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1.
Nat Commun ; 13(1): 1815, 2022 04 05.
Article in English | MEDLINE | ID: mdl-35383192

ABSTRACT

The ability to detect and target ß cells in vivo can substantially refine how diabetes is studied and treated. However, the lack of specific probes still hampers a precise characterization of human ß cell mass and the delivery of therapeutics in clinical settings. Here, we report the identification of two RNA aptamers that specifically and selectively recognize mouse and human ß cells. The putative targets of the two aptamers are transmembrane p24 trafficking protein 6 (TMED6) and clusterin (CLUS). When given systemically in immune deficient mice, these aptamers recognize the human islet graft producing a fluorescent signal proportional to the number of human islets transplanted. These aptamers cross-react with endogenous mouse ß cells and allow monitoring the rejection of mouse islet allografts. Finally, once conjugated to saRNA specific for X-linked inhibitor of apoptosis (XIAP), they can efficiently transfect non-dissociated human islets, prevent early graft loss, and improve the efficacy of human islet transplantation in immunodeficient in mice.


Subject(s)
Aptamers, Nucleotide , Clusterin , Islets of Langerhans Transplantation , Islets of Langerhans , Vesicular Transport Proteins , Animals , Aptamers, Nucleotide/genetics , Clusterin/genetics , Graft Rejection , Humans , Indicators and Reagents , Islets of Langerhans/metabolism , Mice , RNA/metabolism , Vesicular Transport Proteins/genetics
2.
PLoS One ; 11(6): e0157245, 2016.
Article in English | MEDLINE | ID: mdl-27285580

ABSTRACT

BACKGROUND: Allogeneic human islet transplantation is an effective therapy for the treatment of patients with Type 1 Diabetes (T1D). The low number of islet transplants performed worldwide and the different transplantation protocols used limit the identification of the most effective therapeutic options to improve the efficacy of this approach. METHODS: We present a retrospective analysis on the data collected from 44 patients with T1D who underwent islet transplantation at our institute between 2000 and 2007. Several variables were included: recipient demographics and immunological characteristics, donor and transplant characteristics, induction protocols, and additional medical treatment received. Immunosuppression was induced with anti-CD25 (Daclizumab), alone or in association with anti-tumor necrosis factor alpha (TNF-α) treatments (Etanercept or Infliximab), or with anti-CD52 (Alemtuzumab) in association with anti-TNF-α treatments (Etanercept or Infliximab). Subsets of patients were treated with Filgrastim for moderate/severe neutropenia and/or Exenatide for post prandial hyperglycemia. RESULTS: The analysis performed indicates a negative association between graft survival (c-peptide level ≥ 0.3 ng/ml) and islet infusion volume, with the caveat that, the progressive reduction of infusion volumes over the years has been paralleled by improved immunosuppressive protocols. A positive association is instead suggested between graft survival and administration of Exenatide and Filgrastim, alone or in combination. CONCLUSION: This retrospective analysis may be of assistance to further improve long-term outcomes of protocols for transplant of islets and other organs.


Subject(s)
Filgrastim/therapeutic use , Graft Survival/drug effects , Hematologic Agents/therapeutic use , Hypoglycemic Agents/therapeutic use , Islets of Langerhans Transplantation/methods , Islets of Langerhans/drug effects , Peptides/therapeutic use , Venoms/therapeutic use , Adult , Aged , Antibodies, Monoclonal, Humanized/therapeutic use , Daclizumab , Exenatide , Humans , Hyperglycemia/drug therapy , Hyperglycemia/etiology , Immunoglobulin G/therapeutic use , Immunosuppressive Agents/therapeutic use , Islets of Langerhans/physiology , Islets of Langerhans Transplantation/adverse effects , Middle Aged , Neutropenia/drug therapy , Neutropenia/etiology , Retrospective Studies , Transplantation, Homologous
3.
J Diabetes Res ; 2015: 737586, 2015.
Article in English | MEDLINE | ID: mdl-26347378

ABSTRACT

The nonobese diabetic (NOD) mouse represents a well-established experimental model analogous to human type 1 diabetes mellitus (T1D) as it is characterized by progressive autoimmune destruction of pancreatic ß-cells. Experiments were designed to investigate the impact of moderate-intensity training on T1D immunomodulation and inflammation. Under a chronic exercise regime, NOD mice were trained on a treadmill for 12 weeks (12 m/min for 30 min, 5 d/wk) while age-matched, control animals were left untrained. Prior to and upon completion of the training period, fed plasma glucose and immunological soluble factors were monitored. Both groups showed deteriorated glycemic profiles throughout the study although trained mice tended to be more compensated than controls after 10 weeks of training. An exercise-induced weight loss was detected in the trained mice with respect to the controls from week 6. After 12 weeks, IL-6 and MIP-1ß were decreased in the trained animals compared to their baseline values and versus controls, although not significantly. Morphometric analysis of pancreata revealed the presence of larger infiltrates along with decreased α-cells areas in the control mice compared to trained mice. Exercise may exert positive immunomodulation of systemic functions with respect to both T1D and inflammation, but only in a stringent therapeutic window.


Subject(s)
Diabetes Mellitus, Type 1/blood , Hyperglycemia/pathology , Inflammation/pathology , Physical Conditioning, Animal , Animals , Blood Glucose/analysis , Body Weight , Chemokine CCL4/blood , Diabetes Mellitus, Type 1/genetics , Female , Hyperglycemia/therapy , Immunohistochemistry , Inflammation/therapy , Insulin-Secreting Cells/cytology , Interleukin-6/blood , Mice , Mice, Inbred NOD , Pancreas/metabolism
4.
Immun Inflamm Dis ; 2(3): 181-92, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25505552

ABSTRACT

Induction of a T cell mediated immune response is critical for the eradication of viral infections and tumours. Soluble peptide-loaded major histocompatibility complex-Ig ((pep-)MHC-Ig) have been shown to bind their cognate ligands, T cell receptor, with high affinity, and are successfully used to visualize antigen-specific T cells. Furthermore, immobilized (pep-)MHC-Ig can activate and expand antigen-specific T cells in vitro and in vivo. In this study, we investigate the use of (pep-)MHC-Ig as a potential strategy to modulate antigen specific T cell immune responses in vivo. (SIY-)K(b)-Ig immunization, together with the pre-activation by an anti-CD40 monoclonal antibody, is able to stimulate a strong expansion of adoptively transferred 2C transgenic T cells and the formation of long term antigen-specific memory T cells. In addition, mechanistic studies show that the (pep-)MHC-Ig molecules directly activate T cells in vivo without requiring uptake and reprocessing by antigen-presenting cells. Furthermore, B6 mice immunized with (pep-)MHC-Ig molecules inhibit tumour growth in a B16-SIY melanoma prevention model. Thus, soluble (pep-)MHC-Ig molecules represent a powerful tool for active immunotherapy.

5.
Eur J Immunol ; 44(11): 3307-19, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25113564

ABSTRACT

By restraining T-cell activation and promoting Treg-cell expansion, myeloid-derived suppressor cells (MDSCs) and tolerogenic DCs can control self-reactive and antigraft effector T cells in autoimmunity and transplantation. Their therapeutic use and characterization, however, is limited by their scarce availability in the peripheral blood of tumor-free donors. In the present study, we describe and characterize a novel population of human myeloid suppressor cells, named fibrocytic MDSC, which are differentiated from umbilical cord blood precursors by 4-day culture with FDA-approved cytokines (recombinant human-GM-CSF and recombinant human-G-CSF). This MDSC subset, characterized by the expression of MDSC-, DC-, and fibrocyte-associated markers, promotes Treg-cell expansion and induces normoglycemia in a xenogeneic mouse model of Type 1 diabetes. In order to exert their protolerogenic function, fibrocytic MDSCs require direct contact with activated T cells, which leads to the production and secretion of IDO. This new myeloid subset may have an important role in the in vitro and in vivo production of Treg cells for the treatment of autoimmune diseases, and in either the prevention or control of allograft rejection.


Subject(s)
Indoleamine-Pyrrole 2,3,-Dioxygenase/biosynthesis , Lymphocyte Activation/immunology , Myeloid Cells/immunology , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/immunology , Animals , Cell Differentiation/immunology , Cell Line , Cell Proliferation , Diabetes Mellitus, Type 1/immunology , Female , Fetal Blood/cytology , Gene Expression Profiling , Graft Rejection/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , HEK293 Cells , Humans , Immune Tolerance , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Myeloid Cells/cytology , Recombinant Proteins/pharmacology , T-Lymphocytes, Regulatory/transplantation
6.
Genom Data ; 2: 389-92, 2014 Dec.
Article in English | MEDLINE | ID: mdl-26484135

ABSTRACT

Myeloid-derived suppressor cells (MDSCs) have been shown to control self-reactive and anti-graft effector T-cells in autoimmunity and transplantation, but their therapeutic use is limited by their scarce availability in the peripheral blood of tumor-free donors. We isolated and characterized a novel population of myeloid suppressor cells, named fibrocytic MDSC (f-MDSC), which are differentiated from umbilical cord blood (UCB) precursors (Zoso et al., 2014). This MDSC subset promotes regulatory T-cell expansion and induces normoglycemia in a xenogeneic model of type 1 diabetes. Here we describe in details the experimental design and the bioinformatics analyses of the gene expression dataset used to investigate the molecular mechanisms at the base of MDSC tolerogenic and suppressive properties. We also provide an R code to easily access the data and perform the quality controls and basic analyses relevant to this dataset. Raw and pre-processed data are available at Gene Expression Omnibus under accession GSE52376.

7.
Cancer Res ; 72(6): 1373-83, 2012 Mar 15.
Article in English | MEDLINE | ID: mdl-22282665

ABSTRACT

In addition to promoting tumor progression and metastasis by enhancing angiogenesis and invasion, myeloid-derived suppressor cells (MDSC) and tumor-associated macrophage (TAM) also inhibit antitumor T-cell functions and limit the efficacy of immunotherapeutic interventions. Despite the importance of these leukocyte populations, a simple method for their specific depletion has not been developed. In this study, we generated an RNA aptamer that blocks the murine or human IL-4 receptor-α (IL4Rα or CD124) that is critical for MDSC suppression function. In tumor-bearing mice, this anti-IL4Rα aptamer preferentially targeted MDSCs and TAM and unexpectedly promoted their elimination, an effect that was associated with an increased number of tumor-infiltrating T cells and a reduction in tumor growth. Mechanistic investigations of aptamer-triggered apoptosis in MDSCs confirmed the importance of IL4Ra-STAT6 pathway activation in MDSC survival. Our findings define a straightforward strategy to deplete MDSCs and TAMs in vivo, and they strengthen the concept that IL4Rα signaling is pivotal for MDSC survival. More broadly, these findings suggest therapeutic strategies based on IL4Rα signaling blockades to arrest an important cellular mechanism of tumoral immune escape mediated by MDSCs and TAM in cancer.


Subject(s)
Apoptosis/drug effects , Aptamers, Nucleotide/pharmacology , Carcinoma/drug therapy , Disease Progression , Mammary Neoplasms, Experimental/drug therapy , Receptors, Cell Surface/antagonists & inhibitors , Animals , Apoptosis/immunology , Carcinoma/immunology , Cell Line, Tumor , Humans , Interleukin-4 Receptor alpha Subunit/antagonists & inhibitors , Interleukin-4 Receptor alpha Subunit/immunology , Mammary Neoplasms, Experimental/immunology , Mice , Mice, Inbred BALB C , Myeloid Cells/drug effects , Myeloid Cells/immunology , Receptors, Cell Surface/immunology , STAT6 Transcription Factor/metabolism , Tumor Escape/drug effects , Tumor Escape/immunology
8.
Cancer Res ; 69(24): 9376-84, 2009 Dec 15.
Article in English | MEDLINE | ID: mdl-19934317

ABSTRACT

The development of effective antitumor immune responses is normally constrained by low-avidity, tumor-specific CTLs that are unable to eradicate the tumor. Strategies to rescue antitumor activity of low-avidity melanoma-specific CTLs in vivo may improve immunotherapy efficacy. To boost the in vivo effectiveness of low-avidity CTLs, we immunized mice bearing lung melanoma metastases with artificial antigen-presenting cells (aAPC), made by covalently coupling (pep)MHC-Ig dimers and B7.1-Ig molecules to magnetic beads. aAPC treatment induced significant tumor reduction in a mouse telomerase antigen system, and complete tumor eradication in a mouse TRP-2 antigen system, when low-avidity CTLs specific for these antigens were adoptively transferred. In addition, in an in vivo treatment model of subcutaneous melanoma, aAPC injection also augmented the activity of adoptively transferred CTLs and significantly delayed tumor growth. In vivo tumor clearance due to aAPC administration correlated with in situ proliferation of the transferred CTL. In vitro studies showed that aAPC effectively stimulated cytokine release, enhanced CTL-mediated lysis, and TCR downregulation in low-avidity CTLs. Therefore, in vivo aAPC administration represents a potentially novel approach to improve cancer immunotherapy.


Subject(s)
Antigen-Presenting Cells/immunology , Immunotherapy, Adoptive/methods , Leukemia, Experimental/therapy , Melanoma, Experimental/therapy , T-Lymphocytes/immunology , Animals , Antibody Affinity , Cell Growth Processes/immunology , Female , Leukemia, Experimental/immunology , Lymphocyte Activation , Melanoma, Experimental/immunology , Mice , Mice, Inbred C57BL , T-Lymphocytes, Cytotoxic/immunology
9.
Anticancer Res ; 29(8): 2875-84, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19661290

ABSTRACT

BACKGROUND: To identify immunosuppressive elements present in ovarian cancer associated ascites. PATIENTS AND METHODS: Ascites and plasma were obtained from ovarian, primary peritoneal or fallopian tube cancer patients. Surface markers were identified by fluorescence-activated cell sorting (FACS). Cytokine and chemokine concentrations were measured with LINCOplex microarrays. Antigen-specific T-lymphocytes from ascites and plasma were expanded with artificial antigen-presenting cells (aAPC). Cell-mediated immune response was assessed with chromium release assays. RESULTS: Samples were collected from 37 patients with advanced ovarian cancer. FACS was performed on 27 ascites specimens. A low CD4/CD8 ratio (<1.6) was seen in 13 patient samples and associated with significantly improved overall survival (p=0.040). LINCOplex evaluation of 22 paired ascites and plasma samples demonstrated significantly elevated levels of IL-6, IL-8, IL-10, IL-15, IP-10, MCP-1, MIP-1beta and VEGF and significantly reduced levels of IL-2, IL-5, IL-7, IL-17, PDGF-BB, and RANTES in ascites compared to plasma (p<0.05). Autologous ovarian cancer cell lysis with T-lymphocytes from ascites was limited. Although aAPC stimulation resulted in effective expansion of antigen specific T-cells from peripheral lymphocytes (35-fold), only limited expansion was noted from ascites-derived lymphocytes (10-fold). CONCLUSION: Ovarian cancer-associated ascites may provide an immunosuppressive environment. A high CD4/CD8 ratio, which may indicate the presence of regulatory T-cells, is associated with poor outcome. Reduced IL-2 and elevated IL-6 and IL-10 levels favor a Th2 inhibitory immune response. This immunosuppressive climate may explain our observation of non responsiveness in ascites derived T-cells.


Subject(s)
Ascites/immunology , Fallopian Tube Neoplasms/immunology , Ovarian Neoplasms/immunology , Peritoneal Neoplasms/immunology , T-Lymphocytes, Regulatory/immunology , Adenocarcinoma, Clear Cell/immunology , Adenocarcinoma, Clear Cell/metabolism , Adenocarcinoma, Clear Cell/secondary , Adult , Aged , Aged, 80 and over , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Antigen-Presenting Cells/pathology , Ascites/metabolism , Ascites/pathology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/pathology , Chromium/metabolism , Cystadenocarcinoma, Serous/immunology , Cystadenocarcinoma, Serous/metabolism , Cystadenocarcinoma, Serous/secondary , Cytokines/metabolism , Endometrial Neoplasms/immunology , Endometrial Neoplasms/metabolism , Endometrial Neoplasms/secondary , Fallopian Tube Neoplasms/metabolism , Fallopian Tube Neoplasms/pathology , Female , Flow Cytometry , Humans , Middle Aged , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Peritoneal Neoplasms/metabolism , Peritoneal Neoplasms/pathology , Prognosis , Survival Rate
10.
Blood ; 111(7): 3546-52, 2008 Apr 01.
Article in English | MEDLINE | ID: mdl-18096763

ABSTRACT

Several cell-based immunotherapy strategies have been developed to specifically modulate T cell-mediated immune responses. These methods frequently rely on the utilization of tolerogenic cell-based antigen-presenting cells (APCs). However, APCs are highly sensitive to cytotoxic T-cell responses, thus limiting their therapeutic capacity. Here, we describe a novel bead-based approach to modulate T-cell responses in an antigen-specific fashion. We have generated killer artificial APCs (kappaaAPCs) by coupling an apoptosis-inducing alpha-Fas (CD95) IgM mAb together with HLA-A2 Ig molecules onto beads. These kappaaAPCs deplete targeted antigen-specific T cells in a Fas/Fas ligand (FasL)-dependent fashion. T-cell depletion in cocultures is rapidly initiated (30 minutes), dependent on the amount of kappaaAPCs and independent of activation-induced cell death (AICD). kappaaAPCs represent a novel technology that can control T cell-mediated immune responses, and therefore has potential for use in treatment of autoimmune diseases and allograft rejection.


