Potential of mesenchymal- and cardiac progenitor cells for therapeutic targeting of B-cells and antibody responses in end-stage heart failure

Upon myocardial damage, the release of cardiac proteins induces a strong antibody-mediated immune response, which can lead to adverse cardiac remodeling and eventually heart failure (HF). Stem cell therapy using mesenchymal stromal cells (MSCs) or cardiomyocyte progenitor cells (CPCs) previously showed beneficial effects on cardiac function despite low engraftment in the heart. Paracrine mediators are likely of great importance, where, for example, MSC-derived extracellular vesicles (EVs) also show immunosuppressive properties in vitro. However, the limited capacity of MSCs to differentiate into cardiac cells and the sufficient scaling of MSC-derived EVs remain a challenge to clinical translation. Therefore, we investigated the immunosuppressive actions of endogenous CPCs and CPC-derived EVs on antibody production in vitro, using both healthy controls and end-stage HF patients. Both MSCs and CPCs strongly inhibit lymphocyte proliferation and antibody production in vitro. Furthermore, CPC-derived EVs significantly lowered the levels of IgG1, IgG4, and IgM, especially when administered for longer duration. In line with previous findings, plasma cells of end-stage HF patients showed high production of IgG3, which can be inhibited by MSCs in vitro. MSCs and CPCs inhibit in vitro antibody production of both healthy and end-stage HF-derived immune cells. CPC-derived paracrine factors, such as EVs, show similar effects, but do not provide the complete immunosuppressive capacity of CPCs. The strongest immunosuppressive effects were observed using MSCs, suggesting that MSCs might be the best candidates for therapeutic targeting of B-cell responses in HF.

Introduction Cardiovascular disease (CVD) is the most common cause of death globally with almost 18 million deaths per year [1]. A prominent CVD-subtype is ischemic heart disease (IHD), which is characterized by myocardial cell death due to prolonged ischemia [2]. After subsequent reperfusion strategies, further myocardial damage is initiated by the release of cardiac proteins, which can induce an inflammatory response [3,4]. Activated T-and B lymphocytes significantly contribute to adverse cardiac remodeling via the production of pro-inflammatory cytokines and antibodies [5][6][7], which can progress to severe heart failure (HF) [6,[8][9][10].
Currently, progenitor cell therapy is gaining a lot of interest in order to regenerate the damaged heart due to their regenerative properties and the ability to differentiate into other cell types [11][12][13]. Mesenchymal stromal cells (MSCs) improve cardiac function by reducing scar size and increasing left ventricular ejection fraction (LVEF) with 2-4% [14,15]. However, engraftment of these cells in the heart is relatively poor, where less than 10% of the injected cells remain at the site of injection [16,17]. In addition, the few remaining cells rarely differentiate into cardiac cells [18]. In addition to their regenerative capacity, MSCs have also been shown to suppress inflammatory responses, antibody production, and fibrosis, mostly in a paracrine manner [19,20]. Important paracrine mediators are extracellular vesicles (EVs), small lipid bi-layered vesicles containing lipids, small RNAs and proteins, which are able to influence many processes including inflammation [21,22]. Multiple studies investigated the therapeutic potency of MSCs and MSC derived EVs in cardiovascular disease [13,23,24]. MSC-derived EVs were found to reduce infarct size and infiltration of immune cells into the affected myocardium after myocardial infarction (MI) in animal models [25]. These findings suggest that the use of MSC-derived EVs might be a promising strategy to restore cardiac function, however, technical difficulties in large scale production and purification of MSC-EV are still limiting the translation to the clinic [19,26]. Considering the developmental origin of endogenous cardiac-derived progenitor cells (CPCs), these cells might prove better candidates for cell therapy for cardiac repair. Endogenous CPCs were previously tested in several clinical trials where they improved cardiac function [12,27], especially when combined with MSCs [28,29]. CPCs also have immunosuppressive properties, for example by inhibiting T-cell proliferation, which is partly mediated by paracrine factors [30]. CPC-derived EVs are proposed to be of great importance as paracrine mediators of these cells [31][32][33]. However, the immunosuppressive capacity of CPCs or CPC-derived EVs on B cells and antibody-mediated immune responses has not been elucidated yet. Therefore, we investigated the in vitro inhibitory actions of CPCs and CPC-derived EVs on lymphocyte proliferation and the production of immunoglobulin subclasses, using immune cells from healthy controls and end-stage HF patients.

Isolation of CPC-derived extracellular vesicles and Western blotting
CPC-derived EVs were isolated using size-exclusion chromatography (SEC), as previously described [36]. In brief, fetal-derived CPCs were cultured until they reached a confluency of 80-90%, after which the medium was replaced with serum-free medium (M-199, Gibco 31150-022).

Isolation of peripheral mononuclear cells
Peripheral blood mononuclear cells (PBMCs) were isolated from fresh whole blood samples of healthy controls or end-stage HF patients, in compliance with the declaration of Helsinki and under approval of the Medical Ethics Committee Utrecht (METC, reference number 12/387). Written informed consent for collection and biobanking of blood samples was obtained. Endstage HF derived PBMCs were obtained from blood samples prior to heart transplantation. PBMCs were isolated using Ficoll-plaque PLUS gradient (GE life sciences, 17-1440-03) according to the manufacturers protocol. A total of 2,5x10 5 PBMCs were added per well (48-wells plate) in RPMI-1640 medium (Lonza, BE12-702F) supplemented with 10% fetal bovine serum and 1% PenStrep.

Lymphocyte proliferation
Flow cytometry (Gallios, Beckmann Coulter) was used to assess lymphocyte proliferation. Prior to co-culture, PBMCs were labeled with 1.5mM carboxyfluorescein succinimidyl ester (CFSE, Sigma, 21888) as described previously [33]. In brief, PBMCs were incubated with CFSE for 10 min at 37˚C in a dark shaking bath. After 10 minutes, 5% of FBS was used to prevent further uptake. After two washing steps with PBS, PBMCs were incubated for 30 min with fluorescent antibodies, including CD3 for T cells (Brilliant Violet 510, Biolegend, 317332) and CD19 for B cells (PE/Cy5, Biolegend 302210). After washing with PBS, PBMCs were incubated for 30 min with a fixable viability dye (eFluor506, Bioscience, 65-0866-14) to exclude dead cells. Prior to culture, general cell composition per donor was assessed by measuring the percentage of CD3+ T cells and CD19+ B cells, to ensure that the cell populations were similar between the different donors at baseline. Lymphocyte proliferation was calculated by measuring CFSE intensity and the number of cells present in each division as described before [33]. Since we encountered some donor variations in the absolute number of proliferating cells in the stimulation assays, the stimulated PBMC condition was considered as maximum response and defined as 100% proliferation (ratio = 1) and used for normalization of the data per donor and per experiment. Data was analyzed using Kaluza Analysis Software (Beckman Coulter, version 1.3).

Immunoglobulin multiplex
The levels of IgM and IgG subclasses (IgG1, IgG2, IgG3, IgG4) in the co-cultures (5x diluted) were measured using a Bio-Plex Pro™ human isotyping immunoassay 6-plex (Bio-Rad, 171A3100M) according to manufacturer's instructions and were all within the detection limit of the assay. Immunoglobulin levels in the supernatant after co-culture with MSC/CPC or CPC-derived EVs were calculated using internal standards included in the assay. Immunoglobulin levels are represented as relative production, with the stimulated PBMC condition defined as 100% antibody production (ratio = 1) and used for normalization of the data per donor and experiment.

Statistics
Statistical analysis and data representation were performed using IBM SPSS Statistics 21 and Graphpad Prism (GraphPad Software Inc. version 8.01, San Diego CA, USA). Normal data distribution was tested using the Kolmogorov-Smirnov test. Group comparison was performed by a one-way ANOVA or Kruskal-Wallis test, corrected for multiple comparison testing. Each individual PBMC donor is considered as an independent individual experimental number (n), ranging from 2-8 donors per experiment. Data was considered significant with two-tailed p-values <0.05 and is presented as mean ± SEM.

Progenitor cells suppress lymphocyte proliferation upon cell-cell contact
To investigate the immunosuppressive effects of progenitor cells on the proliferation of lymphocytes, a co-culture using MSCs or CPCs was performed (Fig 1). To represent normal lymphocyte activation by antigen-presenting cells, the total PBMC population was used. After 10 days of co-culture, large clusters of proliferating T cells were visible upon stimulation with IL-2 and PMA. These large clusters were smaller or even absent when PBMCs were cultured in the presence of MSCs or CPCs (Fig 1A). Flow cytometry was used to measure CFSE intensity and to assess lymphocyte proliferation (Fig 1B and 1C). FACS plots clearly showed active cell proliferation upon stimulation with IL-2 and PMA and suppression of proliferation when PBMCs were cultured with MSCs or CPCs. Quantification showed that both MSCs and CPCs significantly decreased proliferation of lymphocytes by 64±18.6% and 19±12.5% respectively (MSC p<0.0001, CPC p<0.05).

