RESUMO
NSCLC arises in the complex environment of chronic inflammation. Depending on lung immune polarization, infiltrating immune cells may either promote or suppress tumor growth. Despite the importance of the immune microenvironment, current staging techniques for NSCLC do not take into consideration the immune milieu in which the neoplasms arise. T-cell subset content was compared between paired tumor-bearing and contralateral lungs, patient and control peripheral blood. The relationship between T-cell subset distribution and survival were evaluated. CD4 and CD8+ T cells were subsetted by CD45RA/CD27 and analyzed for expression of activation, adhesion, and homing markers. Strikingly, T-cell content was indistinguishable between lungs. Compared with peripheral blood, naïve CD4 and CD8 T cells were rare in BAL. CD4+ BAL T cells showed increased CD95 (higher apoptotic potential) and CD103 expression (epithelial adhesion), but decreased CD38 (activation) and CCR7 expression (lymph node homing). CD8+ BAL T cells showed increased CD103 expression and decreased CD28 expression (co-stimulation). Differences in CD28, CD95, and CCR7 expression were more pronounced within memory cells, while differences in CD4+ CD103 expression were more prominent in effector/memory cells. Of these populations, the absence of lung CD4 T cells with an effector-like phenotype (CD45RA+/CD27-) emerged as a predictor of favorable outcome. Patients with a low proportion (≤0.44%) had 90% 5-year survival (n = 10, median survival 2,343 days), compared with 0% (n = 9, median survival 516 days) of patients with a higher proportion. Further study is required to confirm this association prospectively and define the function of this subpopulation.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/cirurgia , Subpopulações de Linfócitos T/imunologia , Idoso , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Subpopulações de Linfócitos T/patologiaRESUMO
Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.
Assuntos
Testes Imunológicos de Citotoxicidade/métodos , Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Células Matadoras Ativadas por Linfocina/imunologia , Células Matadoras Naturais/imunologia , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Complexo CD3/análise , Antígeno CD56/análise , Linhagem Celular Tumoral , Radioisótopos de Cromo/metabolismo , Dactinomicina/análogos & derivados , Dactinomicina/química , Corantes Fluorescentes/química , Proteínas Ligadas por GPI , Granzimas/análise , Humanos , Células K562 , Células Matadoras Ativadas por Linfocina/química , Células Matadoras Ativadas por Linfocina/metabolismo , Células Matadoras Naturais/química , Células Matadoras Naturais/metabolismo , Cinética , Lectinas Tipo C , Leucócitos Mononucleares/imunologia , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Receptores de IgG/análise , Reprodutibilidade dos Testes , Subpopulações de Linfócitos T/química , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/química , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Fatores de TempoRESUMO
BACKGROUND: The Epstein-Barr virus (EBV) establishes and maintains latent infection in B lymphocytes of the healthy adults. Lymphocytes remain viable during red blood cell (RBC) storage. The effect of RBC storage on the stability of EBV-infected B lymphocytes and EBV genome is not known. STUDY DESIGN AND METHODS: Eight randomly selected non-leukoreduced AS-5 RBC units were stored for 42 days under standard blood bank refrigerated at 1-6 degrees C. Cell count and EBV genomes in CD19+ B lymphocytes were measured in fresh products and weekly for 6 weeks. Total white blood cells (CD45+), T lymphocyte (CD3+), and B lymphocyte (CD19+) were quantified by a single platform flow cytometric assay. EBV genomes were quantified by real-time polymerase chain reaction using DNA purified from CD19+ B cells. RESULTS: Viable white blood cell, T and B lymphocytes followed a biphasic decline curve during RBC storage consisting of a steep steady decline during the first 3 weeks followed by a plateau for the remainder of the storage. At the end of the RBC shelf-life, 19% of the original T and B cells remained viable. EBV genomes per 10(5) CD19+ B lymphocytes remained constant during RBC storage. However, the total EBV genomes in the RBC units decline by more than 80% of their original value at the end of RBC storage due to loss of viable B lymphocytes. CONCLUSIONS: The results indicate that lymphocytes and EBV latently infected B cells can survive the normal storage conditions for RBC.
Assuntos
Remoção de Componentes Sanguíneos/métodos , Transfusão de Eritrócitos/métodos , Herpesvirus Humano 4/fisiologia , Linfócitos/fisiologia , Linfócitos/virologia , Viabilidade Microbiana , Antígenos CD19 , Complexo CD3 , DNA Viral/análise , Armazenamento de Medicamentos , Citometria de Fluxo , Herpesvirus Humano 4/isolamento & purificação , Humanos , Antígenos Comuns de Leucócito , Contagem de Linfócitos , Reação em Cadeia da Polimerase/métodosRESUMO
ABC transporters are highly conserved and represent a major protective mechanism for barrier tissues as well as adult tissue stem cells. Emerging data support the existence of a cancer stem cell that shares features of tissue stem cells, including the ability to self-renew and undergo dysregulated differentiation. Here we show that a rare population of cells coexpressing MDR transporters and stem cell markers is a common feature across therapy-naive epithelial cancers as well as normal epithelial tissue. MDR+ and MDR- candidate tumor stem and progenitor populations were all capable of generating highly anaplastic transplantable human tumors in NOD/SCID. The finding that rare cells bearing stem cell markers and having intrinsic MDR expression and activity are already present within the tumorigenic compartment before treatment with cytotoxic agents is of critical importance to cancer therapy. Just as damaged normal epithelial tissues regenerate after chemotherapy by virtue of highly protected resting tissue stem cells, the existence of malignant counterparts in therapy-naive epithelial cancers suggests a common mechanism by which normal and tumor stem cells protect themselves against toxic injury.
Assuntos
Neoplasias Pulmonares/patologia , Células-Tronco/citologia , Células Epiteliais/citologia , Células Epiteliais/imunologia , Citometria de Fluxo , Humanos , Imunofenotipagem , Neoplasias Pulmonares/imunologia , Células-Tronco/imunologiaRESUMO
BACKGROUND: P-glycoprotein (P-gp) is a member of the ABC transporter superfamily. P-gp activity can be detected by measuring efflux of fluorescent substrates such as rhodamine 123 (R123). Our objectives were to evaluate P-gp activity in T cells freshly isolated from bronchoalveolar lavage (BAL) and to develop a strategy to distinguish between basal, in vitro substrate-induced, and total P-gp activities. METHODS: Cells were obtained from blood (n = 44) and BAL (n = 34), stained for expression of CD3, CD4, CD8, and CD14, and incubated with R123 (0.13 microM) +/- cyclosporine (5 microM), a specific P-gp inhibitor. P-gp activity was detected as median fluorescence intensity (MFI) and percentage of cells falling below a pre-established cutoff. RESULTS: BAL T cells displayed significant basal P-gp activity, which was most apparent when measured as percentage below the cutoff. Induced activity (difference between P-gp activity measured after load and efflux) was determined equally well when using the MFI or the percentage below cutoff parameter. Total activity was represented by the efflux parameters (MFI or percentage below cutoff) or by the activity-time area under the curve (AUC) method. The two efflux parameters correlated well but were insensitive to the time-dependent nature of dye efflux. In the AUC method, two samples with identical R123 brightness or percentage below cutoff after dye efflux can have very different total activities, depending on their basal activity. The AUC method was also most sensitive in distinguishing between P-gp activity in peripheral blood and resident lung T cells. Application of this methodology to the comparison of P-gp activity in BAL and peripheral CD8+ T cells best revealed the elevated total P-gp activity in BAL T cells. CONCLUSIONS: We have systematically evaluated several methodologies for analysis of P-gp activity and arrived at a novel and robust strategy amenable to standardization and evaluation of the effects of P-gp modulators in vivo and in vitro.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Líquido da Lavagem Broncoalveolar/imunologia , Citometria de Fluxo/métodos , Subpopulações de Linfócitos T/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Lavagem Broncoalveolar , Líquido da Lavagem Broncoalveolar/citologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Ciclosporina/farmacologia , Fluorescência , Humanos , Leucócitos Mononucleares/ultraestrutura , Rodamina 123/metabolismo , Subpopulações de Linfócitos T/citologiaRESUMO
