sBCs were generated using methods consistent with our previous publications (34). Undifferentiated hPSC Mel1^INS-GFP reporter cells (35) were maintained on hES qualified Matrigel (Corning #354277) in mTeSR+ media (STEMCELL Technologies #05826). Differentiation to stem cell derived beta like cells (sBC) was carried out in suspension‐based, bioreactor magnetic stirring system (Reprocell #ABBWVS03A-6, #ABBWVDW-1013, #ABBWBP03N0S-6) as follows. Confluent hPSC cultures were dissociated into single‐cell suspension by incubation with TrypLE (Gibco #12-604-021) for 8 min at 37 C. Detached cells were quenched with mTESR+ media. Cells were then counted using a MoxiGo II cell counter (Orflow), followed by seeding 0.5 × 10⁶ cells/ml in mTeSR+ media supplemented with 10 μM ROCK inhibitor (Y-27632, R&D Systems #1254-50). Bioreactors were placed on a magnetic stirring system set at 60 rpm in a cell culture incubator with 5% CO₂ to induce sphere formation for 48 hr. To induce definitive endoderm differentiation, spheres were collected in a 50 ml Falcon tube, allowed to settle by gravity, washed once with RPMI (Gibco #11-875-093) + 0.2% FBS, and re‐suspended in d1 media [RPMI containing 0.2% FBS, 1:5,000 ITS (Gibco #41400-045), 100ng/ml Activin A (R&D Systems #338-AC-01M), and 3 μM CHIR99021 (STEMCELL Technologies #72054)]. Differentiation media was changed daily by letting spheres settle by gravity for 3-10 min. Most supernatant was removed by aspiration; fresh media was added, and bioreactors were placed back on stirrer system. sBC differentiation was based on published protocol (5) with modifications as outlined below. Differentiation medias are as: day 2-3: RPMI containing 0.2% FBS, 1:2,000 ITS, and 100 ng/ml Activin A; d4-5: RPMI containing 2% FBS, 1:1,000 ITS, and 50 ng/ml KGF (Prepotech #100-19-1MG); d6: DMEM with 4.5g/L D-glucose (Gibco #11960-044) containing 1:100 SM1 (STEMCELL Technologies #5711), 1:100 NEAA (Gibco #11140-050), 1mM Sodium Pyruvate (Gibco #11360-070), 1:100 GlutaMAX (Gibco #35050-061), 3 nM TTNPB, (R&D Systems #0761), 250 nM Sant-1 (R&D Systems #1974), 250 nM LDN (STEMCELL Technologies #72149), 30 nM PMA (Sigma Aldrich #P1585-1MG), 50 μg/ml 2-phospho-L-ascorbic acid trisodium salt (VitC) (Sigma #49752-10G); d7: DMEM containing 1:100 SM1, 1:100 NEAA, 1mM Sodium Pyruvate, 1:100 GlutaMAX, 3nM TTNPB, and 50 μg/ml VitC; d8-9: DMEM containing 1:100 SM1, 1:100 NEAA, 1mM Sodium Pyruvate, 1:100 GlutaMAX, 100 ng/ml EGF (R&D Systems #236-EG-01M), 50 ng/ml KGF, and 50 μg/ml VitC; d10-16: DMEM containing 2% fraction V BSA, 1:100 NEAA, 1mM Sodium Pyruvate, 1:100 GlutaMAX, 1:100 ITS, 10ug/ml Heparin (Sigma #H3149-250KU), 2 mM N-Acetyl-L-cysteine (Cysteine) (Sigma #A9165-25G), 10 μM Zinc sulfate heptahydrate (Zinc) (Sigma #Z0251-100g), 1x BME, 10 μM Alk5i II RepSox (R&D Systems #3742/50), 1 μM 3,3’,5-Triiodo-L-thyronine sodium salt (T3) (Sigma #T6397), 0.5 μM LDN, 1 μM Gamma Secretase Inhibitor XX (XXi) (AsisChem #ASIS-0149) and 1:250 1M NaOH to adjust pH to ~7.4; d17-23: CMRL (Gibco #11530-037) containing 1% BSA, 1:100 NEAA, 1 mM Sodium Pyruvate, 1:100 GlutaMAX, 10ug/ml Heparin, 2mM Cysteine, 10uM Zinc, 1x BME, 10 μM Alk5i II RepSox, 1 μM T3, 50 μg/ml VitC, and 1:250 NaOH to adjust pH to ~7.4. All medias also contained 1x PenStrep.

