Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 17 de 17
Filter
1.
Transl Psychiatry ; 13(1): 221, 2023 Jun 23.
Article in English | MEDLINE | ID: mdl-37353479

ABSTRACT

Bipolar affective disorder (BPAD) are life-long disorders that account for significant morbidity in afflicted patients. The etiology of BPAD is complex, combining genetic and environmental factors to increase the risk of disease. Genetic studies have pointed toward cytoskeletal dysfunction as a potential molecular mechanism through which BPAD may arise and have implicated proteins that regulate the cytoskeleton as risk factors. Microtubule actin crosslinking factor 1 (MACF1) is a giant cytoskeletal crosslinking protein that can coordinate the different aspects of the mammalian cytoskeleton with a wide variety of actions. In this review, we seek to highlight the functions of MACF1 in the nervous system and the molecular mechanisms leading to BPAD pathogenesis. We also offer a brief perspective on MACF1 and the role it may be playing in lithium's mechanism of action in treating BPAD.


Subject(s)
Bipolar Disorder , Microfilament Proteins , Humans , Actins/metabolism , Bipolar Disorder/drug therapy , Bipolar Disorder/metabolism , Cytoskeleton/metabolism , Lithium/pharmacology , Lithium/therapeutic use , Microfilament Proteins/metabolism , Microfilament Proteins/therapeutic use , Microtubules/metabolism
2.
Mol Imaging Biol ; 24(3): 425-433, 2022 06.
Article in English | MEDLINE | ID: mdl-34694528

ABSTRACT

PURPOSE: Despite unprecedented responses to immune checkpoint inhibitors and targeted therapy in melanoma, a major subset of patients progresses and have few effective salvage options. We have previously demonstrated robust, selective uptake of the peptidomimetic LLP2A labeled with Cu-64 ([64Cu]-LLP2A) for positron emission tomography (PET) imaging in subcutaneous and metastatic models of B16F10 murine melanoma. LLP2A binds with high affinity to very late antigen-4 (VLA-4, integrin α4ß1), a transmembrane protein overexpressed in melanoma and other cancers that facilitates tumor growth and metastasis. Yet B16F10 fails to faithfully reflect human melanoma biology, as it lacks certain oncogenic driver mutations, including BRAF mutations found in ≥ 50 % of clinical specimens. Here, we evaluated the PET tracer [64Cu]-CB-TE1A1P-PEG4-LLP2A ([64Cu]-LLP2A) in novel, translational BRAFV600E mutant melanoma models differing in VLA-4 expression-BPR (VLA-4-) and BPRα (VLA-4+). PROCEDURES: BPR cells were transduced with α4 (CD49d) to overexpress intact cell surface VLA-4 (BPRα). The binding affinity of [64Cu]-LLP2A to BPR and BPRα cells was determined by saturation binding assays. [64Cu]-LLP2A internalization into B16F10, BPR, and BPRα cells was quantified via a plate-based assay. Tracer biodistribution and PET/CT imaging were evaluated in mice bearing subcutaneous BPR and BPRα tumors. RESULTS: [64Cu]-LLP2A demonstrated high binding affinity to BPRα (Kd = 1.4 nM) but indeterminate binding to BPR cells. VLA-4+ BPRα and B16F10 displayed comparable time-dependent [64Cu]-LLP2A internalization, whereas BPR internalization was undetectable. PET/CT showed increased tracer uptake in BPRα tumors vs. BPR tumors in vivo, which was validated by significantly greater (p < 0.0001) BPRα tumor uptake in biodistribution analyses. CONCLUSIONS: [64Cu]-LLP2A discriminates BPRα (VLA-4+) vs. BPR (VLA-4-) melanomas in vivo, supporting translation of these BRAF-mutated melanoma models via prospective imaging and theranostic studies. These results extend the utility of LLP2A to selectively target clinically relevant and therapy-resistant tumor variants toward its use for therapeutic patient care.


Subject(s)
Integrin alpha4beta1 , Melanoma , Animals , Cell Line, Tumor , Copper Radioisotopes , Disease Models, Animal , Humans , Integrin alpha4beta1/metabolism , Melanoma/diagnostic imaging , Melanoma/genetics , Mice , Positron Emission Tomography Computed Tomography , Positron-Emission Tomography/methods , Prospective Studies , Proto-Oncogene Proteins B-raf/genetics , Tissue Distribution
3.
J Immunother Cancer ; 9(11)2021 11.
Article in English | MEDLINE | ID: mdl-34782430

ABSTRACT

BACKGROUND: A first-in-human, randomized pilot phase II clinical trial combining vaccines targeting overexpressed, non-mutated tumor blood vessel antigens (TBVA) and tyrosine kinase inhibitor dasatinib was conducted in human leukocyte antigen (HLA)-A2+ patients with advanced melanoma. METHODS: Patient monocyte-derived type-1-polarized dendritic cells were loaded with HLA-A2-presented peptides derived from TBVA (DLK1, EphA2, HBB, NRP1, RGS5, TEM1) and injected intradermally as a vaccine into the upper extremities every other week. Patients were randomized into one of two treatment arms receiving oral dasatinib (70 mg two times per day) beginning in week 5 (Arm A) or in week 1 (Arm B). Trial endpoints included T cell response to vaccine peptides (interferon-γ enzyme-linked immunosorbent spot), objective clinical response (Response Evaluation Criteria in Solid Tumors V.1.1) and exploratory tumor, blood and serum profiling of immune-associated genes/proteins. RESULTS: Sixteen patients with advanced-stage cutaneous (n=10), mucosal (n=1) or uveal (n=5) melanoma were accrued, 15 of whom had previously progressed on programmed cell death protein 1 (PD-1) blockade. Of 13 evaluable patients, 6 patients developed specific peripheral blood T cell responses against ≥3 vaccine-associated peptides, with further evidence of epitope spreading. All six patients with specific CD8+ T cell response to vaccine-targeted antigens exhibited evidence of T cell receptor (TCR) convergence in association with preferred clinical outcomes (four partial response and two stabilization of disease (SD)). Seven patients failed to respond to vaccination (one SD and six progressive disease). Patients in Arm B (immediate dasatinib) outperformed those in Arm A (delayed dasatinib) for immune response rate (IRR; 66.7% vs 28.6%), objective response rate (ORR) (66.7% vs 0%), overall survival (median 15.45 vs 3.47 months; p=0.0086) and progression-free survival (median 7.87 vs 1.97 months; p=0.063). IRR (80% vs 25%) and ORR (60% vs 12.5%) was greater for females versus male patients. Tumors in patients exhibiting response to treatment displayed (1) evidence of innate and adaptive immune-mediated inflammation and TCR convergence at baseline, (2) on-treatment transcriptional changes associated with reduced hypoxia/acidosis/glycolysis, and (3) increased inflammatory immune cell infiltration and tertiary lymphoid structure neogenesis. CONCLUSIONS: Combined vaccination against TBVA plus dasatinib was safe and resulted in coordinating immunologic and/or objective clinical responses in 6/13 (46%) evaluable patients with melanoma, particularly those initiating treatment with both agents. TRIAL REGISTRATION NUMBER: NCT01876212.


Subject(s)
Antigens, Neoplasm/therapeutic use , Antineoplastic Agents/therapeutic use , Cancer Vaccines/therapeutic use , Dasatinib/therapeutic use , Dendritic Cells/metabolism , Melanoma/drug therapy , Antineoplastic Agents/pharmacology , Cancer Vaccines/pharmacology , Dasatinib/pharmacology , Female , Humans , Male , Melanoma/pathology , Pilot Projects , Prospective Studies
4.
Front Immunol ; 12: 629519, 2021.
Article in English | MEDLINE | ID: mdl-33746966

ABSTRACT

Recent advances in immunotherapy have enabled rapid evolution of novel interventional approaches designed to reinvigorate and expand patient immune responses against cancer. An emerging approach in cancer immunology involves the conditional induction of tertiary lymphoid structures (TLS), which are non-encapsulated ectopic lymphoid structures forming at sites of chronic, pathologic inflammation. Cutaneous melanoma (CM), a highly-immunogenic form of solid cancer, continues to rise in both incidence and mortality rate, with recent reports supporting a positive correlation between the presence of TLS in melanoma and beneficial treatment outcomes amongst advanced-stage patients. In this context, TLS in CM are postulated to serve as dynamic centers for the initiation of robust anti-tumor responses within affected regions of active disease. Given their potential importance to patient outcome, significant effort has been recently devoted to gaining a better understanding of TLS neogenesis and the influence these lymphoid organs exert within the tumor microenvironment. Here, we briefly review TLS structure, function, and response to treatment in the setting of CM. To uncover potential tumor-intrinsic mechanisms that regulate TLS formation, we have taken the novel perspective of evaluating TLS induction in melanomas impacted by common driver mutations in BRAF, PTEN, NRAS, KIT, PRDM1, and MITF. Through analysis of The Cancer Genome Atlas (TCGA), we show expression of DNA repair proteins (DRPs) including BRCA1, PAXIP, ERCC1, ERCC2, ERCC3, MSH2, and PMS2 to be negatively correlated with expression of pro-TLS genes, suggesting DRP loss may favor TLS development in support of improved patient outcome and patient response to interventional immunotherapy.


Subject(s)
Biomarkers, Tumor/genetics , Melanoma/genetics , Mutation , Skin Neoplasms/genetics , Tertiary Lymphoid Structures/immunology , Animals , Humans , Immunotherapy , Melanoma/immunology , Melanoma/pathology , Melanoma/therapy , Prognosis , Skin Neoplasms/immunology , Skin Neoplasms/pathology , Skin Neoplasms/therapy , Tertiary Lymphoid Structures/pathology , Tumor Microenvironment
5.
J Immunother Cancer ; 9(2)2021 02.
Article in English | MEDLINE | ID: mdl-33526609

ABSTRACT

BACKGROUND: The degree of immune infiltration in tumors, especially CD8+ T cells, greatly impacts patient disease course and response to interventional immunotherapy. Enhancement of tumor infiltrating lymphocyte (TIL) is a critical element of efficacious therapy and one that may be achieved via administration of agents that promote tumor vascular normalization (VN) and/or induce the development of tertiary lymphoid structures (TLS) within the tumor microenvironment (TME). METHODS: Low-dose stimulator of interferon genes (STING) agonist ADU S-100 (5 µg/mouse) was delivered intratumorally to established subcutaneous B16.F10 melanomas on days 10, 14 and 17 post-tumor inoculation. Treated and control tumors were isolated at various time points to assess transcriptional changes associated with VN and TLS formation via quantitative PCR (qPCR), with corollary immune cell composition changes in isolated tissues determined using flow cytometry and immunofluorescence microscopy. In vitro assays were performed on CD11c+ BMDCs treated with 2.5 µg/mL ADU S-100 or CD11c+ DCs isolated from tumor digests and associated transcriptional changes analyzed via qPCR or profiled using DNA microarrays. For T cell repertoireß-CDR3 analyses, T cell CDR3 was sequenced from gDNA isolated from splenocytes and enzymatically digested tumors. RESULTS: We report that activation of STING within the TME leads to slowed melanoma growth in association with increased production of antiangiogenic factors including Tnfsf15 (Vegi) and Cxcl10, and TLS-inducing factors including Ccl19, Ccl21, Lta, Ltb and Light. Therapeutic responses resulting from intratumoral STING activation were characterized by improved VN, enhanced tumor infiltration by CD8+ T cells and CD11c+ DCs and local TLS neogenesis, all of which were dependent on host expression of STING. Consistent with a central role for DC in TLS formation, ADU S-100-activated mCD11c+ DCs also exhibited upregulated expression of TLS promoting factors including lymphotoxin-α (LTA), interleukin (IL)-36, inflammatory chemokines and type I interferons in vitro and in vivo. TLS formation in ADU S-100-treated mice was associated with the development of a highly oligoclonal TIL repertoire enriched in expanded T cell clonotypes unique to the TME and not detected in the periphery. CONCLUSIONS: Our data support the premise that i.t. delivery of low-dose STING agonist promotes VN and a proinflammatory TME supportive of TLS formation, enrichment in the TIL repertoire and tumor growth control.


Subject(s)
Antineoplastic Agents/pharmacology , CD8-Positive T-Lymphocytes/drug effects , Dendritic Cells/drug effects , Lymphocytes, Tumor-Infiltrating/metabolism , Melanoma, Experimental/drug therapy , Membrane Proteins/agonists , Neovascularization, Pathologic , Skin Neoplasms/drug therapy , Tertiary Lymphoid Structures/metabolism , Angiogenic Proteins/genetics , Angiogenic Proteins/metabolism , Animals , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Cytokines/genetics , Cytokines/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Female , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Melanoma, Experimental/immunology , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Signal Transduction , Skin Neoplasms/immunology , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Tertiary Lymphoid Structures/immunology , Tertiary Lymphoid Structures/pathology , Tumor Burden/drug effects , Tumor Microenvironment
6.
SLAS Discov ; 26(5): 712-729, 2021 06.
Article in English | MEDLINE | ID: mdl-33208016