A subset of circulating T cells (CD8(+)CD45RO(-)CD27(-)) with a naïve phenotype, but mediating effector function, is considered to play an important role in host antitumour defence. To investigate the attributes of these effector T cells in patients with squamous cell carcinoma (SCC) of the head and neck cancer, venous blood was obtained from 39 individuals with cancer and 45 normal controls (NC). Peripheral blood mononuclear cells were isolated, stained with labelled monoclonal antibodies specific for CD8, CD45RO, CD45RA, CD62L, CD27, TCR-zeta as well as isotype controls and examined by multicolour flow cytometry. Annexin V binding to CD8(+) T cells and PMA/ionomycin-induced IFN-gamma expression were also evaluated in patients and NC. The proportions of CD45RA(+)CD45RO(-) (naïve) and CD45RA(-)CD45RO(+) (memory) cells were found to be comparable within the CD8(+) T-cell subset. However, relative to NC, the frequency of effector CD8(+)CD45RO(-)CD27(-) cells was strikingly increased in all SCC patients regardless of the disease status (P=0.0003). The proportion of these cells was found to increase with age in both patients and NC. In NC, stimulated IFN-gamma expression was largely restricted to CD8(+)CD45RO(-)CD27(+) cells, while in patients CD8(+)CD45RO(-)CD27(-) expressed IFN-gamma after ex vivo stimulation. Expression of the TCR-associated zeta chain was decreased or absent in freshly isolated CD8(+)CD45RO(-)CD27(-) T cells in patients (P<0.0001). Annexin V was found to bind to a higher proportion of circulating CD8(+) T cells in patients than NC (P<0.006), and significantly more Annexin V(+) T cells were present in the effector (P<0.0059) than the naïve subset within the CD8(+)CD45RO(-) compartment. The data indicate that the expanded CD8(+)CD45RO(-)CD27(-) T cells, which contain precursors of IFN-gamma-producing T cells, are zeta-negative and sensitive to apoptosis in the circulation of patients with HNC.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Carcinoma de Células Escamosas/imunologia , Neoplasias de Cabeça e Pescoço/imunologia , Antígenos Comuns de Leucócito/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Anexina A5/metabolismo , Apoptose , Linfócitos T CD8-Positivos/patologia , Carcinógenos/farmacologia , Carcinoma de Células Escamosas/patologia , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Citometria de Fluxo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Memória Imunológica , Interferon gama/metabolismo , Ionomicina/farmacologia , Ionóforos/farmacologia , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/imunologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
BACKGROUND: PBPC or BM is increasingly being harvested in remission for possible use in the event of relapse. Although the value of this approach has not been demonstrated, the long-term storage of progenitor cell components has become commonplace in many facilities. METHODS: We used multi-parameter flow cytometry to determine the viability of 11 long-term cryopreserved BM components (mean = 11.8 years) in liquid phase nitrogen. The components, prepared for autotransplantation but deaccessioned after confirming patient death, were carefully thawed, washed, and assayed immediately. The flow cytometry assay was performed according to the ISHAGE protocol, modified by the addition of 7AAD for analysis of progenitor viability (CD45+ CD34+ 7AAD-) and total leukocyte viability (CD45+ 7AAD-). In addition, total viability was assessed by fluorescence microscopy using acridine orange dye exclusion; granulocyte-monocyte colony-forming units (CFU-GM) were measured after 14 days culture. RESULTS: Leukocyte viability by flow cytometry and fluorescence microscopy agreed well (r2 = 0.55, slope = 0.83, P < 0.0005 by linear regression). CFU-GM did not correlate with CD34% or any of the viability parameters. Compared with short-term stored (mean = 33 days) PBPC assayed at infusion, long-term stored BM had a comparable percentage of CD34+ cells, comparable CFU-GM activity, increased CD34 viability, but decreased total cell viability, the latter most likely due to an increased proportion of differentiated myeloid cells. DISCUSSION: The results indicate that BM products can be cryopreserved for more than a decade without apparent loss of progenitor activity, as measured by these laboratory surrogates. This agrees with clinical anecdotes describing successful engraftment with long-term stored BM, and argues that expiration dates cannot be set for cryopreserved hematopoietic stem-cell components stored in liquid phase nitrogen.
Assuntos
Criopreservação , Células-Tronco Hematopoéticas/citologia , Antígenos CD34/metabolismo , Sobrevivência Celular , Citometria de Fluxo/métodos , Células-Tronco Hematopoéticas/imunologia , Leucócitos/citologia , Leucócitos/imunologia , TempoRESUMO
BACKGROUND: Considerable interest has focused recently on murine CD8alpha- and CD8alpha+ dendritic cell (DC) subsets, because of their roles in initiating and regulating immune responses. Attention has also centered on their presumed human counterparts, DC1 and DC2, respectively, and their precursors. Identification and quantification of these subsets in the blood may be crucial to understanding and monitoring of their immunologic significance, particularly in humans, where blood may be the only tissue readily or routinely available. METHODS: Leukocytes were isolated from anticoagulated human or mouse (C57BL/10J) blood using conventional procedures. Four-color, rare-event, flow cytometric analysis was used to identify DC1 precursors (pDC1; lineage [lin]- CD4+ CD11c+ HLA-DR+) or DC2 precursors (pDC2; lin- CD4+ CD11c- CD123(hi) [IL-3Ralpha(hi)] HLA-DR+) in normal humans. In mice, CD8alpha+ (CD11b(lo), CD11c+) and CD8alpha- (CD11b(hi), CD11c+) DC subsets were identified both in normal animals and after administration of the potent DC growth factor, fms-like tyrosine kinase 3 ligand (Flt3L). RESULTS: All human subjects examined had discrete populations of pDC1 and pDC2 comprising approximately 0.6% and 0.1% of blood mononuclear cells. CD8alpha- and CD8alpha+ DC constituted approximately 0.75% and 0.2%, respectively, of blood mononuclear cells in normal mice, and 12% and 0.5%, respectively, in Flt3L-treated animals. Flt3L administration substantially increased the absolute numbers of circulating CD11c+ DC by approximately 200-fold. CONCLUSIONS: In addition to pDC1 and CD8alpha- DC, pDC2 and CD8alpha+ DC can be identified in normal human or mouse blood, respectively. Monitoring and isolation or characterization of these cells may provide novel insights into their functional significance in transplantation and other clinical conditions.
Assuntos
Células Sanguíneas/imunologia , Células Dendríticas/imunologia , Adjuvantes Imunológicos/farmacologia , Adulto , Animais , Antígenos CD8/sangue , Feminino , Citometria de Fluxo , Humanos , Contagem de Linfócitos , Masculino , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Plasmócitos/imunologia , Células-Tronco/imunologiaRESUMO
The mission of the recently established Immune Tolerance Network includes the development of protocols for the induction of transplant tolerance in organ allograft recipients and the development of assays that correlate with and may be predictive of the tolerant state. The state of clinical organ transplant tolerance seems to already exist in a small minority of conventionally immunosuppressed liver and, more rarely, kidney transplant patients. Immunosuppressive drug therapy has been withdrawn from these patients for a variety of reasons, including protocolized weaning for a uniquely large group of liver patients at the University of Pittsburgh. In this study, we propose to evaluate the validity of a variety of in vitro immunologic and molecular biologic tests that may correlate with, and be predictive of, the state of organ transplant tolerance in stable liver patients off immunosuppression. Only peripheral blood will be available for the execution of these tests. Both adult and pediatric liver graft recipients will be studied, in comparison to appropriate controls. We shall examine circulating dendritic cell (DC) subsets [precursor (p) DC1 and p DC2] including cells of donor origin, and assess both the frequency and function of donor-reactive T cells by ELISPOT and by trans-vivo delayed-type hypersensitivity analysis in a surrogate murine model. Cytokine gene polymorphism and alloantibody titers will also be investigated. It is anticipated that the results obtained may provide physicians with a tolerance assay "profile" that may determine those patients from whom immunosuppressive therapy may be safely withdrawn.
