Fig 1.
Flow chart of enrolment of CKD patients and randomization to receive primary hepatitis B vaccination with either four doses of 20mcg or 40mcg.
Table 1.
Patients demographics according to vaccine groups.
Fig 2.
(a-c). Peripheral blood lymphocyte analysis performed on baseline samples collected from healthy controls (HC; blue) and CKD patients (black) who received hepatitis B vaccination. Absolute counts (HC, n = 12; CKD, n = 25) for total lymphocytes (a), B cell subsets (b), and T cells subsets (c). (d). Memory T cell subsets (HC, n = 6; CKD, n = 16). CM, central memory; EM, effector memory; mem B, memory B cells; sw mem B, switched memory B cells; trans, transitional B cells. All analyses performed using Mann-Whitney U test, ****p<0.0001, ***p<0.001, **p<0.01. Bonferroni correction for α<0.05, p<0.003.
Fig 3.
Post–vaccination HBsAb titers.
(a). Post-vaccination HBsAb titer for patients (CKD, black symbols; n = 49) and healthy controls (HC; blue symbols, n = 11) who completed primary hepatitis B vaccination schedule. Horizontal dotted line represents working range of serological assay. Horizontal bars indicate medians. Statistical analyses performed using Mann-Whitney U test; *p = 0.0498; **p = 0.0028. Bonferroni correction for α<0.05, p<0.03. (b). Rank correlation analysis (Spearman’s test) of age and post vaccination HBsAb titer in healthy controls (n = 11) and CKD patients (n = 49), for both 20mcg CKD group (n = 24) and 40mcg CKD group (n = 25).
Fig 4.
Baseline cTFH subsets in CKD and healthy controls.
(a). Baseline absolute numbers of CXCR5+memory CD4+ T cells in healthy controls (HC, n = 16, blue) and CKD patients (n = 24, black), including those who subsequently received either HBV or influenza vaccine. (b). Baseline proportions of CXCR5+CD45RA- cells expressed as a percentage of CD4+ T cells, in healthy controls (HC, n = 16) and CKD patients (n = 24). (c). Baseline cTFH subsets according to CXCR3 and CCR6 surface expression in healthy controls (HC, n = 16) and CKD patients (n = 24). **p<0.01; Bonferroni correction for α<0.05, p<0.016. (d). Baseline cTFH subsets analyzed according to subsequent serological response (R, serological responder (HBsAb ≥10mIU/ml); NR, serological non-responder (HBsAb <10mIU/ml)) in healthy controls (HC, n = 6, blue) and CKD patients (n = 19, black) who received HBV. Horizontal bars represent medians. Analysis performed using Mann-Whitney U test: *p<0.05. **p<0.01; Bonferroni correction for α<0.05, p<0.009.
Fig 5.
Vaccine induced change in PD-1 expression on CXCR5+ cTFH, and CCR7+ and CCR7- cTFH.
(a). Gating strategy to determine PD-1 MFI on CXCR5+ memory CD4+ T cells before and after vaccination. (b). Change in PD-1 MFI on CXCR5+ memory CD4+ T cells after vaccination according to vaccine group. (c). Gating strategy to determine PD-1 MFI according to CCR7 expression in CXCR5+ memory CD4+ T cells. Baseline, grey; Day 7, cerise; CCR7-, d7, orange; CCR7+ d7, blue. (d-e). Vaccine-induced change in PD-1 MFI on (d) CCR7- and (e) CCR7+ compartments according to vaccine group. CKD, HBV (blue squares, 20mcg n = 9, 40mcg n = 11); HC, HBV (n = 6, blue circles); CKD, Fluvax (blue circles, n = 5); HC, Fluvax (black circles, n = 10). Analysis performed using Mann-Whitney U test, horizontal bars represent medians; *p = 0.0120, Bonferroni correction for α<0.05, p<0.025.
Fig 6.
PD-1 is upregulated on CXCR3+CCR6- cTFH after vaccination, but not after HBV in CKD patients (a). Gating strategy to determine PD-1 MFI on CXCR5+ memory CD4+ T cells before (grey box and histograms) and after (colored box and histogram) vaccination, according to CXCR3 and CCR6 expression (CXCR3+CCR6- compartment shown here), followed by CCR7 expression. (b-c). Change in PD-1 MFI in CCR7+ (b) and CCR7- (c) cTFH compartments after vaccination. HC who received Fluvax (n = 10, blue circles) or HBV (n = 6, blue squares). CKD patients who received HBV (20mcg n = 9, 40mcg n = 11; black squares) or Fluvax (black circles, n = 5). Horizontal bars represent medians. *p<0.05, Bonferroni correction for α<0.05, p<0.025.
Fig 7.
Plasmablasts analysis following HBV.
(a-b). Representative FACS analysis for plasmablast enumeration (a) and isotyping (b) before and 7 days after recombinant hepatitis B surface antigen vaccination. (c). Plasmablast responses for total and isotyped PB. (d). Total and IgG plasmablast responses according to HBV dose schedule (20mcg or 40mcg schedule) in CKD patients. HC, healthy controls (blue), n = 10; CKD, chronic kidney disease patients (black), n = 21; PB, plasmablasts; Δ = change in PB (day 7 minus PB at baseline, when expressed as a percentage of total B cells).
Fig 8.
Plasmablast response after seasonal influenza vaccine.
(a-b). Representative FACS analysis for plasmablast enumeration (a) and isotyping (b) before and 7 days after Fluvax. (c). Plasmablast responses for total and isotyped PB, following either Fluvax (circles) or HBV (squares), in either healthy controls (HC, blue) or CKD patients (black). Horizontal bars represent medians. Analyses performed using Mann-Whitney U test. *p<0.05, **p<0.01, Bonferroni correction for α<0.05, p<0.02. HC, healthy controls; CKD, chronic kidney disease; PB, plasmablasts; Δ PB, change in plasmablasts, from baseline to day 7, expressed as a percentage of B cells.
Fig 9.
Upregulation of PD-1 on CXCR3+CCR6-CXCR5+ cTFH after Fluvax in healthy controls.
(a-b). Relation between IgG PB plasmablast responses and either CCR7- (a) or CCR7+ (b) CXCR3+ CCR6- CD4+ cTFH cells in healthy controls (HC, blue) or CKD (black) after either Fluvax (circles, upper panels) or HBV (squares, lower panels). Δ IgG PB, change in IgG plasmablasts. Δ PD-1, change in mean fluorescence intensity of PD-1. All analyses performed using Mann-Whitney U test. Correlation analyses performed using Spearman r test.