ABSTRACT

We describe the development, optimization, and validation of 384-well growth inhibition assays for six patient-derived melanoma cell lines (PDMCLs), three wild type (WT) for BRAF and three with V600E-BRAF mutations. We conducted a pilot drug combination (DC) high-throughput screening (HTS) of 45 pairwise 4×4 DC matrices prepared from 10 drugs in the PDMCL assays: two B-Raf inhibitors (BRAFi), a MEK inhibitor (MEKi), and a methylation agent approved for melanoma; cytotoxic topoisomerase II and DNA methyltransferase chemotherapies; and drugs targeting the base excision DNA repair enzyme APE1 (apurinic/apyrimidinic endonuclease-1/redox effector factor-1), SRC family tyrosine kinases, the heat shock protein 90 (HSP90) molecular chaperone, and histone deacetylases.Pairwise DCs between dasatinib and three drugs approved for melanoma therapy-dabrafenib, vemurafenib, or trametinib-were flagged as synergistic in PDMCLs. Exposure to fixed DC ratios of the SRC inhibitor dasatinib with the BRAFis or MEKis interacted synergistically to increase PDMCL sensitivity to growth inhibition and enhance cytotoxicity independently of PDMCL BRAF status. These DCs synergistically inhibited the growth of mouse melanoma cell lines that either were dabrafenib-sensitive or had acquired resistance to dabrafenib with cross resistance to vemurafenib, trametinib, and dasatinib. Dasatinib DCs with dabrafenib, vemurafenib, or trametinib activated apoptosis and increased cell death in melanoma cells independently of their BRAF status or their drug resistance phenotypes. These preclinical in vitro studies provide a data-driven rationale for the further investigation of DCs between dasatinib and BRAFis or MEKis as candidates for melanoma combination therapies with the potential to improve outcomes and/or prevent or delay the emergence of disease resistance.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/drug effects , Drug Screening Assays, Antitumor/methods , High-Throughput Screening Assays/methods , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor/standards , Drug Synergism , High-Throughput Screening Assays/standards , Humans , Melanoma/drug therapy , Mice , Reproducibility of Results
7.
Carcinogenesis ; 41(5): 625-633, 2020 07 10.
Article in English | MEDLINE | ID: mdl-31245809

ABSTRACT

Circulating tumor-derived exosomes (TEX) interact with a variety of cells in cancer-bearing hosts, leading to cellular reprogramming which promotes disease progression. To study TEX effects on the development of solid tumors, immunosuppressive exosomes carrying PD-L1 and FasL were isolated from supernatants of murine or human HNSCC cell lines. TEX were delivered (IV) to immunocompetent C57BL/6 mice bearing premalignant oral/esophageal lesions induced by the carcinogen, 4-nitroquinoline 1-oxide (4NQO). Progression of the premalignant oropharyngeal lesions to malignant tumors was monitored. A single TEX injection increased the number of developing tumors (6.2 versus 3.2 in control mice injected with phosphate-buffered saline; P < 0.0002) and overall tumor burden per mouse (P < 0.037). The numbers of CD4+ and CD8+ T lymphocytes infiltrating the developing tumors were coordinately reduced (P < 0.01) in mice injected with SCCVII-derived TEX relative to controls. Notably, TEX isolated from mouse or human tumors had similar effects on tumor development and immune cells. A single IV injection of TEX was sufficient to condition mice harboring premalignant OSCC lesions for accelerated tumor progression in concert with reduced immune cell migration to the tumor.


Subject(s)
Carcinogenesis/pathology , Carcinoma, Squamous Cell/pathology , Cell Proliferation , Exosomes/pathology , Mouth Neoplasms/pathology , 4-Nitroquinoline-1-oxide/toxicity , Animals , Apoptosis , B7-H1 Antigen/metabolism , Carcinogenesis/chemically induced , Carcinogenesis/metabolism , Carcinogens/toxicity , Carcinoma, Squamous Cell/chemically induced , Carcinoma, Squamous Cell/metabolism , Exosomes/drug effects , Exosomes/metabolism , Female , Humans , Mice , Mice, Inbred C57BL , Mouth Neoplasms/chemically induced , Mouth Neoplasms/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
8.
J Control Release ; 318: 270-278, 2020 02.
Article in English | MEDLINE | ID: mdl-31866503

