RESUMO
Prognosis of primary refractory and relapsed pediatric B-lineage acute lymphoblastic leukemia (ALL) is very poor. Relapse rates significantly correlate with persistent minimal residual disease (MRD). In MRD, favorable effector-target ratios prevail and thus this situation might be optimally suited for immunotherapy with antibodies recruiting immunological effector cells. We here report on the generation, preclinical characterization and first clinical application in B-lineage ALL of an Fc-optimized CD19 antibody. This third-generation antibody (4G7SDIE) mediated enhanced antibody-dependent cellular cytotoxicity (ADCC) against leukemic blasts with effector cells from healthy volunteers and B-lineage ALL patients. The antibody was produced in a university-owned production unit and was applied on a compassionate use basis to 14 pediatric patients with refractory and relapsed B-lineage ALL at the stage of MRD. In 10/14 patients, MRD was reduced by ≥ 1 log or below the patient-individual detection limit, and 5/14 patients have achieved ongoing complete molecular remission with a median leukemia-free survival of 428 days. Two additional patients died in complete molecular remission due to complications not related to antibody therapy. Besides profound in vivo B-cell depletion, side effects were negligible. A clinical phase 1/2 study to further assess the therapeutic activity of 4G7SDIE is in preparation.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD19 , Fragmentos Fc das Imunoglobulinas , Neoplasia Residual/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Adolescente , Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD19/imunologia , Antígenos CD19/metabolismo , Linhagem Celular Tumoral , Criança , Pré-Escolar , Terapia Combinada , Monitoramento de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Feminino , Expressão Gênica , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Estimativa de Kaplan-Meier , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Retratamento , Resultado do Tratamento , Adulto JovemRESUMO
FLT3 is a receptor-tyrosine-kinase that is expressed on leukemic cells of the myeloid and lymphoid lineage rather specifically. We here report on the construction and selection of bispecific FLT3 X CD3 antibodies in a new recombinant format, termed Fabsc, that resembles the normal antibody structure more closely than the well-established bispecific single chain (bssc)-format. Our preferred antibody, which emerged from an initial selection procedure utilizing different FLT3- and CD3-antibodies, contains the FLT3-antibody 4G8 and the CD3-antibody UCHT1. The 4G8 X UCHT1 Fabsc-antibody was found to be superior to a bssc-antibody with identical specificities with respect to (i) affinity to the target antigen FLT3, (ii) production yield by transfected cells, and (iii) the diminished formation of aggregates. T-cell activation in the presence and absence of cultured leukemic cells and killing of these cells was comparable for both molecules. In addition, the 4G8 X UCHT1 Fabsc-antibody was found to induce T-cell activation and efficient killing of leukemic blasts in primary peripheral blood mononuclear cell (PBMC) cultures of acute myeloid leukemia (AML) patients. In these experiments, the bispecific molecule was clearly superior to an Fc-optimized monospecific FLT3-antibody described previously, indicating that within PBMC of AML patients the recruitment of T cells is more effective than that of natural killer cells.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Complexo CD3/imunologia , Leucemia Experimental/terapia , Tirosina Quinase 3 Semelhante a fms/imunologia , Animais , Camundongos , Camundongos Endogâmicos C57BLRESUMO
iNKT cells are a particular lymphocyte population with potent immunomodulatory capa-city; by promoting or suppressing immune responses against infections, tumors, and autoimmunity, iNKT cells are a promising target for immunotherapy. The hallmark of iNKT cells is the expression of a semiinvariant TCR (with an invariant α-chain comprising AV14 and AJ18 gene segments), which recognizes glycolipids presented by CD1d. Here, we identified iNKT cells for the first time in the rat using rat CD1d-dimers and PLZF staining. Importantly, in terms of frequencies (1.05% ± 0.52 SD of all intrahepatic αß T cells), coreceptor expression and in vitro expansion features, iNKT cells from F344 inbred rats more closely resemble human iNKT cells than their mouse counterparts. In contrast, in LEW inbred rats, which are often used as models for organ-specific autoimmune diseases, iNKT cell numbers are near or below the detection limit. Interestingly, the usage of members of the rat AV14 gene family differed between F344 and LEW inbred rats. In conclusion, the similarities between F344 rat and human iNKT cells and the nearly absent iNKT cells in LEW rats make the rat a promising animal model for the study of iNKT cell-based therapies and of iNKT-cell biology.
Assuntos
Células T Matadoras Naturais/imunologia , Animais , Antígenos CD1d/imunologia , Citocinas/imunologia , Galactosilceramidas/imunologia , Humanos , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos F344/imunologia , Ratos Endogâmicos Lew , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
A non-coding hexanucleotide repeat expansion (HRE) in C9orf72 is a common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) acting through a loss of function mechanism due to haploinsufficiency of C9orf72 or a gain of function mediated by aggregates of bidirectionally transcribed HRE-RNAs translated into di-peptide repeat (DPR) proteins. To fully understand regulation of C9orf72 expression we surveyed the C9orf72 locus using Cap Analysis of Gene Expression sequence data (CAGEseq). We observed C9orf72 was generally lowly expressed with the exception of a subset of myeloid cells, particularly CD14+ monocytes that showed up to seven fold higher expression as compared to central nervous system (CNS) and other tissues. The expression profile at the C9orf72 locus showed a complex architecture with differential expression of the transcription start sites (TSSs) for the annotated C9orf72 transcripts between myeloid and CNS tissues suggesting cell and/or tissue specific functions. We further detected novel TSSs in both the sense and antisense strand at the C9orf72 locus and confirmed their existence in brain tissues and CD14+ monocytes. Interestingly, our experiments showed a consistent decrease of C9orf72 coding transcripts not only in brain tissue and monocytes from C9orf72-HRE patients, but also in brains from MAPT and GRN mutation carriers together with an increase in antisense transcripts suggesting these could play a role in regulation of C9orf72. We found that the non-HRE related expression changes cannot be explained by promoter methylation but by the presence of the C9orf72-HRE risk haplotype and unknown functional interactions between C9orf72, MAPT and GRN.
Assuntos
Sistema Nervoso Central/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mutação/genética , Células Mieloides/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas tau/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteína C9orf72 , Bases de Dados Factuais/normas , Bases de Dados Factuais/estatística & dados numéricos , Demência Frontotemporal/metabolismo , Humanos , Receptores de Lipopolissacarídeos/metabolismo , ProgranulinasRESUMO
In order to execute their immune functions, leukocytes interact with a broad range of cell types through cell surface receptors, such as those of the immunoglobulin and C-type lectin families, or indirectly through soluble factors. The characterization of activating and inhibitory counterparts of NK cell receptors on myeloid cells, as well as the identification of their physiological ligands, has provided new insights into the underlying mechanisms of immunity and homeostasis. Here, we describe methodology that can be employed to screen for endogenous ligands of type-II C-type lectin-like receptors using reporter cells and Fc fusion proteins.
Assuntos
Ligantes , Receptores Semelhantes a Lectina de Células NK/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , Receptores Semelhantes a Lectina de Células NK/genéticaRESUMO
BACKGROUND: CD1d is a nonpolymorphic MHC class I-like molecule which presents nonpeptide ligands, e.g. glycolipids, to NKT cells. These cells are known to have multiple effects on innate and adaptive immune responses and on the development of pathological conditions. In order to analyze CD1d expression and function in the rat, the first rat CD1d-specific monoclonal antibodies (mAbs) were generated. METHODOLOGY/PRINCIPAL FINDINGS: Two mAbs, WTH-1 and WTH-2, were generated which bound equally well to cell surface-expressed rat and mouse CD1d. Their non-overlapping epitopes were mapped to the CD1d heavy chain. Flow cytometry and immunohistological analyses revealed a nearly identical degree and pattern of CD1d expression for hematopoieitic cells of both species. Notable is also the detection of CD1d protein in mouse and rat Paneth cells as well as the extremely high CD1d expression in acinar exocrine cells of the rat pancreas and the expression of CD4 on rat marginal zone B cells. Both mAbs blocked α-galactosylceramide recognition by primary rat and mouse NKT cells. Interestingly, the two mAbs differed in their impact on the activation of various autoreactive T cell hybridomas, including the XV19.2 hybridoma whose activation was enhanced by the WTH-1 mAb. CONCLUSIONS/SIGNIFICANCE: The two novel monoclonal antibodies described in this study, allowed the analysis of CD1d expression and CD1d-restricted T cell responses in the rat for the first time. Moreover, they provided new insights into mechanisms of CD1d-restricted antigen recognition. While CD1d expression by hematopoietic cells of mice and rats was extremely similar, CD1d protein was detected at not yet described sites of non-lymphatic tissues such as the rat exocrine pancreas and Paneth cells. The latter is of special relevance given the recently reported defects of Paneth cells in CD1d(-/-) mice, which resulted in an altered composition of the gut flora.
Assuntos
Anticorpos Monoclonais/análise , Antígenos CD1/genética , Expressão Gênica , Ativação Linfocitária , Células T Matadoras Naturais/imunologia , Celulas de Paneth/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos CD1/imunologia , Linhagem Celular , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/química , Pâncreas Exócrino , Celulas de Paneth/química , Ratos , Ratos Endogâmicos LewRESUMO
Inhibitory receptors are required for the control of cellular activation and they play essential roles in regulating homeostasis and immunity. We previously identified a human inhibitory C-type lectin-like receptor, MICL (CLEC12A), a heavily glycosylated monomer predominantly expressed on myeloid cells. Here we characterise the murine homolog of MICL (mMICL), and demonstrate that the receptor is structurally and functionally similar to the human orthologue (hMICL), although there are some notable differences. mMICL is expressed as a dimer and is not heavily glycosylated; however, like hMICL, the receptor can recruit inhibitory phosphatases upon activation, and is down-regulated on leukocytes following stimulation with selected TLR agonists. Using novel monoclonal antibodies, we demonstrate that, like the human receptor, mMICL is predominantly expressed by myeloid cells. However, mMICL is also expressed by B cells and CD8+ T cells in peripheral blood, and NK cells in the bone marrow. Finally, we show that mMICL recognises an endogenous ligand in a variety of murine tissues, suggesting that the receptor plays a role in homeostasis.
Assuntos
Lectinas Tipo C/metabolismo , Receptores Mitogênicos/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Lectinas Tipo C/química , Lectinas Tipo C/genética , Ligantes , Camundongos , Dados de Sequência Molecular , Ratos , Receptores Mitogênicos/química , Receptores Mitogênicos/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Recognition of microbial components by germ-line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal beta-glucan receptor, Dectin-1, which collaborates through an undefined mechanism with Toll-like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin-1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF, MIP-1alpha and MIP-2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kappaB (IkappaB), enhancing NFkappaB nuclear translocation. These findings establish the first example of Syk- and MyD88-coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents.
Assuntos
Citocinas/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas Tirosina Quinases/fisiologia , Receptor 2 Toll-Like/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Humanos , Proteínas I-kappa B/metabolismo , Mediadores da Inflamação/fisiologia , Lectinas Tipo C , Ligantes , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/patologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Quinase SykRESUMO
Invariant NKT cells (iNKT cells) are characterized by a semi-invariant TCR comprising an invariant alpha-chain paired with beta-chains with limited BV gene usage which are specific for complexes of CD1d and glycolipid Ags like alpha-galactosylceramide (alpha-GalCer). iNKT cells can be visualized with alpha-GalCer-loaded CD1d tetramers, and the binding of mouse CD1d tetramers to mouse as well as to human iNKT cells suggests a high degree of conservation in recognition of glycolipid Ags between species. Surprisingly, mouse CD1d tetramers failed to stain a discrete cell population among F344/Crl rat liver lymphocytes, although comprised iNKT cells are indicated by IL-4 and IFN-gamma secretion after alpha-GalCer stimulation. The arising hypothesis that rat iNKT TCR recognizes alpha-GalCer only if presented by syngeneic CD1d was then tested with the help of newly generated rat and mouse iNKT TCR-transduced cell lines. Cells expressing mouse iNKT TCR reacted to alpha-GalCer presented by rat or mouse CD1d and efficiently bound alpha-GalCer-loaded mouse CD1d tetramers. In contrast, cells expressing rat iNKT TCR responded only to alpha-GalCer presented by syngeneic CD1d and bound mouse CD1d tetramers only poorly or not at all. Finally, CD1d-dependent alpha-GalCer reactivity and binding of mouse CD1d tetramers was tested for cells expressing iNKT TCR comprising either rat or mouse AV14 (Valpha14) alpha-chains and wild-type or mutated BV8S2 (Vbeta8.2) beta-chains. The results confirmed the need of syngeneic CD1d as restriction element for rat iNKT TCR and identified the CDR2 of BV8S2 as an essential site for ligand recognition by iNKT TCR.
Assuntos
Apresentação de Antígeno , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/genética , Antígenos CD1/genética , Antígenos CD1/imunologia , Antígenos CD1/metabolismo , Antígenos CD1d , Linhagem Celular , Células Cultivadas , Galactosilceramidas/administração & dosagem , Galactosilceramidas/imunologia , Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Imunofenotipagem , Fígado/citologia , Fígado/imunologia , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Ligação Proteica/genética , Ligação Proteica/imunologia , Ratos , Ratos Endogâmicos F344 , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Especificidade da EspécieRESUMO
Host defence against pathogens requires the recognition of conserved microbial molecules, or 'pathogen-associated molecular patterns' (PAMPs), by their receptors termed 'pattern recognition receptors' (PRRs), represented most notably by toll-like receptors (TLRs) and C-type lectins. The 'non-classical' C-type lectins (these that lack the residues involved in calcium binding, required for carbohydrate binding) are traditionally thought of as being restricted to natural killer (NK) or T cells, playing important roles in immune surveillance. In recent years, however, a growing number of these receptors have been identified on myeloid cells, both of human and mouse origin. In contrast to their NK counterparts that primarily control cellular activation through recognition of major histocompatibility antigen (MHC) class I and related molecules, the myeloid-expressed receptors appear to have a far more diverse range of functions and ligands, including those of exogenous origin. Some of C-type lectin-like molecules possess activating/inhibitory signalling motifs that trigger downstream signalling events, suggesting the role for these receptors as positive/negative regulators of granulocyte and monocyte functions. With the exception of a few myeloid NK-like lectins, the natural ligands for most of these receptors remain unidentified, making it difficult to define their functions in normal physiological, inflammatory or pathological conditions. Importantly, in some cases, these novel C-type lectin-like lectins, encoded by genes from the same gene cluster, can act as receptor/ligand pairs, additionally contributing to the regulation of myeloid cell functions or their interaction with other (like NK) cell types. However, the relevance and importance of such interactions still needs to be assessed. Although few of the myeloid-expressed C-type lectins have been characterized in detail, we review here each of these receptors and highlight their prospective roles in innate and adaptive immunity.
Assuntos
Doenças Transmissíveis/imunologia , Proteínas de Membrana/imunologia , Células Mieloides/imunologia , Proteínas do Tecido Nervoso/imunologia , Receptores Depuradores Classe E/imunologia , Animais , Humanos , Imunidade Inata , Células Matadoras Naturais/imunologia , Lectinas Tipo C , Ligantes , Receptores de Reconhecimento de Padrão/imunologia , Transdução de Sinais/imunologiaRESUMO
C-type lectins are the most diverse and prevalent lectin family in immunity. Particular interest has recently been attracted by the C-type lectin-like receptors on NK cells, which appear to regulate the activation/inhibitory balance of these cells, controlling cytotoxicity and cytokine production. We previously identified a human C-type lectin-like receptor, closely related to both the beta-glucan receptor and the lectin-like receptor for oxidized-LDL, named MICL (myeloid inhibitory C-type lectin-like receptor), which we had shown using chimeric analysis to function as an inhibitory receptor. Using a novel MICL-specific monoclonal antibody, we show here that human MICL is expressed primarily on myeloid cells, including granulocytes, monocytes, macrophages, and dendritic cells. Although MICL was highly N-glycosylated in primary cells, the level of glycosylation was found to vary between cell types. MICL surface expression was down-regulated during inflammatory/activation conditions in vitro, as well as during an in vivo model of acute inflammation, which we characterize here. This suggests that human MICL may be involved in the control of myeloid cell activation during inflammation.