RESUMO
BACKGROUND: The detection of insulin autoantibodies (IAA) aids in the prediction of autoimmune diabetes development. However, the long-standing, gold standard 125I-insulin radiobinding assay (RBA) has low reproducibility between laboratories, long sample processing times and requires the use of newly synthesized radiolabeled insulin for each set of assays. Therefore, a rapid, non-radioactive, and reproducible assay is highly desirable. METHODS: We have developed electrochemiluminescence (ECL)-based assays that fulfill these criteria in the measurement of IAA and anti-insulin antibodies (IA) in non-obese diabetic (NOD) mice and in type 1 diabetic individuals, respectively. Using the murine IAA ECL assay, we examined the correlation between IAA, histopathological insulitis, and blood glucose in a cohort of female NOD mice from 4 up to 36 weeks of age. We developed a human IA ECL assay that we compared to conventional RBA and validated using samples from 34 diabetic and 59 non-diabetic individuals in three independent laboratories. RESULTS: Our ECL assays were rapid and sensitive with a broad dynamic range and low background. In the NOD mouse model, IAA levels measured by ECL were positively correlated with insulitis severity, and the values measured at 8-10 weeks of age were predictive of diabetes onset. Using human serum and plasma samples, our IA ECL assay yielded reproducible and accurate results with an average sensitivity of 84% at 95% specificity with no statistically significant difference between laboratories. CONCLUSIONS: These novel, non-radioactive ECL-based assays should facilitate reliable and fast detection of antibodies to insulin and its precursors sera and plasma in a standardized manner between laboratories in both research and clinical settings. Our next step is to evaluate the human IA assay in the detection of IAA in prediabetic subjects or those at risk of type 1 diabetes and to develop similar assays for other autoantibodies that together are predictive for the diagnosis of this common disorder, in order to improve prediction and facilitate future therapeutic trials.
Assuntos
Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/diagnóstico , Eletroquímica/métodos , Anticorpos Anti-Insulina/sangue , Células Secretoras de Insulina/patologia , Medições Luminescentes/métodos , Animais , Autoanticorpos/sangue , Diabetes Mellitus Tipo 1/patologia , Progressão da Doença , Feminino , Humanos , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Curva ROC , Ensaio Radioligante , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
We previously reported that naive, tumor-specific CD8(+) (TcR-I) T cells transferred into prostate tumor-bearing mice traffic to the prostate where they become tolerized. We now report that TcR-I cells suppress the proliferation of naive T cells. This suppression is mediated at least in part by secreted factors, and the suppressive activity can be blocked by Abs directed against TGF-beta. We further report that TcR-I cells must infiltrate the prostate to acquire suppressive activity. Delivery of tumor-specific CD4(+) T cells prevents the conversion of TcR-I cells into suppressor cells. Taken together, our findings may have critical implications for sustaining T cell responsiveness during immunotherapy, as the development of suppressor cells in the tumor microenvironment may eliminate the potency of T cells primed in the periphery or delivered during adoptive immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias da Próstata/imunologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/imunologia , Animais , Anticorpos/imunologia , Linfócitos T CD8-Positivos/transplante , Tolerância Imunológica , Imunoterapia Adotiva , Linfócitos do Interstício Tumoral/transplante , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias da Próstata/terapia , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: This study assessed the Granzyme B (GrB) ELISPOT as a viable alternative to the 51Cr-release assay for measuring cytotoxic activity of innate immune effector cells. We strategically selected the GrB ELISPOT assay