RESUMEN
Appropriate activation of dendritic cells (DC) is essential for successful active vaccination and induction of cell-mediated immunity. The scarcity of precursor cells, as well as long culture methods, have hampered wide-scale application of DC vaccines derived from CD34(+) precursors, despite their suggested superior efficacy over the more commonly applied monocyte-derived DC (MoDC). Here, employing the CD34(+)/CD14(+) AML-derived human DC progenitor cell line MUTZ3, we show that cytostatic anthraquinone-derivatives (i.e., the anthracenedione mitoxantrone and the related anthracyclin doxorubicin) induce rapid differentiation of CD34(+) DC precursors into functional antigen-presenting cells (APC) in a three-day protocol. The drugs were found to act specifically on CD34(+), and not on CD14(+) DC precursors. Importantly, these observations were confirmed for primary CD34(+) and CD14(+) DC precursors from peripheral blood. Mitoxantrone-generated DC were fully differentiated within three days and after an additional 24 h of maturation, were as capable as standard 9-day differentiated and matured DC to migrate toward the lymph node-homing chemokines CCL19 and CCL21, to induce primary allogeneic T cell proliferation, and to prime functional MART1-specific CD8(+) T lymphocytes. Our finding that anthraquinone-derivatives like mitoxantrone support rapid high-efficiency differentiation of DC precursors may have consequences for in vitro production of DC vaccines as well as for novel immunochemotherapy strategies.
Asunto(s)
Vacunas contra el Cáncer , Células Dendríticas/metabolismo , Inmunoterapia , Células Progenitoras Mieloides/metabolismo , Neoplasias/inmunología , Antraciclinas/farmacología , Antraquinonas/farmacología , Antígenos CD34/metabolismo , Antineoplásicos/farmacología , Diferenciación Celular , Línea Celular , Quimiocina CCL19/metabolismo , Quimiocina CCL21/metabolismo , Citostáticos/farmacología , Células Dendríticas/inmunología , Células Dendríticas/patología , Células Dendríticas/trasplante , Humanos , Inmunidad Celular , Receptores de Lipopolisacáridos/metabolismo , Células Progenitoras Mieloides/inmunología , Células Progenitoras Mieloides/patología , Neoplasias/terapiaRESUMEN
Adenovirus (Ad)-based vaccines are considered for cancer immunotherapy, yet, detailed knowledge on their mechanism of action and optimal delivery route for anti-tumor efficacy is lacking. Here, we compared the anti-tumor efficacy of an Ad-based melanoma vaccine after intradermal, intravenous, intranasal or intraperitoneal delivery in the B16F10 melanoma model. The intradermal route induced superior systemic anti-melanoma immunity which was MyD88 signaling-dependent. Predominant transduction of non-professional antigen-presenting cells at the dermal vaccination sites and draining lymph nodes, suggested a role for cross-presentation, which was confirmed in vitro. We conclude that the dermis provides an optimal route of entry for Ad-based vaccines for high-efficacy systemic anti-tumor immunization and that this immunization likely involves cross-priming events in the draining lymph nodes.
Asunto(s)
Vacunas contra el Cáncer/administración & dosificación , Reactividad Cruzada , Melanoma Experimental/terapia , Factor 88 de Diferenciación Mieloide/metabolismo , Adenoviridae/genética , Animales , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Inyecciones Intradérmicas , Inyecciones Intravenosas , Ganglios Linfáticos/inmunología , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Targeted delivery of tumor antigen genes to dendritic cells (DCs) using adenoviral (Ad) vectors holds great potential for cancer immunotherapy. We previously showed that CD40 targeting of Ad vectors enhanced specific transduction of DC in human skin, while simultaneously ensuring their stable maturation and superior allogeneic T-cell stimulatory capacity. In this study, we evaluated whether CD40-targeted Ad encoding the full-length melanoma antigen recognized by T cells-1 (CD40-Ad-MART-1) could be used to efficiently and selectively transduce conventional and plasmacytoid DC to prime melanoma-specific CD8(+) T-effector cells in human melanoma-draining sentinel lymph nodes (SLNs). CD40 targeting of Ad was achieved using a bispecific fusion protein, binding and neutralizing the Ad fiber knob through soluble coxsackie and adenovirus receptor while retargeting the virus to hCD40 through the tumor necrosis factor-like domain of mCD40L. Selective transduction of conventional and plasmacytoid DC subsets by CD40-Ad was observed in suspensions of human melanoma-draining SLN. Moreover, CD40-Ad-MART-1 enhanced the expansion of functional MART-1-specific CD8(+) T cells from SLN with concomitant decreases in CD4:CD8 T-cell ratios and CD4(+)CD25(hi)FoxP3(+) regulatory T-cell rates. Additional studies revealed that transduction and activation of monocyte-derived DCs with CD40-Ad-MART-1 significantly enhanced their priming efficiency of functional CD8(+) effector T cells with high avidity. These findings provide preclinical evidence of possible efficacy of this approach for cancer immunotherapy.