Production of IgM and different IgG subclasses is suppressed by cardiacderived progenitor cells
Next to reduced cell proliferation, MSCs are also able to inhibit several immune cell functions, such as antibody secretion [38]. To examine whether this also holds true for CPCs, we collected the supernatant after 10 days of co-culture and measured the levels of different immunoglobulin subclasses (Fig 2A). Since it is known that the age of the donor can affect their inhibitory potency [39,40], both fetal and adult MSCs and CPCs were included. Adult and fetal-derived MSCs significantly inhibited antibody production from stimulated PBMCs (Fig  2B-2F). Fetal and adult MSC significantly reduced the production of IgM (aMSC = 0.005±0.0 fMSC = 0.02±0.0; p<0.0001), IgG1 (aMSC = 0.24±0.06, fMSC = 0.28±0.06; p<0.0001), IgG3

CPC-derived extracellular vesicles suppress antibody production, but are not as effective as direct cell-cell interaction when using CPCs
To explore whether the suppressive capacity of CPC on antibody production is mediated by paracrine factors, we assessed the potential of CPC-derived EVs (Fig 3). We experienced that it is technically challenging to obtain sufficient MSC-derived EVs using SEC. Therefore, we only included CPC-derived EVs in our co-cultures. Prior to co-culture, EVs were characterized based on size distribution and the presence or absence of protein markers [37]. Isolated EVs showed a representative size distribution profile with the highest peak at approximately 90 nm (Fig 3A). In line with previous findings [36], WB analysis showed that CPC-derived EVs were enriched for the typical EV proteins Alix, CD81, and CD63. Calnexin was only detectable in the cell lysate, thereby confirming the absence of contaminations with other membrane compartments (Fig 3B). An amount of 1x10 μg or 3x10 μg (every 3 days of co-culture) was added to the PBMC cultures (Fig 3C). After 10 days of co-culture, antibody secretion was significantly suppressed by EVs (Fig 3D). The production of IgM, IgG1, and IgG4 was significantly decreased using the 3x dose of CPC-derived EVs (IgM = 0.35±0.05; p<0.05, IgG1 = 0.57±0.03; p<0.05, and IgG4 = 0.66±0.0; p = 0.03), thereby indicating that long term suppression is more effective than a single dose of EVs. However, the inhibitory effect was most robust when adding CPCs and not CPC-derived EVs, with strongest suppressive effects on the release of IgG1 (0.59±0.1; p<0.05), IgG2 (0.23±0.06; p = 0.02), IgG4 (0.53±0.03; p = 0.01) and IgM (0.17±0.03; p<0.01).

