Therapeutic Potential of TLR8 Agonist GS‐9688 (Selgantolimod) in Chronic Hepatitis B: Remodeling of Antiviral and Regulatory Mediators

Background and Aims GS‐9688 (selgantolimod) is a toll‐like receptor 8 agonist in clinical development for the treatment of chronic hepatitis B (CHB). Antiviral activity of GS‐9688 has previously been evaluated in vitro in HBV‐infected hepatocytes and in vivo in the woodchuck model of CHB. Here we evaluated the potential of GS‐9688 to boost responses contributing to viral control and to modulate regulatory mediators. Approach and Results We characterized the effect of GS‐9688 on immune cell subsets in vitro in peripheral blood mononuclear cells of healthy controls and patients with CHB. GS‐9688 activated dendritic cells and mononuclear phagocytes to produce IL‐12 and other immunomodulatory mediators, inducing a comparable cytokine profile in healthy controls and patients with CHB. GS‐9688 increased the frequency of activated natural killer (NK) cells, mucosal‐associated invariant T cells, CD4+ follicular helper T cells, and, in about 50% of patients, HBV‐specific CD8+ T cells expressing interferon‐γ. Moreover, in vitro stimulation with GS‐9688 induced NK‐cell expression of interferon‐γ and TNF‐α, and promoted hepatocyte lysis. We also assessed whether GS‐9688 inhibited immunosuppressive cell subsets that might enhance antiviral efficacy. Stimulation with GS‐9688 reduced the frequency of CD4+ regulatory T cells and monocytic myeloid‐derived suppressor cells (MDSCs). Residual MDSCs expressed higher levels of negative immune regulators, galectin‐9 and programmed death‐ligand 1. Conversely, GS‐9688 induced an expansion of immunoregulatory TNF‐related apoptosis‐inducing ligand+ NK cells and degranulation of arginase‐I+ polymorphonuclear MDSCs. Conclusions GS‐9688 induces cytokines in human peripheral blood mononuclear cells that are able to activate antiviral effector function by multiple immune mediators (HBV‐specific CD8+ T cells, CD4+ follicular helper T cells, NK cells, and mucosal‐associated invariant T cells). Although reducing the frequency of some immunoregulatory subsets, it enhances the immunosuppressive potential of others, highlighting potential biomarkers and immunotherapeutic targets to optimize the antiviral efficacy of GS‐9688.

causes more than 800,000 deaths a year due to HBV-related complications such as cirrhosis and HCC. (1) In the absence of novel treatment strategies, it is projected that new cases of CHB will rise to three million per year by 2030. (2) Current approved therapies include long-term antiviral suppression with nucleos(t)ide analogues and pegylated interferonα (IFNα). These treatments reduce viremia and improve patient outcomes but are rarely curative. (3) As a consequence, there is a pressing need for novel immunotherapeutic strategies to supplement existing direct-acting antivirals to achieve durable immune control.
Control of HBV is dependent on the coordinated action of both innate and adaptive immunity. (4) A major obstacle to HBV clearance in CHB is a dysfunctional adaptive response, characterized by a profound state of immune exhaustion and HBV-specific T-cell depletion. (5,6) The mechanism by which this dysfunction occurs is multifaceted, but likely driven by a combination of ongoing high-dose antigenic stimulation and the tolerogenic liver environment. Moreover, while the natural killer (NK) cell compartment can exert direct and indirect antiviral activity, they may also restrict effective antiviral immunity by deleting apoptosisprone HBV-specific T cells. Specifically, NK cells in the HBV-infected liver up-regulate the death-ligand TNF-related apoptosis-inducing ligand (TRAIL), enabling them to engage with and eliminate T cells expressing the reciprocal receptor, TRAIL-receptor 2 (TRAIL-R2). (7,8) Another mechanism contributing to the suppression of HBV immunity is the expansion of arginase + polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), which deplete key nutrients required by T cells for proliferation and function. (9) One therapeutic approach in clinical development for CHB is to engage innate immune receptors such as toll-like receptors (TLRs), which can exert both direct and indirect effects on the antiviral T-cell and B-cell response. A previous therapeutic strategy attempted to harness TLR7 signaling using the agonist GS-9620 (vesatolimod). GS-9620 induced sustained antiviral responses in animal models, (10,11) but failed to show therapeutic efficacy in patients with CHB. (12,13) More recently, attention has turned to TLR8 activation due to its anticipated ability to stimulate host immunity through the induction of pro-inflammatory and immunomodulatory cytokines. Human TLR8 is predominantly expressed on the endosomal membrane of monocytes, macrophages, conventional dendritic cells (cDCs), and CD4 + regulatory T cells (T REG ), and allows cells to respond to infection through detection of viral single-stranded RNA. (14)(15)(16) GS-9688 (selgantolimod) is an oral selective small-molecule agonist of TLR8 in development for CHB. In vitro studies have demonstrated that cytokines induced in human peripheral blood mononuclear cells (PBMCs) by GS-9688 reduce viral parameters in HBV-infected primary human hepatocytes. (17) Furthermore, GS-9688 treatment was well tolerated and induced a sustained antiviral response in a subset of woodchuck hepatitis virus-infected woodchucks. (18) Although these preclinical studies provide some insight into the antiviral efficacy of GS-9688, they did not provide insight into the mode of action of GS-9688 on human immune cells.
Accordingly, in this study we have evaluated the immunomodulatory effects of GS-9688 on various immune cells in vitro using freshly isolated peripheral leukocytes from healthy controls and patients with CHB, to understand its therapeutic potential for CHB.

StUDy CoHoRt
All participants were anti-HCV and anti-HIV antibody negative. Healthy controls were additionally anti-HBV negative. Participants with CHB were stratified by HBV viral load (IU/mL; determined by real-time PCR), HBsAg titer (IU/mL; determined by Architect; Abbott Laboratories, London, United Kingdom), HBeAg positivity, and serum alanine transaminase (ALT; IU/L) where appropriate (Supporting Table S1).

INVeStIgatIoNal DRUg
GS-9688 (selgantolimod) is a small-molecule TLR8 agonist manufactured by Gilead Sciences Inc. (Foster City, CA). (17) Concentrations used are denoted in the figures/figure legends.

MUltIpaRaMetRIC FloW CytoMetRy
For all flow cytometric analysis, cells were washed before staining in 1 × PBS. Cells were first stained with a fixable cell viability dye then stained with mAbs (Supporting Table S3) in brilliant violet buffer (BD Biosciences) for 30 minutes. Once stained, cells were washed and fixed with BDCytofix (BD Biosciences) for 20 minutes at 4°C. For intracellular antigens, cells were fixed and permeabilized using BDCytofix/Cytoperm and further stained with mAbs against intracellular antigens in 0.1% wt/vol Saponin (Sigma-Aldrich). For intranuclear antigens, cells were fixed and permeabilized using the human forkhead box P3 (FOXP3) buffer (BD Biosciences), and further stained with mAbs targeting intranuclear antigens in 1 × PBS for 30 minutes at 4°C. Samples were acquired on a LSRFortessaX20, and data analyzed using FlowJo (v.10.4.1; BD Biosciences). Single stain controls and anti-mouse immunoglobulin G (IgG) CompBeads (BD Biosciences) were used where appropriate.

aSSeSSMeNt oF MoNoNUCleaR pHagoCyteS, cDCs, aND plaSMaCytoID DeNDRItIC Cell pHeNotype aND FUNCtIoN
A total of 1 × 10 6 PBMCs/well were seeded in 96well flat-bottom plates in cRPMI with GS-9688 or ≤0.1% DMSO vehicle control at 37°C. After 2 hours, cells were treated with 50% vol/vol BDGolgiPlug (BD Biosciences) diluted in 1 × PBS and incubated at 37°C for 6 hours. Cells were then harvested and stained for flow cytometric analysis.

Hepg2 lySIS aSSay
NK cell cytolytic activity was assessed using a fluorescence-based killing assay (DELFIA ETDA Cytotoxicity Assay; PerkinElmer, Waltham, MA). PBMCs were treated with GS-9688 or ≤0.1% DMSO vehicle control at 37°C. After 16 hours, NK cells were isolated by negative selection (StemCell Technologies, Kent, WA). HepG2 cells (HCC cell line [HB-8065]; ATCC, Manassas, VA) were loaded with the intracellular-specific fluorescence-enhancing ligand BATDA and incubated for 30 minutes. HepG2 were co-cultured with pretreated, purified NK cells at a 1:10 target-to-effector ratio in V-bottomed plates and incubated for a further 2 hours. Culture supernatants were then harvested and extracellular release of BATDA was assessed by fluorescence activity using Europium solution, measured by a time-resolved fluorometer (VICTOR, PerkinElmer).

aSSeSSMeNt oF t Reg aND CD4+ FollICUlaR HelpeR t-Cell FReQUeNCy, pHeNotype, aND FUNCtIoN
A total of 0.5-1 × 10 6 PBMCs/well were seeded in 96-well round-bottom plates in cRPMI and stimulated with GS-9688 or ≤0.1% DMSO vehicle control at 37°C for 7 days. Cells were maintained in 500 IU/mL rhIL-2, continually supplemented throughout. After 7 days, cells were harvested and analyzed by flow cytometry.

aSSeSSMeNt oF MDSC FReQUeNCy, pHeNotype, aND FUNCtIoN
A total of 1 × 10 6 freshly isolated PBMCs were stained to determine ex vivo frequencies of MDSC subsets by flow cytometry. To determine the effect of GS-9688 on MDSC frequencies in the short term, 1-2 × 10 6 PBMCs/well were seeded in 48-well plates in cRPMI and cultured for 24 hours with GS-9688 or ≤0.1% DMSO vehicle control at 37°C. Following incubation, cells were harvested for analysis by flow cytometry. Culture supernatants were stored at −80° for analysis of extracellular arginase-I by ELISA (Hycult Biotech, Uden, the Netherlands).

StatIStICal aNalySIS
The E max defines the maximum cytokine level induced by GS-9688. The minimal effective concentration (MEC) defines the concentration of GS-9688 corresponding to three-fold induction of cytokine above background (DMSO control). EC 50 defines the concentration of GS-9688 giving 50% of the maximal response. MEC and EC 50 were calculated using the Pipeline Pilot v.9.2 (BIOVIA, San Diego, CA). All statistical analyses were performed in Prism (v.7.0e; GraphPad Software, San Diego, CA); tests used are indicated in figure legends (Wilcoxon signed-rank t test; Kruskal-Wallis test; Friedman test [ANOVA] with Dunn's post hoc test; and ANOVA with Sidak's post hoc test). All tests were carried out as two-tailed tests. Significant differences are denoted in all figures and defined as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

IN VITRO StIMUlatIoN oF HUMaN pBMC FRoM HealtHy CoNtRolS aND patIeNtS WItH CHB WItH gS-9688 pRoDUCeS IMMUNoMoDUlatoRy MeDIatoRS
To investigate the therapeutic potential of the TLR8 agonist GS-9688, we first assessed its in vitro activity and potency using human PBMCs isolated from 10 healthy controls. PBMCs were stimulated with GS-9688, and cell culture supernatants were evaluated by Luminex array (Fig. 1A). Among the cytokines tested, GS-9688 induced the immunomodulatory cytokine IL-12p40, in addition to the antiviral cytokines IL-6, TNFα, and interferon-g (IFNγ) (  Fig. S1A). Conversely, GS-9688 induced little to no IFNα, a prototypical cytokine predominantly produced following engagement of TLR7, (19,20) consistent with its selectivity for human TLR8 (17) (Fig. 1B,C and Supporting Fig. S1A). Importantly, there was no significant difference in response in PBMCs from 10 age-matched patients with CHB, with comparable MEC, EC 50 , and E max for all cytokines tested ( Fig. 1C and Supporting Fig. S1A).

gS-9688 aCtIVateS cDCs aND MoNoNUCleaR pHagoCyteS to pRoDUCe Il-12p40 aND tNFα
To investigate the cellular source of relevant immunomodulatory cytokines, we examined the effect of GS-9688 on circulating myeloid cells. Human TLR8 is expressed primarily by cDCs and mononuclear phagocytes (MNPs), but is absent on plasmacytoid dendritic cells (pDCs). (14)(15)(16) Short-term stimulation of PBMCs with GS-9688 increased production of IL-12p40 by Lin1 − HLA-DR + CD123 − CD11c + cDCs, in a dose-dependent manner (Fig. 1D). Likewise, TNFα was produced by cDCs and Lin1 + CD14 + MNPs in response to GS-9688 (Fig. 1E). The cDCs, pDCs, and MNPs produced minimal IFNα when treated with GS-9688, consistent with selective activation of TLR8 by GS-9688 (Supporting Fig. S1B). The activation and induction of immunomodulatory cytokines was supported by augmented expression of the activation marker CD40 on both cDCs and MNPs (Supporting Fig. S1C). Collectively, these data demonstrate that GS-9688 activates cDCs and MNPs and is a potent inducer of the immunomodulatory cytokines IL-12 and TNFα in vitro.

FReQUeNCIeS oF FUNCtIoNal HBV-SpeCIFIC CD8 + t CellS INCReaSe IN a SUBSet oF patIeNtS WItH CHB aFteR IN VITRO StIMUlatIoN WItH gS-9688
CD8 + T cells are critical for the control of HBV infection, mediated in part through their capacity for noncytolytic inhibition of HBV replication by the secretion of antiviral cytokines. (21)(22)(23) It is increasingly recognized that local cytokine environments can shape adaptive immune responses. Therefore, we tested the capacity of the cytokines induced by GS-9688 to indirectly modulate the frequency of functional HBVspecific CD8 + T cells in response to HBV-OLP using PBMCs isolated from patients with CHB (clinical parameters at the time of sampling in Supporting Table S1 and gating strategy in Supporting Fig.  S2A). In 13 of 28 patients examined, treatment with GS-9688 increased the frequency of CD8 + T cells producing the antiviral cytokine IFNγ in response to HBcAg ( Fig. 2A,B). A similar proportion of patients showed no increase in the number of IFNγ + HBVspecific CD8 + T cells following stimulation with GS-9688 (Fig. 2B). Interestingly, de novo IFNγ responses induced by GS-9688 were detected in 3 patients who lacked a detectable HBV-specific response to HBV-core OLP stimulation alone. Similarly, when using a panel of HLA-restricted peptide multimers (Supporting Table S2) to detect HBV-specific CD8 + T cells after peptide expansion, the response to GS-9688 was heterogenous. Here, 9 of 23 patients showed a substantial increase in the proportion of detectable HBV-specific CD8 + T cells (Fig. 2C). Notably, the mean fluorescence intensity (MFI) of IFNγ from the HBV-specific IFNγ + CD8 + T-cell pool showed a modest increase, suggesting a greater production of this antiviral cytokine per cell in response to HBVcore OLP in the presence of GS-9688 (Fig. 2D). Furthermore, when assessing the frequency of polyfunctional CD8 + T cells, we observed an increase in the proportion of CD8 + T cells producing both IFNγ and TNFα in 10 patients (36%; Fig. 2E). In contrast, the percentage of CD8 + T cells degranulating in response to HBV-core OLP only increased in 3 (16%) patients following treatment with GS-9688 (Supporting Fig. S2B). Taken together, these data support the potential of GS-9688 to enhance the noncytolytic effector function of pre-existing endogenous HBV-specific CD8 + T cells, and to increase the frequency of antiviral T cells in a subset of patients.
Finally, we explored whether the heterogeneity in the magnitude of the HBV-specific T-cell response to GS-9688 was associated with any clinical parameters in our cohort. In doing so, we noted that a baseline HBsAg titer less than 2,000 IU/mL was associated with an increase in the proportion of IFNγ-producing HBV-specific T cells in the presence of GS-9688 (Fig. 2F). In contrast, most patients denoted "non-responders" (who did not have an increase in the frequency of IFNγ + HBV-specific CD8 + T cells with GS-9688) had a baseline HBsAg titer greater than 2,000 IU/mL (Fig. 2F). No clear association in responsiveness was seen with serum ALT levels or HBV viral load (Supporting Fig. S2C).  Supporting Table S1. Error bars represent the mean ± SEM. **P < 0.01.

tReatMeNt oF pBMCs WItH gS-9688 poteNtIateS tHe aCtIVatIoN aND FUNCtIoN oF aNtIVIRal eFFeCtoRS
Innate effector cells such as NK cells play a dual role in the setting of viral infection. Although these immune mediators can exert antiviral activity by direct or indirect effects (e.g., through modulation of T-cell responses), emerging data underscore mechanisms by which they limit antiviral responses through the inhibition or killing of antigen-specific T cells. In CHB, NK cells can also become defective in their capacity to produce IFNγ and TNFα, resulting in an inability to effectively exert noncytolytic activity in the HBVinfected liver. (24,25) To address the functional impact of GS-9688 on NK cells in vitro, we determined the activation status of the global NK cell population in response to GS-9688 treatment of PBMCs from healthy controls and patients with CHB. GS-9688 activated NK cells in a dose-dependent manner, increasing the expression of the activation markers CD69, HLA-DR, and CD38 (gating strategy; Supporting Fig. S3A and Fig. 3A). Activation was evident on both the immature cytokine producing CD56 bright population, which has been described to be preferentially enriched in the liver, (26) and the cytotoxic, more mature CD56 dim subset (Supporting Fig. S3B,C). In line with their activation, treatment with GS-9688 augmented the noncytolytic effector function of NK cells, confirmed by an increased production of TNFα and IFNγ (Fig. 3B). As expected, the increase in cytokine production driven by GS-9688 was largely attributable to the CD56 bright population (Supporting Fig. S3D). NK cells do not express TLR8 but can be activated by TLR8 agonists through the production of IL-12 and IL-18. (27) Consistent with these data, neutralization of IL-12/IL-18 abrogated the IFNγ production seen in response to GS-9688 treatment in the 6 individuals tested, at all concentrations of GS-9688 (Fig. 3B). This finding supports a role for GS-9688induced cytokines in promoting NK-cell antiviral function.
Although IFNγ production by NK cells can promote antiviral T-cell responses and inhibit viral replication in hepatocytes, additional anti-HBV activity of NK cells is attributable to their ability to directly eliminate infected hepatocytes. (25) In line with this, GS-9688 enhanced the cytolytic capacity of NK cells, as indicated by increased expression of perforin and granzyme B (Fig. 3C) and the propensity for degranulation, as denoted by CD107a mobilization (Fig.  3D). Moreover, GS-9688-treated, purified NK cells also increased lysis of the HepG2 hepatoma cell line (Fig. 3E). Collectively, these data demonstrate that GS-9688 enhances both the noncytolytic and cytolytic effector functions of NK cells in vitro.
Although a role for MAITs (non-classical T cells characterized by expression of the invariant chain Vα7.2) in CHB remains unclear, they are highly enriched in the liver and are potent producers of IFNγ in response to TLR8 agonism. (28) GS-9688 activated MAITs in PBMCs, increasing the expression of IFNγ and granzyme B (Supporting Fig.  S4A-C). Analogous to NK cells, this increase in the noncytolytic and cytolytic potential of MAITs by GS-9688 was IL-12/IL-18-dependent in 2 healthy controls (Supporting Fig. S4B,C).
In CHB, NK cells can also mediate negative regulatory effects by up-regulating death ligands such as TRAIL to kill HBV-specific T cells selectively expressing TRAIL-R2. (7,8) Furthermore, TRAILexpressing NK cells have the potential to directly kill hepatocytes. (7) GS-9688 treatment induced a significant increase in TRAIL-expressing NK cells (Fig.  3F), which was most evident on the CD56 bright subset ( Fig. 3F and Supporting Fig. S3E).
Together, these data demonstrate that GS-9688 can enhance the cytolytic and noncytolytic activity of multiple innate effectors in vitro.

IN VITRO gS-9688 ReDUCeS tHe FReQUeNCy oF t Reg aND INCReaSeS tHe FReQUeNCy oF CIRCUlatINg FollICUlaR HelpeR CD4 + t CellS
Follicular helper CD4 + T cells (T FH ) play a key role in promoting adaptive immunity through the generation of long-lived plasma cells. (29,30) In contrast, CD4 + T REG cells are potent immunosuppressors able to down-regulate adaptive immunity, with previous studies reporting increases in the proportion of circulating and liver-resident T REG in patients, that actively inhibit the antiviral response and contributes to chronicity in CHB. (31) Importantly, recent studies have highlighted the capacity of TLR8 agonists to both reduce the suppressive capacity of T REG in a model of antitumor immunity (15) and enhance T FH differentiation. (32) Thus, GS-9688 was examined for its effect on the frequency and phenotype of circulating T FH (cT FH ) and T REG cells by quantifying their proportions in PBMCs isolated from patients with CHB or healthy controls (T REG : CD4 + CD25 hi CD127 lo FOXP3 + ; cT FH : CD4 + CXCR5 + PD-1 + ; gating strategy found in Supporting Fig. S5A) after in vitro culture.
Although the overall proportion of CD4 + T cells remained unchanged (Supporting Fig. S5B), there was an increase in the frequency of cT FH in response to GS-9688 at all doses tested (Fig. 4A). Furthermore, GS-9688 altered the phenotype of cT FH by increasing the expression of inducible T-cell co-stimulator (ICOS), a marker for T FH differentiation and function (33) (Fig. 4B). Conversely, treatment with GS-9688 induced a dose-dependent decrease in the frequency of circulating T REG (Fig. 4C). Despite reducing the frequency of T REG , GS-9688 had little impact on the immunosuppressive potential of the remaining T REG population on the basis of unaltered expression of the negative regulator, cytotoxic T-lymphocyte-associated protein 4 (CTLA4; Fig.  4D) and CD39, the rate-limiting enzyme in the generation of immunosuppressive adenosine (Fig.  4E). Although the potential for suppression by the remaining T REG population was unaffected by GS-9688, the ability of GS-9688 to decrease the frequency of T REG suggests that it may act to reduce the overall capacity of T REG to limit HBV-specific T-cell responses in vivo.