ABSTRACT

Melanoma is the deadliest type of skin cancer with one of the fastest increasing incidence rates among solid tumors. The use of checkpoint inhibitors (e.g. αPD-1 antibody) has recently emerged as a viable alternative to conventional modes of therapy. However, increasing evidence points towards the need for a tumor priming step to improve intratumoral immune cell infiltration. IL-12 is an immune-activating cytokine with such potential and was explored in earlier clinical trials as a highly concentrated systemic infusion. This unfortunately led to severe adverse effects. From this perspective, the localization and gradual release of such a potent immunotherapeutic agent in the tumor microenvironment is desired. This manuscript reports the use of a heparin-based complex coacervate to deliver IL-12, in which heparin-binding motifs on IL-12 allow for its effective encapsulation. IL-12-encapsulated complex coacervates significantly improved the bioactivity of IL-12 and provided protection from proteolytic cleavage in-vitro. Indeed, a single injection of IL-12 coacervate significantly inhibits the in-vivo growth of treated and untreated, contralateral tumor growth in a syngeneic B16F10 mouse melanoma model. Furthermore, tumors in mice receiving IL-12 complex coacervate treatment displayed increased infiltration by natural killer (NK) cells and CD8α+ T cells, and a decreased presence of CD4+Foxp3+ regulatory T cells. This study provides proof-of-concept data supporting the use of complex coacervates for sustained delivery of immunostimulatory proteins as an effective therapeutic strategy against disseminated tumors.


Subject(s)
Melanoma, Experimental , Skin Neoplasms , Animals , Interleukin-12 , Killer Cells, Natural , Melanoma, Experimental/drug therapy , Mice , Mice, Inbred C57BL , Skin Neoplasms/drug therapy , Tumor Microenvironment
9.
J Nucl Med ; 59(12): 1843-1849, 2018 12.
Article in English | MEDLINE | ID: mdl-29959213

ABSTRACT

Very late antigen-4 (VLA-4; also known as integrin α4ß1) is expressed at high levels in aggressive and metastatic melanoma tumors and may provide an ideal target for imaging and targeted radionuclide therapy (TRT). 177Lu-DOTA-PEG4-LLP2A (177Lu-LLP2A) is a TRT that shows high affinity for VLA-4 and high uptake in B16F10 mouse melanoma tumors in vivo. Here, we report efficacy studies of 177Lu-LLP2A, alone and combined with immune checkpoint inhibitors (ICIs) (anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies), in B16F10 tumor-bearing mice. Methods: Tumor cells (1 × 106) were implanted subcutaneously in C57BL/6 mice. After 8-10 d, the mice were randomized into 8 groups. 177Lu-LLP2A was injected intravenously on day 8 or 9 (single dose), and ICI antibodies were administered intraperitoneally in 3 doses. Tumor growth was monitored over time via calipers. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining for apoptosis was performed on fixed tumors. In a separate study, Cy3-LLP2A or Cy3-scrambled LLP2A was injected in tumor-bearing mice, and tumors were collected 4 h after injection and then analyzed by flow cytometry and immunofluorescence microscopy using different immune cell markers. Results: TRT alone showed efficacy comparable to the dual-ICI anti-PD-1 + anti-CTLA-4 or anti-PD-L1 + anti-CTLA-4, whereas TRT + ICIs significantly enhanced survival. TUNEL staining showed that the highest levels of apoptosis were in the TRT + ICI groups. In addition to targeting tumor cells, TRT also bound immune cells in the tumor microenvironment. Flow cytometry data showed that the tumors consisted of about 77% tumor cells and fibroblasts (CD45-negative/CD49d-positive) and about 23% immune cells (CD45-positive/CD49d-positive) and that immune cells expressed higher levels of VLA-4. Cy3-LLP2A and CD49d colocalized with macrophages (CD68), T cells (CD8, CD4), and B cells (CD19). Immunohistochemical analysis identified a significant colocalization of Cy3-LLP2A and CD68. Conclusion: Combination treatment with TRT + ICIs targets both tumor cells and immune cells and has potential as a therapeutic agent in patients with metastatic melanoma.


Subject(s)
Integrin alpha4beta1/antagonists & inhibitors , Lutetium/therapeutic use , Melanoma, Experimental/immunology , Melanoma, Experimental/radiotherapy , Radioimmunotherapy/methods , Radioisotopes/therapeutic use , Radiopharmaceuticals/therapeutic use , Animals , B7-H1 Antigen/antagonists & inhibitors , CTLA-4 Antigen/antagonists & inhibitors , Cell Line, Tumor , Dipeptides/chemistry , Female , Gallium Radioisotopes , Heterocyclic Compounds, 1-Ring/chemistry , Humans , Lutetium/pharmacokinetics , Male , Melanoma, Experimental/diagnostic imaging , Mice , Mice, Inbred C57BL , Molecular Targeted Therapy/methods , Phenylurea Compounds/chemistry , Polyethylene Glycols/chemistry , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Radioisotopes/pharmacokinetics , Radiopharmaceuticals/chemistry , Radiopharmaceuticals/pharmacokinetics , Radiotherapy Dosage , Tumor Protein, Translationally-Controlled 1
10.
Immunotherapy ; 8(10): 1205-18, 2016 10.
Article in English | MEDLINE | ID: mdl-27605069

ABSTRACT

Dendritic cells (DCs) are potent inducers of adaptive immunity and their clinical use in cancer vaccine formulations remains an area of active translational and clinical investigation. Although cancer vaccines applied as monotherapies have had a modest history of clinical success, there is great enthusiasm for novel therapeutic strategies combining DC-based cancer vaccines with agents that 'normalize' immune function in the tumor microenvironment (TME). Broadly, these combination vaccines are designed to antagonize/remove immunosuppressive networks within the TME that serve to limit the antitumor action of vaccine-induced T cells and/or to condition the TME to facilitate the recruitment and optimal function and durability of vaccine-induced T cells. Such combination regimens are expected to dramatically enhance the clinical potency of DC-based cancer vaccine platforms.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Cancer Vaccines/immunology , Dendritic Cells/transplantation , Immunotherapy/methods , Neoplasms/therapy , T-Lymphocytes/immunology , Animals , Combined Modality Therapy , Costimulatory and Inhibitory T-Cell Receptors/immunology , Dendritic Cells/immunology , Glucocorticoid-Induced TNFR-Related Protein/immunology , Humans , Lymphocyte Activation , Monocytes/immunology , Neoplasms/immunology , Tumor Microenvironment
12.
Oncoimmunology ; 5(6): e1146841, 2016 Jun.
Article in English | MEDLINE | ID: mdl-27471613

ABSTRACT

Dendritic cells (DC) play a pivotal role in the induction and regulation of immune responses. In cancer, DC-based vaccines have proven to be safe and to elicit protective and therapeutic immunological responses. Recently, we showed that specific mTORC2 (mechanistic target of rapamycin complex 2) deficiency in DC enhances their ability to promote Th1 and Th17 responses after LPS stimulation. In the present study, bone marrow-derived mTORC2-deficient (Rictor(-/-)) DC were evaluated as a therapeutic modality in the murine B16 melanoma model. Consistent with their pro-inflammatory profile (enhanced IL-12p70 production and low PD-L1 expression versus control DC), intratumoral (i.t.) injection of LPS-activated Rictor(-/-) DC slowed B16 melanoma growth markedly in WT C57BL/6 recipient mice. This antitumor effect was abrogated when Rictor(-/-) DC were injected i.t. into B16-bearing Rag(-/-) mice, and also after selective CD8(+) T cell depletion in wild-type hosts in vivo, indicating that CD8(+) T cells were the principal regulators of tumor growth after Rictor(-/-) DC injection. I.t. administration of Rictor(-/-) DC also reduced the frequency of myeloid-derived suppressor cells within tumors, and enhanced numbers of IFNγ(+) and granzyme-B(+) cytotoxic CD8(+) T cells both in the spleens and tumors of treated animals. These data suggest that selective inhibition of mTORC2 activity in activated DC augments their pro-inflammatory and T cell stimulatory profile, in association with their enhanced capacity to promote protective CD8(+) T cell responses in vivo, leading to slowed B16 melanoma progression. These novel findings may contribute to the design of more effective DC-based vaccines for cancer immunotherapy.

13.
mBio ; 7(3)2016 05 31.
Article in English | MEDLINE | ID: mdl-27247230

ABSTRACT

UNLABELLED: Curing HIV-1 infection will require elimination of persistent cellular reservoirs that harbor latent virus in the face of combination antiretroviral therapy (cART). Proposed immunotherapeutic strategies to cure HIV-1 infection include enhancing lysis of these infected cells by cytotoxic T lymphocytes (CTL). A major challenge in this strategy is overcoming viral immune escape variants that have evaded host immune control. Here we report that naive CD8(+) T cells from chronic HIV-1-infected participants on long-term cART can be primed by dendritic cells (DC). These DC must be mature, produce high levels of interleukin 12p70 (IL-12p70), be responsive to CD40 ligand (CD40L), and be loaded with inactivated, autologous HIV-1. These DC-primed CD8(+) T cell responders produced high levels of gamma interferon (IFN-γ) in response to a broad range of both conserved and variable regions of Gag and effectively killed CD4(+) T cell targets that were either infected with the autologous latent reservoir-associated virus or loaded with autologous Gag peptides. In contrast, HIV-1-specific memory CD8(+) T cells stimulated with autologous HIV-1-loaded DC produced IFN-γ in response to a narrow range of conserved and variable Gag peptides compared to the primed T cells and most notably, displayed significantly lower cytolytic function. Our findings highlight the need to selectively induce new HIV-1-specific CTL from naive precursors while avoiding activation of existing, dysfunctional memory T cells in potential curative immunotherapeutic strategies for HIV-1 infection. IMPORTANCE: Current immunotherapeutic approaches aim to enhance antiviral immunity against the HIV-1 reservoir; however, it has yet to be shown whether T cells from persons on cART can recognize and eliminate virus-infected cells. We show that in persons on cART a personalized medicine approach using their dendritic cells to stimulate their naive T cells induces potent effector CTL in vitro that recognize and eradicate HIV-1-infected CD4(+) T cells. Additionally, we show that the same stimulation of existing memory T cells results in cytokine secretion but limited effector function. Our study demonstrates that the naive T cell repertoire can recognize persistent HIV-1 during cART and supports immunotherapy strategies for an HIV-1 cure that targets naive T cells, rather than existing, dysfunctional, memory T cells.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , HIV Infections/immunology , HIV-1/immunology , Lymphocyte Activation , T-Lymphocytes, Cytotoxic/immunology , Antiretroviral Therapy, Highly Active , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , CD40 Ligand/immunology , Cytotoxicity, Immunologic , Dendritic Cells/immunology , Dendritic Cells/virology , Gene Products, gag/immunology , HIV Infections/drug therapy , Humans , Immunologic Memory , Interferon-gamma/biosynthesis , Interferon-gamma/immunology , Interleukin-12/biosynthesis , Interleukin-12/immunology
14.
J Immunol ; 194(3): 1047-56, 2015 Feb 01.
Article in English | MEDLINE | ID: mdl-25548234

ABSTRACT

The ability of dendritic cells (DC) to mediate CD4(+) T cell help for cellular immunity is guided by instructive signals received during DC maturation, as well as the resulting pattern of DC responsiveness to the Th signal, CD40L. Furthermore, the professional transfer of antigenic information from migratory DC to lymph node-residing DC is critical for the effective induction of cellular immune responses. In this study we report that, in addition to their enhanced IL-12p70 producing capacity, human DC matured in the presence of inflammatory mediators of type 1 immunity are uniquely programmed to form networks of tunneling nanotube-like structures in response to CD40L-expressing Th cells or rCD40L. This immunologic process of DC reticulation facilitates intercellular trafficking of endosome-associated vesicles and Ag, but also pathogens such HIV-1, and is regulated by the opposing roles of IFN-γ and IL-4. The initiation of DC reticulation represents a novel helper function of CD40L and a superior mechanism of intercellular communication possessed by type 1 polarized DC, as well as a target for exploitation by pathogens to enhance direct cell-to-cell spread.


Subject(s)
CD40 Ligand/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Biological Transport , CD40 Ligand/pharmacology , Cell Communication , Cells, Cultured , Coculture Techniques , Dendritic Cells/drug effects , Dendritic Cells/microbiology , Dendritic Cells/virology , Humans , Inflammation Mediators/metabolism , Lymphocyte Activation/immunology , Signal Transduction , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/metabolism
15.
J Immunol ; 191(5): 2570-80, 2013 Sep 01.
Article in English | MEDLINE | ID: mdl-23913962

ABSTRACT

The ability of HIV-1 to rapidly accumulate mutations provides the virus with an effective means of escaping CD8(+) CTL responses. In this study, we describe how subtle alterations in CTL epitopes expressed by naturally occurring HIV-1 variants can result in an incomplete escape from CTL recognition, providing the virus with a selective advantage. Rather than paralyzing the CTL response, these epitope modifications selectively induce the CTL to produce proinflammatory cytokines in the absence of target killing. Importantly, instead of dampening the immune response through CTL elimination of variant Ag-expressing immature dendritic cells (DC), a positive CTL-to-DC immune feedback loop dominates whereby the immature DC differentiate into mature proinflammatory DC. Moreover, these CTL-programmed DC exhibit a superior capacity to mediate HIV-1 trans-infection of T cells. This discordant induction of CTL helper activity in the absence of killing most likely contributes to the chronic immune activation associated with HIV-1 infection, and can be used by HIV-1 to promote viral dissemination and persistence. Our findings highlight the need to address the detrimental potential of eliciting dysfunctional cross-reactive memory CTL responses when designing and implementing anti-HIV-1 immunotherapies.


