1. Volume 132, Issue 7, Pages 2328-2339 (June 2007)
Increased Regulatory T Cells Correlate With CD8 T-Cell Impairment and Poor Survival in Hepatocellular Carcinoma Patients 
Junliang Fu, Dongping Xu, Zhenwen Liu, Ming Shi, Ping Zhao, Baoyun Fu, Zheng Zhang, Huiyin Yang, Hui Zhang, Chunbao Zhou, Jinxia Yao, Lei Jin, Huifen Wang, Yongping Yang, Yang-Xing Fu, Fu-Sheng Wang 
Gastroenterology 
Volume 132, Issue 7, Pages (June 2007)
DOI: /j.gastro
Copyright © 2007 AGA Institute Terms and Conditions

2. Figure 1
Circulating CD4+CD25+FoxP3+ Treg are increased significantly in HCC patients. (A) Treg are gated from CD25+FoxP3+ subsets of CD3+CD4+ T cells. More than 90% of magnetic bead–purified Treg by CD4-negative CD25-positive double selections expressed intracellular FoxP3, which have a representative phenotypic profile of CD45RO, CD62L, CTLA-4 expression. (B) Representative prevalence of CD4+CD25+FoxP3+ Treg from individual subjects in 3 studied groups. CD25+FoxP3+ Treg were gated from a CD4+ subset of CD3+ T cells. (C) Statistical analysis shows that the frequency of CD4+CD25+FoxP3+ Treg in HCC patients is significantly higher than in LC patients and normal controls. (D) Prevalence of CD4+CD25+FoxP3+ Treg increases with progressive stages in HCC patients. (E) Representative expression of FoxP3 in CD4+CD25lowT cells from individual subjects in 3 studied groups. (F) Statistical analysis shows that FoxP3 expression of CD4+CD25low T cells in HCC patients is significantly higher than in LC patients and normal controls. (G) FoxP3 expression of CD4+CD25low T cells increases with progressive stages in HCC patients. Data in C, D, F, and G are expressed as box plots, in which the horizontal lines illustrate the 25th, 50th, and 75th percentiles. Vertical lines represent the 10th and 90th percentiles. nc, normal controls.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
Immunohistochemical staining shows differential accumulation of FoxP3+ and CD8+lymphocytes between tumor and nontumor or normal liver tissues. Representative results from paraffin-embedded tumor and nontumor specimens from an HCC patient and from liver tissue of a healthy donor as normal control (nc). (A and C) Single staining for FoxP3 and CD8, respectively. Positive cells are stained red, as shown by arrows. Most tumor-infiltrating CD8+ cells distribute in the peritumor region (as shown in F and the circled area in C) rather than in the tumor-in-situ region. (B and C) Comparison of the intratumor region (it) with the nontumor region (nt) shows that the FoxP3+ cell number increased significantly, whereas the CD8+ cell number decreased significantly. (E) Double staining of CD8 (red, on cell membrane) and FoxP3 (blue, in cell nucleus). Contact of CD8+ cells and FoxP3+ cells could be identified in (E) tumor in situ and (F) peritumor regions.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
CD4+CD25+FoxP3+Treg are highly enriched in TIL of advanced-stage HCC patients. (A) Upper panel shows the resected tumors from livers of representative HCC patients at stage I and III phases, respectively. (B) Representative prevalence of CD4+CD25+FoxP3+Treg and CD4+ T cells from TIL/NIL of individual HCC patient at stage I or stage III or from liver-infiltrating lymphocytes of normal controls. (C) Statistical analysis shows that the frequencies of CD4+CD25+FoxP3+ Treg and CD4+ T cells are significantly higher in TIL than in NIL or liver-infiltrating lymphocytes, whereas the frequency of CD8+ T cells is significantly lower in TIL than NIL. The data were from 11 HCC patients and 5 normal controls. nc, normal controls; lil, liver-infiltrating lymphocytes.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
Differential expression of CD25, FoxP3, CTLA-4, and HLA-DR between TIL and NIL of HCC patients. (A) CD4+CD25high and CD4+CD25lowT cells in TIL were 38.9% and 37.0%, respectively, which are much higher than their NIL counterparts, in which the CD4+CD25- subset is overwhelmingly dominant with a very low proportion of CD4+CD25high T cells. (B and C) Representative FoxP3, CTLA-4, and HLA-DR expression profiles in CD4+ T-cell subsets that differ in CD25 expression. Expression levels of FoxP3, CTLA-4, and HLA-DR were much higher in TIL than in NIL.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
GrA, GrB, and perforin expression decreases in TIL rather than PB of HCC patients. (A) There is no obvious difference in the expression of GrA, GrB, and perforin in circulating CD8+ T cells between HCC patients (n = 52) and normal controls (n = 21). (B) Expression profile of the 3 cytolytic enzymes between TIL and NIL of a representative HCC patient. (C) GrA, GrB, and perforin expression all are reduced significantly in TIL compared with the NIL of HCC patients at various stages (n = 11).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 6
Treg suppress degranulation of CD8+ T cells of HCC patients. The degranulation marker, CD107a, was analyzed using flow cytometry. (A) HCC patients show a significant decrease of CD107a expression on CD8+ T cells compared with normal controls after 5 hours of anti-CD3/CD28 stimulation (6.8% vs 14.8%, P < .001), and the decrease was relatively smaller in patients at stage I than patients at stage II and III. (B) Dynamic analysis shows that CD107a expression is much lower in HCC patients than in normal controls at 3, 5, and 7 hours after stimulation. (C) When Treg are depleted from PBMCs of HCC patients, the CD107a expression is restored. (D) Residual GrA, GrB, and perforin were examined after 5 hours of stimulation with anti-CD3/CD28. ■ normal controls (n = 8), ▨ HCC patients (n = 8). Approximately 28% GrA, 36% GrB, and 30% perforin of CD8+ T cells was released in HCC patients, which is much lower than in normal control cells that released more than 70% of all 3 enzymes. Percentage degranulation of CD8+ T cells was calculated using the formula: 1 – (residual enzymes after a 5-h stimulation with anti-CD3/CD28/constitutive expression of enzymes before stimulation) × 100%. (E) Representative fluorescence-activated cell sorter profiles of 3 enzymes individually expressed in CD8+ T cells of an HCC patient before and after 5 hours of stimulation with anti-CD3/CD28.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

