Intratumoral expression of IL-12 from lentiviral or RNA vectors acts synergistically with TLR4 agonist (GLA) to generate anti-tumor immunological memory

Systemic interleukin-12 (IL12) anti-tumor therapy is highly potent but has had limited utility in the clinic due to severe toxicity. Here, we present two IL12-expressing vector platforms, both of which can overcome the deficiencies of previous systemic IL12 therapies: 1) an integrating lentiviral vector, and 2) a self-replicating messenger RNA formulated with polyethyleneimine. Intratumoral administration of either IL12 vector platform resulted in recruitment of immune cells, including effector T cells and dendritic cells, and the complete remission of established tumors in multiple murine models. Furthermore, concurrent intratumoral administration of the synthetic TLR4 agonist glucopyranosyl lipid A formulated in a stable emulsion (GLA-SE) induced systemic memory T cell responses that mediated complete protection against tumor rechallenge in all survivor mice (8/8 rechallenged mice), whereas only 2/6 total rechallenged mice treated with intratrumoral IL12 monotherapy rejected the rechallenge. Taken together, expression of vectorized IL12 in combination with a TLR4 agonist represents a varied approach to broaden the applicability of intratumoral immune therapies of solid tumors.

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Influenza Virus Vaccination Induces Interleukin-12/23 Receptor β1 (IL-12/23Rβ1)-Independent Production of Gamma Interferon (IFN-γ) and Humoral Immunity in Patients with Genetic Deficiencies in IL-12/23Rβ1 or IFN-γ Receptor I

Clinical and Vaccine Immunology ◽

10.1128/cvi.00090-08 ◽

2008 ◽

Vol 15 (8) ◽

pp. 1171-1175 ◽

Cited By ~ 8

Author(s):

Tjitske de Boer ◽

Jaap T. van Dissel ◽

Taco W. J. Kuijpers ◽

Guus F. Rimmelzwaan ◽

Frank P. Kroon ◽

...

Keyword(s):

Influenza Virus ◽

T Cell ◽

Seasonal Influenza ◽

Gamma Interferon ◽

Interleukin 12 ◽

Virus Vaccination ◽

Cell Responses ◽

Antibody Titers ◽

Ifn Γ ◽

Striking Contrast

ABSTRACT To investigate whether protective immune responses can be induced in the absence of normal interleukin-12/23/gamma interferon (IL-12/23/IFN-γ) axis signaling, we vaccinated with the seasonal influenza virus subunit vaccine two patients with complete IL-12/23 receptor β1 (IL-12/23Rβ1) deficiencies, two patients with partial IFN-γ receptor I (pIFN-γRI) deficiencies, and five healthy controls. Blood samples were analyzed before, 7 days after, and 28 days after vaccination. In most cases, antibody titers reached protective levels. Moreover, although T-cell responses in patients were lower than those observed in controls, significant influenza virus-specific T-cell proliferation, IFN-γ production, and numbers of IFN-γ-producing cells were found in all patients 7 days after the vaccination. Interestingly, influenza virus-specific IFN-γ responses were IL-12/23 independent, in striking contrast to mycobacterium-induced IFN-γ production. In conclusion, influenza virus vaccination induces IL-12/23-independent IFN-γ production by T cells and can result in sufficient humoral protection in both IL-12/23Rβ1- and pIFN-γRI-deficient individuals.

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Effects of Interleukin-12 and Interleukin-15 on Measles-Specific T-Cell Responses in Vaccinated Infants

Viral Immunology ◽

10.1089/vim.2007.0113 ◽

2008 ◽

Vol 21 (2) ◽

pp. 163-172 ◽

Cited By ~ 12

Author(s):

Hayley A. Gans ◽

Linda L. Yasukawa ◽

Cathryn Z. Zhang ◽

Rima Hanna Wakim ◽

Mary Rinki ◽

...

Keyword(s):

T Cell ◽

T Cell Responses ◽

Interleukin 12 ◽

Cell Responses ◽

Interleukin 15

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Incorporation of the TLR4 Agonist Monophosphoryl Lipid A Into the Bilayer of DDA/TDB Liposomes: Physico-Chemical Characterization and Induction of CD8+ T-Cell Responses In Vivo

Pharmaceutical Research ◽

10.1007/s11095-010-0301-9 ◽

2010 ◽

Vol 28 (3) ◽

pp. 553-562 ◽

Cited By ~ 33

Author(s):

Pernille Nordly ◽

Else Marie Agger ◽

Peter Andersen ◽

Hanne Mørck Nielsen ◽

Camilla Foged

Keyword(s):

T Cell ◽

Lipid A ◽

Chemical Characterization ◽

Cd8 T Cell ◽

Cell Responses ◽

Monophosphoryl Lipid A ◽

Monophosphoryl Lipid ◽

Physico Chemical ◽

Tlr4 Agonist

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m6A demethylase ALKBH5 controls CD4+ T cell pathogenicity and promotes autoimmunity

Science Advances ◽

10.1126/sciadv.abg0470 ◽

2021 ◽

Vol 7 (25) ◽

pp. eabg0470

Author(s):

Jing Zhou ◽

Xingli Zhang ◽

Jiajia Hu ◽

Rihao Qu ◽

Zhibin Yu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Messenger Rna ◽

Interferon Γ ◽

Autoimmune Encephalomyelitis ◽

Cell Responses ◽

Chemokine Ligand ◽

The Central Nervous System ◽

Chemokine Ligand 2

N6-methyladenosine (m6A) modification is dynamically regulated by “writer” and “eraser” enzymes. m6A “writers” have been shown to ensure the homeostasis of CD4+ T cells, but the “erasers” functioning in T cells is poorly understood. Here, we reported that m6A eraser AlkB homolog 5 (ALKBH5), but not FTO, maintains the ability of naïve CD4+ T cells to induce adoptive transfer colitis. In addition, T cell–specific ablation of ALKBH5 confers protection against experimental autoimmune encephalomyelitis. During the induced neuroinflammation, ALKBH5 deficiency increased m6A modification on interferon-γ and C-X-C motif chemokine ligand 2 messenger RNA (mRNA), thus decreasing their mRNA stability and protein expression in CD4+ T cells. These modifications resulted in attenuated CD4+ T cell responses and diminished recruitment of neutrophils into the central nervous system. Our findings reveal an unexpected specific role of ALKBH5 as an m6A eraser in controlling the pathogenicity of CD4+ T cells during autoimmunity.

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SARS-CoV-2 mRNA vaccines induce a robust germinal centre reaction in humans

10.21203/rs.3.rs-310773/v1 ◽

2021 ◽

Cited By ~ 2

Author(s):

Ali Ellebedy ◽

Jackson Turner ◽

Jane O'Halloran ◽

Elizaveta Kalaidina ◽

Wooseob Kim ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Messenger Rna ◽

Neutralizing Antibodies ◽

Germinal Centre ◽

S Protein ◽

Fine Needle Aspirates ◽

Cell Responses ◽

Cell Clones ◽

Shared Epitopes

Abstract Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) messenger RNA (mRNA)-based vaccines are ~95% effective in preventing coronavirus disease 2019. However, the dynamics of antibody secreting plasmablasts (PBs) and germinal centre (GC) B cells induced by these vaccines in SARS-CoV-2 naïve and antigen-experienced humans remains unclear. Here we examined peripheral blood and/or lymph node (LN) antigen-specific B cell responses in 32 individuals who received two doses of BNT162b2, an mRNA-based vaccine encoding the full-length SARS-CoV-2 spike (S) gene. Circulating IgG- and IgA-secreting PBs targeting the S protein peaked one week after the second immunization then declined and were undetectable three weeks later. PB responses coincided with maximal levels of serum anti-S binding and neutralizing antibodies to a historical strain as well as emerging variants, especially in individuals previously infected with SARS-CoV-2, who produced the most robust serological responses. Fine needle aspirates of draining axillary LNs identified GC B cells that bind S protein in all participants sampled after primary immunization. GC responses increased after boosting and were detectable in two distinct LNs in several participants. Remarkably, high frequencies of S-binding GC B cells and PBs were maintained in draining LNs for up to seven weeks after first immunization, with a substantial fraction of the PB pool class-switched to IgA. GC B cell-derived monoclonal antibodies predominantly targeted the RBD, with fewer clones binding to the N-terminal domain or shared epitopes within the S proteins of human betacoronaviruses OC43 and HKU1. Our studies demonstrate that SARS-CoV-2 mRNA-based vaccination of humans induces a robust and persistent GC B cell response that engages pre-existing as well as new B cell clones, which enables generation of high-affinity, broad, and durable humoral immunity.

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A "prime-pull" immunotherapy approach using a lentiviral vector and intratumoral TLR4 agonist redirects cytotoxic T cells

Journal for ImmunoTherapy of Cancer ◽

10.1186/2051-1426-2-s3-p190 ◽

2014 ◽

Vol 2 (S3) ◽

Author(s):

Tina C Albershardt ◽

Andrea J Parsons ◽

Patrick Flynn ◽

Peter Berglund ◽

Jan ter Meulen

Keyword(s):

T Cells ◽

Lentiviral Vector ◽

Cytotoxic T Cells ◽

Tlr4 Agonist

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Mitoxantrone-Loaded Nanoparticles for Magnetically Controlled Tumor Therapy–Induction of Tumor Cell Death, Release of Danger Signals and Activation of Immune Cells

Pharmaceutics ◽

10.3390/pharmaceutics12100923 ◽

2020 ◽

Vol 12 (10) ◽

pp. 923

Author(s):

Teresa Ratschker ◽

Laura Egenberger ◽

Magdalena Alev ◽

Lisa Zschiesche ◽

Julia Band ◽

...

Keyword(s):

Cell Death ◽

Immune System ◽

Immune Cells ◽

Checkpoint Inhibitors ◽

Tumor Therapy ◽

Superparamagnetic Iron Oxide ◽

Dependent Manner ◽

Tumor Cell Death ◽

Immune Therapies

Stimulating the patient’s immune system represents a promising therapeutic strategy to fight cancer. However, low immunogenicity of the tumor cells within an immune suppressive milieu often leads to weak anti-tumor immune responses. Additionally, the immune system may be impaired by accompanying aggressive chemotherapies. We show that mitoxantrone, bound to superparamagnetic iron oxide nanoparticles (SPIONs) as the transport system, can be magnetically accumulated in adherent HT-29 colon carcinoma cells, thereby inducing the same cell death phenotype as its soluble counterpart, a chemotherapeutic agent and prototypic inductor of immunogenic cell death. The nanoparticle-loaded drug induces cell cycle stop, apoptosis and secondary necrosis in a dose- and time-dependent manner comparable to the free drug. Cell death was accompanied by the release of interleukin-8 and damage-associated molecular patterns (DAMPs) such as HSP70 and ATP, which fostered chemotactic migration of monocytes and maturation of dendritic cells. We furthermore ensured absence of endotoxin contaminations and compatibility with erythrocytes and platelets and investigated the influence on plasma coagulation in vitro. Summarizing, with magnetic enrichment, mitoxantrone can be accumulated at the desired place, sparing healthy peripheral cells and tissues, such as immune cells. Conserving immune competence in cancer patients in the future might allow combined therapeutic approaches with immune therapies (e.g., checkpoint inhibitors).

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Epigenetic Control of C/EBPa by Noncoding RNAs.

Blood ◽

10.1182/blood.v114.22.3644.3644 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3644-3644

Author(s):

Annalisa Di Ruscio ◽

Alexander K Ebralidze ◽

Francesco D'Alò ◽

Maria Teresa Voso ◽

Giuseppe Leone ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Messenger Rna ◽

Noncoding Rna ◽

Gene Locus ◽

Conflicts Of Interest ◽

Lentiviral Vector ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Factor C

Abstract Abstract 3644 Poster Board III-580 Little is currently known about the role of noncoding RNA transcripts (ncRNA) in gene regulation; although most, and perhaps all, gene loci express such transcripts. Our previous results with the PU.1 gene locus showed a shared transcription factor complex and chromatin configuration requirements for biogenesis of both messenger and ncRNAs. These ncRNAs were localized within the nuclear and cytoplasmic compartments. Disrupting ncRNAs in the cytoplasmic cellular fraction results in increased PU.1 mRNA and protein. Recently, we have focused on the C/EBPa gene locus and observed extensive noncoding transcription. The transcription factor C/EBPa plays a pivotal role in hematopoietic stem cell (HSC) commitment and differentiation. Expression of the C/EBPa gene is tightly regulated during normal hematopoietic development, and dysregulation of C/EBPa expression can lead to lung cancer and leukemia. However, little is known about how the C/EBPa gene is regulated in vivo. In this study, we characterize ncRNAs derived from the C/EBPa locus and demonstrate their functional role in regulation of C/EBPa gene expression. First, northern blot analysis and RT PCR determined a predominantly nuclear localization of the C/EBPa ncRNAs. Second, strand-specific quantitative RT PCR demonstrated a concordant expression of coding and noncoding C/EBPa transcripts. Next, we investigated the results of ablation of ncRNAs using a lentiviral vector containing ncRNA-targeting shRNAs on the expression of the C/EBPa gene. We have observed that reduced levels of ncRNAs leads to a significant downregulation of the expression of coding messenger RNA. These data strongly suggest that C/EBPa ncRNAs play an important role in maintaining optimal expression of the C/EBPa gene at different stages of hematopoiesis and makes targeting noncoding transcripts a novel and attractive tool in correcting aberrant gene expression levels. Disclosures: No relevant conflicts of interest to declare.

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