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1.
J Transl Med ; 11: 79, 2013 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-23531320

RESUMEN

BACKGROUND: Despite availability of efficient treatment regimens for early stage colorectal cancer, treatment regimens for late stage colorectal cancer are generally not effective and thus need improvement. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) strains is a promising new strategy for therapy of a variety of human cancers. METHODS: Oncolytic efficacy of replication-competent vaccinia virus GLV-1h68 was analyzed in both, cell cultures and subcutaneous xenograft tumor models. RESULTS: In this study we demonstrated for the first time that the replication-competent recombinant VACV GLV-1h68 efficiently infected, replicated in, and subsequently lysed various human colorectal cancer lines (Colo 205, HCT-15, HCT-116, HT-29, and SW-620) derived from patients at all four stages of disease. Additionally, in tumor xenograft models in athymic nude mice, a single injection of intravenously administered GLV-1h68 significantly inhibited tumor growth of two different human colorectal cell line tumors (Duke's type A-stage HCT-116 and Duke's type C-stage SW-620), significantly improving survival compared to untreated mice. Expression of the viral marker gene ruc-gfp allowed for real-time analysis of the virus infection in cell cultures and in mice. GLV-1h68 treatment was well-tolerated in all animals and viral replication was confined to the tumor. GLV-1h68 treatment elicited a significant up-regulation of murine immune-related antigens like IFN-γ, IP-10, MCP-1, MCP-3, MCP-5, RANTES and TNF-γ and a greater infiltration of macrophages and NK cells in tumors as compared to untreated controls. CONCLUSION: The anti-tumor activity observed against colorectal cancer cells in these studies was a result of direct viral oncolysis by GLV-1h68 and inflammation-mediated innate immune responses. The therapeutic effects occurred in tumors regardless of the stage of disease from which the cells were derived. Thus, the recombinant vaccinia virus GLV-1h68 has the potential to treat colorectal cancers independently of the stage of progression.


Asunto(s)
Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Viroterapia Oncolítica/métodos , Virus Oncolíticos/genética , Virus Vaccinia/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Chlorocebus aethiops , Progresión de la Enfermedad , Humanos , Inyecciones Intravenosas , Macrófagos/metabolismo , Masculino , Ratones , Ratones Desnudos , Microscopía Fluorescente , Trasplante de Neoplasias
2.
J Immunol ; 187(1): 126-32, 2011 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-21632715

RESUMEN

Sustained intratumoral delivery of IL-12 and GM-CSF can overcome tumor immune suppression and promote T cell-dependent eradication of established disease in murine tumor models. However, the antitumor effector response is transient and rapidly followed by a T suppressor cell rebound. The mechanisms that control the switch from an effector to a regulatory response in this model have not been defined. Because dendritic cells (DC) can mediate both effector and suppressor T cell priming, DC activity was monitored in the tumors and the tumor-draining lymph nodes (TDLN) of IL-12/GM-CSF-treated mice. The studies demonstrated that therapy promoted the recruitment of immunogenic DC (iDC) to tumors with subsequent migration to the TDLN within 24-48 h of treatment. Longer-term monitoring revealed that iDC converted to an IDO-positive tolerogenic phenotype in the TDLN between days 2 and 7. Specifically, day 7 DC lost the ability to prime CD8(+) T cells but preferentially induced CD4(+)Foxp3(+) T cells. The functional switch was reversible, as inhibition of IDO with 1-methyl tryptophan restored immunogenic function to tolerogenic DC. All posttherapy immunological activity was strictly associated with conventional myeloid DC, and no functional changes were observed in the plasmacytoid DC subset throughout treatment. Importantly, the initial recruitment and activation of iDC as well as the subsequent switch to tolerogenic activity were both driven by IFN-γ, revealing the dichotomous role of this cytokine in regulating IL-12-mediated antitumor T cell immunity.


Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/patología , Interferón gamma/fisiología , Interleucina-12/administración & dosificación , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Escape del Tumor/inmunología , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/inmunología , Técnicas de Cocultivo , Células Dendríticas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Tolerancia Inmunológica/genética , Interferón gamma/deficiencia , Interferón gamma/genética , Interleucina-12/fisiología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/prevención & control , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Alveolos Pulmonares/inmunología , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Subgrupos de Linfocitos T/metabolismo , Escape del Tumor/genética
3.
J Immunol ; 187(8): 4109-18, 2011 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-21908736

RESUMEN

Intratumoral delivery of IL-12 and GM-CSF induces local and systemic antitumor CD8(+) T cell activation and tumor kill. However, the effector response is transient and is rapidly countered by CD4(+) Foxp3(+) T suppressor cell expansion. To determine whether depletion of the pre-existing T suppressor cell pool prior to treatment could diminish posttherapy regulatory cell resurgence, FVBneuN mice bearing advanced spontaneous mammary tumors were treated with cyclophosphamide (CY) 1 d before IL-12/GM-CSF therapy. Administration of CY mediated a significant delay in the post-IL-12/GM-CSF T suppressor cell rebound, resulting in a 7-fold increase in the CD8(+) CTL/T suppressor cell ratio, a 3-fold enhancement of CTL cytotoxicity, and an extension of the effector window from 3 to 7 d. In long-term therapy studies, chronic chemoimmunotherapy promoted a dramatic enhancement of tumor regression, resulting in complete cure in 44% of the mice receiving CY plus IL-12/GM-CSF. Tumor eradication in the chronic therapy setting was associated with the ability to repeatedly rescue and maintain cytotoxic CD8(+) T cell activity. These findings demonstrated that chronic administration of CY in conjunction with immune therapy enhances the initial induction of antitumor T effector cells and, more importantly, sustains their cytotoxic activity over the long-term via persistent blockade of homeostatic counter-regulation.


Asunto(s)
Ciclofosfamida/administración & dosificación , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Inmunoterapia/métodos , Interleucina-12/administración & dosificación , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Separación Celular , Quimioterapia , Femenino , Citometría de Flujo , Inmunosupresores/administración & dosificación , Neoplasias Mamarias Experimentales/inmunología , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología
4.
J Transl Med ; 10: 152, 2012 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-22846631

RESUMEN

BACKGROUND: Myeloid-derived Suppressor Cells (MDSC) have been identified as tumor-induced immature myeloid cells (IMC) with potent immune suppressive activity in cancer. Whereas strict phenotypic classification of MDSC has been challenging due to the highly heterogeneous nature of cell surface marker expression, use of functional markers such as Arginase and inducible nitric oxide synthase (iNOS) may represent a better categorization strategy. In this study we investigated whether iNOS could be utilized as a specific marker for the identification of a more informative homogenous MDSC subset. METHODS: Single-cell suspensions from tumors and other organs were prepared essentially by enzymatic digestion. Flow cytometric analysis was performed on a four-color flow cytometer. Morphology, intracellular structure and localization of iNOS(+) ring cells in the tumor were determined by cytospin analysis, immunofluorescence microscopy and immunohistochemistry, respectively. For functional analysis, iNOS(+) ring subset were sorted and tested in vitro cell culture experiments. Pharmacologic inhibition of iNOS was performed both in vivo and in vitro. RESULTS: The results showed that intracellular iNOS staining distinguished a granular iNOS(+) SSC(hi) CD11b(+) Gr-1(dim) F4/80(+) subset with ring-shaped nuclei (ring cells) among the CD11b(+) Gr-1(+) cell populations found in tumors. The intensity of the ring cell infiltrate correlated with tumor size and these cells constituted the second major tumor-infiltrating leukocyte subset found in established tumors. Although phenotypic analysis demonstrated that ring cells shared characteristics with tumor-associated macrophages (TAM), morphological analysis revealed a neutrophil-like appearance as detected by cytospin and immunofluorescence microscopy analysis. The presence of distinct iNOS filled granule-like structures located next to the cell membrane suggested that iNOS was stored in pre-formed vesicles and available for rapid release upon activation. Tumor biopsies showed large areas with infiltrating ring cells primarily surrounding necrotic areas. Importantly, these cells significantly impaired CD8(+) T-cell proliferation and induced apoptotic death. The intratumoral accumulation and suppressive activity of ring cells could be blocked through pharmacologic inhibition of iNOS, demonstrating the critical role of this enzyme in mediating both the differentiation and the activity of these cells. CONCLUSIONS: In this study, iNOS expression was linked to a homogeneous subset; ring cells with a particular phenotype and immune suppressive function, in a common and well-established murine tumor model; 4T-1. Since the absence of a Gr-1 homolog in humans has made the identification of MDSC much more challenging, use of iNOS as a functional marker of MDSC may also have clinical importance.