because GrB is a hallmark effector molecule of cell-mediated destruction of target cells. METHODS: We optimized the GrB ELISPOT assay using the human-derived TALL-104 cytotoxic cell line as effectors against K562 target cells. Titration studies were performed to assess whether the ELISPOT assay could accurately enumerate the number of GrB-secreting effector cells. TALL-104 were treated with various secretion inhibitors and utilized in the GrB ELISPOT to determine if GrB measured in the ELISPOT was due to degranulation of effector cells. Additionally, CD107a expression on effector cells after effector-target interaction was utilized to further confirm the mechanism of GrB release by TALL-104 and lymphokine-activated killer (LAK) cells. Direct comparisons between the GrB ELISPOT, the IFN-gamma ELISPOT and the standard 51Cr-release assays were made using human LAK cells. RESULTS: Titration studies demonstrated a strong correlation between the number of TALL-104 and LAK effector cells and the number of GrB spots per well. GrB secretion was detectable within 10 min of effector-target contact with optimal secretion observed at 3-4 h; in contrast, optimal IFN-gamma secretion was not observed until 24 h. The protein secretion inhibitor, brefeldin A, did not inhibit the release of GrB but did abrogate IFN-gamma production by TALL-104 cells. GrB secretion was abrogated by BAPTA-AM (1,2-bis-(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid tetra(acetoxymethyl) ester), which sequesters intracellular Ca2+, thereby preventing degranulation. The number of effector cells expressing the degranulation associated glycoprotein CD107a increased after interaction with target cells and correlated with the stimulated release of GrB measured in the ELISPOT assay. CONCLUSIONS: Because of its high sensitivity and ability to estimate cytotoxic effector cell frequency, the GrB ELISPOT assay is a viable alternative to the 51Cr-release assay to measure MHC non-restricted cytotoxic activity of innate immune cells. Compared to the IFN-gamma ELISPOT assay, the GrB ELISPOT may be a more direct measure of cytotoxic cell activity. Because GrB is one of the primary effector molecules in natural killer (NK) cell-mediated killing, detection and enumeration of GrB secreting effector cells can provide valuable insight with regards to innate immunological responses.
RESUMO
BACKGROUND: The desired outcome of cancer vaccination is to induce a potent T cell response which can specifically recognize and eliminate autologous tumor cells in vivo. Accordingly, immunological assays that demonstrate recognition of native tumor cells (tumor-specific) may be more clinically relevant than assays that demonstrate recognition of tumor protein or peptide (antigen-specific). METHODS: Towards this goal, we adapted the IFN-gamma ELISPOT assay to measure immune responses against autologous primary tumor cells in vaccinated cancer patients. As a model system to develop the assay, we utilized peripheral blood mononuclear cells (PBMC) directly isolated from follicular lymphoma patients vaccinated with tumor-derived idiotype protein. RESULTS: After optimizing several variables, we demonstrated that the modified IFN-gamma ELISPOT assay could be used to reliably and reproducibly determine the tumor-reactive T cell frequency in the PBMC of these patients. The precursor frequency of tumor-reactive T cells was significantly higher in the postvaccine PBMC, compared with prevaccine samples in all patients tested. Furthermore, the specificity of these T cells was established by the lack of reactivity against autologous normal B cells. CONCLUSIONS: These results demonstrate the feasibility of quantitating tumor-specific T cell responses when autologous, primary tumor cells are available as targets.
RESUMO
The Society for Immunotherapy of Cancer (SITC) 2012 Professional Development Session was held as part of the SITC 27th Annual Meeting, Washington, DC, on October 24, 2012. The session was designed as a new opportunity for early career investigators to learn about relevant career development topics in a didactic setting.