Single cell sBC were filtered through cell strainer into FACS 5ml tubes and incubated for 30 min on ice for surface markers or overnight at 4°C for intracellular markers. After incubation, the cells were washed and strained again through cell strainer and resuspended in FACS buffer for analyses on CYTEK Aurora. Analysis and graphs were made using FlowJo software v10.6.2.

Generation of hPSC lines has been described previously (34). Briefly, hPSC Mel1 ^INS-GFP cells were dissociated into single cells using TrypLE incubation at 37°C for 8 min. Cells were then quenched with mTeSR+ media and counted using MoxiGo II cell counter. 2 x 10⁶ cells were transferred into microcentrifuge tubes and washed twice with PBS. Washed cells were then prepared for nucleofection of TALEN mediated knock-in (KI) of a Tet-On PD-L1 inducible system (i) or a CRISPR-Cas9 mediated B2M gene knock-out (ii). Cells were nucleofected in P3 buffer following the Amaxa P3 Primary cell 4D-Nucleofector kit protocol (V4XP-3024) using the CB-150 program. (i) AAVS1-TALEN-L and AAVS1-TALEN-R (gift from Danwei Huangfu, Addgene plasmid # 59025; http://n2t.net/addgene:59025; RRID: Addgene 59025) as well as a Tet-On inducible PD-L1 overexpression plasmid (generated in house) were nucleofected into Mel1 ^INS-GFP hPSC. Nucleofected cells were then plated in 10 cm plates with 10 μM ROCK inhibitor. After 48 hr of plating, puromycin selection (0.5 μg/ml) was performed for 48 hr then removed for 48hr followed by neomycin selection (50 μg/ml) for 6 days. Remaining colonies were picked and amplified for further characterization. (ii) guide RNAs (gRNA) targeting exon 1 and exon 2 of the B2M gene were generated in house and nucleofected along with CRISPR-Cas9. 24hr after plating, cells were selected for 48 hr with puromycin (0.5 μg/ml). Single colonies were picked and amplified for further characterization. Genomic DNA was extracted from targeted colonies and PCR analysis for TALEN KI and B2M KO was performed to identify potential modified clones. Further Sanger sequencing of PCR products confirm B2M mutations in the genome of Mel1 ^INS-GFP hPSC.

The mouse strain NOD-cMHCI^-/–A2 (Jax Stock No: 031856) was provided by Dr. Dave Serreze at The Jackson Laboratory. Mice were maintained in University of Florida Institutional Animal Care Services and genotype was confirmed by PCR using a commercial service (Transnetyx). All experiments involving animals were approved by the University of Florida Animal Care and Use Committee (IACUC no. 201810300). NOD-cMHCI^-/–A2 mice underwent transplant of 1,000 sBCs under the left kidney capsule, similarly to transplantation of pancreatic islets as described (5, 8, 36). Mice were anesthetized with 1.5-2% isoflurane, shaved and cleaned with alternate washes of sterile saline and 2% chlorhexidine. Subcutaneous injection of buprenorphine was administered as post-operative analgesic. Four days prior to transplant, mice were either kept in a standard feed diet (Envigo 2918) or changed to a doxycycline (DOX) infused feed diet (Envigo TD.01306) ad libitum for the remainder of the study. The animals weight was recorded the day of surgery and every week after that.

Diabetes was confirmed by blood glucose > 250 mg/dl on two consecutive measurements by tail tip blood with ACCU-CHEK^® Guide glucose meter. Mice with blood glucose >400 mg/dl were administered with 1-2s unit of Lantus^® long-acting insulin, daily. The age of the mice in the study ranged from 15 weeks to 29 weeks. Two weeks after transplantation the graft-bearing kidneys and mice pancreas were recovered for immunohistochemistry analysis.

The iP-sBCs were genetically engineered to express constitutive firefly luciferase for longitudinal monitoring after transplantation (34). sBCs bioluminescence was tested in vitro in a 96-well polystyrene black plate (Fisher no. 12-566-620) at different densities (0, 25, 50 or 100 clusters). iP and iP + DOX sBCs were incubated for 5 minutes at 37°C in media with or without D-luciferin (150 µg/mL) supplementation, prior to bioluminescence imaging. Bioluminescence was detected with an IVIS^® Spectrum In Vivo Imaging System (IVIS, PerkinElmer, Waltham, MA, USA). For bioluminescence imaging, the system was set to take images without any emission filter (open) and chamber temperature set to 37°C.

