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1.
Immunity ; 55(3): 442-458.e8, 2022 03 08.
Article in English | MEDLINE | ID: mdl-35182483

ABSTRACT

Consecutive exposures to different pathogens are highly prevalent and often alter the host immune response. However, it remains unknown how a secondary bacterial infection affects an ongoing adaptive immune response elicited against primary invading pathogens. We demonstrated that recruitment of Sca-1+ monocytes into lymphoid organs during Salmonella Typhimurium (STm) infection disrupted pre-existing germinal center (GC) reactions. GC responses induced by influenza, plasmodium, or commensals deteriorated following STm infection. GC disruption was independent of the direct bacterial interactions with B cells and instead was induced through recruitment of CCR2-dependent Sca-1+ monocytes into the lymphoid organs. GC collapse was associated with impaired cellular respiration and was dependent on TNFα and IFNƎĀ³, the latter of which was essential for Sca-1+ monocyte differentiation. Monocyte recruitment and GC disruption also occurred during LPS-supplemented vaccination and Listeria monocytogenes infection. Thus, systemic activation of the innate immune response upon severe bacterial infection is induced at the expense of antibody-mediated immunity.


Subject(s)
Bacterial Infections , Listeriosis , B-Lymphocytes , Germinal Center , Humans , Monocytes
2.
Gene Ther ; 31(7-8): 378-390, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38684788

ABSTRACT

Allogeneic cell therapies, such as those involving macrophages or Natural Killer (NK) cells, are of increasing interest for cancer immunotherapy. However, the current techniques for genetically modifying these cell types using lenti- or gamma-retroviral vectors present challenges, such as required cell pre-activation and inefficiency in transduction, which hinder the assessment of preclinical efficacy and clinical translation. In our study, we describe a novel lentiviral pseudotype based on the Koala Retrovirus (KoRV) envelope protein, which we identified based on homology to existing pseudotypes used in cell therapy. Unlike other pseudotyped viral vectors, this KoRV-based envelope demonstrates remarkable efficiency in transducing freshly isolated primary human NK cells directly from blood, as well as freshly obtained monocytes, which were differentiated to M1 macrophages as well as B cells from multiple donors, achieving up to 80% reporter gene expression within three days post-transduction. Importantly, KoRV-based transduction does not compromise the expression of crucial immune cell receptors, nor does it impair immune cell functionality, including NK cell viability, proliferation, cytotoxicity as well as phagocytosis of differentiated macrophages. Preserving immune cell functionality is pivotal for the success of cell-based therapeutics in treating various malignancies. By achieving high transduction rates of freshly isolated immune cells before expansion, our approach enables a streamlined and cost-effective automated production of off-the-shelf cell therapeutics, requiring fewer viral particles and less manufacturing steps. This breakthrough holds the potential to significantly reduce the time and resources required for producing e.g. NK cell therapeutics, expediting their availability to patients in need.


Subject(s)
Genetic Vectors , Killer Cells, Natural , Lentivirus , Macrophages , Transduction, Genetic , Humans , Genetic Vectors/genetics , Killer Cells, Natural/immunology , Lentivirus/genetics , Transduction, Genetic/methods , Macrophages/immunology , Macrophages/metabolism , Gene Transfer Techniques , Monocytes/immunology , Monocytes/metabolism , Genetic Therapy/methods , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Animals
3.
Int J Mol Sci ; 24(4)2023 Feb 04.
Article in English | MEDLINE | ID: mdl-36834542

ABSTRACT

Natural killer (NK) cells are a subset of lymphocytes that offer great potential for cancer immunotherapy due to their natural anti-tumor activity and the possibility to safely transplant cells from healthy donors to patients in a clinical setting. However, the efficacy of cell-based immunotherapies using both T and NK cells is often limited by a poor infiltration of immune cells into solid tumors. Importantly, regulatory immune cell subsets are frequently recruited to tumor sites. In this study, we overexpressed two chemokine receptors, CCR4 and CCR2B, that are naturally found on T regulatory cells and tumor-resident monocytes, respectively, on NK cells. Using the NK cell line NK-92 as well as primary NK cells from peripheral blood, we show that genetically engineered NK cells can be efficiently redirected using chemokine receptors from different immune cell lineages and migrate towards chemokines such as CCL22 or CCL2, without impairing the natural effector functions. This approach has the potential to enhance the therapeutic effect of immunotherapies in solid tumors by directing genetically engineered donor NK cells to tumor sites. As a future therapeutic option, the natural anti-tumor activity of NK cells at the tumor sites can be increased by co-expression of chemokine receptors with chimeric antigen receptors (CAR) or T cell receptors (TCR) on NK cells can be performed in the future.