Asunto(s)
Neoplasias Experimentales/patología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Animales , Citometría de Flujo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Fluorescente , Neoplasias Experimentales/enzimología
5.
Cancer Immunol Immunother ; 60(6): 839-45, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21387108

RESUMEN

Interleukin-12 (IL-12) can promote tumor regression via activation of multiple lymphocytic and myelocytic effectors. Whereas the cytotoxic mechanisms employed by T/NK/NKT cells in IL-12-mediated tumor kill are well defined, the antitumor role of macrophage-produced cytotoxic metabolites has been more controversial. To this end, we investigated the specific role of nitric oxide (NO), a major macrophage effector molecule, in post-IL-12 tumor regression. Analysis of tumors following a single intratumoral injection of slow-release IL-12 microspheres showed an IFNγ-dependent sevenfold increase in inducible nitric oxide synthase (iNOS) expression within 48 h. Flow cytometric analysis of tumor-resident leukocytes and in vivo depletion studies identified CD11b(+) F4/80(+) Gr1(lo) macrophages as the primary source of iNOS. Blocking of post-therapy iNOS activity with N-nitro-L: -arginine methyl ester (L-NAME) dramatically enhanced tumor suppression revealing the inhibitory effect of NO on IL-12-driven antitumor immunity. Superior tumor regression in mice receiving combination treatment was associated with enhanced survival and proliferation of activated tumor-resident CD8+ T-effector/memory cells (Tem). These findings demonstrate that macrophage-produced NO negatively regulates the antitumor activity of IL-12 via its detrimental effects on CD8+ T cells and identify L-NAME as a potent adjuvant in IL-12 therapy of cancer.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Interleucina-12/inmunología , Interleucina-12/farmacología , Óxido Nítrico/inmunología , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/patología , Femenino , Citometría de Flujo , Humanos , Inyecciones Intralesiones , Interleucina-12/antagonistas & inhibidores , Activación de Linfocitos/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/inmunología , Óxido Nítrico Sintasa de Tipo II/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
6.
J Immunol ; 182(7): 4217-25, 2009 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-19299720