Asunto(s)
Adenoviridae/genética , Antígenos CD40/metabolismo , Células Dendríticas/metabolismo , Inmunoterapia , Melanoma/terapia , Linfocitos T Citotóxicos/metabolismo , Antígenos CD4/biosíntesis , Ligando de CD40/genética , Ligando de CD40/inmunología , Ligando de CD40/metabolismo , Citotoxicidad Inmunológica , Células Dendríticas/inmunología , Células Dendríticas/patología , Factores de Transcripción Forkhead/biosíntesis , Humanos , Interferón gamma/metabolismo , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Células K562 , Ganglios Linfáticos/patología , Antígeno MART-1/genética , Antígeno MART-1/inmunología , Antígeno MART-1/metabolismo , Melanoma/inmunología , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/genética , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/patología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología , Transducción GenéticaRESUMEN
Dendritic cell (DC) migration to draining lymph nodes is important for the initiation of an effective immune response. Recently we reported that the human ATP-binding cassette (ABC) transporter multidrug resistance protein 4 (MRP4 and ABCC4) is required for the migration of human DC. Since the ABC transporter MRP1 (ABCC1) was previously shown to play a role in both human and mouse DC migration, we here studied whether Mrp4 is similarly required for DC migration in mice and whether the absence of Mrp4 interferes with the generation of an immune response. Immunological responses were compared in wild-type FVB (FVBwt), FVB Mrp4 knockout (KO) or FVB Mrp4/5 double knockout (dKO) mice. Skin, a preferred immunization site, was analyzed for DC markers, as well as for Mrp1 and Mrp4 expression. Whereas Mrp1 was abundantly present within FVBwt skin, only few Mrp4 expressing cells were detected. In addition, no Mrp4 protein expression was detected on in vitro cultured FVBwt bone marrow-derived DC (BM-DC). DC migration from murine ear skin was unaltered between FVBwt and MRP4/5 dKO animals. The absence of Mrp4 also had no effect on immune responses upon allergen sensitization, immunization or oral tolerance induction. We thus conclude that in contrast to its human counterpart, murine Mrp4 is not involved in DC migration, nor indeed, in the generation of an effective immune response. These data reveal disparities in the physiological role of ABC transporters between species, which may derive from differences in substrate specificity.
Asunto(s)
Células Dendríticas/inmunología , Tolerancia Inmunológica/inmunología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/inmunología , Piel/inmunología , Animales , Movimiento Celular/inmunología , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/metabolismo , Ratones , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Ovalbúmina/inmunología , Piel/citología , Piel/metabolismoRESUMEN
PURPOSE: Impaired immune effector functions in the melanoma sentinel lymph node (SLN) may allow for early metastatic events. Local administration of PF-3512676 (formerly known as CpG 7909) has shown immunostimulatory effects of both dendritic cell and T-cell subsets in the melanoma SLN. Here, we set out to ascertain whether these PF-3512676-induced immunostimulatory effects translate into higher frequencies of melanoma-specific CD8(+) T cells. EXPERIMENTAL DESIGN: Twenty-four stage I to III melanoma patients were randomized to preoperative local administration of either PF-3512676 or saline. CD8(+) T cells from SLN and peripheral blood were tested for reactivity by IFN-gamma ELISPOT assay against several HLA-A1/A2/A3-restricted epitopes derived from various melanoma-associated antigens (MAA) in 21 of 24 enrolled patients. Frequencies of natural killer (NK) cells and frequencies and maturation state of dendritic cell subsets in the SLN were determined by flow cytometry. RESULTS: Melanoma-specific CD8(+) T-cell response rates against >1 MAA epitope in the SLN were 0 of 11 for the saline group versus 5 of 10 for the PF-3512676-administered group (P = 0.012). Of these 5 responding patients, 4 also had a measurable response to >1 MAA epitope in the blood. Increased frequencies in the SLN of both MAA-specific CD8(+) T cells and NK cells correlated to CpG-induced plasmacytoid dendritic cell maturation. CONCLUSIONS: These data show an increase in melanoma-specific CD8(+) T-cell frequencies as well as an increased effector NK cell rate after a single dose of PF-3512676 and thus support the utility of local PF-3512676 administration as adjuvant treatment in early-stage melanoma to try and halt metastatic spread.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inmunoterapia Activa/métodos , Melanoma/tratamiento farmacológico , Oligodesoxirribonucleótidos/administración & dosificación , Neoplasias Cutáneas/tratamiento farmacológico , Antígenos de Neoplasias/inmunología , Quimioterapia Adyuvante , Células Dendríticas/citología , Células Dendríticas/inmunología , Citometría de Flujo , Humanos , Inyecciones Intralesiones , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Melanoma/inmunología , Persona de Mediana Edad , Terapia Neoadyuvante , Biopsia del Ganglio Linfático Centinela , Neoplasias Cutáneas/inmunologíaRESUMEN
PURPOSE: A decrease in the frequency and activation state of dendritic cells in the sentinel lymph node (SLN) has been observed in early stages of melanoma development. This may hinder the generation of effective antitumor T-cell responses and increase the likelihood of metastatic spread. Immunopotentiation of the melanoma SLN may therefore be a valuable adjuvant treatment option. One way to achieve this is through the use of bacterially derived unmethylated cytosine-phosphate-guanine (CpG) DNA sequences that bind Toll-like receptor 9 and activate plasmacytoid dendritic cells (PDC). CpG-activated PDC, in turn, release IFN alpha and may thus boost T-cell and natural killer cell responses as well as activate conventional myeloid dendritic cells (MDC). EXPERIMENTAL DESIGN: We studied the effects of preoperative local administration of the CpG B-type oligodeoxynucleotide (ODN) PF-3512676 (formerly known as CPG 7909) on dendritic cell and T-cell subsets in the SLN of 23 stage I to III melanoma patients, randomized to receive intradermal injections of either PF-3512676 or saline (NaCl 0.9%). RESULTS: PF-3512676 administration resulted in bulkier SLN, higher yields of isolated SLN leukocytes, and activation of BDCA-2(+)CD123(+) PDC as well as of CD1a(+) MDC. In addition, PF-3512676 administration was associated with the presence of a newly identified CD11c(hi)CD123(+)CD83(+)TRAIL(+) mature SLN-MDC subset, an increased release of a variety of inflammatory cytokines, and lower frequencies of CD4(+)CD25(hi)CTLA-4(+)FoxP3(+) regulatory T cells in the SLN. CONCLUSIONS: These findings point to the possible utility of the conditioning of SLN by PF-3512676 as an adjuvant immunotherapeutic modality for early-stage melanoma.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Melanoma/tratamiento farmacológico , Oligodesoxirribonucleótidos/administración & dosificación , Neoplasias Cutáneas/tratamiento farmacológico , Anciano , Quimioterapia Adyuvante , Islas de CpG , Citocinas/metabolismo , Femenino , Humanos , Inmunoterapia , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Masculino , Melanoma/inmunología , Melanoma/patología , Persona de Mediana Edad , Células Mieloides/efectos de los fármacos , Metástasis de la Neoplasia , Cuidados Preoperatorios , Biopsia del Ganglio Linfático Centinela , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Linfocitos T/inmunologíaRESUMEN
PURPOSE: Impaired immune functions in the sentinel lymph node (SLN) may facilitate early metastatic events during melanoma development. Local potentiation of tumor-specific T cell reactivity may be a valuable adjuvant treatment option. EXPERIMENTAL DESIGN: We examined the effect of locally administered granulocyte/macrophage-colony stimulating factor (GM-CSF) on the frequency of tumor-specific CD8+ T cells in the SLN and blood of patients with stage I melanoma. Twelve patients were randomly assigned to preoperative local administration of either recombinant human GM-CSF or NaCl 0.9%. CD8+ T cells from SLN and peripheral blood were tested for reactivity in an IFNgamma ELISPOT assay against the full-length MART-1 antigen and a number of HLA-A1, HLA-A2, and HLA-A3-restricted epitopes derived from a range of melanoma-associated antigens. RESULTS: Melanoma-specific CD8+ T cell response rates in the SLN were one of six for the control group and four of six for the GM-CSF-administered group. Only one patient had detectable tumor-specific CD8+ T cells in the blood, but at lower frequencies than in the SLN. All patients with detectable tumor-specific CD8+ T cells had a percentage of CD1a+ SLN-dendritic cells (DC) above the median (i.e., 0.33%). This association between above median CD1a+ SLN-DC frequencies and tumor antigen-specific CD8+ T cell reactivity was significant in a two-sided Fisher's exact test (P = 0.015). CONCLUSIONS: Locally primed antitumor T cell responses in the SLN are detectable as early as stage I of melanoma development and may be enhanced by GM-CSF-induced increases in SLN-DC frequencies.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Ganglios Linfáticos/inmunología , Melanoma/inmunología , Biopsia del Ganglio Linfático Centinela , Adulto , Anciano , Recuento de Células , Femenino , Antígenos HLA-A/análisis , Humanos , Ganglios Linfáticos/patología , Masculino , Melanoma/patología , Melanoma/cirugía , Persona de Mediana Edad , Estadificación de Neoplasias , Proteínas RecombinantesRESUMEN
Mature and immature myeloid dendritic cells (DCs) are thought to differentially modulate T-cell responses in secondary lymphoid tissues. Although mature DCs are believed to induce T-cell activation under proinflammatory conditions, immature DCs are believed to maintain a state of T-cell tolerance under steady state conditions. However, little is known about the actual activation state of human DCs under these different conditions. Here, we compare the frequency and activation state of human DCs between matched skin and sentinel lymph node (SLN) samples, after intradermal administration of either granulocyte/macrophage colony-stimulating factor (GM-CSF) or saline, at the excision site of stage I primary melanoma. Although DCs remained immature (CD1a+CD83-) and mostly situated in the epidermis of the saline-injected skin (fully consistent with a quiescent steady state), mature (CD1a+CD83+) DC frequencies significantly increased in the GM-CSF-injected skin and correlated with the number of mature DCs in the SLN, indicative of increased DC migration. Interestingly, irrespective of GM-CSF or saline administration, all CD1a+ myeloid DCs in the SLN were phenotypically mature (ie, CD83+). These data are indicative of migration of small numbers of phenotypically mature DCs to lymph nodes under steady state conditions.
Asunto(s)
Células Dendríticas/citología , Células Dendríticas/fisiología , Ganglios Linfáticos/inmunología , Melanoma/inmunología , Neoplasias Cutáneas/inmunología , Piel/inmunología , Adolescente , Adulto , Anciano , Antígenos CD/análisis , Antígenos CD1/análisis , Movimiento Celular , Células Dendríticas/inmunología , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Inmunoglobulinas/análisis , Inmunohistoquímica , Ganglios Linfáticos/patología , Melanoma/patología , Glicoproteínas de Membrana/análisis , Persona de Mediana Edad , Piel/patología , Neoplasias Cutáneas/patología , Cloruro de Sodio/administración & dosificación , Antígeno CD83RESUMEN
The IFN-gamma-inducible and CXCR3-targeting human CXC chemokines CXCL9 (Mig) and CXCL10 (IP10) have potent antitumor activity through attraction of cytotoxic T lymphocytes and inhibition of angiogenesis. The more recently identified CXCR3-targeting chemokine CXCL11 (I-TAC/IP9) proved to be a more potent chemokine than CXCL9 and CXCL10 in vitro, both in chemotaxis assays with CXCR3+ T lymphocytes and in calcium mobilization experiments. However, its antitumor activity in vivo has not been shown so far. To investigate this, mice were challenged with EL4 T-cell lymphoma cells, genetically modified to produce murine CXCL11. Tumor growth curves showed complete rejection of CXCL11-producing tumors but not of control tumors. Tumor infiltrate analysis by flow cytometry showed a clear correlation between rejection of CXCL11-producing tumors and an increase of tumor-infiltrating CD8+CXCR3+ as well as CD8+CXCR3- T lymphocytes. In vivo CD8 T-cell depletion completely abrogated the antitumor effect. No difference in angiogenesis between control and CXCL11-producing tumors was observed. In survivors, rechallenge experiments with wild-type tumor cells suggested development of protective antitumor immunity involving tumor-specific IFN-gamma production by CD8+ T lymphocytes. These experiments show, for the first time, antitumor activity of CXCL11 in vivo, which warrants exploration for its potential role in anticancer immunotherapy.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Quimiocinas CXC/inmunología , Quimiotaxis de Leucocito/inmunología , Linfoma de Células T/inmunología , Neovascularización Patológica/inmunología , Receptores de Quimiocina/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Recuento de Células , Línea Celular Tumoral , Proliferación Celular , Quimiocina CXCL11 , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Endotelio/patología , Femenino , Humanos , Memoria Inmunológica/inmunología , Interferón gamma/metabolismo , Depleción Linfocítica , Linfoma de Células T/patología , Macrófagos/citología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/patología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , Receptores CXCR3 , Linfocitos T/citología , Linfocitos T/inmunología , TransfecciónRESUMEN
The initial tumor-draining lymph node, the sentinel lymph node, not only constitutes the first expected site of micrometastasis but also the first point of contact between tumor-associated antigens and the adaptive immune system. A tumor-induced decrease in the frequency and activation state of sentinel lymph node dendritic cells will impair the generation of effective antitumor T-cell responses and increase the likelihood of metastatic spread. Here, we demonstrate that intradermal administration of granulocyte macrophage-colony stimulating factor around the excision site of stage I primary melanoma tumors increases the number and activation state of dendritic cells in the paracortical areas of the sentinel lymph node and enhances their binding to T cells. We conclude that local treatment of melanoma patients with granulocyte macrophage-colony stimulating factor, before surgery, conditions the sentinel lymph node microenvironment to enhance mature dendritic cell recruitment and hypothesize that this may be more conducive to the generation of T-cell-mediated antitumor immunity.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Células Dendríticas/inmunología , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Ganglios Linfáticos/inmunología , Melanoma/tratamiento farmacológico , Biopsia del Ganglio Linfático Centinela , Adyuvantes Inmunológicos/administración & dosificación , Anciano , Femenino , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Humanos , Inmunohistoquímica , Ganglios Linfáticos/patología , Masculino , Melanoma/inmunología , Persona de Mediana Edad , Método Simple CiegoRESUMEN
Immune responses against tumor antigens will initially occur in the first tumor-draining lymph node, the sentinel node (SN). Because of extensive diagnostic procedures, obtaining a piece of SN to isolate viable immune cells for functional analyses is often impossible. For this reason an alternative method to obtain viable cells from a lymph node (LN) was investigated, ie, scraping LNs with a surgical blade, and compared with dissociation of total LNs. Tumor-draining lymph nodes were retrieved from five oncological patients. The collected dendritic cells and T cells were phenotypically and functionally characterized by flow cytometry and antigen-specific interferon (IFN)-gamma release in an ELISPOT assay. Results were compared between the two isolation methods. Viabilities and phenotypic characteristics of the collected cells were entirely comparable for both methods. T-cell functionality was also comparable between both methods, with equal T-cell expansion factors and similar frequencies of cytotoxic T cells specifically recognizing the M1 matrix protein of Influenza haemophilus or the tumor antigen Her-2/neu. In conclusion, scraping LNs to obtain cells for analysis of immune functions in LNs is feasible and presents a good alternative to dissociation of LNs. Scraping may even be applied to small LNs that a pathologist will submit entirely for histological examination and may thus prove useful in the monitoring of immune responses in SNs.