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1.
Front Pharmacol ; 15: 1380000, 2024.
Article in English | MEDLINE | ID: mdl-38887559

ABSTRACT

Introduction: Interleukin 15 (IL-15) is a potential anticancer agent and numerous engineered IL-15 agonists are currently under clinical investigation. Selective targeting of IL-15 to specific lymphocytes may enhance therapeutic effects while helping to minimize toxicities. Methods: We designed and built a heterodimeric targeted cytokine (TaCk) that consists of an anti-programmed cell death 1 receptor antibody (anti-PD-1) and an engineered IL-15. This "PD1/IL15" selectively delivers IL-15 signaling to lymphocytes expressing PD-1. We then investigated the pharmacokinetic (PK) and pharmacodynamic (PD) effects of PD1/IL15 TaCk on immune cell subsets in cynomolgus monkeys after single and repeat intravenous dose administrations. We used these results to determine the first-in-human (FIH) dose and dosing frequency for early clinical trials. Results: The PD1/IL15 TaCk exhibited a nonlinear multiphasic PK profile, while the untargeted isotype control TaCk, containing an anti-respiratory syncytial virus antibody (RSV/IL15), showed linear and dose proportional PK. The PD1/IL15 TaCk also displayed a considerably prolonged PK (half-life range ∼1.0-4.1 days) compared to wild-type IL-15 (half-life ∼1.1 h), which led to an enhanced cell expansion PD response. The PD was dose-dependent, durable, and selective for PD-1+ lymphocytes. Notably, the dose- and time-dependent PK was attributed to dynamic TMDD resulting from test article-induced lymphocyte expansion upon repeat administration. The recommended first-in-human (FIH) dose of PD1/IL15 TaCk is 0.003 mg/kg, determined based on a minimum anticipated biological effect level (MABEL) approach utilizing a combination of in vitro and preclinical in vivo data. Conclusion: This work provides insight into the complex PK/PD relationship of PD1/IL15 TaCk in monkeys and informs the recommended starting dose and dosing frequency selection to support clinical evaluation of this novel targeted cytokine.

4.
Nature ; 627(8004): 646-655, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38418879

ABSTRACT

Tiragolumab, an anti-TIGIT antibody with an active IgG1κ Fc, demonstrated improved outcomes in the phase 2 CITYSCAPE trial (ClinicalTrials.gov: NCT03563716 ) when combined with atezolizumab (anti-PD-L1) versus atezolizumab alone1. However, there remains little consensus on the mechanism(s) of response with this combination2. Here we find that a high baseline of intratumoural macrophages and regulatory T cells is associated with better outcomes in patients treated with atezolizumab plus tiragolumab but not with atezolizumab alone. Serum sample analysis revealed that macrophage activation is associated with a clinical benefit in patients who received the combination treatment. In mouse tumour models, tiragolumab surrogate antibodies inflamed tumour-associated macrophages, monocytes and dendritic cells through Fcγ receptors (FcγR), in turn driving anti-tumour CD8+ T cells from an exhausted effector-like state to a more memory-like state. These results reveal a mechanism of action through which TIGIT checkpoint inhibitors can remodel immunosuppressive tumour microenvironments, and suggest that FcγR engagement is an important consideration in anti-TIGIT antibody development.


Subject(s)
Antibodies, Monoclonal , Antineoplastic Agents , B7-H1 Antigen , Myeloid Cells , Neoplasms , Receptors, Immunologic , T-Lymphocytes, Regulatory , Animals , Humans , Mice , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/therapeutic use , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/immunology , CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Drug Therapy, Combination , Immune Checkpoint Inhibitors/immunology , Immune Checkpoint Inhibitors/therapeutic use , Macrophage Activation , Myeloid Cells/immunology , Neoplasms/drug therapy , Neoplasms/immunology , Receptors, IgG/immunology , Receptors, Immunologic/immunology , T-Lymphocytes, Regulatory/immunology , Tumor Microenvironment/immunology , Tumor-Associated Macrophages/immunology
5.
J Immunother Cancer ; 10(4)2022 04.
Article in English | MEDLINE | ID: mdl-35379739

