The Immune Monitoring Facility, which is part of the Ludwig Center for Cancer Immunotherapy, is dedicated to providing laboratory services and translational research to support immune monitoring during cancer immunotherapy. Robust immune monitoring assays are essential for characterizing the immune status in patients receiving novel immune-modulating therapies, and the insights gained from studying the immune response in treated patients may help transition promising therapies from the clinical trial phase to standard of care.
A primary focus of the facility is to develop cutting-edge immune-monitoring technology while also standardizing and validating immune assays with rigorous quality-control standards to ensure data reliability. Our facility is equipped with highly specialized technologies to analyze both cellular and soluble biomarkers, including several multiparameter BD Biosciences LSRFortessa flow cytometers, a Meso Scale Discovery (MSD) multiplex immunoassay reader, and a GenePix protein microarray scanner.
We offer consultation regarding optimal immunologic assessments 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 analysis tailored to fit the endpoints of each specific clinical trial. Proposals will be reviewed and prioritized in order to design the appropriate methodology and ensure projects are carried out efficiently and successfully with available resources.
The Immune Monitoring Facility offers the following services and assays:
Processing of peripheral blood mononuclear cells, serum, or plasma from human whole blood and sample cryopreservation
The Immune Monitoring Facility routinely processes and banks clinical samples from a variety of clinical studies. Typically, whole blood is delivered to the facility and processed under standardized protocols for optimal isolation of peripheral blood mononuclear cells and plasma samples, which are then cryopreserved for batched downstream immunological analyses. The identity and storage location of samples are logged and tracked in an electronic clinical database.
Immunophenotypic analysis of T cell activation/exhaustion by multiparameter flow cytometry
Flow-based assays provide valuable phenotypic information about a patient’s immune response at the single-cell level. We can assess the activation and exhaustion status of different T cell populations within human PBMC including CD4+, CD8+, and regulatory T cells. This data can then be used to identify possible correlates with clinical responses. Further details regarding specific markers on available T cell flow panels in the IMF are available upon request.
Enumeration of myeloid-derived suppressor phenotype cells
This is a flow-based phenotypic assay used to measure cell surface expression of HLA-DR, CD14, and other lineage markers used to describe human myeloid derived suppressor cells. PBMCs are stained with a panel of antibodies conjugated with different fluorochromes to allow identification of the CD14+ myeloid subset of cells that are negative for lineage markers of T, B, and NK cells. The myeloid cells are then evaluated for class II HLA-DR expression. MDSC have been described as the subset of CD14+Lin- cells that are negative or low for HLA-DR expression.
Measurement of antigen-specific T cells and their functionality by intracellular cytokine staining (ICS)
This flow-based assay is used to detect functional T cell responses triggered by antigenic stimulation, as read out by cytokine production (e.g., IFN-γ, TNF-α, IL-2) within individual cells. Stimulation can be performed using specific peptide/protein antigens or nonspecific general activating agents such as anti-CD3 monoclonal antibody or staphylococcal enterotoxin B (SEB). Cells are treated with an inhibitor of the secretory pathway permitting accumulation of cytokines inside the cytoplasm. Cytokine production is determined by staining the fixed and permeabilized cells. By combining the intracellular cytokine stain with staining for phenotypic markers, it is possible to determine the type of cells that produce the cytokine as well as the relative level of cytokine produced per cell. Detection of Ag-specific T cells can also be performed by staining cells with labeled peptide/MHC multimers to enumerate the frequencies directly if the HLA/peptide-specific reagents are available.
Serological assays to detect antibodies to tumor-associated antigens and autoantigens
These assays are used to estimate the titer of antibodies specific to one or more tumor-associated antigens, including cancer-testis (CT) antigens such as NY-ESO-1, in human serum or plasma samples. The IMF has a panel of over 20 verified antigens for which tumor serology ELISA assays can be run, allowing monitoring of potential changes in particular tumor antigen-specific antibody responses during the course of cancer immunotherapy. For a broader seromics evaluation of antibody specificities, samples can also be tested in the IMF using commercially available human protein microarrays that are spotted with over 16,000 full-length human self proteins that cover over 80% of the proteome to identify potential autoantibodies that may arise during immune therapy and which may be associated with clinical response or toxicity.
Single and multiplex cytokine analysis
Commercially available, multi-array immunoassay kits from Meso Scale Discovery can be used for the detection of multiple cytokines in serum, plasma, or tissue culture supernatants by electrochemiluminescence detection technology. Kits consist of plates coated with up to ten different antibodies specific for individual cytokines or other protein analytes, allowing simultaneous detection of multiple biomarkers from a single small volume sample. Typical cytokines assessed include a 10-plex panel that measures IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-α.