Development and application of an in vitro assay to assess target-independent B-cell activation by targeted TLR7 immune agonists.

Targeted immune agonist (TIA) comprising a TLR7 agonist conjugated to tumor-targeting antibodies have been shown to induce potent anti-tumor responses in various preclinical models. However, the clinical proof-of-concept of a TIA has been hampered by systemic dose-limiting immune-related toxicities, including rapid induction of anti-drug antibodies in patients. We have developed ELISPOT-based assay to measure activation of antibody-secreting cells (ASCs), intended to simulate the interaction between TIA and peripheral B cells as a tool to pre-clinically de-risk tumor target-independent peripheral B-cell activation by TIA. This method has proven to be robust and has fast turn-around time to evaluate the induction of spontaneous B-cell activation by TIA in a tumor target- and FcγR-independent manner. This novel ASC assay platform may serve as a preclinical tool to de-risk TIAs that can potentially induce immune-related adverse effects in the clinic.

Long-chain polyunsaturated lipids associated with responsiveness to anti-PD-1 therapy are colocalized with immune infiltrates in the tumor microenvironment.

The programmed cell death protein-1 (PD-1) is highly expressed on the surface of antigen-specific exhausted T cells and, upon interaction with its ligand PD-L1, can result in inhibition of the immune response. Anti-PD-1 treatment has been shown to extend survival and result in durable responses in several cancers, yet only a subset of patients benefit from this therapy. Despite the implication of metabolic alteration following cancer immunotherapy, mechanistic associations between antitumor responses and metabolic changes remain unclear. Here, we used desorption electrospray ionization mass spectrometry imaging to examine the lipid profiles of tumor tissue from three syngeneic murine models with varying treatment sensitivity at the baseline and at three time points post-anti-PD-1 therapy. These imaging experiments revealed specific alterations in the lipid profiles associated with the degree of response to treatment and allowed us to identify a significant increase of long-chain polyunsaturated lipids within responsive tumors following anti-PD-1 therapy. Immunofluorescence imaging of tumor tissues also demonstrated that the altered lipid profile associated with treatment response is localized to dense regions of tumor immune infiltrates. Overall, these results indicate that effective anti-PD-1 therapy modulates lipid metabolism in tumor immune infiltrates, and we thereby propose that further investigation of the related immune-metabolic pathways may be useful for better understanding success and failure of anti-PD-1 therapy.

Corrigendum: Effective Anti-tumor Response by TIGIT Blockade Associated With FcγR Engagement and Myeloid Cell Activation.

[This corrects the article DOI: 10.3389/fimmu.2020.573405.].

Effective Anti-tumor Response by TIGIT Blockade Associated With FcγR Engagement and Myeloid Cell Activation.

The molecule "T cell immunoreceptor with immunoglobulin and ITIM domain," or TIGIT, has recently received much attention as a promising target in the treatment of various malignancies. In spite of the quick progression of anti-TIGIT antibodies into clinical testing both as monotherapy and in combination with programmed cell death-1 (PD-1)-directed immune checkpoint blockade, the molecular mechanism behind the observed therapeutic benefits remains poorly understood. Here we demonstrate, using mouse tumor models, that TIGIT blocking antibodies with functional Fc binding potential induce effective anti-tumor response. Our observations reveal that the anti-TIGIT therapeutic effect is not achieved by depletion of intratumoral regulatory T cells (Treg) or any cell population expressing TIGIT, but instead is mediated by possible "reverse activating signals" through FcγRs on myeloid cells, inducing expression of various mediators such as cytokines and chemokines. Furthermore, we discovered an induction of a robust and persistent granzyme B and perforin response, distinct from a predominantly interferon-γ (IFN-γ)-driven anti-PD-1 blockade. Our observations, for the first time, provide the basis for a mechanistic hypothesis involving the requirement of a functional Fc domain of anti-TIGIT monoclonal antibodies, of which various isotypes are currently under intense clinical investigation.

Blockade of BAFF Reshapes the Hepatic B Cell Receptor Repertoire and Attenuates Autoantibody Production in Cholestatic Liver Disease.

Defects in biliary transport proteins, MDR3 in humans and Mdr2 in mice, can lead to a spectrum of cholestatic liver disorders. Although B cell disorders and the aberrant Ab production are the leading extrahepatic manifestations of cholestatic liver diseases, the mechanism underlying this phenomenon is incompletely understood. Using mice with deficiency of Mdr2 that progressively develop cholestatic liver disease, we investigated the contributions of BAFF to aberrant IgG autoantibody production and hepatic fibrosis. In Mdr2-/- mice, hepatic B lymphocytes constitutively produced IgG during fibrosis progression, which correlated with elevated serum levels of BAFF, antinuclear Abs (ANA) and immune complexes. The elevated BAFF and ANA titers were also detected in human patients with primary sclerosing cholangitis and hepatobiliary cholangiopathies. Consistent with the higher BAFF levels, liver-specific selection of the focused BCR IgH repertoire was found on hepatic B cells in Mdr2-/- mice. Interestingly, the administration of anti-BAFF mAb in Mdr2-/- mice altered the BCR repertoire on hepatic B lymphocytes and resulted in reduced ANA and immune complex titers. However, anti-BAFF treatment did not attenuate hepatic fibrosis as measured by collagen deposition, hepatic expressions of collagen-1a, α-smooth muscle actin, and mononuclear cell infiltration (CD11b+ Ly-6chi monocytes and CD11b+ Gr1+ neutrophils). Importantly, depletion of B cells by anti-CD20 mAb reduced both hepatic fibrosis and serum levels of ANA and immune complexes. Our findings implicate B cells as the potential therapeutic targets for hepatic fibrosis and targeting BAFF specifically for attenuating the autoantibody production associated with cholestatic liver disease.

Characterization of murine CEACAM1 in vivo reveals low expression on CD8+ T cells and no tumor growth modulating activity by anti-CEACAM1 mAb CC1.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) has been reported to mediate both tumorigenic and anti-tumor effects in vivo. Blockade of the CEACAM1 signaling pathway has recently been implicated as a novel mechanism for cancer immunotherapy. CC1, a mouse anti-CEACAM1 monoclonal antibody (mAb), has been widely used as a pharmacological tool in preclinical studies to inform on CEACAM1 pathway biology although limited data are available on its CEACAM1 blocking characteristics or pharmacodynamic-pharmacokinetic profiles. We sought to investigate CEACAM1 expression on mouse tumor and immune cells, characterize CC1 mAb binding, and evaluate CC1 in syngeneic mouse oncology models as a monotherapy and in combination with an anti-PD-1 mAb. CEACAM1 expression was observed at high levels on neutrophils, NK cells and myeloid-derived suppressor cells (MDSCs), while the expression on tumor-infiltrating CD8+ T cells was low. Unexpectedly, rather than blocking, CC1 facilitated binding of soluble CEACAM1 to CEACAM1 expressing cells. No anti-tumor effects were observed in CT26, MBT2 or A20 models when tested up to 30 mg/kg dose, a dose that was estimated to achieve >90% target engagement in vivo. Taken together, tumor infiltrating CD8+ T cells express low levels of CEACAM1 and CC1 Ab mediates no or minimal anti-tumor effects in vivo, as a monotherapy or in combination with anti-PD-1 treatment.

Enhancing FcγR-mediated antibody effector function during persistent viral infection.

Persistent viral infections can interfere with FcγR-mediated antibody effector functions by excessive immune complex (IC) formation, resulting in resistance to therapeutic FcγR-dependent antibodies. We and others have previously demonstrated that mice persistently infected with lymphocytic choriomeningitis virus (LCMV) are resistant to a wide range of depleting antibodies due to excessive IC formation. Here, we dissect the mechanisms by which two depleting antibodies overcome the obstacle of endogenous ICs and achieve efficient target cell depletion in persistently infected mice. Efficient antibody-mediated depletion during persistent LCMV infection required increased levels of antibody bound to target cells or use of afucosylated antibodies with increased affinity for FcγRs. Antibodies targeting the highly expressed CD90 antigen or overexpressed human CD20 efficiently depleted their target cells in naïve and persistently infected mice, whereas antibodies directed against less abundant antigens failed to deplete their target cells during persistent LCMV infection. In addition, we demonstrate the superior activity of afucosylated antibodies in the presence of endogenous ICs. We generated afucosylated antibodies directed against CD4 and CD8α, which, in contrast to their parental fucosylated versions, efficiently depleted their respective target cells in persistently infected mice. Efficient antibody-mediated depletion can thus be achieved if therapeutic antibodies can outcompete endogenous ICs for access to FcγRs either by targeting highly expressed antigens or by increased affinity for FcγRs. Our findings have implications for the optimization of therapeutic antibodies and provide strategies to allow efficient FcγR engagement in the presence of competing endogenous ICs in persistent viral infections, autoimmune diseases, and cancer.

Female Bias in Systemic Lupus Erythematosus is Associated with the Differential Expression of X-Linked Toll-Like Receptor 8.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of anti-nuclear antibodies. SLE is one of many autoimmune disorders that have a strong gender bias, with 70-90% of SLE patients being female. Several explanations have been postulated to account for the severity of autoimmune diseases in females, including hormonal, microbiota, and gene dosage differences. X-linked toll-like receptors (TLRs) have recently been implicated in disease progression in females. Our previous studies using the 564Igi mouse model of SLE on a Tlr7 and Tlr9 double knockout background showed that the presence of Tlr8 on both X chromosomes was required for the production of IgG autoantibodies, Ifn-I expression and granulopoiesis in females. Here, we show the results of our investigation into the role of Tlr8 expression in SLE pathogenesis in 564Igi females. Female mice have an increase in serum pathogenic anti-RNA IgG2a and IgG2b autoantibodies. 564Igi mice have also been shown to have an increase in neutrophils in vivo, which are major contributors to Ifn-α expression. Here, we show that neutrophils from C57BL/6 mice express Ifn-α in response to 564 immune complexes and TLR8 activation. Bone marrow-derived macrophages from 564Igi females have a significant increase in Tlr8 expression compared to male-derived cells, and RNA fluorescence in situ hybridization data suggest that Tlr8 may escape X-inactivation in female-derived macrophages. These results propose a model by which females may be more susceptible to SLE pathogenesis due to inefficient inactivation of Tlr8.

Cutting Edge: miR-17-92 Is Required for Both CD4 Th1 and T Follicular Helper Cell Responses during Viral Infection.

Viral infections induce the differentiation of naive CD4 T cells into two distinct lineages, Th1 cells and T follicular helper (TFH) cells. Two recent studies demonstrated that the microRNA cluster miR-17-92 selectively promotes CD4 TFH responses. However, we show in this study that miR-17-92 expression is required for the clonal expansion of both virus-specific Th1 and TFH cells. Upon viral infection, miR-17-92-deficient CD4 T cells showed impaired clonal expansion and subsequent memory formation. Although miR-17-92 deficiency impaired the clonal expansion of both Th1 and TFH cells, the expansion of Th1 cells was more affected. Overexpression of miR-17-92 in CD4 T cells resulted in increased expansion of both virus-specific Th1 and TFH cells but selectively enhanced the Th1 response. Taken together, our data suggest that miR-17-92 is necessary for both Th1 and TFH cells to respond efficiently to viral infections and that the Th1 response is more sensitive to the level of miR-17-92 expression.

Liver fibrosis occurs through dysregulation of MyD88-dependent innate B-cell activity.

UNLABELLED: Chronic liver disease mediated by activation of hepatic stellate cells (HSCs) leads to liver fibrosis. Here, we postulated that the immune regulatory properties of HSCs might promote the profibrogenic activity of B cells. Fibrosis is completely attenuated in carbon tetrachloride-treated, B cell-deficient µMT mice, showing that B cells are required. The retinoic acid produced by HSCs augmented B-cell survival, plasma cell marker CD138 expression, and immunoglobulin G production. These activities were reversed following addition of the retinoic acid inhibitor LE540. Transcriptional profiling of fibrotic liver B cells revealed increased expression of genes related to activation of nuclear factor κ light chain enhancer of activated B cells, proinflammatory cytokine production, and CD40 signaling, suggesting that these B cells are activated and may be acting as inflammatory cells. Biological validation experiments also revealed increased activation (CD44 and CD86 expression), constitutive immunoglobulin G production, and secretion of the proinflammatory cytokines tumor necrosis factor-α, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1α. Likewise, targeted deletion of B-cell-intrinsic myeloid differentiation primary response gene 88 signaling, an innate adaptor with involvement in retinoic acid signaling, resulted in reduced infiltration of migratory CD11c(+) dendritic cells and Ly6C(++) monocytes and, hence, reduced liver pathology. CONCLUSION: Liver fibrosis occurs through a mechanism of HSC-mediated augmentation of innate B-cell activity. These findings highlight B cells as important "first responders" of the intrahepatic immune environment.

Antibody effector functions mediated by Fcγ-receptors are compromised during persistent viral infection.

T cell dysfunction is well documented during chronic viral infections but little is known about functional abnormalities in humoral immunity. Here we report that mice persistently infected with lymphocytic choriomeningitis virus (LCMV) exhibit a severe defect in Fcγ-receptor (FcγR)-mediated antibody effector functions. Using transgenic mice expressing human CD20, we found that chronic LCMV infection impaired the depletion of B cells with rituximab, an anti-CD20 antibody widely used for the treatment of B cell lymphomas. In addition, FcγR-dependent activation of dendritic cells by agonistic anti-CD40 antibody was compromised in chronically infected mice. These defects were due to viral antigen-antibody complexes and not the chronic infection per se, because FcγR-mediated effector functions were normal in persistently infected mice that lacked LCMV-specific antibodies. Our findings have implications for the therapeutic use of antibodies and suggest that high levels of pre-existing immune complexes could limit the effectiveness of antibody therapy in humans.

Autophagy is essential for effector CD8(+) T cell survival and memory formation.

The importance of autophagy in the generation of memory CD8(+) T cells in vivo is not well defined. We report here that autophagy was dynamically regulated in virus-specific CD8(+) T cells during acute infection of mice with lymphocytic choriomeningitis virus. In contrast to the current paradigm, autophagy decreased in activated proliferating effector CD8(+) T cells and was then upregulated when the cells stopped dividing just before the contraction phase. Consistent with those findings, deletion of the gene encoding either of the autophagy-related molecules Atg5 or Atg7 had little to no effect on the proliferation and function of effector cells, but these autophagy-deficient effector cells had survival defects that resulted in compromised formation of memory T cells. Our studies define when autophagy is needed during effector and memory differentiation and warrant reexamination of the relationship between T cell activation and autophagy.

Expression of an anti-RNA autoantibody in a mouse model of SLE increases neutrophil and monocyte numbers as well as IFN-I expression.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of antinucleic acid autoantibodies, high levels of circulating type I interferon (IFN-I), and an IFN-I-dependent elevated expression of activating FcγR. Increases in neutrophils and monocytes are often observed in clinical SLE, but how these contribute to autoantibody and IFN-I production is poorly understood. Here, we analyzed SLE pathogenesis in 564Igi mice, an SLE-model strain carrying gene-targeted heavy and light chain antibody genes encoding an anti-RNA autoantibody in a C57BL/6 background. Similar to human SLE patients, 564Igi mice produce anti-RNA autoantibodies and expanded neutrophil and monocyte populations. These myeloid cells produced IFN-I and exhibit increased FcγRIV expression induced via an IFN-I autocrine loop. A direct effect of IFN-I on 56 Igi BM B cells and neutrophils was supported by their upregulation of "IFN-I signature genes". In addition, 564Igi developing B cells showed upregulated TLR7 resulting in IgG2a/2b class switch recombination and autoantibody production. Our results indicate that the production of anti-RNA autoantibody is sufficient to induce an increase of BM, blood, and spleen IFN-I-producing neutrophils, and suggest a mechanism by which autoantibody and IFN-I contribute to SLE by activating B lymphocytes, neutrophils, and monocyte effector cells in vivo.

Stable cytotoxic T cell escape mutation in hepatitis C virus is linked to maintenance of viral fitness.

Mechanisms by which hepatitis C virus (HCV) evades cellular immunity to establish persistence in chronically infected individuals are not clear. Mutations in human leukocyte antigen (HLA) class I-restricted epitopes targeted by CD8(+) T cells are associated with persistence, but the extent to which these mutations affect viral fitness is not fully understood. Previous work showed that the HCV quasispecies in a persistently infected chimpanzee accumulated multiple mutations in numerous class I epitopes over a period of 7 years. During the acute phase of infection, one representative epitope in the C-terminal region of the NS3/4A helicase, NS3(1629-1637), displayed multiple serial amino acid substitutions in major histocompatibility complex (MHC) anchor and T cell receptor (TCR) contact residues. Only one of these amino acid substitutions at position 9 (P9) of the epitope was stable in the quasispecies. We therefore assessed the effect of each mutation observed during in vivo infection on viral fitness and T cell responses using an HCV subgenomic replicon system and a recently developed in vitro infectious virus cell culture model. Mutation of a position 7 (P7) TCR-contact residue, I1635T, expectedly ablated the T cell response without affecting viral RNA replication or virion production. In contrast, two mutations at the P9 MHC-anchor residue abrogated antigen-specific T cell responses, but additionally decreased viral RNA replication and virion production. The first escape mutation, L1637P, detected in vivo only transiently at 3 mo after infection, decreased viral production, and reverted to the parental sequence in vitro. The second P9 variant, L1637S, which was stable in vivo through 7 years of follow-up, evaded the antigen-specific T cell response and did not revert in vitro despite being less optimal in virion production compared to the parental virus. These studies suggest that HCV escape mutants emerging early in infection are not necessarily stable, but are eventually replaced with variants that achieve a balance between immune evasion and fitness for replication.

Class switch recombination and somatic hypermutation in early mouse B cells are mediated by B cell and Toll-like receptors.

Activation-induced cytidine deaminase (AID) is required for immunoglobulin (Ig) gene class switch recombination (CSR), somatic hypermutation (SHM), and somatic hyperconversion. In general, high AID expression is found in mature B cells that are responding to antigens. However, AID expression and SHM have also been detected in developing B cells from transgenic mice that have a limited Ig repertoire. Here we demonstrate that AID expression, ongoing CSR, and active SHM occur in developing B cells from wild-type mice. Further, our results suggest that somatic variants arising from developing B cells in the bone marrow further diversify in the spleen of unimmunized mice. AID expression in developing B cells is T cell independent but involves engagement of B cell receptors and Toll-like receptors. Early AID expression can increase the preimmune repertoire of developing B cells, may provide an innate population of IgG- and IgA-expressing cells, and could be involved in receptor editing of self-reactive immature B cells.

Toll-like receptor 7-dependent loss of B cell tolerance in pathogenic autoantibody knockin mice.

Systemic lupus erythematosus (SLE) is characterized by the production of autoantibodies that are frequently directed against nucleic acid-associated antigens. To better understand how B cells reactive with such antigens are regulated, we generated a model system in which heavy and light chain genes encoding 564 immunoglobulin have been targeted to the heavy and light chain loci of the nonautoimmune C57BL/6 mouse strain. This antibody recognizes RNA, single-stranded DNA, and nucleosomes. We show that B cells expressing this immunoglobulin were activated, producing class-switched autoantibody in vivo despite the apparently normal induction of anergy. This autoantibody production was largely dependent on Toll-like receptor 7 (TLR7). We further show that production of these autoantibodies was sufficient to cause kidney pathology in these mice. These results demonstrate that the particular threat of nucleic acid-containing autoantigens lies in their ability to bind both antigen receptor and TLR7.

Qualitatively differential regulation of T cell activation and apoptosis by T cell receptor zeta chain ITAMs and their tyrosine residues.

The issue of whether three ITAMs in the TCR zeta chain can transmit qualitatively distinct signals or redundantly amplify TCR-mediated activation signals was extensively investigated using stable hCD8-zeta Jurkat transfectants which contain stepwise deletions of each ITAM or mutations of tyrosine residues in each ITAM of TCR zeta chain. The influence of mutations of each tyrosine residue on reduction of the amount and species of tyrosine phosphorylated proteins recruited to zeta chain was quite distinctive, but they were roughly proportional to the number of functionally intact ITAMs. However, the first N-terminal ITAM had a signaling potential to trigger most intracellular signaling events for T cell activation and apoptosis similar to wild-type CD8-zeta, but this level was substantially reduced in the presence of the first and second N-terminal ITAM together. Mutations of tyrosine residues in first and second N-terminal ITAM significantly impaired most signaling events leading to T cell activation and activation-induced cell death, but phosphorylation of mitogen-activated protein kinases (MAPKs) was differentially impaired in each mutant. The mutation of the first tyrosine residue in C-terminal ITAM did not show any impairment in induction of surface antigens and cell death, but rather increased IL-2 secretion and MAPK phosphorylation. Therefore, in this study we demonstrated that the ITAMs and their tyrosine residues of TCR zeta chain can transmit qualitatively differential intracellular signals upon TCR stimulation through distinctive regulation of recruitment of tyrosine phosphorylated proteins to zeta chain and activation of various MAPKs.

HCV persistence and immune evasion in the absence of memory T cell help.

Spontaneous resolution of hepatitis C virus (HCV) infection in humans usually affords long-term immunity to persistent viremia and associated liver diseases. Here, we report that memory CD4+ Tcells are essential for this protection. Antibody-mediated depletion of CD4+ Tcells before reinfection of two immune chimpanzees resulted in persistent, low-level viremia despite functional intra-hepatic memory CD8+ Tcell responses. Incomplete control of HCV replication by memory CD8+ Tcells in the absence of adequate CD4+ Tcell help was associated with emergence of viral escape mutations in class I major histocompatibility complex-restricted epitopes and failure to resolve HCV infection.

Immunosuppressive effects of tautomycetin in vivo and in vitro via T cell-specific apoptosis induction.

Tautomycetin (TMC) was identified as an immunosuppressor of activated T cells. Inhibition of T cell proliferation with TMC was observed at concentrations 100-fold lower than those needed to achieve maximal inhibition with cyclosporin A (CsA). TMC specifically blocked tyrosine phosphorylation of intracellular signal mediators downstream of Src tyrosine kinases in a T cell-specific manner, leading to apoptosis due to cleavage of Bcl-2, caspase-9, caspase-3, and poly(ADP-ribose) polymerase, but not caspase-1. In TMC-treated rats that received a heterotopic cardiac allograft, the graft survived more than 160 days, comparable to graft survival in allografted rats treated with CsA. Thus, TMC, whose mechanism of action is different from that of CsA or FK506, can be used as a potent T cell-specific immunosuppressor.