Preclinical Analysis of Candidate Anti-Human CD79 Therapeutic Antibodies Using a Humanized CD79 Mouse Model.

The BCR comprises a membrane-bound Ig that is noncovalently associated with a heterodimer of CD79A and CD79B. While the BCR Ig component functions to sense extracellular Ag, CD79 subunits contain cytoplasmic ITAMs that mediate intracellular propagation of BCR signals critical for B cell development, survival, and Ag-induced activation. CD79 is therefore an attractive target for Ab and chimeric Ag receptor T cell therapies for autoimmunity and B cell neoplasia. Although the mouse is an attractive model for preclinical testing, due to its well-defined immune system, an obstacle is the lack of cross-reactivity of candidate therapeutic anti-human mAbs with mouse CD79. To overcome this problem, we generated knockin mice in which the extracellular Ig-like domains of CD79A and CD79B were replaced with human equivalents. In this study, we describe the generation and characterization of mice expressing chimeric CD79 and report studies that demonstrate their utility in preclinical analysis of anti-human CD79 therapy. We demonstrate that human and mouse CD79 extracellular domains are functionally interchangeable, and that anti-human CD79 lacking Fc region effector function does not cause significant B cell depletion, but induces 1) decreased expression of plasma membrane-associated IgM and IgD, 2) uncoupling of BCR-induced tyrosine phosphorylation and calcium mobilization, and 3) increased expression of PTEN, consistent with the levels observed in anergic B cells. Finally, anti-human CD79 treatment prevents disease development in two mouse models of autoimmunity. We also present evidence that anti-human CD79 treatment may inhibit Ab secretion by terminally differentiated plasmablasts and plasma cells in vitro.

Lrp4 is a receptor for Agrin and forms a complex with MuSK.

Neuromuscular synapse formation requires a complex exchange of signals between motor neurons and skeletal muscle fibers, leading to the accumulation of postsynaptic proteins, including acetylcholine receptors in the muscle membrane and specialized release sites, or active zones in the presynaptic nerve terminal. MuSK, a receptor tyrosine kinase that is expressed in skeletal muscle, and Agrin, a motor neuron-derived ligand that stimulates MuSK phosphorylation, play critical roles in synaptic differentiation, as synapses do not form in their absence, and mutations in MuSK or downstream effectors are a major cause of a group of neuromuscular disorders, termed congenital myasthenic syndromes (CMS). How Agrin activates MuSK and stimulates synaptic differentiation is not known and remains a fundamental gap in our understanding of signaling at neuromuscular synapses. Here, we report that Lrp4, a member of the LDLR family, is a receptor for Agrin, forms a complex with MuSK, and mediates MuSK activation by Agrin.

MOG autoantibodies trigger a tightly-controlled FcR and BTK-driven microglia proliferative response.

Autoantibodies are a hallmark of numerous neurological disorders, including multiple sclerosis, autoimmune encephalitides and neuromyelitis optica. Whilst well understood in peripheral myeloid cells, the pathophysiological significance of autoantibody-induced Fc receptor signalling in microglia remains unknown, in part due to the lack of a robust in vivo model. Moreover, the application of therapeutic antibodies for neurodegenerative disease also highlights the importance of understanding Fc receptor signalling in microglia. Here, we describe a novel in vivo experimental paradigm that allows for selective engagement of Fc receptors within the CNS by peripherally injecting anti-myelin oligodendrocyte glycoprotein (MOG) monoclonal antibodies into normal wild-type mice. MOG antigen-bound immunoglobulins were detected throughout the CNS and triggered a rapid and tightly regulated proliferative response in both brain and spinal cord microglia. This microglial response was abrogated when anti-MOG antibodies were deprived of Fc receptor effector function or injected into Fcγ receptor knockout mice and was associated with the downregulation of Fc receptors in microglia, but not peripheral myeloid cells, establishing that this response was dependent on central Fc receptor engagement. Downstream of the Fc receptors, BTK was a required signalling node for this response, as microglia proliferation was amplified in BtkE41K knock-in mice expressing a constitutively active form of the enzyme and blunted in mice treated with a CNS-penetrant small molecule inhibitor of BTK. Finally, this response was associated with transient and stringently regulated changes in gene expression predominantly related to cellular proliferation, which markedly differed from transcriptional programs typically associated with Fc receptor engagement in peripheral myeloid cells. Together, these results establish a physiologically-meaningful functional response to Fc receptor and BTK signalling in microglia, while providing a novel in vivo tool to further dissect the roles of microglia-specific Fc receptor and BTK-driven responses to both pathogenic and therapeutic antibodies in CNS homeostasis and disease.

Kinetics of early T cell receptor signaling regulate the pathway of lytic granule delivery to the secretory domain.

Cytolytic granules mediate killing of virus-infected cells by cytotoxic T lymphocytes. We show here that the granules can take long or short paths to the secretory domain. Both paths utilized the same intracellular molecular events, which have different spatial and temporal arrangements and are regulated by the kinetics of Ca(2+)-mediated signaling. Rapid signaling caused swift granule concentration near the microtubule-organizing center (MTOC) and subsequent delivery by the polarized MTOC directly to the secretory domain-the shortest path. Indolent signaling led to late recruitment of granules that moved along microtubules to the periphery of the synapse and then moved tangentially to fuse at the outer edge of the secretory domain-a longer path. The short pathway is associated with faster granule release and more efficient killing than the long pathway. Thus, the kinetics of early signaling regulates the quality of the T cell cytolytic response.

Interference with Ca(2+) release activated Ca(2+) (CRAC) channel function delays T-cell arrest in vivo.

Entry of lymphocytes into secondary lymphoid organs (SLOs) involves intravascular arrest and intracellular calcium ion ([Ca(2+)]i) elevation. TCR activation triggers increased [Ca(2+)]i and can arrest T-cell motility in vitro. However, the requirement for [Ca(2+)]i elevation in arresting T cells in vivo has not been tested. Here, we have manipulated the Ca(2+) release-activated Ca(2+) (CRAC) channel pathway required for [Ca(2+)]i elevation in T cells through genetic deletion of stromal interaction molecule (STIM) 1 or by expression of a dominant-negative ORAI1 channel subunit (ORAI1-DN). Interestingly, the absence of CRAC did not interfere with homing of naïve CD4(+) T cells to SLOs and only moderately reduced crawling speeds in vivo. T cells expressing ORAI1-DN lacked TCR activation induced [Ca(2+)]i elevation, yet arrested motility similar to control T cells in vitro. In contrast, antigen-specific ORAI1-DN T cells had a twofold delayed onset of arrest following injection of OVA peptide in vivo. CRAC channel function is not required for homing to SLOs, but enhances spatiotemporal coordination of TCR signaling and motility arrest.

Protein kinase C theta regulates stability of the peripheral adhesion ring junction and contributes to the sensitivity of target cell lysis by CTL.

Destruction of virus-infected cells by CTL is an extremely sensitive and efficient process. Our previous data suggest that LFA-1-ICAM-1 interactions in the peripheral supramolecular activation cluster (pSMAC) of the immunological synapse mediate formation of a tight adhesion junction that might contribute to the sensitivity of target cell lysis by CTL. Herein, we compared more (CD8(+)) and less (CD4(+)) effective CTL to understand the molecular events that promote efficient target cell lysis. We found that abrogation of the pSMAC formation significantly impaired the ability of CD8(+) but not CD4(+) CTL to lyse target cells despite having no effect of the amount of released granules by both CD8(+) and CD4(+) CTL. Consistent with this, CD4(+) CTL break their synapses more often than do CD8(+) CTL, which leads to the escape of the cytolytic molecules from the interface. CD4(+) CTL treatment with a protein kinase Ctheta inhibitor increases synapse stability and sensitivity of specific target cell lysis. Thus, formation of a stable pSMAC, which is partially controlled by protein kinase Ctheta, functions to confine the released lytic molecules at the synaptic interface and to enhance the effectiveness of target cell lysis.

Radiation-induced CXCL16 release by breast cancer cells attracts effector T cells.

Recruitment of effector T cells to inflamed peripheral tissues is regulated by chemokines and their receptors, but the factors regulating recruitment to tumors remain largely undefined. Ionizing radiation (IR) therapy is a common treatment modality for breast and other cancers. Used as a cytocidal agent for proliferating cancer cells, IR in combination with immunotherapy has been shown to promote immune-mediated tumor destruction in preclinical studies. In this study we demonstrate that IR markedly enhanced the secretion by mouse and human breast cancer cells of CXCL16, a chemokine that binds to CXCR6 on Th1 and activated CD8 effector T cells, and plays an important role in their recruitment to sites of inflammation. Using a poorly immunogenic mouse model of breast cancer, we found that irradiation increased the migration of CD8(+)CXCR6(+) activated T cells to tumors in vitro and in vivo. CXCR6-deficient mice showed reduced infiltration of tumors by activated CD8 T cells and impaired tumor regression following treatment with local IR to the tumor and Abs blocking the negative regulator of T cell activation, CTLA-4. These results provide the first evidence that IR can induce the secretion by cancer cells of proinflammatory chemotactic factors that recruit antitumor effector T cells. The ability of IR to convert tumors into "inflamed" peripheral tissues could be exploited to overcome obstacles at the effector phase of the antitumor immune response and improve the therapeutic efficacy of immunotherapy.

Human immunodeficiency virus type 1 envelope gp120 induces a stop signal and virological synapse formation in noninfected CD4+ T cells.

Human immunodeficiency virus type 1 (HIV-1)-infected T cells form a virological synapse with noninfected CD4(+) T cells in order to efficiently transfer HIV-1 virions from cell to cell. The virological synapse is a specialized cellular junction that is similar in some respects to the immunological synapse involved in T-cell activation and effector functions mediated by the T-cell antigen receptor. The immunological synapse stops T-cell migration to allow a sustained interaction between T-cells and antigen-presenting cells. Here, we have asked whether HIV-1 envelope gp120 presented on a surface to mimic an HIV-1-infected cell also delivers a stop signal and if this is sufficient to induce a virological synapse. We demonstrate that HIV-1 gp120-presenting surfaces arrested the migration of primary activated CD4 T cells that occurs spontaneously in the presence of ICAM-1 and induced the formation of a virological synapse, which was characterized by segregated supramolecular structures with a central cluster of envelope surrounded by a ring of ICAM-1. The virological synapse was formed transiently, with the initiation of migration within 30 min. Thus, HIV-1 gp120-presenting surfaces induce a transient stop signal and supramolecular segregation in noninfected CD4(+) T cells.

Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids.

We examined the in vivo behavior of liver natural killer T cells (NKT cells) by intravital fluorescence microscopic imaging of mice in which a green fluorescent protein cDNA was used to replace the gene encoding the chemokine receptor CXCR6. NKT cells, which account for most CXCR6(+) cells in liver, were found to crawl within hepatic sinusoids at 10-20 microm/min and to stop upon T cell antigen receptor activation. CXCR6-deficient mice exhibited a selective and severe reduction of CD1d-reactive NKT cells in the liver and decreased susceptibility to T-cell-dependent hepatitis. CXCL16, the cell surface ligand for CXCR6, is expressed on sinusoidal endothelial cells, and CXCR6 deficiency resulted in reduced survival, but not in altered speed or pattern of patrolling of NKT cells. Thus, NKT cells patrol liver sinusoids to provide intravascular immune surveillance, and CXCR6 contributes to liver-based immune responses by regulating their abundance.

Structural and kinetic basis for the selectivity of aducanumab for aggregated forms of amyloid-ß.

Aducanumab, a human-derived antibody targeting amyloid-ß (Aß), is in Phase 3 clinical trials for the treatment of Alzheimer's disease. Biochemical and structural analyses show that aducanumab binds a linear epitope formed by amino acids 3-7 of the Aß peptide. Aducanumab discriminates between monomers and oligomeric or fibrillar aggregates based on weak monovalent affinity, fast binding kinetics and strong avidity for epitope-rich aggregates. Direct comparative studies with analogs of gantenerumab, bapineuzumab and solanezumab demonstrate clear differentiation in the binding properties of these antibodies. The crystal structure of the Fab fragment of aducanumab bound to its epitope peptide reveals that aducanumab binds to the N terminus of Aß in an extended conformation, distinct from those seen in structures with other antibodies that target this immunodominant epitope. Aducanumab recognizes a compact epitope that sits in a shallow pocket on the antibody surface. In silico analyses suggest that aducanumab interacts weakly with the Aß monomer and may accommodate a variety of peptide conformations, further supporting its selectivity for Aß aggregates. Our studies provide a structural rationale for the low affinity of aducanumab for non-pathogenic monomers and its greater selectivity for aggregated forms than is seen for other Aß-targeting antibodies.

Antagonism of HIV-specific CD4+ T cells by C-terminal truncation of a minimum epitope.

Antagonism of T cell responses by variants of the cognate peptide is a potential mechanism of viral escape from immune responses and may play a role in the ability of HIV to evade immune control. We show here a rarely described mechanism of antagonism by a peptide shorter than the minimum length epitope for an HIV p24-specific CD4+ T cell clone. The shorter antagonist peptide-MHC complex bound the T cell receptor (TCR), albeit with lower affinity than the full-length agonist peptide. Prior work showing the crystal structure of the peptide-MHC complex revealed a unique glycine hinge near the C-terminus of the agonist peptide, allowing the generation of full-length antagonist peptide lacking the hinge. These results confirm the dependence of productive TCR engagement on residues spilling out from the C-terminus of the MHC binding groove and show that partial engagement of the TCR with a truncated, low-affinity ligand can result in T cell antagonism.

Identification of Novel Fibrosis Modifiers by In Vivo siRNA Silencing.

Fibrotic diseases contribute to 45% of deaths in the industrialized world, and therefore a better understanding of the pathophysiological mechanisms underlying tissue fibrosis is sorely needed. We aimed to identify novel modifiers of tissue fibrosis expressed by myofibroblasts and their progenitors in their disease microenvironment through RNA silencing in vivo. We leveraged novel biology, targeting genes upregulated during liver and kidney fibrosis in this cell lineage, and employed small interfering RNA (siRNA)-formulated lipid nanoparticles technology to silence these genes in carbon-tetrachloride-induced liver fibrosis in mice. We identified five genes, Egr2, Atp1a2, Fkbp10, Fstl1, and Has2, which modified fibrogenesis based on their silencing, resulting in reduced Col1a1 mRNA levels and collagen accumulation in the liver. These genes fell into different groups based on the effects of their silencing on a transcriptional mini-array and histological outcomes. Silencing of Egr2 had the broadest effects in vivo and also reduced fibrogenic gene expression in a human fibroblast cell line. Prior to our study, Egr2, Atp1a2, and Fkbp10 had not been functionally validated in fibrosis in vivo. Thus, our results provide a major advance over the existing knowledge of fibrogenic pathways. Our study is the first example of a targeted siRNA assay to identify novel fibrosis modifiers in vivo.

Anti-BDCA2 monoclonal antibody inhibits plasmacytoid dendritic cell activation through Fc-dependent and Fc-independent mechanisms.

Type I interferons (IFN-I) are implicated in the pathogenesis of systemic lupus erythematosus (SLE). In SLE, immune complexes bind to the CD32a (FcγRIIa) receptor on the surface of plasmacytoid dendritic cells (pDCs) and stimulate the secretion of IFN-I from pDCs. BDCA2 is a pDC-specific receptor that, when engaged, inhibits the production of IFN-I in human pDCs. BDCA2 engagement, therefore, represents an attractive therapeutic target for inhibiting pDC-derived IFN-I and may be an effective therapy for the treatment of SLE. In this study, we show that 24F4A, a humanized monoclonal antibody (mAb) against BDCA2, engages BDCA2 and leads to its internalization and the consequent inhibition of TLR-induced IFN-I by pDCs in vitro using blood from both healthy and SLE donors. These effects were confirmed in vivo using a single injection of 24F4A in cynomolgus monkeys. 24F4A also inhibited pDC activation by SLE-associated immune complexes (IC). In addition to the inhibitory effect of 24F4A through engagement of BDCA2, the Fc region of 24F4A was critical for potent inhibition of IC-induced IFN-I production through internalization of CD32a. This study highlights the novel therapeutic potential of an effector-competent anti-BDCA2 mAb that demonstrates a dual mechanism to dampen pDC responses for enhanced clinical efficacy in SLE.

Labeling antigen-specific CD4(+) T cells with class II MHC oligomers.

Class I MHC-peptide oligomers (MHC tetramers) have become popular reagents for the detection and characterization of antigen-specific CD8(+) T cells. Class II MHC proteins can be produced by expression in Escherichia coli followed by in vitro folding, or by native expression in insect cells; biotin can be introduced by site-specific chemical modification of cysteine, or by enzymatic modification of a peptide tag; and a variety of fluorescent streptavidin preparations can be used for oligomerization. Here we review methodologies for production of fluorescent oligomers of soluble class II MHC proteins and discuss their use in analysis of antigen-specific CD4(+) T cells. We explore the experimental conditions necessary for efficient staining of CD4(+) T cells using oligomers of class II MHC proteins, and we establish a standard protocol. Finally, we consider complications and challenges associated with these reagents, discuss the interpretation of staining results, and suggest future directions for investigation, in particular the use of MHC oligomers for the study of T cell avidity modulation.

Cutting edge: activation by innate cytokines or microbial antigens can cause arrest of natural killer T cell patrolling of liver sinusoids.

Natural killer T (NKT) cells are innate-like lymphocytes that rapidly secrete large amounts of effector cytokines upon activation. Recognition of alpha-linked glycolipids presented by CD1d leads to the production of IL-4, IFN-gamma, or both, while direct activation by the synergistic action of IL-12 and IL-18 leads to IFN-gamma production only. We previously reported that in vitro cultured dendritic cells can modulate NKT cell activation and, using intravital fluorescence laser scanning microscopy, we reported that the potent stimulation of NKT cells results in arrest within hepatic sinusoids. In this study, we examine the relationship between murine NKT cell patrolling and activation. We report that NKT cell arrest results from activation driven by limiting doses of a bacteria-derived weak agonist, galacturonic acid-containing glycosphingolipid, or a synthetic agonist, alpha-galactosyl ceramide. Interestingly, NKT cell arrest also results from IL-12 and IL-18 synergistic activation. Thus, innate cytokines and natural microbial TCR agonists trigger sinusoidal NKT cell arrest and an effector response.

Peptide-MHC potency governs dynamic interactions between T cells and dendritic cells in lymph nodes.

T cells survey antigen-presenting dendritic cells (DCs) by migrating through DC networks, arresting and maintaining contact with DCs for several hours after encountering high-potency complexes of peptide and major histocompatibility complex (pMHC), leading to T cell activation. The effects of low-potency pMHC complexes on T cells in vivo, however, are unknown, as is the mechanism controlling T cell arrest. Here we evaluated T cell responses in vivo to high-, medium- and low-potency pMHC complexes and found that regardless of potency, pMHC complexes induced upregulation of CD69, anergy and retention of T cells in lymph nodes. However, only high-potency pMHC complexes expressed by DCs induced calcium-dependent T cell deceleration and calcineurin-dependent anergy. The pMHC complexes of lower potency instead induced T cell anergy by a biochemically distinct process that did not affect T cell dynamics.

Opposing effects of PKCtheta and WASp on symmetry breaking and relocation of the immunological synapse.

The immunological synapse (IS) is a junction between the T cell and antigen-presenting cell and is composed of supramolecular activation clusters (SMACs). No studies have been published on naive T cell IS dynamics. Here, we find that IS formation during antigen recognition comprises cycles of stable IS formation and autonomous naive T cell migration. The migration phase is driven by PKCtheta, which is localized to the F-actin-dependent peripheral (p)SMAC. PKCtheta(-/-) T cells formed hyperstable IS in vitro and in vivo and, like WT cells, displayed fast oscillations in the distal SMAC, but they showed reduced slow oscillations in pSMAC integrity. IS reformation is driven by the Wiscott Aldrich Syndrome protein (WASp). WASp(-/-) T cells displayed normal IS formation but were unable to reform IS after migration unless PKCtheta was inhibited. Thus, opposing effects of PKCtheta and WASp control IS stability through pSMAC symmetry breaking and reformation.

