Immunological Monitoring Laboratory (IML)

Services

Organization of the IML

To meet widely diverse service demands, the IML is organized into seven distinct units. Each unit of the IML is designated to one area of responsibility as follows:

(a) Specimen intake/processing
(b) Cytokine laboratory
(c) Cytotoxicity laboratory
(d) Flow cytometry
(e) Tissue culture
(f) Data entry and analysis
(g) Research and development

These units are located next to each other in Suite 1.26 at the Hillman Cancer Center (HCC). The IML shares an expanded laboratory suite with the Cellular Products Laboratory (CPL), and both facilities occupy about 4,000 net square feet of space. The IML is also located close to the UPCI Outpatient Services and most of its users.

A close relationship has been developed over the years between the IML and the UPCI Biostatistics Facility. The IML, working closely with the statisticians, has developed a number of programs for data analysis, including an improved lytic unit transformation program as well as curve-fitting programs for cytokine assays. A jointly written program provides for data verification and checking of outlier results prior to final data analysis for each protocol. The data generated by the IML are computerized, checked for accuracy, and made available to biostatisticians for analysis. Similarly, close interactions have developed with the UPCI Cancer Informatics Services, which is actively involved in developing and validating new software in support of IML activities.

Assays Available in the IML

The laboratory now performs over 35,000 assays per year. Of these, approximately 15,000 are performed on cells freshly separated from peripheral blood or body fluids. The remaining assays are performed on samples cryopreserved by controlled-rate freezing and thawed cells. Whenever possible, the samples tests are batched and tested in one assay to minimize inter-assay variability.

Tests are routinely available for assessing lymphocyte functions, including cytotoxicity (NK cell, CTL, LAK, and ADCC); proliferation microassays (responses to lectins, antigens, superantigens, MLC, MLTC); cytokine production and serum levels by bioassays and immunoassays (ELISA and a multi-plexing Luminex technology) for a variety of cytokines, chemokines, growth factors and growth factor receptors; assays for signaling defects, e.g., expression of the zeta chain in T or NK cells; a range of assays for apoptosis (TUNEL, JAM assay, Annexin V binging, caspase activation); and multi-color flow cytometry analysis for an extensive array of mononuclear cell (MNC) surface markers as well as intracellular proteins in permeabilized MNC or tissue cells (including signal transduction pathway molecules by phosphor-protein flow cytometry). Absolute numbers of lymphocyte subsets are determined by a single-platform flow-based method. Assays for antibody responses to proteins and viruses are preformed. Assays for monocyte and granulocyte functions include cytolysis, H2O2 production, and production of monokines. Single-cell assays routinely available for quantification of antigen-specific T cells include ELISPOT for IFN-γ, IL-2, IL-5, and granzyme B; cytokine flow cytometry (CFC); and tetramer binding. IML tests for detection of contamination by mycoplasma or endotoxin.

To monitor patients treated with anti-tumor vaccines, including tumor peptides, dendritic cells +/- peptides, genetically-modified tumor cells, etc., the IML has developed and has routinely available a panel of assays as follows:

  1. Flow cytometry screen for MHC class I antigens A2 and DR4.
  2. Mixed lymphocyte-tumor cultures (MLTC) to generate T cells from peripheral blood, tumor- infiltrating lymphocyte (TIL), or other body fluids and sites.
  3. Cytotoxicity assays of various types, including standard 4h 51Cr-release assays against tumor cell targets +/- blocking with mAbs, FLOCA (flow cytometry cytotoxicity), and CD107a release.
  4. Proliferation assays with T cells responding to antigens (3H-thymidine uptake and CFSE dilution), including multiple rounds of in vitro sensitization (IVS).
  5. Phenotypic characterization of CD4+ and CD8+, T, DC, NK, NK/T, B, and Treg cells by multi-color flow cytometry.
  6. Cytokine assays, including body fluid levels, production, mRNA, and protein levels, especially for changes in Th1 and Th2 cytokine expression.
  7. ELISPOT, CFC, and tetramer assays for determining changes in the frequency of antigen-specific T cells capable of binding or functionally responding to vaccinating peptides or antigens by secretion of IFN-γ or IL-5 (ELISPOT) or by expression of cytokines (CFC).
  8. Evaluation of expression of signaling molecules in T cells by intracytoplasmic staining and flow cytometry.
  9. Determinations of the frequency of T cells binding Annexin V, and thus undergoing apoptosis in the peripheral circulation, before and after vaccination therapy.
  10. Development of autologous EBV cell lines from PBMC (on request).
  11. Titers of anti-tumor antibodies to a selected number of tumor-associated antigens (TAA).

The Facility has the ability to process large volumes of blood (up to 100 ml per patient) as well as leukapheresis products: it cryopreserves (in a rate-controlled manner) and banks patients' cells for later simultaneous testing of sequentially-collected specimens. Flow cytometry is performed on whole blood or separated cells of various types, including tumor cells.

A tissue culture facility is maintained to assure a supply of cell lines for cytotoxicity and other assays. The Laboratory also is responsible for shipping samples collected from patients on the UPCI protocols for assays not performed at this institution and for ECOG-sponsored protocols for assays performed at other participating institutions.