1. Increase of Intermediate Monocytes in Graft-versus-Host Disease: Correlation with MDR1+Th17.1 Levels and the Effect of Prednisolone and 1α,25-Dihydroxyvitamin D3 
Katharina Reinhardt-Heller, Insa Hirschberg, Peter Lang, Thomas Vogl, Rupert Handgretinger, Wolfgang A. Bethge, Ursula Holzer 
Biology of Blood and Marrow Transplantation 
Volume 23, Issue 12, Pages (December 2017)
DOI: /j.bbmt
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

2. Figure 1
Percentages of monocyte subtypes in patients with GVHD correlated with MDR1+Th17.1 cells. (A) The percentages of classical, intermediate, and nonclassical monocytes were determined in PBMCs from healthy donors (n = 10) and patients with acute GVHD (n = 6) and chronic GVHD (n = 5) by flow cytometry. Data are from 10 independent experiments. Statistical significance was determined using Student's unpaired t-test: *P < .05, **P < .01, ***P < .001. The level of induced MDR1+Th17.1 cells was correlated with the percentages of (B) classical, (C) intermediate, and (D) nonclassical monocytes in the whole monocyte population from patients with acute GVHD. Statistical significance was performed using Pearson's correlation (n = 6).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

3. Figure 2
Influence of monocyte subpopulations on the induction of MDR1+Th17.1 cells. (A) After isolating monocyte subtypes from PBMCs from healthy donors by magnetic cell separation, the purity was assessed by flow cytometry. (B) CD4+ T cells were isolated from healthy donors and were co-cultured with anti-CD3 Ab (10 ng/mL) and monocyte subtypes at a monocyte-to-T cell ratio of 1:4 for 5 days. The induced percentages of MDR1+Th17.1 cells were assessed by flow cytometry. MDR1 expression was determined indirectly by Rh123 efflux. Data are from 8 independent experiments. Statistical significance was determined using Student's paired t-test. *P < .05; **P < .01 (n = 8).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

4. Figure 3
Influence of prednisolone on the induction of MDR1+Th17.1 cells. CD4+ T cells from healthy donors were co-cultured with anti-CD3 Ab (10 ng/mL) and monocytes from healthy donors in the presence or absence of prednisolone (.1 µM) at a monocyte-to-T cell ratio of 1:4 for 5 days. The induced percentages of MDR1+Th17.1 cells were assessed by flow cytometry. MDR1 expression was determined indirectly by Rh123 efflux. Data are from 24 independent experiments. Statistical significance was determined using Student's paired t-test. **P < .01 (n = 24).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

5. Figure 4
Influence of 1α,25-(OH)2D3 on the induction of MDR1+Th17.1 cells. Monocytes from healthy donors were pretreated with 1α,25-(OH)2D3 (.1 µM) for 16 hours. The next day, monocytes were optionally stimulated with S100A8/S100A9 for 4 hours before co-culturing them with CD4+ T cells from healthy donors with anti-CD3 Ab (10 ng/mL) in the presence and absence of prednisolone (.1 µM) at a monocyte-to-T cell ratio of 1:4 for 5 days. The induced percentages of MDR1+Th17.1 cells were assessed by flow cytometry. MDR1 expression was determined indirectly by Rh123 efflux. Data are from 7 independent experiments. Statistical significance was determined using Student's paired t-test. *P < .05 (n = 7).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

6. Figure 5
Percentages of monocyte subpopulations after 1α,25-(OH)2D3 treatment correlated with the level of induced MDR1+Th17.1 cells or the expression of IL-6 in monocytes. Monocytes from healthy donors were pretreated with 1α,25-(OH)2D3 (.1 µM) for 16 hours and the percentages of (A) classical, (B) intermediate, and (C) nonclassical monocytes were determined by flow cytometry. Alternatively, monocytes were co-cultured with CD4+ T cells from healthy donors with anti-CD3 Ab (10 ng/mL) at a monocyte-to-T cell ratio of 1:4 for 5 days. The induced percentages of MDR1+Th17.1 cells were assessed by flow cytometry. MDR1 expression was determined indirectly by Rh123 efflux. The percentages of MDR1+Th17.1 cells induced by 1α,25-(OH)2D3 treated monocytes were correlated with the level of (D) classical, (E) intermediate, and (F) nonclassical monocytes in the whole monocyte population after 1α,25-(OH)2D3 treatment. Additionally, IL-6 expression in monocytes was assessed by real-time PCR analysis using genomic DNA. Cycle threshold values for IL-6 were normalized to the housekeeping gene RPL13A. IL-6 expression levels in monocytes were correlated with the percentages of (G) classical, (H) intermediate, and (I) nonclassical monocytes in the whole monocyte population after 1α,25-(OH)2D3 treatment. Data are from 8 independent experiments. Statistical significance was performed using Pearson's correlation (n = 8).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

7. Figure 6
Influence of 1α,25-(OH)2D3 on JAK2 expression in monocytes and CD74 expression on monocytes. Monocytes from healthy donors were pretreated with 1α,25-(OH)2D3 (.1 µM) for 16 hours. The next day, monocytes were optionally stimulated with S100A8/S100A9 for 4 hours. (A) JAK2 expression in monocytes was assessed by real-time PCR analysis using genomic DNA. Cycle threshold values for JAK2 were normalized to the housekeeping gene RPL13A. Statistical significance was determined using Student's paired t-test: **P < .01 (n = 7). (B) CD74 expression on monocytes was determined by flow cytometry. Therefore, either the whole monocyte population, classical, intermediate, or nonclassical monocytes, were gated. Statistical significance was determined using Student's paired t-test: *P < .05 (n = 7; mean + standard deviation).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions