Immune Monitoring

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.

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.

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.

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. 

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. 

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.