Generation of Retinal Pigment Epithelial Cells Derived from Human Embryonic Stem Cells Lacking Human Leukocyte Antigen Class I and II

Summary Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells could serve as a replacement therapy in advanced stages of age-related macular degeneration. However, allogenic hESC-RPE transplants trigger immune rejection, supporting a strategy to evade their immune recognition. We established single-knockout beta-2 microglobulin (SKO-B2M), class II major histocompatibility complex transactivator (SKO-CIITA) and double-knockout (DKO) hESC lines that were further differentiated into corresponding hESC-RPE lines lacking either surface human leukocyte antigen class I (HLA-I) or HLA-II, or both. Activation of CD4+ and CD8+ T-cells was markedly lower by hESC-RPE DKO cells, while natural killer cell cytotoxic response was not increased. After transplantation of SKO-B2M, SKO-CIITA, or DKO hESC-RPEs in a preclinical rabbit model, donor cell rejection was reduced and delayed. In conclusion, we have developed cell lines that lack both HLA-I and -II antigens, which evoke reduced T-cell responses in vitro together with reduced rejection in a large-eyed animal model.

Although the eye is considered an immune privileged site, immunorejection is frequently observed when transplanting allo-or xenogeneic PSC-RPE to animal models (Ilmarinen et al., 2015(Ilmarinen et al., , 2018McGill et al., 2018;Sohn et al., 2015;Stanzel et al., 2014;Zhang and Bok, 1998). We have previously reported that xenogeneic hESC-RPEs may survive in the subretinal space of albino rabbits for extensive periods under intravitreal steroid immunosuppression, and similar findings were reported for allogeneic RPE derived from induced pluripotent stem cells (iPSCs) transplanted to non-human primates (Petrus-Reurer et al., 2017;Plaza Reyes et al., 2016;Sugita et al., 2017). When rejection occurs it typically involves both the innate immune system, which may activate recipient natural killer (NK) cells by lack of a cognate HLA-I (missing self) (Karre et al., 1986;Ljunggren and Karre, 1990), and the adaptive immune system where an antigen-presenting cell (APC) engulfs the donor cell, processes the mismatched HLA-I and HLA-II molecules into peptides, which in turn are presented and recognized by CD8+ cytotoxic and CD4+ helper T-cells via HLA-I and HLA-II molecules, respectively (indirect allorecognition). In addition, donor cell antigens coupled to HLA-II molecules on the APC activate CD4+ T-cells that also can trigger CD8+ T-cells, NK cells and antibody-producing B cells (Bradley et al., 2002;de Rham and Villard, 2011).
A solution to avoid immune rejection is to use autologous RPEs derived from iPSCs (Mandai et al., 2017). However, such personalized technology is costly and time consuming. An approach suggested to overcome these hurdles has been to establish an iPSC bank of ''superdonors,'' by donor collection or genome-editing techniques, with a specific homozygous or edited HLA haplotype that could match a high proportion of the population (Deuse et al., 2019;Sugita et al., 2016aSugita et al., , 2016bTaylor et al., 2012;Xu et al., 2019).
In this study, we generated and characterized hESCs and hESC-RPEs lacking surface presentation of HLA-I and -II through CRISPR/Cas9 targeting of B2M and CIITA. Our results support this strategy to overcome host-donor mismatch in non-autologous cell-based treatment of AMD and other hESC-derived cell replacement therapies.

B2M and CIITA Loci Edited by CRISPR/Cas9 to Obtain Single-and Double-Knockout Lines
Anchoring of HLA-I molecules to the cell membrane is dependent on the b 2 microglobulin protein and is encoded by the B2M gene (Nathenson et al., 1981). Consequently, loss of B2M leads to failure of HLA-I presentation on the cell surface. CIITA is a well-known HLA-II transactivator that activates HLA-II genes (Masternak et al., 2000). To disrupt their function, we used CRISPR/Cas9 with three sgRNAs targeting exon 1 or 2 of B2M (Figures 1A and S1A), or exon 2 or 3 of CIITA ( Figure 1B), respectively, transfected into HEK293T cells. Insertion/deletions (indels) were detected in all samples, and sgRNAs B2M-1 and CIITA-5 had the highest percentage of cleaved DNA with 38.9% (B2M-1) and 30.5% (CIITA-5) efficiency ( Figure 1C). HS980 hESC line was electroporated with pX459-(EF-1a)-B2M-1 ( Figure S1B) and all single-cell clones were sequenced to determine the specific on-target mutation. Of note, Cas9 protein presence was not detected at day 9 when cells were plated for clonal expansion ( Figure S1C). The hESC single-knockout B2M (hESC SKO-B2M) singlecell clone had a 1-bp insertion predicted to cause a frameshift mutation ( Figure 1D, top chromatogram). After B2M knockout validation, the hESC SKO-B2M clone was electroporated with pX459-(EF-1a)-CIITA-5. An hESC doubleknockout (hESC DKO) B2M and CIITA single-cell clone that had a 1-bp deletion predicted to cause a knockout of CIITA was chosen for further validation ( Figure 1D, bottom chromatogram).
First, we looked for specific changes at sites predicted by both Cas-OFFinder  and E-CRISP (Heigwer et al., 2014). The B2M gRNA generated 19,277 and CIITA gRNA generated 22,618 in silico predicted off-targets, respectively. CRISPR/Cas9-induced changes followed by clonal expansion would be expected to result in allele frequencies in line with heterozygote or homozygote changes, such as 0.5 or 1.0, which we also detected at the on-target sites at the B2M locus (chr15:45003753; C/CT; AF 1.0) and the CIITA locus (chr16:10989283; CA/C; AF 1.0) ( Figure 1E). The only additional three changes were detected at lower allelic frequencies, indicating that these instead were acquired changes during culture and unrelated to the CRISPR/Cas9 targeting. Importantly, neither of these were in any known genes. We also searched predicted off-target loci within copy-number deletions and none of the predicted loci were found within homozygous copy-number deletions (Table S4). Moreover, we identified four and three heterozygous copy-number deletions overlapping with the predicted off-target loci for hESC SKO-B2M and hESC DKO samples, respectively, neither of which were in annotated exonic regions. Using an unsupervised approach, we explored if any SNVs had been introduced into known coding genes. This analysis identified 13 (11 SNPs and 2 indels) and 16 (13 SNPs and 3 indels) somatic SNVs within non-redundant exonic boundaries for hESC WT versus SKO-B2M, and SKO-B2M versus DKO samples, respectively; which, after filtering, resulted in three heterozygote SNVs, which were either silent, within the 3ʹ UTR, or a heterozygote nonsense mutation (Table  1). Functionally, neither of these mutations have been linked to disease or tumorigenicity.
After targeting the B2M locus, we evaluated the HLA-I protein knockout in both hESCs and hESC-RPEs. For that purpose, we decided to increase HLA-I expression by stimulating the cells with interferon gamma (IFN-g). Titration experiments showed that treatment with 100 ng/mL IFN-g for 2 days induced high expression of HLA-I in both hESCs and hESC-RPEs, and 5 days induced HLA-II upregulation in hESC-RPEs ( Figures S2A and S2B).
Differentiated SKO-B2M hESC-RPEs showed characteristic pigmentation, cobblestone morphology, and expression of the epithelial marker ZO-1 ( Figure 2A). Moreover, B2M was lost in SKO-B2M cells and HLA-I was not present on the cell surface, as shown by immunofluorescence and flow cytometry (Figures 2A-2D); although the intracellular molecule was detected in the cytoplasm (Figures 2A and  2C). Gene expression of pluripotency-, RPE-, and HLA-I and -II-related genes in SKO-B2M cells did not differ from hESC-RPE WT, indicating that the targeting did not affect any of the downstream phenotypical properties of hESC-RPE cells ( Figure 2D). DKO hESC-RPEs stimulated with IFN-g for 5 days also showed cobblestone morphology, expression of ZO-1 and intracellular HLA-I but lack of B2M ( Figure 2E). Moreover, neither intra-nor extracellular HLA-II were present in DKO cells as indicated by immunofluorescence and flow cytometry ( Figures 2E and 2F). At a transcriptomic level, pluripotency genes (NANOG and OCT3/4), RPE genes (BEST-1 and RPE-65), and HLA-Irelated genes (HLA-A, -B, -C, except for B2M as for SKO-B2M cells) did not differ from WT. HLA-II-related genes (HLA-DR, -DP, -DQ) showed no expression, whereas CIITA was unaltered in DKO cells ( Figure 2G).

hESC-RPEs Show Immunomodulatory Properties
We then evaluated the immunosuppressive capacity of the cells by co-culturing WT, SKO-B2M, or DKO cells with isolated human peripheral blood mononuclear cells (PBMCs) at a 1:1 ratio, with or without OKT-3 (anti-CD3 monoclonal antibody) stimulation. Notably, addition of hESC-RPEs to unstimulated PBMCs did not induce T-cell proliferation in any of the lines, whereas allogeneic PBMC control induced measurable proliferative responses ( Figure 3A). Upon PBMC stimulation with OKT-3, WT and both SKO-B2M and DKO cells significantly decreased the proliferative response of CD8+ and CD4+ T-cells by 5to 10-fold compared with stimulated PBMCs alone, demonstrating the general immunosuppressive ability of the derived RPE cells ( Figure 3A).

SKO-B2M and DKO hESC-RPEs Exhibit Abolished T-Cell Reactivity In Vitro
Next, we assessed whether the hESC-RPEs could promote Tcell response under inflammatory conditions. hESC-RPEs were pre-stimulated with IFN-g 100 ng/mL for 2 days before co-culturing with PBMCs. This treatment alone did not induce T-cell proliferation (data not shown). However, when the T-cell stimulatory signals interleukin-2 (IL-2) (1 ng/mL) and anti-CD28 antibody (aCD28, 1.25 mg/mL) were added to the co-culture, we observed up to a 4-fold increase in CD8+ T-cell proliferation compared with PBMCs only, at ratios of 1:20 and 1:50 of hESC-RPEs to PBMCs (Figure S3A). Purified CD8+ T-cells but not CD4+ T-cells, showed significant reduction of IFN-g secretion in the presence of SKO-B2M cells compared with WT; whereas IFN-g amounts were reduced by both CD8+ and CD4+ T-cells when cultured with DKO cells, thus confirming abolished activation of cytotoxic and helper T-cells ( Figure 3B).
Further analysis of T-cell ligands expressed on the surface of both hESCs and hESC-RPEs demonstrated that PD-L1, but not PD-L2, was robustly detected in WT and DKO cells both under non-stimulated and stimulated conditions (Figure S3B). In addition, after IFN-g stimulation, a small population in all analyzed hESC-RPE lines expressed the co-stimulatory molecule CD80 but not CD86, which was instead present to a greater degree in hESCs. The expression of co-stimulatory molecules could potentially give hESC-RPE APC properties and, consequently, the ability to mount a T-cell reaction by themselves ( Figure S3B).

SKO-B2M and DKO hESC-RPEs Increase NK Cell Activation but Not Cytotoxicity under Inflammatory Conditions In Vitro
In co-cultures with freshly isolated and overnight IL-2activated human NK cells, we first found that HLA-I levels were induced with hESC-RPE differentiation (data not shown) coinciding with older hESC-RPEs (days d55-d63) being less sensitive to NK cell cytotoxicity than younger cells (days d44-d50) and hESCs at a 10:1 effector:target ratio ( Figure S3C). Degranulation analysis measured by CD107 expression upon co-culture of hESC-RPEs and overnight IL-2-activated NK cells showed a donor-dependent response, stronger in NK donor 1 compared with NK donors 2 and 3 ( Figure 3C). Of note, NK cells from all three donors showed no degranulation in the absence of target cells, and showed robust stimulation with positive controls K562 target cells (erythroid leukemia) or phorbol-myristate-acetate/ionomycin stimulation (chemical stimulation) ( Figure S3D). The edited SKO-B2M and DKO cell lines did not differ in NK cell activation compared with WT in non-inflammatory conditions (Figure 3C). Interestingly, IFN-g pre-stimulation of hESC-RPEs showed a significantly decreased response in all analyzed donors, especially for WT cells relative to their unstimulated samples; and the edited lines showed a significantly increased response compared with their respective stimulated WT hESC-RPE co-cultures ( Figure 3C). In addition, when NK cells were mixed with unstimulated SKO-B2M or DKO cells at a 10:1 effector:target ratio, cytotoxicity levels also showed to be donor dependent, and strikingly similar between WT and edited lines lacking HLA-I or HLA-I and -II molecules ( Figures 3D and S3D). IFN-g (100 ng/mL) pre-stimulation of hESC-RPE for 2 days did (E) Bar graph representing allele frequency in specific chromosomal positions from off-target analysis of whole-genome sequencing data. See also Figure S1, Tables S3, and S4. not show any reproducible significant effect among the three donors, although a trend in reduced cytotoxicity could be appreciated in donor 2, in addition to a general maintenance or decrease in cytotoxicity in the edited lines compared with their respective WT hESC-RPE co-cultures ( Figure 3D). Further evaluation of several NK cell ligands by flow cytometry showed that HLA-I (including HLA-C) was expressed in hESCs and WT cells, and this expression increased after 2 or 5 days of IFN-g stimulation. Other ligands tested included MIC-A/B (recognized by the activating NK cell receptor NKG2D) that showed a slightly higher expression in hESCs than in hESC-RPEs; proliferating cell nuclear antigen (recognized by the activating NK cell receptor NKp30) that was not upregulated in any of the cell types; CD112 (a ligand for the activating receptor DNAM-1 and the inhibitory receptor TIGIT) that was highly expressed in all cell types tested; and CD155 (another ligand for DNAM-1, TIGIT, and CD96, which has been ascribed with both activating and inhibitory functions) that was expressed at lower levels in both SKO-B2M and DKO compared with WT cells and IFN-g stimulatory conditions ( Figure S3E). In addition, WT cells exhibit significant expression levels of CD47, CD55, and CD59, which have been suggested previously to function as inhibitors of NK cell activity (Deuse et al., 2019;Finberg et al., 1992;Marcenaro et al., 2003) ( Figure S4A). Furthermore, analysis of NKG2A expression and several killer-cell immunoglobulin-like receptors (KIR)-KIR2DL1, KIR3DL1 KIR2DL5, KIR3DL2, and KIR2DL2/L3/S2 (clone [CH-L]) that recognize different HLA molecules on the donor NK cells showed a donor distinct pattern ( Figure S4B), which could contribute to the different susceptibility of activation and killing potential of the donor NK cells. As expected, expression of NKG2A (binding to the HLA-E molecule) was consistently elevated in the three donors compared with the remaining KIRs that were analyzed. Because the hESCs we used express ligands for KIR2DL1 (HLA-C*04), KIR2DL2 (HLA-C*07), KIR3DL1 (HLA-A*32, HLA-B*38), KIR3DL2 (HLA-A*68), and NKG2A (HLA-E), we studied the degranulation response of NK cells expressing the specific KIRs by the hESC-RPEs. With our panel of antibodies, we were able to categorize NK cells expressing KIR2DL1, KIR2DL2 (detected by KIR2DL2/L3/S2), KIR3DL1, and NKG2A. As in the analysis of total NK cells, KIR+ NK cells showed variation in CD107a expression between the donors ( Figures  S4B and S4C). However, a reduction in response toward WT cells stimulated with IFN-g could be observed in all KIR+ NK cells from all donors ( Figure S4C). This inhibition was less potent in SKO-B2M and DKO cells, probably due to a failure to upregulate HLA-I in response to IFN-g stimulation.