Subject(s)
Neoplasms , Receptors, Chimeric Antigen , Humans , Immunotherapy, Adoptive , Killer Cells, Natural , Neoplasms/pathology , Receptors, Antigen, T-Cell/metabolism , Receptors, CCR4/metabolism , Receptors, Chemokine/metabolism , Receptors, Chimeric Antigen/metabolism , Receptors, CCR2
4.
Pharmacol Res ; 158: 104682, 2020 08.
Article in English | MEDLINE | ID: mdl-32035162

ABSTRACT

Advanced systemic mastocytosis is a rare and still untreatable disease. Blocking antibodies against inhibitory receptors, also known as "immune checkpoints", have revolutionized anti-cancer treatment. Inhibitory receptors are expressed not only on normal immune cells, including mast cells but also on neoplastic cells. Whether activation of inhibitory receptors through monoclonal antibodies can lead to tumor growth inhibition remains mostly unknown. Here we show that the inhibitory receptor Siglec-7 is expressed by primary neoplastic mast cells in patients with systemic mastocytosis and by mast cell leukemia cell lines. Activation of Siglec-7 by anti-Siglec-7 monoclonal antibody caused phosphorylation of Src homology region 2 domain-containing phosphatase-1 (SHP-1), reduced phosphorylation of KIT and induced growth inhibition in mast cell lines. In SCID-beige mice injected with either the human mast cell line HMC-1.1 and HMC-1.2 or with Siglec-7 transduced B cell lymphoma cells, anti-Siglec-7 monoclonal antibody reduced tumor growth by a mechanism involving Siglec-7 cytoplasmic domains in "preventive" and "treatment" settings. These data demonstrate that activation of Siglec-7 on mast cell lines can inhibit their growth in vitro and in vivo. This might pave the way to additional treatment strategies for mastocytosis.


Subject(s)
Lectins/agonists , Leukemia, Mast-Cell/drug therapy , Adult , Aged , Aged, 80 and over , Animals , Antibodies, Monoclonal/therapeutic use , Antigens, Differentiation, Myelomonocytic , Cell Line, Tumor , Cell Survival/drug effects , Female , Genes, src/drug effects , Humans , Leukemia, Mast-Cell/pathology , Lymphoma, B-Cell/drug therapy , Lymphoma, B-Cell/pathology , Male , Mastocytosis/drug therapy , Mice , Mice, SCID , Middle Aged , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 6/drug effects , Proto-Oncogene Proteins c-kit/drug effects , Xenograft Model Antitumor Assays
5.
J Immunol ; 198(9): 3662-3670, 2017 05 01.
Article in English | MEDLINE | ID: mdl-28356383

ABSTRACT

NK cells are part of the innate immune system, and are able to identify and kill hazardous cells. The discrimination between normal and hazardous cells is possible due to an array of inhibitory and activating receptors. NKG2D is one of the prominent activating receptors expressed by all human NK cells. This receptor binds stress-induced ligands, including human MICA, MICB, and UL16-binding proteins 1-6. The interaction between NKG2D and its ligands facilitates the elimination of cells under cellular stress, such as tumor transformation. However, the mechanisms regulating the expression of these ligands are still not well understood. Under normal conditions, the NKG2D ligands were shown to be posttranscriptionally regulated by cellular microRNAs and RNA-binding proteins (RBPs). Thus far, only the 3' untranslated regions (UTRs) of MICA, MICB, and UL16-binding protein 2 were shown to be regulated by RBPs and microRNAs, usually resulting in their downregulation. In this study we investigated whether MICB expression is controlled by RBPs through its 5'UTR. We used an RNA pull-down assay followed by mass spectrometry and identified vigilin, a ubiquitously expressed multifunctional RNA-binding protein. We demonstrated that vigilin binds and negatively regulates MICB expression through its 5'UTR. Additionally, vigilin downregulation in target cells led to a significant increase in NK cell activation against said target cells. Taken together, we have discovered a novel mode of MICB regulation.


Subject(s)
Histocompatibility Antigens Class I/metabolism , Immunologic Surveillance , Killer Cells, Natural/immunology , RNA-Binding Proteins/metabolism , Stress, Physiological/immunology , 5' Untranslated Regions/genetics , Cell Line, Tumor , Gene Expression Regulation/genetics , Histocompatibility Antigens Class I/genetics , Humans , Ligands , Lymphocyte Activation , NK Cell Lectin-Like Receptor Subfamily K/agonists , Protein Binding , RNA, Small Interfering/genetics , RNA-Binding Proteins/genetics
6.
J Virol ; 90(21): 9608-9617, 2016 Nov 01.
Article in English | MEDLINE | ID: mdl-27535049

ABSTRACT

The Herpesviridae family consists of eight viruses, most of which infect a majority of the human population. One of the less-studied members is human herpesvirus 6 (HHV-6) (Roseolovirus), which causes a mild, well-characterized childhood disease. Primary HHV-6 infection is followed by lifelong latency. Reactivation frequently occurs in immunocompromised patients, such as those suffering from HIV infection or cancer or following transplantation, and causes potentially life-threatening complications. In this study, we investigated the mechanisms that HHV-6 utilizes to remain undetected by natural killer (NK) cells, which are key participants in the innate immune response to infections. We revealed viral mechanisms which downregulate ligands for two powerful activating NK cell receptors: ULBP1, ULBP3, and MICB, which trigger NKG2D, and B7-H6, which activates NKp30. Accordingly, this downregulation impaired the ability of NK cells to recognize HHV-6-infected cells. Thus, we describe for the first time immune evasion mechanisms of HHV-6 that protect lytically infected cells from NK elimination. IMPORTANCE: Human herpesvirus 6 (HHV-6) latently infects a large portion of the human population and can reactivate in humans lacking a functional immune system, such as cancer or AIDS patients. Under these conditions, it can cause life-threatening diseases. To date, the actions and interplay of immune cells, and particularly cells of the innate immune system, during HHV-6 infection are poorly defined. In this study, we aimed to understand how cells undergoing lytic HHV-6 infection interact with natural killer (NK) cells, innate lymphocytes constituting the first line of defense against viral intruders. We show that HHV-6 suppresses the expression of surface proteins that alert the immune cells by triggering two major receptors on NK cells, NKG2D and NKp30. As a consequence, HHV-6 can replicate undetected by the innate immune system and potentially spread infection throughout the body. This study advances the understanding of HHV-6 biology and the measures it uses to successfully escape immune elimination.


Subject(s)
Down-Regulation/immunology , Herpesviridae Infections/immunology , Herpesvirus 6, Human/immunology , Killer Cells, Natural/immunology , Cell Line , HIV Infections/immunology , Humans , Immune Evasion/immunology , Immunity, Innate/immunology , Ligands , Receptors, Natural Killer Cell/immunology , Virus Physiological Phenomena/immunology
7.
Mol Ther ; 23(4): 648-55, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25578618

ABSTRACT

FLT3 is a receptor-tyrosine-kinase that is expressed on leukemic cells of the myeloid and lymphoid lineage rather specifically. We here report on the construction and selection of bispecific FLT3 X CD3 antibodies in a new recombinant format, termed Fabsc, that resembles the normal antibody structure more closely than the well-established bispecific single chain (bssc)-format. Our preferred antibody, which emerged from an initial selection procedure utilizing different FLT3- and CD3-antibodies, contains the FLT3-antibody 4G8 and the CD3-antibody UCHT1. The 4G8 X UCHT1 Fabsc-antibody was found to be superior to a bssc-antibody with identical specificities with respect to (i) affinity to the target antigen FLT3, (ii) production yield by transfected cells, and (iii) the diminished formation of aggregates. T-cell activation in the presence and absence of cultured leukemic cells and killing of these cells was comparable for both molecules. In addition, the 4G8 X UCHT1 Fabsc-antibody was found to induce T-cell activation and efficient killing of leukemic blasts in primary peripheral blood mononuclear cell (PBMC) cultures of acute myeloid leukemia (AML) patients. In these experiments, the bispecific molecule was clearly superior to an Fc-optimized monospecific FLT3-antibody described previously, indicating that within PBMC of AML patients the recruitment of T cells is more effective than that of natural killer cells.


Subject(s)
Antibodies, Bispecific/therapeutic use , CD3 Complex/immunology , Leukemia, Experimental/therapy , fms-Like Tyrosine Kinase 3/immunology , Animals , Mice , Mice, Inbred C57BL
8.
Mol Ther Methods Clin Dev ; 32(3): 101328, 2024 Sep 12.
Article in English | MEDLINE | ID: mdl-39286335

ABSTRACT

Chimeric antigen receptor (CAR) TĀ cell therapies have demonstrated significant successes in treating cancer. Currently, there are six approved CAR TĀ cell products available on the market that target different malignancies of the B cell lineage. However, to overcome the limitations of CAR TĀ cell therapies, other immune cells are being investigated for CAR-based cell therapies. CAR natural killer (NK) cells can be applied as allogeneic cell therapy, providing an economical, safe, and efficient alternative to autologous CAR TĀ cells. To improve CAR research and future in-patient monitoring of cell therapeutics, a simple, reliable, and versatile CAR detection reagent is crucial. As most existing CARs contain a single-chain variable fragment (scFv) with either a Whitlow or a G4S linker site, linker-specific monoclonal antibodies (mAbs) can detect a broad range of CARs. This study demonstrates that these linker-specific mAbs can detect different CAR NK cells inĀ vitro, spiked in whole blood, and within patient-derived tumor spheroids with high specificity and sensitivity, providing an effective and almost universal alternative for scFv-based CAR detection. Additionally, we confirm that linker-specific antibodies can be used for functional testing and enrichment of CAR NK cells, thereby providing a useful research tool to fast-track the development of novel CAR-based therapies.

9.
J Exp Med ; 221(11)2024 Nov 04.
Article in English | MEDLINE | ID: mdl-39297882

ABSTRACT

The establishment of long-lasting immunity against pathogens is facilitated by the germinal center (GC) reaction, during which B cells increase their antibody affinity and differentiate into antibody-secreting cells (ASC) and memory cells. These events involve modifications in chromatin packaging that orchestrate the profound restructuring of gene expression networks that determine cell fate. While several chromatin remodelers were implicated in lymphocyte functions, less is known about SMARCA5. Here, using ribosomal pull-down for analyzing translated genes in GC B cells, coupled with functional experiments in mice, we identified SMARCA5 as a key chromatin remodeler in B cells. While the naive B cell compartment remained unaffected following conditional depletion of Smarca5, effective proliferation during B cell activation, immunoglobulin class switching, and as a result GC formation and ASC differentiation were impaired. Single-cell multiomic sequencing analyses revealed that SMARCA5 is crucial for facilitating the transcriptional modifications and genomic accessibility of genes that support B cell activation and differentiation. These findings offer novel insights into the functions of SMARCA5, which can be targeted in various human pathologies.


Subject(s)
B-Lymphocytes , Cell Differentiation , Chromatin Assembly and Disassembly , Chromosomal Proteins, Non-Histone , Germinal Center , Animals , Germinal Center/immunology , Germinal Center/metabolism , B-Lymphocytes/metabolism , B-Lymphocytes/immunology , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/genetics , Mice , Mice, Inbred C57BL , Lymphocyte Activation/immunology , Immunoglobulin Class Switching/genetics , Adenosine Triphosphatases
10.
Biomedicines ; 12(8)2024 Jul 24.
Article in English | MEDLINE | ID: mdl-39200107

ABSTRACT

For the monitoring of chimeric antigen receptor (CAR) T-cell therapies, antigen-based CAR detection methods are usually applied. However, for each target-antigen, a separate detection system is required. Furthermore, when monitored CAR T-cells in the blood of patients treated with bispecific antibodies or T-cell engagers (bsAbs/BiTEs) recognize the same antigen, these methods produce false-positive results in clinical diagnostics. Anti-CAR-linker monoclonal antibodies (mAbs) targeting the linker sequence between the variable domains of the antigen binding CAR fragment promise a universal and unbiased CAR detection. To test this, we analyzed clinical specimens of all BCMA- and CD19-targeting CAR T-cell products currently approved for clinical use. We found a highly specific and sensitive CAR detection using anti-CAR-linker mAb in blood cells from patients treated with Ide-cel, Tisa-cel, Axi-cel, Brexu-cel, and Liso-cel. For Ide-cel and Tisa-cel, the sensitivity was significantly lower compared to that for antigen-based CAR detection assays. Strikingly, the specificity of anti-CAR linker mAb was not affected by the simultaneous presence of bispecific blinatumomab or teclistamab for Axi-cel, Brexu-cel, Liso-cel, or Ide-cel, respectively. Cilta-cel (containing a monomeric G4S-CAR linker) could not be detected by anti-CAR linker mAb. In conclusion, anti-CAR-linker mAbs are highly specific and useful for CAR T-cell monitoring but are not universally applicable.

11.
Front Immunol ; 14: 1290488, 2023.
Article in English | MEDLINE | ID: mdl-38022580

ABSTRACT

Head and neck squamous cell carcinoma (HNSCC) is a major challenge for current therapies. CAR-T cells have shown promising results in blood cancers, however, their effectiveness against solid tumors remains a hurdle. Recently, CD44v6-directed CAR-T cells demonstrated efficacy in controlling tumor growth in multiple myeloma and solid tumors such as HNSCC, lung and ovarian adenocarcinomas. Apart from CAR-T cells, CAR-NK cells offer a safe and allogenic alternative to autologous CAR-T cell therapy. In this paper, we investigated the capacity of CAR-NK cells redirected against CD44v6 to execute cytotoxicity against HNSCC. Anti-CD44v6 CAR-NK cells were generated from healthy donor peripheral blood-derived NK cells using gamma retroviral vectors (gRVs). The NK cell transduction was optimized by exploring virus envelope proteins derived from the baboon endogenous virus envelope (BaEV), feline leukemia virus (FeLV, termed RD114-TR) and gibbon ape leukemia virus (GaLV), respectively. BaEV pseudotyped gRVs induced the highest transduction rate compared to RD114-TR and GaLV envelopes as measured by EGFP and surface CAR expression of transduced NK cells. CAR-NK cells showed a two- to threefold increase in killing efficacy against various HNSCC cell lines compared to unmodified, cytokine-expanded primary NK cells. Anti-CD44v6 CAR-NK cells were effective in eliminating tumor cell lines with high and low CD44v6 expression levels. Overall, the improved cytotoxicity of CAR-NK cells holds promise for a therapeutic option for the treatment of HNSCC. However, further preclinical trials are necessary to test in vivo efficacy and safety, as well to optimize the treatment regimen of anti-CD44v6 CAR-NK cells against solid tumors.


Subject(s)
Head and Neck Neoplasms , Killer Cells, Natural , Humans , Squamous Cell Carcinoma of Head and Neck/therapy , Squamous Cell Carcinoma of Head and Neck/metabolism , Killer Cells, Natural/metabolism , Immunotherapy/methods , Cell Line, Tumor , Head and Neck Neoplasms/therapy , Head and Neck Neoplasms/metabolism
12.
Front Immunol ; 14: 1270243, 2023.
Article in English | MEDLINE | ID: mdl-38022685

ABSTRACT

Chimeric antigen receptor (CAR)-T cell therapy is a groundbreaking immunotherapy for cancer. However, the intricate and costly manufacturing process remains a hurdle. Improving the transduction rate is a potential avenue to cut down costs and boost therapeutic efficiency. Peptide nanofibrils (PNFs) serve as one such class of transduction enhancers. PNFs bind to negatively charged virions, facilitating their active engagement by cellular protrusions, which enhances virion attachment to cells, leading to increased cellular entry and gene transfer rates. While first-generation PNFs had issues with aggregate formation and potential immunogenicity, our study utilized in silico screening to identify short, endogenous, and non-immunogenic peptides capable of enhancing transduction. This led to the discovery of an 8-mer peptide, RM-8, which forms PNFs that effectively boost T cell transduction rates by various retroviral vectors. A subsequent structure-activity relationship (SAR) analysis refined RM-8, resulting in the D4 derivative. D4 peptide is stable and assembles into smaller PNFs, avoiding large aggregate formation, and demonstrates superior transduction rates in primary T and NK cells. In essence, D4 PNFs present an economical and straightforward nanotechnological tool, ideal for refining ex vivo gene transfer in CAR-T cell production and potentially other advanced therapeutic applications.


Subject(s)
Killer Cells, Natural , T-Lymphocytes , Transduction, Genetic , Peptides , Immunotherapy, Adoptive/methods
13.
Obes Facts ; 16(4): 364-373, 2023.
Article in English | MEDLINE | ID: mdl-37232004

ABSTRACT

INTRODUCTION: Infections are a major problem after left ventricular assist device (LVAD) implantation that affects morbidity, mortality, and the quality of life. Obesity often increases the risk for infection. In the cohort of LVAD patients, it is unknown if obesity affects the immunological parameters involved in viral defense. Therefore, this study investigated whether overweight or obesity affects immunological parameters such as CD8+ T cells and natural killer (NK) cells. METHODS: Immune cell subsets of CD8+ T cells and NK cells were compared between normal-weight (BMI 18.5-24.9 kg/m2, n = 17), pre-obese (BMI 25.0-29.9 kg/m2, n = 24), and obese (BMI ≥30 kg/m2, n = 27) patients. Cell subsets and cytokine serum levels were quantified prior to LVAD implantation and at 3, 6, and 12 months after LVAD implantation. RESULTS: At the end of the first postoperative year, obese patients (31.8% Ā± 2.1%) had a lower proportion of CD8+ T cells than normal-weight patients (42.4% Ā± 4.1%; p = 0.04), and the percentage of CD8+ T cells was negatively correlated with BMI (p = 0.03; r = -0.329). The proportion of circulating NK cells increased after LVAD implantation patients in normal-weight (p = 0.01) and obese patients (p < 0.01). Patients with pre-obesity showed a delayed increase (p < 0.01) 12 months after LVAD implantation. Further, obese patients showed an increase in the percentage of CD57+ NK cells after 6 and 12 months (p = 0.01) of treatment, higher proportions of CD56bright NK cells (p = 0.01), and lower proportions of CD56dim/neg NK cells (p = 0.03) 3 months after LVAD implantation than normal-weight patients. The proportion of CD56bright NK cells positively correlated with BMI (p < 0.01, r = 0.403) 1 year after LVAD implantation. CONCLUSIONS: This study documented that obesity affects CD8+ T cells and subsets of NK cells in patients with LVAD in the first year after LVAD implantation. Lower proportions of CD8+ T cells and CD56dim/neg NK cells and higher proportion of CD56bright NK cells were detected in obese but not in pre-obese and normal-weight LVAD patients during the first year after LVAD implantation. The induced immunological imbalance and phenotypic changes of T and NK cells may influence viral and bacterial immunoreactivity.


Subject(s)
Heart Failure , Quality of Life , Humans , Obesity/complications , Obesity/therapy , Body Mass Index , Retrospective Studies
14.
Front Immunol ; 13: 826074, 2022.
Article in English | MEDLINE | ID: mdl-35237271

ABSTRACT

The human leukocyte antigen (HLA)-G is a non-classical HLA class I molecule, which has distinct features to classical HLA-A, -B, -C antigens, such as a low polymorphism, different splice variants, highly restricted, tightly regulated expression and immune modulatory properties. HLA-G expression in tumor cells and virus-infected cells, as well as the release of soluble HLA-G leads to escape from host immune surveillance. Increased knowledge of the link between HLA-G expression, viral infection and disease progression is urgently required, which highlights the possible use of HLA-G as novel diagnostic and prognostic biomarker for viral infections, but also as therapeutic target. Therefore, this review aims to summarize the expression, regulation, function and impact of HLA-G in the context of different viral infections including virus-associated cancers. The characterization of HLA-G-driven immune escape mechanisms involved in the interactions between host cells and viruses might result in the design of novel immunotherapeutic strategies targeting HLA-G and/or its interaction with its receptors on immune effector cells.


Subject(s)
HLA-G Antigens , Virus Diseases , HLA-A Antigens , HLA-G Antigens/genetics , Humans , Immunologic Surveillance , Polymorphism, Genetic
15.
Front Immunol ; 13: 822298, 2022.
Article in English | MEDLINE | ID: mdl-35371071

ABSTRACT

Cancer immunotherapies utilize the capabilities of the immune system to efficiently target malignant cells. In recent years, chimeric antigen receptor (CAR) equipped T cells showed promising results against B cell lymphomas. Autologous CAR-T cells require patient-specific manufacturing and thus extensive production facilities, resulting in high priced therapies. Along with potentially severe side effects, these are the major drawbacks of CAR-T cells therapies. Natural Killer (NK) cells pose an alternative for CAR equipped immune cells. Since NK cells can be safely transferred from healthy donors to cancer patients, they present a suitable platform for an allogeneic "off-the-shelf" immunotherapy. However, administration of activated NK cells in cancer therapy has until now shown poor anti-cancer responses, especially in solid tumors. Genetic modifications such as CARs promise to enhance recognition of tumor cells, thereby increasing anti-tumor effects and improving clinical efficacy. Although the cell biology of T and NK cells deviates in many aspects, the development of CAR-NK cells frequently follows within the footsteps of CAR-T cells, meaning that T cell technologies are simply adopted to NK cells. In this review, we underline the unique properties of NK cells and their potential in CAR therapies. First, we summarize the characteristics of NK cell biology with a focus on signaling, a fine-tuned interaction of activating and inhibitory receptors. We then discuss why tailored NK cell-specific CAR designs promise superior efficacy compared to designs developed for T cells. We summarize current findings and developments in the CAR-NK landscape: different CAR formats and modifications to optimize signaling, to target a broader pool of antigens or to increase in vivo persistence. Finally, we address challenges beyond NK cell engineering, including expansion and manufacturing, that need to be addressed to pave the way for CAR-NK therapies from the bench to the clinics.


Subject(s)
Neoplasms , Receptors, Chimeric Antigen , Humans , Immunotherapy, Adoptive/methods , Killer Cells, Natural , Receptors, Chimeric Antigen/genetics , Receptors, Natural Killer Cell , T-Lymphocytes
16.
Sci Rep ; 12(1): 5758, 2022 04 06.
Article in English | MEDLINE | ID: mdl-35388061

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causal agent of the COVID-19 pandemic. More than 274 million individuals have suffered from COVID-19 and over five million people have died from this disease so far. Therefore, there is an urgent need for therapeutic drugs. Repurposing FDA approved drugs should be favored since evaluation of safety and efficacy of de-novo drug design are both costly and time consuming. We report that imatinib, an Abl tyrosine kinase inhibitor, robustly decreases SARS-CoV-2 infection and uncover a mechanism of action. We show that imatinib inhibits the infection of SARS-CoV-2 and its surrogate lentivector pseudotype. In latter, imatinib inhibited both routes of viral entry, endocytosis and membrane-fusion. We utilized a system to quantify in real-time cell-cell membrane fusion mediated by the SARS-CoV-2 surface protein, Spike, and its receptor, hACE2, to demonstrate that imatinib inhibits this process in an Abl1 and Abl2 independent manner. Furthermore, cellular thermal shift assay revealed a direct imatinib-Spike interaction that affects Spike susceptibility to trypsin digest. Collectively, our data suggest that imatinib inhibits Spike mediated viral entry by an off-target mechanism. These findings mark imatinib as a promising therapeutic drug in inhibiting the early steps of SARS-CoV-2 infection.


Subject(s)
COVID-19 Drug Treatment , Humans , Imatinib Mesylate/pharmacology , Pandemics , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
17.
Front Immunol ; 12: 705848, 2021.
Article in English | MEDLINE | ID: mdl-34539636

ABSTRACT

Activation and differentiation of B cells depend on extensive rewiring of gene expression networks through changes in chromatin structure and accessibility. The chromatin remodeling complex BAF with its catalytic subunit Brg1 was previously identified as an essential regulator of early B cell development, however, how Brg1 orchestrates gene expression during mature B cell activation is less clear. Here, we find that Brg1 is required for B cell proliferation and germinal center formation through selective interactions with enhancers. Brg1 recruitment to enhancers following B cell activation was associated with increased chromatin accessibility and transcriptional activation of their coupled promoters, thereby regulating the expression of cell cycle-associated genes. Accordingly, Brg1-deficient B cells were unable to mount germinal center reactions and support the formation of class-switched plasma cells. Our findings show that changes in B cell transcriptomes that support B cell proliferation and GC formation depend on enhancer activation by Brg1. Thus, the BAF complex plays a critical role during the onset of the humoral immune response.


Subject(s)
B-Lymphocytes/immunology , Cell Proliferation , DNA Helicases , Enhancer Elements, Genetic , Gene Expression Regulation/immunology , Germinal Center/immunology , Nuclear Proteins , Transcription Factors , Animals , DNA Helicases/genetics , DNA Helicases/immunology , Mice , Mice, Transgenic , Nuclear Proteins/genetics , Nuclear Proteins/immunology , Transcription Factors/genetics , Transcription Factors/immunology
18.
Front Immunol ; 12: 714799, 2021.
Article in English | MEDLINE | ID: mdl-34721381