Subject(s)
Antibodies, Monoclonal/immunology , Antigen-Presenting Cells/immunology , Immune Tolerance , Immunoglobulin M/immunology , Lymphocyte Depletion/methods , Microspheres , T-Lymphocytes, Cytotoxic/immunology , fas Receptor/immunology , Antibodies, Monoclonal/chemistry , Antigen-Presenting Cells/chemistry , Autoimmune Diseases/immunology , Autoimmune Diseases/therapy , Cell Death/immunology , Cells, Cultured , Fas Ligand Protein/immunology , Graft Rejection/immunology , Graft Rejection/therapy , Humans , Immunoglobulin M/chemistry , Time Factors , Transplantation, Homologous , fas Receptor/chemistry
11.
Int J Cancer ; 119(7): 1717-22, 2006 Oct 01.
Article in English | MEDLINE | ID: mdl-16671090

ABSTRACT

We quantified the expression of survivin, both as mRNA in real-time PCR and protein in immunohistochemistry, in tumor samples of 112 patients with esophageal cancer (56 squamous cell carcinomas and 56 adenocarcinomas). Overall survival of squamous cell carcinoma patients with high survivin mRNA levels was significantly less than that of patients with low survivin mRNA levels (p = 0.0033). Distribution pattern of survivin (nuclear vs. cytoplasmic or mixed) was not correlated to survival, while the extent of immunostaining was significantly correlated to survivin mRNA values (p = 0.016) and had prognostic relevance in univariate analysis (p = 0.0012). Cox's proportional-hazard regression model showed that tumor survivin expression in esophageal squamous cell carcinoma was the most important prognostic factor, independent of tumor stage and other histopathological factors, both as mRNA relative value (p = 0.0259) and protein immunostaining (p = 0.0147). In esophageal adenocarcinoma, survivin expression and pattern of distribution had no prognostic relevance. Thus, quantifying survivin expression provides a prognostic marker only for esophageal squamous tumors.


Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Esophageal Neoplasms/metabolism , Esophageal Neoplasms/pathology , Microtubule-Associated Proteins/metabolism , Neoplasm Proteins/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Biomarkers, Tumor/genetics , Carcinoma, Squamous Cell/genetics , Esophageal Neoplasms/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Inhibitor of Apoptosis Proteins , Male , Microtubule-Associated Proteins/genetics , Middle Aged , Neoplasm Proteins/genetics , Prognosis , RNA, Messenger/genetics , Survival Rate , Survivin
12.
Cancer Res ; 66(7): 3754-63, 2006 Apr 01.
Article in English | MEDLINE | ID: mdl-16585202

ABSTRACT

The ubiquitin-proteasome system (UPS) mediates targeted protein degradation. Notably, the UPS determines levels of key checkpoint proteins controlling apoptosis and proliferation by controlling protein half-life. Herein, we show that ovarian carcinoma manifests an overstressed UPS by comparison with normal tissues by accumulation of ubiquitinated proteins despite elevated proteasome levels. Elevated levels of total ubiquitinated proteins and 19S and 20S proteasome subunits are evident in both low-grade and high-grade ovarian carcinoma tissues relative to benign ovarian tumors and in ovarian carcinoma cell lines relative to immortalized surface epithelium. We find that ovarian carcinoma cell lines exhibit greater sensitivity to apoptosis in response to proteasome inhibitors than immortalized ovarian surface epithelial cells. This sensitivity correlates with increased cellular proliferation rate and UPS stress rather than absolute proteasome levels. Proteasomal inhibition in vitro induces cell cycle arrest and the accumulation of p21 and p27 and triggers apoptosis via activation of caspase-3. Furthermore, treatment with the licensed proteasome inhibitor PS-341 slows the growth of ES-2 ovarian carcinoma xenograft in immunodeficient mice. In sum, elevated proliferation and metabolic rate resulting from malignant transformation of the epithelium stresses the UPS and renders ovarian carcinoma more sensitive to apoptosis in response to proteasomal inhibition.


Subject(s)
Apoptosis/drug effects , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Protease Inhibitors/pharmacology , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors , Ubiquitin/metabolism , Animals , Apoptosis/physiology , Boronic Acids/pharmacology , Bortezomib , Caspases/metabolism , Cell Division/drug effects , Cell Line, Tumor , Female , G2 Phase/drug effects , Humans , Leupeptins/pharmacology , Mice , Mice, Nude , Oligopeptides/pharmacology , Ovarian Neoplasms/enzymology , Ovarian Neoplasms/pathology , Proteasome Endopeptidase Complex/biosynthesis , Pyrazines/pharmacology , Xenograft Model Antitumor Assays
13.
J Immunol ; 176(3): 1999-2006, 2006 Feb 01.
Article in English | MEDLINE | ID: mdl-16424232

ABSTRACT

Identification of reliable surrogate predictors for evaluation of cancer vaccine efficacy is a critical issue in immunotherapy. We analyzed quantitative and qualitative CD8+ T cell parameters in a large pool of BALB/c mice that were DNA-vaccinated against P1A self tumor-specific Ag. After immunization, mice were splenectomized and kept alive for a subsequent tumor challenge to correlate results of immune monitoring assays with tumor regression or progression in each individual animal, and to assess the prognostic value of the assays. The parameters tested were 1) percentage of in vivo vaccine-induced tumor-specific CD8+ T cells; 2) results of ELISPOT tests from fresh splenocytes; 3) percentage of tumor-specific CD8+ T cells in culture after in vitro restimulation; 4) in vitro increase of tumor-specific CD8+ T cell population expressed as fold of expansion; and 5) antitumor lytic activity of restimulated cultures. Except for the ELISPOT assay, each parameter tested was shown by univariate statistical analysis to correlate with tumor regression. However, multivariate analysis revealed that only in vitro percentage of Ag-specific CD8+ T cells was an independent prognostic factor that predicted tumor outcome. These findings should be considered in the design of new immune monitoring systems used in cancer immunotherapy studies.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/administration & dosage , Monitoring, Immunologic , Neoplasms, Experimental/diagnosis , Vaccines, DNA/administration & dosage , Animals , Antigens, Neoplasm/immunology , Cancer Vaccines/immunology , Cell Line, Tumor , Cell Proliferation , Cells, Cultured , Cytotoxicity Tests, Immunologic/statistics & numerical data , Enzyme-Linked Immunosorbent Assay/statistics & numerical data , Female , Lymphocyte Count/statistics & numerical data , Mice , Mice, Inbred BALB C , Monitoring, Immunologic/methods , Monitoring, Immunologic/statistics & numerical data , Multivariate Analysis , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/immunology , Predictive Value of Tests , Prognosis , Vaccines, DNA/immunology
14.
J Immunol ; 171(10): 5172-9, 2003 Nov 15.
Article in English | MEDLINE | ID: mdl-14607917

ABSTRACT

The weakly immunogenic murine P1A Ag is a useful experimental model for the development of new vaccination strategies that could potentially be used against human tumors. An i.m. DNA-based immunization procedure, consisting of three inoculations with the P1A-coding pBKCMV-P1A plasmid at 10-day intervals, resulted in CTL generation in all treated BALB/c mice. Surprisingly, gene gun skin bombardment with the pBKCMV-P1A vector did not induce CTL, nor was it protective against a lethal challenge with the syngeneic P1A-positive J558 tumor cell line. To speed up the immunization procedure, we pretreated the tibialis anterior muscles with cardiotoxin, which induces degeneration of myocytes while sparing immature satellite cells. The high muscle-regenerative activity observable after cardiotoxin inoculation was associated with infiltration of inflammatory cells and expression of proinflammatory cytokines. A single pBKCMV-P1A plasmid inoculation in cardiotoxin-treated BALB/c mice allowed for sustained expansion of P1A-specific CTL and the induction of strong lytic activity in <2 wk. Cardiotoxin adjuvanticity could not be replaced by another muscle-degenerating substance, such as bupivacaine, or by MF59, a Th1 response-promoting adjuvant. Although this vaccination schedule failed to induce tumor rejection in all immunized mice, the analysis of CD8 T cell responses at an individual mouse level disclosed that the cytotoxic activity of P1A-specific CTL was correlated to the antitumor efficacy. These results highlight the critical need to identify reliable, specific immunological parameters that may predict success or failure of an immune response against cancer.


Subject(s)
Antigens, Neoplasm/immunology , Cancer Vaccines/genetics , Cancer Vaccines/immunology , Cytotoxicity, Immunologic , Epitopes, T-Lymphocyte/immunology , Mast-Cell Sarcoma/immunology , Mast-Cell Sarcoma/prevention & control , T-Lymphocytes, Cytotoxic/immunology , Adjuvants, Immunologic/administration & dosage , Animals , Antigens, Neoplasm/genetics , Biolistics , Cancer Vaccines/therapeutic use , Cell Division/immunology , Cell Line, Tumor , Cells, Cultured , Cobra Cardiotoxin Proteins/administration & dosage , Dose-Response Relationship, Immunologic , Epitopes, T-Lymphocyte/administration & dosage , Epitopes, T-Lymphocyte/genetics , Female , Immunity, Innate/genetics , Immunization Schedule , Immunohistochemistry , Injections, Intramuscular , Lymphocyte Activation/genetics , Mast-Cell Sarcoma/mortality , Mast-Cell Sarcoma/pathology , Mice , Mice, Inbred BALB C , Muscle, Skeletal/chemistry , Muscle, Skeletal/immunology , Muscle, Skeletal/pathology , Neoplasm Transplantation/immunology , Plasmids , Survival Rate , T-Lymphocytes, Cytotoxic/cytology , Th1 Cells/drug effects , Th1 Cells/immunology , Vaccines, DNA/administration & dosage , Vaccines, DNA/immunology , Vaccines, DNA/therapeutic use
15.
Cancer Res ; 63(9): 2158-63, 2003 May 01.
Article in English | MEDLINE | ID: mdl-12727834

ABSTRACT

The TS/A mouse mammary adenocarcinoma is a poorly immunogenic tumor widely used in preclinical models of cancer immunotherapy. CTLs have often been indicated as important in TS/A tumor destruction, but their generation in this model has been rarely studied, nor have their precise target(s) been identified. We hypothesized that the gp70 Env product of an endogenous murine leukemia virus could be a target antigen for TS/A-specific CTLs and investigated this possibility in four different TS/A cell lines engineered with the genes that encode IFN-alpha, IFN-gamma, interleukin-4, and B7.1, respectively. All tumor cell lines expressed gp70, albeit at different levels, as demonstrated by reverse transcription-PCR analysis. Transfected tumor cells exhibited a delayed growth in vivo, and partial tumor regression. Spleen cells from mice that displayed tumor regression had high percentages of CD8(+) T cells that were specifically stained with L(d) tetramers loaded with gp70(423-431), the antigenic epitope of gp70 protein. Mixed leukocyte-peptide and mixed leukocyte-tumor cultures, set up by stimulating splenocytes with the immunogenic peptide and with transfected TS/A tumor cells, respectively, resulted in similar large increases in tetramer-reactive CD8(+) T cells and showed high lytic activity specific for gp70(423-431). Finally, in a Cold Target Inhibition assay, lytic activity of a mixed leukocyte-tumor culture was inhibited in an overlapping fashion by both the TS/A line used for restimulation and 293L(d) cells loaded with gp70(423-431) peptide, but not by 293L(d) cells pulsed with an irrelevant H-2 L(d) epitope, thus demonstrating that all or most of the cytotoxic activity was directed exclusively against this antigenic epitope.


Subject(s)
Adenocarcinoma/immunology , Immunodominant Epitopes/immunology , Mammary Neoplasms, Experimental/immunology , Retroviridae Proteins, Oncogenic/immunology , Retroviridae/immunology , T-Lymphocytes, Cytotoxic/immunology , Viral Envelope Proteins/immunology , Animals , Antigens, Neoplasm/immunology , B7-1 Antigen/biosynthesis , Female , Interferon-alpha/biosynthesis , Interferon-gamma/biosynthesis , Interleukin-4/biosynthesis , Lymphocyte Activation/immunology , Mammary Neoplasms, Experimental/genetics , Mice , Mice, Inbred BALB C , Retroviridae Proteins, Oncogenic/biosynthesis , Retroviridae Proteins, Oncogenic/genetics , Transfection , Viral Envelope Proteins/biosynthesis , Viral Envelope Proteins/genetics
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