Pluripotent Stem Cell-Derived Hepatocytes Inhibit T Cell Proliferation In Vitro through Tryptophan Starvation

Regenerative medicine aims to replace damaged tissues by stimulating endogenous tissue repair or by transplanting autologous or allogeneic cells. Due to their capacity to produce unlimited numbers of cells of a given cell type, pluripotent stem cells, whether of embryonic origin or induced via the reprogramming of somatic cells, are of considerable therapeutic interest in the regenerative medicine field. However, regardless of the cell type, host immune responses present a barrier to success. The aim of this study was to investigate in vitro the immunological properties of human pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs). These cells expressed MHC class I molecules while they lacked MHC class II and co-stimulatory molecules, such as CD80 and CD86. Following stimulation with IFN-γ, HLCs upregulated CD40, PD-L1 and MHC class I molecules. When co-cultured with allogeneic T cells, HLCs did not induce T cell proliferation; furthermore, when T cells were stimulated via αCD3/CD28 beads, HLCs inhibited their proliferation via IDO1 and tryptophan deprivation. These results demonstrate that PSC-derived HLCs possess immunoregulatory functions, at least in vitro.


Introduction
Transplantation is the treatment of choice for many end-stage organs, such as the liver. However, organ rejection and long-term immunosuppression present a real challenge, and new strategies are needed [1]. One alternative strategy is the transplantation of allogeneic adult hepatocytes, which has been pioneered to reduce the severity of congenital errors of liver metabolism and chronic/acute failure to act as a bridge until the organ becomes available [2]. However, in both animals and humans, sustained liver function following hepatocytes' transplantation has not been achieved [3]. Failure of cell engraftment, activation of the recipient's immune response and the lack of a preconditioned environment play important parts in graft failure [4]. Hepatocytes from renewable and immune matched sources, such as pluripotent stem cells (PSCs), have been proposed as a concrete alternative to primary human hepatocytes to support organ function and regeneration.
Functional hepatocyte-like cells (HLCs) can be produced at scale from both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) [5]. While autologous/syngeneic stem cell-derived products may lack immunogenicity once transplanted in vivo, stem cells generated from genetically different individuals are very likely to lead to the activation of the recipient's immune response [6][7][8][9].
A pioneering study by Guha et al. tested the immunogenicity of terminally differentiated murine hepatocyte cells derived from ESCs and iPSCs in vitro and in vivo [10]. During co-cultures, a lack of T cell response was observed toward undifferentiated syngeneic iPSCs or their differentiated progenies. Consistent with the in vitro results, a lack of T cell infiltration was also observed following transplantation into syngeneic mice. More recently, Cisneros et al. showed that murine HLCs lacking MHC class I molecule expression were targeted in vitro by both syngeneic and allogeneic NK cells [11]. Lastly, in our previous work, human 3D liver tissue derived from ESCs provided critical liver support in two xenotransplantation models, including immunocompetent recipients, without being rejected [12].
To date, the immunogenicity of human HLCs remains poorly understood. Considering that autologous cell therapy would not be commercially viable, understanding the immunogenicity of allogeneic stem cell derived products is crucial for the development of future cellular therapies. Here, we investigated the phenotype of iPSC-HLCs and their potential to induce allogeneic T cell response in vitro.
Where indicated, HLCs as well as adult human hepatocytes have been treated with recombinant human IFN-γ (1000 U/mL, R&D Systems, Minneapolis, MN, USA) for 72 h.

T Cell Isolation and Proliferation
Anonymised healthy donor peripheral blood was obtained from the NHS Blood and Transplant Centre with informed consent and ethical approval (Institutional Review Board of Guy's Hospital; reference 09/H0707/86). CD4 + or CD8 + T cells were enriched from ten different blood donors by using the RosetteSep™ Human CD4 + T Cell Enrichment Cocktail and the RosetteSep™ Human CD8 + T Cell Enrichment Cocktail, respectively (STEMCELL Technologies, Vancouver, BC, Canada).

Flow Cytometry
The phenotypic analysis of HLCs was executed by using the following antibodies: Flow cytometry was performed on LSR Fortessa (BD Biosciences, BD biosciencies, Franklin Lakes, NJ, USA) and analysed with FlowJo software (v10.6.1 Tree Star).

ELISA
Human serum albumin enzyme-linked immunosorbent assay (ELISA) was performed as previously described [16]. Human IL-10 (DY217B) and IFN-γ (DY285B) ELISAs (R&D Systems, Minneapolis, MN, USA) were used to evaluate the concentration of the two cytokines in the supernatant of the co-culture assays.

Statistical Evaluation
Results are expressed as mean ± SD. Comparisons between groups were performed using t-test for two groups or one-way ANOVA with a Dunnett or Bonferroni post-test for more than two groups. Analyses were performed using GraphPad Prism 8 software.

