Fig 1.
Changes in PD-L1 expression in H. pylori infected human stomach and histological grade of HGOs.
H&E staining of biopsies collected from a (A) normal uninfected and (B) H. pylori (HP) infected patient stomach. Immunofluorescence staining for PD-L1 (red) and SPEM marker GSII (cyan) and gastric cancer stem cell marker CD44v9 in (C) uninfected and (D) HP infected patient stomach. Co-localization of PD-L1 (red) and CD44v9 (green) is seen in (E). H&E staining of organoids embedded from (F) control, (G) HP infected, (H) HP+GANT61, and (I) GANT61 treated, and (J) CagA mutant HP strain (ΔCagA) infected FHGOs. Histological grade assigned to each experimental group from (K) FHGOs. *P<0.05 compared to control group by one-way ANOVA, n = 8 individual organoids.
Fig 2.
Metaplastic changes in FHGOs and AHGOs infected with H. pylori.
Histological grade was assigned to experimental groups using Intralumenal Epithelial Neoplasia criteria of (A) 0, (B) 1, (C) 2 or (D) 3. H&E staining of organoids embedded from (E) control, (F) HP infected, (G) HP infected and GANT61, and (H) GANT61 treated, and (I) ΔCagA infected AHGOs. Histological grade assigned to each experimental group from (J) AHGOs. *P<0.05 compared to control group by one-way ANOVA, n = 8 individual organoids.
Fig 3.
Characterization of Helicobacter pylori infected FHGOs and AHGOs.
Immunohistochemistry for HP colonization in sections collected from (A, B) control magnified (C, D) HP and (E, F) ΔCagA infected FHGOs. Immunohistochemistry for gastric cancer stem cell marker CD44v9 in sections collected from (G, H) control magnified (I, J) HP and (K, L) ΔCagA infected FHGOs. Immunohistochemistry of PCNA (proliferating cells) in (M) control, (N) HP infected, (O) HP infected and GANT 61 treated, (P) GANT61 treated and (Q) ΔCagA infected FHGOs. Immunohistochemistry of PCNA in (R) control, (S) HP infected, (T) HP infected and GANT 61 treated, (U) GANT61 treated and (V) ΔCagA infected AHGOs. (W) Quantification of PCNA expressing epithelial cells in FHGOs and AHGOs. *P<0.05 compared to control group by one-way ANOVA, n = 3 area’s averaged for 3 individual organoids.
Fig 4.
Phenotypic characterization of parietal cells in FHGOs and AHGOs.
(A) Immunohistochemistry of H+/K+ ATPase in sections collected from FHGOs. Higher magnification shown in (B). Acridine orange accumulation assay of FHGOs (C) before, (D) after histamine stimulation, and (E) 24 hours after HP infection. (F) Quantification of the ratio shift in F458 (red)/F488 (green) in treated FHGOs and AHGOs. *P<0.05 compared to control group by Student’s T test, n = 3 individual cells. Immunofluorescence staining of sections collected from (G) FHGOs and (H) AHGOs for the H+/K+ ATPase (green) or Shh (red). (I) Western blots for the expression of GAPDH and Shh in control, and HP and ΔCagA infected FHGOs and AHGOs.
Fig 5.
Changes in PD-L1 expression in FHGOs and AHGOs infected with H. pylori.
Immunofluorescence staining for PD-L1 (green), GSII (red) and TFF2 (blue) in (A) uninfected control, (B) GANT61 treated, (C) H. pylori (HP) infected, (D) HP and GANT61 treated and (E) ΔCagA infected FHGOs. (F) Western blot analysis for the expression of PD-L1 using whole cell lysate collected from each treatment group of the FHGOs and AHGOs. (G) Western blots were quantified using ImageJ. *P<0.05 compared to control groups by one-way ANOVA, n = 3 individual organoid experiments.
Fig 6.
Changes in PD-L1, Hedgehog signaling and markers of SPEM in FHGOs and AHGOs infected with H. pylori.
Quantitative RT-PCR analysis for the expression of PD-L1, Shh and SPEM markers clusterin (CLU) and human epididymis 4 (HE4) performed using RNA collected from each experimental group of the (A) FHGOs and (B) AHGOs. Quantitative RT-PCR analysis for the expression of canonical Hedgehog signaling genes Gli1, Ptch 1, Ptch 2 and Hedgehog Interacting Protein (HHIP) was performed using RNA collected from each experimental group of the (C) FHGOs and (D) AHGOs. *P<0.05 compared to control group by two-way ANOVA, n = 3 individual organoid experiments.
Fig 7.
Generation of human-derived gastric epithelial monolayers.
(A) Light micrographs of organoids transferred to monolayers 48 hours after culture. (B) Immunofluorescence of monolayers showing apical expression of surface mucous cell marker UEA1 (green), E-cadherin (E cad, red) and Hoechst (blue). (C) Immunofluorescence of monolayers showing expression of parietal cell marker H+,K+-ATPase (HK, green), E cad (red) and Hoechst (blue). (D) DNA gel from RT-PCR using RNA collected from gastric tissue, glands, organoids and monolayers.
Fig 8.
Phenotypic characterization of parietal cells in gastric epithelial monolayers and 3D gastric organoids.
