Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Nov 15;9(416):eaam7543.
doi: 10.1126/scitranslmed.aam7543.

PD-L1 Genetic Overexpression or Pharmacological Restoration in Hematopoietic Stem and Progenitor Cells Reverses Autoimmune Diabetes

Affiliations
Free PMC article

PD-L1 Genetic Overexpression or Pharmacological Restoration in Hematopoietic Stem and Progenitor Cells Reverses Autoimmune Diabetes

Moufida Ben Nasr et al. Sci Transl Med. .
Free PMC article

Abstract

Immunologically based clinical trials performed thus far have failed to cure type 1 diabetes (T1D), in part because these approaches were nonspecific. Because the disease is driven by autoreactive CD4 T cells, which destroy β cells, transplantation of hematopoietic stem and progenitor cells (HSPCs) has been recently offered as a therapy for T1D. Our transcriptomic profiling of HSPCs revealed that these cells are deficient in programmed death ligand 1 (PD-L1), an important immune checkpoint, in the T1D nonobese diabetic (NOD) mouse model. Notably, the immunoregulatory molecule PD-L1 plays a determinant role in controlling/inhibiting activated T cells and thus maintains immune tolerance. Furthermore, our genome-wide and bioinformatic analysis revealed the existence of a network of microRNAs (miRNAs) controlling PD-L1 expression, and silencing one of key altered miRNAs restored PD-L1 expression in HSPCs. We therefore sought to determine whether restoration of this defect would cure T1D as an alternative to immunosuppression. Genetically engineered or pharmacologically modulated HSPCs overexpressing PD-L1 inhibited the autoimmune response in vitro, reverted diabetes in newly hyperglycemic NOD mice in vivo, and homed to the pancreas of hyperglycemic NOD mice. The PD-L1 expression defect was confirmed in human HSPCs in T1D patients as well, and pharmacologically modulated human HSPCs also inhibited the autoimmune response in vitro. Targeting a specific immune checkpoint defect in HSPCs thus may contribute to establishing a cure for T1D.