8. Figure 7
Treg from a representative HCC individual suppress production of granzyme and perforin of autologous CD8+ T cells. Effectors prepared as PBMCs, PBMCs–Treg, and PBMCs–Treg + Treg were set into 96-well plates and incubated for 48 hours with anti-CD3/CD28 stimulation. The granzymes and perforin generated by CD8+ T cells were increased significantly in the PBMC-Treg group in comparison with the PBMC group, and markedly decreased in the PBMC-Treg + Treg group compared with the PBMC-Treg group.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

9. Figure 8
Treg suppress proliferation and cytokine production of autologous CD8+ T cells. (A) carboxyfluorescein succinimidylester-labeled PBMCs, PBMCs-Treg, PBMCs-Treg + Treg at a ratio of 10:1 and 1:1, or Treg alone were on anti-CD3/CD28 stimulation for 4 days, followed by cytometric analysis of the cell-division cycle. “PBMC-Treg|+Treg” represents results from transwell assay. Data were from a representative HCC patient. (B) CD8+ T cells were cultured with Treg at various ratios on anti-CD3/CD28 stimulation for 5 days. For the last 18 hours the cultures were pulsed with 0.5 μCi/well of [3H]-thymidine. Data were from a representative HCC patient. (C) CD8+ T cells were cultured with Treg at various ratios on anti-CD3/CD28 stimulation for 48 hours. Interferon-γ and tumor necrosis factor-α from the supernatant then were harvested and measured by enzyme-linked immunosorbent assay. Data were from 5 HCC individuals.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

10. Figure 9
Increasing prevalence of circulating CD4+CD25+FoxP3+ Treg predicts decreasing survival of HCC patients. Seventy-five HCC patients were divided into 2 groups by the mean value of circulating CD4+CD25+FoxP3+ Treg frequency as High Treg (n = 31, average Treg frequency: 10.2% ± 1.9%) and Low Treg (n = 44, average Treg frequency: 6.1% ± 1.3%). (A) The High Treg group had significantly poorer survival in comparison with the Low Treg group (stage I: n = 12; 1 and 11 patients in the High and Low Treg groups, respectively; stage II: n = 16; 4 and 12 patients in the High and Low Treg groups, respectively; stage III: n = 47; 21 and 26 patients in the High and Low Treg groups, respectively; P < .001). (B) Independent analysis of patients at stage III showed that the survival time of the High Treg group (n = 21, average Treg frequency: 10.9% ± 1.9%) was significantly shorter than that of the Low Treg group (n = 26, average Treg frequency: 6.4% ± 1.4%) (P = .018). Actual overall survival rates were analyzed by the Kaplan–Meier method and survival was measured in weeks from diagnosis to death. The log-rank test was applied to compare between the groups. Multivariate analysis of prognostic factors for overall survival was made using the Cox proportional hazards model.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions