Immunological biomarker discovery in cure regimens for chronic hepatitis B virus infection.

There have been unprecedented advances in the identification of new treatment targets for chronic hepatitis B that are being developed with the goal of achieving functional cure in patients who would otherwise require lifelong nucleoside analogue treatment. Many of the new investigational therapies either directly target the immune system or are anticipated to impact immunity indirectly through modulation of the viral lifecycle and antigen production. While new viral biomarkers (HBV RNA, HBcAg, small, middle, large HBs isoforms) are proceeding through validation steps in clinical studies, immunological biomarkers are non-existent outside of clinical assays for antibodies to HBs, HBc and HBe. To develop clinically applicable immunological biomarkers to measure mechanisms of action, inform logical combination strategies, and guide clinical management for use and discontinuation of immune-targeting drugs, immune assays must be incorporated into phase I/II clinical trials. This paper will discuss the importance of sample collection, the assays available for immunological analyses, their advantages/disadvantages and suggestions for their implementation in clinical trials. Careful consideration must be given to ensure appropriate immunological studies are included as a primary component of the trial with deeper immunological analysis provided by ancillary studies. Standardising immunological assays and data obtained from clinical trials will identify biomarkers that can be deployed in the clinic, independently of specialised immunology laboratories.

Small molecule AX-024 reduces T cell proliferation independently of CD3ϵ/Nck1 interaction, which is governed by a domain swap in the Nck1-SH3.1 domain.

Activation of the T cell receptor (TCR) results in binding of the adapter protein Nck (noncatalytic region of tyrosine kinase) to the CD3ϵ subunit of the TCR. The interaction was suggested to be important for the amplification of TCR signals and is governed by a proline-rich sequence (PRS) in CD3ϵ that binds to the first Src homology 3 (SH3) domain of Nck (Nck-SH3.1). Inhibition of this protein/protein interaction ameliorated inflammatory symptoms in mouse models of multiple sclerosis, psoriasis, and asthma. A small molecule, AX-024, was reported to inhibit the Nck/CD3ϵ interaction by physically binding to the Nck1-SH3.1 domain, suggesting a route to develop an inhibitor of the Nck1/CD3ϵ interaction for modulating TCR activity in autoimmune and inflammatory diseases. We show here that AX-024 reduces T cell proliferation upon weak TCR stimulation but does not significantly affect phosphorylation of Zap70 (ζ chain of T cell receptor-associated protein kinase 70). We also find that AX-024 is likely not involved in modulating the Nck/TCR interaction but probably has other targets in T cells. An array of biophysical techniques did not detect a direct interaction between AX-024 and Nck-SH3.1 in vitro Crystal structures of the Nck-SH3.1 domain revealed its binding mode to the PRS in CD3ϵ. The SH3 domain tends to generate homodimers through a domain swap. Domain swaps observed previously in other SH3 domains indicate a general propensity of this protein fold to exchange structural elements. The swapped form of Nck-SH3.1 is unable to bind CD3ϵ, possibly representing an inactive form of Nck in cells.

LCMV-specific CD4 T cell dependent polyclonal B-cell activation upon persistent viral infection is short lived and extrafollicular.

Persistent virus infections with non- or poorly cytopathic viruses are commonly associated with B cell dysregulations. These include the induction of hypergammaglobulinemia and the emergence of virus-unspecific antibodies. These seemingly unspecific antibody responses interfere with the virus-specific humoral immunity and contribute to delayed virus control. Whether these virus-unspecific antibodies are induced in the B cell follicle or at extrafollicular sites and whether one specific CD4 T cell subset is involved in the polyclonal B cell activation is unclear. Here we studied virus-unrelated IgG antibody responses against self or foreign antigens in the context of persistent lymphocytic choriomeningitis virus (LCMV) infection. We found that the LCMV-unspecific antibody response is short-lived and induced predominantly at extrafollicular sites and depends on the presence of LCMV-specific CD4 T cells. Our data support a scenario in which activated, virus-specific CD4 T cells provide help to non-specific B cells at extrafollicular sites, supporting the production of virus unspecific IgG antibodies during persistent viral infection.

Estimating the In Vivo Killing Efficacy of Cytotoxic T Lymphocytes across Different Peptide-MHC Complex Densities.

Cytotoxic T lymphocytes (CTLs) are important agents in the control of intracellular pathogens, which specifically recognize and kill infected cells. Recently developed experimental methods allow the estimation of the CTL's efficacy in detecting and clearing infected host cells. One method, the in vivo killing assay, utilizes the adoptive transfer of antigen displaying target cells into the bloodstream of mice. Surprisingly, killing efficacies measured by this method are often much higher than estimates obtained by other methods based on, for instance, the dynamics of escape mutations. In this study, we investigated what fraction of this variation can be explained by differences in peptide loads employed in in vivo killing assays. We addressed this question in mice immunized with lymphocytic choriomeningitis virus (LCMV). We conducted in vivo killing assays varying the loads of the immunodominant epitope GP33 on target cells. Using a mathematical model, we determined the efficacy of effector and memory CTL, as well as CTL in chronically infected mice. We found that the killing efficacy is substantially reduced at lower peptide loads. For physiological peptide loads, our analysis predicts more than a factor 10 lower CTL efficacies than at maximum peptide loads. Assuming that the efficacy scales linearly with the frequency of CTL, a clear hierarchy emerges among the groups across all peptide antigen concentrations. The group of mice with chronic LCMV infections shows a consistently higher killing efficacy per CTL than the acutely infected mouse group, which in turn has a consistently larger efficacy than the memory mouse group. We conclude that CTL killing efficacy dependence on surface epitope frequencies can only partially explain the variation in in vivo killing efficacy estimates across experimental methods and viral systems, which vary about four orders of magnitude. In contrast, peptide load differences can explain at most two orders of magnitude.

Macrophage and T cell produced IL-10 promotes viral chronicity.

Chronic viral infections lead to CD8(+) T cell exhaustion, characterized by impaired cytokine secretion. Presence of the immune-regulatory cytokine IL-10 promotes chronicity of Lymphocytic Choriomeningitis Virus (LCMV) Clone 13 infection, while absence of IL-10/IL-10R signaling early during infection results in viral clearance and higher percentages and numbers of antiviral, cytokine producing T cells. IL-10 is produced by several cell types during LCMV infection but it is currently unclear which cellular sources are responsible for induction of viral chronicity. Here, we demonstrate that although dendritic cells produce IL-10 and overall IL-10 mRNA levels decrease significantly in absence of CD11c(+) cells, absence of IL-10 produced by CD11c(+) cells failed to improve the LCMV-specific T cell response and control of LCMV infection. Similarly, NK cell specific IL-10 deficiency had no positive impact on the LCMV-specific T cell response or viral control, even though high percentages of NK cells produced IL-10 at early time points after infection. Interestingly, we found markedly improved T cell responses and clearance of normally chronic LCMV Clone 13 infection when either myeloid cells or T cells lacked IL-10 production and mice depleted of monocytes/macrophages or CD4(+) T cells exhibited reduced overall levels of IL-10 mRNA. These data suggest that the decision whether LCMV infection becomes chronic or can be cleared critically depends on early CD4(+) T cell and monocyte/macrophage produced IL-10.

Non-neutralizing antibodies protect from chronic LCMV infection independently of activating FcγR or complement.

Chronic viral infections lead to CD8(+) T cell exhaustion, characterized by impaired cytokine secretion. The presence of the immune-regulatory cytokine IL-10 promotes chronic lymphocytic choriomeningitis virus (LCMV) Clone 13 infection in mice, whereas the absence of IL-10/IL-10R signaling early during infection results in viral clearance and higher percentages and numbers of antiviral, cytokine-producing T cells. However, it is currently unclear which cell populations and effector molecules are crucial to protect against chronic infection. In this study, we demonstrate that antiviral, LCMV-binding, non-neutralizing antibodies are needed, in addition to CD4(+) and CD8(+) T cells, to clear a high-dose LCMV infection in mice, in the absence of IL-10. The interaction between CD4(+) T cells and B cells in B-cell follicles via CD40/CD40L, in addition to class switch and/or somatic hypermutation, is crucial for viral control in the absence of IL-10. Interestingly, transfer of LCMV-binding non-neutralizing antibodies protected recipients from chronic infection. In addition, viral clearance in the absence of IL-10R signaling was independent of activating Fcγ receptors and complement. These data highlight that non-neutralizing antibodies effectively contribute to the control of LCMV infection when present prior to infection, suggesting that the induction of neutralizing antibodies is not implicitly necessary for the generation of successful vaccines.

Reversal of chronic to resolved infection by IL-10 blockade is LCMV strain dependent.

Chronic viral infections lead to CD8(+) T-cell exhaustion, characterized by impaired cytokine secretion. The immune-regulatory cytokine IL-10 promotes chronicity of infection with lymphocytic choriomeningitis virus (LCMV) Clone 13, as absence of IL-10 or blocking of IL-10R during early LCMV Clone 13 infection results in viral clearance. Thus, treatment of humans suffering from chronic viral infections with IL-10 neutralizing or IL-10R blocking antibodies was proposed to boost virus-specific T-cell responses to enhance control or even clear the viral infection. Here we demonstrate that although CD4(+) and CD8(+) T cells can produce elevated levels of cytokines in IL-10(-/-) mice early after infection compared with WT mice, IL-10(-/-) mice cannot clear an infection with the quicker replicating LCMV strain Docile, eventually resulting in T-cell exhaustion. These data suggest that the success of IL-10 blockade to control chronic viral infections may critically depend on the virulence of the infecting strain.

IL-10 suppression of NK/DC crosstalk leads to poor priming of MCMV-specific CD4 T cells and prolonged MCMV persistence.

IL-10 is an anti-inflammatory cytokine that regulates the extent of host immunity to infection by exerting suppressive effects on different cell types. Herpes viruses induce IL-10 to modulate the virus-host balance towards their own benefit, resulting in prolonged virus persistence. To define the cellular and molecular players involved in IL-10 modulation of herpes virus-specific immunity, we studied mouse cytomegalovirus (MCMV) infection. Here we demonstrate that IL-10 specifically curtails the MCMV-specific CD4 T cell response by suppressing the bidirectional crosstalk between NK cells and myeloid dendritic cells (DCs). In absence of IL-10, NK cells licensed DCs to effectively prime MCMV-specific CD4 T cells and we defined the pro-inflammatory cytokines IL-12, IFN-γ and TNF-α as well as NK cell activating receptors NKG2D and NCR-1 to regulate this bidirectional NK/DC interplay. Consequently, markedly enhanced priming of MCMV-specific CD4 T cells in Il10(-/-) mice led to faster control of lytic viral replication, but this came at the expense of TNF-α mediated immunopathology. Taken together, our data show that early induction of IL-10 during MCMV infection critically regulates the strength of the innate-adaptive immune cell crosstalk, thereby impacting beneficially on the ensuing virus-host balance for both the virus and the host.

Antigen amount dictates CD8+ T-cell exhaustion during chronic viral infection irrespective of the type of antigen presenting cell.

Chronic viral infections lead to CD8(+) T-cell exhaustion, characterized by impaired cytokine secretion and loss of proliferative capacity. While viral load and T-cell dysfunction correlate, it is currently unclear whether the quality of a cell type presenting antigen determines the degree of T-cell exhaustion or if the overall amount of antigen recognized by T cells promotes exhaustion. We found that chronic lymphocytic chorio-meningitis virus infection led to decreased CD8(+) T-cell exhaustion in DC-MHC class I (MHCI) mice, in which CD8(+) T cells can only recognize antigen on DCs. However, this increase in CD8(+) T-cell function came at the expense of fatal immunopathology. Additional antigen recognition on nonhematopoietic cells in DC-MHCI mice promoted T-cell exhaustion and avoidance of immunopathology. Likewise, increased numbers of antigen-expressing hematopoietic cells, as well as a selective elevation of the number of DCs as the only cell type presenting antigen in DC-MHCI mice, resulted in compromised T-cell function. These results favor a scenario in which the overall amount of antigen exposure, rather than the type of cell engaging with virus-specific CD8(+) T cells, is responsible for their functional exhaustion. Furthermore, exhaustion of virus-specific CD8(+) T cells leads to avoidance of life-threatening immunopathology.

Human Th17- and IgG3-associated autoimmunity induced by a translocating gut pathobiont.

Extraintestinal autoimmune diseases are multifactorial with translocating gut pathobionts implicated as instigators and perpetuators in mice. However, the microbial contributions to autoimmunity in humans remain largely unclear, including whether specific pathological human adaptive immune responses are triggered by such pathobionts. We show here that the translocating pathobiont Enterococcus gallinarum induces human IFNγ + Th17 differentiation and IgG3 subclass switch of anti- E. gallinarum RNA and correlating anti-human RNA autoantibody responses in patients with systemic lupus erythematosus and autoimmune hepatitis. Human Th17 induction by E. gallinarum is cell-contact dependent and involves TLR8-mediated human monocyte activation. In murine gnotobiotic lupus models, E. gallinarum translocation triggers IgG3 anti-RNA autoantibody titers that correlate with renal autoimmune pathophysiology and with disease activity in patients. Overall, we define cellular mechanisms of how a translocating pathobiont induces human T- and B-cell-dependent autoimmune responses, providing a framework for developing host- and microbiota-derived biomarkers and targeted therapies in extraintestinal autoimmune diseases. One Sentence Summary: Translocating pathobiont Enterococcus gallinarum promotes human Th17 and IgG3 autoantibody responses linked to disease activity in autoimmune patients.

Protein engineering of a stable and potent anti-inflammatory IL-37-Fc fusion with enhanced therapeutic potential.

Harnessing the immunomodulatory activity of cytokines is a focus of therapies targeting inflammatory disease. The interleukin (IL)-1 superfamily contains pro-inflammatory and anti-inflammatory members that help orchestrate the immune response in adaptive and innate immunity. Of these molecules, IL-37 has robust anti-inflammatory activity across a range of disease models through inhibition of pro-inflammatory signaling cascades downstream of tumor necrosis factor, IL-1, and toll-like receptor pathways. We find that IL-37 is unstable with a poor pharmacokinetic and manufacturing profile. Here, we present the engineering of IL-37 from an unstable cytokine into an anti-inflammatory molecule with an excellent therapeutic likeness. We overcame these shortcomings through site-directed mutagenesis, the addition of a non-native disulfide bond, and the engineering of IL-37 as an Fc-fusion protein. Our results provide a platform for preclinical testing of IL-37 Fc-fusion proteins. The engineering approaches undertaken herein will apply to the conversion of similar potent yet short-acting cytokines into therapeutics.

How chronic viral infections impact on antigen-specific T-cell responses.

Persistent viral infections are, by definition, associated with ineffective antiviral immunity, in particular those infections caused by viruses that are highly productive and replicative (including HIV, HBV and HCV). The reasons for ineffective antiviral immunity in these types of infections are complex and manifold, and only recently a more comprehensive picture of the parameters responsible for attenuation of immune function is emerging. One reason for poor viral control in these types of infections is the functional deterioration of antiviral T-cell responses and understanding the underlying mechanisms is of key importance. This review summarizes our current knowledge of cell-intrinsic and cell-extrinsic parameters that contribute to T-cell exhaustion during chronic viral infections and discusses related implications for host survival, immunopathology, and control of infection.

Comparison of cytotoxic T lymphocyte efficacy in acute and persistent lymphocytic choriomeningitis virus infection.

Immune responses mediated by cytotoxic T lymphocytes (CTLs) have often been found to be functionally impaired in persistent infections. It is assumed that this impairment contributes to persistence of the infection. In this study, we compare the killing efficacy of CD8(+) T-cell responses in mice acutely and persistently infected with the lymphocytic choriomeningitis virus, using an in vivo CTL killing assay. To infer the killing efficacy of CTLs, we developed a new mathematical model describing the disappearance of peptide-pulsed cells from the blood of the mice over time. We estimate a lower half-life for peptide-pulsed cells in acute infection than in persistent infection, which indicates a higher killing efficacy of the CD8(+) T-cell response in acute infection. However, by controlling for the different levels of CTLs in acutely and persistently infected mice, we find that CTLs in persistent infection are only two times less efficacious than CTLs in acute infections. These results strongly suggest that the in vivo cytotoxicity of CD8(+) T-cell responses in persistent infection is modulated via the number of CTLs rather than their individual functionality.

On the role of the inhibitory receptor LAG-3 in acute and chronic LCMV infection.

Chronic viral infections are often characterized by CD8 T-cell responses with poor cytokine secretion potential and limited expansion of the CD8 T-cell pool, collectively referred to as CD8 T-cell exhaustion. Exhaustion of lymphocytic choriomeningitis virus (LCMV)-specific CD8 T cells was shown to be partially regulated by the inhibitory receptor programmed death 1 (PD-1). Here, we demonstrate that exhausted LCMV-specific CD8 T cells also express the negative regulatory receptor lymphocyte activation gene 3 (LAG-3) which is mainly expressed on cells co-expressing the negative regulatory receptors PD-1 and Tim-3. Expression levels of LAG-3 on anti-viral CD8 T cells remain stable over short-term in vitro stimulations in presence of antigenic peptide. Nevertheless, in vitro and in vivo blockade of LAG-3 did not rescue cytokine production by virus-specific CD8 T cells and did not alter the virus titer in various organs. Likewise, chronic LCMV infection of LAG-3-/- mice led to a comparable degree of T-cell exhaustion as observed in C57BL/6 controls and to similar virus titers. Further, LAG-3 did not influence T-cell activation or cell division during chronic LCMV infection. These data suggest that even though LAG-3 is continuously up-regulated on LCMV-specific exhausted CD8 T cells, it alone does not significantly contribute to T-cell exhaustion.

Interferon-gamma prevents death of bystander neurons during CD8 T cell responses in the brain.

T cells restricted to neurotropic viruses are potentially harmful as their activity may result in the destruction of neurons. In the Borna disease virus (BDV) model, antiviral CD8 T cells entering the brain of infected mice cause neurological disease but no substantial loss of neurons unless the animals lack interferon-gamma (IFN-gamma). We show here that glutamate receptor antagonists failed to prevent BDV-induced neuronal loss in IFN-gamma-deficient mice, suggesting that excitotoxicity resulting from glutamate receptor overstimulation is an unlikely explanation for the neuronal damage. Experiments with IFN-gamma-deficient mice lacking eosinophils indicated that these cells, which specifically accumulate in the infected brains of IFN-gamma-deficient mice, are not responsible for CA1 neuronal death. Interestingly, BDV-induced damage of CA1 neurons was reduced significantly in IFN-gamma-deficient mice lacking perforin, suggesting a key role for CD8 T cells in this pathological process. Specific death of hippocampal CA1 neurons could be triggered by adoptive transfer of BDV-specific CD8 T cells from IFN-gamma-deficient mice into uninfected mice that express transgene-encoded BDV antigen at high level in astrocytes. These results indicate that attack by CD8 T cells that cause the death of CA1 neurons might be directed toward regional astrocytes and that IFN-gamma protects vulnerable CA1 neurons from collateral damage resulting from exposure to potentially toxic substances generated as a result of CD8 T cell-mediated impairment of astrocyte function.

Direct Regulation of the T Cell Antigen Receptor's Activity by Cholesterol.

Biological membranes consist of hundreds of different lipids that together with the embedded transmembrane (TM) proteins organize themselves into small nanodomains. In addition to this function of lipids, TM regions of proteins bind to lipids in a very specific manner, but the function of these TM region-lipid interactions is mostly unknown. In this review, we focus on the role of plasma membrane cholesterol, which directly binds to the αß T cell antigen receptor (TCR), and has at least two opposing functions in αß TCR activation. On the one hand, cholesterol binding to the TM domain of the TCRß subunit keeps the TCR in an inactive, non-signaling conformation by stabilizing this conformation. This assures that the αß T cell remains quiescent in the absence of antigenic peptide-MHC (the TCR's ligand) and decreases the sensitivity of the T cell toward stimulation. On the other hand, cholesterol binding to TCRß leads to an increased formation of TCR nanoclusters, increasing the avidity of the TCRs toward the antigen, thus increasing the sensitivity of the αß T cell. In mouse models, pharmacological increase of the cholesterol concentration in T cells caused an increase in TCR clustering, and thereby enhanced anti-tumor responses. In contrast, the γδ TCR does not bind to cholesterol and might be regulated in a different manner. The goal of this review is to put these seemingly controversial findings on the impact of cholesterol on the αß TCR into perspective.

Antiviral CD8 T cells recognize borna disease virus antigen transgenically expressed in either neurons or astrocytes.

Borna disease virus (BDV) can persistently infect the central nervous system (CNS) of mice. The infection remains nonsymptomatic as long as antiviral CD8 T cells do not infiltrate the infected brain. BDV mainly infects neurons which reportedly carry few, if any, major histocompatibility complex class I molecules on the surface. Therefore, it remains unclear whether T cells can recognize replicating virus in these cells or whether cross-presentation of viral antigen by other cell types is important for immune recognition of BDV. To distinguish between these possibilities, we used two lines of transgenic mice that strongly express the N protein of BDV in either neurons (Neuro-N) or astrocytes (Astro-N). Since these animals are tolerant to the neo-self-antigen, we adoptively transferred T cells with specificity for BDV N. In nontransgenic mice persistently infected with BDV, the transferred cells accumulated in the brain parenchyma along with immune cells of host origin and efficiently induced neurological disease. Neurological disease was also observed if antiviral T cells were injected into the brains of Astro-N or Neuro-N but not nontransgenic control mice. Our results demonstrate that CD8 T cells can recognize foreign antigen on neurons and astrocytes even in the absence of infection or inflammation, indicating that these CNS cell types are playing an active role in immune recognition of viruses.

Recombinant Sendai virus induces T cell immunity against respiratory syncytial virus that is protective in the absence of antibodies.

Respiratory syncytial virus (RSV) causes severe respiratory disease in infants and a vaccine is highly desirable. The fusion (F) protein of RSV is an important vaccine target, but the contribution of F-specific T cells to successful vaccination remains unclear. We studied the immune response to vaccination of mice with a recombinant Sendai virus expressing RSV F (rSeV F). rSeV F induced protective neutralizing antibody and RSV F-specific CTL responses. T cell immunity was stronger than that induced by recombinant vaccinia virus (rVV F), a well characterized reference vector. Vaccination of antibody-deficient mice showed that vaccine-induced RSV F-specific T cells were sufficient for protective immunity. rSeV F induced T cell immunity in the presence of neutralizing antibodies, which did not impair the vaccine response. Although the F protein only contains a subdominant CTL epitope, vaccination with rSeV F is sufficient to induce protective T cell immunity against RSV in mice.

Sustained T follicular helper cell response is essential for control of chronic viral infection.

During chronic viral infections, both CD8 and CD4 T cell responses are functionally compromised. Alongside exhaustion of CD8 T cells during chronic viral infections, it has also been documented that the CD4 T cells have an increased propensity to differentiate toward CXCR5+ T follicular helper cell (TFH) lineage. Whether these TFH cells contribute to the immune response to chronic viral infection has remained unclear. Using chronic lymphocytic choriomeningitis virus (LCMV) infection in conjunction with an in vivo system where TFH cells can be conditionally ablated, we have established that these TFH cells do in fact play an important protective function. Specifically, we demonstrate that these TFH cells are essential for the late emergence of neutralizing LCMV-specific antibodies that keep viral titers in check and ultimately allow mice to clear the virus. By supporting the generation of neutralizing antibodies, we show that sustained activity of TFH cells promotes control of the chronic infection in face of exhausted CD8 T cell responses.