Early T follicular helper cell activity accelerates hepatitis C virus-specific B cell expansion

Abstract

Early appearance of neutralizing antibodies during acute hepatitis C virus (HCV) infection is associated with spontaneous viral clearance. However, the longitudinal changes in antigen-specific memory B cell (MBCs) associated with divergent HCV infection outcomes remain undefined. We characterized longitudinal changes in E2 glycoprotein-specific MBCs from subjects who either spontaneously resolved acute HCV infection or progressed to chronic infection, using single-cell RNA-seq and functional assays. HCV-specific antibodies in plasma from chronically infected subjects recognized multiple E2 genotypes, while those from spontaneous resolvers exhibited variable cross-reactivity to heterotypic E2. E2-specific MBCs from spontaneous resolvers peaked early after infection (4-6 months), following expansion of activated circulating T follicular helper cells (cTfh) expressing interleukin 21. In contrast, E2-specific MBCs from chronically infected subjects, enriched in VH1-69, expanded during persistent infection (> 1 year), in the absence of significantly activated cTfh expansion. Early E2-specific MBCs from spontaneous resolvers produced monoclonal antibodies (mAbs) with fewer somatic hypermutations and lower E2 binding but similar neutralization as mAbs from late E2-specific MBCs of chronically infected subjects. These findings indicate that early cTfh activity accelerates expansion of E2-specific MBCs during acute infection, which might contribute to spontaneous clearance of HCV.

Trial registration: ClinicalTrials.gov NCT01436357.

Keywords: Adaptive immunity; B cells; Hepatitis; Immunology; Infectious disease.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. HCV-specific antibodies in the plasma of chronically infected subjects are more abundant and react to a wider breadth of HCV genotypes than antibodies in plasma from resolvers.

(A and B) Longitudinal anti-E2 and NS3 IgG responses in plasma measured by ELISA and represented as OD450–570 with subtraction of the baseline sample for each subject (baseline > 8 weeks before EDI; early acute, 8 ± 2 weeks after EDI; late acute, 20 ± 4 weeks after EDI; follow-up, > 51 weeks after EDI). Antigens are indicated on top of the graphs. Each symbol represents a single subject. (A) Resolvers (black, n = 8). (B) Chronically infected subjects (red, n = 10). (C) Combined data from A and B, presented as mean ± SD for each group. (D) Heatmaps showing the magnitude of the ELISA response at follow-up against different E2 and NS3 proteins. Infecting HCV genotype for each subject is indicated on the left. Key: blue, low or no response; yellow, medium response; red, maximum response. (E and F) Anti-rubella virus IgG response (E) and HBsAg (F) for resolvers (SR, n = 8) and chronically infected subjects (CI, n = 10). Values for healthy donor group (n = 10) were only available for HBsAg (gray). (C, E, and F) Data are shown as means for each group of subjects and error bars represent SD. Two-way repeated measure ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; NS, P > 0.05.

Figure 2. Plasma from chronically infected subjects neutralizes H77 HCVpp more effectively than plasma from resolvers.

Longitudinal plasma neutralizing activity from resolvers (black, n = 8) and chronically infected subjects (red, n = 10) against H77 HCVpp (A and B), J6/JFH1 HCVpp (D and E), and S52 HCVpp (G and H) at 1:50 dilution, presented as percentage of neutralization relative to baseline (see Figure 1 for time point definitions). (C) Combined data from A and B. (F) Combined data from D and E. (I) Combined data from G and H. All data are presented as the mean ± SD for each group. Dotted line indicates the 50% neutralization threshold. Results are presented as the mean of 3 independent experiments and error bars represent SD. Two-way repeated measure ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; NS, P > 0.05.

Figure 3. E2-specific MBCs undergo earlier expansion in resolvers than in chronically infected subjects.

(A) Representative gating strategy of class-switched, E2-specific MBCs (CD3–CD14–CD16–CD56–CD19+CD27+IgM–E2tet+) in total PBMCs. (B and C) Longitudinal frequencies of E2-specific (J6-E2 Tet+) MBCs in resolvers (B, n = 10, black) or chronically infected (CI) subjects (C, n = 10, red) at indicated time points (see Figure 1). Data from healthy individuals (controls) are gray (n = 5). Threshold of detection of E2-specific B cells (dotted line) was 0.095% (mean detection from healthy individuals + 2 SD) (35). (D) Combined data from B and C, presenting the mean ± SD for each group. (E) Representative gating of resting MBCs (CD71–), activated MBCs (CD71+CD20hiCD38int-lo), antibody-secreting MBCs (CD71+CD20loCD38hi) shown for the total MBCs population (gray contour plots), and HCV E2-specific MBCs (red dots). (F) Phenotypes of E2-specific MBCs in resting (blue), activated (orange), or antibody-secreting (purple) states (cells that did not meet these categories are designated as Other and shown in gray) for resolvers (SR) at late acute (n = 5) and CI subjects (n = 5) at late acute and follow-up time points. (B–D) Data are shown as means for each group of subjects and error bars represent SD. Two-way repeated measure ANOVA with Tukey’s post hoc test, **P < 0.01; ***P < 0.001; NS, P > 0.05.

Figure 4. VH1-69 usage is higher in BCR repertoire of E2-specific MBCs from chronically infected subjects compared with BCR of resolvers.

