Follicular Helper T (TFH) Cell Targeting by TLR8 Signaling For Improving HBsAg-Specific B Cell Response In Chronic Hepatitis B Patients

Identifying signaling pathways that induce B cell response can aid functional cure strategies for chronic hepatitis B infection (CHB). TLR8 activation with ssRNA was shown to enhance follicular helper T cell (TFH) function leading to improved B cell responses in vitro. We investigated whether this mechanism can rescue an exhausted immune response in CHB infection. Effect of TLR8 agonism on supporting cytokines and TFH and B cells were evaluated using ex vivo and in vitro assays. The ability of an oral TLR8 agonist to promote TFH and B cell response was tested in samples from phase 1b clinical trial. TLR8 agonism induced TFH polarizing cytokine IL-12 in monocytes. Treatment of peripheral blood mononuclear cells (PBMCs) from CHB patients with TLR8 agonists induced cytokine IL-21 by TFH cells with enhanced IL-21+BCL-6+ and ICOS+BCL-6+ co-expression. Mechanistically, incubation of isolated naïve CD4+ T cells with TLR8 triggered monocytes resulted in their differentiation into IL-21+ICOS+BCL-6+ TFH in an IL-12 dependent manner. Furthermore, co-culture of these IL-21 producing TFH with autologous naïve B cells led to enhanced memory (CD19+CD27+) and plasma B cell generation (CD19+CD27++CD38+) and IgG production. Importantly, in TFH from CHB patients treated with an oral TLR8 agonist, HBsAg-specific BCL-6, ICOS, IL-21 and CD40L expression and rescue of defective activation induced marker (AIM) response along with partial restoration of HBsAg-specific B cell ELISPOT response was evident. TLR8 agonism can thus enhance HBV-specific B cell responses in CHB patients by improving monocyte-mediated TFH function and may play a role in achieving HBV functional cure.


INTRODUCTION
Hepatitis B virus (HBV) infection remains a major global health burden with over 257 million people worldwide chronically infected. A major barrier to HBV cure with standard nucleos/tide analog (NUC) therapy is the persistent dysfunction of the antiviral (HBV) immune response despite therapy induced viral suppression (1,2). As such identifying immunomodulatory strategies that aid HBs antigen loss/seroconversion, which defines functional cure of infection, is of high significance in HBV cure research (3).
In the past years, HBs antigen (HBsAg)-specific B cells have received attention and several laboratories including ours identified dysfunction in antigen-specific B cell response (4)(5)(6). These B cells have upregulated inhibitory receptor expression and impaired HBsAg-specific IgG production ex vivo. Dysfunctional B cell response can be attributed to intrinsic B cell defects along with insufficient help from CD4 + T cells. T follicular helper (T FH ) cells are the CD4 + T cells that provide signals for antigen specific B cell maturation into a plasma cell, making them essential for the generation of most isotype switched and affinity matured antibodies. Coordinated T FH and B cell response determines acquisition and maintenance of antibody response to prophylactic Abbreviations: CHB, chronic hepatitis B; HBV, hepatitis B virus; T FH , follicular helper T cells; cT FH , circulating follicular helper T cells; PBMCs, peripheral blood mononuclear cells; APCs, antigen-presenting cells; TLR8, toll-like receptor 8; HC, healthy control; GC, germinal center; HBsAg, hepatitis B surface antigen; ssRNA40, single stranded RNA40; mg, milligram; Kg, kilogram; PBS, phosphate-buffered saline, FBS, fetal bovine serum; AIM assay, activation induced marker assay; SEB, staphylococcal enterotoxin B; ELISPOT, enzymelinked immune absorbent spot; ASC, antigen-specific antibody secreting cell; IL, interleukin; LPS, lipopolysaccharide; IMQ, imiquimod; TNF-a, tumor-necrosis factor alpha; MB, memory B; PB, plasma B; PCs, plasma cells; PBs, plasmablasts; NBs, naïve B; RMBs, resting memory B; AMBs, activated memory B; ATMBs, atypical memory B; PMA, phorbol-12-myristat-13-acetate; Ion, ionomycin; LEP, large envelop protein; Stim-stimulation; FACS, fluorescence-activated cell sorting; ICS, intracellular staining; UT, untreated; BL, baseline; IgG, immunoglobulin G; ELISA, enzyme-linked immunosorbent assay; HBV Pep; hepatitis B virus peptide; ANOVA, analysis of variance.
HBsAg based vaccine (7). This response is defective during CHB infection, where an accumulation of activated and phenotypically abnormal T FH dysregulates cytokine profiles (8,9). Dysfunctional antigen-specific T FH response was shown to promote persistence of HBV in a mouse model (9). In samples from CHB patients, impaired T FH response was shown to be due to IL-21 suppression mediated by CTLA-expressing Tregs, which could be restored by inhibiting Tregs with an antibody against CTLA-4 (9). We showed that abnormal T FH persist even in long-term NUC treated CHB patients, possibly explaining the low incidence of anti-HBs seroconversion in these patients (6). Thus, we believe this impaired T FH response is a critical defect associated with hepatitis B virus persistence and resolving the dysfunction will be of significance in resolution of CHB.
Among the immunomodulatory strategies, toll-like receptors are a promising way to correct immune deficiencies. Human liver derived monocytes respond strongly to TLR8 agonism, producing IL-12 and IL-18 (10). IL-12 induced by TLR8 signaling has a potential impact on anti-viral CD8 T cell response (11). Importantly, IL-12 was shown to aid T FH differentiation that led to plasma cell generation in individuals vaccinated with a live attenuated vaccine (12). We investigated whether similar mechanism can restore dysfunctional T FH response and aid exhausted HBsAg-specific B cell response (4-6) present in chronic HBV infection. Here we demonstrate that TLR8 signaling induces differentiation of T FH cells into efficient B cell helpers with potential to promote plasma cell generation and an HBsAg-specific B cell response.

Patients and Samples
For investigating in vitro effects of TLR8 stimulation, peripheral blood samples were available from CHB patients or HBV vaccinated healthy volunteers enrolled in HOPE cohort at University of Maryland, Baltimore. The study protocol was approved by the institutional ethical committee, and all subjects gave written, informed consent. Demographic and clinical details of patients are provided in Table 1.

Flow Cytometry (FACS) and Intracellular Cytokine Staining (ICS)
PBMCs were stained and analyzed by flow cytometry using standard methods (panels listed in Supplementary Table 1). Cells were acquired on a Cytek Aurora multi-color FACS machine and data analysis done by Flow Jo software (Tree Star, San Carlos, California, USA).

Activation Induced Markers (AIM) Assay
For examination of contribution of TLR8 signaling in HBV antigen-specific germinal center like (GC) T FH induction, a cytokine-independent approach was followed (16,17). It is known that formulation of antigen can influence the type and extent of activation markers induced in CD4 + T cells (18). To capture multiple activation markers induced in T FH with TLR8 signaling, here we used combination of HBsAg intact protein and peptide, as is described for other antigens (16,17). Approximately 1-2 x 10 6 PBMCs were aliquoted into 24-well plate and stimulated with HBV envelope peptide (2 µg/mL, JPT) and recombinant human HBsAg subtype adw (10 µg/mL, Fitzgerald). Intact protein antigens or antigenic peptides are used for stimulating antigen specific CD4 T cell response. Staphylococcal enterotoxin B (SEB 1 µg/mL, Toxins Technology) served as positive control and AIM-V medium alone served as untreated negative control (UT). CXCR5-BV605 and CXCR3-PeCy7 antibodies (Biolegend, 1 ml each) were added directly into the cell culture well and also included in the staining panel. After 18h stimulation, PBMCs cultures were washed twice with 1X PBS and followed the FACS staining protocol as stated in the above method using AIM assay panel

T FH Cells Sorting and Naïve B Cells Co-Culture
For T FH sorting, cells recovered after 6 days of monocyte-naïve CD4 co-culture (n=4) were stained with anti-human PD-1-BV421, ICOS-BV510, CXCR5-BV605, CXCR3-PeCy7 and CD4-APC antibodies. T FH cell sorting was carried out on BD FACS Aria III. For naïve B-T FH co-culture, naïve B cells were isolated (Miltenyi Biotec) from autologous PBMCs of CHB patients (n=4) and co-cultured with sorted T FH cells (from above) at 2:1 (B:T FH ) in the presence of SEB (1 µg/mL) for 7 days after which T FH -mediated differentiation of B cells into various subsets was analyzed by flow cytometry.

B Cell ELISPOT Assay
PBMC from CHB patients obtained at baseline (BL, pre-TLR8) and TLR8 (8h post-Selgantolimod) single oral dose were tested. To examine HBsAg-specific antibody secreting B cells, cells were stimulated with polyclonal stimuli R848 (1 µg/mL, Mabtech) and recombinant human IL-2 (10 ng/mL, Mabtech) and cultured for 5 days to effectively induce memory B cell proliferation. B cell ELISPOT assay was conducted as previously described (6).

Luminex Multiplex Immunoassay
Quantification of IL-12p40, IL-18 and IL-21 in cell culture supernatants collected from co-culture experiments was analyzed by Luminex multiplex immunoassay (Luminex LX200 multianalyte system, Bio-Rad Corporation) according to manufacturer protocol.

Enzyme-Linked Immunosorbent Assay (ELISA)
Quantification of human class-switched immunoglobulin (IgG) was made in cell culture supernatants collected from naïve B-T FH cell co-cultures using high sensitivity ELISA kit according to manufacturer's protocol (XpressBio, Express Biotech International).

Statistical Analyses
Statistical analyses were performed using one-way analysis of variance (ANOVA) or Kruskal-Wallis with Dunn's multiple comparisons test. Paired samples between BL (pre) and TLR8 (8h-post) were analyzed by two-tailed paired, or the Wilcoxon signed-rank Student's t test or non-parametric Mann-Whitney U test for comparisons between treatments. A p value of <0.05 was considered significant. Levels of significance are indicated by *: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 and ns (no significance).

In Vitro TLR8 Stimulation Induces Proinflammatory Cytokines in Monocytes From CHB Patients
TLR8 signaling induces IL-12 in monocytes, which supports T FH differentiation in healthy individuals (12). We tested whether this pathway induces T FH polarizing cytokines in samples from CHB patients. PBMCs isolated from CHB patients were stimulated with various TLR agonists for 18-24h or with medium alone (UT) as negative control. The ligation of TLR8 in monocytes by synthetic agonists, single stranded RNA40 (ssRNA40/LyoVec), TL8-506 and TLR7/8 ligand R848 induced proinflammatory cytokines IL-6, IL-12p40, IL-18, TNF-a in CD14 + HLA-DR + activated monocytes ( Figures 1A-D) as analyzed by intracellular cytokine staining using flow cytometry. TLR8 agonist ssRNA40 significantly enhanced the production of cytokines IL-12p40 and IL-18 when compared with other TLR agonists. LPS stimulation induced cytokines IL-6, IL-1b and IL-23p19. Relative frequencies of CD14 + HLA-DR + monocytes producing

TLR8 Agonism Induces T FH Cells Differentiation
Having shown potent induction of T FH polarizing cytokine IL-12p40 (12) in monocytes from CHB samples, we next examined the impact of TLR8 agonism on T FH phenotypes. Circulating CD4 + CXCR5 + cells in peripheral blood share characteristics and functional phenotypes with GC-like T FH cells (20). The expression of signature cytokine IL-21, BCL-6 (a master regulator of T FH cells programing and differentiation) and CD40 ligand (CD40L) in T FH cells provide signals to B cells that are required for Ig class-switching recombination and high affinity antibody productions (21). The expression of inducible co-stimulatory molecule (ICOS) indicates T FH development, migration and their active state (22). We asked whether TLR8induced cytokines modulate these immunophenotypic profiles of circulating T FH cells (cT FH ). CHB patient samples were stimulated with TLR8-specific agonists ssRNA40 or TL8-506 in the presence or absence of mitogenic stimulation using PMA/Ion (required for induction of BCL-6 and ICOS). The sequential gating strategy for T FH cell panel is presented in Supplementary  Figure 2A. Flow plots ( Figure 2A) show that the frequency of IL-21 producing cT FH cells (IL-21 + CXCR5 + ) gated on CD4 + T cells was significantly increased in conditions with ssRNA40 or TL8-506. The data represented for ssRNA40 stim or TL8-506 stim are response found in TLR8 agoniost+PMA/Ion re-stimulated response subtracted with PMA/Ion alone response (Figures 2A-F). The co-expression of GC-like T FH markers IL-21 + BCL-6 + and ICOS + BCL-6 + were significantly increased in ssRNA40 stim or TL8-506 stim conditions ( Figures 2B-F).
Since proinflammatory cytokines IL-12p40 and IL-18 were induced in monocytes in response to TLR8 agonism ( Figure 1), we measured levels of these cytokines in the PBMC culture supernatants. As expected, cultures incubated with TLR8-specific agonists ssRNA40 or TL8-506 markedly induced the secretion of both these cytokines ( Figures 2G, H). Next, we asked whether TLR8-specific signaling modulates subsets of circulating T FH cells in CHB patients. When compared with untreated negative control, PBMCs in conditions with ssRNA40 or TL8-506 had significantly increased frequencies of cT F H subsets cT F H 1 (CXCR3 + CCR6 -) and T F H 1/17 (CXCR3 + CCR6 + ) (Supplementary Figures 2B, D, G), while no differences were observed for global cT FH (CXCR5 + CD4 + gated on CD3 + T lymphocytes) cells, subsets cT FH 2 (CXCR3 -CCR6 -) and cT FH 17 (CXCR3 -CCR6 + ) (Supplementary Figures 2C, E, F). It has been previously reported that cT FH 1 and cT FH 1/17 are the source for IL-21 and IFNg, whereas cT FH 2 and cT FH 17 are source for anti-inflammatory cytokines IL-4, IL-10 and IL-17A. These results collectively suggest that TLR8specific signaling induces IL-21 producing T FH subsets in the setting of CHB.

Selgantolimod Treatment Enhanced T FH Differentiation and Rescued Defective Antigen-Specific Activation of T FH Cells
After in vitro demonstration of T FH differentiation with TLR8 agonism, we tested samples from CHB patients given a single oral dose of Selgantolimod. Surprisingly, in post-Selgantolimod treated samples that had been obtained only 8 hours after dosing, a significant induction of GC-like signature T FH factors IL-21 + BCL-6 + , ICOS + BCL-6 + and ICOS + CD40L + cells were observed (Supplementary Figures 3A-H).
Next, we employed a cytokine-independent method for detection of HBsAg-specific T FH cell activation by measuring the upregulation of activation induced markers (AIM) CD69, CD25, OX40, PD-L1 and CD40L (16,17). Both frequencies of CD3 + CD4 + CXCR5 + CXCR3 -PD-1 + T FH and their activation, indicated by surface expression of OX40 + CD25 + , PD-L1 + CD25 + and CD69 + CD40L + , was increased in vaccinated individuals upon in vitro stimulation with HBV antigens or SEB compared to the negative control ( Figures 3A-F,  Supplementary Figure 4). Expectedly, baseline samples from CHB patients displayed low HBV specific activation of T FH indicating defective response. Importantly, a significant increase in AIM response was evident in 8 hour samples, which was comparable to HBV vaccinated samples ( Figures 3A-F). Additionally, the frequencies of PD-1 + CXCR3 -cT FH cells, but not total CXCR5 + cT FH cells, were significantly higher in samples collected at post-TLR8 time-point (Supplementary Figures 4B, C). These data demonstrate that TLR8-specific signaling can normalize defective antigen specific T FH response.
Previously we reported that atypical memory B cells that expressed high levels of FcRL family receptors were expanded in CHB patients and this sub-population lacks HBsAg-specific B cell response (6). Here we observed that TLR8 stimulation expanded memory B cells with an atypical phenotypic signature (CD19 + CD21 -CD27 -) ( Figure 4C, I). We believe that this subset lacking CD21 and CD27 that is expanded in response   (9,23). It is also known that during malaria, B cells with similar 'atypical' phenotype are in fact functional and produce neutralizing antibodies (24).

