Immune Monitoring Core Facility
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Keck Medical Center of USC
BCIM Services and Assays
Apheresis and blood/serum processing
A very convenient service of the Immune Monitoring Core is the standardized processing and isolation of peripheral blood lymphocytes or serum from tubes of blood or bags from apheresed normal donors or patients. For some studies, isolation and freezing of PBMC is performed for analysis at a later date, for example, when multiple time points are collected from one patient over the course of a trial. For other studies, fresh PBMC are used in functional, descriptive, or molecular assays. PBMC are stored in the vapor phase of large capacity liquid nitrogen tanks. Serum or plasma samples are stored in -80° C ultra-low freezers.
This assay is used to quantify the number of cells in your sample secreting a particular cytokine. It is most commonly used to estimate the proportion of T cells reactive with a particular antigen, since responder cells will secrete IFN-g when encountering their antigen. The IMC can currently evaluate T cell responses to either recall antigens to monitor general immune responsiveness or to a specific antigen of your choice using overlapping sets of peptides. The assay can be performed using whole blood samples taken from cancer patients before, during, and after treatment by immunotherapy. Antibody-coated culture wells are used to plate the responder cells. After in vitro stimulation, captured cytokine is detected by another, enzymatically labeled antibody. Colored spots in the well represent individual cytokine secreting cells. In dual color ELISPOT assays, two different cytokines are detected simultaneously using separate enzymatic labels. The IMC can perform the ELISPOT assay for you, or you can come in with your stained plates to assess the spot number using our state-of-the-art Zeiss ELISPOT analyzer.
MHC Tetramer analysis is used to quantify the number of T cells within a given sample that can recognize a particular antigenic epitope based upon expression of a particular T cell receptor. Antigenic epitopes, particularly those presented in the context of HLA-A2 molecules, are rapidly being identified. Using antigenic peptides bound to four (MHC tetramer) or five (MHC pentamer) copies of the HLA molecule linked to a fluorescent marker, a tool has been created to label T cells responsive to an antigen of interest within your sample. Fluorescently-labeled tetramer-positive cells are detected and quantified using flow cytometry. Cells can also be labeled with fluorescent antibodies to cell surface markers to identify phenotypic markers.
Characterization of immune cell activation and effector phenotypes is an important measure of responses to immunotherapy. Flow cytometry is used to detect and quantify cells stained with fluorescently labeled antibodies. Markers of cellular differentiation and homing status can also be measured. Another method that requires flow cytometric analysis and is frequently used in immune monitoring is intracellular cytokine staining, based on the principle, that immunocytes will generate cytokines upon activation. Thus, we can look for T cells responsive to a peptide of interest by quantification of cytokine producing cells before and after addition of the antigen. In this case we are looking for cytokine molecules that have not yet been secreted by the cells. General markers of immune activation can also be used to assess immune responses in patients.
Cytokine measurements are performed either through traditional ELISA assays or on a Bio-Rad Bio-plex System using the Luminex xMAP® technology. The Luminex xMAP® technology allows for the measurement of up to 100 distinct proteins through a suspension bead array that employs a series of distinct color-coded microspheres to simultaneously detect multiple soluble analytes from a single serum, plasma, tissue culture supernatant, or other bodily fluid sample. Biomarker panels for obesity, cardiovascular disease, cancer, endocrinology, cell signaling, bone metabolism, and immunoglobulin isotyping are also available in addition to a wide selection of cytokines and chemokines. The system uses very small sample volumes and delivers fast and cost-effective results when analyzing multiple proteins. Please contact us to discuss which cytokines can be measured simultaneously and for recommendations for sample preparation.
Lymphocyte functional assays
T cell proliferation in response to an antigen of interest is considered a functional marker for immune reactivity. We measure proliferation by traditional tritiated thymidine incorporation assays, where radiolabeled nucleotides are built into newly synthesized DNA in the S phase of mitosis. Effector functions are measured by looking for actual cytotoxic activity towards target cells of interest. This important parameter of immune reactivity provides a close measure of tumor shrinking ability in cancer patients. Release of chromium by dying radiolabeled target cells serves as a measure of cytotoxicity, as mediated by specific CD8 T cells within your sample. CFSE dye-based proliferation assays can also be performed in conjunction with flow cytometry-based immunophenotyping.
We perform molecular biology assays from nucleic acids (DNA or RNA) extracted from tumor biopsies. Human papillomavirus genotyping is performed using the Roche Linear Array HPV Genotyping and detection kit which is based on amplification of target DNA by PCR and nucleic acid hybridization to probes representing thirty-seven anogenital HPV genotypes. HPV viral load studies can be performed by quantitative real-time PCR. Messenger RNA extracted from tumor biopsies can be analyzed by quantitative RT-PCR for specific transcripts, such as cytokines, cellular markers, or regulatory T cell markers.
Our order form page contains a complete listing of services we currently offer. We also offer custom assay development and optimization prior to study initiation based upon study needs and budget. Experiments can be performed as a one-time service or multiple services such as in a long-term clinical trial. If you have an assay in mind that was not described here, please call us or send us an e-mail to discuss the possibilities.