RÉSUMÉ
BACKGROUND: Cancer immunotherapy approaches that elicit immune cell responses, including T and NK cells, have revolutionized the field of oncology. However, immunosuppressive mechanisms restrain immune cell activation within solid tumors so additional strategies to augment activity are required. METHODS: We identified the co-stimulatory receptor NKG2D as a target based on its expression on a large proportion of CD8+ tumor infiltrating lymphocytes (TILs) from breast cancer patient samples. Human and murine surrogate NKG2D co-stimulatory receptor-bispecifics (CRB) that bind NKG2D on NK and CD8+ T cells as well as HER2 on breast cancer cells (HER2-CRB) were developed as a proof of concept for targeting this signaling axis in vitro and in vivo. RESULTS: HER2-CRB enhanced NK cell activation and cytokine production when co-cultured with HER2 expressing breast cancer cell lines. HER2-CRB when combined with a T cell-dependent-bispecific (TDB) antibody that synthetically activates T cells by crosslinking CD3 to HER2 (HER2-TDB), enhanced T cell cytotoxicity, cytokine production and in vivo antitumor activity. A mouse surrogate HER2-CRB (mHER2-CRB) improved in vivo efficacy of HER2-TDB and augmented NK as well as T cell activation, cytokine production and effector CD8+ T cell differentiation. CONCLUSION: We demonstrate that targeting NKG2D with bispecific antibodies (BsAbs) is an effective approach to augment NK and CD8+ T cell antitumor immune responses. Given the large number of ongoing clinical trials leveraging NK and T cells for cancer immunotherapy, NKG2D-bispecifics have broad combinatorial potential.
Sujet(s)
Tumeurs du sein , Lymphocytes T CD8+ , Cellules tueuses naturelles , Sous-famille K des récepteurs de cellules NK de type lectine , Humains , Animaux , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , Sous-famille K des récepteurs de cellules NK de type lectine/immunologie , Souris , Lymphocytes T CD8+/immunologie , Cellules tueuses naturelles/immunologie , Femelle , Tumeurs du sein/immunologie , Tumeurs du sein/thérapie , Récepteur ErbB-2/immunologie , Lignée cellulaire tumorale , Immunothérapie/méthodes , Activation des lymphocytes/immunologie , Lymphocytes TIL/immunologie , Lymphocytes TIL/métabolismeRÉSUMÉ
Natural killer cells (NK) are the "professional killer" of tumors and play a crucial role in anti-tumor immunotherapy. NK cell desensitization is a key mechanism of tumor immune escape. Dysregulated NKG2D-NKG2DL signaling is a primary driver of this desensitization process. However, the factors that regulate NK cell desensitization remain largely uncharacterized. Here, we present the first report that circular RNA circARAP2 (hsa_circ_0069396) is involved in the soluble MICA (sMICA)-induced NKG2D endocytosis in the NK cell desensitization model. CircARAP2 was upregulated during NK cell desensitization and the loss of circARAP2 alleviated NKG2D endocytosis and NK cell desensitization. Using Chromatin isolation by RNA purification (ChIRP) and RNA pull-down approaches, we identified that RAB5A, a molecular marker of early endosomes, was its downstream target. Notably, transcription factor CTCF was an intermediate functional partner of circARAP2. Mechanistically, we discovered that circARAP2 interacted with CTCF and inhibited the recruitment of CTCF-Polycomb Repressive Complex 2 (PRC2) to the promoter region of RAB5A, thereby erasing histone H3K27 and H3K9 methylation suppression to enhance RAB5A transcription. These data demonstrate that inhibition of circARAP2 effectively alleviates sMICA-induced NKG2D endocytosis and NK cell desensitization, providing a novel target for therapeutic intervention in tumor immune evasion.
Sujet(s)
Facteur de liaison à la séquence CCCTC , Antigènes d'histocompatibilité de classe I , Cellules tueuses naturelles , ARN circulaire , Protéines G rab5 , Cellules tueuses naturelles/immunologie , Cellules tueuses naturelles/métabolisme , Humains , Facteur de liaison à la séquence CCCTC/métabolisme , Facteur de liaison à la séquence CCCTC/génétique , ARN circulaire/génétique , ARN circulaire/métabolisme , Protéines G rab5/métabolisme , Protéines G rab5/génétique , Antigènes d'histocompatibilité de classe I/métabolisme , Antigènes d'histocompatibilité de classe I/génétique , Antigènes d'histocompatibilité de classe I/immunologie , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , Sous-famille K des récepteurs de cellules NK de type lectine/génétique , Endocytose , Endosomes/métabolisme , Souris , AnimauxRÉSUMÉ
Endometriosis, affecting 10% of women, is defined as implantation, survival, and growth of endometrium-like/endometriotic tissue outside the uterine cavity, causing inflammation, infertility, pain, and susceptibility to ovarian cancer. Despite extensive studies, its etiology and pathogenesis are poorly understood and largely unknown. The prevailing view is that the immune system of endometriosis patients fails to clear ectopically disseminated endometrium from retrograde menstruation. Exosomes are small extracellular vesicles that exhibit immunomodulatory properties. We studied the role of endometriotic tissue-secreted exosomes in the pathophysiology of endometriosis. Two exosome-mediated mechanisms known to impair the immune response were investigated: 1) downregulation of NKG2D-mediated cytotoxicity and 2) FasL- and TRAIL-induced apoptosis of activated immune cells. We showed that secreted endometriotic exosomes isolated from supernatants of short-term explant cultures carry the NKG2D ligands MICA/B and ULBP1-3 and the proapoptotic molecules FasL and TRAIL on their surface, i.e., signature molecules of exosome-mediated immune suppression. Acting as decoys, these exosomes downregulate the NKG2D receptor, impair NKG2D-mediated cytotoxicity, and induce apoptosis of activated PBMCs and Jurkat cells through the FasL- and TRAIL pathway. The secreted endometriotic exosomes create an immunosuppressive gradient at the ectopic site, forming a "protective shield" around the endometriotic lesions. This gradient guards the endometriotic lesions against clearance by a cytotoxic attack and creates immunologic privilege by induction of apoptosis in activated immune cells. Taken together, our results provide a plausible, exosome-based mechanistic explanation for the immune dysfunction and the compromised immune surveillance in endometriosis and contribute novel insights into the pathogenesis of this enigmatic disease.
Sujet(s)
Apoptose , Endométriose , Endomètre , Exosomes , Sous-famille K des récepteurs de cellules NK de type lectine , Ligand TRAIL , Humains , Endométriose/immunologie , Endométriose/métabolisme , Endométriose/anatomopathologie , Femelle , Exosomes/métabolisme , Exosomes/immunologie , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , Apoptose/immunologie , Endomètre/immunologie , Endomètre/anatomopathologie , Endomètre/métabolisme , Ligand TRAIL/métabolisme , Ligand TRAIL/immunologie , Régulation négative/immunologie , Ligand de Fas/métabolisme , Ligand de Fas/immunologie , Cytotoxicité immunologique , Adulte , Antigènes d'histocompatibilité de classe I/métabolisme , Antigènes d'histocompatibilité de classe I/immunologieRÉSUMÉ
Introduction: Human Herpesvirus 6B (HHV-6B) impedes host immune responses by downregulating class I MHC molecules (MHC-I), hindering antigen presentation to CD8+ T cells. Downregulation of MHC-I disengages inhibitory receptors on natural killer (NK) cells, resulting in activation and killing of the target cell if NK cell activating receptors such as NKG2D have engaged stress ligands upregulated on the target cells. Previous work has shown that HHV-6B downregulates three MHC-like stress ligands MICB, ULBP1, and ULBP3, which are recognized by NKG2D. The U20 glycoprotein of the related virus HHV-6A has been implicated in the downregulation of ULBP1, but the precise mechanism remains undetermined. Methods: We set out to investigate the role of HHV-6B U20 in modulating NK cell activity. We used HHV-6B U20 expressed as a recombinant protein or transduced into target cells, as well as HHV-6B infection, to investigate binding interactions with NK cell ligands and receptors and to assess effects on NK cell activation. Small-angle X-ray scattering was used to align molecular models derived from machine-learning approaches. Results: We demonstrate that U20 binds directly to ULBP1 with sub-micromolar affinity. Transduction of U20 decreases NKG2D binding to ULBP1 at the cell surface but does not decrease ULBP1 protein levels, either at the cell surface or in toto. HHV-6B infection and soluble U20 have the same effect. Transduction of U20 blocks NK cell activation in response to cell-surface ULBP1. Structural modeling of the U20 - ULBP1 complex indicates some similarities to the m152-RAE1γ complex.
Sujet(s)
Protéines liées au GPI , Herpèsvirus humain de type 6 , Cellules tueuses naturelles , Activation des lymphocytes , Sous-famille K des récepteurs de cellules NK de type lectine , Humains , Cellules tueuses naturelles/immunologie , Cellules tueuses naturelles/métabolisme , Herpèsvirus humain de type 6/immunologie , Protéines liées au GPI/métabolisme , Protéines liées au GPI/immunologie , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , Sous-famille K des récepteurs de cellules NK de type lectine/immunologie , Activation des lymphocytes/immunologie , Liaison aux protéines , Protéines virales/immunologie , Protéines virales/métabolisme , Glycoprotéines/immunologie , Glycoprotéines/métabolisme , Protéines et peptides de signalisation intracellulaireRÉSUMÉ
The short-lived nature and heterogeneity of Natural Killer (NK) cells limit the development of NK cell-based therapies, despite their proven safety and efficacy against cancer. Here, we describe the biological basis, detailed phenotype and function of long-lived anti-tumour human NK cells (CD56highCD16+), obtained without cell sorting or feeder cells, after priming of peripheral blood cells with Bacillus Calmette-Guérin (BCG). Further, we demonstrate that survival doses of a cytokine combination, excluding IL18, administered just weekly to BCG-primed NK cells avoids innate lymphocyte exhaustion and leads to specific long-term proliferation of innate cells that exert potent cytotoxic function against a broad range of solid tumours, mainly through NKG2D. Strikingly, a NKG2C+CD57-FcεRIγ+ NK cell population expands after BCG and cytokine stimulation, independently of HCMV serology. This strategy was exploited to rescue anti-tumour NK cells even from the suppressor environment of cancer patients' bone marrow, demonstrating that BCG confers durable anti-tumour features to NK cells.