Immunogenicity of WT hESC-RPEs upon Subretinal Injection in a Xenograft Model
We have previously shown that WT cells can integrate in the subretinal space of albino rabbits under intravitreal immunosuppression with triamcinolone (TCA) (Plaza Reyes et al., 2016). However, we also noted that integration efficiency varied considerably, and pigmented areas of integrated cells were frequently lost over time, suggestive of immunorejection. We analyzed this in more detail by observing evidence of cell infiltration in the transplanted area on consecutive spectral-domain optical coherence tomography (SD-OCT) scans. As expected, subretinally transplanted WT cells under TCA immunosuppression displayed homogeneous monolayer integration as demonstrated by a distinct pigmented area on multicolor-confocal Total non-redundant exonic regions considered in the analysis: 229,235 (26,201 unique genes). AD, allelic depths for the reference and altered alleles in the order listed; AF, allele fraction of the event in the hESC WT and hESC SKO-B2M (sample 1); or hESC SKO-B2M and hESC DKO (sample 2). See also Figure S1, Tables S3 and S4.
scanning laser ophthalmoscopy and a hyperreflective subretinal layer on SD-OCT ( Figure 4A). However, at later time points, a pronounced mass was progressively formed in the subretinal space that displaced the pigmented donor cell layer and was accompanied by a thickening of the choroid, suggestive of immunorejection ( Figure 4A; Video S1). Histological analysis confirmed that infiltrates consisted of densely packed, mainly mononuclear, inflammatory cells ( Figure 4B). As the reaction progressed, the pigmented donor cells were gradually lost, and the subretinal infiltrate and choroidal thickening regressed, eventually leaving an atrophic retina and choroid ( Figures 4B and 4C). Immune cells from both the innate (NK cells and macrophages) and the adaptive (CD3+ T-cells) immune system were present at different rejection stages compared with non-rejected eyes, whereas B cells (CD79a + ) emerged in early and late stages but not at the onset of rejection ( Figure 4B). Human HLA-I was expressed in the transplanted WT cells even in later stages when cell shape and integrity had deteriorated, whereas HLA-II only appeared positive in cases of more prominent rejection (early and late) ( Figure 4B). We next compared the rejection rates over time of WT transplanted animals using SD-OCT. In non-immunosuppressed animals, rejection was detected in all eyes within 7 days after transplantation, whereas in TCA-treated animals, subretinal infiltration was rarely seen after 1 week, and even after 30 days only half of the transplanted eyes showed signs of rejection ( Figures 4C and 4D). By 90 days, rejection was observed in most of the transplanted eyes, also with TCA immunosuppression.
Immunogenicity of SKO-B2M, SKO-CIITA, and DKO hESC-RPEs upon Subretinal Injection in a Xenograft Model We next assessed the immune response after subretinal transplantation of SKO-B2M and DKO without TCA immunosuppression in the xenogeneic model. Both types of gene-edited cells showed significantly lower rates of early (1 week) rejection compared with WT cells (Figures 5A and 5B). Interestingly, DKO cells did not survive better than SKO-B2M in the in vivo setting ( Figure 5B). To examine the role specifically of HLA-II we also established singleknockout CIITA hESC-RPEs (SKO-CIITA) that lacked HLA-II expression while still being able to present HLA-I ( Figures  S5A-S5D). SKO-CIITA cells also showed a significantly reduced rejection, similar to the cells lacking HLA-I or HLA-I and -II ( Figures 5A and 5B Figure 5C). Without immunosuppressive treatment, anti-human antibodies were detected 7 days after WT transplantation, but were only detectable after 14 days (in 4-fold significantly lower levels compared with WT) in SKO-B2M-, SKO-CIITA-, and DKO-transplanted animals. After 30 and 90 days, anti-human antibodies were widely detected in all transplantation conditions, albeit at lower amounts in the gene-edited compared with WT cells ( Figure 5C). Thus, both early and late xenorejection of either SKO-B2M, SKO-CIITA, or DKO were delayed compared with WT cells.

DISCUSSION
We have shown that targeting B2M and CIITA genes in hESCs using CRISPR/Cas9 technology eliminates cell surface presentation of HLA-I and HLA-II proteins, respectively. In addition, these cells can be further differentiated  into hESC-RPEs that also lack HLA-I, HLA-II, or both HLA-I and -II presentation but retain native RPE cell properties. We also show that SKO-B2M cells reduce CD8+ but not CD4+ T-cell activation, that DKO reduce both CD8+ and CD4+ T-cell activation, and that both lines increase NK cell activation but not cytotoxic activity compared with WT cells under inflammatory conditions. When transplanted into the rabbit xenograft model without immunosuppression, all gene-edited lines reduce early rejection rates and delay anti-human antibody production associated with late rejection. Engineering cells introduces the risk of off-target changes. Nonetheless, it is encouraging that we did not detect any signs of deleterious mutations or deletions in the predicted off-target sites after B2M or CIITA targeting neither defective properties in morphology nor characteristic marker expression in either SKO-B2M or DKO cells, suggesting that the mutational load is modest during our genome editing procedure. Absence of predicted off-target changes may be linked to the fact that we have performed clonal selection, which may eliminate less frequent offtarget editing that may be detected if the initial bulk culture would have been expanded without clonal selection. However, changes in the genome can also occur during clonal selection and subsequent culture. Indeed, we did detect four heterozygote changes in known genes that may be spontaneous or CRISPR/Cas9 induced. Irrespectively, it is important to note that neither of these heterozygous changes have been linked to disease or tumorigenicity.
Despite the immunosuppressive phenotype of RPE cells, our study also shows that they can still induce a T-cell response under certain conditions in vitro: (1) that a specific RPE:immune cell ratio is achieved and (2) that inflammatory/stimulatory molecules, such as IFN-g or IL-2 are present. In our setting, we show that HLA-I and HLA-II mismatched cells can trigger cytotoxic CD8+ and helper CD4+ T-cells, which would eventually lead to both acute and chronic graft rejection. However, if neither HLA molecule is present, T-cell response is clearly avoided. Interestingly, we found a population of approximately 5% among the IFN-g-stimulated hESC-RPEs that showed upregulation of the co-stimulatory ligand CD80, which would confer the ability to induce an immune response without APC.
Our data suggest that the ''missing self'' phenotype (lack of HLA-I or both HLA-I and -II) of the engineered hESC-RPEs does increase the innate NK cell activation of the mismatched HLA-I and/or HLA-II WT cells. Such phenotype was observed only after inflammatory stimulation of the hESC-RPEs, but further studies are needed to explore if this is specific for hESC-RPEs despite their immunosuppressive properties. This finding suggests that the incorporation of an NK cell inhibitory ligand, such as HLA-E or the recently suggested cell surface molecules CD47, CD55, and CD59, may be beneficial to avoid rejection of grafted cells by the innate immune system (Deuse et al., 2019;Finberg et al., 1992;Gornalusse et al., 2017;Marcenaro et al., 2003;Sugita et al., 2018). Interestingly, our derived hESC-RPEs endogenously express high levels of CD47, CD55, and CD59, which could in part explain the lack of difference in NK cell cytotoxicity regardless of the HLA edits. Our degranulation assay showed a distinct difference in responsiveness dependent on donor, which correlated with the outcome of the cytotoxic assay. The level of activation might be subjected to NK cell education, i.e., expression of specific KIRs that recognize the presence of certain HLA molecules, such as HLA-C2, for the 2DL1 KIR present in the donor NK cells (Pegram et al., 2011). However, the degree and differences seen in degranulation and cytotoxicity when HLA-I was absent (SKO-B2M or DKO) or increased (IFN-g stimulation), could be explained by the changes in expression of certain ligands (e.g., CD155), and may be specific to hESC-RPEs. Overall, NK cell activation toward RPE cells was limited to a few donors, and did not result in measurable release of cytokines (IFN-g) (data not shown). In addition, these NK studies show no difference in NK cell cytotoxicity after removal of HLA-I or both HLA-I and -II in normal or inflammatory conditions, thus suggesting that HLA-II has no effect on NK cells and reinforcing the possibility to use these double (B) Bar graphs showing the secretion of IFN-g produced by either CD8+ or CD4+ isolated T-cells from two different donors after 5 days co-culture with WT, SKO-B2M, or DKO cells at 1:20 and 1:50 ratios (hESC-RPE:PBMC) with the presence of IL-2+aCD28 stimulation (and 2 days IFN-g 100 ng/mL hESC-RPE pre-stimulation). CD8+ or CD4+ only were used as negative controls unstimulated (No ST) or IL2+aCD28 stimulated (ST). (C) Bar graphs showing the percentage of NK degranulation by CD107-positive expression in the total NK cells when co-cultured with WT, SKO-B2M, and DKO cells unstimulated or 2 days IFN-g 100 ng/mL pre-stimulation from three different donors. (D) Bar graph showing the percentage of cytotoxicity of the unstimulated or 2 days IFN-g 100 ng/mL pre-stimulated WT, SKO-B2M, or DKO cells (target) measured by chromium release of the killed cells by the freshly isolated and overnight IL-2-stimulated NK cells (effector) from three different donors at 10:1 effector:target ratio. NK cells were freshly isolated from human blood PBMCs, further separated (CD56 MACS isolation kit) and activated with IL-2 overnight before co-culture with hESC-RPEs.  (legend continued on next page) gene-edited cells to maximize the escape from both Tand NK cells. Nonetheless, another possible strategy to counteract HLA-I-and -II-independent NK cell effect could reside on the selection of a donor hESC-RPE line from a cell bank based on reactivity with the recipient NK cells, aiming to avoid activation of NK cells that could elicit strong responses when the specific HLA epitopes are missing in the hESC-RPEs.
Without immunosuppression, donor cells triggered rapid rejection in our xenogeneic transplantation model, whereas TCA-mediated immunosuppression delayed and sometimes prohibited rejection. The degree and time of rejection varied greatly, implying that local factors were involved. For instance, mechanical damage to Bruch's membrane during transplantation could compromise the immune-privileged subretinal space, thus allowing invasion by immune cells as suggested previously (Petrus-Reurer et al., 2017). The cells infiltrating into the graft belonged to both the innate and adaptive immune systems in all stages of rejection. Evaluation of the T-cell subtypes (CD8+ or CD4+) in the infiltrates could not be performed due to lack of reliable immunoreaction with anti-rabbit antibodies but deserves further exploration since a reduction in CD8+ cells after SKO-B2M transplantation, and a reduction of both CD8+ and CD4+ T-cells after DKO transplantation, would be expected, which could explain the milder rejection phenotype compared with WT cells. Contrary to what we expected from the in vitro results, transplantation of DKO cells was not superior to either SKO-B2M or SKO-CIITA, possibly suggesting that either CD4+ T-cells are pre-activated (i.e., rendering HLA-II loss ineffective) or that, despite delayed T-cell-mediated rejection, NK cells may eventually counter react it. The fact that all hESC-RPE lines showed rejection over time may be due to antibodies produced against human cells via indirect allorecognition (macrophages, dendritic cells, or B lymphocytes recognizing grafted cells) that activate CD4+ T-cells (de Rham and Villard, 2011;Fox et al., 2001;McGill et al., 2018;Sohn et al., 2015;Sugita et al., 2017).
Our in vitro and in vivo observations show that DKO cells have the potential to partially evade the adaptive immune system, making these cells attractive candidates as universal donors. Nevertheless, the innate immune system, including NK cells, may still be activated as discussed above. It is important to note that the immune system is an essential first-line defense against tumor formation and infection, and generation of immune evasive cells could also escape this defense mechanism. Therefore, a fail-safe/suicidal cassette system enabling the clearance of engineered cells may have to be integrated before clinical consideration (Liang et al., 2018). Also, minor antigens and sugars that do not require HLA molecule presentation or fragments of dead grafted cells could be also be a source of APC recognition and immune activation.
In this study, donor cells were transplanted in a cross-species model, whereas in a comparable clinical setting donor cells would be allogenic where rejection is expected to be less pronounced (Fox et al., 2001). It is well known from other models that xenogeneic rejection involves not only allogeneic pathways but also innate neutrophil and macrophage responses toward non-self oligosaccharides that precede T-cell activation. The stronger activation of the innate immune system may in turn explain why conventional immunosuppressants are less effective in preventing xenogeneic rejection (Hawthorne et al., 2016). The rabbit immune system is in this respect poorly studied but it is reasonable to infer the involvement of xeno-specific rejection pathways, as suggested by our data. Therefore, our finding that removal of HLA-I, HLA-II, or both HLA-I and -II delayed rejection is encouraging since a reduced rejection, as observed with either gene-edited hESC-RPEs, is also expected to translate into an allogeneic human setting. However, and ideally, the immune reactions demonstrated in this study should also be confirmed in a preclinical allogeneic model, in line with the previously demonstrated observations in non-human primates transplanted with allogeneic WT iPSC-RPEs (Sugita et al., 2017).
In conclusion, we show that WT hESC-RPEs can induce both adaptive and innate immunoresponses in vitro and in vivo, but the loss of surface HLA-I and HLA-II molecules leads to a decreased immunogenicity, thus comprising a first step toward universal-donor cells for cell replacement therapy in AMD and related conditions.

