RESUMEN
BACKGROUND: In this study, we aimed to establish a versatile in vivo model of prostate cancer, which adequately mimics intraprostatic tumor growth, and the natural routes of metastatic spread. In addition, we analyzed the capability of high-resolution ultrasonography (hrUS), in vivo micro-CT (µCT), and 9.4T MRI to monitor tumor growth and the development of lymph node metastases. METHODS: A total of 5 × 105 VCaP cells or 5 × 105 cells of LuCaP136- or LuCaP147 spheroids were injected into the prostate of male CB17-SCID mice (n = 8 for each cell type). During 12 weeks of follow-up, orthotopic tumor growth, and metastatic spread were monitored by repetitive serum-PSA measurements and imaging studies including hrUS, µCT, and 9.4T MRI. At autopsy, primary tumors and metastases were harvested and examined by histology and immunohistochemistry (CK5, CK8, AMACR, AR, Ki67, ERG, and PSA). From imaging results and PSA-measurements, tumor volume doubling time, tumor-specific growth rate, and PSA-density were calculated. RESULTS: All 24 mice developed orthotopic tumors. The tumor growth could be reliably monitored by a combination of hrUS, µCT, MRI, and serum-PSA measurements. In most animals, lymph node metastases could be detected after 12 weeks, which could also be well visualized by hrUS, and MRI. Immunohistochemistry showed positive signals for CK8, AMACR, and AR in all xenograft types. CK5 was negative in VCaP- and focally positive in LuCaP136- and LuCaP147-xenografts. ERG was positive in VCaP- and negative in LuCaP136- and LuCaP147-xenografts. Tumor volume doubling times and tumor-specific growth rates were 21.2 days and 3.9 %/day for VCaP-, 27.6 days and 3.1 %/day for LuCaP136- and 16.2 days and 4.5 %/day for LuCaP147-xenografts, respectively. PSA-densities were 433.9 ng/mL per milliliter tumor for VCaP-, 6.5 ng/mL per milliliter tumor for LuCaP136-, and 11.2 ng/mL per milliliter tumor for LuCaP147-xenografts. CONCLUSIONS: By using different monolayer and 3D spheroid cell cultures in an orthotopic xenograft model, we established an innovative, versatile in vivo model of prostate cancer, which enables the study of both intraprostatic tumor growth as well as metastatic spread to regional lymph nodes. HrUS and MRI are feasible tools to monitor tumor growth and the development of lymph node metastases while these cannot be visualized by µCT.
Asunto(s)
Modelos Animales de Enfermedad , Ganglios Linfáticos/diagnóstico por imagen , Ganglios Linfáticos/patología , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/patología , Animales , Línea Celular Tumoral , Humanos , Imagenología Tridimensional , Metástasis Linfática , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones SCID , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/secundario , Células Tumorales Cultivadas , Ultrasonografía , Microtomografía por Rayos X , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cytotoxic T lymphocytes (CTL) control the replication of human cytomegalovirus (CMV). Previous studies assessed the clonotypic composition of CTL specific for individual immunodominant peptides within a certain HLA context. Such an approach has inherent limitations and may not assess the true clonal CTL response in vivo. Here, the clonotypic composition of CMV-specific CTL was determined in HLA-A2, CMV-seropositive kidney transplant recipients and healthy blood donors after stimulation of peripheral blood mononuclear cells with either a pp65 whole-peptide pool or a single immunodominant peptide. Even after stimulation with the whole peptide pool, CMV-specific CTL remained monoclonal or oligoclonal. Regarding intraindividual variation, the CDR3 motifs of the dominant clones were identical to those observed in CTL generated by the single immunodominant peptide. Sequencing of the CDR3 regions demonstrated significant interindividual variation; however, structural homology was observed for immunodominant clonotypes in three individuals. In conclusion, the highly focused T cell receptor repertoire found after stimulation with either a single immunodominant peptide or a peptide pool demonstrates a pivotal role for immunodominant epitopes in the generation of a clonal repertoire. These results provide new insights into the regulation of CMV clonal dominance and may contribute to the design and monitoring of adoptive immunotherapy.
