The immune monitoring facility, part of the Ludwig Center for Cancer Immunotherapy, is dedicated to cancer immunotherapy and translational research. Alexander Rudensky and Jedd D. Wolchok serve as Director and Associate Director for Clinical Research, respectively. The development of immune monitoring assays is essential to determine the immune responses in patients receiving novel immune therapies and ultimately transitioning these therapies from the clinical trial phase to standard of care. We offer consultation regarding optimal immunologic assessment and assay development for innovative approaches to evaluate immune responses. Our staff will provide expert advice regarding the types of assays for immune monitoring and data interpretation tailored to fit the endpoints of each specific clinical trial.
The ultimate goal of the facility is to continuously develop cutting-edge immune monitoring technology. We are currently participating in a worldwide immune monitoring consortium to standardize and validate immune assays and establish rigorous quality control standards. Our facility is equipped with highly specialized technology such as a 9-color CYAN flow cytometer and an ELISpot reader.
Staff are available for consultation regarding immune monitoring of clinical trials. Proposals will then be reviewed and prioritized by the executive committee in order to design the appropriate methodology to successfully measure the endpoints of the study.
Overview of assays developed, validated, and provided in our center:
- Flow Cytometry for Phenotypic Analysis with FACS
including leukocyte subset analysis, T regulatory cell assays
- Peptide/MHC Tetramer staining
- Enzyme-Linked Immunospot Assay (ELISPOT)
- Intracellular Cytokine Staining (ICS)
- Flow Cytometric Based Cytotoxicity Assays
- Multiplex Cytokine Analysis using BD CBA system
- TCR Vβ Repertoire Analysis using flow cytometry staining
- TCR Vβ LI RT-PCR< using analysis Spectratyping>
Flow Cytometry for Phenotypic Analysis:
Flow-based assays provide valuable information on a patient's immune response. We can assess the immunophenotype and activation status of different cell populations, including CD4+, CD8+, NK, NK T, and regulatory T cells. This technology can be combined with fluorescently labeled MHC class I multimers (tetramer or pentamers) to measure the number of CD4+ or CD8+ T cells that recognize a particular antigenic epitope.
Peptide/MHC Tetramer Staining:
Multimers of peptide/MHC complexes tagged with fluorescent dyes are used as TCR ligands to identify T cells with particular antigenic specificity. This permits detection of individual T cells by flow cytometry. T cell phenotype may also be determined by co-tagging the T cell with tetramer.
Enzyme-Linked Immunospot Assay (ELISPOT):
This assay is used to detect and quantify the number of T cells that secrete a particular cytokine (e.g Interferon-γ) upon recognition of a specific antigen. T cells are cultured with antigen-presenting cells in wells coated with an antibody recognizing a particular cytokine. The coating antibody captures the secreted cytokine and a second cytokine-specific antibody is coupled to a chromogenic substrate and used for detection. Results appear as spots, with each spot corresponding to one cytokine-secreting cell. The number of spots equals the number of cytokine-secreting cells for a specific antigen. However this assay does not determine the amount of cytokine secreted. This assay is highly sensitive (1 cell in 100,000) and can be performed directly ex-vivo using relatively few T cells.
Intracellular Cytokine Staining (ICS):
This assay is used to detect T cell response to specific antigenic stimulation. Stimulation can be performed with mononuclear cells isolated from PBMCs or whole blood. Cells are treated with an inhibitor of the secretory pathway permitting accumulation of cytokines inside the cytoplasm. Cytokine concentration is determined by staining the fixed and permeabilized cells. By combining the intracellular cytokine stain with staining for phenotypic markers and tetramers, it is possible to determine the type of cells that produce the cytokine as well as the concentration of cytokine produced per cell. This assay requires larger sample volumes than the ELISPOT. However, it permits the simultaneous determination of the phenotype of the cytokine-secreting cell as well as the identification of the type of cytokine being produced.
Flow Cytometric Based Cytotoxicity Assays:
This cell-mediated cytotoxicity assay is an alternative to the standard chrominum-51 (51Cr) release assays. Target cells are labeled with the cell tracking dye, CFSE, and incubated with effector cells. 7-AAD, which binds to DNA and can only enter membrane-compromised cells, is used as an indicator of cell death.
Multiplex Cytokine Analysis:
Commercially available kits can be used for the detection of multiple cytokines in serum, plasma or tissue culture supernatants. Kits consist of beads dyed with different concentrations of two fluorophores to generate distinct bead sets. Each bead set is coated with a capture antibody specific for a specific cytokine. A second biotinylated antibody and streptavidin-phycoerythrin (SAV-PE) is used to detect the captured cytokine.
TCR Vβ Repertoire Analysis:
The TCR Vβ repertoire assay is a flow cytometry based assay employing a panel of monoclonal antibodies recognizing approximately 70 percent of known members of the TCR Vβ family. This assay can be combined with staining for other cell surface markers to allow Vâ repertoire analysis on T cell subsets.
TCR Vβ Spectratyping:
Gene expression analysis of the T cell receptor repertoire can provide useful information in evaluating the immune response under a variety of conditions. TCR spectratyping is a multiplex RT-PCR protocol used to analyze T cell repertoire diversity by comparing the relative frequencies of different clonal length products within the CDR3 region of a particular TCR Vβ family. This technique is useful for analysis of clonal compositions in T cell populations, which changes during an immune response and can be dramatically altered in some disease states. This assay provides qualitative information regarding the diversity of the TCR Vβ repertoire, however the flow cytometric Vβ repertoire assay provides quantitative analyses.