Discussion
The post-MI immune response is an important contributor to adverse cardiac remodeling and the development of HF [41][42][43][44]. The release of cardiac proteins upon MI can trigger antibodymediated immune responses, which further induce cardiac damage and heart failure [45][46][47]. Stem cell therapy using progenitor cells, such as MSCs or CPCs, showed promising reparative effects on cardiac function despite poor engraftment in the myocardium [17,48]. This indicates that paracrine mediators, secreted by progenitor cells, can be of great importance. MSCs and MSC-derived EVs also have immunosuppressive properties, for example by lowering antibody production in vitro [49,50]. However, the immunosuppressive capacity of endogenous CPCs and CPC-derived EVs on B cells and antibody production has not been elucidated yet. Consequently in this study, we investigated the immunosuppressive effects of CPCs and CPCderived paracrine mediators on antibody production using immune cells of both healthy controls and end-stage HF patients.
In line with previous findings, we showed that both MSCs and CPCs significantly suppressed proliferation of lymphocytes [30,32,38]. The suppressive effects of MSCs were more effective than CPCs. The suppressive effects of MSCs and CPCs on effector and regulatory T cells have been described before, where several studies show T cell inhibition via PDL-1/PD1 in a direct cell communication manner [32,51]. Moreover, both MSCs and CPCs are also able to suppress CD4+ T helper cell-mediated immune responses [52]. However, the interaction of progenitor cells with B cells is still controversial and this issue has recently gained more interest [53][54][55]. MSCs can inhibit plasma cell formation and subsequent IgG production in a cell-cell contact dependent as well as in an independent manner [38,55]. It is not known whether CPCs are also able to suppress antibody production in vitro. We demonstrated that, similar to MSCs, CPCs effectively suppress antibody production in vitro. We showed that both adult-and fetal-derived CPCs significantly inhibit the levels of IgM, IgG1, and IgG3, of which IgM was most efficiently suppressed, despite variation between different donors. These findings are in line with the effects of MSC, where MSC are known to exert an inhibitory effect on T helper cells, B-cell differentiation and class switching into IgG-producing cells [56,57]. Therefore, we could speculate that CPC might use a similar mechanism, in which IgG production might be suppressed either by inhibiting T-helper cell responses, thereby influencing B-cell activation and antibody production, or by directly influencing B-cell differentiation and subsequent class-switching.
To facilitate clinical translation, we examined if the strong immunosuppressive effects of CPCs and MSCs on antibody production using healthy donors, can be confirmed for IgG production using HF patient-derived PBMCs. MSCs were able to significantly inhibit the production of IgM and all IgG subclasses. For CPCs, the immunosuppressive effects were not as potent compared to MSCs, where CPCs only significantly lowered the production of IgM and IgG2. In end-stage HF, chronically activated immune cells progressively worsen cardiac function, for example by the production of cardiac antibodies [58,59]. Our findings indicate that progenitor cells, preferably MSCs, might be used as therapeutic agents to suppress antibodymediated immune responses as observed in end-stage HF. However, mimicking the physiological immune response in vitro, as observed in end-stage HF patients, is still complicated. Therefore, these findings still have to be validated in vivo.
Part of the immunosuppressive properties of MSCs is mediated by paracrine factors, such as EVs [19,21]. The advantage of using EVs is that they can be used as a cell-free approach, thereby increasing safety, and allowing a longer duration of the treatment [19,26]. However, high variability in quantity and quality in the scaling and production process of MSC-derived EVs has been a limitation [26]. CPC-derived EVs might provide a promising alternative, not only due to their regenerative and immune modulating capacities [60], but also for their culture scalability. CPC-derived EVs have immunosuppressive effects on T cells [30,60], however, the effects on B cells and antibody-mediated responses is not clear. Our findings showed that CPC-derived EVs lower the different immunoglobulin isotypes and subclasses, such as IgM, IgG1, and IgG4. However, the number of EVs needed to reach similar suppressive effects compared to CPCs, remains challenging. In this study, we were only able to test EVs produced by fetal CPCs due to technical difficulties in obtaining sufficient numbers of EVs from adult CPCs. Fetal-derived progenitor cells might exert different effects than adult-derived cells, where, for example, adult-derived MSCs show stronger immunosuppressive capacities relative to fetal-derived MSCs [61]. For CPCs, it has been described that fetal-and adult-derived CPCs have different developmental potentials, and adult CPCs may be more effective in cardiac repair [62,63]. In addition, fetal-derived CPCs are highly proliferative as compared to adultderived cells. Due to this proliferative state CPCs may secrete a different palette of paracrine factors that are more associated to cell cycle rather than immunomodulation. Therefore, the effects of EVs from adult CPCs may differ from fetal-derived CPCs and have to be investigated in future studies. Nonetheless, from our data, it is clear that EVs can be used as immunosuppressive mediators, but do completely cover the strong immunosuppressive effect of CPCs.
In conclusion, we demonstrated immunosuppressive actions of both MSCs and CPCs on lymphocyte proliferation and antibody production, with strongest effects observed when using MSCs. These are partly mediated by EVs, in a time-dependent matter. Lastly, we showed that CPCs and especially MSCs were able to suppress antibody production by patient-derived cells, thereby indicating the therapeutic potential of progenitor cells in HF. Currently, cell therapy using MSCs is no longer the holy grail for true cardiac regeneration and cell replacement therapy, however, MSCs might be promising candidates for targeting the post-MI immune response and HF progression. Future studies should focus on the identification of the cardiac antigens which are targeted by the produced IgGs and on the potential of combination therapies, using both MSCs and CPCs, to simultaneously target cardiac regeneration and antibodymediated immune responses.
Supporting information S1 Fig. Extracellular