Asunto(s)
Citomegalovirus/metabolismo , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/virología , Anticuerpos Monoclonales/química , Antígenos/química , Linfocitos T CD8-positivos/metabolismo , Citomegalovirus/genética , Citometría de Flujo/métodos , Antígenos HLA/metabolismo , Antígeno HLA-A2/biosíntesis , Humanos , Inmunoterapia/métodos , Inmunoterapia Adoptiva/métodos , Trasplante de Riñón/inmunología , Péptidos/química , Fosfoproteínas/química , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas de la Matriz Viral/químicaRESUMEN
PURPOSE: To generate and characterize 3D spheroid suspension cultures from radical prostatectomy (RP) specimens as a versatile model system for organ-confined prostate cancer (PCa). METHODS: Cancerous tissue samples from RP specimens were excised by a uropathologist. Preparation of 3D spheroids was done by mechanical disintegration and limited enzymatic digestion followed by serial filtration through 100 µm- and 40 µm-cell strainers. Thereafter, spheroids were cultured in a modified stem cell medium and characterized by a live/dead assay, whole-spheroid immunohistochemistry (IHC; CK5, CK8, AMACR, PSA, Ki67, AR, αSMA, Vimentin, E-Cadherin) and PSA-measurements in culture medium. Furthermore, their response to pharmaceutical treatment with docetaxel, bicalutamide, enzalutamide and abiraterone was tested. RESULTS: 173 RP cases were included. The median preoperative PSA-level was 16.12 ng/ml [range 0.99;345], the median Gleason score was 7b [6;10]. 64 cases were excluded due to low tumor content in frozen sections (43) or to insufficient spheroid formation (21). In the remaining 109 cases, spheroids formed successfully and stayed viable for up to several months. IHC analysis revealed AR-, CK8-, and AMACR-positivity in nearly all cases, while CK5-positive cells were detectable only occasionally as were α-SMA and Vimentin. E-Cadherin was positive in most cases. Furthermore, spheroids proved to be amenable to cryopreservation. While abiraterone had no effect and docetaxel only a moderate effect, spheroid viability was markedly reduced upon bicalutamide and enzalutamide treatment. CONCLUSIONS: Multicellular 3D spheroids can be generated from patient-derived RP tissue samples and serve as an innovative in vitro model of organ-confined PCa.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Neoplasias de la Próstata/patología , Esferoides Celulares , Células Tumorales Cultivadas , Humanos , MasculinoRESUMEN
BACKGROUND: Despite improved immunosuppression, intestinal transplantation is still complicated by severe rejection episodes. To further improve immunosuppressive concepts, we evaluated an anti-CD4 antibody and an anti-tumor necrosis factor (TNF)-alpha monoclonal antibody for their immunosuppressive efficacy in the standard rat model of intestinal transplantation. METHODS: Intestinal transplantation was performed in the DA to Lewis combination, and recipients were treated perioperatively with either the anti-CD4 antibody RIB5/2 (day -1, 0, postoperative days 1, 2, 4, 7, 10, 14, 17, and 21), the anti-TNF antibody etanercept (60 min before reperfusion, postoperative days 3, 6, and 9) or a combination of both. Survival, histology and expression of immunologic mediator genes on days 3 and 4 after transplantation were investigated. RESULTS: Treatment with anti-CD4 antibody alone (19.71+/-5.94) and the antibody combination (171.58+/-122.76) prolonged survival. The chemokine MIP-1alpha was significantly decreased in both anti-CD4 antibody treatment groups, possibly indicating an additional effect of the TNF-alpha blockade on the immune modulation by RIB5/2. CONCLUSIONS: Our study demonstrated long-term graft survival in short-term treatment with a combination of an anti-CD4 antibody and a TNF-alpha antibody in more than 50% of the recipients of intestinal grafts. Such a combined approach could also be useful in clinical small bowel transplantation.
Asunto(s)
Anticuerpos/inmunología , Anticuerpos/uso terapéutico , Antígenos CD4/inmunología , Supervivencia de Injerto/inmunología , Intestino Delgado/inmunología , Intestino Delgado/trasplante , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , Animales , Peso Corporal , Supervivencia Celular , Intestino Delgado/patología , Linfocitos/citología , ARN Mensajero/genética , Ratas , Tasa de Supervivencia , Factores de Tiempo , Donantes de Tejidos , Trasplante HomólogoRESUMEN
BACKGROUND: Posttransplant lymphoproliferative disorder (PTLD) is a life-threatening complication after organ transplantation. The identification of risk factors for PTLD development is important for disease management. It has been shown that cytokine gene polymorphisms are associated with lymphoma and Epstein-Barr virus (EBV)-associated diseases in nonimmunosuppressed patients. In the present case-control study, we analyzed the impact of -1082 interleukin (IL)-10, -308 tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1 (codon 10, 25), and +874 interferon (IFN)-gamma gene single-nucleotide polymorphisms on the late onset EBV-associated PTLD. METHODS: Out of 1,765 solid organ recipients, 38 patients with late-onset EBV-associated PTLD and 408 matched solid organ recipients were selected and enrolled in the study. Single nucleotide polymorphisms (SNPs) for -1082IL-10, -308TNF-alpha, TGF-beta1 (codon 10, 25), and +874IFN-gamma genes were analyzed by a sequence specific primer polymerase chain reaction and were related to the PTLD development, and the disease course and outcome. RESULTS: The TGF-beta1 (codon 25) GG genotype was detected more frequently in controls than in PTLD patients (odds ratio=0.34, 95% confidence interval: 0.17-0.69, P=0.0022). The frequency of -1082 IL-10 GG genotype was also significantly higher in controls than in PTLD patients (odds ratio=0.5, 95% confidence interval: 0.25-1.0, P=0.044). There were no associations between -308TNF-alpha, TGF-beta1 codon 10, and +874IFN-gamma SNPs and PTLD. Disease course and outcome were not associated with any cytokine SNPs. CONCLUSIONS: Polymorphisms in two key anti-inflammatory cytokines, IL-10 and TGF-beta, are associated with susceptibility to EBV-associated PTLD, suggesting that a shift in pro-/anti-inflammatory response is involved in the pathogenesis of PTLD.
Asunto(s)
Predisposición Genética a la Enfermedad/genética , Interleucina-10/genética , Trastornos Linfoproliferativos/genética , Trasplante de Órganos/efectos adversos , Factor de Crecimiento Transformador beta1/genética , Adulto , Estudios de Casos y Controles , Progresión de la Enfermedad , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/genética , Femenino , Genotipo , Humanos , Interferón gamma/genética , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , Factores de Riesgo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND: Cytokines play a crucial role in different immunopathological conditions. Cytokine secretion is reported to be determined by polymorphisms in the cytokine genes. Since TNF-alfa and IL-10 are involved in regulation of inflammation, and TGF-beta 1 can induce fibrosis and renal insufficiency - dominant features of end-stage renal disease (ESRD), we explored the hypothesis that polymorphisms of these cytokine genes may be possible genetic susceptibility factors for the progression of renal failure. METHODS: We studied the IL-10 (-1082), TNF-alfa (-308), TGF-beta 1 (codon 10;25) gene single nucleotide polymorphisms in 118 healthy donors and 103 patients with ESRD (44 hemodialysis patients with diabetic nephropathy and 59 hemodialysis patients with glomerulonephritis) using PCR-SSP. RESULTS: Significant associations of ESRD with the TGF-beta 1 (codon 10) TT (odds ratio [OR] = 5.31; 95% confidence interval [95% CI], 3.77-7.02; p<0.001) and IL-10 (-1082) GG (OR=2.35; 95% CI, 1.67-3.15; p<0.01) genotypes were found. Statistical analysis of genotype frequencies made separately for the underlying renal disease (diabetes or glomerulo-nephritis) revealed the same linkage trend: TGF-beta 1 (codon 10) TT and IL-10 (-1082) GG were associated with type 2 diabetic nephropathy (p<0.001 and p<0.05, respectively) and chronic glomerulonephritis (p<0.001 and p<0.01, respectively). No significant differences in the TNF-alfa , TGF-beta 1 (codon 25) genotype distribution between healthy controls and patients with diabetic nephropathy- or glomerulonephritis-associated ESRD were detected. CONCLUSIONS: Carriage of the TGF-beta 1 (codon 10) TT and IL-10 (-1082) GG genotypes may increase susceptibility to ESRD in German patients with type 2 diabetes or glomerulonephritis.
Asunto(s)
Citocinas/genética , Nefropatías Diabéticas/genética , Glomerulonefritis/genética , Fallo Renal Crónico/genética , Polimorfismo de Nucleótido Simple , Adulto , Anciano , Nefropatías Diabéticas/complicaciones , Femenino , Alemania , Glomerulonefritis/complicaciones , Humanos , Fallo Renal Crónico/etiología , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Cytokine gene polymorphisms may have an impact on the susceptibility to and progression of chronic periodontitis. In this study, we analyzed the -1082 interleukin-10 (IL-10), -308 tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta 1 (TGF-beta1) (codons 10 and 25), -174IL-6, and +874 interferon-gamma (IFN-gamma) gene single-nucleotide polymorphisms in a cohort of patients with chronic periodontal disease. METHODS: The diagnosis was made on the basis of standardized clinical and radiographic criteria. A total of 122 adult patients with chronic periodontitis and 114 unrelated, ethnically and age-matched white control subjects were genotyped by a polymerase chain reaction-sequence-specific primer. RESULTS: The number of individuals carrying the -174IL-6 CC genotype was significantly higher in the group of patients than in the control group (odds ratio [OR] = 1.896; 95% confidence interval [CI] = 1.106 to 3.250; P = 0.0283). The TGF-beta1 (codon 25) GG (Arg(25)/Arg(25)) genotype was detected more frequently in control subjects than in periodontitis patients (OR = 0.459; 95% CI = 0.230 to 0.920; P = 0.0421). CONCLUSION: The -174IL-6 and TGF-beta1 (codon 25) single-nucleotide polymorphisms are associated with susceptibility to chronic periodontitis in the population studied.