Sujet(s)
Prolifération cellulaire , Cellules tueuses naturelles , Cellules tueuses naturelles/immunologie , Cellules tueuses naturelles/effets des médicaments et des substances chimiques , Humains , Prolifération cellulaire/effets des médicaments et des substances chimiques , Tumeurs/immunologie , Tumeurs/traitement médicamenteux , Vaccin BCG/immunologie , Vaccin BCG/administration et posologie , Mycobacterium bovis/immunologie , Activation des lymphocytes/effets des médicaments et des substances chimiques , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , Interleukines/métabolisme , Antigènes CD56/métabolisme , Sous-famille C des récepteurs de cellules NK de type lectine/métabolismeRÉSUMÉ
Interface residues at sites of protein-protein interaction (PPI) are the focus for affinity optimisation. However, protein hydrophobic cores (HCs) play critical roles and shape the protein surface. We hypothesise that manipulating protein HCs can enhance PPI interaction affinities. A cell stress molecule, major histocompatibility complex class I chain-related protein A (MICA), binds to the natural killer group 2D (NKG2D) homodimer to form three molecule interactions. MICA was used as a study subject to support our hypothesis. We redesigned MICA HCs by directed mutagenesis and isolated high-affinity variants through a newly designed partial-denature panning (PDP) method. A few mutations in MICA HCs increased the NKG2D-MICA interaction affinity by 325-5613-fold. Crystal structures of the NKG2D-MICA variant complexes indicated that mutagenesis of MICA HCs stabilised helical elements for decreasing intermolecular interactive free energy (ΔG) of the NKG2D-MICA heterotrimer. The repacking of MICA HC mutants maintained overall surface residues and the authentic binding specificity of MICA. In conclusion, this study provides a new method for MICA redesign and affinity optimisation through HC manipulation without mutating PPI interface residues. Our study introduces a novel approach to protein manipulation, potentially expanding the toolkit for protein affinity optimisation.
Sujet(s)
Antigènes d'histocompatibilité de classe I , Interactions hydrophobes et hydrophiles , Humains , Antigènes d'histocompatibilité de classe I/métabolisme , Antigènes d'histocompatibilité de classe I/composition chimique , Antigènes d'histocompatibilité de classe I/génétique , Modèles moléculaires , Mutation , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , Sous-famille K des récepteurs de cellules NK de type lectine/composition chimique , Sous-famille K des récepteurs de cellules NK de type lectine/génétique , Liaison aux protéinesRÉSUMÉ
Multiple myeloma is a malignancy characterized by the accumulation of malignant plasma cells in bone marrow and the production of monoclonal immunoglobulin. A hallmark of cancer is the evasion of immune surveillance. Histone deacetylase inhibitors have been shown to promote the expression of silenced molecules and hold potential to increase the anti-MM efficacy of immunotherapy. The aim of the present work was to assess the potential effect of tinostamustine (EDO-S101), a first-in-class alkylating deacetylase inhibitor, in combination with daratumumab, an anti-CD38 monoclonal antibody (mAb), through different preclinical studies. Tinostamustine increases CD38 expression in myeloma cell lines, an effect that occurs in parallel with an increment in CD38 histone H3 acetylation levels. Also, the expression of MICA and MICB, ligands for the NK cell activating receptor NKG2D, augments after tinostamustine treatment in myeloma cell lines and primary myeloma cells. Pretreatment of myeloma cell lines with tinostamustine increased the sensitivity of these cells to daratumumab through its different cytotoxic mechanisms, and the combination of these two drugs showed a higher anti-myeloma effect than individual treatments in ex vivo cultures of myeloma patients' samples. In vivo data confirmed that tinostamustine pretreatment followed by daratumumab administration significantly delayed tumor growth and improved the survival of mice compared to individual treatments. In summary, our results suggest that tinostamustine could be a potential candidate to improve the efficacy of anti-CD38 mAbs.
Sujet(s)
Antigènes CD38 , Anticorps monoclonaux , Myélome multiple , Sous-famille K des récepteurs de cellules NK de type lectine , Animaux , Humains , Souris , Antigènes CD38/effets des médicaments et des substances chimiques , Antigènes CD38/métabolisme , Anticorps monoclonaux/pharmacologie , Anticorps monoclonaux/usage thérapeutique , Lignée cellulaire tumorale , Synergie des médicaments , Antigènes d'histocompatibilité de classe I/métabolisme , Antigènes d'histocompatibilité de classe I/génétique , Inhibiteurs de désacétylase d'histone/pharmacologie , Inhibiteurs de désacétylase d'histone/usage thérapeutique , Glycoprotéines membranaires/effets des médicaments et des substances chimiques , Glycoprotéines membranaires/métabolisme , Myélome multiple/traitement médicamenteux , Myélome multiple/métabolisme , Myélome multiple/anatomopathologie , Sous-famille K des récepteurs de cellules NK de type lectine/effets des médicaments et des substances chimiques , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , Régulation positive/effets des médicaments et des substances chimiques , Tests d'activité antitumorale sur modèle de xénogreffe , Benzimidazoles/pharmacologie , Benzimidazoles/usage thérapeutiqueRÉSUMÉ
OBJECTIVE: Natural killer (NK) cells are an integral part of the staunch defense line against malignant tumors within the tumor microenvironment. Existing research indicates that miRNAs can influence the development of NK cells by negatively modulating gene expression. In this study, we aim to explore how the miR-17-5p in Hepatocellular Carcinoma (HCC) exosomes regulates the killing function of NK cells towards HCC cells through the transcription factor RNX1. METHODS: The exosomes were isolated from HCC tissues and cell lines, followed by a second generation sequencing to compare differential miRNAs. Verification was performed using qRT-PCR and Western blot methods. The mutual interactions between miR-17-5p and RUNX1, as well as between RUNX1 and NKG2D, were authenticated using techniques like luciferase reporter gene assays, Western blotting, and Chromatin Immunoprecipitation (ChIP). The cytotoxic activity of NK cells towards HCC cells in vitro was measured using methods such as RTCA and ELISPOT. The zebrafish xenotransplantation was utilized to assess the in vivo killing capacity of NK cells against HCC cells. RESULTS: The level of miR-17-5p in exosomes from HCC tissue increased compared to adjacent tissues. We verified that RUNX1 was a target of miR-17-5p and that RUNX1 enhances the transcription of NKG2D. MiR-17-5p was found to downregulate the expression of RUNX1 and NKG2D, subsequently reducing the in vitro and in vivo cytotoxic capabilities of NK cells against HCC cells. CONCLUSIONS: The miR-17-5p found within HCC exosomes can target RUNX1, subsequently attenuating the cytotoxic activity of NK cells.
Sujet(s)
Carcinome hépatocellulaire , Sous-unité alpha 2 du facteur CBF , Exosomes , Régulation de l'expression des gènes tumoraux , Cellules tueuses naturelles , Tumeurs du foie , microARN , Sous-famille K des récepteurs de cellules NK de type lectine , microARN/génétique , microARN/métabolisme , Humains , Exosomes/métabolisme , Sous-unité alpha 2 du facteur CBF/génétique , Sous-unité alpha 2 du facteur CBF/métabolisme , Carcinome hépatocellulaire/immunologie , Carcinome hépatocellulaire/génétique , Carcinome hépatocellulaire/anatomopathologie , Animaux , Cellules tueuses naturelles/immunologie , Cellules tueuses naturelles/métabolisme , Tumeurs du foie/immunologie , Tumeurs du foie/génétique , Tumeurs du foie/anatomopathologie , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , Sous-famille K des récepteurs de cellules NK de type lectine/génétique , Lignée cellulaire tumorale , Danio zébré , Régulation négative , Cytotoxicité immunologiqueRÉSUMÉ
Metastatic (m) colorectal cancer (CRC) is an incurable disease with a poor prognosis and thus remains an unmet clinical need. Immune checkpoint blockade (ICB)-based immunotherapy is effective for mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) mCRC patients, but it does not benefit the majority of mCRC patients. NK cells are innate lymphoid cells with potent effector responses against a variety of tumor cells but are frequently dysfunctional in cancer patients. Memory-like (ML) NK cells differentiated after IL-12/IL-15/IL-18 activation overcome many challenges to effective NK cell anti-tumor responses, exhibiting enhanced recognition, function, and in vivo persistence. We hypothesized that ML differentiation enhances the NK cell responses to CRC. Compared to conventional (c) NK cells, ML NK cells displayed increased IFN-γ production against both CRC cell lines and primary patient-derived CRC spheroids. ML NK cells also exhibited improved killing of CRC target cells in vitro in short-term and sustained cytotoxicity assays, as well as in vivo in NSG mice. Mechanistically, enhanced ML NK cell responses were dependent on the activating receptor NKG2D as its blockade significantly decreased ML NK cell functions. Compared to cNK cells, ML NK cells exhibited greater antibody-dependent cytotoxicity when targeted against CRC by cetuximab. ML NK cells from healthy donors and mCRC patients exhibited increased anti-CRC responses. Collectively, our findings demonstrate that ML NK cells exhibit enhanced responses against CRC targets, warranting further investigation in clinical trials for mCRC patients, including those who have failed ICB.
Sujet(s)
Différenciation cellulaire , Tumeurs colorectales , Mémoire immunologique , Cellules tueuses naturelles , Tumeurs colorectales/immunologie , Tumeurs colorectales/anatomopathologie , Tumeurs colorectales/traitement médicamenteux , Cellules tueuses naturelles/immunologie , Cellules tueuses naturelles/effets des médicaments et des substances chimiques , Humains , Animaux , Souris , Différenciation cellulaire/effets des médicaments et des substances chimiques , Lignée cellulaire tumorale , Interféron gamma/métabolisme , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , Souris de lignée NOD , FemelleRÉSUMÉ
Chronic infections induce CD4+ T-cells with cytotoxic functions (CD4 CTLs); at present, it is still unknown whether latent tuberculosis (LTB) and active tuberculosis (ATB) induce CD4 CTLs. Plasma and cells from four patient groups-uninfected contact (UC), LTB, and ATB (divided as sensitive [DS-TB]- or resistant [DR-TB]-drug)-were evaluated by flow cytometry, q-PCR, and proteomics. The data showed that ATB patients had an increased frequency of CD4+ T-cells and a decreased frequency of CD8+ T-cells. The latter displays an exhausted-like profile characterized by CD39, CD279, and TIM-3 expression. ATB had a high frequency of CD4 + perforin+ cells, suggesting a CD4 CTL profile. The expression (at the transcriptional level) of granzyme A, granzyme B, granulysin, and perforin, as well as the genes T-bet (Tbx21) and NKG2D (Klrk1), in enriched CD4+ T-cells, confirmed the cytotoxic signature of CD4+ T-cells during ATB (which was stronger in DS-TB than in DR-TB). Moreover, proteomic analysis revealed the presence of HSP70 (in DS-TB) and annexin A5 (in DR-TB), which are molecules that have been associated with favoring the CD4 CTL profile. Finally, we found that lipids from Mycobacterium tuberculosis increased the presence of CD4 CTLs in DR-TB patients. Our data suggest that ATB is characterized by exhausted-like CD8+ T-cells, which, together with a specific microenvironment, favor the presence of CD4 CTLs.
Sujet(s)
Lymphocytes T CD4+ , Lymphocytes T CD8+ , Granzymes , Récepteur cellulaire-2 du virus de l'hépatite A , Perforine , Tuberculose , Humains , Lymphocytes T CD8+/immunologie , Lymphocytes T CD8+/métabolisme , Lymphocytes T CD4+/immunologie , Lymphocytes T CD4+/métabolisme , Mâle , Granzymes/métabolisme , Granzymes/génétique , Granzymes/immunologie , Perforine/métabolisme , Perforine/génétique , Perforine/immunologie , Adulte , Femelle , Récepteur cellulaire-2 du virus de l'hépatite A/métabolisme , Récepteur cellulaire-2 du virus de l'hépatite A/immunologie , Tuberculose/immunologie , Tuberculose/microbiologie , Tuberculose latente/immunologie , Tuberculose latente/microbiologie , Adulte d'âge moyen , Lymphocytes T cytotoxiques/immunologie , Lymphocytes T cytotoxiques/métabolisme , Mycobacterium tuberculosis/immunologie , Protéines à domaine boîte-T/métabolisme , Protéines à domaine boîte-T/génétique , Protéines à domaine boîte-T/immunologie , Antigènes CD/métabolisme , Antigènes CD/immunologie , Antigènes CD/génétique , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , Sous-famille K des récepteurs de cellules NK de type lectine/immunologie , Sous-famille K des récepteurs de cellules NK de type lectine/génétique , Protéomique/méthodes , Antigènes de différenciation des lymphocytes T , ApyraseRÉSUMÉ
BACKGROUND: Glioblastoma (GBM) almost invariably becomes resistant towards conventional treatment of radiotherapy and temozolomide (TMZ) chemotherapy, partly due to subpopulations of intrinsically resistant glioma stem-like cells (GSC). The oncolytic herpes simplex virus-1 G207 is a promising approach for GBM virotherapy although its efficacy in patients with GBM is often limited. Natural killer group 2 member D ligands (NKG2DLs) are minimally expressed by healthy cells but are upregulated by the DNA damage response (DDR) and in malignant cells with chronic DDR signaling, resulting in innate immune activation. METHODS: We have designed a bispecific T-cell engager (BiTE) capable of cross-linking CD3 on T cells with NKG2DL-expressing GBM cells. We then engineered the G207 virus to express the NKG2D BiTE and secrete it from infected cells. The efficacy of the free BiTE and BiTE delivered by G207 was evaluated in combination with conventional therapies in GBM cells and against patient-derived GSCs in the context of T-cell activation and target cell viability. RESULTS: NKG2D BiTE-mediated cross-linking of GBM cells and T cells causes antigen-independent T-cell activation, pro-inflammatory cytokine release, and tumor cell death, thereby combining direct viral oncolysis with BiTE-mediated cytotoxicity. Surface NKG2DL expression was further elevated on GBM cells following pretreatment with sublethal doses of TMZ and radiation to induce the DDR, increasing sensitivity towards G207-NKG2D BiTE and achieving synergistic cytotoxicity. We also demonstrate a novel strategy for targeting GSCs that are non-permissive to G207 infection but remain sensitive to NKG2D BiTE. CONCLUSIONS: We propose a potential model for targeting GSCs in heterogeneous tumors, whereby differentiated GBM cells infected with G207-NKG2D BiTE produce NKG2D BiTE locally, directing T-cell cytotoxicity towards the GSC subpopulations in the tumor microenvironment.
Sujet(s)
Glioblastome , Sous-famille K des récepteurs de cellules NK de type lectine , Cellules souches tumorales , Thérapie virale de cancers , Humains , Tumeurs du cerveau/thérapie , Tumeurs du cerveau/immunologie , Tumeurs du cerveau/anatomopathologie , Lignée cellulaire tumorale , Glioblastome/traitement médicamenteux , Glioblastome/immunologie , Glioblastome/thérapie , Cellules souches tumorales/effets des médicaments et des substances chimiques , Cellules souches tumorales/métabolisme , Sous-famille K des récepteurs de cellules NK de type lectine/effets des médicaments et des substances chimiques , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , Thérapie virale de cancers/méthodes , Lymphocytes T/effets des médicaments et des substances chimiques , Lymphocytes T/immunologie , Lymphocytes T/métabolismeRÉSUMÉ
Ataxia-telangiectasia (A-T) is a rare disorder caused by genetic defects of A-T mutated (ATM) kinase, a key regulator of stress response, and characterized by neurodegeneration, immunodeficiency, and high incidence of cancer. Here we investigated NK cells in a mouse model of A-T (Atm-/-) showing that they are strongly impaired at killing tumor cells due to a block of early signaling events. On the other hand, in Atm-/- littermates with thymic lymphoma NK cell cytotoxicity is enhanced as compared with ATM-proficient mice, possibly via tumor-produced TNF-α. Results also suggest that expansion of exhausted NKG2D+ NK cells in Atm-/- mice is driven by low-level expression of stress-inducible NKG2D ligands, whereas development of thymoma expressing the high-affinity MULT1 ligand is associated with NKG2D down-regulation on NK cells. These results expand our understanding of immunodeficiency in A-T and encourage exploring NK cell biology in A-T patients in the attempt to identify cancer predictive biomarkers and novel therapeutic targets.