ABSTRACT

The coevolution of the human immune system and herpesviruses led to the emergence and diversification of both cellular danger molecules recognized by immune cells on the one hand and viral countermeasures that prevent the expression of these proteins on infected cells on the other. There are eight ligands for the activating receptor NKG2D in humans - MICA, MICB, ULBP1-6. Several of them are induced and surface-expressed on herpesvirus-infected cells to serve as danger signals to activate the immune system. Therefore, these ligands are frequently targeted for suppression by viral immune evasion mechanisms. Mechanisms to downregulate NKG2D ligands and thereby escape immune recognition have been identified in all other human herpesviruses (HHV), except for HHV-6A. In this study, we identify two HHV-6A encoded immunoevasins, U20 and U21, which suppress the expression of the NKG2D ligands ULBP1 and ULBP3, respectively, during infection. Additionally, MICB is targeted by a so far unexplored viral protein. Due to the diminished NKG2D ligand surface expression on infected cells, recognition of HHV-6A infected cells by innate immune cells is impaired. Importantly, our study indicates that immune escape mechanisms between the related herpesviruses HHV-6A and HHV-6B are evolutionary conserved as the same NKG2D ligands are targeted. Our data contribute an additional piece of evidence for the importance of the NKG2D receptor - NKG2D ligand axis during human herpesvirus infections and sheds light on immune evasion mechanisms of HHV-6A.


Subject(s)
GPI-Linked Proteins/metabolism , Herpesvirus 6, Human/physiology , Host-Pathogen Interactions/immunology , Roseolovirus Infections/immunology , Roseolovirus Infections/metabolism , Roseolovirus Infections/virology , Viral Proteins/metabolism , Flow Cytometry , GPI-Linked Proteins/genetics , Gene Expression Regulation , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Host-Pathogen Interactions/genetics , Humans , Immune Evasion , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Ligands , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Protein Binding
19.
J Exp Med ; 218(10)2021 10 04.
Article in English | MEDLINE | ID: mdl-34402854

ABSTRACT

Long-lasting immunity depends on the generation of protective antibodies through the germinal center (GC) reaction. N6-methyladenosine (m6A) modification of mRNAs by METTL3 activity modulates transcript lifetime primarily through the function of m6A readers; however, the physiological role of this molecular machinery in the GC remains unknown. Here, we show that m6A modifications by METTL3 are required for GC maintenance through the differential functions of m6A readers. Mettl3-deficient GC B cells exhibited reduced cell-cycle progression and decreased expression of proliferation- and oxidative phosphorylation-related genes. The m6A binder, IGF2BP3, was required for stabilization of Myc mRNA and expression of its target genes, whereas the m6A reader, YTHDF2, indirectly regulated the expression of the oxidative phosphorylation gene program. Our findings demonstrate how two independent gene networks that support critical GC functions are modulated by m6A through distinct mRNA binders.


Subject(s)
Germinal Center/physiology , Methyltransferases/metabolism , RNA/metabolism , Adenosine/analogs & derivatives , Adenosine/genetics , Adenosine/metabolism , Animals , B-Lymphocytes/pathology , Cell Cycle/genetics , Gene Expression Regulation , Genes, myc , Germinal Center/pathology , Methylation , Methyltransferases/genetics , Mice, Inbred C57BL , Mice, Transgenic , Oxidative Phosphorylation , RNA/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Smegmamorpha , Spleen/pathology
20.
Viruses ; 12(7)2020 07 20.
Article in English | MEDLINE | ID: mdl-32698530

ABSTRACT

The Pneumoviridae family includes human metapneumovirus (HMPV) and human orthopneumovirus, which is also known as a respiratory syncytial virus (HRSV). These are large enveloped, negative single-strand RNA viruses. HMPV and HRSV are the human members, which commonly infect children. HMPV, which was discovered in 2001, infects most children until the age of five, which causes an influenza-like illness. The interaction of this virus with immune cells is poorly understood. In this study, we show that HMPV evades natural killer (NK) cell attack by downregulating stress-induced ligands for the activating receptor NKG2D including: Major histocompatibility complex (MHC) class I polypeptide-related sequences A and B (MICA, MICB), UL16 binding proteins ULBP2, and ULBP3, but not ULBP1. Mechanistically, we show that the viral protein G is involved in the downregulation of ULBP2 and that the viral protein M2.2 is required for MICA and MICB downregulation. These findings emphasize the importance of NK cells, in general, and NKG2D, in particular, in controlling HMPV infection, which opens new avenues for treating HMPV.


Subject(s)
Killer Cells, Natural/immunology , Metapneumovirus/immunology , NK Cell Lectin-Like Receptor Subfamily K/immunology , Antibodies, Viral/immunology , Blotting, Western , Down-Regulation , Flow Cytometry , Humans , Paramyxoviridae Infections/immunology , Real-Time Polymerase Chain Reaction , Viral Proteins/immunology
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