Asunto(s)
Interferón gamma/genética , Interleucina-10/genética , Interleucina-6/genética , Periodontitis/genética , Factor de Crecimiento Transformador beta1/genética , Factor de Necrosis Tumoral alfa/genética , Adulto , Anciano , Estudios de Casos y Controles , Enfermedad Crónica , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Periodontitis/inmunología , Polimorfismo de Nucleótido Simple , Valores de ReferenciaRESUMEN
Epidemiological data suggests that Epstein-Barr virus may be involved in the pathogenesis of Multiple Sclerosis (MS). We aimed to determine the frequency of CD8+ T cells specific for one EBNA1-derived epitope (HPVGEADYFEY) in cerebrospinal fluid (CSF) and blood of patients with MS and other inflammatory neurological diseases (OIND). The frequency of specific CD8+ T cells was assessed by HLA-class-I-binding pentamers restricted to HLA-B35. The frequency of HPVGEADYFEY-specific CD8+ T cells did neither differ significantly in blood nor CSF in MS compared to OIND, but was consistently higher in CSF compared to blood regardless of diagnosis.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por Virus de Epstein-Barr/líquido cefalorraquídeo , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Esclerosis Múltiple/líquido cefalorraquídeo , Anticuerpos/sangre , Ensayo de Inmunoadsorción Enzimática , Infecciones por Virus de Epstein-Barr/complicaciones , Femenino , Citometría de Flujo , Humanos , Masculino , Esclerosis Múltiple/sangre , Esclerosis Múltiple/complicaciones , Esclerosis Múltiple/virologíaRESUMEN
Recent data suggest valganciclovir (VGC) to be as effective as ganciclovir for cytomegalovirus (CMV) prophylaxis. The objective of this study was to analyze the effect of oral valganciclovir in renal transplant patients with symptomatic CMV infection. Twenty-one patients with symptomatic CMV infection received VGC in doses adjusted to renal function until resolution of CMV antigenemia. The patients were followed for a mean of 5.5 months. During therapy, CMV antigenemia dropped in all patients from pretreatment positive levels of 5.2 +/- 3.7 to negative values of 0.25 +/- 0.2 positive cells/10,000 PBMC (P<0.001). After cessation of therapy, none of patients developed relapse of CMV antigenemia/symptoms within the follow-up. VGC therapy was well tolerated in all patients and no major adverse effects occurred. This pilot trial showed VGC to be safe and highly effective in antiviral therapy after renal transplantation. However, subsequent multicenter clinical trials for treatment of CMV disease are necessary.
Asunto(s)
Antivirales/uso terapéutico , Infecciones por Citomegalovirus/tratamiento farmacológico , Ganciclovir/análogos & derivados , Ganciclovir/uso terapéutico , Trasplante de Riñón/fisiología , Complicaciones Posoperatorias/virología , Infecciones por Citomegalovirus/epidemiología , Humanos , Proyectos Piloto , Estudios Retrospectivos , ValganciclovirRESUMEN
Chronically immunosuppressed patients, like solid-organ-transplant (SOT) recipients, are at increased risk for severe human cytomegalovirus (HCMV) infection. Despite the availability of effective antiviral drugs, lasting control of remaining viruses is dependent on an effective T-cell immunity. So in some patients conventional antiviral therapy cannot control the infection and prolonged virostatic therapy is limited by its side effects and the development of viral resistance. Selective reconstitution of cellular immunity by adoptive transfer of HCMV-specific T cells derived from healthy donors is a safe and effective approach in hematopoietic stem cell transplant recipients. The aim of this study was to determine whether functional HCMV-specific T cells can also be generated from chronically immunosuppressed patients. Autologous CD4+/8+ T-cell lines directed against the HCMV protein IE-1 were generated from the peripheral blood of SOT patients using a recently developed modular protocol easily applicable to good-manufacturing-practice conditions. T-cell lines from SOT patients showed similar features as cells from healthy donors regarding phenotype, functionality (HCMV-specific killing, gene expression pattern, and cytokine secretion), IE-1 epitope recognition, and dominance of distinct T-cell receptor V beta families. Most importantly, this protocol also allowed the generation of T-cell lines from immunosuppressed patients with HCMV infection/chronic HCMV disease. Our data suggest the potential of this autologous approach for the treatment of SOT recipients suffering from HCMV infection/disease poorly responding to virostatic therapy.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Infecciones por Citomegalovirus/terapia , Citomegalovirus/inmunología , Proteínas Inmediatas-Precoces/inmunología , Inmunoterapia Adoptiva , Linfocitos T CD4-Positivos/trasplante , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/trasplante , Linfocitos T CD8-positivos/virología , Citocinas/inmunología , Citocinas/metabolismo , Citotoxicidad Inmunológica/inmunología , Mapeo Epitopo , Perfilación de la Expresión Génica , Humanos , Trasplante de Riñón/inmunología , Trasplante de Pulmón/inmunologíaRESUMEN
Mannose-binding lectin (MBL) mediates important functions within the innate immune system, and its deficiency was associated with infectious complications. However, in adults without concomitant immunodeficiency the clinical relevance of MBL deficiency remains controversial. We analyzed the distribution of MBL deficiency and its association with concomitant immunodeficiency in 228 adult Caucasian patients with a history of recurrent and/or severe infections. Two hundred forty-one unrelated Caucasians without recurrent or severe infections served as control subjects. The frequency of severe MBL deficiency (plasma levels Asunto(s)
Síndromes de Inmunodeficiencia/complicaciones
, Síndromes de Inmunodeficiencia/inmunología
, Lectina de Unión a Manosa/deficiencia
, Adolescente
, Adulto
, Anciano
, Anciano de 80 o más Años
, Femenino
, Humanos
, Síndromes de Inmunodeficiencia/epidemiología
, Infecciones/sangre
, Infecciones/inmunología
, Masculino
, Lectina de Unión a Manosa/sangre
, Persona de Mediana Edad
, Prevalencia
, Recurrencia
, Estudios Retrospectivos
, Adulto Joven
RESUMEN
BACKGROUND: Post-transplant lymphoproliferative disease (PTLD) is a severe complication in transplant recipients. Detection of increased Epstein-Barr viral (EBV) load in the peripheral blood acts as a surrogate marker for increased risk of PTLD development. We prospectively monitored EBV load, immunosuppression and PTLD in pediatric heart transplant (HTx) patients to determine risk factors for an increased EBV load and risk of PTLD. METHODS: Forty-one pediatric heart transplant recipients were included and underwent prospective monitoring of their immunosuppression and ethylene-diamine tetraacetic acid (EDTA) blood sampling for EBV load (copies/microg DNA) measurement using quantitative real-time polymerase chain reaction (PCR; TaqMan) during January 2001 to December 2006. RESULTS: EBV load was measurable in 70% and was significantly increased (>2,000 copies/microg DNA) in 35% of the patients, with a median EBV load of 5,100 (range 0 to 50,665 copies/microg DNA). Increased EBV load was detected in patients receiving CsA-azathioprine or more than two doses of anti-thymocyte globulin (ATG) and in those <10 years of age, without any significant differences in CsA blood levels. Lowest or negative EBV load was measured in patients receiving CsA-mycophenolate mofetil (MMF) or CsA only. CsA blood levels were not predictable for increased EBV load or PTLD. Six patients developed a EBV-associated B-cell lymphoma (PTLD), among whom 4 (67%) were receiving CsA-azathioprine. CONCLUSIONS: Frequent EBV load monitoring identifies patients at high risk for PTLD development. Azathioprine and ATG are major risk factors for increased EBV load and PTLD and patients may benefit from a change of immunosuppression in addition to pre-emptive anti-viral or anti-tumor strategies.
Asunto(s)
ADN Viral/análisis , Infecciones por Virus de Epstein-Barr/virología , Rechazo de Injerto/prevención & control , Trasplante de Corazón/efectos adversos , Herpesvirus Humano 4/genética , Terapia de Inmunosupresión/efectos adversos , Trastornos Linfoproliferativos/virología , Adolescente , Adulto , Niño , Preescolar , Infecciones por Virus de Epstein-Barr/etiología , Femenino , Estudios de Seguimiento , Rechazo de Injerto/complicaciones , Humanos , Inmunosupresores/efectos adversos , Lactante , Trastornos Linfoproliferativos/etiología , Masculino , Complicaciones Posoperatorias , Pronóstico , Estudios Retrospectivos , Carga ViralRESUMEN
BACKGROUND: The effects of different cell culture media on expansion efficiency and alterations in T-cell receptor V beta (TRBV) expression of in vitro expanded lymphocytes are not well established. MATERIAL/METHODS: Low numbers of CD3+ T cells from peripheral blood lymphocytes of healthy donors were subjected to polyclonal in vitro expansion in the presence of autologous CD3-depleted mononuclear cells as feeder cells (FCs) and their numbers and TRBV expressions were compared in media containing human (HS-RPMI) or fetal bovine serum (FBS-RPMI), Panserin413, or X-Vivo 15TM designed for lymphocyte culture. RESULTS: During three courses of restimulation within 28 days with CD3-antibody (OKT-3), IL-2, and initial CD3+, T-cell: FC ratios of 1:50 lowered to 1:5 and T cells expanded more than 1,000-fold in the media containing complete sera. Loss of cluster formation, associated with expansion failure, was only observed in cultures using synthetic media and resulted in only about 70-fold expansion. Whereas TRVB expression as determined by real-time PCR was substantially altered after 14 days of culture in X-Vivo 15, at day 28 only T cells from long-term culture in HS-RPMI presented the initial TRBV composition. CONCLUSIONS: Culture media have substantial impact on in vitro T-cell expansion. In the presence of FCs, medium containing human serum is superior to synthetic media and FBS-RPMI for long-term culture regarding T-cell number and TRBV repertoire. In contrast, the synthetic media Panserin413 and XVivo15 show lower expansion efficiency and reproducibility and, as RPMI1640+10%FBS, can contribute to overstimulation of certain TRBVs at advanced culture time points.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Linfocitos T/fisiología , Animales , Complejo CD3/biosíntesis , Bovinos , Células Cultivadas , Medio de Cultivo Libre de Suero/farmacología , Cartilla de ADN/química , Humanos , Inmunohistoquímica/métodos , Activación de Linfocitos , Macrófagos/citología , Monocitos/citología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Adoptive immunotherapy with in vitro generated Epstein-Barr virus (EBV)-specific T cells is a safe and effective treatment in patients with EBV-related complications after transplantation. More frequent use of EBV-specific T cells is held back by their cost and time-intensive generation under good manufacturing practice (GMP) conditions. Currently, EBV-specific T cells are produced by repetitive stimulation of peripheral blood mononuclear cells with EBV-infected lymphoblastoid cell lines (LCLs), a protocol that requires several open GMP-handling steps. The aim of the present study was to improve T-cell generation under GMP conditions. We introduce a novel generation protocol that replaces repetitive with short-term LCL stimulation of PMBCs. Vital and formalin-fixed LCLs were used to further increase biosafety. Stimulated T cells were selected by the clinically approved cytokine secretion assay followed by nonspecific expansion. Sufficient numbers of EBV-specific T-cell lines were generated with all protocols. Specific recognition and killing of EBV-infected targets was found and was independent of the generation protocol applied. The novel protocol based on formalin-fixed cells, selection, and expansion reduced open GMP-handling steps and increased biosafety. Furthermore, fixation will allow the use of transgenic LCLs (eg, with cytomegalovirus or tumor antigens) and thereby facilitate the generation of antigen-specific T cells directed against pathogens other than EBV.
Asunto(s)
Formaldehído/química , Herpesvirus Humano 4/inmunología , Inmunoterapia Adoptiva/métodos , Linfocitos T/inmunología , Fijación del Tejido/métodos , Complejo CD3/análisis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular , Línea Celular Tumoral , Técnicas de Cocultivo/métodos , Pruebas Inmunológicas de Citotoxicidad , Epítopos de Linfocito T/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Humanos , Inmunofenotipificación , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Leucocitos Mononucleares/inmunología , Activación de Linfocitos/inmunología , Neoplasias/inmunología , Neoplasias/patología , Neoplasias/terapia , Oligopéptidos/inmunología , Linfocitos T/química , Linfocitos T/citología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Proteínas de la Matriz Viral/inmunologíaRESUMEN
Donor-reactive cellular sensitization does not routinely suggest humoral sensitization and vice versa, but both predict poor kidney transplant outcome. Irrespective of donor reactivity, panel-reactive antibody (PRA) screening identifies patients who are at enhanced risk. Therefore, it was hypothesized that panel-reactive memory T cell reactivity (PRT) might be an additional risk assessment factor of dialysis patients who are on the transplant waiting list. IFN-gamma-enzyme-linked immunosorbent spot memory T cell frequencies were determined in 10 healthy volunteers and 41 hemodialysis patients using for stimulation an allogeneic cell bank (ACB) from 17 healthy individuals who represented the most frequent white HLA antigens. Positive responses to ACB were analogous to PRA defined as percentage of positive assays of the ACB sets. Hemodialysis patients expressed higher PRT levels compared with healthy volunteers. Five of 10 PRT++ patients were PRA negative, and only four of 10 PRA++ patients exhibited PRT reactivity, suggesting independence of humoral and cellular sensitization. Pretransplantation PRT testing of recipients might improve individual risk assessment to make individualized therapy decisions.
Asunto(s)
Prueba de Histocompatibilidad/métodos , Isoanticuerpos/metabolismo , Fallo Renal Crónico/inmunología , Fallo Renal Crónico/metabolismo , Diálisis Renal , Linfocitos T/inmunología , Adulto , Anciano , Bancos de Muestras Biológicas , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Rechazo de Injerto , Humanos , Fallo Renal Crónico/terapia , Trasplante de Riñón , Masculino , Persona de Mediana Edad , Medición de RiesgoRESUMEN
We have investigated the in vivo safety, efficacy, and persistence of autologous Epstein Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) for the treatment of solid organ transplant (SOT) recipients at high risk for EBV-associated posttransplantation lymphoproliferative disease (PTLD). EBV-CTLs generated from 35 patients expanded with normal kinetics contained both CD8 and CD4 lymphocytes and produced significant specific killing of autologous EBV-transformed B lymphoblastoid cell lines (LCLs). Twelve SOT recipients at high risk for PTLD, or with active disease, received autologous CTL infusions without toxicity. Real-time polymerase chain reaction (PCR) monitoring of EBV-DNA showed a transient increase in plasma EBV-DNA suggestive of lysis of EBV-infected cells, although there was no consistent decrease in virus load in peripheral-blood mononuclear cells. Interferon-gamma enzyme-linked immunospot (ELISPOT) assay and tetramer analysis showed an increase in the frequency of EBV-responsive T cells, which returned to preinfusion levels after 2 to 6 months. None of the treated patients developed PTLD. One patient with liver PTLD showed a complete response, and one with ocular disease has had a partial response stable for over one year. These data are consistent with an expansion and persistence of adoptively transferred EBV-CTLs that is limited in the presence of continued immunosuppression but that nonetheless produces clinically useful antiviral activity.
Asunto(s)
Rechazo de Injerto/inmunología , Herpesvirus Humano 4/inmunología , Transfusión de Linfocitos , Linfocitos T Citotóxicos/trasplante , Inmunología del Trasplante , Antígenos Virales/sangre , Preescolar , Femenino , Rechazo de Injerto/prevención & control , Humanos , Lactante , Masculino , Estudios Retrospectivos , Trasplante AutólogoRESUMEN
Efficient protocols to generate cytomegalovirus (CMV)-specific T cells are required for adoptive immunotherapy. Recombinant Epstein-Barr virus (EBV) vectors called mini-EBV can be used to establish permanent B cell lines in a single step, which present the CMV antigen pp65 in a constitutive manner. These B cell lines, coined pp65 mini-LCL, were successfully used to reactivate and expand CMV-specific cytotoxic T cells. Here we evaluate this pp65 mini-EBV system in closer detail, focusing on (1) the quantification of T cells with specific effector function and (2) the identification of CMV-specific CD4(+) helper T cells. The co-expansion of various functional CMV epitope specificities was demonstrated by IFN-gamma enzyme-linked immunospot assay (ELISPOT) assays and HLA-peptide tetramer staining. Single-cell cloning resulted in both CD4(+) and CD8(+) T cell clones, the majority of which was CMV specific. Thus, mini-LCL present the pp65 antigen on HLA class I and II, mobilizing both arms of the T cell response. Using a peptide library covering the pp65 sequence for further analysis of T cell clones, we identified new pp65 CD8(+) and CD4(+) T cell epitopes.
Asunto(s)
Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Citomegalovirus/inmunología , Epítopos de Linfocito T/inmunología , Herpesvirus Humano 4/fisiología , Activación de Linfocitos/inmunología , Adulto , Presentación de Antígeno/inmunología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/virología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular Transformada , Células Clonales , Citomegalovirus/genética , Antígenos HLA/inmunología , Humanos , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/inmunología , Proteínas de la Matriz Viral/metabolismoRESUMEN
Recently, we demonstrated the capacity of allo-specific gene-engineered T lymphocytes as transport vehicle for therapeutic transgenes into allografts. In this study, the influence of viral IL-10 as therapeutic transgene was addressed. Lewis rat T-cell lines specific for DA rat alloantigens were engineered to express vIL-10 by using a retroviral gene expression system. Like T regulatory 1 cells, vIL-10 transgenic T lymphocytes express the phenotype CD4(+)25(+) and secrete, in addition to vIL-10, rat IL-10 and IFN-gamma but no IL-4. First, the capacity of vIL-10 transgenic T-cell lines to modulate alloantigen-specific immune responses was evaluated in vitro. In comparison to control MLR with no transgenic cells or equal numbers of control T(EGFP)-lymphocytes, the proliferation as well as production of IFN-gamma by naive responder cells were significantly diminished. Despite this regulatory capacity in vitro, T(vIL-10)-lymphocytes were not able, either alone or in combination with suboptimal doses of Cyclosporine A, to prolong the survival of either DA rat cardiac or renal allografts in Lewis rat recipients. These data demonstrate that intra-graft IL-10 over-expression is not sufficient to prolong allograft survival in a high-responder strain combination and that the regulatory capacity of T cells in vitro does not predict their in vivo efficiency.
Asunto(s)
Interleucina-10/inmunología , Isoantígenos/inmunología , Trasplante de Órganos , Linfocitos T/inmunología , Transducción Genética , Animales , Ensayo de Inmunoadsorción Enzimática , Tolerancia Inmunológica/inmunología , Masculino , Ratas , Ratas Endogámicas Lew , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Adoptive immunotherapy with antigen-specific T cells has been successfully used to treat certain infectious diseases and cancers. Although more patients may profit from T cell therapy, its more frequent use is restricted by limitations in current T cell generation strategies. The most commonly applied peptide-based approaches rely on the knowledge of relevant epitopes. Therefore, T cells cannot be generated for diseases with unknown epitopes or for patients with unfavorable HLA types. We developed a peptide-based approach for HLA type-independent generation of specific T cells against various proteins. It is based on short-time stimulation with peptide libraries that cover most CD4(+) and CD8(+) T cell epitopes of given proteins. The procedure requires no prior knowledge of epitopes because libraries are synthesized solely on the basis of the protein's amino acid sequence. Stimulation is followed by immunomagnetic selection of activated IFN-gamma-secreting cells and nonspecific expansion. To evaluate the protocol, we generated autologous T cells specific for a well-characterized antigen, the human cytomegalovirus phosphoprotein 65 (pp65). Generated T cell lines consisted of pp65-specific CD4(+) and CD8(+) lymphocytes that displayed antigen-specific killing and proliferation. The protocol combines the biosafety of peptide-based approaches with HLA type independence and may help to advance adoptive immunotherapy in the future.
Asunto(s)
Epítopos de Linfocito T/inmunología , Antígenos HLA/inmunología , Inmunoterapia Adoptiva , Linfocitos T/inmunología , Proliferación Celular , Epítopos , Humanos , Inmunoterapia Adoptiva/métodos , Activación de Linfocitos/inmunología , Factores de TiempoRESUMEN
So far, gene therapy in transplantation mainly focuses on the expression of therapeutic proteins in the graft itself. Insufficient transfection and inflammatory responses that are due to the immunogenicity of multiple vector systems are often limiting factors in these approaches. The potential of genetically modified T lymphocytes was investigated as a delivery system for therapeutic transgenes to transplanted organs as a way to circumvent immunogenicity and efficiency problems in a rat transplant model. Gene-engineering of alloantigen-specific rat T cell lines was performed by using a Moloney murine leukemia virus (MoMuLV)-based enhanced green fluorescence protein (EGFP) encoding retroviral transduction system. The ex vivo gene-modified lymphocytes were adoptively transferred into syngeneic rats carrying allogeneic, syngeneic, or third party kidneys. Homing behavior, activation level, and transgene expression of the adoptively given cells were monitored. The T(EGFP) lymphocytes infiltrated the transplanted kidneys in an antigen-specific manner. High numbers of alloantigen-specific T lymphocytes accumulated exclusively in allografts but not in syngeneic or third party grafts. Flow cytometric analysis revealed that only T(EGFP) lymphocytes found in allografts had an activated phenotype that resulted in higher transgene expression. Alloantigen-specific homing, activation, and transgene expression are important prerequisites for the guarantee of localized delivery and expression of transgenic proteins by gene-engineered T lymphocytes. Antigen-specific gene-targeting strategies using ex vivo modified T lymphocytes with donor specificity are a novel approach to the delivery of therapeutic transgenes in transplantation.