(A–C) Circos diagrams showing relative frequencies of V gene pairs from heavy and light chains of pooled E2-specific MBCs from resolvers (SR, n = 6 subjects, 219 pooled cells, A) at late acute stage and chronically infected (CI) subjects (n = 5) at late acute (123 pooled cells, B) and follow-up (403 pooled cells, C) time points. The width of each ribbon indicates the frequency of the VH–VK/VL pairing. The length of the arc corresponds to V gene frequency. (D and E) Relative abundances of heavy (D) or light (E) chain V genes in BCRs of pooled E2-specific MBCs from resolvers (dark gray) at late acute and CI subjects at late acute (white, red border) or follow-up (solid red) time points, presented as mean percentages of repertoire ± SD. Two-way ANOVA with Bonferroni’s post hoc test (D and E), unpaired, Mann-Whitney U test (F). *P < 0.05; **P < 0.01; ***P < 0.001; NS, P > 0.05.

Figure 5. E2-specific mAbs from resolvers neutralize HCVpp as efficiently as mAbs from chronically infected subjects despite weaker J6 E2 binding.

(A) Relative binding strength of pooled mAbs from resolvers (SR, black circles, n = 3 subjects, 10 mAbs per subject) and CI subjects (red squares, n = 3 subjects, 10 mAbs per subject) to J6 E2 protein, quantified by biolayer interferometry and expressed as mean log EC50–1 ± SD. (B and C) Neutralization against J6/JFH1 (B) or H77 (C) HCVpp of pooled mAbs from resolvers or CI subjects. Dotted line indicates the threshold of IC50–1 = 0.02. (D and E) Comparison of neutralizing activity against H77 and J6/JFH1 by individual mAbs from resolvers (D) and CI subjects (E). (F and G) Correlation between J6/JFH1 HCVpp neutralization and binding abilities by individual mAbs of resolvers (F) or CI subjects (G). Results for binding experiments represent means of pooled mAbs and error bars represent SD. Results for neutralization experiments represent the mean of 3 independent experiments. Unpaired, Mann-Whitney U test (A–D), Spearman’s correlation (E–H). **P < 0.01; ***P < 0.001; NS, P > 0.05. In all graphs, each dot represents a single mAb.

Figure 6. mAbs from resolvers at late acute stage have fewer somatic hypermutation than mAbs from chronically infected subjects at follow-up.

(A–D) SHMs in pooled, single E2-specific MBCs (n = 219) from resolvers (n = 6 subjects, gray violins) and CI subjects (n = 123 cells, n = 5 subjects, white violins with red border) at late acute and CI subjects (n = 5) at follow-up (n = 403 cells, red violins) time points, presented as the number of nucleotide substitutions (A and B) and amino acid mutations (C and D) in heavy (A and C) and light (B and D) chains plotted as absolute number of mutations. (E and F) Correlation between mAbs neutralization combined rank (rank of 1 = best neutralizer, rank of 114 = worst neutralizer, established from H77 and J6/JFH1 neutralization, see Figure 5) and number of SHMs in CDR3H and CDR3L regions of mAbs derived from resolvers (n = 3 subjects, 30 mAbs, E) or chronically infected subjects (n = 3, 30 mAbs, F). (G and H) Number of SHMs in CDR3 regions of all cloned mAbs (G) or the top neutralizers (H, combined rank < 50). (E–H) Each dot represents the rank value for an individual mAb. Results represent means of pooled mAbs and error bars represent SD 1-way ANOVA with Tukey’s post hoc test (A–D) or paired Student’s t test (G and H). *P < 0.05; **P < 0.01; ***P < 0.001; NS, P > 0.05.

Figure 7. Activated cTfh cells expand early during acute infection in resolvers but not in chronically infected subjects.

(A) Representative gating strategy of total cTfh cells (CD3+CD4+CD45RA–CXCR5+PD1+FoxP3–), activated cTfh cells (ICOS+), total cTfr cells (CD3+CD4+CD45RA–CXCR5+PD1+FoxP3+), and activated cTfr cells (ICOS+), from PBMCs of HCV-infected subjects. (B) Frequencies of total cTfh cells at different time points in resolvers (n = 10, black) or chronically infected subjects (n = 10, red; see Figure 1). Data from 5 healthy donors (controls) are shown in gray. (C) Frequencies of activated cTfh cells at different time points. (D) Ratio of activated cTfh over cTfr cells at different time points, shown as fold-change from baseline. (E) Frequencies of activated Th1-like (CXCR3+) cTfh cells at different time points. (F) Frequencies of activated Th2-like (CXCR3–) cTfh cells at different time points. (G) Representative gating strategy of cytokine-producing or CD40L-expressing, activated cTfh cells before (no stim, top) and after PMA and ionomycin stimulation (5 hours, bottom). (H–J) Frequencies of ICOS+IL-21+ cells (H), ICOS+CD40L+ cells (I), and ICOS+IFN-γ+ (J) cTfh cells at different time points. (K) Summary heatmaps of the main data from this study. Each component (row) indicates the intensity of response of the indicated test (far left) at the indicated time point (far right). Each square provides data for one resolver (left) or chronically infected subject (right). An X indicates no response (values of 0); N/A, subject for which the test could not be done (no baseline blood sample available). Blue, low or no response; yellow, medium response; red, maximum response. Data are shown as means for each group of subjects and error bars represent SD. Two-way repeated measure ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; NS, P > 0.05.