gS-9688 SIgNIFICaNtly DeCReaSeS MoNoCytIC MDSC FReQUeNCIeS, WHIle tRIggeRINg a ReleaSe oF aRgINaSe-I FRoM pMN-MDSCs
MDSCs are immature myeloid progenitors that exert potent immune regulation through the manipulation of nutrient availability and expression of inhibitory ligands like programmed death-ligand 1 (PD-L1). (34) The MDSC compartment consists of two populations, polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs (M-MDSCs). (34) Arginase-I expressing PMN-MDSCs accumulate in patients with CHB, where they limit the proliferation and function of bystander and HBV-specific T cells. (9) Different TLR8 agonists were recently shown to reduce M-MDSC frequencies in vitro by selectively inducing their apoptosis and limiting their immunosuppressive activity. (35,36) Consequently, we assessed the capacity of GS-9688 to modulate the frequency and functionality of MDSC.
First, we assessed the effect of GS-9688 on the frequency of MDSC subsets in patients with CHB, defining PMN-MDSCs as CD11b + CD33 + HLA-DR lo CD14 − CD15 + and M-MDSCs as CD11b + CD33 + HLA-DR lo CD14 + CD15 − (gating strategy found in Supporting Fig. S6A). The ex vivo frequency of each MDSC subset was quantified as a percentage of the immature myeloid compartment (CD11b hi CD33 + ), revealing that M-MDSCs are the predominant subset, accounting for approximately 45% of immature myeloid precursors (Supporting Fig. S6B). In line with previous reports with other TLR8 agonists, short-term culture with GS-9688 led to a reduction in the frequency of M-MDSCs ( Fig. 5A and Supporting Fig. S6C). Although we noted a marked reduction in M-MDSCs, this coincided with a relative increase in the proportion of immature myeloid cells with a PMN-MDSC phenotype (Supporting Fig. S6C), the absolute frequency of PMN-MDSCs remained unchanged when evaluated as a percentage of total leukocytes (Fig. 5A).
GS-9688 induced MDSC activation, as determined by increased expression of CD80 (Fig. 5B) and altered the immunosuppressive potential of both subsets in vitro. Short-term GS-9688 exposure decreased expression of intracellular arginase-I in PMN-MDSCs, while simultaneously increasing its expression in M-MDSCs, albeit to a much lower level than the PMN-MDSCs (Fig. 5C). Expression of CD63, a measure of azurophilic granule mobilization to the cell surface, remained unchanged on PMN-MDSCs but dramatically increased on M-MDSCs (Fig. 5D). These data are suggestive of active degranulation of MDSC in vitro triggered by GS-9688, supported by an increased release of extracellular arginase-I into the culture media (Fig. 5E). In addition, we observed an increase in expression of the negative regulators galectin-9 (on both subsets of MDSCs) and PD-L1 (on the remaining M-MDSCs), which could conceivably result in enhanced suppression of T-cell immunoglobulin and mucin domain containing-3 (Tim-3)-expressing or programmed cell death protein 1 (PD-1)-expressing HBV-specific T cells (6,37) (Fig. 5F,G).