Sujet(s)
Protéines mutées dans l'ataxie-télangiectasie , Cellules tueuses naturelles , Sous-famille K des récepteurs de cellules NK de type lectine , Animaux , Cellules tueuses naturelles/immunologie , Protéines mutées dans l'ataxie-télangiectasie/génétique , Protéines mutées dans l'ataxie-télangiectasie/métabolisme , Sous-famille K des récepteurs de cellules NK de type lectine/génétique , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , Souris , Ataxie-télangiectasie/génétique , Ataxie-télangiectasie/immunologie , Souris knockout , Souris de lignée C57BL , Thymome/immunologie , Thymome/génétique , Facteur de nécrose tumorale alpha/métabolisme , Facteur de nécrose tumorale alpha/immunologie , Cytotoxicité immunologique , Tumeurs du thymus/immunologie , Tumeurs du thymus/génétique , Transduction du signal , Protéines membranaires , Antigènes d'histocompatibilité de classe IRÉSUMÉ
In our previous studies, a novel cryothermal therapy (CTT) was developed to induce systemic long-term anti-tumor immunity. Natural killer (NK) cells were found to play an important role in CTT-induced long-term immune-mediated tumor control at the late stage after CTT, but the underlying mechanism is unclear. Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that have potent immunosuppressive effects on T cells and weaken the long-term benefits of immunotherapy. Consequently, overcoming MDSC immunosuppression is essential for maintaining the long-term efficacy of immunotherapy. In this study, we revealed that NK cells considerably diminish MDSC accumulation at the late stage after CTT, boost T cell production, increase T cell activation, and promote MDSC maturation, culminating in Th1-dominant CD4+ T cell differentiation and enhancing NK and CD8+ T cell cytotoxicity. Additionally, NK cells activate ERK signaling in MDSCs through NKG2D-ligand interaction to increase the activity of tumor necrosis factor (TNF)-α converting enzyme (TACE)-cleaved membrane TNF-α. Furthermore, Increased TACE activity releases more soluble TNF-α from MDSCs to promote MDSC maturation. In our studies, we propose a novel mechanism by which NK cells can overcome MDSC-induced immunosuppression and maintain CTT-induced persistent anti-tumor immunity, providing a prospective therapeutic option to improve the performance of cancer immunotherapy.
Sujet(s)
Cellules tueuses naturelles , Cellules myéloïdes suppressives , Sous-famille K des récepteurs de cellules NK de type lectine , Facteur de nécrose tumorale alpha , Cellules myéloïdes suppressives/métabolisme , Cellules myéloïdes suppressives/immunologie , Cellules tueuses naturelles/immunologie , Cellules tueuses naturelles/métabolisme , Animaux , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , Souris , Facteur de nécrose tumorale alpha/métabolisme , Souris de lignée C57BL , Activation des lymphocytes/immunologie , Différenciation cellulaire , Ligands , Protéine ADAM17/métabolismeRÉSUMÉ
BACKGROUND: Psoriasis is a chronic immune-mediated skin condition. Although biologic treatments are effective in controlling psoriasis, some patients do not respond or lose response to these therapies. Thus, new strategies for psoriasis treatment are still urgently needed. Double-negative T cells (DNT) play a significant immunoregulatory role in autoimmune diseases. In this study, we aimed to evaluate the protective effect of DNT in psoriasis and explore the underlying mechanism. METHODS: We conducted a single adoptive transfer of DNT into an imiquimod (IMQ)-induced psoriasis mouse model through tail vein injection. The skin inflammation and IL-17A producing γδ T cells were evaluated. RESULTS: DNT administration significantly reduced the inflammatory response in mouse skin, characterized by decreased skin folds, scales, and red patches. After DNT treatment, the secretion of IL-17A by RORc+ γδlow T cells in the skin was selectively suppressed, resulting in an amelioration of skin inflammation. Transcriptomic data suggested heightened expression of NKG2D ligands in γδlow T cells within the mouse model of psoriasis induced by IMQ. When blocking the NKG2D ligand and NKG2D (expressed by DNT) interaction, the cytotoxic efficacy of DNT against RORc+IL17A+ γδlow T cells was attenuated. Using Ccr5-/- DNT for treatment yielded evidence that DNT migrates into inflamed skin tissue and fails to protect IMQ-induced skin lesions. CONCLUSIONS: DNT could migrate to inflamed skin tissue through CCR5, selectively inhibit IL-17-producing γδlow T cells and finally ameliorate mouse psoriasis. Our study provides feasibility for using immune cell therapy for the prevention and treatment of psoriasis in the clinic.
Sujet(s)
Interleukine-17 , Psoriasis , Humains , Souris , Animaux , Interleukine-17/métabolisme , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , Psoriasis/thérapie , Peau/anatomopathologie , Imiquimod/effets indésirables , Imiquimod/métabolisme , Inflammation/anatomopathologie , Lymphocytes T/métabolisme , Modèles animaux de maladie humaineRÉSUMÉ
NK cells are cytotoxic innate lymphocytes that play a critical role in antitumor immunity. NK cells recognize target cells by using a repertoire of activating NK receptors and exert the effector functions. Although the magnitude of activation signals through activating NK receptors controls NK cell function, it has not been fully understood how these activating signals are modulated in NK cells. In this study, we found that a scaffold protein, THEMIS2, inhibits activating NK receptor signaling. Overexpression of THEMIS2 attenuated the effector function of human NK cells, whereas knockdown of THEMIS2 enhanced it. Mechanistically, THEMIS2 binds to GRB2 and phosphorylated SHP-1 and SHP-2 at the proximity of activating NK receptors DNAM-1 and NKG2D. Knockdown of THEMIS2 in primary human NK cells promoted the effector functions. Furthermore, Themis2-deficient mice showed low metastatic burden in an NK cell-dependent manner. These findings demonstrate that THEMIS2 has an inhibitory role in the antitumor activity of NK cells, suggesting that THEMIS2 might be a potential therapeutic target for NK cell-mediated cancer immunotherapy.