EXPERIMENTAL PROCEDURES
Cell Culture and Differentiation hESC line HS980 was established and cultured under xeno-free and defined conditions on rhLN-521, and passaged as described previously (Rodin et al., 2014a(Rodin et al., , 2014b. For differentiation, cells were plated at a density of 2.4 3 10 4 cells/cm 2 on 20 mg/mL hrLN-111coated dishes using NutriStem hESC XF medium and Rho-kinase inhibitor during the first 24 h. NutriStem hESC XF without basic fibroblast growth factor and transforming growth factor b was then replaced and from day 6 after plating, 100 ng/mL of activin A was added to the medium for a total of 5 weeks. Genome Editing of hESCs hESC line HS980 was modified at B2M and CIITA loci using CRISPR/ Cas9 plasmid constructs. First, high cutting efficiency sgRNAs were identified through screening in HEK293T. Second, gene editing in hESCs was first done at the B2M locus, followed by puromycin selection and clonal expansion. An SKO-B2M clone was then edited at the CIITA locus, followed by puromycin selection and clonal expansion. For both of the generated cell lines, on-target edits were determined by Sanger sequencing, and the characterization was based on detection of HLA-I and -II proteins under inflammatory conditions (IFN-g, 100 ng/mL). Finally, potential off-target edits were analyzed by whole-genome sequencing.

T-Cell and NK Cell Co-cultures with hESC-RPE Lines
PBMCs were isolated from buffy coats by density gradient centrifugation. PBMCs were either stained using the CellTrace CFSE Cell Proliferation Kit; or CD4+, CD8+, and CD56+ negatively isolated with MACS beads and co-cultured with day 30 unstimulated or 2 day IFN-g (100 ng/mL) pre-stimulated irradiated or chromiumlabeled hESC-RPEs plated at a cell density range of 1 3 10 3 (1:500) to 5.5 3 10 5 (1:1) cells/cm 2 depending on the assay. Overnight IL-2-activated hNK cells were used for 51 Cr release and degranulation assays and assay-dependent stimulatory molecules were added. Co-cultures were maintained for 4 h (degranulation, cytotoxicity) or 5 days (T-cell proliferation) at 37 C.
Cells were fed three times a week and kept for 5 weeks. Monolayers were then trypsinized using TrypLE Select (ThermoFisher Scientific, 12563-011) for 10 min at 37°C, 5% CO2. The enzyme was carefully removed and the cells were collected in fresh pre-warmed NutriStem hESC XF medium without bFGF and TGFβ (Biologicial Industries, 06-5100-01-1A) by gentle pipetting to obtain a single cell suspension. The cells were centrifuged at 300g for 4 min, the pellet was resuspended, passed through a cell strainer (ø 40 μm, VWR, 732-2757) and cells were seeded on LN-coated dishes (hrLN-521 20 μg/mL, Biolamina) at a cell density of 7x10 4 cells/cm 2 . Re-plated cells were fed three times a week during the subsequent four weeks with the same differentiation medium referred above.

Promoter Exchange of pX459
The EF-1a promoter has been shown to be more stable in embryonic stem cells, which would ensure optimal expression of Cas9 and GFP or puromycin-resistance, respectively, from the plasmid (Liu et al., 2009) Firstly, the CMV promoter of pX458 (addgene no. 48138) and pX459 (addgene #62988) was exchanged for EF-1a (Fig S1A and S1B)
Annealed and PNK treated gRNA oligonucleotides were ligated into pX459-EF-1a using T4 DNA ligase (ThermoFisher Scientific, K1423) and the mixture was transformed into STBL3 bacteria (ThermoFisher Scientific, C737303) and plated on agar plates containing 100 µg/mL ampicillin. Single colonies were picked and expanded in LB broth with ampicillin (100 µg/mL) overnight and plasmid DNA was extracted using QIAprep Spin Miniprep Kit (QIAGEN, 27104) according to the manufacturer's protocol.
Transfection success was estimated through GFP intensity from the pX458_EF-1a-Cas9_U6-sgRNA. The transfected cells were plated on 6-well plates and were allowed to recover until confluent before harvesting the cells for gDNA extraction and mutation detection. Cells in one out of the two wells were then used for genomic DNA extraction and Sanger sequencing of the region surrounding the edit using B2M primers F2 and R1 (Table S2)  contained indels and five remained unedited.

Mutation Detection using SURVEYOR Nuclease Assay
Genomic DNA extraction was performed using QuickExtractTM DNA extraction solution (Lucigen, QE09050) and 1 µL of the extracted DNA was utilized for PCR amplification using Herculase II Fusion Enzyme (Agilent, 600677) and primers F1 and R1 for sgRNAs 1 and 2 located in exon 1 and primers F3 and R3 for sgRNA-3 located in exon 2 in the B2M locus, (Table S2). Primers F2 and R2 amplified a region around sgRNAs 5 and 9 and primers F3 and R4 amplified a region surrounding sgRNA 4 in the CIITA locus (Table S2). Mutation detection was performed using SURVEYOR Nuclease Assay (IDT, 706020) according to the manufacturer's protocol. The mismatch analysis was carried out according to the published protocol by Ran et al. (2013). Indel percentage was calculated according to 100*1-√((1-((b+c))/((a+b+c)))), where a symbolizes the integrated intensity of the unedited DNA fragment, and b and c represent the intensities for each cleaved fragment (Ran et al., 2013).

Detection of Cas9 in Cells using Fluorescence Activated Cell Sorting (FACS)
HS980 hESCs were cultured and electroporated at nine, four and one days before analysis through FACS. Cells were selected for 24h with 0.5µg/ml puromycin (ThermoFisher

Pre-processing of Whole-Genome Sequencing Reads
Whole genome DNA sequencing reads (paired-end; 150bp) of human embryonic stem cell line HS980, hESC SKO-B2M and hESC DKO cells were aligned to the human reference genome (NCBI reference genome GRCh37 based "human_g1k_v37") using the Burrows-Wheeler Aligner (BWA-0.7.12) (Li and Durbin, 2010). Sequencing data is available upon request. Duplicate reads in reference genome aligned BAM files were marked using Picard 2.9.0 "MarkDuplicates" utility. Next, we followed the "GATK Best Practice" guidelines (DePristo et al., 2011;Van der Auwera et al., 2013) using GATK 3.7 toolkit and performed local realignments and base quality recalibration with default parameters. GATK resource bundle "b37" that include data sets from HapMap, Omni, Mills Indels, 1000 Genome Indels and dbSNP v138 databases were used in this analysis. Further, germline SNVs and indels were identified using HaplotypeCaller with default parameters.

