RESUMEN
BACKGROUND: To describe an oncolytic adenovirus (OAd) encoding SP-SA-E7-4-1BBL that is capable of inducing tumor regression in therapeutic assays. Herein, we tested whether the antitumor effect is given by the induction of a tumor-specific immune response, as well as the minimum dose needed to elicit antitumor protection and monitor the OAd biodistribution over time. METHODS AND RESULTS: C57BL/6 mice (n = 5) per group were immunized twice with OAds encoding SP-SA-E7-4-1BBL, SA-E7-4-1BBL, or SP-SA-4-1BBL and challenged with TC-1 cancer cells. The DNA construct SP-SA-E7-4-1BBL was employed as a control via biolistic or PBS injection. Groups without tumor development at 47 days were rechallenged with TC-1 cells, and follow-up lasted until day 90. The minimum dose of OAd to induce the antitumor effect was established by immunization using serial dilution doses. The cytometry bead assay and the ELISpot assay were used to evaluate cytokine release in response to ex vivo antigenic stimulation. The distribution profile of the OAd vaccine was evaluated in the different organs by histological, immunohistochemical and qPCR analyses. The OAd SP-SA-E7-4-1BBL-immunized mice did not develop tumors even in a rechallenge. A protective antitumor effect was observed from a dose that is one hundredth of most reports of adenoviral vaccines. Immunization with OAd increases Interferon-gamma-producing cells in response to antigen stimulation. OAd was detected in tumors over time, with significant morphological changes, contrary to nontumor tissues. CONCLUSIONS: The OAd SP-SA-E7-4-1BBL vaccine confers a prophylactic, safe, long-lasting, and antigen-dependent antitumor effect mediated by a Th1 antitumor immune response.
Asunto(s)
Vacunas contra el Cáncer , Neoplasias , Animales , Ratones , Papillomavirus Humano 16 , Ligando 4-1BB/genética , Ligando 4-1BB/farmacología , Distribución Tisular , Ratones Endogámicos C57BL , Adenoviridae/genética , Inmunidad , Neoplasias/terapiaRESUMEN
Tumor-specific tumor-infiltrating lymphocytes (TILs) can be in vitro expanded and have the ability to induce complete and durable tumor regression in some patients with melanoma following adoptive cell therapy (ACT). In this preclinical study, we investigated the feasibility of expanding TIL from sarcomas, as well as performing functional in vitro analyses on these. TILs were expanded in vitro by the use of IL2 stimulation with or without the addition of 4-1BB and CD3 antibodies. Phenotypical and functional analyses were mainly performed by flow cytometry. TILs were expanded from 25 of 28 (89%) tumor samples from patients with 9 different sarcoma subtypes. TILs were predominantly αß T-cells of effector memory subtype with CD4+ dominance. In particular, CD8+ TIL highly expressed LAG3 and to a lesser degree PD-1 and BTLA. In total, 10 of 20 TIL cultures demonstrated in vitro recognition of autologous tumor. In some cases, the fraction of tumor-reactive T cells was more than 20%. 4-1BB stimulation augmented expansion kinetics and favored CD8+ occurrence. In conclusion, TIL expansion from sarcoma is feasible and expanded TILs highly express LAG3 and comprise multifunctional tumor-reactive T-cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Técnicas de Cultivo de Célula/métodos , Linfocitos Infiltrantes de Tumor/inmunología , Sarcoma/inmunología , Ligando 4-1BB/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos T CD8-positivos/efectos de los fármacos , Femenino , Humanos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Masculino , Persona de Mediana Edad , Células Tumorales CultivadasRESUMEN
Although many anticancer agents for gastric cancer have been developed, the prognosis for many patients remains poor. Recently, costimulatory immune molecules that reactivate antitumor immune responses by utilizing the host immune system have attracted attention as new therapeutic strategies. CD137 is a costimulatory molecule that reportedly potentiates the antitumor activity of tumor-targeting monoclonal antibodies (mAbs) by enhancing antibody-dependent cellular cytotoxicity. However, it remains unclear whether CD137 stimulates tumor-regulatory activity in gastric cancer. In this study, we investigated the antitumor effects of CD137 stimulation on gastric cancer cells administered tumor-targeting mAbs. Our results showed that human natural killer (NK) cells were activated by expressing CD137 after encountering trastuzumab-coated gastric cancer cells, and that stimulation of activated NK cells in the presence of trastuzumab and recombinant human CD137 ligand (rhCD137L) enhanced cytotoxicity and release of cytokines (IFN-γ, TNF, granzyme A, or granzyme B) as compared with activated NK cells with trastuzumab alone (p < 0.05). By combination treatment with rhCD137L, similar effects were obtained regarding cancer cell cytotoxicity in the presence of cetuximab (p < 0.01). Moreover, we revealed that CD137 expression was dependent upon the affinity between the Fc portion of the antibodies and FcγRIIIa of NK cells based on results indicating that human IgG1 and IgG3 subclasses enhanced CD137 expression (p < 0.001). These results confirmed that FcγRIIIA polymorphisms (158 V/V) enhanced CD137 expression to a greater degree than 158 F polymorphisms (p = 0.014). Our results suggested that CD137 stimulation could promote the effects of tumor-targeting mAbs in gastric cancer, and that further investigation of antibody binding affinity and in vivo activities might improve therapeutic strategies related to the treatment of gastric cancer patients.
Asunto(s)
Ligando 4-1BB/farmacología , Antineoplásicos Inmunológicos/farmacología , Células Asesinas Naturales/inmunología , Neoplasias Gástricas/inmunología , Trastuzumab/farmacología , Ligando 4-1BB/genética , Ligando 4-1BB/metabolismo , Anticuerpos Monoclonales Humanizados/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Sinergismo Farmacológico , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Neoplasias Gástricas/tratamiento farmacológico , Regulación hacia ArribaRESUMEN
OBJECTIVE: Tumor necrosis factor superfamily member 9 (TNFSF9), also known as 4-1BBL and CD137L, has been implicated in cancer immunotherapy due to its function as a T-cell co-stimulator. We aimed to investigate the role of TNFSF9 in the cancer pathogenesis in hepatocellular carcinoma (HCC). METHODS: TNFSF9 expression was examined by immunohistochemistry in 106 pairs of HCC and adjacent non-tumorous tissues, and by quantitative polymerase chain reaction and Western blot in HCC cell lines. The impact of TNFSF9 on the proliferation, migration and invasion of HCC cells was determined using the 3-(4,5-diethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) and transwell assays in vitro. We also assessed the influence of TNFSF9 on the growth and metastasis of HCC tumors in an orthotopic mouse model of human HCC. RESULTS: TNFSF9 expression was downregulated in approximately 70% of HCC tissues. A decreased expression of TNFSF9 was also consistently observed in all the four HCC cell lines. Either the overexpression of TNFSF9 or treatment with recombinant TNFSF9 protein could significantly inhibit the proliferation, migration and invasion of Huh7 and SMMC-7721 HCC cells in vitro. The inhibitory effect of TNFSF9 on HCC was further confirmed in vivo. Mice orthotopically transplanted with TNFSF9-overexpressing Huh7 cells developed significantly smaller tumors with less intrahepatic metastasis and distant metastasis compared with the control group. CONCLUSIONS: TNFSF9 may be a tumor suppressor in HCC. Based on its immune stimulatory aspect and the tumor inhibition property, TNFSF9 may be a promising therapeutic target for HCC.
Asunto(s)
Ligando 4-1BB/fisiología , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Ligando 4-1BB/genética , Ligando 4-1BB/farmacología , Animales , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/secundario , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Regulación hacia Abajo , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Masculino , Ratones Endogámicos BALB C , Invasividad Neoplásica , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In this issue of Blood, Shah et al describe the onset of severe acute graft-versus-host disease (GVHD) in 5 of 9 patients who received infusions of ex vivo expanded donor natural killer (NK) cells after HLA-matched hematopoietic stem cell transplantation (HSCT).
