1. Volume 135, Issue 6, Pages 2119-2127 (December 2008)
Myeloid Dendritic Cells of Patients With Chronic HCV Infection Induce Proliferation of Regulatory T Lymphocytes 
Angela Dolganiuc, Edward Paek, Karen Kodys, Joanne Thomas, Gyongyi Szabo 
Gastroenterology 
Volume 135, Issue 6, Pages (December 2008)
DOI: /j.gastro
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2. Figure 1
Myeloid dendritic cells of HCV patients differentially inhibit proliferation of CD4+ T cells depending on CD25 expression in an IL-2- and IL-10-dependent manner. (A) Monocyte-derived dendritic cells of controls (Control) or cHCV patients (HCV-DC) were incubated with allogeneic control CD4+ T cells for 5-day MLR without or with anti-IL-10 antibodies (10 μg/mL) or recombinant IL-2 (25 μg/mL), and the proliferation was assessed by the incorporation of 3H-thymidine (3H-Td) during the last 16 hours of assay. The average counts per minute (cpm) ± SE from n = 4 are shown; asterisk (*) indicates P < .05. (B and C) The cell-free supernatants of MLR culture as above (A) were analyzed for IL-10 (B) or IL-2 (C) content using specific ELISA. *Indicates P < .05. (D) BDCA1+ MDCs of controls and HCV patients were included in MLR with allogeneic CD4+ T cells as described above (A). (E and F) The cell-free supernatants of MLR culture with BDCA1+ MDCs (D) were analyzed for IL-10 (E) or IL-2 (F) content using specific ELISA. *Indicates P < .05. (G) The monocyte-derived and BDCA1+ MDCs of controls (open bars, n = 4) or cHCV patients (solid bars, n = 4) were incubated with allogeneic CD4+ depleted of CD25+ population (ie, CD4+CD25− T cells) as described above (A). The proliferation was assessed by the incorporation of 3H-thymidine (3H-Td) during the last 16 hours of assay; the average counts per minute (cpm) ± SE from n = 4 are shown; the asterisk (*) indicates P < .05. (H) The cell-free supernatants of MLR culture described in G were analyzed for IL-2 content using specific ELISA. *Indicates P < .05.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Dendritic cells of HCV patients selectively stimulate proliferation of CD4+ CD25+FoxP3+ Tregs. (A) The MLR was set up with CFSE-labeled allogeneic control CD4+ T cells as responders and control or HCV mo-DCs or BDCA1+MDC as stimulators. At the end of MLR, T cells were labeled with specific anti-CD25 and anti-FoxP3 antibodies, and the CD4+CD25+FoxP3+ population was analyzed for CFSE content using flow cytometry. CFSE-labeled T cells cultured without DCs are included as controls (top histogram); the gate represents CFSElow cells suggestive of proliferation and was carried over to all experimental samples. Representative histograms from 4 individuals in each group out of n = 6 are shown. (B) The content of MLR containing monocyte-derived or BDCA1+MDC of control individuals (Control, open bar) or HCV patients (HCV, solid bar) and allogeneic control CD4+ T cells was analyzed for the expression of FoxP3 using specific primers in real-time PCR. The data were normalized to 18S housekeeping control. The FoxP3 content in MLR with control DCs was arbitrarily considered as equal to 1, and the fold change in FoxP3 in the MLR with HCV-DC was calculated. *Indicates a value of P < .05, n = 10 for mo-DCs and n = 4 for MDCs. (C) The CD4+ T cells were included in “primary” MLR with mo-DCs of control or HCV patients, as described in Figure 1A. After 5 days of primary MLR, the CD4+CD25+ T cells were separated from CD4+CD25− T cells with magnetic beads, and similar numbers of CD4+CD25+ and CD4+CD25− T cells at different ratios (1:40, 1:20, 1:5) were included in a “secondary” MLR with normal mo-DCs. The proliferation was assessed by the incorporation of 3H-thymidine (3H-Td) during the last 16 hours of the secondary MLR assay. The proliferation of CD4+CD25− T cells during secondary MLR was considered as 100%, and the fraction of proliferation of CD4+CD25+/CD4+CD25− T cells at a different ratio was calculated as MLR index. The mean index ± SE from n = 4 is shown; the asterisk (*) indicates P < .05 compared with corresponding CD4+CD25− T cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

