Figure 1.
RA-responsive mononuclear cells are increased in areas adjacent to GI-GVHD tissue damage. (A) RARα protein expression measured by flow cytometry in human PBMCs allostimulated in the presence or absence of exogenous RA. Example pseudodot plots are shown. Cells with high expression of RARα are shown in Region 1 (R1). (B) RARα protein expression in human PBMCs allostimulated in the presence or absence of exogenous RA. Results depict 18 independent experiments. (C) Identification of RARαhi mononuclear cells in GI biopsies using IHC staining for RARα (upper panel, brown) with hematoxylin counterstaining (blue). After antigen retrieval, tissue was stained with rabbit anti-human RARα antibody (Clone F-9; Santa Cruz Biotechnology) for 40 minutes at room temperature. Sections were then stained using the Biogenex SuperSensitive Polymer-HRP IHC Kit and diaminobenzidine (DAB) chromogen and were counterstained with hematoxylin. Mononuclear cells were identified by size and nuclear staining characteristics (middle panel, blue dots), and RARα staining intensity was digitized using the Ariol Olympus microscope and automated image analysis system (middle panel, red). A threshold of RARα staining intensity was set to identify cells with high RARα expression (lower panel, yellow dots). RARαhi mononuclear cells (lower panel, black arrows) were distinguished from mononuclear cells with lower expression of RARα (lower panel, green arrows). Scale bars, 10 μm. (D) Correlation between RARαhi mononuclear cell numbers and cellular RA-binding protein CRABPI/II+ cell numbers in upper and lower GI biopsies from allogeneic hematopoietic stem cell transplantation (allo-HSCT) patients. Solid lines denote linear regression and dotted lines denote 95% confidence intervals. P and r values are for Pearson correlation. (E) Representative GI biopsy stained for RARα using conventional IHC and DAB chromogen. Subcrypt regions are outlined in black and are indicated by arrows. Scale bar, 50 μm. (F) Enrichment of RARαhi mononuclear cell numbers enumerated using the Ariol automated system in subcrypt regions of GI biopsies. (G) Representative GI biopsies from healthy controls and allo-HSCT patients with and without histologic GVHD were stained for RARα using conventional IHC and DAB chromogen. Scale bars, 50 μm. Black arrows depict mononuclear cells with high expression of RARα. (H) RARαhi mononuclear cell numbers in subcrypts of GI biopsies from allo-HSCT patients and healthy controls with and without histologic GVHD. (I) Receiver operating characteristic curves for subcrypt RARαhi cell numbers, RARαhi cell intensity, and CRABPI/II+ cell numbers and the presence of histologic GVHD. (J) RARαhi mononuclear cell numbers in subcrypts of upper (left) and lower (right) GI biopsies with and without histologic GVHD. Horizontal lines on graphs depict medians. AUC, area under the curve; FSC, forward scatter; LR, likelihood ratio; ns, not significant. *P < .05; **P < .01; ***P < .001; ****P < .0001, Mann-Whitney U tests (B,F) and analysis of variance (ANOVA) tests with correction for multiple comparisons (H,J).

RA-responsive mononuclear cells are increased in areas adjacent to GI-GVHD tissue damage. (A) RARα protein expression measured by flow cytometry in human PBMCs allostimulated in the presence or absence of exogenous RA. Example pseudodot plots are shown. Cells with high expression of RARα are shown in Region 1 (R1). (B) RARα protein expression in human PBMCs allostimulated in the presence or absence of exogenous RA. Results depict 18 independent experiments. (C) Identification of RARαhi mononuclear cells in GI biopsies using IHC staining for RARα (upper panel, brown) with hematoxylin counterstaining (blue). After antigen retrieval, tissue was stained with rabbit anti-human RARα antibody (Clone F-9; Santa Cruz Biotechnology) for 40 minutes at room temperature. Sections were then stained using the Biogenex SuperSensitive Polymer-HRP IHC Kit and diaminobenzidine (DAB) chromogen and were counterstained with hematoxylin. Mononuclear cells were identified by size and nuclear staining characteristics (middle panel, blue dots), and RARα staining intensity was digitized using the Ariol Olympus microscope and automated image analysis system (middle panel, red). A threshold of RARα staining intensity was set to identify cells with high RARα expression (lower panel, yellow dots). RARαhi mononuclear cells (lower panel, black arrows) were distinguished from mononuclear cells with lower expression of RARα (lower panel, green arrows). Scale bars, 10 μm. (D) Correlation between RARαhi mononuclear cell numbers and cellular RA-binding protein CRABPI/II+ cell numbers in upper and lower GI biopsies from allogeneic hematopoietic stem cell transplantation (allo-HSCT) patients. Solid lines denote linear regression and dotted lines denote 95% confidence intervals. P and r values are for Pearson correlation. (E) Representative GI biopsy stained for RARα using conventional IHC and DAB chromogen. Subcrypt regions are outlined in black and are indicated by arrows. Scale bar, 50 μm. (F) Enrichment of RARαhi mononuclear cell numbers enumerated using the Ariol automated system in subcrypt regions of GI biopsies. (G) Representative GI biopsies from healthy controls and allo-HSCT patients with and without histologic GVHD were stained for RARα using conventional IHC and DAB chromogen. Scale bars, 50 μm. Black arrows depict mononuclear cells with high expression of RARα. (H) RARαhi mononuclear cell numbers in subcrypts of GI biopsies from allo-HSCT patients and healthy controls with and without histologic GVHD. (I) Receiver operating characteristic curves for subcrypt RARαhi cell numbers, RARαhi cell intensity, and CRABPI/II+ cell numbers and the presence of histologic GVHD. (J) RARαhi mononuclear cell numbers in subcrypts of upper (left) and lower (right) GI biopsies with and without histologic GVHD. Horizontal lines on graphs depict medians. AUC, area under the curve; FSC, forward scatter; LR, likelihood ratio; ns, not significant. *P < .05; **P < .01; ***P < .001; ****P < .0001, Mann-Whitney U tests (B,F) and analysis of variance (ANOVA) tests with correction for multiple comparisons (H,J).

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