Identification of Somatic SNVs and Copy Number Deletions
We considered non-redundant whole exonic intervals (GRCh37 gene annotation) and predicted off-target sites for SNV analysis. Separate list of potential off-targets sites for hESC SKO-B2M and hESC DKO experiments were created using Cas-OFFinder  (up to nine mismatches allowed and up to 2bp bulge formation of either RNA or DNA) and E-CRISP (Heigwer et al., 2014) (not allowed for any 5' mismatches to be ignored and tolerated edit distance to the target sequence set to three) online prediction tools. We used 'NGG' as PAM motif sequence and 5'-GGCCGAGATGTCTCGCTCCG-3' and 5'-GAGATTGAGCTCTACTCAGG-3', as B2M and CIITA sgRNA sequences, respectively, for off-target sites prediction. All exonic intervals and predicted off-target sites (75 bases flanking both ends) were searched for SNVs using GATK 3.7 MuTect2 utility (Cibulskis et al., 2013) with default parameters. SNVs with allele frequency (AF) ≥ 0.25 and read depth (DP) > 10 were considered for further analysis. Somatic copy number deletions were then considered since in principle a copy number deletion refers to loss of large genomic segment and a predicted off-target site could be completely missed if it is within copy number deleted analysis. Overlapping study of predicted off-targets with copy number deletions was performed using UCSC Galaxy bed-tools "intersect intervals" utility.

Quantitative Real-time PCR
Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen, 74106) and treated with RNase-free DNase (Qiagen, 79254) and RNaseH (ThermoFisher Scientific,18021071) according to manufacturer's protocol. cDNA was synthesized using 1 μg of total RNA in a 20 μL reaction mixture containing random hexamers (ThermoFisher Scientific, N8080127) and Superscript III reverse transcriptase (ThermoFisher Scientific, 18080085), according to the manufacturer's instructions.
Taq-polymerase (ThermoFisher Scientific, 4304437) together with Taqman probes Samples were subjected to real-time PCR amplification protocol on StepOneTM real-time PCR System (Applied Biosystems). Biological triplicates were performed for every condition and technical triplicates were carried out for each sample. Results are presented as mean±SEM (standard error of the mean).
The membrane was then washed 3 x 10 min with 0.05% Tween-20 (Sigma, T8787) in DPBS (ThermoFisher Scientific, 14190-094) before the membrane was imaged using LI-COR analyzed using a Cytoflex flow cytometer (Beckman Coulter). Analysis of the data was carried out using FlowJo v.10 software (Tree Star). Results are presented as mean±SEM (standard error of the mean).

Animals
After approval by the Northern Stockholm Animal Experimental Ethics Committee (DNR N25/14), female New Zealand white albino rabbits (provided by Lidköpings rabbit farm, Lidköping, Sweden) aged 5 months and weighing 3.5 to 4.0 kg were used in this study. All experiments were conducted in accordance with the Statement for the Use of Animals in Ophthalmic and Vision Research.
25G trocars were inserted 1 mm from the limbus and an infusion cannula was connected to the lower temporal trocar. The cell suspension was drawn into a 1 mL syringe connected to an extension tube and a 38G polytip cannula (MedOne Surgical Inc, 3219 and 3223).
Without infusion or prior vitrectomy the cannula was inserted through the upper temporal trocar. After proper tip positioning, ascertained by a focal whitening of the retina, 50 μL of cell suspension (equivalent to 50,000 cells) was injected slowly subretinally approximately 6 mm below the inferior margin of the optic nerve head, forming a uniform bleb that was clearly visible under the operating microscope. To minimize reflux, the tip was maintained within the bleb during the injection. After instrument removal light pressure was applied to the selfsealing suture-less sclerotomies. 2 mg (100 μL) of intravitreal triamcinolone (Triescence 40 mg/mL, Alcon Nordic A/S, 412915) was administered a day prior to the surgery, and no post-surgical antibiotics were given. For the TCA cohort, intravitreal triamcinolone was readministered every 3 months, if required.

Multimodal Real-time Imaging and Choroidal and Subretinal Thickness Measurements
Multimodal real-time imaging was performed as described before (Bartuma et al., 2015).
Briefly, anesthetized animals were placed in an adjustable mount (Spectralis HRA +

Statistical Analysis
For statistical analyses, Chi-squared test was used to compare differences in rejection rates in the three subretinally transplanted groups with WT, SKO-B2M, SKO-CIITA and DKO cells.
ANOVA (one-way or two-way) and posthoc multiple comparisons using Tukey test were performed to assess the in vitro differences of the different knock out lines upon co-culture with immune cells, and to assess anti-human antibody presence upon subretinal injection in the xenograft model.