Enhanced Immunomodulation in Inflammatory Environments Favors Human Cardiac Mesenchymal Stromal-Like Cells for Allogeneic Cell Therapies

Abstract

Rising numbers of patients with cardiovascular diseases and limited availability of donor hearts require new and improved therapy strategies. Human atrial appendage-derived cells (hAACs) are promising candidates for an allogeneic cell-based treatment. In this study, we evaluated their inductive and modulatory capacity regarding immune responses and underlying key mechanisms in vitro. For this, cryopreserved hAACs were either cultured in the presence of interferon-gamma (IFNγ) or left unstimulated. The expression of characteristic mesenchymal stromal cell markers (CD29, CD44, CD73, CD105, CD166) was revealed by flow cytometry that also highlighted a predominant negativity for CD90. A low immunogeneic phenotype in an inflammatory milieu was shown by lacking expression of co-stimulatory molecules and upregulation of the inhibitory ligands PD-L1 and PD-L2, despite de novo expression of HLA-DR. Co-cultures of hAACs with allogeneic peripheral blood mononuclear cells, proved their low immunogeneic state by absence of induced T cell proliferation and activation. Additionally, elevated levels of IL-1β, IL-33, and IL-10 were detectable in those cell culture supernatants. Furthermore, the immunomodulatory potential of hAACs was assessed in co-cultures with αCD3/αCD28-activated peripheral blood mononuclear cells. Here, a strong inhibition of T cell proliferation and reduction of pro-inflammatory cytokines (IFNγ, TNFα, TNFβ, IL-17A, IL-2) were observable after pre-stimulation of hAACs with IFNγ. Transwell experiments confirmed that mostly soluble factors are responsible for these suppressive effects. We were able to identify indolamin-2,3-dioxygenase (IDO) as a potential key player through a genome-wide gene expression analysis and could demonstrate its involvement in the observed immunological responses. While the application of blocking antibodies against both PD-1 ligands did not affect the immunomodulation by hAACs, 1-methyl-L-tryptophan as specific inhibitor of IDO was able to restore proliferation and to lower apoptosis of T cells. In conclusion, hAACs represent a cardiac-derived mesenchymal stromal-like cell type with a high potential for the application in an allogeneic setting, since they do not trigger T cell responses and even increase their immunomodulatory potential in inflammatory environments.

Keywords: IDO; IFNγ; cardiac-derived cells; immunogenicity; immunomodulation; inflammation.

Figure 1

Characteristics of human atrial appendage-derived cells (hAAC). (A) The general procedure to generate hAACs is shown: right atrial appendages were harvested from patients during open-heart surgery. The dissected tissue was minced into small pieces and cultured. Subsequently, the fragment‘s outgrowth was harvested, immuno-magnetically sorted as CD90^low cells and seeded again for further expansion of the desired purified hAAC product. The cells were cryopreserved and long-term stored for later experimental use. (B) Representative immunofluorescence images show the characteristic morphology of one hAAC donor in a cell culture plate after 12 h of incubation. Cell membranes (orange) were stained with wheat germ agglutinin (WGA) and nuclei (blue) with 4,6-diamidin-2-phenylindol (DAPI). Scale bar represents 50 μm. (C) Generated hAACs were harvested by treatment with trypsin after passage four and stained with human-specific antibodies against surface markers characteristic for cells of mesenchymal origin (CD29, CD44, CD73, CD105, CD166), markers for exclusion of hematopoietic contaminants (CD14, CD31, CD34, CD45) and the cardiac progenitor marker c-Kit (CD117). Representative flow cytometry histograms of one hAAC donor are shown as fluorescence intensity against cell counts for all tested markers, indicating the mean of fluorescence intensity (MFI) of positive cells (blue) compared to the unlabeled controls (gray) for each marker. (D) The percentage of remaining CD90⁺ cells in the hAAC cell product is shown for one representative donor in a dot plot of CD90 against CD29.

