PMC

Anti-HBsAg titers. Four weeks after receipt of the last HBV vaccination, anti-HBsAg titers were measured. On the y axis, anti-HBs titers (IU/L) are depicted, and on the x axis, the healthy volunteers (▪) and patients with ESRD (□). The dotted lines represent the <10 IU/L (nonresponders) and >100 IU/L (good responders).

Peak responses for HBsAg-specific IL-2–and IFN-γ–producing cells. Peak responses of absolute numbers of HBsAg-specific IL-2–and IFN-γ–producing naive (A and B), central memory (C and D), and effector memory (E and F) CD4⁺ T lymphocytes obtained within 4 wk after HBsAg vaccination. Absolute peak numbers of cells producing HBsAg-specific IL-2 and IFN-γ are displayed for healthy control subjects (▪) and patients with ESRD (□).

Levels of HBsAg-specific cytokines produced after re-stimulation. PBMC, obtained 2 wk after receipt of the last HBV booster vaccination, were stimulated with HBsAg (5 μg/ml; squares) or polyclonal with PHA (1 μg/ml; triangles). At day 6, the cells were re-stimulated for 48 h using fresh PBMC (1 × 10⁵) with or without the various stimuli. The closed symbols represent healthy control subjects, and the open symbols represent the patients with ESRD.

(A and B) PHA-induced IL-2–producing (A) and IFN-γ–producing (B) CD4⁺ T lymphocytes in study population. Similar as described in , percentages of IL-2–producing (A) and IFN-γ–producing CD4⁺ T cells induced by PHA were determined. On the y axis, the percentages of cytokine producing cells (mean ± SEM) are displayed and on the x axis, the different CD4⁺ T cell subsets. ▪, signals observed for healthy volunteers; □, signals for patients with ESRD. *Significantly (P < 0.05) different from healthy volunteers.

(A and B) Peak responses for both percentages (A) and absolute numbers (B) of HBsAg-specific IL-2 effector memory CD4⁺ T cells correlate with anti-HBsAg titers. Peak responses for HBsAg-specific percentages and absolute numbers of IL-2–producing effector memory CD4⁺ T cells are associated with anti-HBsAg titers measured 4 wk after the last HBV vaccination. On the x axis, anti-HBs titers (IU/L) are depicted, and on the y axis, percentages (A) and absolute numbers (B) of HBsAg-specific IL-2–producing effector memory CD4⁺ T cells are displayed. ▪, Responses for healthy volunteers; □, responses for patients with ESRD. Spearman rank correlation was calculated using SPSS for Windows.

Kinetics of HBsAg-specific proliferation. At the various intervals, freshly isolated PBMC were stimulated with medium alone and HBsAg (5 μg/ml) for 6 d. At day 6, proliferation was determined by ³H-thymidine incorporation and is expressed as counts per minute (cpm). Subsequently, stimulation indices (SI) were calculated by dividing the HBsAg-induced proliferation by that induced by culture medium alone. On the x axis, various time intervals after the last HBV vaccination are depicted and on the y axis, proliferation expressed as SI. ▪, Responses for healthy volunteers; □, responses for patients with ESRD. *Significantly (P < 0.05) different from healthy volunteers.

Typical example of flowcytometric analysis of IL-2–producing Tem of healthy volunteer (D through F) and a hemodialysis patient (G through I) 2 wk after the last booster of HBV vaccination. A typical example of the flowcytometric analysis of cytokine-producing T cells. (A and B) The lymphocyte population (A) was gated first (R1), and subsequently the CD4⁺ T lymphocytes (R2) were gated within these lymphocytes (B). (C) Next, we identified the proportions of CD4⁺ T lymphocytes (R1+R2) in the various subsets using a dotplot of CCR7 versus CD45RO. Naive CD4⁺ T cells are displayed in region 3; central memory CD4⁺ T cells in region 4 and region 5 displays the effector memory CD4⁺ T population. (D through I) Next, the IL-2 signals observed for CD4⁺ Tem in presence of co-stimulation only (D and G), co-stimulation with HBsAg (E and H), and co-stimulation with PHA (F and I) are plotted for a healthy volunteer and a hemodialysis patient 2 wk after the last HBV vaccination. Percentages represent IL-2–producing cells.

Kinetics of HBsAg-specific IL-2–and IFN-γ–producing CD4⁺ T lymphocytes in the study population. PBMC were stimulated with αCD28 and αCD49d and HBsAg for 6 h in the presence of Brefeldin A for the last 5 h of incubation. Thereafter, the percentage of IL-2⁺ (A, C, E, and G) or IFN-γ⁺ (B, D, F, and H) cells was determined for the different T cell subsets. T cell subsets were defined by expression of cell surface markers CD4, CD45RO, and CCR7. The following subsets were defined Tnaive (CD4⁺CD45RO⁻CCR7⁺), Tcm cells (CD4⁺CD45RO⁺CCR7⁺), and Tem cells (CD4⁺CD45RO⁺CCR7⁻). ▪, Signals observed for healthy volunteers; □, signals observed for patients with ESRD. On the x axis, the various time intervals are depicted, and on the y axis, mean ± SEM percentages of HBsAg-specific cytokine-producing CD4⁺ (A and B), Tnaive (C and D), Tcm (E and F), and Tem (G and H) cells are displayed, respectively. *Significantly (P < 0.05) different from healthy volunteers.