RESUMEN
BACKGROUND AIMS: For patients needing allogeneic stem cell transplantation but lacking a major histocompatibility complex (MHC)-matched donor, haplo-identical (family) donors may be an alternative. Stringent T-cell depletion required in these cases to avoid lethal graft-versus-host disease (GVHD) can delay immune reconstitution, thus impairing defense against virus reactivation and attenuating graft-versus-leukemia (GVL) activity. Several groups reported that GVHD is caused by cells residing within the naive (CD45RA+) T-cell compartment and proposed use of CD45RA-depleted donor lymphocyte infusion (DLI) to accelerate immune reconstitution. We developed and tested the performance of a CD45RA depletion module for the automatic cell-processing device CliniMACS Prodigy and investigated quality attributes of the generated products. METHODS: Unstimulated apheresis products from random volunteer donors were depleted of CD45RA+ cells on CliniMACS Prodigy, using Good Manufacturing Practice (GMP)-compliant reagents and methods throughout. Using phenotypic and functional in vitro assays, we assessed the cellular constitution of CD45RA-depleted products, including T-cell subset analyses, immunological memory function and allo-reactivity. RESULTS: Selections were technically uneventful and proceeded automatically with minimal hands-on time beyond tubing set installation. Products were near-qualitatively CD45RA+ depleted, that is, largely devoid of CD45RA+ T cells but also of almost all B and natural killer cells. Naive and effector as well as γ/δ T cells were greatly reduced. The CD4:CD8 ratio was fivefold increased. Mixed lymphocyte reaction assays of the product against third-party leukocytes revealed reduced allo-reactivity compared to starting material. Anti-pathogen responses were retained. DISCUSSION: The novel, closed, fully GMP-compatible process on Prodigy generates highly CD45RA-depleted cellular products predicted to be clinically meaningfully depleted of GvH reactivity.
Asunto(s)
Enfermedad Injerto contra Huésped/prevención & control , Memoria Inmunológica/fisiología , Inmunoterapia Adoptiva , Antígenos Comunes de Leucocito/metabolismo , Depleción Linfocítica , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/trasplante , Adulto , Automatización de Laboratorios , Células Cultivadas , Femenino , Enfermedad Injerto contra Huésped/inmunología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/métodos , Prueba de Histocompatibilidad , Humanos , Separación Inmunomagnética/instrumentación , Separación Inmunomagnética/métodos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Leucaféresis/instrumentación , Leucaféresis/métodos , Prueba de Cultivo Mixto de Linfocitos , Depleción Linfocítica/instrumentación , Depleción Linfocítica/métodos , Masculino , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Donantes de Tejidos , Trasplante Homólogo , Adulto JovenRESUMEN
EMR3 is a member of the epidermal growth factor-seven-transmembrane (EGF-TM7) family of adhesion class TM7 receptors. This family also comprises CD97, EMR1, EMR2, and EMR4. To characterize human EMR3 at the protein level, we generated Armenian hamster mAb. Using the mAb 3D7, we here demonstrate that EMR3, like other EGF-TM7 receptors, is expressed at the cell surface as a heterodimeric molecule consisting of a long extracellular alpha-chain, which possesses at its N-terminus EGF-like domains and a membrane-spanning beta-chain. Flow cytometric analysis revealed that all types of myeloid cells express EMR3. In peripheral blood, the highest expression of EMR3 was found on granulocytes. More mature CD16(+) monocytes express high levels of EMR3, and CD16(-) monocytes and myeloid dendritic cells (DC) are EMR3(dim/low). Lymphocytes and plasmacytoid DC are EMR3(-). It is interesting that in contrast with CD97 and EMR2, CD34(+)CD33(-)/CD38(-) committed hematopoietic stem cells and CD34(+)CD33(+)/CD38(+) progenitors in bone marrow do not express EMR3. In vitro differentiation of HL-60 cells and CD34(+) progenitor cells revealed that EMR3 is only up-regulated during late granulopoiesis. These results demonstrate that the expression of EGF-TM7 receptors on myeloid cells is differentially regulated. EMR3 is the first family member found mainly on granulocytes.
Asunto(s)
Biomarcadores , Granulocitos/inmunología , Receptores Acoplados a Proteínas G/inmunología , Receptores de Péptidos/inmunología , Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Citometría de Flujo , Células HL-60 , Humanos , Monocitos/inmunología , Neutrófilos/inmunología , Receptores Acoplados a Proteínas G/biosíntesis , Receptores de IgG/inmunología , Receptores de Péptidos/biosíntesis , Células Madre/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
The epidermal growth factor (EGF)-TM7 receptors CD97, EMR1, EMR2, EMR3, and EMR4 form a group of adhesion class heptahelical molecules predominantly expressed by cells of the immune system. These receptors bind cellular ligands through EGF-like domains, localized N-terminal to a large extracellular region. Remarkably, EMR2 possesses a chimeric structure with a seven-span transmembrane (TM7) region most related to EMR3 and an EGF domain region nearly identical to CD97. By comparing EGF-TM7 receptors in primates and dogs, we identified an intriguing pattern of concerted evolution, apparently mediated by gene conversion, among EMR2 and the oppositely orientated and physically adjacent genes CD97 and EMR3. This concerted evolution has continuously maintained the chimeric structure of EMR2 since early mammal radiation. Most highly conserved between EMR2 and CD97 is the fourth EGF domain, which mediates binding to chondroitin sulfate, a ligand specificity shared by both receptors. Another ligand, CD55, is bound effectively only by CD97. We show that different molecular mechanisms (mutations vs. alternative splicing) prevent CD55 binding by EMR2 in hominoids. Our findings illustrate how various and partially opposing evolutionary events have shaped the structure and ligand specificity of a modern mammalian gene family.
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Evolución Molecular , Receptores Acoplados a Proteínas G/genética , Secuencia de Aminoácidos , Animales , Callithrix , Perros , Macaca mulatta , Datos de Secuencia Molecular , Pan troglodytes , Papio hamadryas , Pongo pygmaeus , Conformación Proteica , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G/química , SaguinusRESUMEN
The EGF-TM7 receptors CD97 and EMR2 are heptahelical molecules predominantly expressed on leukocytes. A characteristic of these receptors is their ability to interact with cellular ligands via the N-terminal epidermal growth factor (EGF)-like domains. The first two EGF domains of CD97 (but not EMR2) bind CD55 (decay-accelerating factor), while the fourth EGF domain of both CD97 and EMR2 interacts with the glycosaminoglycan chondroitin sulfate (CS). Using fluorescent beads coated with soluble recombinant CD97 and EMR2 protein, and isoform-specific monoclonal antibodies, we have determined the cellular and molecular characteristics of the interaction with CS. The fourth EGF domain of CD97 and EMR2 is expressed on activated lymphocytes and myeloid cells, whereas the ligand is specifically found on B cells within the peripheral blood. The interaction between CD97/EMR2 and CS may therefore play a role in the interaction of activated T cells, dendritic cells, and macrophages with B cells.
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Linfocitos B/metabolismo , Sulfatos de Condroitina/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Leucocitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Anticuerpos Monoclonales/metabolismo , Antígenos CD , Antígenos CD55/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Humanos , Ligandos , Activación de Linfocitos , Macrófagos/metabolismo , Células Mieloides/metabolismo , Isoformas de Proteínas , Receptores Acoplados a Proteínas GRESUMEN
The EGF-TM7 family is a group of class B seven-span transmembrane (TM7) receptors expressed predominantly by cells of the immune system. Family members CD97, EMR1, EMR2, EMR3, and ETL are characterized by an extended extracellular region with a variable number of N-terminal epidermal growth factor (EGF)-like domains coupled to a TM7 domain by a stalk. The EGF domain region of the recently identified EMR2 differs from that of CD97 in only 6 out of 236 amino acids. Although small, this difference has been shown to alter ligand specificity. To analyze the structure and cellular distribution of EMR2, a specific monoclonal antibody (2A1) was generated. Use of 2A1 has demonstrated EMR2, like CD97, to be expressed as a heterodimeric receptor consisting of an extracellular alpha part and a TM7/cytoplasmic beta part. Analysis of EMR2 expression on primary blood leukocytes, on hematopoietic cells lines, and in situ revealed a myeloid-restricted profile. Highest expression levels were detected on the more mature CD16(+) blood monocytes, on macrophages, and on BDCA-3(+) myeloid DC, whereas little if any expression was found on granulocytes. Unlike CD97, no expression was observed on resting or activated lymphocytes. Different expression patterns and the inability of EMR2 to interact with the CD97 ligand CD55 indicate that the molecular twins EMR2 and CD97 likely have nonredundant functions.
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Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/metabolismo , Células Mieloides/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Anticuerpos Monoclonales/inmunología , Antígenos CD , Diferenciación Celular , Línea Celular , Células Cultivadas , Células Dendríticas/química , Células Dendríticas/metabolismo , Dimerización , Factor de Crecimiento Epidérmico/inmunología , Granulocitos/inmunología , Granulocitos/metabolismo , Humanos , Lectinas/análisis , Linfocitos/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/análisis , Monocitos/inmunología , Monocitos/metabolismo , Receptores Acoplados a Proteínas G , Distribución TisularRESUMEN
Despite high remission rates after therapy, 60% to 70% of patients with acute myeloid leukemia (AML) do not survive 5 years after their initial diagnosis. The main cause of treatment failures may be insufficient eradication of a subpopulation of leukemic stem-like cells (LSC), which are thought to be responsible for relapse by giving rise to more differentiated leukemic progenitors (LP). To address the need for therapeutic targets in LSCs, we compared microRNA (miRNA) expression patterns in highly enriched healthy CD34(+)CD38(-) hematopoietic stem cells (HSC), CD34(+)CD38(-) LSCs, and CD34(+)CD38(+) LPs, all derived from the same patients' bone marrow (BM) specimens. In this manner, we identified multiple differentially expressed miRNAs, in particular miR-126, which was highly expressed in HSCs and increased in LSCs compared with LPs, consistent with a stem-like cell function. High miR-126 expression in AML was associated with poor survival, higher chance of relapse, and expression of genes present in LSC/HSC signatures. Notably, attenuating miR-126 expression in AML cells reduced in vitro cell growth by inducing apoptosis, but did not affect the survival of normal BM in which it instead enhanced expansion of HSCs. Furthermore, targeting miR-126 in LSCs and LPs reduced their clonogenic capacity and eliminated leukemic cells, again in the absence of similar inhibitory effects on normal BM cells. Our results define miR-126 as a therapeutic focus to specifically eradicate LSCs and improve AML outcome.
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ADP-Ribosil Ciclasa 1/análisis , Antígenos CD34/análisis , Células Madre Hematopoyéticas/metabolismo , Leucemia Mieloide Aguda/terapia , MicroARNs/fisiología , Apoptosis , Supervivencia Celular , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , MicroARNs/análisis , MicroARNs/antagonistas & inhibidores , Células Madre Neoplásicas/metabolismo , Pronóstico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Classical macrophage activation is inhibited by the CD200 receptor (CD200R). Here, we show that CD200R expression was specifically induced on human in vitro polarized macrophages of the alternatively activated M2a subtype, generated by incubation with IL-4 or IL-13. In mice, peritoneal M2 macrophages, elicited during infection with the parasites Taenia crassiceps or Trypanosoma brucei brucei, expressed increased CD200R levels compared to those derived from uninfected mice. However, in vitrostimulation of mouse peritoneal macrophages and T. crassiceps infection in IL-4-/- and IL-4R-/- mice showed that, in contrast to humans, induction of CD200R in mice was not IL-4 or IL-13 dependent. Our data identify CD200R as a suitable marker for alternatively activated macrophages in humans and corroborate observations of distinct species- and/or site-specific mechanisms regulating macrophage polarization in mouse and man.
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Antígenos CD/metabolismo , Interleucina-13/inmunología , Interleucina-4/inmunología , Activación de Macrófagos/inmunología , Macrófagos Peritoneales/inmunología , Animales , Humanos , Macrófagos Peritoneales/parasitología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Taenia/inmunología , Teniasis/inmunología , Teniasis/parasitología , Trypanosoma brucei brucei/inmunología , Tripanosomiasis Africana/inmunología , Tripanosomiasis Africana/parasitologíaRESUMEN
The members of the epidermal growth factor (EGF)-transmembrane (TM)7 family of adhesion class G-protein coupled receptors are abundantly expressed by cells of the myeloid lineage. A detailed investigation of their expression by functional subsets of activated macrophages is still lacking. Therefore, we determined the expression of CD97, EGF module-containing mucin-like receptor (EMR)2 and EMR3 by monocyte-derived macrophages experimentally polarized in vitro. This was compared to three types of disease-associated lipid-laden macrophages displaying an alternatively activated phenotype in situ. Polarization in vitro towards classically activated M1 versus alternatively activated M2 extremes of macrophage activation did not result in a congruent regulation of EGF-TM7 receptor mRNA and protein except for a down-regulation of CD97 by IL-10. In contrast, macrophages handling lipid overload in vivo displayed differences in the expression of CD97 and EMR2. While foamy macrophages in atherosclerotic vessels expressed both CD97 and EMR2, foam cells in multiple sclerosis brain expressed CD97, but only little EMR2. Foam cell formation in vitro by oxidized LDL and myelin did not affect CD97 or EMR2 expression. Gaucher spleen cells accumulating glucosylceramide expressed very high levels of CD97 and EMR2. These findings indicate that complex cellular expression programmes rather than activation modes regulate the expression of EGF-TM7 receptors in macrophages.
Asunto(s)
Antígenos CD/inmunología , Aterosclerosis/inmunología , Enfermedad de Gaucher/inmunología , Regulación de la Expresión Génica , Macrófagos/inmunología , Glicoproteínas de Membrana/inmunología , Esclerosis Múltiple/inmunología , Receptores Acoplados a Proteínas G/inmunología , Aterosclerosis/genética , Arterias Carótidas/inmunología , Citometría de Flujo , Enfermedad de Gaucher/genética , Inmunohistoquímica , Esclerosis Múltiple/genética , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
CD97 is a large heptahelical EGF-TM7 receptor broadly expressed on hematopoietic cells as three isoforms with respectively three, four, or five epidermal growth factor (EGF)-like domains. We here describe the expression characteristics of CD97 on human lymphocyte subsets. We found CD97 to be present on all lymphocytes in blood and lymphoid tissue. Expression of CD97 on B cells was lower compared to T and NK cells and did not differ between B-cell subsets. In CD4(+) T cells, CD97 expression was higher on memory cells compared to naive cells. In CD8(+) T and NK cells, we found a downregulation of CD97 on cytolytic effector cells. Stimulation through CD3 and CD28 resulted in a rapid upregulation of CD97 in all T-cell subsets within 2-4h. A link between CD97 expression and lymphocyte proliferation was established in NK cells, which markedly upregulated CD97 in response to IL-2 and IL-15. Mixed lymphocyte cultures revealed a limited ability of the stalk region-specific monoclonal antibody CLB-CD97/3 to inhibit CD8(+) and CD4(+) allogeneic T-cell proliferation.
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Anticuerpos/inmunología , Antígenos CD/inmunología , Activación de Linfocitos , Glicoproteínas de Membrana/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunología , Antígenos CD28/inmunología , Antígenos CD28/metabolismo , Complejo CD3/inmunología , Complejo CD3/metabolismo , Humanos , Interleucina-15/inmunología , Interleucina-15/metabolismo , Interleucina-2/inmunología , Interleucina-2/metabolismo , Receptores Acoplados a Proteínas G , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Regulación hacia Arriba/inmunologíaRESUMEN
The EGF-TM7 F4/80 is a defining marker of murine macrophage populations. Applying flow cytometric analysis using the newly generated mAb A10, and quantitative real-time PCR, we here report the surprising observation that the human ortholog of F4/80, EGF-like module containing mucin-like hormone receptor (EMR)1, is absent on mononuclear phagocytic cells including monocytes, macrophages, and myeloid dendritic cells. Unexpectedly, we found that EMR1 expression is restricted to eosinophilic granulocytes, where expression is overlapping with the eotaxin receptor CCR3 and the immunoglobulin-like lectin Siglec-8. Absence on other leukocytes, including basophils, implies that EMR1 is a highly specific marker for eosinophils in humans.
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Antígenos de Diferenciación/metabolismo , Eosinófilos/inmunología , Eosinófilos/metabolismo , Glicoproteínas de Membrana/metabolismo , Mucinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Homología Estructural de Proteína , Animales , Anticuerpos Monoclonales/biosíntesis , Antígenos de Diferenciación/química , Biomarcadores/metabolismo , Células CHO , Proteínas de Unión al Calcio , Células Cultivadas , Cricetinae , Cricetulus , Humanos , Macaca mulatta , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Ratones , Mucinas/biosíntesis , Mucinas/química , Mucinas/genética , Pan troglodytes , Papio , Pongo pygmaeus , Receptores Acoplados a Proteínas G/biosíntesis , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , SaguinusRESUMEN
The receptor tyrosine kinase MET is overexpressed in human colorectal adenomas and carcinomas, suggesting an instrumental role for MET signaling in the onset and progression of colorectal cancer. To corroborate this role, animal models are needed. To study the expression of Met in the normal and neoplastic mouse intestine, we generated an Armenian hamster monoclonal antibody against mouse Met. By using this antibody in immunohistochemical studies, we observed strong Met expression in fetal mouse intestinal epithelial cells. In contrast, in the intestines of adult mice, Met expression was very low whereas the protein was undetectable on the neoplastic epithelium of intestinal adenomas in Apc+/min mice. By immunoblotting, we were also unable to detect Met in intestinal adenomas, whereas Met mRNA levels in microdissected adenomas were very low. The absence of detectable Met protein expression in adenomas of Apc+/min mice contrasts sharply with the vast overexpression of the protein in adenomas of humans with familial adenomatous polyposis or sporadic colorectal carcinomas. Our results imply that deregulation of Wnt signaling in mouse--unlike in human--intestinal epithelium does not result in Met overexpression. Our findings thus reveal important interspecies differences in the regulation of Met expression during intestinal tumorigenesis.