Figure 2

Immune phenotype of cryopreserved hAACs under constitutive and inflammatory conditions. (A) Representative histogram overlays for one hAAC donor display the expression pattern for immunologically-relevant surface markers (HLA-ABC, HLA-E, HLA-DR, CD80, CD86, PD-L1, PD-L2). Cells were cultured for 48 h without additional stimulation (hAAC; light red line) or in presence of 100 ng/mL human interferon-gamma (IFNγ hAAC; dark red line). After harvest by application of trypsin, cells were stained with human-specific antibodies and analyzed by flow cytometry. Fluorescence intensity of marker expression is presented compared to the unlabeled control (Control; filled gray curve). (B) Summarized hAAC surface marker expression data are presented as normalized mean of fluorescence intensities (MFI), that are calculated based on the respective controls (set to one; dashed black line), and shown as mean + SEM (n = 6; three independent experiments with six different hAAC donors). Differences between unstimulated hAACs and IFNγ hAACs were considered significant when ^**p ≤ 0.01 with the Mann-Whitney t-test.

Figure 3

hAACs maintain a low-immunogeneic profile even in an inflammatory environment. (A) The experimental setup for analyzing hAACs immunogenicity in immune cell co-cultures is illustrated: mesenchymal stromal cells from the umbilical cord (MSCs) as well as human umbilical vein endothelial cells (HUVECs) served, respectively, as cellular controls [MSC (-); HUVEC (+)] and were cultured along with hAACs for 48 h in the presence or absence of 100 ng/mL IFNγ. The cells were gamma-irradiated with 60 Gray before carboxyfluorescein succinimidyl ester (CFSE)-labeled, human leukocyte antigen (HLA)-mismatched peripheral blood mononuclear cells (PBMCs) were either added to the adherent cell cultures of all three cell types or left alone as control. After 4 days of incubation, supernatants were taken for cytokine detection using the Legendplex™ human inflammation panel and after 7 days PBMCs were harvested, stained with human immune cell specific antibodies and analyzed flow cytometrically. Levels of CD4⁺ and CD8⁺ T cell proliferation were detected by determining reduced CFSE signal intensity (black square). (B) Representative flow cytometry plots of proliferated CD4⁺ and CD8⁺ T cells are shown for all PBMC co-culture groups and PBMCs only (Control). (C) Summarized proliferation data for CD4⁺ and CD8⁺ T cells are presented as mean ± SEM (n = 6; three independent experiments with six different hAAC donor). Groups were considered significantly different compared to the Control when ^*p ≤ 0.05; ^**p ≤ 0.01; ^***p ≤ 0.001 with Kruskal Wallis ANOVA and Dunn's post-test. (D) Measured cytokine levels in [pg/mL] for TNFα, IL-10, IL-1β, and IL-33 are shown as mean ± SEM (n = 6). Cytokine levels of the PBMC control groups are depicted as dotted gray line. Groups were considered significantly different when ^*p ≤ 0.05; ^**p ≤ 0.01; ^***p ≤ 0.001 with Kruskal Wallis ANOVA and Dunn's post-test. Differences between treatments were considered significant when #p ≤ 0.05; ##p ≤ 0.01 with ordinary two-way ANOVA and Sidak's post-test.

Figure 4

IFNγ pre-stimulation enhances the immune-modulatory capacity of hAACs. CFSE-labeled PBMCs were activated with anti-CD3/anti-CD28 antibodies (+αCD3/αCD28) and cultured alone (Control) or in the presence of unstimulated or IFNγ-stimulated hAACs, HUVECs or MSCs for 72 h. Cells were harvested, stained with human-specific antibodies and analyzed by flow cytometry for T cell proliferation, based on reduced CFSE signal intensity. Percentages of CD4⁺ or CD8⁺ proliferated cells for all experimental groups are shown as representative dot plots (A) and as summarized data with mean ± SEM (B) (n = 6–9; four independent experiments with seven different hAAC donors). (C) Experimental settings were repeated with hAACs under transwell culture conditions to evaluate a contact-dependent mode of action in the observed immune-modulatory effects (n = 7; three independent experiments with six different hAAC donors). (D) Supernatants of the direct immune cell co-cultures with either unstimulated or IFNγ-treated hAACs were analyzed with a Luminex bead kit for their content of IFNγ, TNFα, TNFβ, MDC, IL-1β, IL-2, IL-10, and IL-17A. Summarized data for cytokine concentrations [pg/mL] are presented as mean ± SEM (n = 6; three independent experiments with six different hAAC donors). Groups were considered significantly different when ^*p ≤ 0.05; ^**p ≤ 0.01; ^***p ≤ 0.001 with Kruskal Wallis ANOVA and Dunn's post-test.