Acridine orange accumulation in of gastric epithelial monolayers (A) before, and (B) after histamine stimulation, (C) before and (D) after HP infection. Acridine orange accumulation of 3D gastric organoids (E) before and after histamine stimulation, and (F) after a 24 hour HP infection. (G) Quantification of the ratio shift in F458 (red)/F488 (green) in treated gastric epithelial monolayers during treatment (Histamine) or HP infection (H. pylori), and during histamine treatment of 3D gastric organoids (huFGO). *P<0.05 compared to control group by one-way ANOVA, n = 3 individual cells. Immunofluorescence staining for H. pylori (green), H+/K+ ATPase positive parietal cells (red) and Shh (cyan) in (H) control, or (I) HP infected gastric epithelial monolayers. (J) Western blots for the expression of GAPDH and Shh in control, HP and ΔCagA infected gastric epithelial monolayers.
Fig 9.
Changes in PD-L1 expression in response to H. pylori using human-derived gastric epithelial cell monolayers.
Immunofluorescence stain of gastric epithelial monolayers collected from (A) control, (B) H. pylori (HP), (C) HP + GANT61 treated and (D) GANT61 treated monolayers for E cadherin (E cad, red) and PD-L1 (blue). (E) Quantitative RT-PCR analysis for the expression of PD-L1, Shh and canonical Hedgehog signaling genes Gli1, Ptch 1, Ptch 2 and Hedgehog Interacting Protein (HHIP) was performed using RNA collected from each experimental group. *P<0.05 compared to control group by two-way ANOVA, n = 3 individual organoid experiments.
Fig 10.
Changes in SPEM markers, and Shh in response to H. pylori infection using human-derived gastric epithelial cell monolayers.
Immunofluorescence stain for PD-L1 (green), GSII (red) and TFF2 (blue) of (A) control, (B) H. pylori (HP) infected, (C) HP+GANT61 treated, and (D) GANT61 treated monolayers. (E) Quantitative RT-PCR analysis for the expression of SPEM markers CLU and HE4 was performed using RNA collected from each experimental group. *P<0.05 compared to control group by two-way ANOVA, n = 3 individual organoid experiments. Flow cytometric analysis of cells co-expressing Shh and the H+/K+ ATPase, the mucus neck cell marker GSII and Pepsinogen A (PgA), or GSII and PD-L1 in (F) the unstained population, and (G) 0, (H) 6, (I) 12, and (J) 24 hours post HP infection. Quantification of the percent of (K) H+/K+ ATPase positive cells that co-express SHH, (L) GSII positive cells that co-express PgA, and (M) GSII positive cells that co-express PD-L1, 0, 6, 12 and 24 hours post-infection with HP. *P<0.05 compared to control group by one-way ANOVA, n = 3 individual organoid experiments.
Fig 11.
Organoid/dendritic cell and cytotoxic T lymphocyte co-cultures.
(A) Schematic diagram demonstrating the co-culture of human-derived gastric organoids (huFGOs) and autologous dendritic cells (DCs) and cytotoxic T lymphocytes (CTLs). Representative flow cytometric dot plots of (B) unstained control sample, and (C) HLA-DRhigh dendritic cells. (D) Quantitative flow cytometric analysis for the percentage of live CD8+ve CTLs extracted from co-cultures expressing PD-1, IL-2 and IFNγ. *P<0.05 compared to control group by two-way ANOVA, n = 3 individual organoid experiments. (E) Percentage of CD8+ non-proliferating, low, moderately and high proliferating cells determined by CFSE staining *P<0.05 compared to control group by two-way ANOVA, n = 3 individual organoid experiments, and (F) proliferative index. Quantitative flow cytometric analysis of control or HP infected (G) % live EpCam positive cells, (H) %EpCam Positive cells expressing PD-L1. *P<0.05 compared to control group by one-way ANOVA, n = 3 individual organoid experiments.
Fig 12.
Changes in epithelial cell viability in human-derived organoid/immune cell co-cultures infected with H. pylori.
Light micrographs, immunofluorescence staining for E cadherin (E cad, green) and CD8 (red), and flow cytometric dot plots for percentage of Zombie (dead) PD-L1+ve cells in (A-C) control, (D-F) PD-1Inh treated, (G-I) H. pylori (HP) infected, and (J-L) HP+PD-1Inh treated organoid/immune cell co-cultures, and (M-O) PD-1Inh treated organoids and (P-R) HP infected organoids. (S) Flow cytometric analysis for the percentage of PD-L1 positive cells from control, PD-1I, HP infected and HP+PD-1I treated co-cultures. (T) Flow cytometric analysis for the percentage of PD-L1+ve gated Zombie (dead) cells from control, PD-1I, HP infected and HP+PD-1I treated co-cultures. *P<0.05 compared to control group by one-way ANOVA, n = 3 individual organoid experiments.
Fig 13.
Proposed model of H. pylori induced PD-L1 expression in the gastric epithelium.
(A) Shh secretion is induced from the acid secreting parietal cells in response to H. pylori infection that is driven by a CagA dependent mechanism. (B) We propose that Shh induces the expression of PD-L1 on GSII/PgA transdifferentiated cells. PD-L1 then interacts with PD-1 on the surface of CTLs and shuts down the CTL effector function which may lead to the survival of these transdifferentiated metaplastic cells. (C) The addition of PD-1Inh blocks the interaction between PD-1 on CTLs and PD-L1 on transdifferentiated/SPEM cells allowing the CTLs to destroy these cells.