TLR8 Signaling Promotes IL-12-Dependent GC-Like T FH Cell Differentiation and Improved IL-21 Production
In order to discern the mechanism behind TLR8-mediated differentiation and proliferation of T FH cells, we performed coculture experiments of isolated monocytes and CD4 + T cells.   Figures 6A-C).
In humans, TLR8 pathway-specific secretion of proinflammatory cytokine IL-12 sends the signal responsible for IL-21 production and drives the T FH cell differentiation (12,25). In CHB patients, T FH cell frequencies are high due to hyperactivation and these cells lack IL-21 cytokine production. We therefore explored whether TLR8-specific cytokine induction will promote circulating T FH cell differentiation and recover defective IL-21 production in PBMCs of CHB patients. IL-12p40 and IL-18 secretion in monocytes and IL-21 production from newly generated T FH cells were quantitatively analyzed in supernatants of co-culture experiments and naïve CD4 + T cell culture by Luminex multiplex immunoassay. As expected, IL-12 neutralization (ssRNA40+a-IL-12 and more clearly TL8-506+a-IL-12) in CD14 + monocytes-naïve CD4 + T cells co-culture significantly decreased IL-21 production as well as the co-expression of GC-like T FH markers IL-21 + ICOS + and ICOS + PD-1 + , on cT FH cells (CD3 + CD4 + CXCR5 + ); IL-18 neutralization (ssRNA40+a-IL-18 or TL8-506+a-IL-18) did not have similar effect ( Figures 5A-C and Supplementary Figures 6A-C). Correspondingly, the secretion of cytokine IL-21 was significantly reduced in co-cultures pre-treated with IL-12 neutralizing antibody (ssRNA40+a-IL-12 and more notably with TL8-506+a-IL-12). IL-18 blocking again did not have an effect on IL-21 production in either ssRNA40+a-IL-18 or TL8-506+a-IL-18 antibody treatment conditions (Figures 5D-F). IL-18 production remained intact with a-IL-12 treatments. To further justify that TLR8-specific IL-12 cytokine-dependent signal is responsible for T FH cell differentiation and IL-21 cytokine production, we tested naïve CD4+ T cells isolated from PBMCs of HC and CHB patients that were left untreated or directly incubated with recombinant human IL-12 or IL-18 and cultured for 5 days in the absence of CD14 + monocytes. The frequency of IL-21 producing T FH cells was significantly increased when incubated with rhIL-12, but not with rhIL-18 ( Figures 5A-F and Supplementary Figure 6F). It is important to note here that effect of TLR8 was not specific to CXCR5 + T FH and the agonist activated CD4 + T cells including CXCR5cells in an IL-12 dependent manner (Supplementary Figures 6D, E). Taken together, these data demonstrate that TLR8-specific IL-12-induction in monocytes plays a critical role in the T FH cells differentiation, which can restore the deficient IL-21 production in CHB.

T FH Cells Differentiated by TLR8 Agonists Support The Generation of Memory B and Plasma B Cells
To evaluate the utility of newly generated T FH cells as true helpers of B cells, we sorted CD4 + CXCR5 + CXCR3 -PD-1 + ICOS + T FH generated with UT, ssRNA40 or TL8-506 pre-exposed CD14 + monocytes and assessed their ability to promote plasma B cell differentiation after co-culture with autologous naïve B cells for 7 days. T FH generated from TLR8 primed monocytes enhanced the differentiation of global memory B (CD27 + ), plasma B (CD27 ++ CD38 + ), long-lived antibodyproducing plasma cells (CD27 + + CD38 -CD138 + ) and plasmablasts (PBs) (CD27 ++ CD38 + CD138 -), whereas T FH derived from UT or naïve CD4 + T cells co-cultured with B cells failed to induce memory, plasma B and long-live plasma cell differentiation (Figures 6A-J).
Additionally, significantly higher frequencies of IgG + (CD19 + CD27 + ) memory B cells and robust IgG production in TL8-506 primed T FH -naïve B co-culture condition compared to naïve B without or with naïve CD4 + T or UT_T FH coculture conditions was present (Figures 6I, J). Flow plot in Figure 6D shows ssRNA40 and TL8-506-primed T FH -naïve B co-culture conditions resulted in a reduction in naïve B (CD19 + CD21 + CD27 -) and increase in resting memory B (CD19 + CD21 + CD27 + ), activated memory B (CD19 + CD21 -CD27 + ) and atypical memory B (CD19 + CD21 -CD27 -) cell populations, relative to non-TLR8 conditions. Thus T FH differentiated with TLR8-treatment support the generation of memory B cells and their subsets in CHB samples.