ABSTRACT

Recent advances in understanding the roles of immune checkpoints in allowing tumors to circumvent the immune system have led to successful therapeutic strategies that have fundamentally changed oncology practice. Thus far, immunotherapies against only two checkpoint targets have been approved, CTLA-4 and PD-L1/PD-1. Antibody blockade of these targets enhances the function of antitumor T cells at least in part by relieving inhibition of the T cell costimulatory receptor CD28. These successes have stimulated considerable interest in identifying other pathways that may bte targeted alone or together with existing immunotherapies. One such immune checkpoint axis is comprised of members of the PVR/nectin family that includes the inhibitory receptor T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory domains (TIGIT). Interestingly, TIGIT acts to regulate the activity of a second costimulatory receptor CD226 that works in parallel to CD28. There are currently over two dozen TIGIT-directed blocking antibodies in various phases of clinical development, testament to the promise of modulating this pathway to enhance antitumor immune responses. In this review, we discuss the role of TIGIT as a checkpoint inhibitor, its interplay with the activating counter-receptor CD226, and its status as the next advance in cancer immunotherapy.


Subject(s)
Immune Checkpoint Inhibitors , Neoplasms , Antigens, Differentiation, T-Lymphocyte , Humans , Immunotherapy , Neoplasms/drug therapy , Neoplasms/metabolism , Receptors, Immunologic , Receptors, Virus , T-Lymphocytes
6.
Immunity ; 55(3): 512-526.e9, 2022 03 08.
Article in English | MEDLINE | ID: mdl-35263569

ABSTRACT

Dual blockade of the PD-1 and TIGIT coinhibitory receptors on T cells shows promising early results in cancer patients. Here, we studied the mechanisms whereby PD-1 and/or TIGIT blockade modulate anti-tumor CD8+ T cells. Although PD-1 and TIGIT are thought to regulate different costimulatory receptors (CD28 and CD226), effectiveness of PD-1 or TIGIT inhibition in preclinical tumor models was reduced in the absence of CD226. CD226 expression associated with clinical benefit in patients with non-small cell lung carcinoma (NSCLC) treated with anti-PD-L1 antibody atezolizumab. CD226 and CD28 were co-expressed on NSCLC infiltrating CD8+ T cells poised for expansion. Mechanistically, PD-1 inhibited phosphorylation of both CD226 and CD28 via its ITIM-containing intracellular domain (ICD); TIGIT's ICD was dispensable, with TIGIT restricting CD226 co-stimulation by blocking interaction with their common ligand PVR (CD155). Thus, full restoration of CD226 signaling, and optimal anti-tumor CD8+ T cell responses, requires blockade of TIGIT and PD-1, providing a mechanistic rationale for combinatorial targeting in the clinic.


Subject(s)
CD8-Positive T-Lymphocytes , Neoplasms , Antigens, Differentiation, T-Lymphocyte/metabolism , CD28 Antigens/metabolism , Humans , Neoplasms/metabolism , Programmed Cell Death 1 Receptor/metabolism , Receptors, Immunologic/metabolism
7.
Nature ; 579(7798): 274-278, 2020 03.
Article in English | MEDLINE | ID: mdl-32103181

ABSTRACT

Despite the resounding clinical success in cancer treatment of antibodies that block the interaction of PD1 with its ligand PDL11, the mechanisms involved remain unknown. A major limitation to understanding the origin and fate of T cells in tumour immunity is the lack of quantitative information on the distribution of individual clonotypes of T cells in patients with cancer. Here, by performing deep single-cell sequencing of RNA and T cell receptors in patients with different types of cancer, we survey the profiles of various populations of T cells and T cell receptors in tumours, normal adjacent tissue, and peripheral blood. We find clear evidence of clonotypic expansion of effector-like T cells not only within the tumour but also in normal adjacent tissue. Patients with gene signatures of such clonotypic expansion respond best to anti-PDL1 therapy. Notably, expanded clonotypes found in the tumour and normal adjacent tissue can also typically be detected in peripheral blood, which suggests a convenient approach to patient identification. Analyses of our data together with several external datasets suggest that intratumoural T cells, especially in responsive patients, are replenished with fresh, non-exhausted replacement cells from sites outside the tumour, suggesting continued activity of the cancer immunity cycle in these patients, the acceleration of which may be associated with clinical response.


Subject(s)
Lymphocytes, Tumor-Infiltrating/cytology , Lymphocytes, Tumor-Infiltrating/metabolism , Neoplasms/pathology , Pharmacogenomic Variants , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/cytology , Antibodies, Monoclonal, Humanized/therapeutic use , Antineoplastic Agents/therapeutic use , Clone Cells , Humans , Neoplasms/drug therapy , Neoplasms/immunology , T-Lymphocytes/metabolism , Transcriptome
8.
Eur J Immunol ; 50(6): 891-902, 2020 06.
Article in English | MEDLINE | ID: mdl-32043568

ABSTRACT

CD96 is a member of the poliovirus receptor (PVR, CD155)-nectin family that includes T cell Ig and ITIM domain (TIGIT) and CD226. While CD96, TIGIT, and CD226 have important roles in regulating NK cell activity, and TIGIT and CD226 have also been shown to regulate T cell responses, it is unclear whether CD96 has inhibitory or stimulatory function in CD8+ T cells. Here, we demonstrate that CD96 has co-stimulatory function on CD8+ T cells. Crosslinking of CD96 on human or mouse CD8+ T cells induced activation, effector cytokine production, and proliferation. CD96 was found to transduce its activating signal through the MEK-ERK pathway. CD96-mediated signaling led to increased frequencies of NUR77- and T-bet-expressing CD8+ T cells and enhanced cytotoxic effector activity, indicating that CD96 can modulate effector T cell differentiation. Antibody blockade of CD96 or genetic ablation of CD96 expression on CD8+ T cells impaired expression of transcription factors and proinflammatory cytokines associated with CD8+ T cell activation in in vivo models. Taken together, CD96 has a co-stimulatory role in CD8+ T cell activation and effector function.


Subject(s)
Antigens, CD/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Lymphocyte Activation , MAP Kinase Signaling System/immunology , Models, Immunological , Animals , Antigens, CD/genetics , Cell Differentiation/genetics , Cell Line, Tumor , Humans , MAP Kinase Signaling System/genetics , Mice , Mice, Knockout
9.
Cell Rep ; 27(1): 269-281.e4, 2019 04 02.
Article in English | MEDLINE | ID: mdl-30943407

ABSTRACT

Myeloid-derived suppressor cells (MDSCs) are found in most cancer malignancies and support tumorigenesis by suppressing immunity and promoting tumor growth. Here we identify the bromodomain (BRD) of CBP/EP300 as a critical regulator of H3K27 acetylation (H3K27ac) in MDSCs across promoters and enhancers of pro-tumorigenic target genes. In preclinical tumor models, in vivo administration of a CBP/EP300-BRD inhibitor (CBP/EP300-BRDi) alters intratumoral MDSCs and attenuates established tumor growth in immunocompetent tumor-bearing mice, as well as in MDSC-dependent xenograft models. Inhibition of CBP/EP300-BRD redirects tumor-associated MDSCs from a suppressive to an inflammatory phenotype through downregulation of STAT pathway-related genes and inhibition of Arg1 and iNOS. Similarly, CBP/EP300-BRDi decreases differentiation and suppressive function of human MDSCs in vitro. Our findings uncover a role of CBP/EP300-BRD in intratumoral MDSCs that may be targeted therapeutically to boost anti-tumor immunity.


Subject(s)
Carcinogenesis/metabolism , Histones/metabolism , Myeloid Cells/metabolism , p300-CBP Transcription Factors/metabolism , Acetylation , Animals , Arginase/genetics , Arginase/metabolism , Cell Line, Tumor , Cells, Cultured , Enhancer Elements, Genetic , Humans , Mice , Mice, Inbred BALB C , Mice, SCID , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Promoter Regions, Genetic , Protein Domains , STAT Transcription Factors/metabolism , p300-CBP Transcription Factors/chemistry
10.
Proc Natl Acad Sci U S A ; 115(50): E11731-E11740, 2018 12 11.
Article in English | MEDLINE | ID: mdl-30504141

ABSTRACT

Natural killer (NK) cell recognition of tumor cells is mediated through activating receptors such as CD226, with suppression of effector functions often controlled by negative regulatory transcription factors such as FOXO1. Here we show that CD226 regulation of NK cell cytotoxicity is facilitated through inactivation of FOXO1. Gene-expression analysis of NK cells isolated from syngeneic tumors grown in wild-type or CD226-deficient mice revealed dysregulated expression of FOXO1-regulated genes in the absence of CD226. In vitro cytotoxicity and stimulation assays demonstrated that CD226 is required for optimal killing of tumor target cells, with engagement of its ligand CD155 resulting in phosphorylation of FOXO1. CD226 deficiency or anti-CD226 antibody blockade impaired cytotoxicity with concomitant compromised inactivation of FOXO1. Furthermore, inhibitors of FOXO1 phosphorylation abrogated CD226-mediated signaling and effector responses. These results define a pathway by which CD226 exerts control of NK cell responses against tumors.


Subject(s)
Antigens, Differentiation, T-Lymphocyte/metabolism , Forkhead Box Protein O1/antagonists & inhibitors , Forkhead Box Protein O1/metabolism , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Animals , Antigens, Differentiation, T-Lymphocyte/genetics , Cell Line, Tumor , Cytotoxicity, Immunologic , Gene Expression Regulation, Neoplastic , Humans , Ligands , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Melanoma, Experimental/genetics , Melanoma, Experimental/immunology , Melanoma, Experimental/metabolism , Mice , Mice, Knockout , Nectins/metabolism , Phosphorylation , Receptors, Virus/metabolism , Signal Transduction/immunology
11.
PLoS One ; 12(7): e0180154, 2017.
Article in English | MEDLINE | ID: mdl-28683073

ABSTRACT

Ion channels regulate a variety of physiological processes and represent an important class of drug target. Among the many methods of studying ion channel function, patch clamp electrophysiology is considered the gold standard by providing the ultimate precision and flexibility. However, its utility in ion channel drug discovery is impeded by low throughput. Additionally, characterization of endogenous ion channels in primary cells remains technical challenging. In recent years, many automated patch clamp (APC) platforms have been developed to overcome these challenges, albeit with varying throughput, data quality and success rate. In this study, we utilized SyncroPatch 768PE, one of the latest generation APC platforms which conducts parallel recording from two-384 modules with giga-seal data quality, to push these 2 boundaries. By optimizing various cell patching parameters and a two-step voltage protocol, we developed a high throughput APC assay for the voltage-gated sodium channel Nav1.7. By testing a group of Nav1.7 reference compounds' IC50, this assay was proved to be highly consistent with manual patch clamp (R > 0.9). In a pilot screening of 10,000 compounds, the success rate, defined by > 500 MΩ seal resistance and >500 pA peak current, was 79%. The assay was robust with daily throughput ~ 6,000 data points and Z' factor 0.72. Using the same platform, we also successfully recorded endogenous voltage-gated potassium channel Kv1.3 in primary T cells. Together, our data suggest that SyncroPatch 768PE provides a powerful platform for ion channel research and drug discovery.


Subject(s)
High-Throughput Screening Assays/methods , Membrane Potentials/physiology , NAV1.7 Voltage-Gated Sodium Channel/metabolism , Patch-Clamp Techniques/methods , Potassium Channel Blockers/pharmacology , Sodium Channel Blockers/pharmacology , Animals , CHO Cells , Cricetulus , Drug Evaluation, Preclinical , Gene Expression , High-Throughput Screening Assays/instrumentation , Kv1.3 Potassium Channel/deficiency , Kv1.3 Potassium Channel/genetics , NAV1.1 Voltage-Gated Sodium Channel/genetics , NAV1.1 Voltage-Gated Sodium Channel/metabolism , NAV1.2 Voltage-Gated Sodium Channel/genetics , NAV1.2 Voltage-Gated Sodium Channel/metabolism , NAV1.3 Voltage-Gated Sodium Channel/genetics , NAV1.3 Voltage-Gated Sodium Channel/metabolism , NAV1.4 Voltage-Gated Sodium Channel/genetics , NAV1.4 Voltage-Gated Sodium Channel/metabolism , NAV1.5 Voltage-Gated Sodium Channel/genetics , NAV1.5 Voltage-Gated Sodium Channel/metabolism , NAV1.6 Voltage-Gated Sodium Channel/genetics , NAV1.6 Voltage-Gated Sodium Channel/metabolism , NAV1.7 Voltage-Gated Sodium Channel/genetics , Patch-Clamp Techniques/instrumentation , Primary Cell Culture , Rats , Sodium Channels/genetics , Sodium Channels/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Transgenes
12.
Nat Commun ; 8: 14644, 2017 03 01.
Article in English | MEDLINE | ID: mdl-28248292

ABSTRACT

Voltage-gated Kv1.3 and Ca2+-dependent KCa3.1 are the most prevalent K+ channels expressed by human and rat T cells. Despite the preferential upregulation of Kv1.3 over KCa3.1 on autoantigen-experienced effector memory T cells, whether Kv1.3 is required for their induction and function is unclear. Here we show, using Kv1.3-deficient rats, that Kv1.3 is involved in the development of chronically activated antigen-specific T cells. Several immune responses are normal in Kv1.3 knockout (KO) rats, suggesting that KCa3.1 can compensate for the absence of Kv1.3 under these specific settings. However, experiments with Kv1.3 KO rats and Kv1.3 siRNA knockdown or channel-specific inhibition of human T cells show that maximal T-cell responses against autoantigen or repeated tetanus toxoid stimulations require both Kv1.3 and KCa3.1. Finally, our data also suggest that T-cell dependency on Kv1.3 or KCa3.1 might be irreversibly modulated by antigen exposure.


Subject(s)
Epitopes/immunology , Immunologic Memory , Intermediate-Conductance Calcium-Activated Potassium Channels/metabolism , Kv1.3 Potassium Channel/metabolism , T-Lymphocytes/metabolism , Animals , Gene Knockdown Techniques , Humans , Immunity/drug effects , Immunologic Memory/drug effects , Intermediate-Conductance Calcium-Activated Potassium Channels/deficiency , Lymphocyte Activation/immunology , Phenotype , Potassium Channel Blockers/pharmacology , RNA, Small Interfering/metabolism , Rats , T-Lymphocytes/drug effects
13.
Trends Immunol ; 38(1): 20-28, 2017 01.
Article in English | MEDLINE | ID: mdl-27793572

ABSTRACT

Immunotherapies that harness the activity of the immune system against tumors are proving to be an effective therapeutic approach in multiple malignancies. Indeed, through accumulation of genetic mutations, many tumors express antigens that can potentially elicit specific tumor immunity. However, tumors can also suppress these responses by activating negative regulatory pathways and checkpoints such as PD-1/PD-L1 and CTLA-4. Blocking these checkpoints on T cells has provided dramatic clinical benefit, but only a subset of patients exhibit clear and durable responses, suggesting that other mechanisms must be limiting the immune response. We discuss here the role of TIGIT, an inhibitory receptor expressed by lymphocytes, in limiting antitumor responses and we review its mechanisms of action during the cancer immunity cycle.


Subject(s)
Immunity, Cellular , Immunotherapy/methods , Neoplasms/immunology , Receptors, Immunologic/metabolism , T-Lymphocytes/immunology , Animals , Antibodies, Monoclonal/therapeutic use , Costimulatory and Inhibitory T-Cell Receptors/immunology , Costimulatory and Inhibitory T-Cell Receptors/metabolism , Humans , Neoplasms/therapy , Receptors, Immunologic/immunology
14.
J Biol Chem ; 290(50): 29732-41, 2015 Dec 11.
Article in English | MEDLINE | ID: mdl-26491012

ABSTRACT

The pharmacokinetic (PK) behavior of monoclonal antibodies in cynomolgus monkeys (cynos) is generally translatable to that in humans. Unfortunately, about 39% of the antibodies evaluated for PKs in cynos have fast nonspecific (or non-target-mediated) clearance (in-house data). An empirical model relating variable region (Fv) charge and hydrophobicity to cyno nonspecific clearance was developed to gauge the risk an antibody would have for fast nonspecific clearance in the monkey. The purpose of this study was to evaluate the predictability of this empirical model on cyno nonspecific clearance with antibodies specifically engineered to have either high or low Fv charge. These amino acid changes were made in the Fv region of two test antibodies, humAb4D5-8 and anti-lymphotoxin α. The humAb4D5-8 has a typical nonspecific clearance in cynos, and by making it more positively charged, the antibody acquires fast nonspecific clearance, and making it less positively charged did not impact its clearance. Anti-lymphotoxin α has fast nonspecific clearance in cynos, and making it more positively charged caused it to clear even faster, whereas making it less positively charged caused it to clear slower and within the typical range. These trends in clearance were also observed in two other preclinical species, mice and rats. The effect of modifying Fv charge on subcutaneous bioavailability was also examined, and in general bioavailability was inversely related to the direction of the Fv charge change. Thus, modifying Fv charge appears to impact antibody PKs, and the changes tended to correlate with those predicted by the empirical model.


Subject(s)
Immunoglobulin Variable Region/immunology , Pharmacokinetics , Animals , Enzyme-Linked Immunosorbent Assay , Immunoglobulin Variable Region/chemistry , Macaca fascicularis , Risk Assessment
15.
Proc Natl Acad Sci U S A ; 110(49): 19896-901, 2013 Dec 03.
Article in English | MEDLINE | ID: mdl-24248355

ABSTRACT

Homotrimeric TNF superfamily ligands signal by inducing trimers of their cognate receptors. As a biologically active heterotrimer, Lymphotoxin(LT)α1ß2 is unique in the TNF superfamily. How the three unique potential receptor-binding interfaces in LTα1ß2 trigger signaling via LTß Receptor (LTßR) resulting in lymphoid organogenesis and propagation of inflammatory signals is poorly understood. Here we show that LTα1ß2 possesses two binding sites for LTßR with distinct affinities and that dimerization of LTßR by LTα1ß2 is necessary and sufficient for signal transduction. The crystal structure of a complex formed by LTα1ß2, LTßR, and the fab fragment of an antibody that blocks LTßR activation reveals the lower affinity receptor-binding site. Mutations targeting each potential receptor-binding site in an engineered single-chain variant of LTα1ß2 reveal the high-affinity site. NF-κB reporter assays further validate that disruption of receptor interactions at either site is sufficient to prevent signaling via LTßR.


Subject(s)
Cytokines/chemistry , Lymphotoxin alpha1, beta2 Heterotrimer/metabolism , Lymphotoxin beta Receptor/metabolism , Multiprotein Complexes/immunology , Signal Transduction/immunology , Chromatography, Gel , Cytokines/immunology , Dimerization , Humans , Multiprotein Complexes/metabolism
16.
Microbes Infect ; 15(10-11): 677-87, 2013.
Article in English | MEDLINE | ID: mdl-23850656

ABSTRACT

Herpes simplex virus 1 infection of the eye can result in stromal keratitis, a chronic immunoinflammatory lesion that is a significant cause of human blindness. A key to controlling the severity of lesions is to identify cellular and molecular events responsible for tissue damage. This report evaluates the role of lymphotoxin-α, a proinflammatory cytokine that could be involved during stromal keratitis. We demonstrate that after infection, both lymphotoxin-α and lymphotoxin-ß transcripts are detectable at high levels 48 h postinfection, suggesting roles for the secreted homotrimer lymphotoxin-α3 and the membrane-bound lymphotoxin-α1ß2 heterotrimer in stromal keratitis. Using a corneal stromal fibroblast cell line, lymphotoxin-α3 and lymphotoxin-α1ß2 were found to have proinflammatory roles by stimulating production of chemokines. Treatment of mice with a depleting anti-lymphotoxin-α mAb during the clinical phase of the disease significantly attenuated stromal keratitis lesions. In treated mice, expression of proinflammatory molecules and chemokines was reduced, as were numbers of cornea-infiltrating proinflammatory cells, particularly Th1 cells. The protective effect of anti-lymphotoxin-α mAb was highly reduced with a mutant version of the mAb that lacks Fc receptor binding activity, indicating that depletion of lymphotoxin-expressing cells was mainly responsible for efficacy, with LT-α3 contributing minimally to inflammation. These data demonstrate that lymphotoxin-expressing cells, such as Th1 cells, mediate stromal keratitis.


Subject(s)
Herpesvirus 1, Human/immunology , Keratitis, Herpetic/pathology , Keratitis, Herpetic/virology , Lymphotoxin-alpha/immunology , Animals , Cell Line , Disease Models, Animal , Female , Fibroblasts/immunology , Fibroblasts/virology , Lymphotoxin-alpha/antagonists & inhibitors , Mice , Mice, Inbred C57BL , Th1 Cells/immunology
17.
PLoS One ; 8(12): e83457, 2013.
Article in English | MEDLINE | ID: mdl-24386204

ABSTRACT

Epstein-Barr virus induced receptor 2 (EBI2), a Gαi-coupled G protein-coupled receptor, is a chemotactic receptor for B, T and dendritic cells (DC). Genetic studies have also implicated EBI2 as a regulator of an interferon regulatory factor 7 (IRF7)-driven inflammatory network (IDIN) associated with autoimmune diseases, although the corollary in primary type I IFN-producing cells has not been reported. Here we demonstrate that EBI2 negatively regulates type I IFN responses in plasmacytoid DC (pDCs) and CD11b(+) myeloid cells. Activation of EBI2(-/-) pDCs and CD11b(+) cells with various TLR ligands induced elevated type I IFN production compared to wild-type cells. Moreover, in vivo challenge with endosomal TLR agonists or infection with lymphocytic choriomeningitis virus elicited more type I IFNs and proinflammatory cytokines in EBI2(-/-) mice compared to normal mice. Elevated systemic cytokines occurred despite impaired ability of EBI2-deficient pDCs and CD11b(+) cells to migrate from the blood to the spleen and peritoneal cavity under homeostatic conditions. As reported for other immune cells, pDC migration was dependent on the ligand for EBI2, 7α,25-dihydroxycholesterol. Consistent with a cell intrinsic role for EBI2, type I IFN-producing cells from EBI2-deficient mice expressed higher levels of IRF7 and IDIN genes. Together these data suggest a negative regulatory role for EBI2 in balancing TLR-mediated responses to foreign and to self nucleic acids that may precipitate autoimmunity.


Subject(s)
Dendritic Cells/metabolism , Interferon Type I/metabolism , Myeloid Cells/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Chemotaxis/drug effects , Chemotaxis/genetics , Chemotaxis/immunology , Dendritic Cells/immunology , Female , Gene Expression , Interferon Regulatory Factor-7/metabolism , Ligands , Lymphocytic Choriomeningitis/immunology , Lymphocytic Choriomeningitis/metabolism , Lymphocytic choriomeningitis virus/immunology , Mice , Mice, Knockout , Myeloid Cells/immunology , Peritoneal Cavity , Pertussis Toxin/administration & dosage , Receptors, G-Protein-Coupled/genetics , Signal Transduction , Spleen/immunology , Spleen/metabolism , Toll-Like Receptors/metabolism
18.
PLoS One ; 7(3): e33106, 2012.
Article in English | MEDLINE | ID: mdl-22427961

ABSTRACT

Graft-versus-host disease (GVHD) is a major barrier to successful allogeneic hematopoietic cell transplantation and is largely mediated by activated donor lymphocytes. Lymphotoxin (LT)-α is expressed by subsets of activated T and B cells, and studies in preclinical models demonstrated that targeted depletion of these cells with a mouse anti-LT-α monoclonal antibody (mAb) was efficacious in inhibiting inflammation and autoimmune disease. Here we demonstrate that LT-α is also upregulated on activated human donor lymphocytes in a xenogeneic model of GVHD and targeted depletion of these donor cells ameliorated GVHD. A depleting humanized anti-LT-α mAb, designated MLTA3698A, was generated that specifically binds to LT-α in both the soluble and membrane-bound forms, and elicits antibody-dependent cellular cytotoxicity (ADCC) activity in vitro. Using a human peripheral blood mononuclear cell transplanted SCID (Hu-SCID) mouse model of GVHD, the anti-human LT-α mAb specifically depleted activated LT-expressing human donor T and B cells, resulting in prolonged survival of the mice. A mutation in the Fc region, rendering the mAb incapable of mediating ADCC, abolished all in vitro and in vivo effects. These data support a role for using a depleting anti-LT-α antibody in treating immune diseases such as GVHD and autoimmune diseases.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antibody-Dependent Cell Cytotoxicity/drug effects , Graft vs Host Disease/immunology , Graft vs Host Disease/prevention & control , Lymphocytes/immunology , Lymphotoxin-alpha/deficiency , Transplantation, Homologous/adverse effects , Animals , Antibodies, Monoclonal/immunology , Antibody-Dependent Cell Cytotoxicity/immunology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Lymphotoxin-alpha/immunology , Mice , Mice, SCID , Surface Plasmon Resonance , Transplantation, Homologous/immunology
19.
J Immunol ; 186(2): 1279-88, 2011 Jan 15.
Article in English | MEDLINE | ID: mdl-21160042

ABSTRACT

IL-1R-associated kinases (IRAKs) are important mediators of MyD88-dependent signaling by the TLR/IL-1R superfamily and facilitate inflammatory responses. IRAK4 and IRAK1 function as active kinases and as scaffolds for protein-protein interactions. We report that although IRAK1/4 kinase activity is essential for human plasmacytoid dendritic cell (pDC) activation, it is dispensable in B, T, dendritic, and monocytic cells, which is in contrast with an essential active kinase role in comparable mouse cell types. An IRAK1/4 kinase inhibitor abrogated TLR7/9-induced IFN-α responses in both mouse and human pDCs, but other human immune cell populations activated via TLR7/9 or IL-1R were refractory to IRAK4 kinase inhibition. Gene ablation experiments using small interfering RNA demonstrated an essential scaffolding role for IRAK1 and IRAK4 in MyD88-dependent signaling. Finally, we demonstrate that autoimmune patient (systemic lupus erythematosus and rheumatoid arthritis) serum activates both pDC and B cells, but IRAK1/4 kinase inhibition affects only the pDC response, underscoring the differential IRAK1/4 functional requirements in human immune cells. These data reveal important species differences and elaborate cell type requirements for IRAK1/4 kinase activity.


Subject(s)
Antigen-Antibody Complex/blood , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/pathology , Interleukin-1 Receptor-Associated Kinases/physiology , Lupus Erythematosus, Systemic/immunology , Lupus Erythematosus, Systemic/pathology , Animals , Arthritis, Rheumatoid/enzymology , Autoantibodies/biosynthesis , Autoantibodies/blood , B-Lymphocyte Subsets/enzymology , B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/pathology , Cells, Cultured , Dendritic Cells/enzymology , Dendritic Cells/immunology , Dendritic Cells/pathology , Female , Humans , Interleukin-17/biosynthesis , Interleukin-17/blood , Lupus Erythematosus, Systemic/enzymology , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Monocytes/enzymology , Monocytes/immunology , Monocytes/pathology , Signal Transduction/immunology , Th1 Cells/enzymology , Th1 Cells/immunology , Toll-Like Receptor 9/physiology
20.
Nat Med ; 15(7): 766-73, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19561618

ABSTRACT

Uncontrolled T helper type 1 (T(H)1) and T(H)17 cells are associated with autoimmune responses. We identify surface lymphotoxin-alpha (LT-alpha) as common to T(H)0, T(H)1 and T(H)17 cells and employ a unique strategy to target these subsets using a depleting monoclonal antibody (mAb) directed to surface LT-alpha. Depleting LT-alpha-specific mAb inhibited T cell-mediated models of delayed-type hypersensitivity and experimental autoimmune encephalomyelitis. In collagen-induced arthritis (CIA), preventive and therapeutic administration of LT-alpha-specific mAb inhibited disease, and immunoablated T cells expressing interleukin-17 (IL-17), interferon-gamma and tumor necrosis factor-alpha (TNF-alpha), whereas decoy lymphotoxin-beta receptor (LT-betaR) fusion protein had no effect. A mutation in the Fc tail, rendering the antibody incapable of Fcgamma receptor binding and antibody-dependent cellular cytotoxicity activity, abolished all in vivo effects. Efficacy in CIA was preceded by a loss of rheumatoid-associated cytokines IL-6, IL-1beta and TNF-alpha within joints. These data indicate that depleting LT-alpha-expressing lymphocytes with LT-alpha-specific mAb may be beneficial in the treatment of autoimmune disease.


Subject(s)
Autoimmune Diseases/therapy , Interleukin-17/physiology , Lymphocyte Depletion , Lymphotoxin-alpha/antagonists & inhibitors , Th1 Cells/immunology , Animals , Antibodies, Monoclonal/therapeutic use , Arthritis, Experimental/therapy , Autoimmune Diseases/etiology , Autoimmune Diseases/immunology , Inflammation/etiology , Mice , Mice, Inbred DBA
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