Coordinated expansion of memory T follicular helper and B cells mediates spontaneous clearance of HCV reinfection

Introduction Follicular helper T cells are essential for helping in the maturation of B cells and the production of neutralizing antibodies (NAbs) during primary viral infections. However, their role during recall responses is unclear. Here, we used hepatitis C virus (HCV) reinfection in humans as a model to study the recall collaborative interaction between circulating CD4 T follicular helper cells (cTfh) and memory B cells (MBCs) leading to the generation of NAbs. Methods We evaluated this interaction longitudinally in subjects who have spontaneously resolved primary HCV infection during a subsequent reinfection episode that resulted in either another spontaneous resolution (SR/SR, n = 14) or chronic infection (SR/CI, n = 8). Results Both groups exhibited virus-specific memory T cells that expanded upon reinfection. However, early expansion of activated cTfh (CD4+CXCR5+PD-1+ICOS+FoxP3−) occurred in SR/SR only. The frequency of activated cTfh negatively correlated with time post-infection. Concomitantly, NAbs and HCV-specific MBCs (CD19+CD27+IgM−E2-Tet+) peaked during the early acute phase in SR/SR but not in SR/CI. Finally, the frequency of the activated cTfh1 (CXCR3+CCR6−) subset correlated with the neutralization breadth and potency of NAbs. Conclusion These results underscore a key role for early activation of cTfh1 cells in helping antigen-specific B cells to produce NAbs that mediate the clearance of HCV reinfection.


Introduction
T follicular helper (Tfh) cells play a crucial role in helping B cells and in the formation of germinal centers, affinity maturation, and development of plasma cells and memory B cells (MBCs) during primary viral infections and vaccinations (1,2).However, their role during recall responses and their correlation with the production of neutralizing antibodies upon reinfection are understudied (3).Hepatitis C virus (HCV) infection represents an ideal model to study this question with two dichotomous outcomes where approximately 30% of acutely infected individuals resolve spontaneously while the rest develop chronic infection.Despite the resolution of primary HCV infection, people who inject drugs (PWID) remain at high risk of HCV exposure and reinfection (4)(5)(6)(7)(8), thus representing a natural experimental rechallenge framework to study memory immune responses against human viral infection.Spontaneous resolution of primary HCV is associated with both Tcell and antibody responses.However, although the resolution of primary infection leads to the generation of long-lived memory T cells (9, 10), antibody responses proceed to decline rapidly (10-12).Virus-specific circulating T follicular helper (cTfh) CD4 + T cells expand during acute HCV (13) and the frequencies of CXCR3 + cTfh (cTfh1) positively correlate with the magnitude and breadth of HCV-neutralizing antibody responses (14).We have demonstrated that early expansion of activated cTfh1 expressing interleukin 21 (IL-21), CD40L, and interferon-g (IFN-g) is associated with the expansion of HCV-specific MBCs in spontaneous resolvers of acute HCV infection (11).
Early expansion of HCV-specific MBCs and the production of neutralizing antibodies (NAbs) are associated with clearance of acute primary HCV infection and reinfection (11,12,(15)(16)(17)(18)(19)(20)(21).NAbs with exceptionally high neutralization breadth and potency were identified in HCV elite neutralizers and are associated with the use of the VH1-69 heavy-chain gene segment (22,23).HCV reinfection and repeated exposure to viruses with antigenically related, antibody-sensitive E1E2s glycoproteins lead to the generation of potent broadly neutralizing antibodies (bNAbs) (16).Resolution of HCV reinfection is also associated with expansion of HCV-specific CD4 and CD8 T cells (18,24).We have demonstrated that spontaneous resolution of HCV reinfection is associated with an early plasma cell transcriptomic signature, variable levels of NAbs, and early expansion of HCV-specific MBCs and CD8 T cells (17), indicating that cooperative effort between NAbs and T cells is required for long-term protection against HCV.How much of this response is driven by memory CD4 T-cell help and Tfh-MBC interaction upon reinfection has not been studied thus far.
Here, we investigated the longitudinal expansion of activated cTfh and HCV-specific MBCs during HCV reinfection.We observed the earlier expansion of activated cTfh and HCVspecific MBCs in resolvers as opposed to chronic subjects.Furthermore, the frequencies of activated cTfh1 were associated with the neutralization breadth and potency of antibodies in resolvers.Our data suggest a cooperative role for activated HCVspecific cTfh cells and NAbs in the clearance of HCV during re-exposure.

