RESUMEN
Sialic acid-binding immunoglobulin-type lectins (Siglecs) are expressed on the majority of white blood cells of the immune system and play critical roles in immune cell signaling. Through recognition of sialic acid-containing glycans as ligands, they help the immune system distinguish between self and nonself. Because of their restricted cell type expression and roles as checkpoints in immune cell responses in human diseases such as cancer, asthma, allergy, neurodegeneration, and autoimmune diseases they have gained attention as targets for therapeutic interventions. In this review we describe the Siglec family, its roles in regulation of immune cell signaling, current efforts to define its roles in disease processes, and approaches to target Siglecs for treatment of human disease.
Asunto(s)
Susceptibilidad a Enfermedades , Proteínas de Punto de Control Inmunitario/genética , Proteínas de Punto de Control Inmunitario/metabolismo , Inmunomodulación , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Transducción de Señal , Animales , Biomarcadores , Humanos , Sistema Inmunológico/inmunología , Sistema Inmunológico/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismoRESUMEN
The absence of cancer-restricted surface markers is a major impediment to antigen-specific immunotherapy using chimeric antigen receptor (CAR) T cells. For example, targeting the canonical myeloid marker CD33 in acute myeloid leukemia (AML) results in toxicity from destruction of normal myeloid cells. We hypothesized that a leukemia-specific antigen could be created by deleting CD33 from normal hematopoietic stem and progenitor cells (HSPCs), thereby generating a hematopoietic system resistant to CD33-targeted therapy and enabling specific targeting of AML with CAR T cells. We generated CD33-deficient human HSPCs and demonstrated normal engraftment and differentiation in immunodeficient mice. Autologous CD33 KO HSPC transplantation in rhesus macaques demonstrated long-term multilineage engraftment of gene-edited cells with normal myeloid function. CD33-deficient cells were impervious to CD33-targeting CAR T cells, allowing for efficient elimination of leukemia without myelotoxicity. These studies illuminate a novel approach to antigen-specific immunotherapy by genetically engineering the host to avoid on-target, off-tumor toxicity.
Asunto(s)
Células Madre Hematopoyéticas/citología , Inmunoterapia/métodos , Leucemia Mieloide Aguda/terapia , ARN Guía de Kinetoplastida/genética , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genética , Linfocitos T/inmunología , Animales , Diferenciación Celular , Línea Celular Tumoral , Linaje de la Célula , Electroporación , Femenino , Hematopoyesis , Humanos , Leucemia Mieloide Aguda/inmunología , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Trasplante de Neoplasias , Especies Reactivas de Oxígeno , Linfocitos T/citologíaRESUMEN
Siglecs (sialic acid-binding immunoglobulin-like lectins) are a family of vertebrate glycan-binding cell-surface proteins. The majority mediate cellular inhibitory activity once engaged by specific ligands or ligand-mimicking molecules. As a result, Siglec engagement is now of interest as a strategy to therapeutically dampen unwanted cellular responses. When considering allergic inflammation, human eosinophils and mast cells express overlapping but distinct patterns of Siglecs. For example, Siglec-6 is selectively and prominently expressed on mast cells while Siglec-8 is highly specific for both eosinophils and mast cells. This review will focus on a subset of Siglecs and their various endogenous or synthetic sialoside ligands that regulate eosinophil and mast cell function and survival. It will also summarize how certain Siglecs have become the focus of novel therapies for allergic and other eosinophil- and mast cell-related diseases.
Asunto(s)
Eosinófilos , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico , Humanos , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Mastocitos , Antígenos CD/química , LigandosRESUMEN
BACKGROUND: Sialic acid-binding immunoglobulin-like lectin-3 (Siglec-3 [CD33]) is a major Siglec expressed on human mast cells and basophils; engagement of CD33 leads to inhibition of cellular signaling via immunoreceptor tyrosine-based inhibitory motifs. OBJECTIVE: We sought to inhibit human basophil degranulation by simultaneously recruiting inhibitory CD33 to the IgE-FcεRI complex by using monoclonal anti-IgE directly conjugated to CD33 ligand (CD33L). METHODS: Direct and indirect basophil activation tests (BATs) were used to assess both antigen-specific (peanut) and antigen-nonspecific (polyclonal anti-IgE) stimulation. Whole blood from donors with allergy was used for direct BAT, whereas blood from donors with nonfood allergy was passively sensitized with plasma from donors with peanut allergy in the indirect BAT. Blood was incubated with anti-IgE-CD33L or controls for 1 hour or overnight and then stimulated with peanut, polyclonal anti-IgE, or N-formylmethionyl-leucyl-phenylalanine for 30 minutes. Degranulation was determined by measuring CD63 expression on the basophil surface by flow cytometry. RESULTS: Incubation for 1 hour with anti-IgE-CD33L significantly reduced basophil degranulation after both allergen-induced (peanut) and polyclonal anti-IgE stimulation, with further suppression after overnight incubation with anti-IgE-CD33L. As expected, anti-IgE-CD33L did not block basophil degranulation due to N-formylmethionyl-leucyl-phenylalanine, providing evidence that this inhibition is IgE pathway-specific. Finally, CD33L is necessary for this suppression, as monoclonal anti-IgE without CD33L was unable to reduce basophil degranulation. CONCLUSIONS: Pretreating human basophils with anti-IgE-CD33L significantly suppressed basophil degranulation through the IgE-FcεRI complex. The ability to abrogate IgE-mediated basophil degranulation is of particular interest, as treatment with anti-IgE-CD33L before antigen exposure could have broad implications for the treatment of food, drug, and environmental allergies.
Asunto(s)
Basófilos , Degranulación de la Célula , Inmunoglobulina E , Lectina 3 Similar a Ig de Unión al Ácido Siálico , Humanos , Basófilos/inmunología , Inmunoglobulina E/inmunología , Degranulación de la Célula/inmunología , Lectina 3 Similar a Ig de Unión al Ácido Siálico/inmunología , Lectina 3 Similar a Ig de Unión al Ácido Siálico/metabolismo , Tetraspanina 30/inmunología , Tetraspanina 30/metabolismo , Receptores de IgE/inmunología , Receptores de IgE/metabolismo , Hipersensibilidad al Cacahuete/inmunología , Prueba de Desgranulación de los Basófilos , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Antiidiotipos/farmacologíaRESUMEN
Siglecs are a family of sialic acid-binding receptors expressed by cells of the immune system and a few other cell types capable of modulating immune cell functions upon recognition of sialoglycan ligands. While human Siglecs primarily bind to sialic acid residues on diverse types of glycoproteins and glycolipids that constitute the sialome, their fine binding specificities for elaborated complex glycan structures and the contribution of the glycoconjugate and protein context for recognition of sialoglycans at the cell surface are not fully elucidated. Here, we generated a library of isogenic human HEK293 cells with combinatorial loss/gain of individual sialyltransferase genes and the introduction of sulfotransferases for display of the human sialome and to dissect Siglec interactions in the natural context of glycoconjugates at the cell surface. We found that Siglec-4/7/15 all have distinct binding preferences for sialylated GalNAc-type O-glycans but exhibit selectivity for patterns of O-glycans as presented on distinct protein sequences. We discovered that the sulfotransferase CHST1 drives sialoglycan binding of Siglec-3/8/7/15 and that sulfation can impact the preferences for binding to O-glycan patterns. In particular, the branched Neu5Acα2-3(6-O-sulfo)Galß1-4GlcNAc (6'-Su-SLacNAc) epitope was discovered as the binding epitope for Siglec-3 (CD33) implicated in late-onset Alzheimer's disease. The cell-based display of the human sialome provides a versatile discovery platform that enables dissection of the genetic and biosynthetic basis for the Siglec glycan interactome and other sialic acid-binding proteins.
Asunto(s)
Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Análisis de Matrices Tisulares/métodos , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Mucina-1 , Polisacáridos/metabolismo , Sialiltransferasas/genética , Sialiltransferasas/metabolismoRESUMEN
The myelomonocytic receptor CD33 (Siglec-3) inhibits innate immune reactivity by extracellular V-set domain recognition of sialic acid (Sia)-containing "self-associated molecular patterns" (SAMPs). We earlier showed that V-set domain-deficient CD33-variant allele, protective against late-onset Alzheimer's Disease (LOAD), is derived and specific to the hominin lineage. We now report multiple hominin-specific CD33 V-set domain mutations. Due to hominin-specific, fixed loss-of-function mutation in the CMAH gene, humans lack N-glycolylneuraminic acid (Neu5Gc), the preferred Sia-ligand of ancestral CD33. Mutational analysis and molecular dynamics (MD)-simulations indicate that fixed change in amino acid 21 of hominin V-set domain and conformational changes related to His45 corrected for Neu5Gc-loss by switching to N-acetylneuraminic acid (Neu5Ac)-recognition. We show that human-specific pathogens Neisseria gonorrhoeae and Group B Streptococcus selectively bind human CD33 (huCD33) as part of immune-evasive molecular mimicry of host SAMPs and that this binding is significantly impacted by amino acid 21 modification. In addition to LOAD-protective CD33 alleles, humans harbor derived, population-universal, cognition-protective variants at several other loci. Interestingly, 11 of 13 SNPs in these human genes (including CD33) are not shared by genomes of archaic hominins: Neanderthals and Denisovans. We present a plausible evolutionary scenario to compile, correlate, and comprehend existing knowledge about huCD33-evolution and suggest that grandmothering emerged in humans.
Asunto(s)
Abuelos , Hominidae , Alelos , Aminoácidos , Animales , Cognición , Hominidae/genética , HumanosRESUMEN
Standard agarose gel electrophoresis is a widely used method to analyse diversity of nucleic acids. Certain conditions, however, may give rise to artefactual bands. We report on artefactual bands frequently occurring, especially when partially homologous nucleic acids, such as splicing variants of DNA transcripts, are analysed simultaneously. Interestingly, to some extent agarose concentration may influence the occurrence of artefactual bands.
Asunto(s)
ADN , Ácidos Nucleicos , Sefarosa , Electroforesis en Gel de Agar/métodosRESUMEN
The structure of a sialoglycan can be translated into to a biological response when it binds to a specific endogenous lectin. Among endogenous sialic acid-binding lectins in humans are those comprising the 15-member Siglec family, most of which are expressed on overlapping sets of immune cells. Endogenous Siglec ligands are sialoglycolipids (gangliosides) and/or sialoglycoproteins, on cell surfaces or in the extracellular milieu, that bind to and initiate signaling by cell surface Siglecs. In the nervous system, where gangliosides are the predominant sialoglycans, Siglec-4 (myelin-associated glycoprotein) on myelinating cells binds to gangliosides GD1a and GT1b on nerve cell axons to ensure stable and productive axon-myelin interactions. In the immune system, Siglec-7 on natural killer cells binds to gangliosides GD3 and GD2 to inhibit immune signaling. Expression of GD3 and GD2 on cancer cells can lead to tumor immune evasion. Siglec-1 (sialoadhesin, CD169) on macrophages binds to gangliosides on tumors and enveloped viruses. This may enhance antigen presentation in some cases, or increase viral distribution in others. Several other Siglecs bind to gangliosides in vitro, the biological significance of which has yet to be fully established. Gangliosides, which are found on all human cells and tissues in cell-specific distributions, are functional Siglec ligands with varied roles driving Siglec-mediated signaling.
Asunto(s)
Gangliósidos , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico , Humanos , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Ligandos , Gangliósidos/metabolismo , Neuronas/metabolismoRESUMEN
We have reported previously that CD33hi myeloid-derived suppressor cells (MDSCs) play a direct role in the pathogenesis of myelodysplastic syndromes (MDSs) and that their sustained activation contributes to hematopoietic and immune impairment, including modulation of PD1/PDL1. MDSCs can also limit the clinical activity of immune checkpoint inhibition in solid malignancies. We hypothesized that depletion of MDSCs may ameliorate resistance to checkpoint inhibitors and, hence, targeted them with AMV564 combined with anti-PD1 in MDS bone marrow (BM) mononuclear cells (MNCs) enhanced activation of cytotoxic T cells. AMV564 was active in vivo in a leukemia xenograft model when co-administered with healthy donor peripheral blood MNCs (PBMCs). Our findings provide a strong rationale for clinical investigation of AMV564 as a single agent or in combination with an anti-PD1 antibody and in particular for treatment of cancers resistant to checkpoint inhibitors.
Asunto(s)
Anticuerpos Biespecíficos , Antineoplásicos , Melanoma , Síndromes Mielodisplásicos , Células Supresoras de Origen Mieloide , Animales , Anticuerpos Biespecíficos/farmacología , Antineoplásicos/farmacología , Humanos , Melanoma/tratamiento farmacológico , Síndromes Mielodisplásicos/tratamiento farmacológico , Lectina 3 Similar a Ig de Unión al Ácido Siálico , Linfocitos TRESUMEN
Chimeric antigen receptor natural killer (CAR-NK) cells have remarkable cytotoxicity against hematologic malignancies; however, they may also attack normal cells sharing the target antigen. Since human leukocyte antigen DR (HLA-DR) is reportedly lost or downregulated in a substantial proportion of hematologic malignancies, presumably a mechanism to escape immune surveillance, we hypothesize that the anti-cancer specificity of CAR-NK cells can be enhanced by activating them against cancer antigens while inhibiting them against HLA-DR. Here, we report the development of an anti-HLA-DR inhibitory CAR (iCAR) that can effectively suppress NK cell activation against HLA-DR-expressing cells. We show that dual CAR-NK cells, which co-express the anti-CD19 or CD33 activating CAR and the anti-HLA-DR iCAR, can preferentially target HLA-DR-negative cells over HLA-DR-positive cells in vitro. We find that the HLA-DR-mediated inhibition is positively correlated with both iCAR and HLA-DR densities. We also find that HLA-DR-expressing surrounding cells do not affect the target selectivity of dual CAR-NK cells. Finally, we confirm that HLA-DR-positive cells are resistant to dual CAR-NK cell-mediated killing in a xenograft mouse model. Our approach holds great promise for enhancing CAR-NK and CAR-T cell specificity against malignancies with HLA-DR loss.
Asunto(s)
Neoplasias Hematológicas , Neoplasias , Receptores Quiméricos de Antígenos , Animales , Línea Celular Tumoral , Antígenos HLA-DR/genética , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/terapia , Humanos , Inmunoterapia Adoptiva , Complejo Hierro-Dextran , Ratones , Receptores Quiméricos de Antígenos/genéticaRESUMEN
BACKGROUND: Microglial activation is considered to assume a role in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). To date, the relationship between ALS and the rs3865444 polymorphism of the cluster of differentiation 33 (CD33) has not been explored. The current report aimed to investigate the potential connection between CD33 rs3865444 and ALS. METHODS: Patients diagnosed with sporadic ALS according to the revised El Escorial criteria, as well as age and sex matched community controls, were enrolled. Two evenly numbered, age and sex matched groups of 155 participants each were genotyped. RESULTS: No association was found between rs3865444 and ALS [log-additive odds ratio (OR) = 0.83 (0.57, 1.22), over-dominant OR = 0.86 (0.55, 1.36), recessive OR = 0.73 (0.25, 2.17), dominant OR = 0.82 (0.52, 1.29), co-dominant OR1 = 0.68 (0.23, 2.05) and co-dominant OR2 = 0.84 (0.53, 1.33)]. Moreover, no relationship was established between rs3865444 and the age of ALS onset based on both unadjusted and sex adjusted Cox-proportional hazards models. Finally, no association between rs3865444 and ALS was found in subgroup analyses based on the site of ALS onset (bulbar or spinal) and sex. CONCLUSIONS: The current analysis is the first to report that rs3865444 is not linked to ALS. Larger multi-racial studies are required to confirm these findings.
Asunto(s)
Esclerosis Amiotrófica Lateral , Humanos , Esclerosis Amiotrófica Lateral/genética , Estudios de Casos y Controles , Lectina 3 Similar a Ig de Unión al Ácido SiálicoRESUMEN
Immunotherapies, such as chimeric antigen receptor (CAR) modified T cells and antibody-drug conjugates (ADCs), have revolutionized the treatment of cancer, especially of lymphoid malignancies. The application of targeted immunotherapy to patients with acute myeloid leukemia (AML) has been limited in particular by the lack of a tumor-specific target antigen. Gemtuzumab ozogamicin (GO), an ADC targeting CD33, is the only approved immunotherapeutic agent in AML. In our study, we introduce a CD33-directed third-generation CAR T-cell product (3G.CAR33-T) for the treatment of patients with AML. 3G.CAR33-T cells could be expanded up to the end-of-culture, that is, 17 days after transduction, and displayed significant cytokine secretion and robust cytotoxic activity when incubated with CD33-positive cells including cell lines, drug-resistant cells, primary blasts as well as normal hematopoietic stem and progenitor cells (HSPCs). When compared to second-generation CAR33-T cells, 3G.CAR33-T cells exhibited higher viability, increased proliferation and stronger cytotoxicity. Also, GO exerted strong antileukemia activity against CD33-positive AML cells. Upon genomic deletion of CD33 in HSPCs, 3G.CAR33-T cells and GO preferentially killed wildtype leukemia cells, while sparing CD33-deficient HSPCs. Our data provide evidence for the applicability of CD33-targeted immunotherapies in AML and its potential implementation in CD33 genome-edited stem cell transplantation approaches.
Asunto(s)
Gemtuzumab/uso terapéutico , Trasplante de Células Madre Hematopoyéticas , Inmunoterapia Adoptiva , Leucemia Mieloide Aguda/terapia , Receptores Quiméricos de Antígenos/inmunología , Lectina 3 Similar a Ig de Unión al Ácido Siálico/inmunología , Edición Génica , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/patología , Lectina 3 Similar a Ig de Unión al Ácido Siálico/análisis , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genéticaRESUMEN
BACKGROUND AIMS: Efforts to safely and effectively treat acute myeloid leukemia (AML) by targeting a single leukemia-associated antigen with chimeric antigen receptor (CAR) T cells have met with limited success, due in part to heterogeneous expression of myeloid antigens. The authors hypothesized that T cells expressing CARs directed toward two different AML-associated antigens would eradicate tumors and prevent relapse. METHODS: For co-transduction with the authors' previously optimized CLL-1 CAR currently in clinical study (NCT04219163), the authors generated two CARs targeting either CD123 or CD33. The authors then tested the anti-tumor activity of T cells expressing each of the three CARs either alone or after co-transduction. The authors analyzed CAR T-cell phenotype, expansion and transduction efficacy and assessed function by in vitro and in vivo activity against AML cell lines expressing high (MOLM-13: CD123 high, CD33 high, CLL-1 intermediate), intermediate (HL-60: CD123 low, CD33 intermediate, CLL-1 intermediate/high) or low (KG-1a: CD123 low, CD33 low, CLL-1 low) levels of the target antigens. RESULTS: The in vitro benefit of dual expression was most evident when the target cell line expressed low antigen levels (KG-1a). Mechanistically, dual expression was associated with higher pCD3z levels in T cells compared with single CAR T cells on exposure to KG-1a (P < 0.0001). In vivo, combinatorial targeting with CD123 or CD33 and CLL-1 CAR T cells improved tumor control and animal survival for all lines (KG-1a, MOLM-13 and HL-60); no antigen escape was detected in residual tumors. CONCLUSIONS: Overall, these findings demonstrate that combinatorial targeting of CD33 or CD123 and CLL-1 with CAR T cells can control growth of heterogeneous AML tumors.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Leucemia Mieloide Aguda , Animales , Línea Celular Tumoral , Inmunoterapia Adoptiva , Subunidad alfa del Receptor de Interleucina-3 , Leucemia Mieloide Aguda/terapia , Linfocitos TRESUMEN
BACKGROUND: The aim of this study was to analyze the level of CD33 expression in patients with newly diagnosed AML and determine its correlation with clinical characteristics. METHODS: Samples were collected for analysis from AML patients at diagnosis. We evaluated the level of CD33 expression by flow cytometry analysis of bone marrow. Chi-square or t- tests were used to assess the association between the high and low CD33 expression groups. Survival curves were generated by the Kaplan-Meier and Cox regression model method. RESULTS: In this study we evaluated the level of CD33 expression in de novo patients diagnosed from November 2013 until January 2019. The mean value of 73.4% was used as the cutoff for the two groups. Statistical analysis revealed that 53 of the 86 (61.2%) AML patients were above the mean. Although there was no statistical significance between CD33 expression level and gene mutation, FLT3 mutation (P = 0.002) and NPM1 mutation (P = 0.001) were more likely to be seen in the high CD33 group. The overall survival (OS) was worse in the high CD33 group (39.0 m vs. 16.7 m, x2 = 13.06, P < 0.001). The Cox survival regression display that the CD33 is independent prognostic marker (HR =0.233,p = 0.008). Univariate analysis showed that the high expression of CD33 was an unfavorable prognostic factor. Of the 86 patients, CD33-high was closely related to the patients with normal karyotype (x2 = 4.891,P = 0.027), high white blood cell count (WBC, t = 2.804, P = 0.007), and a high ratio of primitive cells (t = 2.851, P = 0.005). CONCLUSIONS: These findings provide a strong rationale for targeting CD33 in combination with chemotherapy, which can be considered a promising therapeutic strategy for AML.
Asunto(s)
Médula Ósea/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Lectina 3 Similar a Ig de Unión al Ácido Siálico/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Femenino , Citometría de Flujo , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Mutación , Nucleofosmina/genética , Pronóstico , Modelos de Riesgos Proporcionales , Tasa de Supervivencia , Adulto Joven , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
BACKGROUND/AIMS: In despite of conflicting results among different ethnic groups, the rs3865444 of CD33 gene has previously been identified as a risk factor for late-onset Alzheimer's disease (LOAD).This study was aimed to evaluate the association between rs3865444 SNP with LOAD occurrence, and to investigate whether CD33 mRNA expression will change in the leukocytes of peripheral blood in LOAD patients. METHODS: The rs3865444 polymorphism was genotyped in 233 LOAD and 238 control subjects using the Tetra-ARMS-PCR method. CD33 mRNAs expression in leukocytes were assessed and analyzed using the real-time qPCR method. We used in silico approach to analyze potential effects imparted by rs3865444 polymorphism in LOAD pathogenesis. RESULTS: Our results show a significant increase in CD33 mRNA expression levels in white blood cells of LOAD patients, however, the association between CD33 rs3865444 polymorphism and LOAD was found to be not significant. We also noticed that LOAD patients with the C/A genotype had higher CD33 mRNA levels in their peripheral blood than those of the control group. CONCLUSIONS: rs3865444, located upstream of the 5'CD33 coding region, might positively influence CD33 mRNAs expression in leukocytes of LOAD versus healthy people. This is likely to happen through interfering rs3865444 (C) with the functional activity of several other transcription factors given that rs3865444 is in linkage disequilibrium with other functional polymorphisms in this coding region according to an in silico study. We propose that CD33 mRNAs elevation in peripheral immune cells - as a potential biomarker in LOAD - is related to peripheral immune system impairment.
Asunto(s)
Enfermedad de Alzheimer , Predisposición Genética a la Enfermedad , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Humanos , Leucocitos/metabolismo , Polimorfismo de Nucleótido Simple , ARN Mensajero/genética , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genéticaRESUMEN
Antigen-directed immunotherapies for acute myeloid leukemia (AML), such as chimeric antigen receptor T cells (CAR-Ts) or antibody-drug conjugates (ADCs), are associated with severe toxicities due to the lack of unique targetable antigens that can distinguish leukemic cells from normal myeloid cells or myeloid progenitors. Here, we present an approach to treat AML by targeting the lineage-specific myeloid antigen CD33. Our approach combines CD33-targeted CAR-T cells, or the ADC Gemtuzumab Ozogamicin with the transplantation of hematopoietic stem cells that have been engineered to ablate CD33 expression using genomic engineering methods. We show highly efficient genetic ablation of CD33 antigen using CRISPR/Cas9 technology in human stem/progenitor cells (HSPC) and provide evidence that the deletion of CD33 in HSPC doesn't impair their ability to engraft and to repopulate a functional multilineage hematopoietic system in vivo. Whole-genome sequencing and RNA sequencing analysis revealed no detectable off-target mutagenesis and no loss of functional p53 pathways. Using a human AML cell line (HL-60), we modeled a postremission marrow with minimal residual disease and showed that the transplantation of CD33-ablated HSPCs with CD33-targeted immunotherapy leads to leukemia clearance, without myelosuppression, as demonstrated by the engraftment and recovery of multilineage descendants of CD33-ablated HSPCs. Our study thus contributes to the advancement of targeted immunotherapy and could be replicated in other malignancies.
RESUMEN
The CD33 gene encodes for a member of the sialic-acid-binding immunoglobulin-type lectin (Siglec) family, and is one of the top-ranked Alzheimer's disease (AD) risk genes identified by genome-wide association studies (GWAS). Many CD33 polymorphisms are associated with an increased risk of AD, but the function and potential mechanism of many CD33 single-nucleotide polymorphisms (SNPs) in promoting AD have yet to be elucidated. We recently identified the CD33 SNP rs2455069-A>G (R69G) in a familial form of dementia. Here, we demonstrate an association between the G allele of the rs2455069 gene variant and the presence of AD in a cohort of 195 patients from southern Italy. We carried out in silico analysis of the 3D structures of CD33 carrying the identified SNP to provide insights into its functional effect. Structural models of the CD33 variant carrying the R69G amino acid change were compared to the CD33 wild type, and used for the docking analysis using sialic acid as the ligand. Our analysis demonstrated that the CD33-R69G variant may bind sialic acid at additional binding sites compared to the wild type, thus potentially increasing its affinity/specificity for this molecule. Our results led to a new hypothesis of rs2455069-A>G SNP as a risk factor for AD, suggesting that a long-term cumulative effect of the CD33-R69G variant results from the binding of sialic acid, acting as an enhancer of the CD33 inhibitory effects on amyloid plaque degradation.
Asunto(s)
Enfermedad de Alzheimer , Polimorfismo de Nucleótido Simple , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Microglía/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genéticaRESUMEN
Genome-wide association studies demonstrated that polymorphisms in the CD33/sialic acid-binding immunoglobulin-like lectin 3 gene are associated with late-onset Alzheimer's disease (AD). CD33 is expressed on myeloid immune cells and mediates inhibitory signaling through protein tyrosine phosphatases, but the exact function of CD33 in microglia is still unknown. Here, we analyzed CD33 knockout human THP1 macrophages and human induced pluripotent stem cell-derived microglia for immunoreceptor tyrosine-based activation motif pathway activation, cytokine transcription, phagocytosis, and phagocytosis-associated oxidative burst. Transcriptome analysis of the macrophage lines showed that knockout of CD33 as well as knockdown of the CD33 signaling-associated protein tyrosine phosphatase, nonreceptor type 6 (PTPN6) led to constitutive activation of inflammation-related pathways. Moreover, deletion of CD33 or expression of Exon 2-deleted CD33 (CD33ΔE2 /CD33m) led to increased phosphorylation of the kinases spleen tyrosine kinase (SYK) and extracellular signal-regulated kinase 1 and 2 (ERK1 and 2). Transcript analysis by quantitative real-time polymerase chain reaction confirmed increased levels of interleukin (IL) 1B, IL8, and IL10 after knockout of CD33 in macrophages and microglia. In addition, upregulation of the gene transcripts of the AD-associated phosphatase INPP5D was observed after knockout of CD33. Functional analysis of macrophages and microglia showed that phagocytosis of aggregated amyloid-ß1-42 and bacterial particles were increased after knockout of CD33 or CD33ΔE2 expression and knockdown of PTPN6. Furthermore, the phagocytic oxidative burst during uptake of amyloid-ß1-42 or bacterial particles was increased after CD33 knockout but not in CD33ΔE2 -expressing microglia. In summary, deletion of CD33 or expression of CD33ΔE2 in human macrophages and microglia resulted in putative beneficial phagocytosis of amyloid ß1-42 , but potentially detrimental oxidative burst and inflammation, which was absent in CD33ΔE2 -expressing microglia.
Asunto(s)
Enfermedad de Alzheimer , Células Madre Pluripotentes Inducidas , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides , Estudio de Asociación del Genoma Completo , Humanos , Inflamación , Microglía , Fenotipo , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genéticaRESUMEN
CD33 is a Siglec (sialic acid-binding immunoglobulin-type lectin) receptor on microglia. Human CD33 can be alternatively spliced into two isoforms: the long isoform (CD33M) and a shorter isoform (CD33m) that lacks the sialic acid-binding site. CD33m appears to protect against Alzheimer's disease; however, it remains unclear how. To investigate potential mechanisms by which CD33m may confer protection, we expressed the CD33m and CD33M isoforms of human CD33 in mouse BV-2 and human CHME3 microglial cells and assessed microglia functions. In the BV-2 cells, CD33M inhibited microglial phagocytosis of beads, synapses, debris and dead cells, while CD33m increased phagocytosis of beads, debris and cells. RNAi knockdown of the endogenous mouse CD33 increased phagocytosis and prevented CD33m's (but not CD33M's) effect on phagocytosis. CD33M increased cell attachment but inhibited cell proliferation, while CD33m did the opposite. We also found that CD33M inhibited cell migration. In human CHME3 cells, CD33M increased cell attachment, but inhibited phagocytosis, proliferation and migration, whereas CD33m did the opposite. We conclude that CD33M inhibits microglial phagocytosis, inhibits migration and increases adhesion, while CD33m increases phagocytosis, proliferation and inhibits adhesion. Thus, CD33m might protect against Alzheimer's disease by increasing microglial proliferation, movement and phagocytosis of debris and dead cells.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Microglía/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genética , Enfermedad de Alzheimer/genética , Animales , Línea Celular , Encefalitis/genética , Técnicas de Silenciamiento del Gen , Variación Genética , Humanos , Ratones , Neuraminidasa/química , Interferencia de ARN , Lectina 3 Similar a Ig de Unión al Ácido Siálico/metabolismoRESUMEN
Similar to pediatric acute myeloid leukemia (AML) the subgroup of biphenotypic acute lymphoblastic leukemia (ALL) is a rare complex entity with adverse outcome, characterized by the surface expression of CD33. Despite novel and promising anti-CD19 targeted immunotherapies such as chimeric antigen receptor T cells and bispecific anti-CD19/CD3 antibodies, relapse and resistance remain a major challenge in about 30% to 60% of patients. To investigate the potential role of the fully humanized bispecific antibody CD16 × CD33 (BiKE) in children with CD33+ acute leukemia, we tested whether the reagent was able to boost NK cell effector functions against CD33+ AML and biphenotypic ALL blasts. Stimulation of primary NK cells from healthy volunteers with 16 × 33 BiKE led to increased cytotoxicity, degranulation and cytokine production against CD33+ cell lines. Moreover, BiKE treatment significantly increased degranulation, IFN-γ and TNF-α production against primary ALL and AML targets. Importantly, also NK cells from leukemic patients profited from restoration of effector functions by BiKE treatment, albeit to a lesser extent than NK cells from healthy donors. In particular, those patients with low perforin and granzyme expression showed compromised cytotoxic function even in the presence of BiKE. In patients with intrinsic NK cell deficiency, combination therapy of CD16xCD33 BiKE and allogeneic NK cells might thus be a promising therapeutic approach. Taken together, CD16xCD33 BiKE successfully increased NK cell effector functions against pediatric AML and biphenotypic ALL blasts and constitutes a promising new option for supporting maintenance therapy or "bridging" consolidation chemotherapy before hematopoietic stem cell transplantation.