RESUMO
MPhi and DC are key elements in the control of tissue homeostasis and response to insult. In this work, we demonstrate that MPhi and DC are the major producers of the phenylalanine catabolizing enzyme IL-4-induced gene 1 (IL4I1) under inflammatory conditions. IL4I1 was first described in B cells, which indeed can produce IL4I1 in vitro, although at much lower levels. In vivo, IL4I1 is highly expressed by MPhi and DC of Th1 granulomas (sarcoidosis, tuberculosis) but poorly detected in Th2 granulomas (schistosomiasis). In vitro, expression of the enzyme is induced in mononuclear phagocytes by various pro-inflammatory stimuli through the activation of the transcription factors NF-kappaB and/or STAT1. B cells also express IL4I1 in response to NF-kappaB-activating stimuli such as CD40L; however, in contrast to myeloid cells, B cells are insensitive to IFN-gamma but respond to stimulation of the IL-4/STAT6 axis. As we show that the expression of IL4I1 by a monocytic cell line inhibits T-cell proliferation and production of IFN-gamma and inflammatory cytokines, we propose that IL4I1 participates in the downregulation of Th1 inflammation in vivo.
Assuntos
Linfócitos B/metabolismo , Flavoproteínas/biossíntese , Sistema Fagocitário Mononuclear/metabolismo , Células Th1/metabolismo , Células Th2/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/patologia , Ligante de CD40/farmacologia , Linhagem Celular , Proliferação de Células , Técnicas de Cocultura , Flavoproteínas/genética , Flavoproteínas/imunologia , Humanos , Tolerância Imunológica , Inflamação , Interferon gama/farmacologia , Interleucina-4/imunologia , Interleucina-4/metabolismo , L-Aminoácido Oxidase , Sistema Fagocitário Mononuclear/efeitos dos fármacos , Sistema Fagocitário Mononuclear/imunologia , Sistema Fagocitário Mononuclear/patologia , NF-kappa B/metabolismo , RNA Interferente Pequeno/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT6/metabolismo , Células Th1/imunologia , Células Th1/patologia , Células Th2/imunologia , Células Th2/patologiaRESUMO
The Wilms tumor antigen, WT1, is associated with several human cancers, including leukemia. We evaluated WT1 as an immunotherapeutic target using our proven DNA fusion vaccine design, p.DOM-peptide, encoding a minimal tumor-derived major histocompatibility complex (MHC) class I-binding epitope downstream of a foreign sequence of tetanus toxin. Three p.DOM-peptide vaccines, each encoding a different WT1-derived, HLA-A2-restricted epitope, induced cytotoxic T lymphocytes (CTLs) in humanized transgenic mice expressing chimeric HLA-A2, without affecting hematopoietic stem cells. Mouse CTLs killed human leukemia cells in vitro, indicating peptide processing/presentation. Low numbers of T cells specific for these epitopes have been described in cancer patients. Expanded human T cells specific for each epitope were lytic in vitro. Focusing on human WT1(37-45)-specific cells, the most avid of the murine responses, we demonstrated lysis of primary leukemias, underscoring their clinical relevance. Finally, we showed that these human CTL kill target cells transfected with the relevant p.DOM-peptide DNA vaccine, confirming that WT1-derived epitopes are presented to T cells similarly by tumors and following DNA vaccination. Together, these data link mouse and human studies to suggest that rationally designed DNA vaccines encoding WT1-derived epitopes, particularly WT1(37-45), have the potential to induce/expand functional tumor-specific cytotoxic responses in cancer patients.
Assuntos
Linfócitos T/imunologia , Vacinação , Vacinas de DNA/imunologia , Proteínas WT1/imunologia , Animais , Apresentação de Antígeno/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Citotoxicidade Imunológica , Antígenos HLA-A/imunologia , Antígeno HLA-A2 , Saúde , Hematopoese , Humanos , Leucemia/patologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Peptídeos/imunologia , Linfócitos T/citologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Doadores de TecidosRESUMO
Therapeutic vaccination aims at a strong stimulation of antigen-specific CD8(+) T-cells, so that they differentiate into effectors active in vivo against antigenic targets. Two adenovirus vectors (Ad) encoding two HLA-A*0201-restricted HIV epitope sequences (pol 476 and pol 589) were constructed. The Ad differ by the presence or absence of a ubiquitin monomer sequence (AdUb(+) and AdUb(-)). The effect of transgene product ubiquitination was analyzed on (1) in vivo, the immunization of Ad vaccinated HLA-A*0201 humanized HHD mice and (2) in vitro, the presentation of the transgene encoded peptides by transduced human dendritic cells (DC). In vivo, we found that immunization of humanized HHD mice with AdUb(+) elicited a transgene product-specific interferon (INF)-gamma CD8(+) T-cell response detectable by enzyme-linked immunospot (ELISPOT), whereas the AdUb(-) construction did not. Antigen-specific cytotoxic T lymphocytes (CTL) were also generated in HHD mice immunized with AdUb(+) and not with AdUb(-). In vitro, using human AdUb(+)-transduced DC, a sizeable expansion of pol 476 and pol 589 tetramer positive CD8(+) T cells as well as CD8(+) CTL were obtained in healthy donors. Compared to AdUb(-)-transduced DC, AdUb(+)-transduced DC triggered a higher number of pol 476-specific IFN-gamma-secreting CD8(+) T cells. In agreement, AdUb(+) transduced DC, used as target in a (51)Cr-release assay, were more efficiently lysed by peptide-specific CTL than AdUb(-)-transduced DC. In conclusion, the addition of an ubiquitin sequence to the adenoviral transgene, used as an antigen source, resulted in both in vivo enhanced CD8(+) T-cell immunogenicity in HHD mice and in vitro increased HLA class I-restricted presentation of encoded peptides by human DC.
Assuntos
Adenovírus Humanos/genética , Células Dendríticas/imunologia , Técnicas de Transferência de Genes , Antígenos de Histocompatibilidade Classe I/imunologia , Imunoterapia Ativa/métodos , Ubiquitina/metabolismo , Adenovírus Humanos/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Epitopos , Terapia Genética , Humanos , Interferon gama/imunologia , Células K562 , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Transdução Genética , TransgenesRESUMO
Anti-Hu syndrome is a paraneoplastic neurologic disease seemingly associated with an efficient antitumoral immune response against HuD protein expressed by both small cell lung cancer (SCLC) and neurons. Since anti-Hu antibodies are not pathogenic, and oligoclonal CD8(+) T cells infiltrate neoplastic and nervous tissues, we examined MHC class I-restricted immunogenicity of human HuD. Among 14 HuD-derived peptides potentially immunogenic in HLA-A*0201 restriction, 10 had actual in vitro binding capacity to the HLA molecule, 8 elicited specific cytotoxic T lymphocytes (CTLs) in a humanized murine model after peptidic vaccination, 2 also elicited specific CTLs in healthy humans, and 1 was naturally processed and presented to the immune system.
Assuntos
Antígenos de Neoplasias/imunologia , Citotoxicidade Imunológica/imunologia , Proteínas do Tecido Nervoso/imunologia , Síndromes Paraneoplásicas do Sistema Nervoso/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas de Ligação a RNA/imunologia , Animais , Apresentação de Antígeno , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/metabolismo , Linhagem Celular Tumoral , Proteínas ELAV , Proteína Semelhante a ELAV 4 , Antígenos HLA-A/imunologia , Antígenos HLA-A/metabolismo , Antígeno HLA-A2 , Humanos , Injeções Intramusculares , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/administração & dosagem , Proteínas do Tecido Nervoso/metabolismo , Síndromes Paraneoplásicas do Sistema Nervoso/genética , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/imunologia , Proteínas de Ligação a RNA/administração & dosagem , Proteínas de Ligação a RNA/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Interleukin-4-induced gene 1 (IL4I1) was first described as a B-cell IL4-inducible gene and is highly expressed in primary mediastinal B-cell lymphomas. We established stable HEK293 clones expressing human and mouse IL4I1 to examine their biochemical properties and function. Both proteins were secreted into the culture medium, and we observed the secretion of endogenous human IL4I1 (hIL4I1) protein in a mediastinal lymphoma B-cell line, MedB-1. We showed that IL4I1 has l-amino acid oxidase activity, optimal at physiological pH and primarily directed toward phenylalanine. Immunohistochemical analysis of secondary lymphoid organs showed staining of germinal center macrophages and inflammatory myeloid cells. In vitro, functional enzyme was highest in mature dendritic cells (DCs), suggesting a role in antigen-presenting cell/T-lymphocyte cross-talk. Indeed, hIL4I1 inhibited the proliferation of CD3-stimulated T lymphocytes with a similar effect on CD4(+) and CD8(+) T cells. In contrast, memory T cells were more strongly affected by hIL4I1 and its catabolite H(2)O(2) than naive T cells. hIL4I1 inhibitory effect was dependent on enzymatic activity and H(2)O(2) production and associated with a transient down-regulation of TCRzeta expression. Altogether these data suggest IL4I1 as a new immunomodulatory enzyme produced by DCs.
Assuntos
Aminoácido Oxirredutases/imunologia , Proliferação de Células , Células Dendríticas/química , L-Aminoácido Oxidase/imunologia , Linfócitos T/citologia , Aminoácido Oxirredutases/biossíntese , Aminoácido Oxirredutases/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Peróxido de Hidrogênio/metabolismo , Imuno-Histoquímica , L-Aminoácido Oxidase/biossíntese , L-Aminoácido Oxidase/metabolismo , Linfoma de Células B/patologia , Macrófagos/química , Camundongos , Células Mieloides/químicaRESUMO
Lymphoma-derived immunoglobulin idiotype (Id) is a tumour-specific antigen used for antitumour vaccination in follicular lymphoma (FL). However, FL Ids are subject to hypermutation and subclones may escape antitumour cytotoxic T-cell response. To investigate the intraclonal epitope diversity, we sequenced the FL heavy chain gene (consensus Id gene) and subclones of 24 patients. The derived polypeptide sequences were analysed by bioinformatics for human leucocyte antigen (HLA)-A0201-restricted epitope prediction. Epitopes were classified according to BIMAS and SYFPEITHI scores. Surprisingly in these highly mutated polypeptides, the epitopes concentrated in short hotspots in the conserved framework regions (FRs), both in HLA-A0201(+) and HLA-A0201(-) patients. Similar hotspots have been observed by others in chronic lymphocytic leukaemia Ids which in contrast to FL have low mutation frequency. FR3 amino acids 78-88 displayed the best-score epitopes in Ids containing a VH3-family segment, the most represented in FL Ids. Such VH3-FR3(78-88) epitopes were previously demonstrated as immunogenic. Modifications of the epitope pattern in subclones of HLA-A0201(+) patients were generally absent from high-score peptides, including VH3-FR3(78-88) epitopes (83% unmodified). Therefore, no tendency for loss of HLA-A0201-restricted epitopes was evidenced and, given their limited intraclonal diversity, VH3-FR3(78-88) epitopes may provide a useful target for the induction of cytotoxic response in Id-vaccinated FL patients.
Assuntos
Epitopos de Linfócito T , Antígenos HLA-A , Idiótipos de Imunoglobulinas/genética , Linfoma Folicular/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Células Clonais , Sequência Consenso , Mapeamento de Epitopos , Feminino , Genes de Cadeia Pesada de Imunoglobulina , Genótipo , Antígeno HLA-A2 , Humanos , Linfoma Folicular/genética , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
BACKGROUND: Changes in cell surface glycosylation that accompany apoptosis are thought to be involved in the recognition and removal of apoptotic cells by phagocytes, but in most instances these changes are ill defined. To improve our understanding of this phenomenon, we designed a trivariate flow cytometry procedure that allows direct comparison of cell surface glycosylation in apoptotic and viable cells. METHODS: The annexin V/propidium iodide assay has been adapted for cell surface glycosylation analysis by combining the use of these two reagents with biotinylated lectins, and this has been used to investigate camptothecin-induced apoptosis in U-937 cells. RESULTS: Although numerous lectins are potent inducers of apoptosis, we found that it is possible to determine lectin concentrations that produce interpretable data without inducing significant cytotoxicity even when using apoptogenic lectins. That apoptosis is associated with a marked decrease in cell surface sialylation was confirmed by using the sialic acid-specific lectins Maackia amurensis agglutinin and Sambucus nigra agglutinin. These observations were corroborated by lectin blotting analysis with the same lectins. CONCLUSIONS: Species- and cell-dependent altered glycosylation patterns are likely to be associated with different modes of apoptosis. The easy and versatile method described in this report should be useful for exploring this field.
Assuntos
Apoptose/fisiologia , Membrana Celular/metabolismo , Citometria de Fluxo/métodos , Glicosilação , Lectinas/metabolismo , Western Blotting , Humanos , Células U937RESUMO
Successful immunization requires that mature dendritic cells (mDCs) prime T cells in secondary lymphoid tissue (LT). Previously, the authors have shown that LT T cell activation has an increased costimulatory threshold for a proliferative response as compared with peripheral blood (PB) T cells. Therefore, to optimize mDC immunogenicity, DC maturation was studied using LT T cells as responders. While mDCs obtained with soluble CD40Ligand (sCD40L) or a sCD40L/IFNgamma combination similarly expressed the CD83 and CCR7 molecules on their membrane, only the latter secreted IL-12. sCD40L/IFNgamma mDCs, as compared with sCD40L mDCs, enhanced allogeneic LT T cell proliferation, LT CD4+ cell IFNgamma production and LT CD8+ cell cytotoxicity. Enhancement could be predominantly ascribed to IL-12 secreted by sCD40L/IFNgamma mDCs and to additional costimulatory signals as shown remarkably in the IFNgamma response when IL-12 was neutralized. Therefore, in addition to their membrane phenotype, mDCs to be used in immunization protocols should be assessed for IL-12 secretion as a surrogate marker for an optimum costimulatory potential.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Interleucina-12/imunologia , Ativação Linfocitária/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Ligante de CD40/imunologia , Linfócitos T CD8-Positivos/citologia , Divisão Celular , Células Cultivadas , Citotoxicidade Imunológica , Células Dendríticas/citologia , Humanos , Imunofenotipagem , Interferon gama/biossíntese , Interleucina-12/biossíntese , Interleucina-12/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Tecido Linfoide/citologia , CamundongosRESUMO
The MAL mRNA was initially identified during T-cell development and was later found in myelin-forming cells and certain polarized epithelial cell lines. It encodes a proteolipid believed to participate in membrane microdomains stabilization, transport machinery and signal transduction. Using a differential display reverse-transcription approach, we identified MAL as a distinct molecular marker of primary mediastinal large B-cell lymphoma compared with nonmediastinal diffuse large B-cell lymphomas. In the present study, we used immunohistochemistry to extend MAL expression analysis to normal lymphoid tissues; to 185 lymphomas representing most B, T, and Hodgkin lymphoma entities; and to the primary mediastinal large B-cell lymphoma derived B-cell line MedB-1. In addition, B and T cells from peripheral blood, tonsil, and spleen were analyzed by flow cytometry. Our results show that MAL is highly expressed in thymocytes, in a large percentage of peripheral CD4 T cells, and in a lower proportion of CD8 peripheral T cells. In the normal B-cell compartment, MAL expression appears to be restricted to a minor subpopulation of thymic medullary B cells and to occasional mature plasma cells located in the interfollicular areas of tonsil and lymph nodes. Among B-cell lymphomas (n = 110), MAL expression in tumor cells was observed in 21/33 primary mediastinal large B-cell lymphomas (70%) and in 3/5 plasmacytoma/myeloma, but not in all other B-cell lymphomas with the exception of 1/33 nonmediastinal diffuse large B-cell lymphomas. The MedB-1 B-cell line was also MAL positive. Among T-cell neoplasms, MAL was highly expressed in lymphoblastic tumors (5/6), whereas mature T-cell lymphomas were essentially MAL negative (27/28). Among 41 Hodgkin lymphomas, 3 nodular-sclerosing cases with mediastinal involvement showed MAL-positive Reed Sternberg cells. In conclusion, this study further supports thymic B cells as the putative normal counterpart of primary mediastinal large B-cell lymphomas and supports MAL as a distinct molecular marker of this lymphoma subtype among diffuse large B-cell lymphomas.
Assuntos
Linfoma de Células B/patologia , Neoplasias do Mediastino/patologia , Proteínas de Membrana Transportadoras , Proteínas da Mielina , Proteolipídeos/biossíntese , Antígenos CD/análise , Biomarcadores Tumorais/análise , Antígenos CD79 , Citometria de Fluxo , Humanos , Imuno-Histoquímica/métodos , Linfonodos/química , Linfonodos/patologia , Linfócitos/química , Linfoma de Células B/metabolismo , Neoplasias do Mediastino/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina , Tonsila Palatina/química , Tonsila Palatina/patologia , Receptores de Antígenos de Linfócitos B/análise , Baço/química , Baço/patologia , Timo/química , Timo/patologiaRESUMO
Clinical studies in hairy cell leukaemia (HCL) have linked the frequent occurrence of infections due to intracellular pathogens and a profound monocytopenia. More recently, dendritic cells (DC), a subset of which are related to monocytes, were shown to be the professional antigen-presenting cells which stimulate the adaptive immune response. Using membrane markers and flow cytometry, we determined in peripheral blood whether various DC subsets and monocytes were impaired in HCL. Lymphoid and myeloid DC were virtually absent in five HCL patients with active disease. After treatment, both DC and monocytes recovered slowly. The decrease in DC suggests that defective antigen presentation could affect susceptibility to intracellular pathogens in HCL.
Assuntos
Células Dendríticas/patologia , Leucemia de Células Pilosas/imunologia , Adulto , Idoso , Apresentação de Antígeno , Antineoplásicos/uso terapêutico , Contagem de Células , Cladribina/uso terapêutico , Células Dendríticas/efeitos dos fármacos , Citometria de Fluxo , Seguimentos , Humanos , Leucemia de Células Pilosas/tratamento farmacológico , Pessoa de Meia-IdadeRESUMO
BACKGROUND: This study evaluated the ability of a modified cell separator (Cobe Spectra Apheresis) system to isolate monocytes (MOs) by elutriation. The evaluation was performed in two independent international laboratories. The capacity of collected MOs to differentiate into dendritic cells (DCs) was also assessed. STUDY DESIGN AND METHODS: MNCs from platelet apheresis residues were elutriated on a modified cell separator (Cobe Spectra Apheresis system) using a custom disposable set. Cells were separated according to their size and density. Recovery and purity of the collected cell product were evaluated by impedance counting and flow cytometry. DCs were differentiated in culture from the elutriated MOs and characterized by their surface markers and stimulatory capacity in a mixed WBC reaction assay. RESULTS: Six apheresis mononuclear cell products were used by each laboratory. The separation was achieved in less than 1 hour. Collected MOs had the potential to differentiate into DCs. CONCLUSION: The modified cell separator is an easy and fast device to obtain highly enriched MOs with a DC differentiation potential. The system is closed and employs a single-use disposable set and is more amenable to good tissue practice. This method could become a valuable tool for DC-based active immunotherapy.