RESUMO
In this study, we evaluated the applicability of a flow cytometry-based cytotoxicity (FC) assay previously developed by our laboratory, for monitoring cancer vaccine trials. The assay simultaneously measures effector cell degranulation and target cell death. Clinically relevant samples consisted of frozen peripheral blood mononuclear cells (PBMC) from vaccinated melanoma patients with known response to the melanoma peptide g209. Both PBMC and 7 day in vitro-stimulated lymphocyte from patient samples were used as effector cells in the FC assay. Activity against the relevant g209 and the control g280 peptide measured in the FC assay was directly compared with results obtained from the Granzyme B enzyme-linked immunosorbent spot assay and the standard 51Cr-release assay run in tandem. The FC assay detected low or no activity when PBMC were used as effector cells. Using cytotoxic T lymphocytes as effector cells, little or no effector cell degranulation or cytotoxicity was measured in the FC assay in prevaccination samples. After vaccination, an increase in both degranulation and target cell death could be determined when target cells were pulsed with g209. No or low reactivity was found against g280 at any time point. Our findings exhibited excellent correlation between CD107a expression and GrB secretion and also Annexin V binding to target cells and specific lysis measured in the 51Cr-release assay. Results obtained from the FC assay were highly reproducible. Therefore, the FC assay may be applied to vaccine trial monitoring and allows the measurement of effector cell degranulation and target cell death simultaneously in a single sample.
Assuntos
Vacinas Anticâncer/uso terapêutico , Testes Imunológicos de Citotoxicidade , Citometria de Fluxo/métodos , Melanoma/terapia , Monitorização Imunológica/métodos , Neoplasias Cutâneas/terapia , Vacinas Anticâncer/imunologia , Degranulação Celular/imunologia , Ensaios Clínicos como Assunto , Citotoxicidade Imunológica , Ensaio de Imunoadsorção Enzimática , Granzimas/imunologia , Humanos , Melanoma/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Neoplasias Cutâneas/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
We reported previously that tumor-specific CD8(+) T cells (TcR-I) become tolerant in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. In this study, we show that CD4(+) TcR transgenic (TcR-II) T cells transferred into TRAMP mice became activated in lymph nodes, trafficked to the prostate, and initially functioned as T(H)1 cells. Although a single cotransfer of TcR-II cells delayed TcR-I cell tolerization, repeated transfer of TcR-II cells was required to prevent TcR-I cell tolerization and significantly slowed progression of TRAMP prostate tumors. After transfer of TcR-II cells, dendritic cells within the tumor expressed higher levels of costimulatory molecules and displayed an enhanced ability to stimulate proliferation of naive T cells. Blockade of CD40-CD40L interactions during TcR-II transfer resulted in a profound reduction in dendritic cell stimulatory capacity and a partial loss of TcR-I effector functions and tumor immunity. These data show that sustained provision of activated tumor-specific CD4(+) T cells alters the immunosuppressive tumor microenvironment, ultimately leading to the control of tumor growth. These findings will assist in the design of more effective immunotherapeutic approaches for cancer.
Assuntos
Adenocarcinoma/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Neoplasias da Próstata/imunologia , Adenocarcinoma/terapia , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos CD40/antagonistas & inibidores , Antígenos CD40/imunologia , Ligante de CD40/antagonistas & inibidores , Ligante de CD40/imunologia , Tolerância Imunológica , Imunoterapia Adotiva , Linfonodos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Neoplasias da Próstata/terapiaRESUMO
Comprehensive evaluation of cell-mediated cytotoxicity is very important, especially in the clinical setting, when a surrogate immunologic endpoint that correlates with a clinical outcome needs to be defined. With the objective of simultaneously evaluating target cell death and effector cell frequency, the authors combined the measuring of the expression of the degranulation marker CD107a by effector cells with the apoptosis marker annexin V binding to target cells. Using human cytotoxic T lymphocytes, the authors found a significant incubation time-dependent increase of surface CD107a expression on effector cells due to degranulation. A parallel increase of annexin V binding to target cells due to target cell apoptosis was also found. These two parameters have shown excellent correlation in all effector/target cell systems studied. To find possible connections between effector cell degranulation (CD107a expression), granzyme B secretion, and target cell death (annexin V binding), the GrB ELISPOT assay and flow cytometric assay were performed and excellent cross-correlation was found between all three parameters. The specificity of the assay was also shown. These data show that this novel assay allows measurement of cytolytic cell activation and frequency as well as target cell death in the same sample.