Assuntos
Tolerância Imunológica , Imunoconjugados , Transplante de Fígado/imunologia , Abatacepte , Antígenos CD , Antígenos de Diferenciação/genética , Antígenos CD40/genética , Antígeno CTLA-4 , Citocinas/genética , Células Dendríticas/fisiologia , Humanos , Hipersensibilidade Tardia , Isoantígenos/imunologia , Polimorfismo GenéticoRESUMO
BACKGROUND: Major histocompatibility complex (MHC)-peptide tetrameric complexes (tetramers) are valuable tools for detecting and characterizing peptide-specific T cells. Because the frequency of these cells is generally very low, it may be difficult to discriminate between nonspecific and specific tetramer binding. METHODS: A four-color flow cytometric assay that simultaneously measures tetramer, CD3, CD8, and CD14 was used to investigate the sensitivity and specificity of MHC class I tetramer staining. This was accomplished by using the influenza virus matrix protein peptide, GILGFVFTL (FLU), as a model recall antigen and the human immunodeficiency virus (HIV) reverse transcriptase peptide, ILKEPVHGV (HIV), as a model novel antigen. Peripheral blood mononuclear cells (PBMC) from 31 HLA-A2.1(+) and 10 HLA-A2.1(-) healthy individuals were stained with the tetramers. RESULTS: The lower limit of detection was established at approximately 1/8,000. In HLA-A2(+) PMBC, frequencies of tetramer-positive CD8(+) T cells were log normally distributed and were high for FLU (1/910) but low for HIV (1/6,067). A novel competition assay, in which tetramer binding was shown to diminish subsequent staining with anti-CD3 antibody, was used to confirm the specificity of tetramer binding to the T-cell receptor (TCR) complex. The competition assay was validated by evaluating several anti-CD3 antibodies and showing that in PBMC from HLA-A2(-) subjects, spurious tetramer-positive events (1/20,000) failed to compete with CD3 binding. For the "recall" FLU tetramer, the degree of competition was proportional to the frequency, suggesting a selection of high avidity cells. Although CD3 competition was also highly correlated with the intensity of tetramer staining, competition allowed the identification of false positive cases with relatively high tetramer staining intensity. CONCLUSION: The data indicate that competition of CD3 binding allows confirmation of the specificity of tetramer binding to the TCR, extending the usefulness of tetramers in the frequency analysis of peptide-specific T lymphocytes.
Assuntos
Complexo CD3/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Antígenos Virais/química , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Ligação Competitiva , Complexo CD3/análise , Complexo CD3/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citometria de Fluxo/métodos , HIV/imunologia , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/imunologia , Transcriptase Reversa do HIV/metabolismo , Antígeno HLA-A2/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Antígenos Comuns de Leucócito/análise , Substâncias Macromoleculares , Orthomyxoviridae/imunologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Sensibilidade e Especificidade , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/imunologia , Proteínas da Matriz Viral/metabolismoRESUMO
BACKGROUND: WBC-reduced platelet components may be prepared by filtration or apheresis processing. Both methods have previously been shown to result in a residual total WBC content <5 x 10(6) per component. However, there may be differences in the efficacy of these techniques for removing certain WBC subsets. STUDY DESIGN AND METHODS: Two multiparameter flow cytometric assays were developed and validated to perform WBC analysis on WBC-reduced platelets collected with two apheresis instruments (Amicus and COBE Spectra) and on 6 units of filtered pooled random-donor platelet concentrates. RESULTS: All components contained <1 x 10(5) WBCs. The COBE Spectra and Amicus apheresis platelet components contained more WBCs than did filtered pooled platelets (p<0.05). Lymphocytes (T and B), monocytes, and granulocytes were identified in all components. Granulocyte content was lowest in the Amicus components and filtered pools. Monocytes were lowest in filtered pools. Amicus platelet components had fewer granulocytes and monocytes than the COBE Spectra platelets. Amicus and COBE Spectra components contained more lymphocytes than the filtered pools. CONCLUSION: Multiparameter flow cytometry can be used to quantify and characterize WBCs in WBC-reduced platelet components. WBC reduction by filtration or apheresis was highly effective. WBCs from each subset were identified in all components. Although filtered pools had the lowest numbers of WBCs, the very low numbers observed in all components suggests that the absolute quantitative differences in WBC subset content are of questionable clinical significance.
Assuntos
Citometria de Fluxo/métodos , Plaquetoferese/métodos , Contagem de Células Sanguíneas , Plaquetas/citologia , Humanos , Sensibilidade e EspecificidadeRESUMO
Human dendritic cells (DC) have polarized responses to chemokines as a function of maturation state, but the effect of maturation on DC trafficking in vivo is not known. We have addressed this question in a highly relevant rhesus macaque model. We demonstrate that immature and CD40 ligand-matured monocyte-derived DC have characteristic phenotypic and functional differences in vitro. In particular, immature DC express CC chemokine receptor 5 (CCR5) and migrate in response to macrophage inflammatory protein-1alpha (MIP-1alpha), whereas mature DC switch expression to CCR7 and respond exclusively to MIP-3beta and 6Ckine. Mature DC transduced to express a marker gene localized to lymph nodes after intradermal injection, constituting 1.5% of lymph node DC. In contrast, cutaneous DC transfected in situ via gene gun were detected in the draining lymph node at a 20-fold lower frequency. Unexpectedly, the state of maturation at the time of injection had no influence on the proportion of DC that localized to draining lymph nodes, as labeled immature and mature DC were detected in equal numbers. Immature DC that trafficked to lymph nodes underwent a significant up-regulation of CD86 expression indicative of spontaneous maturation. Moreover, immature DC exited completely from the dermis within 36 h of injection, whereas mature DC persisted in large numbers associated with a marked inflammatory infiltrate. We conclude that in vitro maturation is not a requirement for effective migration of DC in vivo and suggest that administration of Ag-loaded immature DC that undergo natural maturation following injection may be preferred for DC-based immunotherapy.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/imunologia , Monócitos/imunologia , Vacinas/imunologia , Transferência Adotiva/métodos , Animais , Antígenos CD40/metabolismo , Ligante de CD40 , Contagem de Células , Diferenciação Celular/imunologia , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Células Dendríticas/transplante , Vetores Genéticos/imunologia , Proteínas de Fluorescência Verde , Humanos , Imunofenotipagem , Injeções Intradérmicas , Ligantes , Proteínas Luminescentes/administração & dosagem , Proteínas Luminescentes/genética , Proteínas Luminescentes/imunologia , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/metabolismo , Macaca mulatta , Glicoproteínas de Membrana/farmacologia , Monócitos/citologia , Pinocitose/imunologia , Receptores de Quimiocinas/biossíntese , Pele/citologia , Pele/imunologia , TransfecçãoRESUMO
We developed a murine IgG1 mAb, 5G9, following immunization of a BALB/c mouse with Daudi cells. By immunoprecipitation, 5G9 reacted with a 220-kDa Ag on Daudi cells, which reduced to four subunits (55, 65, 80, and 85 kDa). mAb 5G9 bound to 40-60% of peripheral blood B cells, weakly reacted with monocytes and granulocytes, and did not bind to erythrocytes, platelets, T cells, or NK cells. mAb 5G9 brightly stained scattered cells in human tonsil sections, which appeared to be dendritic cells (DC) by morphology. mAb 5G9 also stained scattered cells in cytospin slides of monocyte-derived DC with long, thin, beaded membrane processes, morphologically distinct from other monocyte-derived DC. Positive selection of blood mononuclear cells with mAb 5G9 and sheep anti-mouse IgG Dynabeads demonstrated an enriched population of DC. By flow cytometry analysis, these cells were CD19, CD20, CD22, CD40, CD44, CD83, CD86, IgD, and HLA-Dr positive and either kappa- or lambda-L chain positive. They did not express CD3, CD4, CD5, CD10, CD11b, CD13, CD25, CD56, CD14, CD33, or CD64. Isolated 5G9+ cells were potent APCs in allogeneic MLR, compared with 5G9- PBMC, 5G9- B cells, monocytes, and monocytes cultured in IL-4 and GM-CSF for 24 h. mAb 5G9 defines a novel peripheral blood cell with B cell phenotype and DC morphology and function: DC-like B cells. The significance of this cell in immune responses requires further study.
Assuntos
Anticorpos Monoclonais/sangue , Antígenos de Superfície/imunologia , Subpopulações de Linfócitos B/imunologia , Células Dendríticas/imunologia , Adulto , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Subpopulações de Linfócitos B/química , Subpopulações de Linfócitos B/citologia , Células Cultivadas , Células Dendríticas/química , Células Dendríticas/citologia , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Separação Imunomagnética , Isoantígenos/imunologia , Ativação Linfocitária/imunologia , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Células Tumorais CultivadasRESUMO
Factors influencing mobilization and engraftment of PBSC were analyzed in 38 patients with metastatic breast cancer who were undergoing PBSC transplantation. None of these patients had had previous chemotherapy for metastatic disease. PBSC were mobilized with cyclophosphamide (CY) and G-CSF (n = 21) or CY and etoposide (CY-etoposide) and G-CSF (n = 17). All received cyclophosphamide 6000 mg/m2, thiotepa 500 mg/m2, and carboplatin 800 mg/m2 (CTCb) as preparative regimen. PBSC infusion was followed by G-CSF at 5 microg/kg in 30 patients or 10 microg/kg in 8 patients. A median number of 27 x 10(6) CD34+ cells/kg was obtained with a median of four aphereses. Previous chemotherapy, radiation therapy, marrow disease, time from previous chemotherapy to mobilization, and type of mobilization regimen did not have a statistically significant effect on collection efficiency (CE). CE was defined as the total number of CD34+ collected/number of collections. Engraftment was rapid, with patients reaching a neutrophil count of 0.5 x 10(9)/L a median of 9 days (range 7-23) and a platelet count of 20 x 10(9)/L a median of 12 days (range 8-28) after transplantation. Shorter times to platelet recovery were associated with a higher number of CD34+ cells infused (p = 0.012), CY mobilization (p = 0.033), and a lower number of prior chemotherapy cycles (p = 0.022). When the number of CD34+ cells was included in the proportional hazard model, no other variables were found to be significant predictors of platelet engraftment. Time to neutrophil recovery was negatively associated with the dose of G-CSF used after transplantation (p = 0.036) CD34 cell dose is an important predictor of engraftment kinetics. A posttransplant dose of G-CSF improves neutrophil recovery. For patients with metastatic breast cancer and no previous chemotherapy for metastatic disease, we have no evidence for a difference between CY and CY-Etoposide as the mobilization regimen.
Assuntos
Neoplasias da Mama/terapia , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Adulto , Antígenos CD34 , Contagem de Células Sanguíneas , Neoplasias da Mama/patologia , Feminino , Sobrevivência de Enxerto , Humanos , Pessoa de Meia-Idade , Metástase NeoplásicaRESUMO
A central question in hematopoiesis is how cell-cycling behavior changes during the emergence of the differentiated state. To further understand what genetic regulators might couple proliferation status to differentiation, we studied the expression of the cell-cycle inhibitors p21 and p27 during the in vitro differentiation of normal CD34(+) blast cells along the myeloid lineage. We find p27 but not p21 to be expressed in freshly harvested resting CD34(+) cells. Thereafter, p21 levels peak concurrent with cellular proliferation and then decline in expression as cells undergo terminal differentiation. In contrast, p27 levels are fairly constant but the subcellular localization of p27 changes from nuclear expression to predominantly cytoplasmic expression and finally to perinuclear localization at progressive stages of differentiation. This report discusses the implications of these findings.
Assuntos
Proteínas de Ciclo Celular , Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/genética , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Supressoras de Tumor , Antígenos CD/análise , Antígenos CD34/análise , Diferenciação Celular , Ciclina A/genética , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Inibidores Enzimáticos , Sangue Fetal/citologia , Citometria de Fluxo , Humanos , Recém-Nascido , Frações Subcelulares/metabolismoRESUMO
Cytomegalovirus (CMV) disease continues to be a major problem for lung transplant recipients. In CMV-seropositive individuals, we detected two types of CMV-specific responses: a self-restricted response stimulated by soluble CMV antigen (sCMV-Ag) and a non-self-restricted response induced by CMV-infected cells (cCMV-Ag). Lung transplant recipients who develop the CMV-specific self-restricted T helper response have a low risk of recurrent CMV disease. In contrast, during CMV disease, lung transplant recipients exhibit only the non-self-restricted T helper responses. We characterized the T cell activation and the kinetics of cytokine production of sorted CD4+ and CD8+ T cells from PBLs of CMV seropositive donors. The two types of CMV antigens induced cytokine production in both T cell subsets. We also performed competitive RT-PCR for Granzyme B (GB) in BAL cells of lung transplant recipients prior to, during and following CMV disease. CMV disease was associated with increase in GB gene expression when was accompanied by acute cellular rejection while it remained low in patients with CMV disease that did not have a complicated course. In summary, CMV-activated T cells within the allograft may produce inflammatory cytokines and effector molecules that may promote allograft rejection.
Assuntos
Citocinas/biossíntese , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Transplante de Pulmão/efeitos adversos , Linfócitos T Auxiliares-Indutores/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígenos Virais/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Infecções por Citomegalovirus/virologia , Granzimas , Humanos , Memória Imunológica , Lectinas Tipo C , Ativação Linfocitária , Complicações Pós-Operatórias/imunologia , Complicações Pós-Operatórias/virologia , RNA Mensageiro/metabolismo , Receptores de Interleucina-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/metabolismo , Subpopulações de Linfócitos T/imunologiaRESUMO
Studies of circulating T (CD3(+)) lymphocytes have shown that on a population basis T-cell numbers remain stable for many years after HIV-1 infection (blind T-cell homeostasis), but decline rapidly beginning approximately 1.5-2.5 years before the onset of clinical AIDS. We derived a general method for defining the loss of homeostasis on the individual level and for determining the prevalence of homeostasis loss according to HIV status and the occurrence of AIDS in more than 5,000 men enrolled in the Multicenter AIDS Cohort Study. We used a segmented regression model for log10 CD3(+) cell counts that included separate T-cell trajectories before and after a time (the T-cell inflection point) where the loss of T-cell homeostasis was most likely to have occurred. The average slope of CD3(+) lymphocyte counts before the inflection point was close to zero for HIV- and HIV+ men, consistent with blind T-cell homeostasis. After the inflection point, the HIV+ individuals who developed AIDS generally showed a dramatic decline in CD3(+) cell counts relative to HIV- men and HIV+ men not developing AIDS. A CD3(+) cell decline of greater than 10 percent per year was present in 77% of HIV+ men developing AIDS but in only 23% of HIV+ men with no onset of AIDS. Our findings at the individual level support the blind T-cell homeostasis hypothesis and provide strong evidence that the loss of homeostasis is an important mechanism in the pathogenesis of the severe immunodeficiency that characterizes the late stages of HIV infection.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Infecções por HIV/imunologia , Linfócitos T/imunologia , Complexo CD3/imunologia , Estudos de Coortes , Progressão da Doença , HIV-1 , Humanos , MasculinoRESUMO
We previously reported that blind T cell homeostasis, in which the total T cell count is maintained but the CD4(+) and CD8(+) subset composition of the T cells can vary, fails approximately 1.5 to 2.5 years before the onset of AIDS. The present study was premised on the hypothesis that if failure of T cell homeostasis (i.e., a decline in total T cell counts) is important in the pathogenesis of AIDS, it should be a significant predictor of AIDS after controlling for the CD4(+) lymphocyte count. Data from 1556 homosexual men with sufficient sequential T cell subset measurements were evaluated, representing 11,988 person-visits in men with known clinical outcomes over a period of more than 10 years. Using regression models that incorporated CD4(+) lymphocyte count and HIV-related symptoms (fever, thrush), it was determined that a yearly decline of more than 300 T cells/microliter of peripheral blood was an independent predictor of the onset of AIDS for subjects with CD4(+) lymphocyte counts of <500 cells/microliter. The results support an important role for failure of T cell homeostasis in the pathogenesis of AIDS.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , HIV-1 , Linfócitos T/citologia , Linfócitos T/imunologia , Síndrome da Imunodeficiência Adquirida/sangue , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Estudos de Coortes , Seguimentos , Infecções por HIV/sangue , Infecções por HIV/imunologia , Soropositividade para HIV , Homeostase , Humanos , MasculinoRESUMO
Treatment of cells with granulocyte colony-stimulating factor (G-CSF) leads to tyrosine phosphorylation of cellular proteins. G-CSF stimulates both the activation of protein tyrosine kinases Lyn, Jak1, and Jak2 and the association of these enzymes with the G-CSF receptor. Wild-type, lyn-deficient, and syk-deficient chicken B lymphocyte cell lines were transfected with the human G-CSF receptor, and stable transfectants were studied. G-CSF-dependent tyrosyl phosphorylation of Jak1 and Jak2 occurred in all three cell lines. Wild-type and syk-deficient transfectants responded to G-CSF in a dose-responsive fashion with increased thymidine incorporation, but none of the clones of lyn-deficient transfectants did. Ectopic expression of Lyn, but not that of c-Src, in the lyn-deficient cells restored their mitogenic responsiveness to G-CSF. Ectopic expression in wild-type cells of the kinase-inactive form of Lyn, but not of the kinase-inactive form of Jak2, inhibited thymidine incorporation in response to G-CSF. These studies show that the absence of Lyn results in the loss of mitogenic signaling in the G-CSF signaling pathway and that activation of Jak1 or Jak2 is not sufficient to cause mitogenesis.