Human study participants
Study subjects were recruited among PWID who were participating in the Montreal Hepatitis C cohort (HEPCO, study protocol approval number: SL 05.014).All subjects were HIVnegative.HCV reinfection was defined by an HCV-positive RNA test following two consecutive negative tests >30 days apart (Cobas Ampliprep/Cobas TaqMan HCV Qualitative Test, version 2.0; limit of detection: 15 IU/ml).The median between the last negative and first positive HCV RNA test was used to determine the estimated date of reinfection (EDI).Study subjects were considered spontaneous resolvers of reinfection if HCV RNA was negative at 6 months post-EDI, while chronics were defined by a positive test.

IFN-g enzyme-linked immunospot assay
HCV-specific T-cell responses were measured using an IFN-g enzyme-linked immunospot (ELISpot) assay, as previously described (25) with an input of 2 × 10 5 PBMCs/well against 11 pools of overlapping peptides spanning the entire HCV polyprotein corresponding to genotype (Gt) 1a (H77), 1b (J4), or 3a (K3a/650) sequences (BEI Resources, Manassas, VA, USA).For some subjects, we used the ELISpot Flex Human IFN-g (ALP) kit (Mabtech, Cincinnati, OH, USA).All assays were performed directly ex vivo on frozen PBMCs.Specific spot-forming cells (SFCs) were calculated as the mean number of spots in test wells minus the mean number of spots in negative control wells and normalized to SFC/10 6 PBMCs.Early and late acute time points used for this assay differed from the ones mentioned in Supplementary Table 1 for subjects SR/SR-4, SR/SR-6, and SR/CI-2.Pre-reinfection time points of SR/CI-1 and SR/CI-2 were also different.

Flow cytometry
Cryopreserved PBMCs were thawed and washed twice with RPMI 10 and then washed twice with FACS buffer (PBS 1×, 1% FBS, 0.01% sodium azide).For the identification of E2-specific MBCs, cells were incubated for 10 min at RT with Human BD Fc Block (BD Biosciences, Franklin Lakes, NJ, USA) and then stained with biotinylated E2 tetramers for 30 min at RT. Cells were then washed twice with FACS buffer and stained for 30 min at 4°C with surface markers (see Supplementary Table 2: Panel #1 for antibodies) and viability dye [LIVE/DEAD fixable aqua dead cell stain kit (Thermo Fisher Scientific)] to identify live cells.Following two washes with FACS buffer, cells were fixed using 1% formaldehyde (Millipore Sigma).For cTfh phenotyping, cells were stained for 30 min at 4°C with surface markers (see Supplementary Table 2: Panel #2 for antibodies) and viability dye.Cells were washed again and permeabilized using FoxP3 fixation and permeabilization buffer (Thermo Fisher Scientific) for 20 min at 4°C.Intracellular staining was performed for 30 min at 4°C.Cells were then washed twice with PermWash buffer and fixed using 1% formaldehyde.Multiparameter flow cytometry was performed at the flow cytometry core of the CRCHUM using a BD LSRFortessa instrument equipped with five lasers [UV (355 nm), violet (450 nm), blue (488 nm), yellow-green (561 nm), and red (640 nm)] and the FACSDiva version 9.2 (BD Biosciences).FCS data files were analyzed using FlowJo (version 10.8.1 for Mac; BD Biosciences).