Subject(s)
Dendritic Cells/immunology , Epitopes, T-Lymphocyte/immunology , HIV Infections/immunology , HIV-1/immunology , T-Lymphocytes, Cytotoxic/immunology , Coculture Techniques , Flow Cytometry , Humans , Lymphocyte Activation/immunology , Microscopy, Electron, Scanning , T-Lymphocytes, Cytotoxic/virology
16.
Immunobiology ; 215(12): 941-8, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20356650

ABSTRACT

IgE-mediated food allergies are an important cause of life-threatening hypersensitivity reactions. Orally administered peanut antigens mixed with the mucosal adjuvant cholera toxin (CT) induce a strong peanut extract (PE)-specific serum IgE response that is correlated with T-helper type 1 (Th1) and type 2 (Th2)-like T-cell responses. This study was conducted to determine if respiratory enteric orphan virus (reovirus), a non-pathogenic virus that induces robust Th1-mediated mucosal and systemic responses could modulate induction of PE-specific allergic responses when co-administered with PE. Young mice were orally exposed to PE mixed with CT, reovirus, or both CT and reovirus. As expected, CT promoted PE-specific serum IgE, IgG1, and IgG2a and intestinal IgA production as well as splenic Th1- and Th2-associated cytokine recall responses. Reovirus did not alter PE-specific serum IgE and IgG1 levels, but substantially increased the PE-specific IgG2a response when co-administered with PE with or without CT. Additionally, reovirus significantly decreased the percentage of the Peyer's patch CD8+ T-cells and Foxp3+CD4+ T-regulatory cells when co-administered with PE. These results demonstrate that an acute mucosal reovirus infection and subsequent Th1 immune response is capable of modulating the Th1/Th2 controlled humoral response to PE. The reovirus-mediated increase in the PE-specific IgG2a antibody response may have therapeutic implications as increased levels of non-allergenic PE-specific IgG2a could block PE antigens from binding to IgE-sensitized mast cells.


Subject(s)
Arachis/immunology , Immunoglobulin G/immunology , Peanut Hypersensitivity/immunology , Reoviridae Infections/immunology , Animals , Arachis/chemistry , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/microbiology , Cell Line , Cells, Cultured , Cholera Toxin/immunology , Cholera Toxin/pharmacology , Cytokines/immunology , Cytokines/metabolism , Disease Models, Animal , Female , Flow Cytometry , Humans , Immunoglobulin A/immunology , Intestinal Mucosa/drug effects , Intestinal Mucosa/immunology , Intestinal Mucosa/virology , Intestine, Small/drug effects , Intestine, Small/immunology , Intestine, Small/virology , Mice , Mice, Inbred C3H , Peyer's Patches/immunology , Peyer's Patches/virology , Plant Extracts/immunology , Plant Extracts/pharmacology , Spleen/cytology , Spleen/drug effects , Spleen/metabolism , Th1 Cells/drug effects , Th1 Cells/immunology , Tissue Culture Techniques
17.
J Immunol Methods ; 314(1-2): 30-7, 2006 Jul 31.
Article in English | MEDLINE | ID: mdl-16822520

ABSTRACT

Respiratory enteric orphan virus (reovirus) has been used to study many aspects of the biology and genetics of viruses, viral infection, pathogenesis, and the immune response to virus infection. This report describes the functional activity of virus labeled with Alexa Fluor 488, a stable fluorescent dye. Matrix assisted laser desorption-time of flight analysis indicated that Alexa Fluor 488 labeled the outer capsid proteins of reovirus. Labeled virus bound to murine L929 fibroblasts as determined by flow cytometry and fluorescence microscopy, and the specificity of binding were demonstrated by competitive inhibition with non-labeled virus. Labeled reovirus induced apoptosis and cytopathic effect in infected L929 cells. Mice infected with labeled virus mounted robust serum antibody and CD8(+) T-cell responses, indicating that labeled virus retained immunogenicity in vivo. These results indicate that Alexa Fluor 488-labeled virus provides a powerful new tool to analyze reovirus infection in vitro and in vivo.


Subject(s)
Mammalian orthoreovirus 3/chemistry , Reoviridae Infections/immunology , Staining and Labeling/methods , Succinimides/chemistry , Virion/chemistry , Animals , Capsid Proteins/chemistry , Fluorescent Dyes/chemistry , Male , Mammalian orthoreovirus 3/immunology , Mammalian orthoreovirus 3/pathogenicity , Mice , Mice, Inbred C3H , Mice, Inbred C57BL
SELECTION OF CITATIONS
SEARCH DETAIL
...