Figures

Fig. 1.
Fig. 1.. PD-L1 is down-regulated In HSPCs from NOD mice.
(A and B) Transcriptomic profiling of KLS cells obtained from bone marrow of NOD and C57BL/6 mice; n = 3 samples per group were evaluated. Statistical analysis was performed also by using the software available (RT2 profiler PCR Array Data Analysis, Qiagen). TNF-α, tumor necrosis factor–α. (C) Bar graph representing mRNA expression of PD-L1 as measured by quantitative RT-PCR (qRT-PCR) in KL cells, collected from bone marrow of C57BL/6 and NOD mice. All samples were run in triplicate and normalized to expression of the housekeeping gene GAPDH. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D to G) Representative flow cytometric analysis and quantitative bar graphs assessing PD-L1 expression in four populations of HSPCs in C57BL/6 and NOD mice. (H) Representative flow cytometry and quantitative bar graphs of PD-L1 expression in KL cells obtained from the bone marrow of C57BL/6 and NOD mice at different ages; n = 3 mice per group were evaluated and for statistical analysis, one-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test for group comparisons between C57BL/6 and NOD mice. Lin, Lineage; Ab, antibody; Hglc, hyperglycemic. (I) Representative flow cytometric analysis and quantitative bar graphs of PD-L1 expression in KL cells and in other nonprogenitor cells from bone marrow or spleen respectively obtained from C57BL/6 and NOD mice; n = 3 mice per group were evaluated. (J and K) Confocal imaging and quantification of bone marrow sections of C57BL/6 and NOD mice for c-kit (shown in red) and PD-L1 (shown in green) staining (n = 5 sections per strain); the quantification of the orange-stained bone marrow element was performed by ImageJ, and statistical significance was performed using two-tailed unpaired t test with Welch’s correction. Histology magnification, ×63. Scale bar, 40 μm. (L) Western blotting and quantitative bar graphs confirming reduced PD-L1 protein expression in KL cells obtained from bone marrow of C57BL/6 and NOD mice (n = 3 samples per group), with GAPDH used as an internal control. Data are expressed as means ± SEM. Data are representative of at least n = 3 mice. *P < 0.05; **P <0.01; ***P < 0.001.
Fig. 2.
Fig. 2.. Mechanism of PD-L1 down-regulation in NOD HSPCs.
(A) Bar graphs depicting percentage of PD-L1+ cells within KL cells isolated from bone marrow of C57BL/6 and NOD mice and cultured for 3 days in normal glucose, in 20 mM, or in 35 mM high glucose. Experiments were run in triplicate. NG, normal glucose; HG-20, 20 mM high glucose; HG-35, 35 mM high glucose. (B) Proliferation rates of carboxy-fluorescein succinimidyl ester (CFSE)–labeled KL cells obtained from C57BL/6 and NOD bone marrow at baseline and after 1 and 3 days of culture. (C) Frequency of apoptosis of KL cells obtained from bone marrow of C57BL/6 and NOD mice at baseline and after 1 and 3 days of culture. For statistical analysis in (A) to (C), one-way ANOVA followed by Bonferroni multiple comparison test for group comparisons between C57BL/6 and NOD mice; in (A): P = not significant (ns); in (B): #P < 0.0001 versus all except NOD-day 0 (D0) (P = ns); in (C): §P < 0.0001 versus all except NOD-D0 (P = ns) and NOD-D1 (P < 0.001); #P < 0.0001 versus all except NOD–D1 (P =ns). 7-AAD, 7-aminoactinomycin D. (D) MA plot for GWAS performed in KLS cells obtained from bone marrow of C57BL/6 compared to NOD mice; MA-plot, log2 normalized expression levels of expression for KLS cells from C57BL/6 (y axis) in comparison with KLS cells from NOD (x axis). Avg, average. (E and F) List of miRNAs significantly up-regulated (E) and down-regulated (F) in KLS cells obtained from the bone marrow of NOD as compared to C57BL/6 mice. (G) miRNA network controlling PD-L1 gene expression generated with MGI. (H and I) KL cells obtained from bone marrow of NOD mice were cultured in the presence of miR-1905 inhibitor, and wild-type (WT) (untreated KL) were used as controls. qRT-PCR of miR-1905 (H) and qRT-PCR of PD-L1 (I) are shown. Experiments were run in triplicate, and statistical significance was determined using two-tailed unpaired Student’s t test. (J) Western blotting and quantitative bar graphs of PD-L1 protein expression in KL cells obtained from bone marrow of NOD mice cultured in the presence of miR-1905 inhibitor, and WT (untreated KL) were used as controls, with GAPDH used as an internal control. Experiments were run in triplicate, and statistical significance was determined using two-tailed unpaired Student’s t test (P= 0.0005). (K) Methylation status of the PD-L1 locus in KLS cells obtained from the bone marrow of C57BL/6 and NOD mice. Experiments were run in triplicate, and statistical significance comparing methylated CpG% of C57BL/6 to NOD was determined using two-tailed unpaired Student’s t test. Data are expressed as means ± SEM. Data are representative of at least n = 3 mice. *P < 0.05; **P <0.01; ***P < 0.0001.
Fig. 3.
Fig. 3.. Genetically engineered PD-Ll.Tg KL cells abrogate the autoimmune response in vitro and revert diabetes in hyperglycemic NOD mice in vivo.
(A) Freshly isolated murine KL cells were transduced with PD-L1 lentiviral particles, and 24 hours after transduction, cells were collected for fluorescence-activated cell sorting (FACS) analysis; representative flow cytometric analysis and quantitative bar graph of KL cells obtained from bone marrow of NOD mice before and after transduction with PD-L1 lentivirus. Experiments were run in triplicate, and statistical significance comparing WT to Tg postlentiviral PD-L1 transduction was determined by using two-tailed unpaired t test. (B) Confocal imaging of KL cells obtained from bone marrow of NOD mice pre- and postlentiviral PD-L1 transduction confirmed PD-L1 up-regulation. Histology magnification, ×63. Scale bar, 50 μm. DAPI, 4’,6- diamidino-2-phenylindole. (C and D) MA plot and PD-L1 fold change for gene expression level in KL cells obtained from bone marrow of NOD mice transduced with PD-L1 lentivirus as compared to mock-transduced KL cells, demonstrating PD-L1 up-regulation. Experiments were run in triplicate, and statistical analysis was performed using pairwise ANOVA test. (E and F) Representative flow cytometric analysis and quantitative bar graph of IFN-γ+CD4+ T cells isolated from NOD-BDC2.5 T cell receptor (TCR) Tg mice stimulated with BDC2.5 peptide in the presence of DCs (control) or upon coculture with untransduced KL cells (WT), with PD-L1.Tg KL cells (at different ratios), or with PD-L1.Tg KL cells pretreated with anti–PD-L1–blocking mAb, with the isotype control also shown. αPD-L1, anti–PD-L1–blocking mAb. (G and H) Representative flow cytometric analysis and quantitative bar graph of IFN-γ+CD8+ T cells isolated from NOD-8.3 TCR Tg mice stimulated with IGRP peptide in the presence of DCs (control), or upon coculture with WTKL cells, with PD-L1.Tg KL cells (at different ratios), or with PD-L1.Tg KL cells pretreated with PD-L1–blocking mAb. Experiments were run in triplicate, and statistical significance was determined by using two-tailed unpaired t test; #P < 0.05 versus all except Tg 1:10 and anti–PD-L1–blocking mAb (P = ns); §P < 0.05 versus all. (I and J) Representative flow cytometric analysis and quantitative bar graph of IFN-γ+CD4+ T cells isolated from normoglycemic NOD mice stimulated with soluble anti-CD3/anti-CD28 (control), or upon coculture with WT KL cells, with PD-L1.Tg KL cells (at different ratios), or with PD-L1.Tg KL cells pretreated with PD-L1–blocking mAb. PD-L1.Tg KL cells strongly abrogate the CD4- and CD8-restricted autoimmune response and anti–CD3/CD28-dependent T cell stimulation in vitro. All experiments were run at least in triplicate, and statistical significance was determined using two-tailed unpaired t test; #P < 0.05 versus all except WT (P = ns); §P < 0.05 versus all. (K and L) Naive CD4+CD25 T cells isolated from BDC2.5 TCR Tg NOD mice or CD8+ T cells from 8.3 TCR Tg NOD mice and stimulated with BDC2.5 or IGRP islet peptides and CD11c+ DCs were cocultured with KL cells (WT) or PD-L1.Tg KL cells, and the rate of apoptosis of CD4 or CD8T cells was assessed by flow cytometry. PD-L1.Tg KL cells’ effect on cell death in autoreactive CD4+ and CD8+T cells as compared to WT KL cells. Experiments were run in triplicate, and statistical significance was determined by using two-tailed unpaired t test. (M) Quantitative bar graphs for lymphoid and myeloid markers of isolated KL cells before and after lentiviral transduction. GFP+, green fluorescent protein. (N) Newly hyperglycemic NOD mice were treated with WT KL cells, with PD-L1.Tg KL cells, and with doxycycline or were left untreated. (O) Representative immunohistochemical hematoxylin and eosin (H&E) analysis and CD3/insulin staining in serial pancreatic islet tissue sections from PD-L1.Tg KL cell–treated or untreated newly hyperglycemic NOD mice. Histology magnification, ×20. Scale bars, 200 mm. *P < 0.05; **P <0.01; ***P < 0.0001.
Fig. 4.
Fig. 4.. Genetically engineered PD-L1.Tg KL cells traffic to the pancreas in hyperglycemic NOD mice.
(A) Insulitis score: n = 9 sections per group were analyzed. Unt, untreated. (B) OVA rechallenge test. Data are representative of one experiment performed in three mice per group, and statistical significance was determined by using two-tailed unpaired t test. Unimm Unt, unimmunized untreated; Imm Unt, immunized untreated; Imm WT, immunized treated with KL; Imm Tg, immunized treated with PD-L1.Tg KL cells. (C) Immunophenotypic analysis of lymphocytes isolated from spleens by flow cytometry of FoxP3+ regulatory T cells (Tregs) in PD-L1.Tg KL cell–treated NOD mice as compared to untreated NOD mice. Data are representative of one experiment performed in three mice per group, and statistical significance was determined by using two-tailed unpaired t test. (D) Quantification of IFN-γ-producing cells (with number of spots normalized for background) in an ex vivo assay, in which splenocytes were challenged with islet peptides [BDC2.5, IGRP, glutamic acid decarboxylase 65 (GAD-65), and insulin] 40 days after treatment in newly hyperglycemic PD-L1.Tg KL cell–treated NOD mice or in untreated hyperglycemic NOD mice. Data are expressed as means ± SEM, and statistical significance was determined by using two-tailed unpaired t test. Data are representative of at least n = 3 mice. *P < 005; **P < 0.01; ***P < 0.001. #P < 0.05 versus all; §P < 0.05 versus all. (E and G) Representative flow cytometric analysis and quantitative bar graphs of ZsGreen+PD-L1. Tg KL cells in the pancreas of hyperglycemic (Hyper) and normoglycemic (Normo) NOD mice at 1,7, and 14 days after treatment with PD-L1.Tg KL cells. Experiments were run in triplicate [in (F): in duplicate], and statistical significance was determined by using two-tailed unpaired t test. (F and H) Quantification of ZsGreen mRNA in the pancreas of hyperglycemic and normoglycemic NOD mice by qRT-PCR after treatment with PD-L1.Tg KL cells. (I and K) Bar graphs depicting flow cytometric quantification of ZsGreen+PD-L1.Tg KL cells and (J and L) quantification of ZsGreen mRNA by qRT-PCR in the bone marrow of hyperglycemic and normoglycemic NOD mice, after treatment with PD-L1.Tg KL cells. Experiments were run in triplicate [in (M): in duplicate], and statistical significance was determined by using two-tailed unpaired t test. (M and O) Bar graphs for flow cytometric quantification of ZsGreen+PD-L1.Tg KL cells and (N and P) quantification of ZsGreen mRNA by qRT-PCR in the spleen of hyperglycemic and normoglycemic NOD mice. (Q and R) Confocal imaging of pancreatic sections obtained from normoglycemic or hyperglycemic NOD mice after 1,7, and 14 days after treatment with ZsGreen+PD-L1.Tg KL cells. Histology magnification, ×63 in all confocal images. Scale bars, 5 μm. (S and T) Luminescent images of NOD mice adoptively transferred with luciferase+PD-L1.Tg KL cells after 1 and 7 days of treatment. Data are expressed as means ± SEM. Data are representative of at least n = 2 mice. Statistical significance was determined using two-tailed unpaired t test. *P < 0.05; **P < 0.01; ***P < 0.001. CTRL, control.
Fig. 5.
Fig. 5.. pKL cells abrogate the autoimmune response in vitro.
(A to C) Results of screening of small molecules tested for their ability to up-regulate PD-L1 [mean fluorescence intensity (MFI)] on mobilized CD34+ cells obtained from healthy controls, the three-color coding shown in (C) represents lowest PD-L1 MFI values (orange), median PD-L1 MFI values (yellow), and highest PD-L1 MFI values (green). TLR, toll-like receptor; wp, well plate. (D and E) PD-L1 expression (mRNA and MFI) fold change was quantified for each component of the small-molecule cocktail tested singularly or in combination. Tx, treatment. (F) Representative flow cytometric analysis and quantitative bar graph of PD-L1 expression on KL cells from NOD mice pre- and postpharmacological modulation with a cocktail of small molecules (n = 3 from two independent experiments), and statistical significance was performed by using twotailed unpaired t test. (G) Confocal imaging of KL cells pre- and postmodulation with cocktail of small molecules, showing DAPI (in blue) and PD-L1 (in red) staining. Histology magnification, ×63. Scale bar, 50 μm. (H and I) MA plot and fold change for gene expression in KL cells obtained from bone marrow of NOD mice and pKL as compared to unmodulated KL cells (Vehicle-KL cells, WT) n = 3 samples per condition, and statistical significance was performed using pairwise ANOVA test. (J and K) Representative flow cytometric analysis and quantitative bar graph for IFN-γ+CD4+ T cells isolated from NOD-BDC2.5 TCRTg mice and stimulated with BDC2.5 peptide in the presence of DCs (Controls) or upon coculture with unmodulated KL (WT), with pKL cells (at different ratios), or with pKL cells pretreated with anti–PD-L1–blocking mAb with the isotype control also shown; n = 3 samples per condition, and statistical significance was performed using two-tailed unpaired t test. #P < 0.05 versus all; §P < 0.05 versus all except Tg. 1:10. (L) Bar graph for flow cytometric quantification of IFN-γ+CD4+T cells after coculture of CD4+ T cells isolated from NOD-BDC2.5 TCR Tg mice stimulated with BDC2.5 peptide in the presence of DCs (control) or upon coculture with unmodulated KL cells (WT), with pKL cells, with PD-L1.Tg KL cells, or with CD4+CD25+ T regulatory cells; n = 4 samples per condition were used, and statistical significance was performed using two-tailed unpaired t test; #P < 0.05 versus all. (M and N) Representative flow cytometric analysis and quantitative bar graph for IFN-γ+CD8+ T cells isolated from NOD-8.3 TCR Tg mice and stimulated with IGRP peptide in the presence of DCs (control) or upon coculture with unmodulated KL cells (WT), with pKL cells (at different ratios), or with pKL cells pretreated with anti–PD-L1–blocking mAb. Experiments were run in triplicate (n = 3 for controls, all the rest n ≥ 5), and statistical analysis was performed using two-tailed unpaired t test. (O) Bar graph for flow cytometric quantification of IFN-γ+CD8+T cells after coculture of CD8+T cells isolated from NOD-8.35 TCR Tg mice stimulated by IGRP peptide in the presence of DCs (control) or upon coculture with unmodulated KL cells(WT), with pKL cells, with PD-L1.Tg KL cells, or with CD4+CD25+T regulatory cells. Experiments were run in triplicate (n = 4 for controls and WT, all the rest n = 3), and statistical analysis was performed using two-tailed unpaired t test (P and Q) Representative flow cytometric analysis and quantitative bar graph for IFN-γ+CD4+ T cells isolated from NOD mice and stimulated with soluble anti-CD3/anti-CD28 (control) or upon coculture with unmodulated KL cells (WT), with pKL cells (at different ratios), or with pKL cells pretreated with PD-L1 -blocking/-neutralizing mAb. Experiments were run in triplicate [n = 3 for all conditions except for anti–PD-Ll (n = 4)], and statistical analysis was performed using two-tailed unpaired t test; in (R): #P < 0.05 versus all except anti–PD-Ll–blocking mAb and pKL 1:10 (P = ns); §P < 0.05 versus all. (R) Bar graph depicting flow cytometric quantification of IFN-γ+CD4+ Tcells within CD4+CD25 T cells isolated from NOD mice and stimulated with soluble anti-CD3/anti-CD28 (control) or upon coculture with WT, with pKL, with PD-L1.Tg KL cells, or with CD4+CD25+ T regulatory cells. Experiments were run in triplicate [n = 3 for all conditions, except for WT (n = 4)], and statistical analysis was performed using two-tailed unpaired t test. Data are expressed as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.0001. #P < 0.05 versus all; §P < 0.05 versus all.
Fig. 6.
Fig. 6.. pKL cells revert hyperglycemia in NOD mice in vivo.
(A and B) Newly hyperglycemic NOD mice were treated with WT cells, pKL cells, or Tg cells. Data are representative of 10 untreated mice, 10 mice treated with WT cells, 10 mice treated with pKL cells, and 15 mice treated with Tg cells. Incidence of diabetes in all groups of NOD mice (untreated, WT-treated, pKL-treated, and Tg-treated mice) was compared using the log-rank (Mantel-Cox) test;#P <0.0001 versus all, P < 0.0001 for Tg versus WT, and P <0.05 for pKL versus WT. (C) Quantification of IFN-γ–producing cells in an in vitro assay, in which splenocytes isolated from newly hyperglycemic pKL-treated NOD mice or in untreated hyperglycemic NOD mice 40 days after treatment were challenged with islet peptides after treatment with pKL, normalized for background. Experiments were run in triplicate, and statistical analysis was performed using two-tailed unpaired t test. (D) Immunophenotype of lymphocytes isolated from spleen of pKL-treated newly hyperglycemic NOD mice showed an increase in the percentage of FoxP3+ regulatory T cells. Experiments were run in triplicate (n = 3 pKL-treatedand n = 5 untreated), and statistical analysis was performed using two-tailed unpaired t test. (E to G) Bar graph showing the levels of pro- (IL-2/IL-6) and anti-inflammatory (IL-4) cytokines as measured by Luminex in the serum of untreated or pKL-treated newly hyperglycemic NOD mice at baseline and at 7 days after treatment. Experiments were run in triplicate per group, and condition (D0 and D7) and statistical analysis were performed using two-tailed unpaired t test. (H) OVA rechallenge. Experiments were run in triplicate per group, and statistical analysis were performed using two-tailed unpaired t test, #P < 0.05 versus all. (I) Insulitis score in untreated and pKL-treated newly hyperglycemic NOD mice. (J and K) Representative immunohistochemical H&E analysis and CD3/insulin staining in serial pancreatic islet tissue sections from pKL-treated or untreated NOD mice; n = 9 sections per group were analyzed. Histology magnification, ×20. Scale bars, 200 μm. Data are expressed as means ± SEM. Data are representative of at least n = 3 mice. *P < 0.05; **P < 0.01; ***P < 0.0001; #P < 0.05 versus all.
Fig. 7.
Fig. 7.. The PD-L1 defect is evident in HSPCs from T1D patients.
(A) Representative flow cytometric and quantitative bar graph of PD-L1+CD34+ cells from patients with T1D as compared to healthy controls (n = 10 from each group), and statistical significance was performed by using two-tailed unpaired t test. (B) Western blot analysis and (C) qRT-PCR confirmed the PD-L1 defect in CD34+ cells from T1D patients (n = 3 in each group), and statistical significance was performed by using two-tailed unpaired t test. (D and E) Confocal imaging and quantitative bar graph of bone marrow sections obtained from T1D patients and healthy controls showing PD-L1 (green) and CD34 (red) staining; the quantification of the orange-stained bone marrow element was performed by ImageJ. Data are representative of n = 5 sections per group, and statistical significance was performed using two-tailed unpaired t test with Welch’s correction. Histology magnification, ×63. Scale bar, 40 μm. (F) Bar graph depicting the percentage of PD-L1 on CD34+ cells obtained from peripheral blood ofT1D patients or healthy controls at baseline or cultured for 3 days in normal glucose, in 20 mM, or in 35 mM high glucose (n = 3 samples from each group), and statistical significance was performed using two-tailed unpaired t test; #P < 0.05 versus all except T1D HG-35 and CTRL HG-20; §P < 0.05 versus all except T1D HG-35 (ns) and T1D HG-20 (ns). (G) CFSE-based proliferation assay of peripheral CD34+ cells obtained from T1D and healthy control patients at baseline and after 1 and 3 days of culture (n = 3 samples from each group), and statistical significance was performed using two-tailed unpaired t test; #P < 0.0001 versus all except T1D-D0; §P < 0.0001 versus all except CTRL-D0 (ns). (H) Frequency of apoptosis of CD34+ cells obtained from T1D and healthy control patients at baseline and after 1 and 3 days of culture. Experiments were run in triplicate, and statistical significance was performed using two-tailed unpaired t test. (I) Table of human miRNAs, discovered by bioinformatic approach, involved in the regulation of PD-L1 expression. (J) qRT-PCR showed differentially expressed miRNA in human CD34+ cells obtained from T1D patients as compared to controls (at least n = 5 samples from each group), and statistical significance was performed using two-tailed unpaired t test with Welch’s correction. (K) DNA methylation status of the PD-L1 gene promoter in peripheral CD34+ cells obtained from T1D patients as compared to healthy controls (n = 2 samples from each group). Statistical significance was performed using two-tailed unpaired t test with Welch’s correction. (L) Representative flow cytometric and quantitative bar graph of PD-L1 expression on peripheral CD34+ cells from T1D patients pre- and postpharmacological modulation with a cocktail of small molecules. Experiments were run in triplicate, and statistical significance was performed using two-tailed unpaired t test. (M) Confocal imaging of PD-L1 expression on CD34+ cells from T1D patients pre- and postpharmacological modulation. Histology magnification, ×63. Scale bar, 50 μm. (N) PD-L1 expression fold change in pCD34+ after 24 hours and in vehicle-treated CD34+ cells as assessed by RT-PCR. Experiments were run in duplicate, and statistical significance was performed using two-tailed unpaired t test. (O to R) Quantification of IFN-γ–producing cells where human PBMCs from T1D were challenged with IA-2 in the presence of unmodulated CD34+ or pCD34+ cells with or without an anti–PD-L1–blocking mAb. Experiments were run in triplicate, and statistical significance was performed using two-tailed unpaired t test. Experiments were performed at least in triplicate; in (R): Data related to anti–PD-L1 treatment were performed in duplicate, and statistical significance was performed using two-tailed unpaired t test. (S) Quantification of IFN-γ–producing cells where human PBMCs from T1D already stimulated with anti-CD3/anti-CD28 were cocultured in the presence of unmodulated CD34+ or pCD34+ cells with or without an anti–PD-L1–blocking mAb. Experiments were run in duplicate, and statistical significance was performed using two-tailed unpaired t test. Data are expressed as means ± SEM. *P < 0.05; **P <0.01; ***P < 0.001. #P < 0.05 versus all.

Comment in

Similar articles

See all similar articles

Cited by 13 articles

See all "Cited by" articles
Feedback