Activation-induced markers assay
Cryopreserved PBMCs were thawed and then washed twice with RPMI 5 [RPMI medium supplemented with 5% human Serum (Wisent) and 1% penicillin/streptomycin (Wisent)].Cells (1 to 4 × 10 6 ) were then incubated in 1 ml of RPMI 5 for 3 h at 37°C, 5% CO 2 in 5 ml of polypropylene push cap tubes (Thermo Fisher Scientific).Antibodies for the chemokine receptors CCR4, CCR6, CCR7, CXCR3, and CXCR5 were added to the culture, and the cells were further incubated for 15 min at 37°C, 5% CO 2 , followed by the addition of a CD40 blocking antibody (Miltenyi, Gaithersburg, MD, USA) to prevent the interaction of CD40L with CD40 and its subsequent downregulation.Cells were then stimulated for 18 h at 37°C, 5% CO 2 with 1 µg/ml of HCV overlapping peptide pools showing the highest response in ELISpot.Cells stimulated with 1 µg/ml of staphylococcal enterotoxin B (SEB) (Toxin Technology Inc., Sarasota, FL, USA) and unstimulated cells served as positive and negative controls, respectively.Cells were then collected and transferred to a 96-well V-bottom plate, washed twice with FACS buffer, and stained for 30 min at 4°C with surface markers (see Supplementary Table 2: Panel #3 for antibodies) and viability dye.Following two washes, cells were fixed using 1% formaldehyde.Multiparameter flow cytometry was performed as described above.
All samples were tested in duplicates.The difference in optical densities (OD450-570) was determined by subtracting the mean absorbance at 570 nm (background) from the mean absorbance at 450 nm.Absorbance measurements were performed on a Synergy 4 Microplate Reader (BioTek Instruments, Winooski, VT, USA).
Cell lines and cell culture CD81-knockout (KO) human embryonic kidney 293T (HEK 293T) (kindly provided by Drs.Joe Grove, University of Glasgow and Justin Bailey, Johns Hopkins) and human hepatoma Huh-7 (kindly provided by Dr. Charles Rice, The Rockefeller University) cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% FBS and maintained at 37°C and 5% CO 2 .

HCVpp neutralization assays
Neutralization assays were performed as previously described elsewhere (27).Huh-7 cells were plated on wells of sterile 96-well white plates (Thermo Fisher Scientific) at a density of 1.5 × 10 4 cells/ well.The next day, plasma aliquots were incubated at 56°C for 30 min to inactivate the complement and centrifuged at 1,200×g for 5 min.Fixed plasma dilutions (1:25) were added to an equal volume of either UKNP1.11.6, 1a154, UKNP4.2.2, 1a72, 1b58, UKNP3.1.2,or UKNP1.18.1 E1E2 HCVpp or mock HCVpp.Dilutions of HCVnegative and HCV-positive (chronically infected donor) plasma were included as negative and positive controls, respectively.Plasma-HCVpp complexes (final plasma dilution 1:50) were incubated at 37°C for 1 h.A volume of 100 µl of plasma-HCVpp mixture was added to cell monolayers in duplicate and incubated for 5 to 6 h at 37°C and 5%CO 2 , after which the mixture was discarded and replaced with phenol red-free DMEM (Wisent) supplemented with 10% FBS and 4 mM of L-glutamine (Wisent).After 48 h, cells were lysed in 50 ml of 1× Luciferase cell culture lysis buffer (Promega, Madison, WI, USA) at RT. Luminescence was detected by adding 50 µl of luciferase reagent (Promega) and measuring relative light units (RLU) on a Synergy 4 Microplate Reader (BioTek Instruments).Percent neutralization was calculated using the following equation: % neutralization = [1 − (RLU infection time point − RLU mock )/(RLU negative − RLU mock )].Neutralizing breadth was defined as the number of HCVpp (of the 7 HCVpp used) neutralized with a % neutralization >50%, and neutralizing potency was calculated as the geometric mean of the % neutralization from the assays against 7 HCVpp for a given time point.

Statistics
Statistical analyses were performed with Prism version 10.1.0(GraphPad, Boston, MA, USA).Details of the tests (including the number of data points (n) and P-values) are provided in each figure legend.Differences between groups in longitudinal analyses were determined by two-way repeated measure ANOVA with Tukey's post-hoc test.Comparisons that did not include multiple time points were examined by the two-tailed Mann-Whitney U test.Correlations between variables were examined using Spearman's test.Non-linear regression was used to calculate antibody titers.For all statistical tests, P-values less than 0.05 were considered significant.

Study design and characteristics of HCVreinfected subjects
We examined the HCV-specific immune response during documented HCV reinfection episodes after spontaneous resolution in a group of PWID participating in the Montreal Hepatitis C cohort (HEPCO) study as described in the Materials and methods (29, 30) (n = 22; Table 1).Of these reinfection cases, 14 subjects spontaneously resolved the reinfection episode, hereinafter termed resolvers or SR/ SR, while eight subjects developed chronic infection, hereinafter termed chronics or SR/CI.The detailed demographics and clinical characteristics of the subjects are summarized in Supplementary Table 1.We analyzed the response at key time points during reinfection: pre-reinfection (variable), early acute (<3 months post-EDI), late acute (3-7 months post-EDI), and follow-up (>7 months post-EDI).Most SR/SR (64.2%) and SR/CI (62.5%) were reinfected with HCV Gt 1, while only 1 of 14 SR/SR and 2 of 8 SR/CI were reinfected with HCV Gt 3a (Table 1).Both groups had pre-existing virus-specific memory T-cell responses that expanded during reinfection as measured in IFN-g ELISpot assay against overlapping peptide pools covering the entire HCV polyprotein.During early acute reinfection, both groups had comparable total T-cell responses that were sustained during the late acute time point primarily in SR/ SR (Supplementary Figure 1).