4. Figure 3
Dendritic cells of HBV but not those of NASH patients exhibit poor allostimulatory capacity and selectively stimulate proliferation of CD4+ CD25+FoxP3+ Tregs. (A) The mo-DCs of control (n = 4) and HBV patients (n = 3) and those of NASH patients (n = 4) were employed as stimulators in a 5-day MLR assay with allogeneic control CD4+ T cells. The proliferation was assessed by the incorporation of 3H-thymidine (3H-Td) during the last 16 hours of assay. The average counts per minute (cpm) ± SE are show; the asterisk (*) indicates P < .05. (B) The MLR was set up with CFSE-labeled allogeneic control CD4+ T cells as responders and control or HBV or NASH mo-DCs as stimulators. At the end of MLR, T cells were labeled with specific anti-CD25 and anti-FoxP3 antibodies, and the CD4+CD25+FoxP3+ population was analyzed for CFSE content using flow cytometry. The gate represents CFSElow cells, suggestive of T-cell proliferation, and it was set-up as described in Figure 2A. Three representative histograms out of n = 4 for NASH patients and n = 3 for HBV are shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

5. Figure 4
HCV-infected patients have higher frequency of CD4+CD25+ T cells compared with controls. (A) The frequency of CD4+CD25+ T cells in peripheral blood mononuclear cells (PBMCs) of controls and cHCV patients was assessed by flow cytometry analysis. The mean percent positive cells ± SE is shown. (B) Equal amounts of RNA from equal numbers of PBMCs of controls and cHCV patient were analyzed for FoxP3 content using specific primers in real-time PCR. The data were normalized to 18S housekeeping gene, the FoxP3 content in control PBMCs was arbitrarily considered as equal to 1, and the fold increase in FoxP3 content of HCV cells was calculated. The mean fold change ± SD is shown. (C) Equal amounts of RNA from CD4+CD25− and CD4+CD25+ T cells of controls and cHCV patients were analyzed for FoxP3 content using specific primers in real-time PCR. The data were normalized to 18S housekeeping gene, the FoxP3 content in CD4+CD25+ T cells of controls was arbitrarily considered as equal to 1, and the fold increase in FoxP3 content in all other groups was calculated. The mean fold change ± SD is shown. (D) The CD4+CD25− and the CD4+CD25+ T cells were separated from peripheral blood of controls (T cells of Control, open bars) or HCV-infected patients (solid bars) and included in allogeneic MLR with control DCs, as indicated. A 1:10 and 1:20 ratio of CD4+CD25−/CD4+CD25+ T cells was analyzed. The proliferation was assessed by the incorporation of 3H-thymidine (3H-Td) during the last 16 hours of assay. *Indicates a value of P < .05 compared with corresponding CD4+CD25− T cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

6. Figure 5
HCV DC induced expression of costimulatory molecules during MLR. Monocyte-derived DCs of control or cHCV patients (HCV-DC) were incubated with allogeneic control CD4+ T cells; CD4+ T cells alone were used as controls. At the end of MLR, the RNA was extracted and analyzed for PDL2 (CD273) (A), ICOS-L (B), CD276 (C), PD-L1 (CD274) (D), BTLA (E), ICOS (F), and PD-1 (G) expression. The values were normalized to 18S gene, the content of CD4+ T cells group was considered as equal to 1, and the fold change in all MLR groups was calculated from n = 8. (H) The CD4+ T cells were included in MLR with control or HCV mo-DCs in the presence or absence of anti-PD-1 antibody. The proliferation was assessed by the incorporation of 3H-thymidine (3H-Td) during the last 16 hours of the secondary MLR assay. The proliferation without anti-PD-1 antibody was considered as 100%, and the fraction of proliferation in the presence of antibodies was calculated as the MLR index. The mean index ± SE from n = 3 is shown; the asterisk (*) indicates P > .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

7. Figure 6
Myeloid DCs of HCV patients support proliferation but not de novo induction of FoxP3-expressing T cells. The content of MLR containing BDCA1+ MDCs or mo-DC of controls or HCV patients and allogeneic control CD4+ or CD4+CD25− T cells, as indicated, was analyzed for the expression of FoxP3 using specific primers in real-time PCR. The data were normalized to 18S housekeeping control. The T cells alone were included as controls. The FoxP3 content of the total CD4+ T cells was arbitrarily considered as equal to 1, and the fold change of FoxP3 content in all experimental groups was calculated. The average fold change ± SE from n = 4 for HCV mo-DC and n = 3 from BDCA1+ MDCs is shown; the asterisk (*) indicates P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions