RESUMO
AIMS: Mesenchymal stromal cells (MSC) derived from adult bone marrow or adipose tissue offer the potential to open a new frontier in medicine. MSC are involved in modulating immune response and tissue repair in vitro and in vivo. Experimental evidence and preliminary clinical studies have demonstrated that MSC exhibit an important immunomodulatory function in patients with graft versus host disease (GVHD) following allogeneic hematopoietic stem cell transplantation. The immunosuppressive properties of MSC have already been exploited in the clinical setting. However the precise mechanisms are being still investigated. METHODS: We examined the immunosuppressive function of MSC by coculturing them with stimulated HLA incompatible allogeneic lymphocytes in a mixed lymphocyte culture test. The metabolic and proliferative activity of lymphocytes was determined by MTT test. RESULTS: After stimulation with alloantigens the presence of MSC caused significant decrease of absorbance levels by 62% (P<0.01), 26% (P<0.01) and 6% (P=0.0437) in comparison to positive control depending on the MSC/lymphocyte ratio (1:5, 1:50, 1:500). The mitogenic stimulation of lymphocytes with fMLP or PHA was also significantly reduced during MSC cocultivation. The absorbance was reduced by 42% (P<0.001) and 67% (P<0.001). CONCLUSIONS: Allogeneic bone marrow is an ideal source of MSC for clinical application. The experiments confirmed the dose-dependent inhibitory effect of MSC on lymphocyte proliferation triggered by cellular or mitogenic stimulation. The mixed lymphocyte culture test offers a simple method for characterization and verification of the immunosuppressive potential of MSC, being prepared for clinical use.
Assuntos
Tolerância Imunológica/imunologia , Isoantígenos/fisiologia , Linfócitos/imunologia , Células-Tronco Mesenquimais/imunologia , Adulto , Proliferação de Células/fisiologia , Células Cultivadas , Doença Enxerto-Hospedeiro/imunologia , Antígenos HLA/imunologia , Humanos , Técnicas In Vitro , Linfócitos/citologiaRESUMO
Detection and isolation of viable alloreactive T cells at the single-cell level requires a cell surface marker induced specifically upon T cell receptor activation. In this study, a member of the tumour necrosis factor receptor (TNFR)-family, CD137 (4-1BB) was investigated for its potential to identify the total pool of circulating alloreactive T cells. Optimal conditions for sensitive and specific detection of allogeneic-induced CD137 expression on circulating T cells were established. Thereafter, CD137(+) alloreactive T cells were phenotypically and functionally characterized by multi-parameter flow cytometry. Alloantigen-induced CD137 expression identified both alloreactive CD8(+) T cells (mean ± standard error of the mean: 0·21 ± 0·07%) and alloreactive CD4(+) T cells (0·21 ± 0·05%). CD137(+) alloreactive T cells were detected in different T cell subsets, including naive T cells, but were found preferentially in CD28(+) T cells and not in the terminally differentiated T cell subset. Upon allogeneic (re-)stimulation, the cytokine-producing as well as proliferative capacity of T cells resided mainly within the CD137-expressing fraction. About 10% of the CD137(+) alloreactive T cells produced any combination of interferon (IFN)-γ, interleukin (IL)-2 and TNF-α. Polyfunctional alloreactive T cells, defined by multiple cytokine expression, were observed infrequently. In conclusion, activation-induced CD137 expression is a fast assay allowing for detection and functional analysis of the total alloreactive T cell compartment at the single-cell level by multi-parameter flow cytometry.
Assuntos
Citometria de Fluxo/métodos , Isoantígenos/fisiologia , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos T/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/biossíntese , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , Citometria de Fluxo/normas , Humanos , Imunofenotipagem/métodos , Imunofenotipagem/normas , Teste de Cultura Mista de Linfócitos/métodos , Teste de Cultura Mista de Linfócitos/normas , Depleção Linfocítica , Sensibilidade e Especificidade , Células-Tronco/citologia , Células-Tronco/imunologia , Células-Tronco/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genéticaRESUMO
Proteinase 3 (PR3) is a major autoantigen in anti-neutrophil cytoplasmic antibodies (ANCA)-associated systemic vasculitis (AASV), and the proportion of neutrophils expressing PR3 on their membrane (mPR3+) is increased in AASV. We have shown recently that mPR3 and CD177 are expressed on the same cells in healthy individuals. In this study we try to elucidate mechanisms behind the increased mPR3 expression in AASV and its relationship to CD177. All neutrophils in all individuals were either double-positive or double-negative for mPR3 and CD177. The proportion of double-positive neutrophils was increased significantly in AASV and systemic lupus erythematosus patients. The proportion of mPR3+/CD177+ cells was not correlated to general inflammation, renal function, age, sex, drug treatment and levels of circulating PR3. AASV patients had normal levels of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. Pro-PR3 was found to constitute 10% of circulating PR3 but none of the mPR3. We found increased mRNA levels of both PR3 and CD177 in AASV, but they did not correlate with the proportion of double-positive cells. In cells sorted based on membrane expression, CD177-mRNA was several-fold higher in mPR3+ cells. When exogenous PR3 was added to CD177-transfected U937 cells, only CD177+ cells bound PR3 to their membrane. In conclusion, the increased membrane expression of PR3 found in AASV is not linked directly to circulating PR3 or PR3 gene transcription, but is dependent upon CD177 expression and correlated with the transcription of the CD177 gene.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/enzimologia , Membrana Celular/metabolismo , Isoantígenos/fisiologia , Glicoproteínas de Membrana/fisiologia , Mieloblastina/biossíntese , Neutrófilos/enzimologia , Receptores de Superfície Celular/fisiologia , Adulto , Idoso , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/sangue , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Artrite Reumatoide/sangue , Artrite Reumatoide/enzimologia , Artrite Reumatoide/imunologia , Doadores de Sangue , Indução Enzimática , Feminino , Proteínas Ligadas por GPI , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/sangue , Fator Estimulador de Colônias de Granulócitos e Macrófagos/sangue , Hemoglobinúria Paroxística/sangue , Hemoglobinúria Paroxística/enzimologia , Hemoglobinúria Paroxística/imunologia , Humanos , Isoantígenos/biossíntese , Isoantígenos/genética , Transplante de Rim , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/enzimologia , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Mieloblastina/genética , Mieloblastina/farmacologia , Neutrófilos/imunologia , RNA Mensageiro/biossíntese , RNA Mensageiro/sangue , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transcrição Gênica , Células U937/efeitos dos fármacos , Células U937/enzimologiaRESUMO
Neutrophil alloantigens are involved in a variety of clinical conditions including immune neutropenias, transfusion-related acute lung injury (TRALI), refractoriness to granulocyte transfusions and febrile transfusion reactions. In the last decade, considerable progress has been made in the characterization of the implicated antigens. Currently, seven antigens are assigned to five human neutrophil antigen (HNA) systems. The HNA-1a, HNA-1b and HNA-1c antigens have been identified as polymorphic forms of the neutrophil Fcγ receptor IIIb (CD16b), encoded by three alleles. Recently, the primary structure of the HNA-2a antigen was elucidated and the HNA-2a-bearing glycoprotein was identified as a member of the Ly-6/uPAR superfamily, which has been clustered as CD177. The HNA-3a antigen is located on a 70-95 kDa glycoprotein; however, its molecular basis is still unknown. Finally, the HNA-4a and HNA-5a antigens were found to be caused by single nucleotide mutations in the αM (CD11b) and αL (CD11a) subunits of the leucocyte adhesion molecules (β2 integrins). Molecular and biochemical characterization of neutrophil antigenshave expanded our diagnostic tools by the introduction of genotyping techniques and immunoassays for antibody identification. Further studies in the field of neutrophil immunology will facilitate the prevention and management of transfusion reactions and immune diseases caused by neutrophil antibodies.
Os aloantígenos de neutrófilos estão associados a várias condições clínicas como neutropenias imunes, insuficiência pulmonar relacionada à transfusão (TRALI), refratariedade à transfusão de granulócitos, e reações transfusionais febris. Na última década, foi observado considerável progresso na caracterização dos aloantígenos envolvidos nestas condições clínicas. Atualmente sete antígenos estão incluídos em cinco sistemas de antígenos de neutrófilo humano (HNA). Os antígenos HNA-1a, HNA-1b e HNA-1c foram identificados como formas polimórficas do receptor Fcγ RIIIb (CD16b), codificados por três alelos. Recentemente, a estrutura primária do antígeno HNA-2a foi elucidada e a glicoproteína carreadora do antígeno foi identificada como um membro da superfamília Ly-6/uPARe designada como CD177. O antígeno HNA-3a está localizadoem uma glicoproteína de 70-90 kDa, entretanto sua base molecular ainda é desconhecida. Finalmente, os antígenos HNA-4ae HNA-5a são resultantes de mutações de um único nucleotídeo nas subunidades αM (CD11b) and αL (CD11a) das moléculas de adesão de leucócitos (β2 integrinas). A caracterização molecular e bioquímica dos antígenos neutrofílicos permitiu a expansão das ferramentas diagnósticas pela introdução de técnicas de genotipagem e imunoensaios para a identificação de anticorpos. Novos estudos envolvendo a imunologia de granulócitos serão de grande valor para a prevenção e tratamento de reações transfusionais e doenças imunes causadas por aloanticorpos de neutrófilos.
Assuntos
Humanos , Isoantígenos/genética , Neutrófilos/imunologia , Autoanticorpos/imunologia , Genótipo , Isoantígenos/imunologia , Isoantígenos/fisiologia , FenótipoRESUMO
Prv-1 is a hematopoietic cell surface receptor that has been shown to be overexpressed in patients diagnosed with polycythemia vera (PV) and essential thrombocythemia (ET), yet its cellular function remains unclear. In this study, we assessed the role of Prv-1 in thrombopoietin (Tpo)/Mpl signaling with the goal of identifying molecular mechanisms which augment Tpo-induced proliferation. By engineering the cytokine-dependent hematopoietic cell line BaF3 to express both Prv-1 and wild-type or mutant forms of Mpl, we were able to follow the time course of Tpo-dependent proliferation. We report that the overexpression of Prv-1 increased Tpo as well as IL-3-induced proliferation of BaF3/Mpl and BaF3 cells. Cells co-expressing Prv-1 and an Mpl receptor containing a Box 1 motif mutation, which fails to activate Jak2, was completely deficient in Tpo-dependent proliferation. In addition, BaF3 and BaF3/Prv-1 cells stimulated with IL-3 in the presence of the Jak2 inhibitor, AG490, abrogated the proliferative response, indicating that Prv-1 requires a functional Jak2 for its signaling activities. Western blot analysis showed an increase in Tpo and IL-3-induced Stat3 and Stat5 tyrosine phosphorylation in BaF3/Mpl and BaF3 cells expressing Prv-1. These results indicate a novel function for Prv-1 as a signaling molecule in cytokine signaling cascades and may lead to a greater understanding of the mechanism of overexpression of Prv-1 in myeloproliferative disorders.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Interleucina-3/farmacologia , Isoantígenos/fisiologia , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Trombopoetina/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proteínas Ligadas por GPI , Sistema Hematopoético/citologia , Humanos , Fosforilação , Policitemia Vera/etiologia , Receptores de Trombopoetina/fisiologia , Fatores de Transcrição STAT/metabolismo , Trombocitemia Essencial/etiologiaRESUMO
Programmed cell death-1 (PD-1, CD279) and its widely expressed, inducible ligand, PD-L1 (CD274), together dampen T cell activation, but whether they are essential for allograft tolerance is unknown. We show, using gene-deficient mice and blocking mAbs in wild-type mice, that costimulation blockade is ineffectual in PD-1(-/-) or PD-L1(-/-) allograft recipients, or in wild-type allograft recipients treated with anti-PD-1 or anti-PD-L1 mAb. Alloreactive PD-1(-/-) CD4 and CD8 T cells had enhanced proliferation and cytokine production compared to wild-type controls, and anergy could not be induced in PD-1-deficient CD4 T cells. We conclude that without inhibitory signals from PD-1 ligation, alloantigen-induced T cell proliferation and expansion cannot be regulated by costimulation blockade, and peripheral tolerance induction cannot occur.
Assuntos
Antígenos de Superfície/fisiologia , Proteínas Reguladoras de Apoptose/fisiologia , Antígeno B7-1/fisiologia , Glicoproteínas de Membrana/fisiologia , Peptídeos/fisiologia , Tolerância ao Transplante/imunologia , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-H1 , Proliferação de Células , Células Cultivadas , Anergia Clonal/genética , Anergia Clonal/imunologia , Sobrevivência de Enxerto/genética , Sobrevivência de Enxerto/imunologia , Transplante de Coração/imunologia , Isoantígenos/fisiologia , Ligantes , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Peptídeos/metabolismo , Receptor de Morte Celular Programada 1 , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Tolerância ao Transplante/genética , Transplante HomólogoRESUMO
Antineutrophil cytoplasmic antibodies (ANCAs) with specificity for proteinase 3 (PR3) are central to a form of ANCA-associated vasculitis. Membrane PR3 (mPR3) is expressed only on a subset of neutrophils. The aim of this study was to determine the mechanism of PR3 surface expression on human neutrophils. Neutrophils were isolated from patients and healthy controls, and hematopoietic stem cells from cord blood served as a model of neutrophil differentiation. Surface expression was analyzed by flow cytometry and confocal microscopy, and proteins were analyzed by Western blot experiments. Neutrophil subsets were separated by magnetic cell sorting. Transfection experiments were carried out in HEK293 and HL60 cell lines. Using neutrophils from healthy donors, patients with vasculitis, and neutrophilic differentiated stem cells we found that mPR3 display was restricted to cells expressing neutrophil glycoprotein NB1, a glycosylphosphatidylinositol (GPI)-linked surface receptor. mPR3 expression was decreased by enzymatic removal of GPI anchors from cell membranes and was absent in a patient with paroxysmal nocturnal hemoglobinuria. PR3 and NB1 coimmunoprecipitated from and colocalized on the neutrophil plasma membrane. Transfection with NB1 resulted in specific PR3 surface binding in different cell types. We conclude that PR3 membrane expression on neutrophils is mediated by the NB1 receptor.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Antígenos de Superfície/metabolismo , Isoantígenos/fisiologia , Glicoproteínas de Membrana/fisiologia , Mieloblastina/metabolismo , Neutrófilos/enzimologia , Receptores de Superfície Celular/fisiologia , Membrana Celular/metabolismo , Células Cultivadas , Sangue Fetal/citologia , Proteínas Ligadas por GPI , Glicosilfosfatidilinositóis/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunoprecipitação , Técnicas In Vitro , Mieloblastina/imunologia , Neutrófilos/metabolismo , TransfecçãoRESUMO
BACKGROUND: CD26 is a T-cell co-stimulator, and interacts with adenosine deaminase, human immunodeficiency virus (HIV) Tat-1 protein and extracellular matrix. It possesses dipeptidylpeptidase IV (DPP IV) catalytic activity, which is linked to its co-stimulatory efficacy. We investigated the effect of specific DPP IV systemic activity inhibition on acute pulmonary rejection. METHODS: Rat single-lung transplantation (Tx) was performed (LBNF1/LEW donor/recipient) in two groups (n = 12). Group I (n = 6) received daily treatment with a Pro-Pro-diphenylphosphonate derivative (AB197), and Group II served as an untreated control. At Day 5 post-Tx, ventilatory parameters, cytotoxicity and mixed lymphocyte reaction were analyzed and staining for ISHLT rejection grade and proliferating cell nuclear antigen (PCNA) was performed. RESULTS: Treatment with AB192 abrogated acute rejection and preserved pulmonary function up to Day 5 post-Tx for PO2 (Group II: 24.9 +/- 6.9 mm Hg; Group I: 149.5 +/- 24.3 mm Hg; p < 0.001), PCO2 (Group II: 53.3 +/- 13.6 mm Hg; Group I: 39.0 +/- 9.8 mm Hg; p < 0.05) and peak airway pressure (Group II: 50.7 +/- 17.2 mm Hg; Group I: 20.2 +/- 10.0 mm Hg; p < 0.01). Controls showed moderate/severe rejection (ISHLT Grade A2 or 3), grafts from inhibited hosts revealed no/mild rejection (Grade A0 to 2: Group II: 2.8 +/- 0.3; Group I: 1.25 +/- 1.0; p < 0.005). Proliferating cell nuclear antigen (PCNA) staining of rejection-associated cellular infiltrates showed a significant reduction in positivity in perivascular infiltrates (34 +/- 11.5%; p < 0.05) and bronchial surface epithelium (31.7 +/- 10.6%; p < 0.05) in Group I vs Group II (55.9 +/- 8.4% and 57.2 +/- 4.5%). CONCLUSIONS: Irreversible enzymatic inhibition of DPP IV has been shown to abrogate acute pulmonary rejection, maintain pulmonary function, and preserve histomorphologic architecture. These results extend earlier findings and illustrate the role of CD26/DPP IV in alloantigen-mediated immune responses.
Assuntos
Dipeptidil Peptidase 4/fisiologia , Inibidores Enzimáticos/uso terapêutico , Rejeição de Enxerto/tratamento farmacológico , Rejeição de Enxerto/enzimologia , Transplante de Pulmão/fisiologia , Animais , Proliferação de Células/efeitos dos fármacos , Dipeptídeos/farmacologia , Dipeptidil Peptidase 4/sangue , Inibidores Enzimáticos/farmacologia , Rejeição de Enxerto/prevenção & controle , Isoantígenos/fisiologia , Pulmão/enzimologia , Pulmão/patologia , Pulmão/fisiopatologia , Transplante de Pulmão/imunologia , Transplante de Pulmão/patologia , Masculino , Ratos , Ratos EndogâmicosRESUMO
Intense interest has centered around the role of a subset of regulatory T cells, CD4+CD25+ Treg, in controlling the development of autoimmune disorders, allograft rejection, infection, malignancy, and allergy. We previously reported that MD1, a molecule known to be important in regulation of expression of RP105, also was important in regulating alloimmunity, and that blockade of expression of MD1 diminished graft rejection in vivo. One mechanism by which an MD1-RP105 complex exerts an effect on immune responses is through interference with an LPS-derived signal delivered through the CD14-MD-2-TLR4 complex. We show below that LPS signaling for Treg induction occurs at higher LPS thresholds that for effector T cell responses. In addition, blockade of MD1 functional activity in dendritic cells (using anti-MD1 mAbs, MD1 antisense deoxyoligonucleotides, or responder cells from mice with deletion of the MD1 gene), resulted in elevated Treg induction in response to allogeneic stimulation (in vivo or in vitro) in the presence of LPS. These data offer one mechanistic explanation for the augmented immunosuppression described following anti-MD1 treatment.
Assuntos
Antígenos de Superfície/biossíntese , Diferenciação Celular/imunologia , Glicoproteínas de Membrana/biossíntese , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Animais , Anticorpos Monoclonais/fisiologia , Antígenos de Superfície/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Ilhas de CpG/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Sobrevivência de Enxerto/genética , Sobrevivência de Enxerto/imunologia , Inibidores do Crescimento/fisiologia , Isoantígenos/fisiologia , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Ativação Linfocitária/imunologia , Teste de Cultura Mista de Linfócitos , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/farmacologia , Transplante de Pele/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/metabolismo , Regulação para Cima/imunologiaRESUMO
We have characterized three different programs of activation for alloreactive CD8 T cells expressing the BM3.3 TCR, their elicitation depending on the characteristics of the stimulating peptide/MHC complex. The high-affinity interaction between the TCR and the K(b)-associated endogenous peptide pBM1 (INFDFNTI) induced a complete differentiation program into effector cells correlated with sustained ERK activation. The K(bm8) variant elicited a partial activation program with delayed T cell proliferation, poor CTL activity and undetectable ERK phosphorylation; this resulted from a low-avidity interaction of TCR BM3.3 with a newly identified endogenous peptide, pBM8 (SQYYYNSL). Interestingly, mismatched pBM1/K(bm8) complexes induced a split response in BM3.3 T cells, with total reconstitution of T cell proliferation but defective generation of CTL activity that was correlated with strong but shortened ERK phosphorylation. Crystal structures highlight the molecular basis for the higher stability of pBM8/K(bm8) compared to pBM1/K(bm8) complexes that exist in two conformers. This study illustrates the importance of the stability of both peptide/MHC and peptide/MHC-TCR interactions for induction of sustained signaling required to induce optimal CTL effector functions. Subtle allelic structural variations, amplified by peptide selection, may thus orient distinct outcomes of alloreactive TCR-based therapies.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígenos H-2/imunologia , Antígenos de Histocompatibilidade/imunologia , Isoantígenos/fisiologia , Ativação Linfocitária/imunologia , Peptídeos/fisiologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Células Clonais , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
The qualitative nature of immune responses induced by dendritic cells (DCs) is influenced by the balance of pro-inflammatory (e.g. IL-12) and anti-inflammatory (e.g. IL-10) cytokines that they secrete. Evidence to date suggests that IL-12 and IL-10 secretion is reciprocally regulated and that IL-10 inhibits IL-12 secretion. This study identifies a population of resting, immature rat bone marrow-derived DCs (BMDCs) which secretes IL-10, the IL-12(p70) heterodimer and the free IL-12(p40) subunit, the latter in vast excess of IL-12(p70). Counter-intuitively, activation with LPS induces the secretion of high and equivalent levels of IL-10 and IL-12(p40), but only quantitatively small increases in IL-12(p70). Neutralization of IL-10 increased the secretion of IL-12(p40) by resting BMDCs, but decreased IL-12(p40) secretion by LPS-activated BMDCs. Pre-incubation of resting BMDCs for 24h with neutralizing antibody to IL-10 reduced the subsequent secretion of IL-10 in allogeneic cultures of Lewis CD3(+) T cells with resting and LPS-activated Wistar BMDCs, and enhanced IL-12(p40) secretion in allogeneic cultures with LPS-activated BMDCs. IL-10 neutralization had no effect on the levels of IL-12(p70), IFN-gamma or IL-4 in allogeneic cultures. In summary, this study has identified a population of rat BMDCs that secretes low levels of bioactive IL-12(p70), but high levels of IL-10 and IL-12(p40). These findings argue against the concept that there is a reciprocal relationship between IL-10 and IL-12 secretion. They might also have implications for understanding the role of DCs in post-activation qualitative skewing of immune responses.
Assuntos
Células da Medula Óssea/citologia , Células Dendríticas/citologia , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Animais , Células da Medula Óssea/metabolismo , Complexo CD3/análise , Citocinas/metabolismo , Células Dendríticas/metabolismo , Interferon gama/metabolismo , Subunidade p40 da Interleucina-12 , Isoantígenos/imunologia , Isoantígenos/fisiologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Testes de Neutralização , Subunidades Proteicas/metabolismo , Coelhos , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
An increase in the level of polycythemia rubra vera -1 (PRV-1) mRNA has been reported in some myeloproliferative disorders. We have studied the effects of PRV-1 on cell proliferation and cell survival. In cell growth assays, the number of heterologous cells expressing PRV-1 increased faster than sham-transfected cells, a difference that was more pronounced in serum-free media. Even after 5 days of exposure to serum-free media, cells expressing PRV-1 continued to proliferate, whereas the control cells ceased to proliferate. We conclude that PRV-1 is a pro-proliferation molecule, and hypothesize that its overexpression may have a role in the pathogenesis of myeloproliferative disorders.
Assuntos
Isoantígenos/fisiologia , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Células CHO , Proliferação de Células , Sobrevivência Celular , Cricetinae , Proteínas Ligadas por GPI , Substâncias de Crescimento/farmacologia , Isoantígenos/genética , Glicoproteínas de Membrana/genética , Receptores de Superfície Celular/genética , TransfecçãoRESUMO
CTL eliminate cells infected with intracellular pathogens and tumor cells by two distinct mechanisms mediated by Fas ligand (FasL) and lytic granules that contain perforin and granzymes. In this study we show that an epoxycyclohexenone derivative,(2R,3R,4S)-2,3-epoxy-4-hydroxy-5-hydroxymethyl-6-(1E)-propenyl-cyclohex-5-en-1-one (ECH) specifically inhibits the FasL-dependent killing pathway in CTL-mediated cytotoxicity. Recently, we have reported that ECH blocks activation of procaspase-8 in the death-inducing signaling complex and thereby prevents apoptosis induced by anti-Fas Ab or soluble FasL. Consistent with this finding, ECH profoundly inhibited Fas-mediated DNA fragmentation and cytolysis of target cells induced by perforin-negative mouse CD4+ CTL and alloantigen-specific mouse CD8+ CTL pretreated with an inhibitor of vacuolar type H+-ATPase concanamycin A that selectively induces inactivation and proteolytic degradation of perforin in lytic granules. However, ECH barely influenced perforin/granzyme-dependent DNA fragmentation and cytolysis of target cells mediated by alloantigen-specific mouse CD8+ CTL. The components of lytic granules and the granule exocytosis pathway upon CD3 stimulation were also insensitive to ECH. In conclusion, our present results demonstrate that ECH is a specific nonpeptide inhibitor of FasL-dependent apoptosis in CTL-mediated cytotoxicity. Therefore, ECH can be used as a bioprobe to evaluate the contributions of two distinct killing pathways in various CTL-target settings.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/imunologia , Cicloexanonas/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Compostos de Epóxi/farmacologia , Imunossupressores/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , Linfócitos T Citotóxicos/imunologia , Animais , Linhagem Celular Tumoral , Células Clonais , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/imunologia , Grânulos Citoplasmáticos/metabolismo , Testes Imunológicos de Citotoxicidade/métodos , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/imunologia , Epitopos de Linfócito T/fisiologia , Exocitose/efeitos dos fármacos , Exocitose/imunologia , Proteína Ligante Fas , Isoantígenos/fisiologia , Leucemia L5178 , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Perforina , Proteínas Citotóxicas Formadoras de Poros , Solubilidade , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
Blockade of the CD40-CD154 costimulatory pathway can inhibit CD4(+) T cell-mediated alloimmune responses. The aim of this study was to define the in vivo requirement for CD40-CD154 costimulation by CD4(+) T cells that respond to alloantigen following direct recognition. We used TCR-transgenic CD4(+) T cells that are reactive to the MHC class II alloantigen, H2A(s). An experimental in vivo model was established that allowed direct comparison of the fate of a trace population of H2A(s)-reactive CD4(+) T cells when challenged with different forms of H2A(s+) alloantigen under conditions of CD40-CD154 costimulation blockade. In this study, we demonstrate that an i.v. infusion of H2A(s+) leukocytes in combination with anti-CD154 therapy rapidly deletes H2A(s)-reactive CD4(+) T cells. In contrast, following transplantation of an H2A(s+) cardiac allograft, H2A(s)-reactive CD4(+) T cell responses were unaffected by blocking CD40-CD154 interactions. Consistent with these findings, combined treatment with donor leukocytes and anti-CD154 therapy was found to be more effective in prolonging the survival of cardiac allografts compared with CD154 mAb treatment alone. The dominant mechanism by which donor leukocyte infusion and anti-CD154 therapy facilitate allograft acceptance is deletion of donor-reactive direct pathway T cells. No evidence for the generation of regulatory cells by this combined therapy was found. Taken together, these results clearly demonstrate that naive alloreactive CD4(+) T cells have distinct requirements for CD40-CD154 costimulation depending on the form and microenvironment of primary alloantigen contact.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Antígenos CD40/fisiologia , Ligante de CD40/fisiologia , Isoantígenos/metabolismo , Transdução de Sinais/imunologia , Transferência Adotiva , Animais , Anticorpos Bloqueadores/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/transplante , Antígenos CD40/imunologia , Ligante de CD40/imunologia , Divisão Celular/imunologia , Deleção Clonal , Terapia Combinada , Sobrevivência de Enxerto/imunologia , Antígenos H-2/biossíntese , Antígenos H-2/imunologia , Transplante de Coração/imunologia , Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe II/imunologia , Histonas , Imunofenotipagem , Imunossupressores/uso terapêutico , Isoantígenos/fisiologia , Ativação Linfocitária , Transfusão de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Nus , Camundongos Transgênicos , Baço/imunologia , Baço/metabolismo , Baço/transplante , Condicionamento Pré-TransplanteRESUMO
The TCR repertoire of a normal animal is shaped in the thymus by ligand-specific positive- and negative-selection events. These processes are believed to be determined at the single-cell level primarily by the affinity of the TCR-ligand interactions. The relationships among all the variables involved are still unknown due to the complexity of the interactions and the lack of quantitative analysis of those parameters. In this study, we developed a quantitative model of thymic selection that provides estimates of the fractions of positively and negatively selected thymocytes in the cortex and in the medulla, as well as upper-bound ranges for the number of selecting ligands required for the generation of a normal diverse TCR repertoire. Fitting the model to current estimates of positive- and negative-selected thymocytes leads to specific predictions. The results indicate the following: 1) the bulk of thymocyte death takes place in the cortex, and it is due to neglect; 2) the probability of a thymocyte to be negatively selected in the cortex is at least 10-fold lower than in the medulla; 3) <60 ligands are involved in cortical positive selection; and 4) negative selection in the medulla is constrained by a large diversity of selecting ligands on medullary APCs.
Assuntos
Isoantígenos/fisiologia , Modelos Imunológicos , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Timo/citologia , Timo/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Diferenciação Celular/imunologia , Sobrevivência Celular/imunologia , Biologia Computacional/métodos , Biologia Computacional/estatística & dados numéricos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Contagem de Linfócitos , Camundongos , Camundongos Transgênicos , Peptídeos/imunologia , Peptídeos/metabolismo , Probabilidade , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/metabolismo , Timo/metabolismoRESUMO
We identify and consider some characteristics of a peptide antagonist for the Ag-specific receptor on 2C cells (the 2C TCR). The peptide, GNYSFYAL (called GNY), binds to H-2K(b), and a very high-resolution crystal structure of the GNY-K(b) complex at 1.35 A is described. Although the GNY peptide does not bind to L(d), the potency of GNY-K(b) as an antagonist is evident from its ability to specifically inhibit 2C TCR-mediated reactions to an allogenic agonist complex (QLSPFPFDL-L(d)), as well as to a syngeneic agonist complex (SIYRYYGL-K(b)). The crystal structure and the activities of alanine-substituted peptide variants point to the properties of the peptide P4 side chain and the conformation of the Tyr-P6 side chain as the structural determinants of GNYSFYAL antagonist activity.
Assuntos
Isoantígenos/fisiologia , Oligopeptídeos/química , Oligopeptídeos/fisiologia , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/fisiologia , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/imunologia , Animais , Arginina/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células Clonais , Cristalografia por Raios X , Testes Imunológicos de Citotoxicidade , Feminino , Antígenos H-2/metabolismo , Antígeno de Histocompatibilidade H-2D , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oligopeptídeos/metabolismo , Ligação Proteica/imunologia , Receptores de Antígenos de Linfócitos T/agonistas , Serina/metabolismo , Relação Estrutura-AtividadeRESUMO
Regulatory T cells (Treg) are important in maintaining tolerance to self tissues. As both CD28 and CTLA-4 molecules are implicated in the function of Treg, we investigated the ability of their two natural ligands, CD80 and CD86, to influence the Treg-suppressive capacity. During T cell responses to alloantigens expressed on dendritic cells, we observed that Abs against CD86 potently enhanced suppression by CD4(+)CD25(+) Treg. In contrast, blocking CD80 enhanced proliferative responses by impairing Treg suppression. Intriguingly, the relative expression levels of CD80 and CD86 on dendritic cells are modulated during progression from an immature to a mature state, and this correlates with the ability of Treg to suppress responses. Our data show that CD80 and CD86 have opposing functions through CD28 and CTLA-4 on Treg, an observation that has significant implications for manipulation of immune responses and tolerance in vivo.
Assuntos
Adjuvantes Imunológicos/fisiologia , Antígenos CD/fisiologia , Antígeno B7-1/fisiologia , Glicoproteínas de Membrana/fisiologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Antígenos CD/imunologia , Antígenos de Diferenciação/fisiologia , Antígeno B7-2 , Antígenos CD28/fisiologia , Antígeno CTLA-4 , Diferenciação Celular/imunologia , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Imunossupressores/farmacologia , Isoantígenos/biossíntese , Isoantígenos/fisiologia , Teste de Cultura Mista de Linfócitos , Glicoproteínas de Membrana/imunologia , Receptores de Interleucina-2/biossíntese , Tolerância a Antígenos Próprios/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
The capacity of naturally occurring autoreactive CD25+CD4+ regulatory T cells (Treg) to control immune responses both in vivo and in vitro is now well established. It has been demonstrated that these cells undergo positive selection within the thymus and appear to enter the periphery as committed CD25+CD4+ Treg. We have shown previously that CD25+CD4+ Treg with the capacity to prevent skin allograft rejection can be generated by pretreatment with donor alloantigen under the cover of anti-CD4 therapy. Here we demonstrate that this process does not require an intact thymus. Furthermore, generation of these Treg is not dependent on the expansion of CD25+CD4+ thymic emigrants, because depletion of CD25+ cells before pretreatment does not prevent Treg development, and Treg can be generated from CD25-CD4+ precursors. Taken together, these results clearly demonstrate that CD25+CD4+ Treg can be generated in the periphery from CD25-CD4+ precursors in a pathway distinct to that by which naturally occurring autoreactive CD25+CD4+ Treg develop. These observations may have important implications for the design of protocols, both experimental and clinical, for the induction of tolerance to autoantigens or alloantigens in adults with limited thymic function.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Isoantígenos/fisiologia , Receptores de Interleucina-2/biossíntese , Células-Tronco/imunologia , Subpopulações de Linfócitos T/imunologia , Timo/citologia , Timo/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Transfusão de Sangue , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/transplante , Diferenciação Celular/imunologia , Movimento Celular/imunologia , Isoantígenos/administração & dosagem , Isoantígenos/biossíntese , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Receptores de Interleucina-2/administração & dosagem , Receptores de Interleucina-2/imunologia , Transplante de Pele/imunologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/transplante , Timo/metabolismoRESUMO
Trans-differentiation of stem cells shows promise for use in tissue repair medicine. Although poorly defined, mesenchymal stem cells (MSC) appear useful for applications in repair medicine. Despite the low frequency of MSC, they are relatively easy to expand. The expression of MHC class II on MSC, however, could deter their use in repair medicine, since these molecules could stimulate an allogeneic host response. This study sought to compare the immune stimulatory and suppressive effects of MSC. Primary human MSC were cultured from bone marrow aspirates and then passaged at least three times before use in assays. Morphologically, MSC were symmetrical; were SH2(+), MHC class II(+), CD45(-), CD44(+), CD31(-), CD14(-), proly-4-hydroxylase(-); and showed normal karyotype patterns and elevated telomerase activities. MSC elicited significant stimulatory responses when cocultured with allogeneic PBMC. Despite the production of different types of growth factors, allogeneic effects of MSC could not be explained by the production of these growth factors. One-way MLR reactions were significantly blunted by third-party MSC. Similar suppression was not observed for responses to three different recall Ags. Based on these functional differences by MSC in responses to allo- and recall Ags, we examined whether MSC could exert veto-like functions. We showed that MSC could blunt the cytotoxic effects of allogeneic-induced effectors to mitogen-activated targets. The results showed that although MSC elicited allogeneic responses in a model that mimics a graft-vs-host reaction, they also exerted veto-like activity, but caused no effect on responses to recall Ags.
Assuntos
Memória Imunológica , Isoantígenos/fisiologia , Células-Tronco/imunologia , Adulto , Antígenos CD/imunologia , Biópsia por Agulha , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/efeitos da radiação , Sistema Livre de Células/imunologia , Sistema Livre de Células/metabolismo , Células Cultivadas , Citocinas/biossíntese , Citocinas/genética , Testes Imunológicos de Citotoxicidade , Endoglina , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Formaldeído/farmacologia , Raios gama , Antígenos de Histocompatibilidade Classe II/biossíntese , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/efeitos da radiação , Memória Imunológica/efeitos dos fármacos , Memória Imunológica/efeitos da radiação , Teste de Cultura Mista de Linfócitos/métodos , Mesoderma/citologia , Mesoderma/imunologia , Proteínas do Tecido Nervoso/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Polímeros/farmacologia , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Receptores de Superfície Celular , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Células-Tronco/efeitos da radiação , Molécula 1 de Adesão de Célula Vascular/imunologiaRESUMO
The plasma membrane is compartmentalized into microdomains and the association/dissociation of receptors and signaling molecules with/from these membrane domains is a major principle for regulation of signal transduction. By following the reorganization of microdomains on living cells and performing biochemical studies, we show that Ab targeting of the T cell activation-associated Ag CD147 prevents TCR stimulation-dependent reorganization and clustering of microdomains. Triggering CD147 induces a displacement of the GPI-anchored coreceptors CD48 and CD59 from microdomains in human T lymphocytes. This perturbation of microdomains is accompanied by a selective inhibition of TCR-mediated T cell proliferation. The CD147-inhibited cells secret normal levels of IL-2 but acquire reduced amounts of the IL-2 receptor alpha-chain CD25. These results indicate that negative regulating signals can modulate microdomains and suggest a general mechanism for inhibition of receptor signaling.