Resolvers' early cTfh are HCV-specific
To confirm that the expanded cTfh are virus-specific, we performed an activation-induced markers (AIM) assay at early acute in two SR/SR (SR/SR-9 and SR/SR-10) and two SR/CI subjects (SR/CI-3 and SR/CI-5).We used the peptide pools that gave the highest response in the IFN-g ELISpot assay, in addition to E2 as it is the main target of NAbs that require Tfh help.HCV-specific (AIM + ) CD4 T cells were identified as CD14 − CD19 − CD3 + CD4 + CD8 − CD69 + T cells expressing the activation markers CD40L and/or OX40.The representative gating strategy is presented in Figure 2A.SR/SR subjects exhibited significantly higher frequencies of HCV-specific CD4 T cells (P = 0.026) than SR/CI subjects (Figure 2B).

Early expansion of HCV-specific MBCs in resolvers during reinfection
Given the early expansion of activated HCV-specific cTfh observed in the SR/SR group and their essential role in helping B cells, we investigated the longitudinal dynamics of E2-specific, classswitched MBCs in both groups using HCV glycoprotein E2 tetramers (26) as previously described (17).E2-specific MBCs were identified as CD3 − CD14 − CD16 − CD56 − CD19 + IgM − CD27 + Tetramer APC + and PE + (Figure 3A).We observed early, transient, and significant expansion of E2-specific MBCs in SR/SR subjects as compared to follow-up time points (P = 0.0075) (Figure 3B), and the frequency of E2-specific MBCs remained significantly higher than the follow-up time points during the late acute stage of reinfection.During early acute reinfection, 10 out of 14 SR/SR exhibited elevated levels of E2specific MBCs above the detection threshold.In contrast, expansion of E2-specific MBCs in SR/CI was delayed until the follow-up time points and did not reach the level of significance, with only three subjects surpassing the threshold of detection at early acute reinfection, two of which already had detectable levels at the prereinfection time point (Figure 3C).Thus, SR/SR exhibited higher and earlier expansion of E2-specific MBCs compared to the SR/CI subjects (Figure 3D).

Early antibody titers in resolvers correlate with the frequency of E2-specific MBCs
To assess whether expansion of HCV-specific B cells is associated with enhanced production of anti-HCV antibodies, we evaluated the levels of IgG in plasma targeting HCV proteins by ELISA.We performed four-fold serial plasma dilutions to determine the antibody titer at 50% binding, defined as the dilution showing 50% binding to H77-E2 Gt 1a (Supplementary Figure 2A), J6-E2 Gt 2a (Supplementary Figure 2B), and H77-NS3 Gt 1a as a control (Supplementary Figure 2C).At early acute reinfection, SR/SR showed higher titers of IgG than SR/CI against H77-E2 and J6-E2 proteins, 3-fold and 2.5-fold, respectively (Figures 4A, B).However, these differences were not significant.Titers of anti-E2 Gt 2a in SR/SR decreased significantly (P = 0.0079) from early acute to follow-up time points (Figure 4B).In contrast, SR/CI had higher antibody titers against the H77-NS3 protein compared to SR/SR at all times during reinfection (Figure 4C).Overall, SR/CI reached the highest antibody titers at follow-up when chronic HCV infection was already established.Together, results indicate that SR/SR develops earlier and higher antibody titers against E2 protein, the target for NAbs that may contribute to viral clearance.

Broad and potent neutralization occurs earlier in resolvers compared to chronics
Next, we investigated whether the high titers of HCV-specific antibodies were reflected in the plasma neutralization activity.We performed neutralization assays with seven HCVpp using a standardized panel (28,37) harboring envelope glycoproteins E1E2 from HCV Gt 1a, 1b, 3a and 4a and representing different tiers of neutralization sensitivity (28,37).The detailed results are presented in Figure 5 and summarized in Figure 4D.Seven out of 14 SR/SR neutralized >50% UKNP1.11.6 (Gt 1a) HCVpp from tier 1 (most sensitive to neutralization) compared to only two out of eight SR/CI at early acute reinfection (Figure 4D).However, there was no significant difference in the percent neutralization between SR/SR and SR/CI at this time point.The highest percent neutralization against this HCVpp by SR/SR (98.31%) occurred at early acute, while in the SR/CI, the highest neutralization (96.20%) was not achieved until the follow-up time point (Supplementary Figure 5A).Neutralization of tier 2 (1a154-H77 Gt 1a) and tier 3 (UKNP4.2.2 Gt 4a, 1a72 Gt 1a, and 1b58 Gt 1b) HCVpp followed the same trend, where SR/SR showed a higher percentage of neutralization at early acute compared to follow-up, while SR/CI reached the highest percent of neutralization only late during reinfection at follow-up time points (Supplementary Figures 5B-E).Regarding tier 4, the most resistant to neutralization represented by UKNP1.5G).We further integrated the neutralization results to define neutralization breadth and potency.Neutralization breadth, which is the number of HCVpp neutralized >50%, peaked at early acute in the SR/SR and declined with time (Figure 4E).In contrast, neutralization breadth in SR/CI exhibited slower kinetics, where it increased significantly only at the follow-up time points (P = 0.0365 and 0.0263 compared to early and late acute, respectively).Neutralization potency, which is the geometric mean of % neutralization, demonstrated a similar trend (Figure 4F), where the neutralization potency in SR/SR peaked at early acute, but only increased significantly in SR/CI much later at follow-up (P = 0.0460 compared to late acute).The early neutralization breadth and potency correlated positively and significantly in SR/SR with the levels anti-E2 Gt 1a (Supplementary Figure 6A, breadth: r = 0.8742, P = 0.0002 and potency: r = 0.8736, P = 0.0002) and anti-E2 Gt 2a (Supplementary Figure 6B, breadth: r = 0.7635, P = 0.0037 and potency: r = 0.7473, P = 0.0046) but not in SR/CI (Supplementary Figures 6C, D, respectively).These findings suggest that anti-E2 antibodies generated early with high titers in resolvers mediate HCVpp neutralization with high breadth and potency, thus potentially contributing to HCV clearance upon reinfection.

Activated cTfh1 and E2-specific MBCs correlate with neutralization breadth and potency during early acute reinfection in resolvers
Because of the early coordinated expansion of cTfh and E2specific MBCs along with the elevated plasma neutralization activity in the SR/SR, we examined the correlations between these responses at early acute.Overall, we observed more significant positive associations between the different early acute immune parameters in SR/SR as compared to SR/CI as summarized in Figure 5A and detailed in Supplementary Figures 7, 8. Notably, the frequencies of E2-specific MBCs correlated with neutralization breadth and potency in SR/SR but not SR/CI (Figures 5B, C).The frequencies of activated cTfh1 (ICOS + FoxP3 − CXCR3 + CCR6 − ) also correlated positively with plasma neutralizing activity in the SR/SR but not SR/ CI (Figures 5D, E).Furthermore, at early acute reinfection, the frequencies of total activated cTfh from SR/SR but not SR/CI showed positive but non-significant correlation with anti-E2 antibodies (Supplementary Figures 7A, B), E2-specific MBCs (Supplementary Figures 7C, D), and neutralization breadth and potency (Supplementary Figures 7E, F).Similarly, activated cTfh1 from SR/SR but not SR/CI showed a positive non-significant correlation with anti-E2 antibodies (Supplementary Figures 8A,  B) and E2-specific MBCs (Supplementary Figures 8C, D) at early acute reinfection.Altogether, these results further support that early activation of cTfh with a Th1 phenotype along with early expansion of E2-specific MBCs may drive the production of HCV-specific NAbs leading to the clearance of HCV reinfection.