Following transplantation, the sBC grafts’ viability was longitudinally monitored by transdermal bioluminescence. A fresh stock solution of D-luciferin (PerkinElmer no. 122799) was prepared at a concentration of 30 mg/mL and filtered through a 0.22μm Spin-X^® centrifuge tube filter (Costar no. 8160) at each intervention. Mice were anesthetized with 1.5-2% isoflurane and sterile ocular lubricant was applied. Hair on the left lateral side of the mice was removed with depilatory cream. D-Luciferin was injected subcutaneously at 75 mg/kg five minutes prior to imaging. Transdermal bioluminescence was captured with an IVIS^® Spectrum In Vivo Imaging System. For bioluminescence imaging, the system was set to take images without any emission filter (open) and sequential imaging was performed until signal peaked. Transplants were longitudinally monitored at days 1, 3, 7 or 9, and 14 after surgery.

In vivo bioluminescence imaging data was analyzed using PerkinElmer, Inc Living Image^® 4.5.5 software. The total flux (p/s) was estimated with a square region of interest (ROI) over the left kidney region. A corrected total flux (CTF) was determined by measuring the background signal in the leg and applying the following formula:

Graft-bearing kidneys and pancreas were fixed with 3.2% paraformaldehyde (Thermo Fisher no. 047377) at 4°C in PBS overnight followed by washes with PBS. The tissues were immersed in 15% w/v sucrose in PBS overnight, followed by 30% w/v sucrose overnight then snap frozen in Optimal Cutting Temperature (O.C.T) compound (Thermo Fisher) using 2-methylbutane (>99% purity) chilled with liquid nitrogen. Frozen tissue blocks were cut with a cryostat into serial 10 μm sections and mounted on Superfrost Plus microscope slides (Thermo Fisher).

Cryosections were incubated twice for 15 minutes in 0.3% Triton X-100. Samples were then blocked and permeabilized in PBS with 0.3% Triton X-100 + 10% donkey serum for 1 hour at room temperature. Primary antibodies were incubated overnight in PBS with 0.3% Triton X-100 + 1% donkey serum at 4°C. CF488A-, CF568A- and 650 Dylight-conjugated secondary antibodies were incubated at 1:200 dilutions in PBS with 0.3% Triton X-100 for 1 hour at room temperature. Coverslips were mounted with ProLong™ Gold with DAPI (Thermo Fisher).

The following primary antibodies were used for immunofluorescence staining with their respective dilution: Guinea pig-anti-insulin (Dako no. A0564, 1:1000), Rat-anti-mouse CD3 (Biolegend no. 100209, 1:50), Rabbit-anti-human PD-L1 (Abcam no. ab205921, 1:200), Rat-anti-mouse CD45 (Sigma Aldrich no. 05-1416, 1:100), Rat-anti-mouse FOXP3 (Biolegend no. 126401, 1:150), Goat-anti-mouse CD4 (Fisher no. AF554SP, 1:200) and Rabbit-anti-laminin (Abcam no. ab11575, 1:300). CF, Alexa and Dylight secondary antibodies were acquired from Sigma-Aldrich and Invitrogen, respectively.

Histochemical samples of the graft-bearing kidneys were analyzed for the number of insulin positive cells (Ins⁺ cells), PD-L1⁺ and CD3⁺ cells. The whole graft section in the kidneys was imaged by tile scanning on a Leica SP8 Confocal microscope with a 20x/0.75 numerical aperture Plan-Apochromat air objective.

All image samples were analyzed using Fiji software (ImageJ). Prior to analysis, a max intensity projection (MIP) of the z-stack was generated. Image channels were split and the CD3⁺ channel was subtracted from the Ins⁺ channel with the image calculator. PD-L1⁺ channel was binarized with threshold set automatically using the moments model. An ROI (ROI-1) was hand drawn around the inner graft area using the CD3⁺ channel as reference. The number of Ins⁺ and PD-L1⁺ cells per unit area in ROI-1 was measured using the Fiji Plug-In StarDist 2D with the following settings: [Model: Versatile (fluorescence nuclei); Normalize Image; Percentile (1.0 - 99.8); Probability/Score (0.7); Overlap (0.4)] (37). Manual counting was used if the number of cells was less than 15 total to avoid a StarDist error.

A second ROI (ROI-2) was hand drawn on the CD3⁺ channel around the outer graft area. The number of CD3⁺ cells per unit area in ROI-2 was determined using StarDist 2D with the following settings: [Model: Versatile (fluorescence nuclei); Normalize Image; Percentile (1.0 - 99.8); Probability/Score (0.6); Overlap (0.4)] (37).