Selgantolimod Treatment Enhanced B Cell Responses
To assess whether this mechanism of enhancing T FH response impacts B cell response in vivo, we next examined the effect of Selgantolimod oral treatment in paired samples from before and 8 hours after treatment. For this experiment, in addition to phase 1b clinical samples, clinical samples from phase 1a trial in CHB negative healthy subjects with known positive HBV vaccination status were tested for HBsAg-specific IgG and total IgG with ELISPOT assays. We defined responders as those who exhibited an increase of ≥1.5-fold SFU/ml after Selgantolimod administration. Using this as criteria, there were 4/11 responders for HBs specific and 6/13 responders for total IgG response among phase 1a samples ( Figures 7A-C). In samples from CHB patients (phase 1b), HBs specific IgG spots increased in 6/17 subjects and total IgG in 5/18 subjects (Figures 7D-F).
Most subjects that showed improvement in spots had lower baseline IgG response, though there was no clear cutoff for determining responders. These outcomes suggest that TLR8 treatment has potential to augment memory B cell response in a subset of CHB subjects. It will be important to identify characteristics of responders or non-responders; however, here we did not see positive correlation between T FH response and ELISPOT response in the small number of responders.

DISCUSSION
In this investigation we demonstrated that triggering of TLR8 induces IL-12 production from monocytes, which in turn leads to differentiation of CD4 + T cells into IL-21 producing T FH in peripheral blood samples from CHB patients. Accordingly, co-culture of these differentiated T FH with autologous B cells resulted in B cell differentiation into plasma cells and promoted IgG production. Finally, in a fraction of CHB patients treated with a selective TLR8 agonist, improved HBsAg-specific T FH and B cell responses were observed. Therefore, our study established that TLR8 signaling has potential to restore a critical defect in T-B interaction that is necessary for robust B cell response.
Humoral immunity requires interaction between B cells and specialized populations of CD4 + T cells, the CD4 + CXCR5 +
follicular helper (T FH ) cells, the latter help generate memory B cells and long-lived plasma cells (26); both these immune cell types are dysfunctional in CHB patients. Since HBcAg-specific antibodies are generated during infection, this defect is selective to HBsAg-specific cells, potentially due to high levels of circulating HBsAg present in patients (27). Indeed, HBs specific but not HBc specific B cells show characteristics of impaired cells characterized by atypical memory phenotype and poor differentiation into antibody secreting cells (5,6,28,29). At the same time, T FH have dysregulated response to HBsAg that associates with HBV persistence (30). Our results also demonstrate that HBsAg-specific T FH response is defective in CHB patients relative to HBV vaccinated controls. We show here that IL-12 induction by monocytes in response to TLR8 signaling was able to rescue these defective T FH . Specifically, signaling through TLR8 resulted in differentiation of T FH into phenotypes consistent with bona fide helpers of B cells i.e., PD-1 + BCL-6 + ICOS + IL-21 + , in samples from CHB patients. In vitro use of Selgantolimod resulted in a similar increase in frequency of circulating PD-1 + T FH in another study (31). We have only studied peripheral T FH here due to limitations in access to lymphoid cells from patients. As such this may not entirely reflect the impact on antibody generation process, which occurs in secondary lymphoid organs, specifically in structures called germinal centers. However, specific subsets of circulating T FH are identified which share similar phenotypes, transcriptional and  functional characteristics with germinal center T FH cells (20); this peripheral population allows an easily accessible means to examine phenotypic and functional state of these cells during diseases and to investigate effects of interventions on them (21). Canonical T FH differentiation starts with dendritic cell priming of naïve CD4 + T cell whereupon receiving cytokine signals, expression of CXCR5 on CD4 + T cell allows early T FH to migrate to T-B cell border and undergo further differentiation. Among DC derived cytokines, IL-12 and TGF-b1 are most efficient at inducing human naïve CD4 + T cells to express T FH molecules CXCR5, ICOS, IL-21 and BCL-6 (32-36). Recently, it was discovered that TLR8 signaling results in differentiation of functional T FH ; the intact ssRNA in attenuated vaccines triggers TLR8 on monocytes, inducing IL-12 production, which in turn differentiates naïve CD4 + T cells into functional T FH . These T FH cells support plasma cell generation and IgG production from B cells (12). CHB infection is associated with significant immune dysregulation, whether TLR8 agonism will lead to TFH differentiation and subsequent B cell help was therefore not a given. Reports have shown both functional (10) and defective TLR8 response (37) in CHB. However, in vitro testing of Selgantolimod