Upon reinfection or booster vaccination, virus-specific MBCs can
into ASCs or re-enter the germinal center to undergo further affinity maturation with the help of Tfh cells (1).In our study, we detected the early expansion of E2-specific MBCs that differentiated into ASCs in resolvers.Notably, resolvers with detectable ASCs also showed the highest frequencies of activated cTfh, suggesting coordinated expansion of both populations.In contrast, chronics displayed delayed kinetics of E2-specific MBC expansion with a dominant activated phenotype, but no ASCs were detected.Compared to primary HCV infection (11), expansion of E2specific MBCs upon reinfection occurred earlier and at nearly six-fold higher frequencies (up to 2.5% in reinfection versus 0.4% in primary infection).These findings suggest rapid reactivation of HCV-specific MBCs possibly due to efficient HCV-specific memory T-cell help generated following clearance of primary infection.Indeed, the upregulation of transcriptomic plasma cell signatures was associated with the clearance of HCV secondary infection (17).Interestingly, frequencies of E2-specific MBCs correlated with the levels of anti-E2 antibodies in resolvers and chronics at early acute reinfection as described during primary infection (48).Thus, E2specific MBC expansion led to higher titers of anti-E2 antibodies in resolvers compared to chronics at early acute reinfection.It is possible that similar to reports during chronic lymphocytic choriomeningitis virus (LCMV) infection, the production of type I IFN by CD8 T cells or monocytes may have driven the differentiation of B cells into short-lived ASCs with a limited early antibody response (49-51).Plasma neutralization activity was also boosted in resolvers upon reinfection, as reported by previous studies (16, 20, 21, 52), and is probably mediated by the higher titers of anti-E2.Indeed, our observations are in line with a recently reported mechanism in which early NAbs exert selection pressure that leads to E2 substitutions followed by loss of viral fitness contributing to clearance of HCV (15, 53).Virus sequencing may resolve some of these questions; however, the low-level viremia and rapid clearance upon reinfection remain a major obstacle to this approach.Overall, resolvers showed earlier and higher plasma neutralization breadth and potency that declined with time.This early plasma neutralization activity is strongly correlated with frequencies of E2-specific MBC and cTfh1 cells in resolvers, which has been previously for other viral infections or vaccines (38,(54)(55)(56)).
Both T-and B-cell responses contribute to the clearance of HCV reinfection, but very few studies have examined both simultaneously.Resolution of HCV reinfection was associated with expansion of virus-specific memory CD4 and CD8 T cells (17,18,24,57) and was dependent upon cross-recognition of the reinfection virus sequence by pre-existing memory T cells (24).When both responses were examined, different patterns and kinetics of T-and B-cell responses were detected in resolving reinfections (18).In the present study, we also observed heterogeneity within the cTfh, B cells, and NAb responses in resolvers.Similarly, two recent studies reported variable patterns of NAbs during HCV reinfection (20,21).Altogether, these data suggest different paths to HCV clearance where each arm of the adaptive immune response may contribute to various degrees.Although we could not assess viral sequence in the present study, it is tempting to speculate that in the case of viral escape from the CD8 T cells, help from Tfh to support the production of NAbs and functional MBCs (20) becomes more crucial in the control of virus replication.
There are a few limitations to this study.First, because of the limited availability of the samples, we focused primarily on the analysis of AIM + cells at the early acute time points where resolvers showed higher frequencies of activated cTfh and E2-specific MBCs.Second, because we used J6-E2 (Gt 2a) tetramers, we may have missed MBCs recognizing non-conserved epitopes in the E2 protein.The use of the full-length envelope protein (E1E2) would be of interest in future studies to enhance the detection of HCV-specific MBCs.Third, we did not define the epitopes and structural characteristics of NAbs isolated from resolvers versus chronics that may influence neutralization efficacy.However, data from previous studies identified potent NAbs targeting similar epitopes in resolvers and chronics (53,(58)(59)(60).So far, data suggest that it is the timing of the appearance of NAbs rather than their quality that is the main determinant of spontaneous clearance of HCV infection and reinfection.
Our results suggest an important role of Tfh cells during recall responses to human HCV infection, and cTfh1 cells provide help to MBCs for the generation of potent NAbs that contribute to the rapid clearance of reinfection upon re-exposure.Future studies examining the molecular mechanisms implicated in preferential expansion of these subsets in resolvers should provide potential targets for enhancing the immune response to next-generation vaccines against HCV and other viruses.
The funders had no role in study design, data collection analysis, decision to publish, or preparation of the manuscript.

TABLE 1
Summary of the subjects' demographics and clinical characteristics.
ND, not determined.