Characterising iPSC-HLC Function and Immune Modulatory Molecule Expression
In order to understand the immunogenicity of iPSC-HLCs, we first evaluated whether specific markers of hepatocellular differentiation were maintained on HCLs in a proinflammatory microenvironment. We selected HNF4a, as it is a transcription factor essential for hepatocyte specification [17], and SERPINA, which is an alpha-1-antitrypsin enzyme produced specifically by hepatocytes [18]. At baseline, HNF4a and SERPINA gene expression in HLCs were similar to adult hepatocytes ( Figure 1A,B), while undifferentiated iPSCs were negative for the expression of these two genes ( Figure 1A,B). When HLCs were treated with IFN-γ, the gene expression of HNF4a and SERPINA was not modified. Similarly, albumin production remained unchanged following IFN-γ treatment in both HLCs and adult hepatocytes ( Figure 1C,D). In addition, IFN-γ treatment did not affect the polyhedral shape of HLCs ( Figure 1E). Taken together, these data suggest that HLCs' differentiation state, function and morphology were not altered following IFN-γ treatment. Due to the link with the gut via the portal vein, the liver is constantly exposed to gut-derived bacterial products. The detection of pathogen-associated molecular patterns (PAMPs) is mediated by the pattern recognition receptors (PRRs), including the Toll-like receptors (TLRs) that play a crucial role in maintaining hepatic immune homeostasis. Dysregulation in TLR signalling can lead to the production of proinflammatory cytokines and interferons favouring chronic liver diseases and fibrosis [19]. As human-cultured hepatocytes express the mRNA of all the TLRs at low levels [20], we aim to understand whether HLCs show the same expression pattern. HLCs express lower levels of TLR-2, -4, -5, -7 and -9 compared to adult hepatocytes, as shown in Supplementary Figure S1. We then evaluated the expression level of molecules involved in the activation/inhibition of cells contributing to adaptive immunity, namely CD4 + and CD8 + T cells. Specifically, we tested by flow cytometry the expression of HLA-ABC (major histocompatibility complex (MHC) class I), HLA-DR (MHC class II) and co-stimulatory/co-inhibitory receptors such as CD86, CD80, CD40 and PD-L1 before and after exposure to IFN-γ. HLCs constitutively expressed HLA-ABC with no expression of HLA-DR and co-stimulatory molecules (CD80 and CD86) ( Figure 1F, Supplementary Figures S2 and S3). Of note, IFN-γ treatment increased the levels of PD-L1, CD40 and HLA-ABC molecules ( Figure 1F and G, Supplementary Figure S2).
Altogether, our results indicate that even after IFN-γ sensing, HLCs do not express the co-stimulatory receptors required to activate the T cells.

Allogeneic iPSC-HLCs Inhibit the T Cell Immune Response
Recent reports have shown that different stem cell-derived products do not induce T cell proliferation in vitro [21,22]. Furthermore, another study has reported that adult human hepatocytes display immunosuppressive properties when co-cultured with human PBMCs [23]. To fully understand whether HLCs could induce an allogeneic immune response, CD8 + and CD4 + T cells were co-cultured with allogeneic HLCs treated or not with IFN-γ. HLCs did not induce CD8 + or CD4 + T cell proliferation (Figure 2A-C and Supplementary Figure S4). Next, we evaluated whether they were able to suppress T cell proliferation following polyclonal activation. Indeed, HLCs were able to suppress the proliferation of both CD8 + and CD4 + T cells activated with αCD3/CD28 beads (Figure 2A-C and Supplementary Figure S4).
The production of IL-10 and IFN-γ during the co-cultures was also analysed. We observed that IL-10 was increased in CD4 + T cells co-cultured with HLCs compared to CD4 + T cells cultured with αCD3/CD28 beads, although HLCs per se did not produce IL-10 ( Figure 2D). In contrast, IFN-γ levels during the co-cultures were dramatically reduced compared to cultures of CD4 + T cells activated with αCD3/CD28 beads ( Figure 2D). These results suggest that HLCs can induce a tolerogenic environment in vitro by increasing IL-10 production, suppressing T cell proliferation and their ability to release IFN-γ.
To evaluate whether the inhibition of T cell proliferation exerted by HLCs was dependent on cell-to-cell contact, HLCs treated or not with IFN-γ were separated from either CD8 + or CD4 + T cells by a transwell system. Untreated HLCs were able to suppress T cell proliferation during cell-to-cell contact, while this capacity was lost when the transwell system was used (Figure 2A-C). Of note, when T cells were co-cultured with HLCs treated with IFN-γ, T cell proliferation was inhibited even across the transwell insert. These findings imply that soluble inhibitory factors contributed to T cell suppression when HLCs were treated with IFN-γ. To dissect the mechanisms behind the inhibition of cell proliferation in the presence of HLCs, neutralising antibodies specific to IL-10 receptor (IL-10R), CD40 and PD-L1 were used during the co-cultures. The results shown in Figure 2E demonstrated that both the expression of PD-L1 and CD40 by HLCs and the presence of IL-10 in the co-culture did not contribute to the inhibition of CD4 + T cell proliferation. This observation was extended to the co-culture of HLCs with CD8 + T cells to investigate whether the recognition of MHC class I molecules by CD8 + T cells could lead to different results from CD4 + T cells. However, the addition of blocking antibodies specific to PD-L1, CD40 and IL-10 did not revert the lack of T cell proliferation that HLCs mediated (Supplementary Figure S5).
Another possible mechanism responsible for the inhibition of cell proliferation seen in our co-culture involves the conversion of conventional CD4 + T cells into regulatory T cells, as shown with iPSC-derived RPE cells [24]. A lower expression of FOXP3 was observed in stimulated T cells when cultured in the presence of iPSC-HLCs compared to T cells cultured with αCD3/CD28 beads only ( Figure 2F and Supplementary Figure S6). In addition, the presence of the different neutralising antibodies did not modify the levels of FOXP3 expression ( Figure 2F and Supplementary Figure S6).