RESUMEN

Sustained delivery of IL-12 and GM-CSF to tumors induces the activation of tumor-resident CD8(+) T effector/memory cells (Tem) followed by cytotoxic CD8(+) T effector cell expansion. To determine whether the secondary effectors expanded from tumor-associated Tem or were primed de novo, activation kinetics of tumor-draining lymph node (TDLN) CD8(+) T cells were analyzed. Treatment promoted a 4-fold increase in the numbers of TDLN CD8(+) T cells displaying a CD69(+)CCR5(+)CD62L(-) periphery-homing effector phenotype by day 4 posttherapy. Pulse labeling of tumor and TDLN T cells with BrdU confirmed that proliferation occurred exclusively within the draining lymph nodes between days 1 and 4 with subsequent migration of primed CD8(+) T effectors to tumors on day 7. Day 4 CD8(+) T effector cells preferentially homed to and lysed experimental, but not control, tumors, establishing tumor specificity. To determine whether the secondary CD8(+) T effector cell response was dependent on activation of tumor-resident CD8(+) Tem, mice that were selectively depleted of tumor-infiltrating CD8(+) T cells were treated and monitored for T effector priming. In the absence of tumor-resident CD8(+) Tem, T effector cell expansion was completely abrogated in the TDLN, revealing that restoration of CD8(+) Tem function was critical to the induction of secondary T effectors. T cell priming failed to occur in IFN-gamma or perforin knockout mice, demonstrating that the requirement for Tem activation was associated with induction of Tem cytotoxicity. These data confirm that intratumoral IL-12 plus GM-CSF induces de novo priming of tumor-specific CD8(+) T effector cells in the TDLN and establish the critical role of preexisting intratumoral CD8(+) Tem in driving this process.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Activación de Linfocitos/inmunología , Neoplasias Experimentales/inmunología , Traslado Adoptivo , Animales , Proliferación Celular , Quimiotaxis de Leucocito/inmunología , Femenino , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Interleucina-12/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados
7.
J Immunol ; 183(12): 7656-60, 2009 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-19923444

RESUMEN

Tumor-resident CD8(+) T cells display a quiescent effector/memory phenotype that is maintained in part by infiltrating CD4(+) CD25(+) Foxp3(+) T-suppressor cells. Intratumoral delivery of IL-12, in contrast, can restore cytotoxic function to tumor-associated CD8(+) T cells and induce the apoptotic death of T-suppressor cells. Depletion of CD8(+) T cells from tumors before IL-12 treatment resulted in the abrogation of treatment-mediated T-suppressor cell apoptosis revealing a link between CD8(+) T cell activation and T-suppressor elimination. Furthermore, IL-12 failed to induce T-suppressor cell loss in IFN-gamma- or FasL-deficient mice demonstrating a requirement for IFN-gamma and FasL in this process. Adoptive transfer of wild-type CD8(+) T cells to FasL-knockout mice restored posttherapy T-suppressor cell elimination from tumors establishing that expression of FasL on CD8(+) T cells was sufficient to promote T-suppressor cell death. IL-12 failed to induce FasL on T-effectors in IFN-gamma-knockout mice demonstrating a requirement for IFN-gamma in FasL up-regulation. Adoptive transfer of wild-type CD8(+) T cells induced T-suppressor cell death in IFN-gamma-knockout mice confirming that autocrine IFN-gamma was sufficient for CD8(+) T cell FasL expression. These findings reveal a mechanism by which cytotoxic T cells can abrogate regulatory cell activity.


Asunto(s)
Apoptosis/inmunología , Linfocitos T CD8-positivos/inmunología , Carcinoma de Células Escamosas/inmunología , Citotoxicidad Inmunológica , Proteína Ligando Fas/fisiología , Memoria Inmunológica , Neoplasias Pulmonares/inmunología , Activación de Linfocitos/inmunología , Linfocitos T Reguladores/inmunología , Animales , Apoptosis/genética , Antígenos CD4/biosíntesis , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Citotoxicidad Inmunológica/genética , Proteína Ligando Fas/deficiencia , Proteína Ligando Fas/genética , Factores de Transcripción Forkhead/biosíntesis , Memoria Inmunológica/genética , Interleucina-12/fisiología , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Neoplasias Pulmonares/patología , Activación de Linfocitos/genética , Depleción Linfocítica , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Alveolos Pulmonares/inmunología , Alveolos Pulmonares/patología , Linfocitos T Reguladores/patología
8.
Hum Gene Ther ; 17(3): 334-46, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16544982

RESUMEN

Vaccination with autologous tumor cells genetically modified to express costimulatory molecules has shown utility for cancer immunotherapy in preclinical and limited clinical settings. Given the complicated nature of gene therapy, a practical alternative approach has been designed that relies on modification of the cell membrane with biotin and its "decoration" with a chimeric protein composed of the functional portion of human CD80 and core streptavidin (CD80-SA). We tested whether primary tumor cells resected from cancer patients can be decorated with CD80-SA and whether such cells serve as antigen-presenting cells (APCs) to generate autologous T cell responses ex vivo. Tumors and peripheral blood lymphocytes (PBLs) were collected from 14 lung, 9 colon, and 2 breast "treatment-naive" cancer patients presenting various clinical stages of the disease. Tumors were mechanically processed, irradiated, decorated with CD80-SA or control streptavidin (SA) protein, and used as APCs in ex vivo autologous T cell-proliferative and cytotoxicity assays. All tumor samples were modified with CD80-SA, albeit with various degrees of decoration ranging from 21.8 to 100%. CD80- SA-decorated cells generated significant proliferative responses in autologous T cells from 9 of 16 evaluable patients (p < 0.05). Proliferative responses were CD80-SA specific and heterogeneous, with stimulation indices ranging from 0.25 to 45. In 15 of 15 evaluable patients, CD80-SA-specific cytotoxic T cell responses against autologous tumors were generated, 11 of which were significant, with specific killing ranging from 5 to 70%. Taken together, these data demonstrate that primary tumor cells can be effectively decorated with CD80-SA and that such cells serve as APCs to induce autologous antitumor T cell responses.


Asunto(s)
Células Presentadoras de Antígenos/inmunología , Antígeno B7-1/inmunología , Inmunoterapia , Neoplasias/inmunología , Linfocitos T/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estreptavidina , Linfocitos T Citotóxicos/inmunología , Células Tumorales Cultivadas
9.
Arch Immunol Ther Exp (Warsz) ; 58(6): 399-405, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20872283

RESUMEN

Tumor-resident T cells display a functionally impaired effector/memory (Tem) phenotype. Sustained intratumoral administration of IL-12, on the other hand, can restore cytolytic function to pre-existing CD8+ Tem, resulting in effective tumor kill. Whereas cytotoxic T lymphocytes (CTL) are generally assumed to mediate tumor regression via direct tumor cytotoxicity, recent work revealed that activated CD8+ Tem mobilize a systemic, multi-component effector cascade that includes both innate and adaptive immune mechanisms. Here we summarize these mechanisms, review how tumor-resident CD8+ Tem orchestrate this cascade and discuss the potential clinical implications of these findings.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Interleucina-12/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias/inmunología , Escape del Tumor , Animales , Citotoxicidad Inmunológica , Humanos , Memoria Inmunológica , Inmunoterapia/métodos , Neoplasias/terapia
10.
Cancer Res ; 70(1): 129-38, 2010 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-20028855

RESUMEN

Sustained intratumoral delivery of interleukin-12 (IL-12) and granulocyte macrophage colony-stimulating factor induces tumor regression via restoration of tumor-resident CD8+ T-effector/memory cell cytotoxicity and subsequent repriming of a secondary CD8+ T-effector cell response in tumor-draining lymph nodes (TDLN). However, treatment-induced T-effector activity is transient and is accompanied with a CD4+ CD25+ Foxp3+ T-suppressor cell rebound. Molecular and cellular changes in posttherapy tumor microenvironment and TDLN were monitored to elucidate the mechanism of counterregulation. Real-time PCR analysis revealed a 5-fold enhancement of indoleamine 2,3-dioxygenase (IDO) expression in the tumor and the TDLN after treatment. IDO induction required IFNgamma and persisted for up to 7 days. Administration of the IDO inhibitor D-1-methyl tryptophan concurrent with treatment resulted in a dramatic enhancement of tumor regression. Enhanced efficacy was associated with a diminished T-suppressor cell rebound, revealing a link between IDO activity and posttherapy regulation. Further analysis established that abrogation of the regulatory counterresponse resulted in a 10-fold increase in the intratumoral CD8+ T-cell to CD4+ Foxp3+ T-cell ratio. The ratio of proliferating CD8+ T-effector to CD4+ Foxp3+ T-suppressor cells was prognostic for efficacy of tumor suppression in individual mice. IFNgamma-dependent IDO induction and T-suppressor cell expansion were primarily driven by IL-12. These findings show a critical role for IDO in the regulation of IL-12-mediated antitumor immune responses.


Asunto(s)
Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Interferón gamma/inmunología , Interleucina-12/inmunología , Ratones Noqueados/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Citometría de Flujo , Tolerancia Inmunológica/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
11.
Cancer Immunol Immunother ; 57(7): 997-1005, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18049819

RESUMEN

Local delivery of IL-12 and GM-CSF to advanced primary tumors results in T- and NK-cell-dependent cure of disseminated disease in a murine spontaneous lung metastasis model. Post-therapy functional dynamics of cytotoxic T- and NK-cells were analyzed in primary and metastatic tumors to determine the specific roles of each subset in tumor eradication. Time-dependent depletion of CD8+ T and NK-cells demonstrated that CD8+ T-cells were critical to eradication of metastatic tumors within 3 days of treatment, but not later. In contrast, NK-cells were found to be essential to tumor regression for at least 10 days after cytokine delivery. Analysis of tumor-infiltrating lymphocyte populations in post-therapy primary tumors demonstrated that treatment resulted in the activation of tumor-associated CD8+ T-cells within 24 h as determined by IFNgamma and perforin production. T-cell activity peaked between days 1 and 3 and subsided rapidly thereafter. Activation was not accompanied with an increase in cell numbers suggesting that treatment mobilized pre-existing T-effector/memory cells without inducing proliferation. In contrast, therapy resulted in a > or = 3-fold enhancement of both the quantity and the cytotoxic activity of NK-cells in primary and metastatic tumors on day 3 post-therapy. NK-cell activity was also transient and subsided to pre-therapy levels by day 5. Depletion of CD4+ and CD8+ T-cells prior to treatment completely abrogated NK-cell infiltration into primary and metastatic tumors demonstrating the strict dependence of NK-cell recruitment on pre-existing T-effector/memory cells. Treatment failed to induce significant NK-cell infiltration in IFNgamma-knockout mice establishing the central role of IFNgamma in NK-cell chemotaxis to tumors. These data show that transient activation of tumor-associated T-effector/memory and NK-cells, but not long-term CD8+ T-cell responses, are critical to suppression of metastatic disease in this model; and reveal a novel role for preexisting adaptive T-cell immunity in the recruitment of innate effectors to tumors.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Asesinas Naturales/inmunología , Neoplasias Pulmonares/terapia , Animales , Citotoxicidad Inmunológica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Memoria Inmunológica , Inmunoterapia , Interleucina-12/uso terapéutico , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Activación de Linfocitos , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Metástasis de la Neoplasia
12.
Artículo en Inglés | MEDLINE | ID: mdl-18220947

RESUMEN

Cytokine therapy can induce tumor regression in cancer patients but systemic administration of cytokines is accompanied with severe toxicity. Loco-regional delivery represents an effective and less toxic alternative to systemic injection. However; the requirement for frequent repeated injections of recombinant cytokine or the logistical difficulties associated with gene-modification have limited wide-spread use of loco-regional therapy. A simpler alternative local delivery strategy involves the use of controlled-release cytokine depot formulations. These formulations provide the advantage that physiological doses of cytokines are directly released to the tumor microenvironment in a sustained manner. Anti-tumor efficacy of IL-2; IL-12; GM-CSF or TNFalpha-encapsulated polymer microspheres has been evaluated in syngeneic murine and human tumor /SCID mouse xenograft models. A single intra-tumoral injection of these formulations; particularly that of IL-12 in combination with GM-CSF or TNFalpha; promoted the regression of established primary tumors; induced systemic anti-tumor T- and NK-cell responses and achieved complete eradication of disseminated disease. Cellular and molecular analysis of post-therapy tumor microenvironment demonstrated that treatment promoted the activation of tumor-associated T-effector/memory cells; the elimination of CD4+ CD25+ Foxp3+ T-suppressors and the de novo priming of tumor-specific CD8+ T-effector cells. Long-term monitoring of post-therapy tumors revealed that reversal of intra-tumoral immune suppression was transient and that T-suppressor cells rapidly re-infiltrated tumors. Repeated treatment resurrected anti-tumor activity; however, therapeutic efficacy declined with each treatment cycle. The observed loss of therapeutic efficacy was associated with a progressive intensification of the post-treatment T-suppressor cell rebound. In contrast; depletion of T-suppressor cells with low dose chemotherapy prior to each cycle of treatment resulted in a dramatic enhancement of long-term therapeutic efficacy leading to complete remissions. Clinical implications of these findings are discussed herein.


Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Citocinas/administración & dosificación , Neoplasias/terapia , Animales , Ensayos Clínicos como Asunto , Citocinas/inmunología , Preparaciones de Acción Retardada , Humanos , Neoplasias/inmunología
13.
Exp Mol Pathol ; 80(3): 252-61, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16487512

RESUMEN

Effective aggregation of cell surface immune receptors with their ligands is critical in promoting humoral and cellular immune responses. Simulation of these interactions using soluble multimeric ligands having potent adjuvant effects may prove an effective alternative to agonistic antibodies as immunotherapeutics. Multimeric ligands may effectively engage their receptors, leading to aggregation and effective signal transduction. We exploited the structural characteristics of streptavidin (SA) for the generation of multimeric chimeric proteins. Streptavidin forms stable tetramers and oligomers under physiological conditions, and, as such, chimeric molecules with SA are expected to possess similar features. Two chimeric molecules consisting of the extracellular domains of human and mouse CD40L and a modified form of core streptavidin were generated. These proteins form stable oligomers that could only be dissociated into monomers by heating at 100 degrees C, but not 60 degrees C, under denaturing conditions. The chimeric proteins vigorously stimulated B cells, monocytes, and dendritic cells for the production of cytokines and chemokines and upregulation of immunostimulatory molecules. The use of SA as a chaperon presents a novel approach to generate multimeric immunological molecules with potent activities and their use as potential therapeutics for the treatment of cancer and other immune-based disorders.


Asunto(s)
Ligando de CD40/química , Ligando de CD40/inmunología , Chaperonas Moleculares/química , Estreptavidina/química , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Western Blotting , Ligando de CD40/farmacología , Línea Celular Tumoral , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Ratones , Chaperonas Moleculares/inmunología , Chaperonas Moleculares/farmacología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Polímeros/síntesis química , Polímeros/química , Polímeros/farmacología , Estreptavidina/inmunología , Estreptavidina/farmacología , Transfección
14.
J Immunol ; 176(12): 7325-34, 2006 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-16751376

RESUMEN

A single intratumoral injection of IL-12 and GM-CSF-encapsulated microspheres induces the complete regression of advanced spontaneous tumors in her-2/neu transgenic mice. However, tumor regression in this model is transient and long-term cure is not achieved due to recurrence. Posttherapy molecular analysis of immune activation/suppression markers within the tumor microenvironment demonstrated a dramatic up-regulation of IFN-gamma and a concomitant down-regulation of Forkhead/winged-helix protein 3 (Foxp3), TGFbeta, and IL-10 expression. Therapy-induced reversion of immune suppression was transient since all three markers of suppression recovered rapidly and surpassed pretherapy levels by day 7 after treatment, resulting in tumor resurgence. Repeated treatment enhanced short-term tumor regression, but did not augment long-term survival. Serial long-term analysis demonstrated that although chronic stimulation enhanced the IFN-gamma response, this was countered by a parallel increase in Foxp3, TGFbeta, and IL-10 expression. Analysis of tumor-infiltrating T lymphocyte populations showed that the expression of Foxp3 and IL-10 was associated with CD4(+)CD25(+) T cells. Repeated treatment resulted in a progressive increase in tumor-infiltrating CD4(+)CD25(+)Foxp3(+) T suppressor cells establishing their role in long-term neutralization of antitumor activity. Analysis of tumor-infiltrating CD8(+) T cells demonstrated that although treatment enhanced IFN-gamma production, antitumor cytotoxicity was diminished. Monitoring of CD8(+) T cells that specifically recognized a dominant MHC class I her-2/neu peptide showed a dramatic increase in tetramer-specific CD8(+) T cells after the first treatment; however, continuous therapy resulted in the loss of this population. These results demonstrate that both enhanced suppressor activity and deletion of tumor-specific T cells are responsible for the progressive loss of efficacy that is associated with chronic immune therapy.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Genes erbB-2 , Inmunoterapia Activa , Depleción Linfocítica , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/terapia , Linfocitos T Reguladores/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/uso terapéutico , Animales , Linfocitos T CD8-positivos/patología , Movimiento Celular/inmunología , Citotoxicidad Inmunológica/genética , Epítopos de Linfocito T/inmunología , Femenino , Factores de Transcripción Forkhead/biosíntesis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Inmunoterapia Activa/métodos , Inyecciones Intralesiones , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Interleucina-12/administración & dosificación , Interleucina-12/uso terapéutico , Linfocitos Infiltrantes de Tumor/patología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/mortalidad , Ratones , Ratones Transgénicos , Linfocitos T Reguladores/patología , Factor de Crecimiento Transformador beta/biosíntesis
15.
J Immunol ; 177(10): 6962-73, 2006 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-17082611

RESUMEN

A single intratumoral injection of IL-12 and GM-CSF-loaded slow-release microspheres induces T cell-dependent eradication of established primary and metastatic tumors in a murine lung tumor model. To determine how the delivery of cytokines directly to the microenvironment of a tumor nodule induces local and systemic antitumor T cell activity, we characterized therapy-induced phenotypic and functional changes in tumor-infiltrating T cell populations. Analysis of pretherapy tumors demonstrated that advanced primary tumors were infiltrated by CD4+ and CD8+ T cells with an effector/memory phenotype and CD4+CD25+Foxp3+ T suppressor cells. Tumor-associated effector memory CD8+ T cells displayed impaired cytotoxic function, whereas CD4+CD25+Foxp3+ cells effectively inhibited T cell proliferation demonstrating functional integrity. IL-12/GM-CSF treatment promoted a rapid up-regulation of CD43 and CD69 on CD8+ effector/memory T cells, augmented their ability to produce IFN-gamma, and restored granzyme B expression. Importantly, treatment also induced a concomitant and progressive loss of T suppressors from the tumor. Further analysis established that activation of pre-existing effector memory T cells was short-lived and that both the effector/memory and the suppressor T cells became apoptotic within 4 days of treatment. Apoptotic death of pre-existing effector/memory and suppressor T cells was followed by infiltration of the tumor with activated, nonapoptotic CD8+ effector T lymphocytes on day 7 posttherapy. Both CD8+ T cell activation and T suppressor cell purge were mediated primarily by IL-12 and required IFN-gamma. This study provides important insight into how local IL-12 therapy alters the immunosuppressive tumor milieu to one that is immunologically active, ultimately resulting in tumor regression.


Asunto(s)
Adenocarcinoma Bronquioloalveolar/terapia , Apoptosis/inmunología , Linfocitos T CD8-positivos/patología , Movimiento Celular/inmunología , Memoria Inmunológica , Interleucina-12/administración & dosificación , Neoplasias Pulmonares/terapia , Linfocitos Infiltrantes de Tumor/patología , Linfocitos T Reguladores/inmunología , Adenocarcinoma Bronquioloalveolar/inmunología , Adenocarcinoma Bronquioloalveolar/patología , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/uso terapéutico , Muerte Celular/inmunología , Línea Celular Tumoral , Células Cultivadas , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Inyecciones Intralesiones , Interleucina-12/uso terapéutico , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Microesferas , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología
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