showed comparable levels of cytokines in PBMC from healthy and CHB subjects (31). In clinical trial of this agonist in healthy subjects, we previously showed IL-12p40, IL-12p70, IL-1RA and IL-18 induction in serum (14), here we show T FH polarizing cytokines, IL-12 is induced with the agonist in monocytes from CHB patients which led to naïve CD4 + T cells to differentiated into CXCR5 + BCL-6 + ICOS + IL-21 + T FH . A significant finding here is demonstration of HBsAg-specific T FH response (ICOS + BCL-6 + T FH and upregulation of AIM markers) in CHB patients treated with a single dose of Selgantolimod. IL-12p40 and IL-12-p70 are induced within 4 hours after oral administration of this agonist (14); we hypothesize that IL-12p70 provided requisite signal to naïve CD4 + T cells in treated patients, which was manifested by the expression of T FH -specific growth factors when PBMC were tested in our in vitro culture assays. Mechanistically, TLR8 agonism induced T FH with phenotypes bona fide helpers which aided plasma cell generation and IgG response. In our study this was reflected in results from naïve B-T FH co-culture and B cell ELISPOT assays, which demonstrated enhanced total IgG and HBsAg-specific B cell responses after TLR8 agonist treatment. It may seem surprising that an improvement in B cell response was present in an acute treatment setting (8 hours) with a single dose of Selgantolimod. However, it is important to note that these B cell responses were not measured ex vivo, rather the ELISPOT assay measures memory B cell responses generated after short-term cultures of 5 days. We believe that Selgantolimod treatment generated favorable conditions, such as effective T FH response and cytokine IL-21, which aided these improved B cell responses in vitro.
These finding have significance for use of TLR8 agonism for hepatotropic HBV; liver derived cells respond to TLR8 agonism by inducing IL-12 and IL-18 (10). In a woodchuck model of chronic HBV, this same TLR8 agonist resulted in sustained antiviral response and loss of HBs antigen (38). This model uses a surrogate virus and the translatability of these findings to CHB in humans is uncertain, however, the same agonist resulted in similar induction of cytokines IL-12p40, IL-12p70, IL-1b, IL-6, IL-18 and TNF-a in vivo in humans (14,13,38) and in cultures (here). In a separate study, IL-12, IL-1b and IFN-b induced by ssRNA were shown critical for T FH differentiation, this effect was mediated by TLR signaling adaptor TRIF (39).
There are certain limitations of our study. For more precise evaluation of antigen specific T FH and B cells, use of class II tetramers and HBsAg specific B cell probes, respectively, is needed. It is out of the scope for this investigation due to limited availability of frozen PBMC from these previously completed clinical trials for further experiments. It is also important to show whether an increase in the IgG secreting B cells (ASCs) observed in ELISPOT from some patients correlates with enhanced monocytic IL-12 production or with expression of GC-like T FH growth factors (IL-21 + BCL-6 + , IL-21 + CD40L + and ICOS + BCL-6 + ) or B cell phenotypes. However, due to small sample size it was not possible to perform such correlation analyses in 'responders' and 'non-responders' to TLR8 agonism. A focus on peripheral examination of T-B responses with lack of any investigation into tissue or lymph node responses is another limitation here. T FH -B cell responses will be studied in the ongoing Phase 2 clinical trial, which could establish the true nature of T FH -B cell interactions in restoring clinically relevant anti-HBs response in CHB patients.
Our data has significance for our understanding of impaired HBV-specific protective immunity in CHB patients. Strategies to achieve a functional cure are focused on restoring HBs-antibody responses in CHB patients. HBs antigen-based vaccine candidates have shown mixed results when tested in clinical trials in chronically infected patients (40). Our data support further testing of TLR8 agonism in HBV functional cure approaches that include a combination of antiviral and immune modulatory agents.

DATA AVAILABILITY STATEMENT
The original data presented in the study are included in the article and Supplementary Material. Further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by University of Maryland, Baltimore. The patients/ participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
NA designed methods, performed the experiments, analyzed data and prepared results. LT provided clinical expertise and provided CHB samples. SKT, DC, JW, and SF provided samples from Selgantolimod clinical trials and critically edited the manuscript. SK participated in study design, critically edited the manuscript and provided resources. BP designed the study, analyzed and interpreted the results, wrote the manuscript and provided funding. All authors contributed to the article and approved the submitted version.

FUNDING
The study was financially supported by Gilead Sciences, Foster City, CA and IHV, University of Maryland school of Medicine, Baltimore, MD.