HLCs Inhibit Allogeneic T Cell Proliferation by Tryptophan Starvation
Our results suggest that soluble factors may be involved in suppressing T cell proliferation mediated by IFN-γ pre-conditioned HLCs (Figure 2A-C). A previous work showed that the suppression of T cell proliferation by mesenchymal stem cells can be through tryptophan starvation mediated by indoleamine 2,3-dioxygenase-1 (IDO1) [25]. As IFN-γ is a potent IDO1 inducer, we hypothesised that HLCs may regulate T cell proliferation through this mechanism. Our results showed that the expression of IDO1 was increased following IFN-γ treatment ( Figure 3A,B). To directly prove that IDO1 expression in HLCs was indeed involved in the suppression of T cell proliferation, the IDO1 inhibitor 1-L-methyltryptophan (1-LMT) and its non-functional isomer 1-D-methyl-tryptophan (1-DMT) were added to the co-cultures, and T cell proliferation was analysed. In the presence of the IDO1 inhibitor 1-LMT, but not 1-DMT, the suppressive effect of HLCs on CD4 + T cell proliferation was completely abrogated (Figure 3C,D). Moreover, supplementation of L-tryptophan (TRP) completely reversed the effect, with increased T cell proliferation observed ( Figure 3C,E). Similar results have been found with CD8 + T cells (data not shown). These results suggest that IDO1 expression by HLCs contributes to T cell proliferation arrest.
Tryptophan deprivation induces a stress response, which results in downregulation of TCRζ chain in T cells, thus modulating their cell cycle and proliferation [26]. This was confirmed in our co-cultures as we observed a 2-fold reduction in the TCRζ chain expression ( Figure 4A,B) in CD4 + T cells co-cultured with HLCs treated with IFN-γ. Additionally, we observed a reduction in the expression of CD25 molecules, a T cell activation marker ( Figure 4C). In contrast, in the presence of either TRP or 1-LMT to counterbalance the IDO1 effect, the TCRζ chain and CD25 expression were re-established ( Figure 4B,C). Thus, IDO1-mediated tryptophan starvation is a mechanism used by HLCs to downregulate the TCRζ chain and CD25 molecules in CD4 + T cells, which lead to T cell proliferation arrest.

