Immunological Properties of Murine Parthenogenetic Stem Cell-Derived Cardiomyocytes and Engineered Heart Muscle

Abstract

Pluripotent parthenogenetic stem cells (pSCs) can be derived by pharmacological activation of unfertilized oocytes. Homozygosity of the major histocompatibility complex (MHC) in pSCs makes them an attractive cell source for applications in allogeneic tissue repair. This was recently demonstrated for pSC-based tissue-engineered heart repair. A detailed analysis of immunological properties of pSC-derived cardiomyocytes and engineered heart muscle (EHM) thereof is, however, lacking. The aim of this study was to determine baseline and cytokine-inducible MHC class I and MHC class II as well as programmed death ligand-1 (PDL-1) and co-stimulatory protein (CD40, CD80, CD86) expression in pSC-derived cardiomyocytes and pSC-EHM in vitro and in vivo. Cardiomyocytes from an MHC-homologous (H2d/d) pSC-line were enriched to ~90% by making use of a recently developed cardiomyocyte-specific genetic selection protocol. MHC class I and MHC class II expression in cardiomyocytes could only be observed after stimulation with interferon gamma (IFN-γ). PDL-1 was markedly upregulated under IFN-γ. CD40, CD80, and CD86 were expressed at low levels and not upregulated by IFN-γ. EHM constructed from H2d/d cardiomyocytes expressed similarly low levels of MHC class I, MHC class II, and costimulatory molecules under basal conditions. However, in EHM only MHC class I, but not MHC class II, molecules were upregulated after IFN-γ-stimulation. We next employed a cocultivation system with MHC-matched and MHC-mismatched splenocytes and T-cells to analyze the immune stimulatory properties of EHMs. Despite MHC-mismatched conditions, EHM did not induce splenocyte or T-cell proliferation in vitro. To evaluate the immunogenicity of pSC-derived cardiomyocytes in vivo, we implanted pSC-derived embryoid bodies after elimination of non-cardiomyocytes (cardiac bodies) under the kidney capsules of MHC-matched and -mismatched mice. Spontaneous beating of cardiac bodies could be observed for 28 days in the matched and for 7 days in the mismatched conditions. Teratomas formed after 28 days only in the MHC-matched conditions. Immunohistochemistry revealed single clusters of CD3-positive cells in the border zone of the implant in the mismatched conditions with few CD3-positive cells infiltrating the implant. Taken together, MHC-matched pSC-cardiomyocyte allografts show little immune cell activation, offering an explanation for the observed long-term retention of pSC-EHM allografts in the absence of immunosuppression.

CD8+-T Cells With Specificity for a Model Antigen in Cardiomyocytes Can Become Activated After Transverse Aortic Constriction but Do Not Accelerate Progression to Heart Failure

“…In comparison to CD4 + -T cells ( 12 ), CD8 + -T cells with specificity for a model antigen in cardiomyocytes have little impact on the progression of pressure overload-induced heart failure. This observation might be understandable in view of reports that cardiomyocytes largely lack MHC class I molecules under non-inflammatory conditions ( 18 , 27 ) even though they are inducible by pro-inflammatory cytokines ( 18 ) and CTL-mediated killing of cardiomyocytes clearly occurs during viral myocarditis ( 13 ). A low expression level of MHC class I molecules on cardiomyocytes could explain why the OVA-specific CD8 + -T cells had little impact in the cMy-mOVA+OT-I mice after TAC although they reached the OVA-expressing tissue as suggested by the presence of higher numbers of CD3 + -T cells in the myocardium of these mice.…”

“…Collagen was visualized by Sirius Red staining to measure the extent of fibrosis as described previously ( 12 , 17 ). Presence of immune cells in the myocardium was determined by immunohistochemistry ( 12 , 18 ) using anti-CD3 (1:200, MCA1477, rat IgG 1 , ABD Serotec, Oxford, UK), anti-CD4 (1:200, clone 4SM95, rat IgG 1 , eBiosciences, Frankfurt, Germany), anti-CD8 (1:200, clone 4SM15, rat IgG 2a , eBiosciences), anti-CD45R(B220) (1:200, clone RA3-6B2, rat IgG 2a , Biolegend, Fell, Germany), and anti-F4/80 monoclonal antibodies (1:200, clone A3-1, rat IgG 2b , Biolegend), respectively. For all antibodies except anti-F4/80, antigen retrieval was performed by boiling the slides 5 times for 5 min in sodium citrate buffer (10 mmol/L sodium citrate, pH 6, 0.05% Tween 20).…”

T helper cells with specificity for an antigen in cardiomyocytes promote pressure overload-induced progression from hypertrophy to heart failure

“…The Th 1 and Th 17 -polarized T helper cells might interact with macrophages and other antigen presenting cells in the myocardium, which have taken-up OVA released from dying cardiomyocytes as these normally do not express MHC class II molecules even under inflammatory conditions 40 . T helper cells might act either directly via secretion of cytokines on cardiomyocytes and other cells of the myocardium such as cardiac fibroblasts or indirectly via an activation of other immune cells such as macrophages.…”

“…In addition to the total amount of the fibrotic area in the left ventricle, perivascular fibrosis was determined defined as fibrotic area directly associated with vessels of a diameter greater than 100 µm 54 . Infiltration of the myocardium by immune cells was determined by immunohistochemistry as described elsewhere 40 using anti-CD3 (1:200, MCA1477, rat IgG 1 , ABD Serotec, Oxford, UK), anti-CD45R/B220 (1:200, RA3-6B2, rat IgG 2a , Biolegend, Fell, Germany), anti-FOXP3 (1:100, with 0.5% Triton-X100, FJK-16s, rat IgG 2a , eBiosciences, Frankfurt, Germany), and anti-F4/80 monoclonal antibodies (1:200, A3-1, rat IgG 2b , Biolegend), respectively. For CD3 and CD45R/B220 staining, antigen retrieval was performed by boiling the slides 5 times for 5 min in sodium citrate buffer (10 mmol/L sodium citrate, pH 6, 0.05% Tween 20).…”

Identifying and managing the adverse effects of immune checkpoint blockade

“…Studies suggest that the checkpoint pathway is crucial in preventing inflammation within the milieu of the cardiac microenvironment. PD-L1 is expressed in both human and murine heart cells, and expression can be influenced by immune signaling molecules such as IFN-γ (36). Expression of PD-1 and PD-L1 may help regulate the immune microenvironment and suppress overactivation of T cells within the heart.…”