For this analysis 3 to 4 MIPs tile scans of 10 μm thin sections of 2 to 4 recovered graft-bearing kidneys for each group, from which graft was successfully located, were utilized. The inner graft area of the histochemical samples was determined for the analysis as the immediate area enclosing all the insulin positive cells. While, the outer graft area was determined as the area encompassing the whole graft adjacent to the kidney tissue wall minus the inner graft area. All images were processed in the same way.

One-Way ANOVA with multiple comparisons was used to determine significant changes for the flow cytometry analysis. IVIS Imaging bioluminescence data was analyzed by Two-Way ANOVA. Repeated measures from the same mouse were matched by time point and corrected with Geisser-Greenhouse correction method (if ϵ < 0.75). For immunohistochemistry quantification, means between two groups were compared by two tailed Student’s t-test. A confidence level of 95% was considered significant. The statistical test used, P values and definition of n are indicated in the individual figure legends. Error bars display the standard error from the mean (± s.e.m). Statistical analyses were performed in GraphPad Prism 9.3 software.

Programmed death-1 (PD-1) is an inhibitory receptor expressed on the surface of antigen-stimulated T cells that is important for maintenance of tolerance (38). The ligand of PD-1, PD-L1, is expressed on a variety of somatic cell types and is important for regulating the immune system’s response to self-antigen by down-regulating T cell inflammatory activity. Binding of PD-L1 to its receptor PD-1 on activated T cells inhibits T cell activation signals and inhibits CD8⁺ T cell cytotoxicity. In the context of autoimmune diabetes, systemically disrupting PD-1/PD-L1 interactions with a monoclonal antibody or through genetic deficiency accelerates T1D in NOD mice (39). PD-1/PD L1 checkpoint inhibitors used clinically to treat cancer in humans can result in autoimmune side effects including an aggressive form of autoimmune diabetes termed fulminant T1D (40, 41). Thus, augmenting PD-1/PD-L1 signaling in the local vicinity of the islet is a valid strategy to counter islet immune rejection (23).

We previously evaluated the protective capability of sBCs with overexpression of PD-L1 and deletion of HLA surface expression to counteract autoreactive CD8⁺ TCR stimulation in vitro (34). Similarly, we used the PD-L1 inducible system that was integrated into the AAVS1 locus of hPSC that contains a GFP fluorescence reporter under the control of the endogenous insulin promotor (INS-GFP). The transgene cassette also contains a strong CAG promoter driving constitutive expression of the M2rtTA that facilitates the Tet-On–DOX inducible PD-L1 expression system (referred to as iP) under the control of the TRE3G promoter. Site specific integration was possible using transcription activator-like effector nucleases (TALEN) technology ( Figure 1A ). HLA class I surface expression by sBCs was disrupted by knockout (KO) of the beta-2- microglobulin (B2M) gene (referred to as iP-BKO) with CRISPR/Cas9 by two gRNAs directed towards exon 1 and exon 2 of the B2M gene ( Figure 1B ). sBCs were generated from iP and iP-BKO hPSCs employing a 3D suspension based directed differentiation protocol, as previously described (34), and INS-GFP expression was confirmed by live fluorescence imaging ( Figures 1C, D ). After completion of the differentiation protocol, dual-expression of key beta cell markers C-peptide (cPEP, a byproduct of endogenous insulin biosynthesis) and NKX6.1 was determined by flow cytometry ( Figure 1E ). About 40% of total cells expressed cPEP at the end of the differentiation protocol. Furthermore, the percentage of NKX6.1 and cPEP double positive cells is ~25% on average for both lines. Introduction of the PD-L1 inducible system or deletion of all HLA class I surface molecules via beta-2 microglobulin knockout was previously demonstrated to not decrease or impede insulin expression (34). Efficiency of the PD-L1 DOX inducible system and ablation of HLA class I surface expression (BKO) was determined by flow cytometry analysis on iP and iP-BKO sBCs from independent differentiation experiments treated with exogenous DOX and/or interferon-γ (IFNg) cytokine ( Figures 1F, G ). IFNg but not DOX treatment increased HLA class I expression in iP sBCs, while iP-BKO sBCs had essentially no HLA Class I expression in all treatment groups. This data confirms that sBCs upregulate HLA class I when exposed to inflammatory cytokines and complete ablation of HLA class I molecules in iP-BKO sBCs was successful. DOX and IFNg treatments individually increased PD-L1 expression in iP and iP-BKO sBCs. Moreover, combination of both treatments show the highest PD-L1 induction in both iP and iP-BKO sBCs. Taken together, we show quantitative PD-L1 and HLA class I surface expression on sBC with inducible PD-L1 expression in the presence or absence of HLA class I molecules under distinct disease modeling conditions.

To quantify and assess the viability of sBC grafts post-transplantation we utilized the constitutively expressed bioluminescence reporter luciferase ( Figure 1A ). Luciferase is a well-characterized reporter protein commonly used to monitor and quantify tumor growth or cell populations over time in vivo. When luciferase oxidizes its substrate, luciferin, it results in light emission that can be detected with a camera (42). Prior to transplantation, sBC bioluminescence was confirmed in vitro by incubating sBC clusters at different densities with D-luciferin and detected with an IVIS^® Spectrum In Vivo Imaging System. Wells that had no sBCs or were not supplied with D-luciferin did not emit any bioluminescence, as expected ( Supplementary Figure 1 ). Results show that total flux is directly proportional to the number of clusters ( Supplementary Figure 1 ).

The mouse strain NOD-cMHCI^-/–A2 was selected for this study because it develops spontaneous autoimmune diabetes and recapitulates human HLA Class I (24). Due to the HLA-A2 match, human sBC grafts should therefore experience autoimmune pressure from immune recognition of islet antigens but not invoke an immediate xenograft response. A limitation of the model is that NOD-cMHCI^-/–A2 mice could still develop adaptive immunity toward other human antigens including non-classical HLA molecules. However, immune tolerance strategies by others have been shown to be effective at controlling xenoantigen responses in non-autoimmune beta cell transplant settings (23).

One thousand iP or iP-BKO sBC clusters with or without DOX pretreatment in the culture medium were transplanted under the kidney capsule of NOD-cMHCI^-/–A2 mice. Mice in the +DOX group were switched to a DOX infused feed diet four days prior to surgical intervention ( Figure 2 ). The viability of the transplanted sBC grafts under the kidney capsule of mice for experiments was monitored by transdermal bioluminescence for a period of two weeks with IVIS Imaging System ( Figure 2 ). D-luciferin was administered to the mice subcutaneously, then sequential imaging was performed until bioluminescence signal peaked ( Figures 3A, B ). Animal weight was monitored throughout the study and showed no significant differences between experimental groups ( Supplementary Figure 2 ).

A two-way ANOVA of the total photon flux detected longitudinally by IVIS showed a statistical difference (p-value <0.05) for mice transplanted with iP sBCs + DOX feed at day 1 and 3 ( Figure 3C ). The data suggest that sBCs expressing PD-L1 were more resistant to immune rejection in the first 3 days, but that this expression was insufficient for full graft survival. Results from the iP-BKO + DOX sBCs showed a significant improvement in beta cell survival later, at day 9 ( Figure 3D ). Because the differences in survival with PD-L1 expression at the earlier day 3 timepoint were not seen for BKO sBCs, we speculate that the initial mechanism of immune attack is likely to be predominantly HLA- class I driven. When comparing all iP and iP-BKO sBC, mice transplanted with iP-BKO + DOX had the highest number of animals with detectable bioluminescence at late time points ( Figure 3E ). Although luciferase signal dropped below detection limits by day 14 for most mice, one mouse from the iP-BKO + DOX group showed detectable transdermal luminescence at day 14 ( Supplementary Figure 3 ). In addition, IVIS scan of the kidney removed ex vivo at day 16 showed surviving sBC clusters.

To further elucidate the effects of PD-L1 overexpression and HLA Class I KO on the survival of the transplanted sBCs we performed detailed immunohistochemistry on the recovered graft-bearing kidneys. Cryosections sections of the graft area were stained for insulin, CD3, CD45, CD4, FOXP3 and PD-L1. After two weeks in vivo, all groups had significant numbers of surviving insulin⁺ cells in the inner graft region, surrounded by a robust immune infiltration of CD3⁺ ( Figure 4A ; Supplementary Figure 4 ), CD45⁺ cells ( Supplementary Figure 5 ) and CD4⁺ ( Supplementary Figure 6 ). Large numbers of CD3⁺ cells accumulated at the interface between the kidney tissue and the graft site and lower numbers of CD3⁺ cells were observed in the inner graft zone near sBCs. No qualitative differences were observed in the localization of CD3⁺ cells between any of the groups. However, some of our histology samples possesses a bright red autofluorescence near the center of the graft, in the same channel we used for CD3 staining. We believe this autofluorescence stems from cellular debris, as it lacks a cellular morphology and can be observed in unstained control slides ( Supplementary Figure 7 ). Mice were found to be experiencing severe insulitis in the pancreas during the same timeframe as the sBC graft experiment, indicating an active autoimmune response to beta cell antigens ( Supplementary Figure 8 ).

In the first experiment with transplantation of sBCs expressing HLA-A2, DOX treatment doubled the number of surviving insulin⁺ cells in the inner graft region with an average of 213 ± 39 and 438 ± 39 insulin⁺ cells/mm² ± s.e.m. for iP and iP + DOX, respectively ( Figures 4B, C ). DOX-inducible expression of PD-L1 was extremely robust with no PD-L1⁺ cells detected in the graft region for iP and 275 ± 92 PD-L1⁺ cells/mm² ± s.e.m for iP + DOX. There was no difference in the number of CD3⁺ cells between the two groups ( Figures 4D, E ), indicating that DOX treatment and PD-L1 expression did not alter the number of T cells at the time point examined. A reduction in T cell number is not expected because PD-L1 works by inhibiting T cell activation and does not induce T cell death as its name might suggest (38, 43). In vitro, IFNg treatment induces sBC PD-L1 expression ( Figure 1 ). Thus, the lack of PD-L1 expression in vivo in the absence of DOX suggests lower levels of IFNg in the local immune environment. The number of PD-L1 expressing cells was 60% of the number of insulin-expressing cells, consistent with the PD-L1 expression trends observed in vitro ( Figure 1 ). The HLA-A2 matching in this model likely enabled the partial survival of insulin⁺/PD-L1^- sBCs, while DOX-induced PD-L1 increased the number of surviving cells.

In the second experiment with transplantation of BKO sBCs that lack expression of HLA Class-I, the number of surviving insulin⁺ cells increased to 412 ± 69 and 631 ± 59 insulin⁺ cells/mm² ± s.e.m for iP-BKO and iP-BKO + DOX, respectively ( Figures 4F, G ). The increased number of insulin⁺ cells in both groups relative to the first experiment suggests the lack of MHC-I expression may improve sBC survival rates. As before, DOX treatment significantly improved sBC survival and robustly induced PD-L1 expression from 46 ± 25 to 915 ± 57 PD-L1⁺ cells/mm² ± s.e.m for iP-BKO and iP-BKO + DOX, respectively. Higher magnification of the graft better demonstrate insulin⁺ cells co-expressing PD-L1 ( Supplementary Figure 9 ), consistent with the in vitro data ( Figure 1 ). Differently from the first experiment, in the iP-BKO sBCs DOX treatment resulted in a slighter greater number of PD-L1-expressing cells than insulin-expressing cells. There was also a non-zero mean number of PD-L1 expression for the group without DOX. These results suggest a slightly more inflammatory environment (i.e. higher IFNg) in the iP-BKO grafts. The total number of infiltrating CD3⁺ cells remained the same as the previous experiment and was not significantly different for +/- DOX in either experiment ( Figures 4H, I ).

To better characterize the protective ability imparted by PD-L1 overexpression on the transplanted sBCs the presence of regulatory T (Tregs) cells was investigated. Activation of PD-1/PD-L1 pathway play an important role in the promotion of Tregs development and peripheral tolerance (Francisco LM et al., 2009; Fanelli G et al., 2021). Qualitative analysis of the processed samples suggests that PD-L1 overexpression by iP + DOX and iP-BKO + DOX samples resulted in a greater number of FOXP3⁺ cells ( Supplementary Figure 6 ). The FOXP3⁺ cells were primarily found in the areas where abundant CD4⁺ cells are present. These finding suggest that the increased survival of sBCs in the iP + DOX and iP-BKO + DOX can be partly attributed to development and recruitment of Tregs at the graft site.

Additionally, in the interest of assessing the surviving sBCs capacity to regulate glycemia, blood glucose was monitored throughout the study and blood serum was collected from some of the mice at the study end point to evaluate human C-peptide levels. Grafted iP-sBCs were unable to reverse hyperglycemia within the period of the study. Human C-peptide ELISA showed detectable human C-peptide in a small number of mice at the study end point, albeit at low levels. We did not add this data to the manuscript as it was not routinely measured at all timepoints or for all mice, as our study was focused on the immune rejection of the sBCs. Moving forward, a different dosage of iP-sBCs would be evaluated to determine the amount required to have a significant effect on blood glucose in this model.