Discussion
Liver transplantation represents the treatment of choice for several end-stage liver failures. However, the organ availability, as well as the detrimental side effects caused by immunosuppression, reduce the outcome of this life-saving procedure. So far, different strategies have been adopted to increase organ acceptance and induce operational tolerance [27][28][29]. Compared to other organs, the liver has an extraordinary capacity to regenerate; therefore, cell-based regenerative therapies are expected to become an attractive alternative strategy to organ transplantation in the near future. Functional hepatocyte-like cells (HLCs) derived from allogeneic sources can be produced in large numbers from both embryonic stem cells (ESC) and induced PSC (iPSC) and have the potential to be an off-theshelf product. However, similarly to organ transplantation, allogeneic HLCs can activate the host immune system, causing rejection and graft loss. In this study, we have shown that HLCs do express MHC class I molecules but not MHC class II or CD80/CD86 molecules. Furthermore, they upregulate MHC class I molecules, CD40 and PDL1 in the presence of IFN-γ. Finally, these cells were not immunogenic in vitro and showed immunoregulatory capacity by inhibiting T cell proliferation via tryptophan starvation.
In solid organ transplantation, the recognition of donor antigens by recipient T cells via direct/indirect and semi-direct allorecognition triggers a strong immune response [30]. This occurs through the recognition of MHC class I and class II molecules, expressed on either donor or recipient antigen-presenting cells by recipient CD8 + and CD4 + T cells, respectively. Similarly to other stem cell-derived products, HLCs constitutively express MHC-I molecules that are upregulated following IFN-γ exposure [21,31]. Although these cells express MHC class I molecules, CD8 + T cells were not activated, likely due to the lack of co-stimulatory molecules expression ( Figure 1F,G and Figure 2A). However, MHC class I molecules expressed by HLCs can be presented either indirectly or semi-directly following cell infusion, and specific in vivo models are required to test this scenario. To reduce the immunogenicity of both ESC-and iPSC-derived cells, different groups are now generating cell products lacking MHC class I molecules. However, the lack of these molecules can trigger recipient NK cell activation, causing graft loss [31]. To bypass this problem, RPE and hESC neural progenitor cells have been engineered to overexpress MHC molecules acting as NK inhibitors, such as HLA-E, HLA-F and HLA-G [32][33][34][35].
The cell product evaluated in this study did not express HLA-DR even when the cells were exposed to IFN-γ. This represents an advantage over other cellular therapies, such as RPE cells, which, by upregulating HLA-DR following IFN-γ treatment, acquire the ability to directly present the antigens to CD4 + T cells [21,31].
HLCs expressed the co-inhibitory molecule PD-L1, which is further upregulated following IFN-γ. A constitutive expression of PD-L1 by MSC contributed to suppressing CD4 + T cell activation and IL-2 secretion [36]. Furthermore, the upregulation of PD-L1 following IFN-γ stimulation has been also found in ESC-RPE, and it has been proposed as a possible mechanism mediating T cell suppression [21]. The suppressive role of PD-L1 has been also shown in vivo by Yoshihara et al. This group overexpressed PD-L1 on human islet-like cells, ameliorating their survival and function compared to non-engineered cells [37]. However, in our experimental model, PD-L1 did not play a role, as demonstrated by the lack of an effect on the inhibitory function of HLCs when neutralizing anti-PD-L1 antibodies were added to the system ( Figure 2F and Supplementary Figure S5).
Here, we have shown that HLCs have an immunomodulatory function, as they inhibited T cell proliferation. This was not directly due to IL-10 production or Treg induction ( Figure 2 and Supplementary Figure S6). In our system, IDO1-mediated tryptophan starvation downregulated the levels of TCRζ chain and CD25 molecules in CD4 + T cells, which lead to T cell proliferation arrest. IDO-mediated immunomodulatory effect has already been described in mesenchymal stem cells derived from different compartments [25,38] and human iPSC-derived mesoangioblasts [39]. Furthermore, IFN-γ priming enhanced the immunosuppressive properties of human MSCs due to the upregulation of IDO1 [40]. Whether the immunomodulatory capacity of the HLC can be reverted by danger signals, as shown by Zhang et al. with liver sinusoidal endothelial cells [41], is still a matter of debate. Based on our data, HLC-mediated immune tolerance may not be affected by the ligation of TLRs; however, new functional data are necessary to evaluate whether and how HLC respond to TLR stimulation.
In conclusion, our in vitro findings provide some mechanistic understanding of T cell immunomodulation via IDO1 expression and tryptophan starvation by stem cell-derived HLCs. This mechanism may be important for inducing local immune suppression when the cells are infused in vivo. In the future, when reliable humanised models of liver injury and HLC engraftment are established, the immunoregulatory capacities of these cells will be fully examined in vivo.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/cells11010024/s1, Figure S1: TLR-2, -4, -5, -7, -9 gene expression in HLCs and adult hepatocytes (Heps) evaluated by RT-PCR, S2: Representative dot plots showing the expression of the indicated markers in NT and INF-γ-pre-treated HLCs, S3: Expression of CD80, CD86 and HLA-DR in NT and INF-γ-pre-treated HLCs. Data are expressed as mean ±SD of 6 independent experiments with one donor per experiment, S4: Gating strategy used to evaluate the % of divided cells, Figure S5: Quantification for percentage of divided CD8-T cells activated with αCD3/CD28 beads and cocultured with HLCs in the presence of αPD-L1, αCD40 or αIL-10R. Data are expressed as mean ±SD of 3 independent experiments, with one donor per experiment, in duplicate, Figure S6: Representative dot plots of FoxP3 expression in CD4 + cells activated with αCD3/CD28 beads and co-cultured with HLCs in the presence of αPD-L1, αCD40 or αIL-10R and the cognate isotype controls.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author, [G.L.], upon reasonable request.