Sujet(s)
Cellules tueuses naturelles , Transduction du signal , Animaux , Humains , Souris , Lignée cellulaire tumorale , Cytotoxicité immunologique , Cellules tueuses naturelles/immunologie , Activation des lymphocytes/immunologie , Souris de lignée C57BL , Souris knockout , Tumeurs/immunologie , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , Sous-famille K des récepteurs de cellules NK de type lectine/immunologie , Récepteurs immunologiques , Récepteurs de cellules tueuses naturelles/métabolisme , Transduction du signal/immunologieRÉSUMÉ
SARS-CoV-2 and other sarbecoviruses continue to threaten humanity, highlighting the need to characterize common mechanisms of viral immune evasion for pandemic preparedness. Cytotoxic lymphocytes are vital for antiviral immunity and express NKG2D, an activating receptor conserved among mammals that recognizes infection-induced stress ligands (e.g., MIC-A/B). We found that SARS-CoV-2 evades NKG2D recognition by surface downregulation of MIC-A/B via shedding, observed in human lung tissue and COVID-19 patient serum. Systematic testing of SARS-CoV-2 proteins revealed that ORF6, an accessory protein uniquely conserved among sarbecoviruses, was responsible for MIC-A/B downregulation via shedding. Further investigation demonstrated that natural killer (NK) cells efficiently killed SARS-CoV-2-infected cells and limited viral spread. However, inhibition of MIC-A/B shedding with a monoclonal antibody, 7C6, further enhanced NK-cell activity toward SARS-CoV-2-infected cells. Our findings unveil a strategy employed by SARS-CoV-2 to evade cytotoxic immunity, identify the culprit immunevasin shared among sarbecoviruses, and suggest a potential novel antiviral immunotherapy.
Sujet(s)
COVID-19 , Échappement immunitaire , Cellules tueuses naturelles , Sous-famille K des récepteurs de cellules NK de type lectine , SARS-CoV-2 , Humains , SARS-CoV-2/immunologie , Cellules tueuses naturelles/immunologie , Cellules tueuses naturelles/métabolisme , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , COVID-19/immunologie , COVID-19/virologie , Antigènes d'histocompatibilité de classe I/immunologie , Antigènes d'histocompatibilité de classe I/métabolisme , Animaux , Cytotoxicité immunologique , Régulation négative , Poumon/immunologie , Poumon/virologie , Poumon/anatomopathologieRÉSUMÉ
Tumor-associated macrophages (TAM) are abundant in several tumor types and usually correlate with poor prognosis. Previously, we demonstrated that anti-inflammatory macrophages (M2) inhibit NK cell effector functions. Here, we explored the impact of TAM on NK cells in the context of clear-cell renal cell carcinoma (ccRCC). Bioinformatics analysis revealed that an exhausted NK cell signature strongly correlated with an M2 signature. Analysis of TAM from human ccRCC samples confirmed that they exhibited an M2-skewed phenotype and inhibited IFN-γ production by NK cells. Moreover, human M0 macrophages cultured with conditioned media from ccRCC cell lines generated macrophages with an M2-skewed phenotype (TAM-like), which alike TAM, displayed suppressive activity on NK cells. Moreover, TAM depletion in the mouse Renca ccRCC model resulted in delayed tumor growth and reduced volume, accompanied by an increased frequency of IFN-γ-producing tumor-infiltrating NK cells that displayed heightened expression of T-bet and NKG2D and reduced expression of the exhaustion-associated co-inhibitory molecules PD-1 and TIM-3. Therefore, in ccRCC, the tumor microenvironment polarizes TAM toward an immunosuppressive profile that promotes tumor-infiltrating NK cell dysfunction, contributing to tumor progression. In addition, immunotherapy strategies targeting TAM may result in NK cell reinvigoration, thereby counteracting tumor progression.
Sujet(s)
Néphrocarcinome , Interféron gamma , Tumeurs du rein , Cellules tueuses naturelles , Macrophages associés aux tumeurs , Cellules tueuses naturelles/immunologie , Néphrocarcinome/immunologie , Néphrocarcinome/anatomopathologie , Interféron gamma/métabolisme , Interféron gamma/immunologie , Humains , Animaux , Souris , Tumeurs du rein/immunologie , Tumeurs du rein/anatomopathologie , Macrophages associés aux tumeurs/immunologie , Macrophages associés aux tumeurs/métabolisme , Évolution de la maladie , Lignée cellulaire tumorale , Microenvironnement tumoral/immunologie , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , Récepteur cellulaire-2 du virus de l'hépatite A/métabolisme , Récepteur cellulaire-2 du virus de l'hépatite A/immunologie , Récepteur-1 de mort cellulaire programmée/métabolismeRÉSUMÉ
Natural killer (NK) cells exert an indispensable role in innate immune responses against cancer progression, however NK cell dysfunction has been rarely reported in hepatocellular carcinoma (HCC). This study sought to uncover the immunoregulatory mechanisms of tumor-infiltrating NK cells in HCC. A consensus NK cell-based signature (NKS) was constructed using integrative machine learning algorithms based on multi-omics data of HCC patients. HCC tumors had lower numbers of infiltrating NK cells than para-tumor normal liver tissues. Based on the NK cell-associated genes, the NKS was built for HCC prognostic prediction and clinical utilities. Drug targets and novel compounds were then identified for high-NKS groups. RAC1 was confirmed as the hub gene in the NKS genes. RAC1 was upregulated in HCC tumors and positively correlated with shorter survival time. RAC1 overexpression in NK-92 cells facilitated the cancer-killing capacity by the anticancer cytotoxic effectors and the upregulated NKG2D. The survival time of PDX-bearing mice was also prolonged upon NK-92RAC1 cells. Mechanistically, RAC1 interacted with STAT3 and facilitated its activation, thereby enabling its binding to the promoter region of NKG2D and functioning as a transcriptional regulator in NK-92 via molecular docking, Co-IP assay, CHIP and luciferase experiments. Collectively, our study describes a novel function of RAC1 in potentiating NK cell-mediated cytotoxicity against HCC, highlighting the clinical utilities of NKS score and RAC1high NK cell subset in HCC immunotherapy.
Sujet(s)
Carcinome hépatocellulaire , Cellules tueuses naturelles , Tumeurs du foie , Sous-famille K des récepteurs de cellules NK de type lectine , Facteur de transcription STAT-3 , Protéine G rac1 , Tumeurs du foie/immunologie , Tumeurs du foie/génétique , Tumeurs du foie/anatomopathologie , Tumeurs du foie/thérapie , Carcinome hépatocellulaire/immunologie , Carcinome hépatocellulaire/génétique , Carcinome hépatocellulaire/anatomopathologie , Carcinome hépatocellulaire/thérapie , Humains , Cellules tueuses naturelles/immunologie , Cellules tueuses naturelles/métabolisme , Protéine G rac1/génétique , Protéine G rac1/métabolisme , Facteur de transcription STAT-3/métabolisme , Facteur de transcription STAT-3/génétique , Animaux , Souris , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , Sous-famille K des récepteurs de cellules NK de type lectine/génétique , Immunothérapie/méthodes , Lignée cellulaire tumorale , Régulation de l'expression des gènes tumoraux , Mâle , Pronostic , Tests d'activité antitumorale sur modèle de xénogreffe , FemelleRÉSUMÉ
Chimeric antigen receptor (CAR)-modified immune cells have emerged as a promising approach for cancer treatment, but single-target CAR therapy in solid tumors is limited by immune escape caused by tumor antigen heterogeneity and shedding. Natural killer group 2D (NKG2D) is an activating receptor expressed in human NK cells, and its ligands, such as MICA and MICB (MICA/B), are widely expressed in malignant cells and typically absent from healthy tissue. NKG2D plays an important role in anti-tumor immunity, recognizing tumor cells and initiating an anti-tumor response. Therefore, NKG2D-based CAR is a promising CAR candidate. Nevertheless, the shedding of MICA/B hinders the therapeutic efficacy of NKG2D-CARs. Here, we designed a novel CAR by engineering an anti-MICA/B shedding antibody 1D5 into the CAR construct. The engineered NK cells exhibited significantly enhanced cytotoxicity against various MICA/B-expressing tumor cells and were not inhibited by NKG2D antibody or NKG2D-Fc fusion protein, indicating no interference with NKG2D-MICA/B binding. Therefore, the developed 1D5-CAR could be combined with NKG2D-CAR to further improve the obstacles caused by MICA/B shedding.
Sujet(s)
Tumeurs , Récepteurs chimériques pour l'antigène , Humains , Lignée cellulaire tumorale , Antigènes d'histocompatibilité de classe I/immunologie , Antigènes d'histocompatibilité de classe I/métabolisme , Cellules tueuses naturelles , Tumeurs/immunologie , Tumeurs/métabolisme , Sous-famille K des récepteurs de cellules NK de type lectine/génétique , Sous-famille K des récepteurs de cellules NK de type lectine/métabolisme , Récepteurs chimériques pour l'antigène/génétique , Récepteurs chimériques pour l'antigène/métabolisme , Immunothérapie adoptive/méthodesRÉSUMÉ
NK cells in the peripheral blood of severe COVID-19 patients exhibit a unique profile characterized by activation and dysfunction. Previous studies have identified soluble factors, including type I IFN and TGF-ß, that underlie this dysregulation. However, the role of cell-cell interactions in modulating NK cell function during COVID-19 remains unclear. To address this question, we combined cell-cell communication analysis on existing single-cell RNA sequencing data with in vitro primary cell coculture experiments to dissect the mechanisms underlying NK cell dysfunction in COVID-19. We found that NK cells are predicted to interact most strongly with monocytes and that this occurs via both soluble factors and direct interactions. To validate these findings, we performed in vitro cocultures in which NK cells from healthy human donors were incubated with monocytes from COVID-19+ or healthy donors. Coculture of healthy NK cells with monocytes from COVID-19 patients recapitulated aspects of the NK cell phenotype observed in severe COVID-19, including decreased expression of NKG2D, increased expression of activation markers, and increased proliferation. When these experiments were performed in a Transwell setting, we found that only CD56bright CD16- NK cells were activated in the presence of severe COVID-19 patient monocytes. O-link analysis of supernatants from Transwell cocultures revealed that cultures containing severe COVID-19 patient monocytes had significantly elevated levels of proinflammatory cytokines and chemokines, as well as TGF-ß. Collectively, these results demonstrate that interactions between NK cells and monocytes in the peripheral blood of COVID-19 patients contribute to NK cell activation and dysfunction in severe COVID-19.