Asunto(s)
Ligando 4-1BB/farmacología , Neoplasias Gastrointestinales/terapia , Enfermedad Injerto contra Huésped/patología , Interleucina-15/farmacología , Células Asesinas Naturales/trasplante , Trasplante de Células Madre de Sangre Periférica/métodos , Neoplasias Cutáneas/terapia , Femenino , Humanos , MasculinoRESUMEN
BACKGROUND: Activation of T cells against tumors by recruiting co-stimulatory molecules has been an attractive approach for cancer immunotherapy. Reports suggested that targeting different genes in tumors might also boost T cell-mediated tumor destruction. AIMS: We investigated whether in vitro WEE1 gene silencing in MDA-MB-468 and MCF7 breast cancer cell lines could enhance immunopotentiating effects of CD80 and 4-1BBL co-stimulation in human T cells. MATERIALS AND METHODS: WEE1 gene was specifically silenced in the cancer cells using shRNA technology. The co-stimulatory molecules were over-expressed on the surface of the cancer cells by recombinant non-replicative adenoviruses. The immune reaction of T cells in the co-culture with tumor cells was studied. IFN-g production was assessed by intracellular staining of T cells. To assess cytotoxic activity of CD8+ T cells, the CD107a mobilization-degranulation assay was performed. Expression of granzyme B, perforin and fasl were examined by real time PCR. RESULTS: T cell dual co-stimulation led to a significant increase in the frequency of IFN-g producing cells and higher percentages of degranulation in CD8+ T cells. It also resulted in higher expression levels of the cytotoxicity-related genes. WEE1 gene silencing in the target cells alone however, could not produce significant immune reactivation in the cultured T cells. Likewise, the immune responses of T cells neither improved nor suppressed when dually co-stimulated PBMCs were exposed to the cancer cells with silenced WEE1. CONCLUSIONS: In spite of antitumor effects of WEE1 silencing, combination of this approach with immune co-stimulation could not boost the reactivity of cultured T cells against the tested breast cancer cells.
Asunto(s)
Ligando 4-1BB/farmacología , Antígeno B7-1/farmacología , Neoplasias de la Mama/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Silenciador del Gen , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Citocinas/metabolismo , Femenino , Voluntarios Sanos , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Natural killer (NK) cells can enhance engraftment and mediate graft-versus-leukemia following allogeneic hematopoietic stem cell transplantation (HSCT), but the potency of graft-versus-leukemia mediated by naturally reconstituting NK cells following HSCT is limited. Preclinical studies demonstrate that activation of NK cells using interleukin-15 (IL-15) plus 4-1BBL upregulates activating receptor expression and augments killing capacity. In an effort to amplify the beneficial effects of NK cells post-HSCT, we conducted a first-in-human trial of adoptive transfer of donor-derived IL-15/4-1BBL-activated NK cells (aNK-DLI) following HLA-matched, T-cell-depleted (1-2 × 10(4) T cells/kg) nonmyeloablative peripheral blood stem cell transplantation in children and young adults with ultra-high-risk solid tumors. aNK-DLI were CD3(+)-depleted, CD56(+)-selected lymphocytes, cultured for 9 to 11 days with recombinant human IL-15 plus 4-1BBL(+)IL-15Rα(+) artificial antigen-presenting cells. aNK-DLI demonstrated potent killing capacity and displayed high levels of activating receptor expression. Five of 9 transplant recipients experienced acute graft-versus-host disease (GVHD) following aNK-DLI, with grade 4 GVHD observed in 3 subjects. GVHD was more common in matched unrelated donor vs matched sibling donor recipients and was associated with higher donor CD3 chimerism. Given that the T-cell dose was below the threshold required for GVHD in this setting, we conclude that aNK-DLI contributed to the acute GVHD observed, likely by augmenting underlying T-cell alloreactivity. This trial was registered at www.clinicaltrials.gov as #NCT01287104.
Asunto(s)
Ligando 4-1BB/farmacología , Neoplasias Gastrointestinales/terapia , Enfermedad Injerto contra Huésped/patología , Interleucina-15/farmacología , Células Asesinas Naturales/trasplante , Trasplante de Células Madre de Sangre Periférica/métodos , Neoplasias Cutáneas/terapia , Enfermedad Aguda , Adolescente , Traslado Adoptivo , Adulto , Células Cultivadas , Femenino , Neoplasias Gastrointestinales/inmunología , Neoplasias Gastrointestinales/patología , Enfermedad Injerto contra Huésped/inmunología , Efecto Injerto vs Leucemia , Prueba de Histocompatibilidad , Humanos , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/efectos de los fármacos , Depleción Linfocítica , Masculino , Hermanos , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Linfocitos T/citología , Linfocitos T/inmunología , Quimera por Trasplante , Trasplante Homólogo , Insuficiencia del Tratamiento , Donante no EmparentadoRESUMEN
Reports show enhancement of CD8 T cells' activity through CD137 (4-1BB) signal; however, not all data proved similar effect in natural killer (NK) cells. Here, the impact of 4-1BB signal on NK cells' function was assessed during short term cultures. To that end, cytokine-activated NK cells were cocultured with adenovirally transduced MCF-7 stimulator cells expressing 4-1BB ligand. Cellular cytotoxicity, cytokine production, and expression of cytotoxicity related genes were assessed after overnight cultures. Sharp decrease of CD56+ and CD56bright NK cells was demonstrated. 4-1BB neither enhanced cellular degranulation nor improved IFN-γ production although it promoted granzyme B, perforin, and FasL gene expression. 4-1BB signal stimulated higher proportions of CD56bright population to degranulate and express CD107a; however, it could not recover killing activity against K562 targets. Our data could not show major promotion in activity of all NK subpopulations. Due to great heterogeneity of NK cells, more investigation is needed to draw a comprehensive conclusion.
Asunto(s)
Ligando 4-1BB/farmacología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Adenoviridae/genética , Antígeno CD56/metabolismo , Línea Celular Tumoral , Citotoxicidad Inmunológica/inmunología , Proteína Ligando Fas/biosíntesis , Granzimas/biosíntesis , Humanos , Interferón gamma/metabolismo , Células K562 , Proteína 1 de la Membrana Asociada a los Lisosomas/biosíntesis , Células MCF-7 , Perforina/biosíntesis , Transducción de Señal/inmunología , Linfocitos T Citotóxicos/inmunología , Transducción GenéticaRESUMEN
Vaccines based on tumor-associated antigens (TAA) have limited therapeutic efficacy due to their weak immunogenic nature and the various immune evasion mechanisms active in advanced tumors. In an effort to overcome these limitations, we evaluated a combination of the T-cell costimulatory molecule SA-4-1BBL with the TLR4 agonist monophosphoryl lipid A (MPL) as a novel vaccine adjuvant system. In the TC-1 mouse allograft model of human papilloma virus (HPV)-induced cancer, a single administration of this combination adjuvant with HPV E7 protein caused tumor rejection in all tumor-bearing mice. On its own, SA-4-1BBL outperformed MPL in this setting. Against established tumors, two vaccinations were sufficient to elicit rejection in the majority of mice. In the metastatic model of Lewis lung carcinoma, vaccination of the TAA survivin with SA-4-1BBL/MPL yielded superior efficacy against pulmonary metastases. Therapeutic efficacy of SA-4-1BBL/MPL was achieved in the absence of detectable toxicity, correlating with enhanced dendritic cell activation, CD8(+) T-cell function, and an increased intratumoral ratio of CD8(+) T effector cells to CD4(+)FoxP3(+) T regulatory cells. Unexpectedly, use of MPL on its own was associated with unfavorable intratumoral ratios of these T-cell populations, resulting in suboptimal efficacy. The efficacy of MPL monotherapy was restored by depletion of T regulatory cells, whereas eliminating CD8(+) T cells abolished the efficacy of its combination with SA-4-1BBL. Mechanistic investigations showed that IFNγ played a critical role in supporting the therapeutic effect of SA-4-1BBL/MPL. Taken together, our results offer a preclinical proof of concept for the use of a powerful new adjuvant system for TAA-based cancer vaccines.
Asunto(s)
Ligando 4-1BB/farmacología , Adyuvantes Inmunológicos/farmacología , Vacunas contra el Cáncer/inmunología , Lípido A/análogos & derivados , Estreptavidina/farmacología , Animales , Autoinmunidad , Linfocitos T CD8-positivos/inmunología , Interferón gamma/fisiología , Lípido A/farmacología , Neoplasias Pulmonares/secundario , Ratones , Linfocitos T Reguladores/inmunologíaRESUMEN
The lead candidate plague subunit vaccine is the recombinant fusion protein rF1-V adjuvanted with alum. While alum generates Th2 regulated robust humoral responses, immune protection against Yersinia pestis has been shown to also involve Th1 driven cellular responses. Therefore, the rF1-V-based subunit vaccine may benefit from an adjuvant system that generates a mixed Th1 and humoral immune response. We herein assessed the efficacy of a novel SA-4-1BBL costimulatory molecule as a Th1 adjuvant to improve cellular responses generated by the rF1-V vaccine. SA-4-1BBL as a single adjuvant had better efficacy than alum in generating CD4(+) and CD8(+) T cells producing TNFα and IFNγ, signature cytokines for Th1 responses. The combination of SA-4-1BBL with alum further increased this Th1 response as compared with the individual adjuvants. Analysis of the humoral response revealed that SA-4-1BBL as a single adjuvant did not generate a significant Ab response against rF1-V, and SA-4-1BBL in combination with alum did not improve Ab titers. However, the combined adjuvants significantly increased the ratio of Th1 regulated IgG2c in C57BL/6 mice to the Th2 regulated IgG1. Finally, a single vaccination with rF1-V adjuvanted with SA-4-1BBL+alum had better protective efficacy than vaccines containing individual adjuvants. Taken together, these results demonstrate that SA-4-1BBL improves the protective efficacy of the alum adjuvanted lead rF1-V subunit vaccine by generating a more balanced Th1 cellular and humoral immune response. As such, this adjuvant platform may prove efficacious not only for the rF1-V vaccine but also against other infections that require both cellular and humoral immune responses for protection.
Asunto(s)
Ligando 4-1BB/farmacología , Adyuvantes Inmunológicos/farmacología , Vacunas Bacterianas/inmunología , Inmunidad Celular , Células TH1/inmunología , Yersinia pestis , Animales , Anticuerpos Antibacterianos/sangre , Formación de Anticuerpos , Femenino , Inmunoglobulina G/sangre , Ratones Endogámicos C57BL , Peste/prevención & control , Vacunas de Subunidad/inmunologíaRESUMEN
It has been shown that human prostate cancer (PCa) cells induced apoptotic death of the most potent antigen-presenting cells, dendritic cells (DCs), which are responsible for the induction of specific antitumor immune responses. Here, we investigated the function of 4-1BB on protecting DCs from prostate cancer-induced apoptosis with an agonistic mAb to 4-1BB. RM-1 cells and DCs were co-incubated for 48 h and DC apoptosis was assessed by Annexin Vassay. TNF-α and IL-12 production were assessed by enzyme-linked immunosorbent assay (ELISA) and Bcl-2 and Bcl-xL on DCs were analyzed by Western blot. We have shown that co-incubation of RM-1 cells with DCs is accompanied by an increased level of DCs apoptosis. Triggering 4-1BB on DCs resulted in increased resistance of DCs to RM-1 cells-induced apoptosis, which was owing to the up-regulated expression of Bcl-2 and Bcl-xL, and increased secretion of TNF-αand IL-12. These results demonstrate that triggering 4-1BB on DCs could increased resistance of DCs to PCa-induced apoptosis.
Asunto(s)
Ligando 4-1BB/farmacología , Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Células Dendríticas/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Animales , Apoptosis/genética , Línea Celular Tumoral , Células Dendríticas/metabolismo , Femenino , Interleucina-12/genética , Interleucina-12/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
The paucity of costimulation at the tumor site compromises the ability of tumor-specific T cells to eliminate the tumor. Here, we show that bi-specific oligonucleotide aptamer conjugates can deliver costimulatory ligands to tumor cells in situ and enhance antitumor immunity. In poorly immunogenic subcutaneously implanted tumor and lung metastasis models, systemic delivery of an agonistic 4-1BB aptamer ligand conjugated to a prostate specific membrane antigen (PSMA)-binding tumor-targeting aptamer led to inhibition of tumor growth, was more effective than, and synergized with, vaccination, and exhibited a superior therapeutic index compared to costimulation with 4-1BB antibodies. Tumor inhibition was dependent on homing to PSMA-expressing tumor cells and 4-1BB costimulation. Aptamer targeted costimulation is a broadly applicable and clinically feasible approach to enhance the costimulatory environment of disseminated tumor lesions. This study suggests that potentiating naturally occurring antitumor immunity via tumor-targeted costimulation could be an effective approach to elicit protective immunity to control tumor progression in cancer patients.
Asunto(s)
Ligando 4-1BB/farmacología , Aptámeros de Nucleótidos/administración & dosificación , Neoplasias de la Próstata/metabolismo , Ligando 4-1BB/administración & dosificación , Ligando 4-1BB/metabolismo , Animales , Aptámeros de Nucleótidos/metabolismo , Secuencia de Bases , División Celular , Humanos , Masculino , Ratones , Neoplasias de la Próstata/patologíaRESUMEN
One of the consequences of HIV infection is a progressive loss of T cell functions, resulting in decreased cytokine secretion and proliferation and an increased sensitivity to apoptosis. Therefore, successful therapeutic vaccination approaches should aim at restoring the functionality of existing HIV-specific T cells, as well as to efficiently induce potent, HIV-specific T cells from naïve T cells. In this study, we wanted to determine the stimulatory capacity of DCs coelectroporated with mRNA encoding for different costimulatory molecules of the TNFSF, together with HIV antigen-encoding mRNA. We show that DCs electroporated with 4-1BBL can enhance the proliferation, functionality, cytokine production, and survival of HIV-specific CD8(+) T cells. Furthermore, we are the first to show that a combination of 4-1BBL and CD40L overexpression on DCs dramatically enhances CD4(+) and CD8(+) T cell responses. Finally, we demonstrate that signaling through 4-1BB, but not through CD40, can alleviate the suppressive effect of Tregs on CD8(+) T cell proliferation. Thus, the combination of 4-1BBL and CD40L enhances HIV-specific CD8(+) T cell responses in a synergistic way, resulting in enhanced proliferation of CD4(+) and CD8(+) T cell subsets, an increased cytokine secretion, and a reduced sensitivity to Treg-mediated immune suppression.
Asunto(s)
Ligando 4-1BB/farmacología , Ligando de CD40/farmacología , Células Dendríticas/inmunología , Infecciones por VIH/inmunología , VIH/inmunología , Linfocitos T/inmunología , Western Blotting , Antígenos CD40/inmunología , Proliferación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Seropositividad para VIH , Humanos , Activación de Linfocitos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Reguladores/inmunologíaRESUMEN
The therapeutic CD11a antibody Efalizumab interferes with psoriasis pathogenesis by blocking T cell activation and migration. We have performed a detailed analysis on its effects during the activation of human T cells and found that the capability of Efalizumab to inhibit proliferation and cytokine production of T cells critically depends on the quality and quantity of costimulatory signals. Efalizumab potently inhibited the proliferation and cytokine production of human T cells costimulated via ICOS, OX40, CD27 or 4-1BB, but did not significantly inhibit T cells that received stimuli via CD2 or CD28. The capacity of CD2 and CD28 signals to interfere with the T cell inhibitory effects of Efalizumab was also observed upon stimulation of T cells with allogeneic DC. Furthermore, studies with T cells from psoriasis patients indicated that Efalizumab therapy induces inhibition of T cell responses that can be reverted by CD2 or CD28 signals.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígeno CD11a/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Ligando 4-1BB/farmacología , Abatacept , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Presentación de Antígeno/inmunología , Antígenos CD/farmacología , Antígeno B7-1/inmunología , Antígenos CD28/inmunología , Complejo CD3/inmunología , Antígenos CD58/inmunología , Proliferación Celular/efectos de los fármacos , Células Dendríticas/inmunología , Humanos , Inmunoconjugados/farmacología , Inmunosupresores/farmacología , Inmunosupresores/uso terapéutico , Ligando Coestimulador de Linfocitos T Inducibles , Molécula 1 de Adhesión Intercelular/metabolismo , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Interleucina-2/farmacología , Interleucina-4/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Psoriasis/tratamiento farmacológico , Psoriasis/inmunología , Linfocitos T/metabolismoRESUMEN
Major histocompatibility complex (MHC) class I chain-related protein A (MICA), which is a ligand for human NKG2D, is expressed by a variety of epithelial tumor cells and promotes the activation of natural killer (NK), CD8(+) and γδ-T cells. Although ectopic expression of MICA on tumor cells elicits anti-tumor responses, soluble MICA downregulates the activities of lymphocytes. In this study, we showed that recombinant, immobilized MICA (iMICA) molecules coated on plastic wells weakly promote peripheral NK cell activation, secretion of interferon (IFN)-γ and degranulation without inducing apoptosis. In addition, iMICA synergized with IL-15 and soluble 4-1BB ligand (s4-1BBL) to expand NK cells 25- to 42-fold in a 13-day culture, whereas NK cells stimulated only with IL-15 and s4-1BBL expanded 10- to 16-fold. In contrast to NK cells expanded by IL-15 and s4-1BBL stimulation, NK cells expanded long term in the presence of iMICA exhibited increased cytotoxicity against leukemia cells. These results suggest that large numbers of NK cells with high cytotoxicity can be generated by stimulation with IL-15 and s4-1BBL in the presence of iMICA and that these cells can be used for adoptive cancer immunotherapy.
Asunto(s)
Ligando 4-1BB/inmunología , Citotoxicidad Inmunológica , Antígenos de Histocompatibilidad Clase I/inmunología , Proteínas Inmovilizadas/inmunología , Interleucina-15/inmunología , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Ligando 4-1BB/farmacología , Apoptosis/efectos de los fármacos , Antígeno CD56/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citotoxicidad Inmunológica/efectos de los fármacos , Citometría de Flujo , Antígenos de Histocompatibilidad Clase I/farmacología , Humanos , Proteínas Inmovilizadas/farmacología , Interleucina-15/farmacología , Células Asesinas Naturales/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Fenotipo , Receptores de IgG/metabolismo , Receptores de Células Asesinas Naturales/metabolismo , Solubilidad/efectos de los fármacosRESUMEN
OBJECTIVE: To investigate the adjuvant effect of co-stimulatory molecule CD137L on cellular responses to HBsAg DNA vaccination in mice. METHODS: Eukaryotic expression vector containing the full length of mouse CD137L cDNA sequence (pcD137L) was transfected into NIH3T3 cells, and then the expression of CD137L mRNA and protein in the transfected cells were detected by RT-PCR, flow cytometry and immunofluorescence method, respectively. The BALB/c mice were co-immunized with pcD137L and HBsAg DNA vaccine (pcDS) by intramuscular injection. HBsAg-specific activity of splenic cytotoxic T lymphocyte (CTL) in the immunized mice was measured by LDH release assay. The splenic memory CD8+ T cells, and intracellular IFN-gamma and IL-4 of splenic lymphocytes and CD8+ T cells after immunization were detected by flow cytometry. RESULTS: The NIH3T3 cells transfected with pcD137L efficiently expressed mouse CD137L mRNA and protein. HBsAg-specific CTL responses induced by the pcDS plus pcD137L group were much stronger than those induced by pcDS alone at a week after immunization (P<0.05). Compared to mice immunized with pcDS alone, CD44high and CD127(IL-7R) were all significantly up-regulated in memory CD8+ T cells from the mice immunized with pcDS combined CD137L both at a week and 12 weeks after immunization (P<0.05 and P<0.01). The pcDS plus CD137L group also elicited higher levels of IFN-gamma secreted by CD8+ T cells and splenic lymphocytes than pcDS alone at a week, 12 and 13 weeks after immunization, respectively (all P<0.01). CONCLUSION: DNA, viral/immunol; Co-stimulatory molecule CD137L can enhance the Tc1 (type I) cell-mediated immunity, HBsAg-specific CTL and memory responses induced by HBsAg DNA vaccine, and may be an efficient adjuvant in priming HBV-specific T cell response.
Asunto(s)
Ligando 4-1BB/inmunología , Adyuvantes Inmunológicos/farmacología , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/inmunología , Vacunas de ADN/inmunología , Ligando 4-1BB/farmacología , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Vacunación/métodosRESUMEN
4-1BB, a member of the tumor necrosis factor receptor (TNFR) family, binds the 4-1BB ligand (4-1BBL), works as a costimulatory molecule, and regulates T cell-mediated immune responses. Although inflammation is an essential pathological feature of myocarditis, the role of 4-1BB in experimental autoimmune myocarditis (EAM) remains unclear. Lewis rats were immunized on day 0 with purified porcine cardiac myosin to establish EAM. 4-1BB-immunoglobulin (4-1BBIg) was administered intraperitoneally (n=6) a total of 9 times (3 times per week). Rats were killed on day 21 to study effects of 4-1BB/4-1BBL pathway blockade. For controls, isotype-matched human IgG was administered in other EAM rats (n=6). Histologic and echocardiographic examination showed development of EAM attenuated by 4-1BBIg. Suppression of mRNA expression for IL-1alpha, IL-1beta, IL-4, IL-6, and TNF-alpha was noted in the heart tissue treated with 4-1BBIg. Treatment with 4-1BBIg reduced production of Th1-type cytokines, and inhibited T cell proliferation in vitro. In the 4-1BB signaling pathway in splenocytes, 4-1BBIg suppressed JNK, p38, and IkappaB activity but not that of ERK1/2. Blockade of T cell activation through the 4-1BB/4-1BBL pathway regulates development of EAM; therefore, 4-1BB may be an effective target for treating myocarditis.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/prevención & control , Activación de Linfocitos/inmunología , Miocarditis/inmunología , Miocarditis/prevención & control , Linfocitos T/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Ligando 4-1BB/metabolismo , Ligando 4-1BB/farmacología , Animales , Enfermedades Autoinmunes/enzimología , Enfermedades Autoinmunes/patología , Proliferación Celular/efectos de los fármacos , Citocinas/genética , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Quinasa I-kappa B/metabolismo , Inmunohistoquímica , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Activación de Linfocitos/efectos de los fármacos , Masculino , Miocarditis/enzimología , Miocarditis/patología , Miocardio/enzimología , Miocardio/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas Lew , Sus scrofa , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Células TH1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
AIM: To investigate the change of immunoactivity and surface marker of dendritic cells (DCs) derived from bone marrow and induced by rat 4-1BBL. METHODS: Plasmid pIRES2-EGFP-4-1BBL was constructed and tansfected into HepG2 cells by lipofectin-mediated method, and positive cells were screened by G418. The expression of 4-1BBL in cells were detected by RT-PCR, and the expression of EGFP was detected by fluorescence microscopy. Various HepG2 cells were co-cultured with DC for 2 days, and then the expression of MHC-II and CD80 on DC were detected by FCM, and IL-6 and IL-12 levels secreted by DCs were tested by ELISA. RESULTS: The recombinant pIRES2-EGFP-4-1BBL vector was successfully constructed and was steadily expressed in HepG2 cells transfected by pIRES2-EGFP-4-1BBL. Compared with HepG2 cells transfected by pIRES2-EGFP plasmid , recombinant HepG2 cells induced high expression of MHC-II and CD80 and secreted more IL-6 and IL-12 on DCs(P<0.05). CONCLUSION: The recombinant rat 4-1BBL could promote the maturity of DCs derived from bone marrow.
Asunto(s)
Ligando 4-1BB/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Proteínas Recombinantes/inmunología , Ligando 4-1BB/genética , Ligando 4-1BB/metabolismo , Animales , Antígeno B7-1/metabolismo , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Femenino , Genes MHC Clase II , Humanos , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Masculino , Microscopía Fluorescente , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Our previous data have shown a significantly higher tumor response to anti-CD3/anti-Pgp bispecific diabody-mediated immunotherapy for P-glycoprotein (Pgp)-overexpressing K562/A02 cells, but a rapid tumor relapse occurred at 1 week after therapy. In an attempt to overcome tumor recurrence, we supplemented the previous therapy with extracellular domain of human 4-1BBL (ex4-1BBL) to regulate the activation of peripheral blood lymphocyte (PBL). As a result, this combination showed enhanced cytotoxicity in vitro and eradicated the multidrug-resistant xenografts of K562/A02 in nude mice. Furthermore, no tumor recurrence was observed within 100 days after the first treatment. Therefore, when used as an adjuvant, ex4-1BBL may improve the outcome of PBL-based immunotherapy.
Asunto(s)
Ligando 4-1BB/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/inmunología , Anticuerpos Biespecíficos/uso terapéutico , Complejo CD3/inmunología , Inmunoterapia/métodos , Leucemia/terapia , Linfocitos/inmunología , Ligando 4-1BB/biosíntesis , Ligando 4-1BB/genética , Animales , Anticuerpos Biespecíficos/inmunología , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Femenino , Humanos , Interleucina-2/inmunología , Células Jurkat , Células K562 , Leucemia/inmunología , Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Estructura Terciaria de Proteína , Distribución Aleatoria , Organismos Libres de Patógenos Específicos , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
AIM: To investigate the role of the extracellular domain of human 4-1BBL (ex4-1BBL) in regulating the in vitro activities of peripheral blood lymphocytes (PBL). METHODS: Viable cells were quantified with Trypan-blue exclusion assay. CytoTox 96 Nonradioactive Cytotoxicity Assay Kit was used for measuring LDH levels of supernatant. ELISA kit was used for measuring IL-2 level. In vitro cytotoxity of PBL combined with anti-CD3/anti-Pgp bispecific diabody plus ex4-1BBL was analyzed with CytoTox 96 nonradioactive method. RESULTS: Ex4-1BBL can increase the proliferation of PBL, reduce cell death, promote IL-2 secretion, and the experimental group with ex4-1BBL showed obviously enhanced cytotoxic effect toward K562/A02 cells. CONCLUSION: Ex4-1BBL may be an important adjuvant for improving activities of PBL.