Immune Monitoring Grants and Publications

Immune Monitoring Grant Descriptions

Short Grant Description

The Immune Monitoring shared resource provides investigators with a broad range of services and expertise where precise evaluations of the immunologic response are required. Resource services include the production of peptide-MHC Class-I tetramers, unique reagents for accessing antigen-specific T cells, molecular assays for T-cell receptor-related analysis, and cellular assays for determining cellular immune functions.
 

Long Grant Description 

The Immune Monitoring shared resource supports investigators with resources and expertise to evaluate cellular immunologic processes and responses. The IM core provides reagents, molecular and cellular assays, assay development support and research consulting services. These include peptide-MHC tetramers, T-cell receptor spectratyping, QPCR tracking assays for TCR, TREC assays, immunophenotyping by flow cytometry, cytotoxic and lymphoproliferation assays and NK/LAK assays. In addition, the core provides instrumentation support and technical trainings.

The IM shared resource is located in the historic Steam Plant Building and occupies approximately 700 square feet of space including the lab, office and an equipment room. All necessary instrumentation required for processing and analysis are provided. Equipment includes a biosafety cabinet, a CO₂ incubator, a FPLC system, an ELISPOT reader, an ELISA reader, a QPCR system, a thermocycler, a TopCount microplate cell counter, a cell harvester, a luminometer, a microplate washer, two larger and two micro-centrifuges, a -80°C freezer, a -20°C freezer/refrigerator combination, two -20°C freezers, a refrigerator, a 4°C deli case, a liquid nitrogen tank, two microscopes, a water bath, a shaker incubator, two balances, a spectrophotometer, a cell counter, and other small instruments, such as pH meters, heat blocks, vortex mixers, stirrers, picofuges and pipettes.

Service Description

Peptide-MHC, or pMHC, tetramers are custom-made reagents which bind to antigen-specific T-cell receptors. Tetramers are produced by refolding of recombinant MHC proteins and designated epitopic peptides. We have the commonly studied MHC Class-I proteins from humans, mice and macaques. We provide monomers, fluorochrome-conjugated tetramers and Qdot-conjugated multimers.

Spectratyping is a set of multiplex RT-PCR reactions that amplifies all T-cell receptor beta families to reveal the clonal length polymorphism in the CDR3 region. This technique is used to access clonal composition in T-cell populations. Specifically, it is often used to determine clonal expansion, contraction and deletion during immune response and reconstitution. TCR spectratyping can be provided for PBMC samples or for FACS-sorted cell subsets.

The unique feature of each T-cell clone is found in the recombined T-cell receptor sequences. The IM core can identify the clonal TCR beta chain sequence by RT-PCR and direct sequencing of the clonal cell sample. The clonal TCRB sequence can be cloned and used for QPCR analysis. With knowledge of the clonal TCR sequence, specific primers and a probe for quantitative real-time PCR analysis can be designed, allowing the quantitation of the clonal T cells in a cell population. We provide this service in the setting of T-cell adoptive transfers in immunotherapy trials for cancer patients.

TREC is a quantitative real-time PCR analysis of the TCR excision circle, a circular DNA fragment excised from the genome as a result of the TCR rearrangement in the thymus. This circular DNA is not capable of replication and is therefore diluted as T cells divide in the periphery. The relative quantity of the TREC DNA provides a measurement of thymic output of naïve T cells.

The lymphoproliferation assay is the classic method for assessing lymphocyte response to stimulation. Lymphocytes proliferate after incubation in vitro with mitogens and specific antigens, or in a mixed lymphocyte reaction, which is then measured by 3H-thymidine incorporation. The rate of proliferation provides an indication for general lymphocyte activity or responsiveness to specific antigens. The Immune Monitoring core has provided this assay for several clinical trials in accordance with specific study requirements.

Cytotoxic T cells, or CTLs, and natural killer, or NK, cells function as their names indicate by killing their target cells, which can be measured by the ⁵¹Cr-release assay. Specific target cells are loaded with chromium-51, which is then released into the medium when the cells are lysed by specific CTL or NK cells. This functional assay is widely used in studies of antigen-specific CTL and NK activity.

Analyzing cell-surface and intracellular proteins by labeling with fluorochrome-conjugated antibodies and flow cytometry is a common method in immunology. Flow cytometric analysis can be used to enumerate cell subsets according to their different markers, activation, function and other characteristics. Specific cell subsets can be sorted out from a large population of cells using a cell sorter. Antigen-specific T cells can be analyzed after labeling with a specific pMHC tetramer. The IM shared resource provides cellular analysis by flow cytometry in accordance with specific study requirements. For example, we have provided complete peripheral lymphocyte phenotyping of B cells, T cells, monocytes, NK cells and dendritic cells for the evaluation of immune reconstitution after stem cell transplant.

The enzyme-linked immune absorbent spot, or ELISpot, assay is a high-throughput procedure to evaluate antigen-specific T-cell responses on a single-cell basis. In this assay, cells that are responding to antigenic stimulation secrete cytokines which are captured locally by antibodies and visualized by staining in the form of spots. Thus, precise enumeration of responsive cells can be made. The IM core provides training and technical support for investigators performing this assay and using the ELISpot reader. We also provide complete assays of clinical cell or blood samples.