Down the Pipeline
A New Look at Cytotoxicity
Phenotypic and functional assays pertaining to cytotoxicity are a main focus of the DIL, especially in the evaluation of hemophagocytic disorders. Insight into the cytotoxic machinery can guide the diagnostic workup and can help in formulating the search for genetic disorders. Our current approach to the study of cytotoxicity combines immunophenotypic studies – including the perforin / granzyme B assay, the SAP assay and the ALPS panel – with functional assessment through the NK-cell assay and the CTL (cytotoxic T-lymphocyte) assay. In addition, several disease markers can be used to help in the measurement of disease activity and response to therapy. They include; soluble IL-2Ralpha chain (sIL-2Rα), soluble CD163, as well as plasma cytokines.
Several new projects are making their way through the R&D pipeline. In this, and the next newsletter, an update will be given regarding 2 projects. When ready for primetime they will become part of the "Menu".
Extreme Makeover: NK-cell Edition.
Cytotoxicity assays, like any functional lymphocyte assay consist of several components. In a simplified scheme, there is the population of cells to be studied (e.g. NKcells, CTLs), the pathway and / or mechanism of its function (e.g. perforin / granzymemediated killing), a defined population of target cells (e.g. K562 cells) and a read-out method to assess the demise of the target cells. Additional complexities can be added to further determine functional characteristics (e.g. HLA blocking antibodies to interfere in MHC-restricted killing).
The component that plays a central role in the proposed Makeover is the read-out system. Flow cytometry (FCM) will be used in parallel with the (gold standard) 51Chromium release (51Cr) assay. A variety of methods can be used to measure target cell killing using the flow cytometry platform (see examples in side bar).
A general principle of the FCM approach is the use of a combination of fluorescent markers; one to identify the target cell population and another to detect [a stage of] cell death. In addition to eliminating any radioactive compound, flow-based methods transform a bulk assay into an assay in which single cells are studied. The number of additional parameters that can be included in the assay, such as a more detailed characterization of the effector cell population or the visualization of conjugate formation between effector and target cells, is essentially only limited by the hardware (and software) used in the laboratory.
In our NK-cell makeover approach, killing is determined by granzyme B (GranToxiLux™ kit by OncoImmunin, Inc.) in the – fluorescence-tagged – target cells (see side box for other measurements of target cell lysis). The basic premise of this method is shown in the figures on the previous page. Upon activation of cytotoxic lymphocytes by appropriate target cells, granzyme B that is present in an inactive form in lysosomal granules, is transferred (accompanied by other granule contents such as perforin) from effector to target cells.
This process constitutes an early event in the execution of cytotoxic function that can quantitatively be measured and, when combined with the traditional 51Cr-release assay, representing the final phase of target cell lysis, provides a "from start to finish" assessment of NK-cell function. So far, we have seen good correlation between the two methods (see figure above). Interestingly, we found some discrepancies in patient samples that could be reflective of a specific issue in NK-cell function. Thus, it may be relevant to also include a FCM read-out system that measures cell death at a stage comparable to 51Cr release.
Befitting the emphasis of the DIL on disorders of hemophagocytosis, the GranToxiLux™ methodology could help in detecting disorders that are directly linked to the intracellular machinery and constituents needed for cytotoxicity. For example, defects in the transfer of granzyme B from lysosomal granules to the NK-cell membrane and subsequently to the target cells, could be detected by the retention of granzyme B in effector cells. More on this topic in the next newsletter (CD107; turning cytotoxic molecules inside-out).