Discussion
GS-9688 is a potent TLR8 agonist in development for the treatment of CHB. A recent study in the woodchuck model of CHB revealed that a short, finite period of dosing with GS-9688 is well tolerated and able to induce a sustained antiviral response. (18) Here we evaluated the immunomodulatory effect of GS-9688 on various immune cells in vitro using human PBMCs to understand its therapeutic potential for CHB. Initial experiments confirmed GS-9688 selectivity for TLR8, revealing the potent induction of immunomodulatory cytokines, such as IL-12, by cDCs and MNPs, which was preserved in PBMCs from patients with CHB. In interrogating the effects on correlates of immune protection, we showed that GS-9688 enhanced the frequency of HBV-specific CD8 + T cells in a subset of patients. In vitro treatment with GS-9688 boosted the production of antiviral cytokines, but not cytotoxicity, of HBV-specific CD8 + T cells. The potential for GS-9688 to enhance cellular mediators with antiviral potential in CHB was further evidenced by increased activation, improved cytolytic and noncytolytic effector function of NK cells, and MAITs. In the HBV-infected liver, where NK cells are greatly enriched and where the virusspecific T-cell response is limited, it is possible that GS-9688-activated NK cells could perform a significant antiviral role through secretion of cytokines, and enhanced killing of infected hepatocytes. In line with other TLR8 agonists, we confirmed that the increase in antiviral activity by these effectors was driven by induction of IL-12/IL-18. IL-12 has the potential to reverse mitochondrial defects of exhausted HBVspecific CD8 + T cells, allowing for more efficient bioenergetics and enhanced antiviral functionality. (38)(39)(40) Importantly, the therapeutic benefit of IL-12 has been demonstrated in HBV transgenic mice, the woodchuck model of CHB, and in early clinical trials in patients with CHB. (41)(42)(43)(44) Although promising, it is important to note that GS-9688 did not induce an effective HBV-specific CD8 + T-cell response in PBMCs from all patients, which is typical of many immunotherapeutic strategies in development for CHB using diverse patient cohorts. (6,37,45) This is reminiscent of the antiviral response to GS-9688 in the woodchuck model of CHB, in which only half the animals treated with 3 mg/kg GS-9688 had a sustained reduction in viral parameters. (18) Consistent with the woodchuck study, the response of patient HBV-specific CD8 + T cells in vitro was not associated with age, sex, extent of liver inflammation, or viral load. However, the magnitude of the in vitro response was associated with a low-baseline HBsAg titer (<2,000 IU/mL). To elucidate the potential differences in responders and non-responders, it is important to consider the contribution of potent immunoregulatory leukocytes implicated in suppressing T-cell immunity. In this study we show increased expression of the death ligand TRAIL on NK cells, particularly on the CD56 bright subset driven by GS-9688, which may further eliminate the already depleted pool of HBV-specific T cells within the infected liver. (8) Conversely, these TRAIL + NK cells may act as antifibrotic mediators in CHB, killing activated hepatic stellate cells in a TRAIL-dependent manner or by producing hepatoprotective cytokines. (46) A notable finding of this study was GS-9688 modulation of immunosuppressive cells. GS-9688 reduced the frequency of T REG , potent immunosuppressors implicated in CHB. (31) Intriguingly, the decline in T REG numbers was associated with a corresponding expansion and activation of cT FH . The observed GS-9688-driven increase in ICOS expression on cT FH is significant, as ICOS is critical for IL-21 production and the subsequent delivery of improved T-cell help. (47,48) Although not studied here, we hypothesize that such an increase in cT FH and T FH -associated cytokines will improve immune control in CHB by modulating the frequency of memory B cells and the production of affinity-matured class-switched antibodies. Whether this decrease in T REG and expansion and activation of cT FH is sufficient to overcome the observed B-cell dysfunction characteristic of patients with CHB will require further study. (49,50) Various other mechanisms have been identified that play a role in immune dysfunction in CHB, including the increased activity of MDSCs. Consistent with the literature, (35,36) we demonstrate the capacity of GS-9688 to decrease the frequency of M-MDSCs in vitro. However, this decrease was linked to an initial burst of arginase-I release from PMN-MDSCs, which may transiently limit the availability of the conditionally essential amino acid L-arginine, acting as a crucial T-cell rheostat. (9) Of further significance is the capacity of GS-9688 to induce expression of PD-L1 and galectin-9 on the remaining MDSCs, which could limit HBV-specific CD8 + T cells expressing high levels of PD-1 and Tim-3. (6,37) Intrahepatic PD-L1 and galectin-9 levels are increased in viral hepatitis, and our data suggest that they may be further enhanced by TLR8-induced IFNγ. (37,51) Collectively, these data raise the possibility that combination with checkpoint blockade may improve the antiviral response to GS-9688 treatment.
GS-9688 has good absorption and high first-pass hepatic clearance, to limit systemic immune activation. (17) After oral administration of GS-9688, intestinal absorption is expected to induce TLR8 activation in the gut and liver, where the immune composition is markedly different. (26,(52)(53)(54) It is likely that the secretion of immune mediators from the gut into the portal vein will in turn stimulate cells in the liver. Given the fact that the liver harbors a population of transcriptionally distant liver-resident NK cells (26) and memory CD8 + T cells (53,54) future studies will need to focus on characterizing the intrahepatic immune response to GS-9688.
Although the therapeutic efficacy of GS-9688 in combination with nucleos(t)ide treatment remains to be fully evaluated in patients with CHB, it was shown to be safe and generally well-tolerated in a phase 2 placebo-controlled study in virally suppressed patients, with a subset achieving HBsAg and/or HBeAg loss. (55) While phase 2 studies are ongoing (ClinicalTrials.gov NCT03491553/NCT03615066), our work provides important insights into the immunomodulatory effects of GS-9688 and has important implications for the rational design of combination studies with other immunomodulatory agents. Our data also raise the possibility that in vitro profiling of PBMC responses to GS-9688 in patients with CHB may have utility for predicting therapeutic efficacy in patients.