The duration of exposure to HIV modulates the breadth and the magnitude of HIV-specific memory CD4+ T cells.

The impact of exposure to Ag on the development and maintenance of human CD4(+) memory T cells in general and HIV infection in particular is partially understood. In this study, we measured HIV-specific CD4(+) T cell proliferative responses against HIV proteins and derived peptides one year after highly active antiretroviral therapy initiation in 39 HIV-infected patients who initiated therapy at different times following infection. We show that a brief exposure to HIV of <1 month does not allow the generation of significant detectable frequencies of HIV-specific CD4(+) memory T cells. Patients having prolonged cumulative exposure to high viral load due to therapy failures also demonstrated limited HIV-specific CD4(+) T cell responses. In contrast, patients exposed to significant levels of virus for periods ranging from 3 to 18 mo showed brisk and broad HIV-specific CD4(+) T cell responses 1 year following the onset of therapy intervention. We also demonstrate that the nadir CD4(+) T cell count before therapy initiation correlated positively with the breadth and magnitude of these responses. Our findings indicate that the loss of proliferative HIV-specific CD4(+) T cell responses is associated with the systemic progression of the disease and that a brief exposure to HIV does not allow the establishment of detectable frequencies of HIV-specific memory CD4(+) T cells.

Major histocompatibility complex and T cell interactions of a universal T cell epitope from Plasmodium falciparum circumsporozoite protein.

A 20-residue sequence from the C-terminal region of the circumsporozoite protein of the malaria parasite Plasmodium falciparum is considered a universal helper T cell epitope because it is immunogenic in individuals of many major histocompatibility complex (MHC) haplotypes. Subunit vaccines containing T* and the major B cell epitope of the circumsporozoite protein induce high antibody titers to the malaria parasite and significant T cell responses in humans. In this study we have evaluated the specificity of the T* sequence with regard to its binding to the human class II MHC protein DR4 (HLA-DRB1*0401), its interactions with antigen receptors on T cells, and the effect of natural variants of this sequence on its immunogenicity. Computational approaches identified multiple potential DR4-binding epitopes within T*, and experimental binding studies confirmed the following two tight binding epitopes: one located toward the N terminus (the T*-1 epitope) and one at the C terminus (the T*-5 epitope). Immunization of a human DR4 volunteer with a peptide-based vaccine containing the T* sequence elicited CD4+ T cells that recognize each of these epitopes. Here we present an analysis of the immunodominant N-terminal epitope T*-1. T*-1 residues important for interaction with DR4 and with antigen receptors on T*-specific T cells were mapped. MHC tetramers carrying DR4/T*-1 MHC-peptide complexes stained and efficiently stimulated these cells in vitro. T*-1 overlaps a region of the protein that has been described as highly polymorphic; however, the particular T*-1 residues required for anchoring to DR4 were highly conserved in Plasmodium sequences described to date.

Examination of the highly diverse CD4(+) T-cell repertoire directed against an influenza peptide: a step towards TCR proteomics.

We combined several recent technological advances in immunology and molecular biology to identify and sequence a large number of T-cell receptor (TCR) genes specific for a particular antigen. We utilized class II MHC tetramers and interferon-gamma surface capture to isolate from samples of peripheral blood the population of CD4(+) T cells responding to a peptide derived from influenza hemagglutinin and restricted by HLA-DR1. Detailed analysis of hundreds of clones from three different patients revealed an extremely diverse repertoire, with little overlap between patients. We observed no dominant usage of particular Vbeta segments nor any clear CDR3 sequence motif in the responding T cells, but most of the clones appear to utilize acidic residues in the CDR1 and CDR3 regions, presumably to interact with the exposed basic residues in the MHC-peptide complex. This methodology could be expanded to a large scale to identify the generalized rules governing TCR-MHC engagement and factors which shape the T-cell repertoire after vaccination and in autoimmune pathologies.