Figure 5

Whole genome expression analysis and quantitative verification of immunomodulatory gene expression of unstimulated and IFNγ pre-stimulated hAACs. (A) Microarray expression data of unstimulated and IFNγ-stimulated hAACs were normalized and log2-transformed. Thousand probe sets with the highest variances across all samples were selected and used in a principle component analysis, showing PC1 vs. PC2 for n = 3 hAAC donors (#1, #4, #5). (B) Differentially expressed probe sets were determined by fitting linear models to the data and apply Bayesian statistics. P-values were adjusted for multiple testing using false discovery rate. Probe sets with an adjusted p-value below.05 and a minimal absolute log2-foldchange of 1 are shown in the heatmap. The blue column bar indicates the unstimulated and the red bar the IFNγ pre-stimulated hAACs of donors #1, #4, #5. (C) Differentially expressed probe sets were used in an overrepresentation analysis utilizing the gene ontology system. The eight top-ranking results of the category biological process are shown. (D) The differentially expressed immunomodulatory genes IDO1, LGALS9, TLR3, PD-L1, PD-L2, HLA-G, PTGS1 and VCAM1, identified in the global microarray analysis, were validated by qPCR. Values of IFNγ pre-stimulated hAACs were normalized to the unstimulated samples (set to 1; black dotted line) by means of a ΔΔCt analysis and upregulation is shown as mean of relative expression ± SEM for n = 3 hAAC donors (#1, #4, #5).

Figure 6

Inhibition of T cell proliferation by hAACs is indoleamine-2,3-dioxygenase (IDO)-dependent and involves apoptosis. (A) The experimental setup to evaluate the potential involvement of IDO and programmed death-1 (PD-1) ligands in hAAC effects on T cells is shown schematically: hAACs were seeded and stimulated with IFNγ or left unstimulated for 48 h. Before co-cultivation with immune cells, 1-mehtyl-L-tryptophan (1-MT) as specific inhibitor of IDO or blocking antibodies against PD-L1 and PD-L2 (αPD1 ligands) were applied. Subsequently, either CFSE-labeled or unlabeled PBMCs were activated by adding a cocktail of anti-CD3/anti-CD28 (+αCD3/αCD28) antibodies and left alone as controls or cultured with hAACs for 72 h. CD4⁺ or CD8⁺ T cell proliferation levels as well as the percentage of Annexin-V⁺ (Av+) 7-AAD⁺ late apoptotic cells were determined by flow cytometry. (B) The normalized proliferation values for CD4⁺ or CD8⁺ T cell subsets were calculated based on the respective PBMC controls and are presented as mean ± SEM (n = 6–10; four independent experiments with seven different hAAC donors). (C) The normalized percentages of late-apoptotic Annexin-V⁺ (Av⁺) 7-AAD⁺ cells within the CD4⁺ and CD8⁺ T cell subsets were calculated based on the respective PBMC controls and are presented as mean ± SEM (n = 6; two independent experiments with six different hAAC donors). Unstimulated (hAAC) and IFNγ-stimulated hAACs (IFNγ hAAC) within the same treatment were considered significantly different when ^*p ≤ 0.05; ^**p ≤ 0.01 with the Mann-Whitney t-test. Differences between treatments were considered significant when #p ≤ 0.05; ##p ≤ 0.01 with Kruskal Wallis ANOVA and Dunn's post-test.

Figure 7

Potential crosstalk between hAACs and T cells in an inflammatory milieu. The scheme illustrates the impact of IFNγ stimulation on CD90^low hAACs and its implication on the interplay with αCD3/αCD28 activated human T cells. Stimulation of hAACs alone induces upregulation of HLA-class I (HLA-ABC, HLA-E) and de novo expression of HLA-class II molecules (HLA-DR). Even though expression of the inhibitory co-stimulatory molecules PD-L1 and PD-L2 is enhanced on the cell surface, no specific effects in the interaction with activated T cells were detectable. Similarly, the role of the specific secretion of IL-1 family cytokines (IL-1β, IL-33) by hAACs and additional unknown mediators like exosomes remain unclear. However, a global genome-wide expression profile analysis revealed IDO as one of the strongest expressed genes in IFNγ stimulated hAACs. Specific blocking by 1-MT proved the essential involvement of IDO in the interaction with T cells. It mediates the suppression of CD4⁺ and CD8⁺ T cell proliferation, reduces the secretion of pro-inflammatory cytokines (IFNγ, TNFα, TNFβ, IL-17A) and induces apoptosis of both T cell subsets. We hypothesize, that this mode of hAAC interaction might contribute to the resolution of immune responses and limit thereby the effects of adverse remodeling after cardiac injury.