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1.
Immunooncol Technol ; 8: 12-20, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-35757563

RESUMEN

The composition of the commensal microbiota has recently emerged as a key element influencing the efficacy of cancer treatments. It has become apparent that the interplay between the microbiome and immune system within the host influences the response to immunotherapy, particularly immune checkpoint inhibitor therapy. Identifying the key components of the gut microbiota that influence this response is paramount for designing therapeutic interventions to enhance the response to cancer therapy. This review will discuss strategies being considered to modulate the gut microbiota, including fecal microbiota transplantation, administration of defined bacterial isolates as well as bacterial consortia, supplementation with probiotics, and lifestyle modifications such as dietary changes. Understanding the influence of the complex variables of the human microbiota on the effectiveness of cancer therapy will help drive the clinical design of microbial-based interventions in the field of oncology.

2.
Ann Oncol ; 30(4): 589-596, 2019 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-30689736

RESUMEN

BACKGROUND: With increasing anti-PD-1 therapy use in patients with melanoma and other tumor types, there is interest in developing early on-treatment biomarkers that correlate with long-term patient outcome. An understanding of the pathologic features of immune-mediated tumor regression is key in this endeavor. MATERIALS AND METHODS: Histologic features of immune-related pathologic response (irPR) following anti-PD-1 therapy were identified on hematoxylin and eosin (H&E)-stained slides in a discovery cohort of pre- and on-treatment specimens from n = 16 patients with advanced melanoma. These features were used to generate an irPR score [from 0 = no irPR features to 3 = major pathologic response on biopsy (MPRbx, ≤10% residual viable tumor)]. This scoring system was then tested for an association with objective response by RECIST1.1 and overall survival in a prospectively collected validation cohort of pre- and on-treatment biopsies (n = 51 on-treatment at 4-week timepoint) from melanoma patients enrolled on the nivolumab monotherapy arm of CA209-038 (NCT01621490). RESULTS: Specimens from responders in the discovery cohort had features of immune-activation (moderate-high TIL densities, plasma cells) and wound-healing/tissue repair (neovascularization, proliferative fibrosis) compared to nonresponders, (P ≤ 0.021, for each feature). In the validation cohort, increasing irPR score associated with objective response (P = 0.009) and MPRbx associated with increased overall survival (n = 51; HR 0.13; 95%CI, 0.054-0.31, P = 0.015). Neither tumoral necrosis nor pretreatment histologic features were associated with response. Eight of 16 (50%) of patients with stable disease showed irPR features, two of which were MPRbx, indicating a disconnect between pathologic and radiographic features at the 4-week on-therapy timepoint for some patients. CONCLUSIONS: Features of immune-mediated tumor regression on routine H&E-stained biopsy slides from patients with advanced melanoma correlate with objective response to anti-PD-1 and overall survival. An on-therapy biopsy may be particularly clinically useful for informing treatment decisions in patients with radiographic stable disease. This approach is inexpensive, straightforward, and widely available.


Asunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Biomarcadores de Tumor/análisis , Melanoma/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Piel/patología , Adulto , Anciano , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos Inmunológicos/farmacología , Biopsia , Femenino , Humanos , Ipilimumab/farmacología , Ipilimumab/uso terapéutico , Estimación de Kaplan-Meier , Masculino , Melanoma/inmunología , Melanoma/mortalidad , Melanoma/patología , Persona de Mediana Edad , Neoplasia Residual , Nivolumab/farmacología , Nivolumab/uso terapéutico , Receptor de Muerte Celular Programada 1/inmunología , Estudios Prospectivos , Criterios de Evaluación de Respuesta en Tumores Sólidos , Piel/efectos de los fármacos , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología
3.
Nat Commun ; 6: 7458, 2015 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-26109379

RESUMEN

T-cell trafficking at vascular sites has emerged as a key step in antitumour immunity. Chemokines are credited with guiding the multistep recruitment of CD8(+) T cells across tumour vessels. However, the multiplicity of chemokines within tumours has obscured the contributions of individual chemokine receptor/chemokine pairs to this process. Moreover, recent studies have challenged whether T cells require chemokine receptor signalling at effector sites. Here we investigate the hierarchy of chemokine receptor requirements during T-cell trafficking to murine and human melanoma. These studies reveal a non-redundant role for Gαi-coupled CXCR3 in stabilizing intravascular adhesion and extravasation of adoptively transferred CD8(+) effectors that is indispensable for therapeutic efficacy. In contrast, functional CCR2 and CCR5 on CD8(+) effectors fail to support trafficking despite the presence of intratumoral cognate chemokines. Taken together, these studies identify CXCR3-mediated trafficking at the tumour vascular interface as a critical checkpoint to effective T-cell-based cancer immunotherapy.


Asunto(s)
Neoplasias/irrigación sanguínea , Receptores CXCR3/metabolismo , Transducción de Señal/fisiología , Traslado Adoptivo , Animales , Linfocitos T CD8-positivos/fisiología , Movimiento Celular , Femenino , Regulación de la Expresión Génica , Melanoma/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina/genética , Ovalbúmina/metabolismo , Receptores CCR2/genética , Receptores CCR2/metabolismo , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores CXCR3/genética
4.
Ann Oncol ; 21(8): 1712-1717, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20147741

RESUMEN

BACKGROUND: This phase II study evaluated the safety and activity of ipilimumab, a fully human mAb that blocks cytotoxic T-lymphocyte antigen-4, in patients with advanced melanoma. PATIENTS AND METHODS: Patients with previously treated, unresectable stage III/stage IV melanoma received 10 mg/kg ipilimumab every 3 weeks for four cycles (induction) followed by maintenance therapy every 3 months. The primary end point was best overall response rate (BORR) using modified World Health Organization (WHO) criteria. We also carried out an exploratory analysis of proposed immune-related response criteria (irRC). RESULTS: BORR was 5.8% with a disease control rate (DCR) of 27% (N = 155). One- and 2-year survival rates (95% confidence interval) were 47.2% (39.5% to 55.1%) and 32.8% (25.4% to 40.5%), respectively, with a median overall survival of 10.2 months (7.6-16.3). Of 43 patients with disease progression by modified WHO criteria, 12 had disease control by irRC (8% of all treated patients), resulting in a total DCR of 35%. Adverse events (AEs) were largely immune related, occurring mainly in the skin and gastrointestinal tract, with 19% grade 3 and 3.2% grade 4. Immune-related AEs were manageable and generally reversible with corticosteroids. CONCLUSION: Ipilimumab demonstrated clinical activity with encouraging long-term survival in a previously treated advanced melanoma population.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Melanoma/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/efectos adversos , Femenino , Humanos , Ipilimumab , Masculino , Melanoma/patología , Persona de Mediana Edad , Metástasis de la Neoplasia
5.
Apoptosis ; 10(1): 5-11, 2005 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-15711917

RESUMEN

Maintenance of a sufficient population of naive CD8+ T cells in the peripheral lymphoid compartment is critical for immunocompetence. Peripheral T cell number is a function of T cell generation, survival, and death. Homeostasis, a critical balance between survival and death, must exist to prevent either lymphopenia or lymphocytosis. In the current review, we discuss known requirements for the survival of naive peripheral CD8+ T cells as well as mechanisms of death when survival signals are lost. We also discuss associations between survival and homeostasis-driven proliferation, and highlight the gaps in our knowledge of these critical processes.


Asunto(s)
Antígenos CD8/inmunología , Linfocitos T CD8-positivos/inmunología , Tejido Linfoide/inmunología , Animales , Proliferación Celular , Supervivencia Celular/inmunología , Homeostasis/inmunología , Humanos , Modelos Biológicos
6.
Bone Marrow Transplant ; 35(3): 253-60, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15543195

RESUMEN

Nonmyeloablative allogeneic stem cell transplantation (NST) has considerable activity in patients with metastatic renal cell carcinoma (RCC), although there are limited long-term follow-up data. Between February 1999 and May 2003, 18 patients with metastatic RCC underwent 19 matched-sibling NSTs after conditioning with fludarabine and cyclophosphamide with tacrolimus and mycophenolate mofetil as post-transplant immunosuppression. Among the four objective responses, all were partial and have relapsed with a median response duration of 609 days (range, 107-926). All responders are alive at a median of 41 months. Median overall survival for the entire cohort was 14 months. There were four early treatment-related deaths and one late treatment-related death. Eight patients died from progressive disease and five (28%) from treatment-related mortality. Stratifying transplant outcome as early death, intermediate (no response, no early death), or response, the combination of pre-treatment anemia and decreased performance status, was associated with adverse outcome (P = 0.015) and reduced survival (HR 5.4, 95% confidence interval of 1.4 to 21, P = 0.007). Responders demonstrated prolonged survival compared to nonresponders (P = 0.002). NST leads to durable responses in a minority of metastatic RCC patients. Appropriate patient selection is paramount. Anemia and decreased performance status may enable risk stratification.


Asunto(s)
Carcinoma de Células Renales/terapia , Trasplante de Células Madre Hematopoyéticas/métodos , Acondicionamiento Pretrasplante/métodos , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Renales/mortalidad , Carcinoma de Células Renales/patología , Causas de Muerte , Femenino , Estudios de Seguimiento , Trasplante de Células Madre Hematopoyéticas/mortalidad , Histocompatibilidad , Humanos , Inmunosupresores/efectos adversos , Inmunosupresores/uso terapéutico , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Selección de Paciente , Recurrencia , Factores de Riesgo , Hermanos , Tasa de Supervivencia , Acondicionamiento Pretrasplante/mortalidad , Trasplante Homólogo
7.
Ann Oncol ; 15(7): 1109-14, 2004 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15205206

RESUMEN

BACKGROUND: We postulated that in patients with metastatic renal cell carcinoma (RCC) or melanoma, depletion of normal B cells using the anti-CD20 mAb rituximab before treatment with low-dose interleukin (IL)-2 would improve clinical outcome. PATIENTS AND METHODS: Rituximab (375 mg/m(2)) weekly for 4 weeks. IL-2 [11 (million units) daily] s.c., 4 days a week for weeks 5-8, followed by a 2-week rest (weeks 9 and 10). Patients without disease progression continued on IL-2. Disease re-evaluation was performed after rituximab and after every course of IL-2. RESULTS: Fifteen patients with RCC and six with melanoma were enrolled. One patient had a partial response and seven patients had stable disease. Toxicities were similar to those expected with IL-2 alone, and there were no grade 4 events. Circulating B cells were depleted in all patients. The subsequent low-dose IL-2 increased absolute numbers of natural killer cells, activated CD4(+) and activated CD8(+) T cells. Expanded T cells produced interferon-gamma, but not IL-4. Proliferation of peripheral blood lymphocytes to phytohemagglutinin was diminished following rituximab treatment, suggesting that B cells participate in this response in vitro. CONCLUSIONS: Our results suggest that depletion of circulating B cells with rituximab does not increase the response rate, alter the toxicity profile or change the biological activity in response to low-dose IL-2 in patients with RCC or melanoma.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Linfocitos B/metabolismo , Carcinoma de Células Renales/tratamiento farmacológico , Interleucina-2/uso terapéutico , Melanoma/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Anemia/inducido químicamente , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales de Origen Murino , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Linfocitos B/efectos de los fármacos , Femenino , Citometría de Flujo , Humanos , Inmunoglobulinas/efectos de los fármacos , Inmunoglobulinas/metabolismo , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Masculino , Persona de Mediana Edad , Rituximab , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Resultado del Tratamiento
8.
Bone Marrow Transplant ; 33(5): 491-7, 2004 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-14716341

RESUMEN

Nonmyeloablative allogeneic stem cell transplantation (NST) is thought to be an immunologic therapy in which donor T cells mediate a graft-versus-tumor effect. We recently reported the clinical outcome of a phase II trial of NST in metastatic renal cell carcinoma (RCC). However, the immune response correlates of clinical activity remain unknown. We now describe the analysis of T-cell subsets and T-cell cytokine-producing potential for those patients evaluable for immune monitoring. The incidence of graft-versus-host disease (GVHD) correlated with clinical outcome, with all responders exhibiting chronic GVHD. Following initial tapering of immunosuppression, an increase in the total numbers of CD8+ T cells but not CD4+ T cells was observed among responders compared to nonresponders. In addition, a greater ratio of CD8+ to CD4+ T cells producing IFN-gamma and IL-2 was seen in clinical responders at the time when clinical responses were first detected (day 180 after transplantation). Our results support the hypothesis that the antitumor effects of NST may be mediated by IFN-gamma-producing CD8+ T cells, and indicate that isolation of putative tumor antigen-specific T cells, ideally, should be pursued around day +180.


Asunto(s)
Linfocitos T CD8-positivos/citología , Carcinoma de Células Renales/terapia , Trasplante de Células Madre Hematopoyéticas/métodos , Interferón gamma/metabolismo , Neoplasias Renales/terapia , Adulto , Relación CD4-CD8 , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/metabolismo , Carcinoma de Células Renales/inmunología , Estudios de Cohortes , Femenino , Citometría de Flujo , Enfermedad Injerto contra Huésped/epidemiología , Humanos , Inmunoterapia , Incidencia , Neoplasias Renales/inmunología , Masculino , Persona de Mediana Edad , Trasplante Homólogo
9.
J Immunol ; 167(6): 3123-8, 2001 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-11544297

RESUMEN

Studies in Jurkat cells have shown that combined stimulation through the TCR and CD28 is required for activation of c-Jun N-terminal kinase (JNK), suggesting that JNK activity may mediate the costimulatory function of CD28. To examine the role of JNK signaling in CD28 costimulation in normal T cells, murine T cell clones and CD28(+/+) or CD28(-/-) TCR transgenic T cells were used. Although ligation with anti-CD28 mAb augmented JNK activation in Th1 and Th2 clones stimulated with low concentrations of anti-CD3 mAb, higher concentrations of anti-CD3 mAb alone were sufficient for JNK activation even in the absence of anti-CD28. JNK activity was comparably induced in both CD28(+/+) and CD28(-/-) 2C/recombinase-activating gene 2(RAG2)(-/-) T cells stimulated with anti-CD3 mAb alone, and with L(d)/peptide dimers, a direct alphabeta TCR ligand. Moreover, JNK activation was also detected in 2C/RAG2(-/-) T cells stimulated with P815 cells that express the relevant alloantigen L(d) whether or not B7-1 was coexpressed. However, IL-2 production by both Th1 clones and CD28(+/+) 2C/RAG2(-/-) T cells was detected only upon TCR and CD28 coengagement. Thus, CD28 coligation is not necessary, and stimulation through the TCR is sufficient, for JNK activation in normal murine T cells. The concept that JNK mediates the costimulatory function of CD28 needs to be reconsidered.


Asunto(s)
Antígenos CD28/fisiología , Antígenos H-2/inmunología , Interleucina-2/biosíntesis , Activación de Linfocitos/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Receptores de Antígenos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/enzimología , Animales , Anticuerpos Monoclonales/farmacología , Presentación de Antígeno , Complejo CD3/fisiología , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Activación Enzimática , Inducción Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Antígeno de Histocompatibilidad H-2D , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-2/genética , Proteínas Quinasas JNK Activadas por Mitógenos , Sarcoma de Mastocitos/patología , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Oligopéptidos/fisiología , Fosforilación , Procesamiento Proteico-Postraduccional , Transducción de Señal , Células TH1/efectos de los fármacos , Células TH1/enzimología , Células TH1/inmunología , Células Th2/efectos de los fármacos , Células Th2/enzimología , Células Th2/inmunología
10.
J Immunol ; 167(5): 2555-60, 2001 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-11509595

RESUMEN

Ag-specific immune tolerance in clinical organ transplantation is currently an unrealized but critical goal of transplant biology. The specificity and avidity of multimerized MHC-peptide complexes suggests their potential ability to modulate T cell sensitization and effector functions. In this study, we examined the ability of MHC-peptide dimers to modulate T cell function both in vitro and in vivo. Soluble MHC dimers induced modulation of surface TCR expression and inhibited T cell cytolytic activity at nanomolar concentrations in vitro. Furthermore, engagement of TCR by soluble dimers resulted in phosphorylation of the TCR zeta-chain and recruitment and phosphorylation of zeta-associated protein-70 to the signaling complex, the latter of which increased upon dimer cross-linking. Significantly, Ag-specific inhibition of an alloreactive TCR-transgenic T cell population in vivo resulted in consequent outgrowth of an allogeneic tumor. The prolonged Ag-specific suppression of expansion and/or effector function of cognate T cells in vivo suggests that soluble MHC dimers may be a means of inducing sustained Ag-specific T cell unresponsiveness in vivo.


Asunto(s)
Antígenos H-2/metabolismo , Linfocitos T Citotóxicos/inmunología , Animales , Citotoxicidad Inmunológica , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Antígenos H-2/química , Humanos , Tolerancia Inmunológica , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Proteínas Nucleares , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T Citotóxicos/metabolismo , Inmunología del Trasplante , Proteína Tirosina Quinasa ZAP-70
11.
Int Immunol ; 13(5): 625-32, 2001 May.
Artículo en Inglés | MEDLINE | ID: mdl-11312250

RESUMEN

Epitope spreading has been best characterized as an exacerbating factor in CD4(+) T cell-dependent autoimmune disease models and is believed to occur via presentation of antigens liberated by tissue destruction initiated by CD4(+) T cells specific for a primary epitope. The growing evidence that exogenous antigens can also be processed and presented by class I MHC molecules has suggested that epitope spreading could occur for CD8(+) cytotoxic T lymphocyte (CTL) responses as well. In the context of anti-tumor immunity, expansion of a CTL response to include secondary epitopes could improve the efficacy of therapeutic vaccines. To determine directly whether epitope spreading can occur during an anti-tumor immune response, two defined class I MHC-binding peptides in the P815 tumor model were utilized. We observed that immunization against the single tumor peptide, P1A, followed by rejection of a P1A(+) tumor, subsequently yielded CTL activity and tumor protection against a P1A(-) tumor variant. P1A immunized mice that subsequently rejected tumor challenge developed CTL against a second defined epitope, P1E. These results indicate that, as for class II-restricted peptides in autoimmune disease, epitope spreading can occur for class I-restricted peptides during tumor rejection. A broadened CTL response may help eliminate outgrowth of antigen-negative tumor variants.


Asunto(s)
Antígenos de Neoplasias/administración & dosificación , Sarcoma de Mastocitos/inmunología , Animales , Presentación de Antígeno , Antígenos de Neoplasias/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/farmacología , Epítopos/administración & dosificación , Epítopos/metabolismo , Femenino , Rechazo de Injerto , Antígenos de Histocompatibilidad Clase I/metabolismo , Interleucina-12/administración & dosificación , Sarcoma de Mastocitos/terapia , Ratones , Ratones Endogámicos DBA , Trasplante de Neoplasias , Células Tumorales Cultivadas
12.
Clin Cancer Res ; 7(3 Suppl): 895s-901s, 2001 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-11300489

RESUMEN

Vaccination with dendritic cells (DCs) pulsed with tumor antigen peptides has shown promise in the treatment of melanoma. Interleukin (IL)-12 production by DCs is a key component for their efficacy. Murine studies have shown that IL-12 promotes potent antitumor immunization when coadministered with peptides loaded onto other class I MHC+ cells, thus bypassing the need to use DCs. The easiest cell source to obtain in large quantity from human patients is peripheral blood mononuclear cells (PBMCs). A Phase I clinical trial was thus performed in patients with metastatic melanoma using immunization with autologous PBMCs pulsed with a MAGE-3 or a MelanA peptide, coadministered with various doses of recombinant human (rh)IL-12. Patients receiving low-to-moderate doses of rhIL-12 developed increased specific CD8+ T-cell responses. Of the eight patients showing increased immunity, six had evidence of clinical activity, with one complete, one partial, one minor, and three mixed responses observed. In two patients with mixed responses, growing tumors were found to lack expression of the antigen used to immunize. Thus, vaccination with peptide-pulsed PBMCs plus rhIL-12 induces specific immunity and has clinical activity, without the need to generate DCs. Outgrowth of antigen-negative tumors argues for the future development of polyepitope vaccines.


Asunto(s)
Antígenos de Neoplasias , Antígeno HLA-A2/biosíntesis , Interleucina-12/metabolismo , Leucocitos Mononucleares/metabolismo , Melanoma/inmunología , Melanoma/metabolismo , Melanoma/terapia , Proteínas de Neoplasias/uso terapéutico , Proteínas Recombinantes/metabolismo , Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer , Células Cultivadas , Epítopos , Femenino , Humanos , Interferón gamma/metabolismo , Antígeno MART-1 , Masculino , Melanoma/mortalidad , Metástasis de la Neoplasia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/citología , Factores de Tiempo
14.
J Immunol ; 166(6): 3900-7, 2001 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-11238634

RESUMEN

To examine the role of CTLA-4 in controlling Ag-specific CD8(+) T cell activation, TCR-transgenic/CTLA-4 wild-type or -deficient mice were generated in a recombination-activating gene 2-deficient background. Naive T cells from these mice responded comparably whether or not CTLA-4 was expressed. In contrast, primed T cells responded more vigorously if they lacked CTLA-4 expression. We took advantage of the difference between naive and primed T cell responses to approach the mechanism of CTLA-4 function. Single-cell analyses demonstrated that a greater fraction of CTLA-4-deficient cells responded to a fixed dose of Ag compared with CTLA-4-expressing cells, whereas the magnitude of response per cell was comparable. A shift in the dose-response curve to APCs was also observed such that fewer APCs were required to activate CTLA-4-deficient T cells to produce intracellular IFN-gamma and to proliferate. These results suggest that CTLA-4 controls the threshold of productive TCR signaling. Biochemical analysis comparing stimulated naive and primed TCR-transgenic cells revealed no obvious differences in expression of total CTLA-4, tyrosine-phosphorylated CTLA-4, and associated Src homology domain 2-containing protein tyrosine phosphatase. Thus, the biochemical mechanism explaining the differential inhibitory effect of CTLA-4 on naive and primed CD8(+) T cells remains unclear.


Asunto(s)
Antígenos de Diferenciación/genética , Linfocitos T CD8-positivos/inmunología , Inmunoconjugados , Inmunosupresores/farmacología , Activación de Linfocitos/genética , Proteínas Tirosina Fosfatasas/fisiología , Receptores de Antígenos de Linfocitos T/genética , Dominios Homologos src/inmunología , Abatacept , Adyuvantes Inmunológicos/biosíntesis , Adyuvantes Inmunológicos/deficiencia , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/fisiología , Animales , Antígenos CD , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/fisiología , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/metabolismo , Antígeno CTLA-4 , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Inmunización , Inmunosupresores/metabolismo , Interfase/genética , Interfase/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Ratones Transgénicos , Transfección , Células Tumorales Cultivadas , Dominios Homologos src/genética
16.
Curr Protoc Immunol ; Appendix 3: Appendix 3E, 2001 May.
Artículo en Inglés | MEDLINE | ID: mdl-18432657

RESUMEN

Mycoplasma contamination is a serious and frequent problem in the culture laboratory. Although mycoplasma contamination may be suspected by the failure of cells to thrive, the formal diagnosis rests on the detection of adenosine phosphorylase secretion by infected cell lines. This appendix describes how to test for mycoplasma contamination, and also presents methods for antibiotic treatment of infected cultures.


Asunto(s)
Técnicas Citológicas/métodos , Infecciones por Mycoplasma/microbiología , Mycoplasma/aislamiento & purificación , Técnicas de Cultivo de Célula , Mycoplasma/enzimología , Purina-Nucleósido Fosforilasa/análisis
17.
Curr Protoc Immunol ; Chapter 20: Unit 20.4, 2001 May.
Artículo en Inglés | MEDLINE | ID: mdl-18432777

RESUMEN

This unit presents an experimental tumor model which has led to pivotal advances in tumor immunology culminating in the preclinical development of human cancer vaccines for melanoma. The model employs the use of the P815 mastocytoma cell line. Although the P815 cell line belongs to the mast cell lineage, it offers several advantages for in vivo experimentation of the tumor-host relationship. It grows progressively in the majority of syngeneic DBA/2 mice and can be implanted either intraperitoneally or subcutaneously. Moreover, immunogeneic variants have been created yielding tumors that are spontaneously rejected BB a behavior that has provided a context in which to study the immunologically relevant molecules and cells that dictate a successful anti-tumor response.


Asunto(s)
Modelos Animales de Enfermedad , Mastocitoma , Animales , Ascitis/inmunología , Vacunas contra el Cáncer , Línea Celular Tumoral , Citotoxicidad Inmunológica , Humanos , Inmunoterapia , Mastocitoma/genética , Mastocitoma/inmunología , Mastocitoma/patología , Ratones , Trasplante de Neoplasias , Linfocitos T Citotóxicos/inmunología
18.
Proc Natl Acad Sci U S A ; 97(25): 13784-9, 2000 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-11095726

RESUMEN

The inability to easily and efficiently introduce genes into primary T cells has hampered the investigation of the pathways controlling T cell fate. To enable adenoviral-mediated gene transfer into normal naive T cells, transgenic (Tg) mice expressing the coxsackie/adenovirus receptor (CAR) in their T cell compartment were constructed. Whereas naive T cells are resistant to adenoviral infection, Tg expression of CAR on T cells greatly facilitates adenoviral-mediated gene expression ex vivo, in vivo, and in differentiated T helper cells. Thus we have developed a technology for efficient gene delivery to naive T cells. By using adenoviral vectors encoding specific inhibitors, we show that G1 cyclin-dependent kinase, NF-kappaB, and caspase activities are required for the proliferation of primary T cells. In addition, by expressing Bcl-x(L) protein at a level that closely approximates mitogen-induced levels, we demonstrate that Bcl-x(L) expression is sufficient to account for mitogen-mediated survival of primary T cells. Thus, adenoviral-mediated gene delivery to CAR Tg T cells should be useful for the analysis of many genes controlling T cell fate.


Asunto(s)
Adenoviridae/genética , Enterovirus/genética , Técnicas de Transferencia de Gen , Receptores Virales/genética , Linfocitos T/metabolismo , Animales , División Celular , Vectores Genéticos , Humanos , Ratones , Ratones Transgénicos , Recombinación Genética , Linfocitos T/citología , Transducción Genética
19.
J Immunol ; 165(11): 6024-8, 2000 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-11086033

RESUMEN

Experimental evidence suggests that a type 1 T cell response may result in optimal tumor rejection in vivo. This phenotype is determined in part by cytokines that influence T cell differentiation. In transplantable tumor models such as P1.HTR, tumors grow progressively despite expression of defined tumor Ags. We hypothesized that this failure to reject may be due to poor generation of a type 1 phenotype, through a dominant influence of the type 2-promoting cytokines IL-4 and/or IL-13. This hypothesis was tested by implanting P1.HTR tumors into mice deficient in Stat6. In contrast to progressive growth of P1.HTR tumors in wild-type mice, and aggressive growth even of IL-12-transfected P1.HTR in Stat1(-/-) mice, P1.HTR was spontaneously rejected by Stat6(-/-) mice. Rejection was accompanied by augmented tumor-specific IFN-gamma production and CTL activity. These results suggest that pharmacologic inhibition of Stat6 signaling could potentiate anti-tumor immunity in vivo.


Asunto(s)
Antígenos de Neoplasias/inmunología , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Sarcoma de Mastocitos/genética , Sarcoma de Mastocitos/inmunología , Transactivadores/deficiencia , Transactivadores/genética , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Citotoxicidad Inmunológica/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Interferón gamma/biosíntesis , Interleucina-12/genética , Leucemia L1210 , Ratones , Ratones Endogámicos DBA , Ratones Noqueados , Trasplante de Neoplasias , Factor de Transcripción STAT1 , Factor de Transcripción STAT6 , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Transfección , Células Tumorales Cultivadas
20.
J Immunol ; 164(9): 4433-42, 2000 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-10779742

RESUMEN

CTLA-4 (CD152) engagement can down-regulate T cell activation and promote the induction of immune tolerance. However, the strategy of attenuating T cell activation by engaging CTLA-4 has been limited by sharing of its natural ligands with the costimulatory protein CD28. In the present study, a CTLA-4-specific single-chain Ab (scFv) was developed and expressed on the cell surface to promote selective engagement of this regulatory molecule. Transfectants expressing anti-CTLA-4 scFv at their surface bound soluble CTLA-4 but not soluble CD28. Coexpression of anti-CTLA-4 scFv with anti-CD3epsilon and anti-CD28 scFvs on artificial APCs reduced the proliferation and IL-2 production by resting and preactivated bulk T cells as well as CD4+ and CD8+ T cell subsets. Importantly, expression of anti-CTLA-4 scFv on the same cell surface as the TCR ligand was essential for the inhibitory effects of CTLA-4-specific ligation. CTLA-4-mediated inhibition of tyrosine phosphorylation of components of the proximal TCR signaling apparatus was similarly dependent on coexpression of TCR and CTLA-4 ligands on the same surface. These findings support a predominant role for CTLA-4 function in the modification of the proximal TCR signal. Using T cells from DO11.10 and 2C TCR transgenic mice, negative regulatory effects of selective CTLA-4 ligation were also demonstrated during the stimulation of Ag-specific CD4+ and CD8+ T cells by MHC/peptide complexes. Together these studies demonstrate that selective ligation of CTLA-4 using a membrane-bound scFv results in attenuated T cell responses only when coengaged with the TCR during T cell/APC interaction and define an approach to harnessing the immunomodulatory potential of CTLA-4-specific ligation.


Asunto(s)
Anticuerpos Bloqueadores/farmacología , Antígenos de Diferenciación/inmunología , Inmunoconjugados , Región Variable de Inmunoglobulina/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Abatacept , Secuencia de Aminoácidos , Animales , Anticuerpos Bloqueadores/biosíntesis , Anticuerpos Bloqueadores/genética , Anticuerpos Bloqueadores/metabolismo , Especificidad de Anticuerpos/genética , Antígenos CD , Antígenos de Diferenciación/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Antígeno CTLA-4 , Línea Celular , Citocinas/biosíntesis , Citocinas/metabolismo , Femenino , Humanos , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/metabolismo , Interfase/genética , Interfase/inmunología , Ligandos , Activación de Linfocitos/genética , Complejo Mayor de Histocompatibilidad/genética , Complejo Mayor de Histocompatibilidad/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/inmunología , Receptores de Antígenos de Linfocitos B/biosíntesis , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunología , Transfección , Células Tumorales Cultivadas
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