (B) CD28 expression by peripheral-blood CD4+ T cells. Tconv. (C) Detection of mAb 3D11 on and (D) CD28 expression by CD4+ T cells of spleen and mediastinal lymph nodes (med. LN) on day 7 after MI. Repeated measures two-way ANOVA followed by Tukey’s post-hoc test. 0.05 ≤ p < 0.1 are shown as numbers. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Figure 5. Elevated numbers of Treg in the border zone after mAb 3D11 infusion. Flow cytometry of cardiac CD4+ T cells did not show overt changes after mAb 3D11 infusion. (A) Proportion of CD4+ T cells amongst cardiac leukocytes. (B) Frequencies of Treg amongst cardiac CD4+ T cells. (C) Proportion of Ki-67+ cells amongst cardiac Treg and (D) Tconv. Tissue sections of infarcted hearts were stained for CD4 and Foxp3 expression as indicated. (E) Overview showing the infarct core, the border zone and the remote myocardium seven days after MI. (F) Absolute numbers Treg and (G) Tconv per field of view (715 μm x 530 μm) and (H) %Treg of CD4+ T cells. Up to six fields of view were analysed. 3D11: Conventional anti-CD28 mAb 1 mg/kg bw; MOPC-21: control mAb. Stats: A-D: Repeated measures two-way ANOVA followed by Tukey’s post-hoc test. F-H: Comparison values from different areas: Two-tailed paired Wilcoxon test; MOPC versus 3D11: Two-tailed unpaired Wilcoxon test - all not significant. *p < 0.05.