Immunology services for the development
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The specific or adaptive immune system comprises T and B lymphocytes. FOCUS Immunology provides assays to analyze the activity of these cells.

Cytokine secretion or proliferation is assessed in bulk cultures, or alternatively at the single cell level by single cell analysis techniques. The latter techniques offered by FOCUS Immunology are EliSpot or ICS (Intracellular Cytokine Staining) to analyze cytokine secretion on the single cell level or the flow cytometry-based CFSE assay to determine proliferation of individual T or B cells.

Prior to analysis the cells may be stimulated polyclonally by using PHA, PMA + Ionomycin, or antibodies directed against CD3 and CD28. Or the cells may be stimulated in an antigen-specific manner by using CMV (Cytomegalovirus) antigens and Tetanus Toxoid.

For many of the assays it is possible to select different cell populations to start with such as whole blood, purified peripheral blood mononuclear cells (PBMC), or selected lymphoid subpopulations such as B cells, T cells, CD4+cells or regulatory T cells and so on.

Specifically, FOCUS Immunology performs the following tests to determine effects of compounds on cells of the specific immune system:

(i) Proliferation assays to determine the proliferation rate of T or B lymphocytes. Upon antigen-specific or non-specific stimulation, cell proliferation will be determined either in bulk cultures by metabolic assays (MTS or ATP-dependent) or by measuring DNA synthesis (BrdU incorporation).

Alternatively, cell proliferation may be determined on the single cell level using the CFSE assay. Hereby, cells are labelled with CFDA-SE (carboxyfluorescein diacetate succinimidyl ester), a cell-permeable, non-fluorescent compound, which is converted by intracellular esterases to the fluorescent and non-membran-permeable CFSE (carboxyfluorescein succinimidyl ester). This molecule is highly stable and is distributed during cell division evenly to the daughter generations. Thus, flow cytometry allows to track proliferation of stimulated lymphocytes through consecutive generations.

(ii) Flow cytometry assays to analyze functional states of lymphocytes. T and B lymphocytes are not only characterized by lineage-specific markers such as CD3 for T cells and CD19 or CD20 for B cells but also by a continuously growing panel to distinguish subpopulations. CD4 and CD8 for helper and cytotoxic T cells and CD4,CD25 for regulatory T cells are just a few examples.

Additionally, cells of the lymphoid lineage acquire specific activation markers upon stimulation, which are not expressed on resting cells. Among these are CD69, CD25 and CD86.

A combination of lineage and activation markers allows analyzing the functional states of lymphoid cells on the single cell level, which thus allows analyzing the effects test-compounds on the functionality of lymphoid subpopulation.

FOCUS Immunology uses a dual-laser FACSCalibur from BD Biosciences to analyze up to four different markers in parallel allowing a detailed analysis of cell surface and/or intracellular markers.

(iii) Analysis of cytokine induction. Upon antigen-specific or non-specific stimulation cytokine production will be measured using either of three assays:

(a) ELISA, to determine the total amount of cytokine(s) secreted into the cell culture medium,

(b) EliSpot, to determine the frequency of cells secreting (a) particular cytokine(s), and

(c) Intracellular Cytokine Staining (ICS), to measure multiple cytokines and correlate their expression on the single cell level with lineage markers.