Down the Pipeline
CD107a: Turning Cytotoxicity Inside-Out
In the last Newsletter, a flow cytometric (FCM) assay to measure cytotoxicity was introduced (granzyme B transfer assay). This assay is based on transfer of granzyme B from effector cell to target cell. Because of the multi-parameter capabilities of the FCM platform, the assay lends itself to measure more than just killing of target cells.
Measuring NK-cell function is an integral component in the evaluation of hemophagocytic disorders (e.g. HLH). Insight into the cytotoxic machinery can guide the diagnostic workup and can help in formulating the search for genetic disorders. To this effect, functional assays are commonly combined with the perforin/granzyme B assay.
Originally developed to screen for genetic defects in the gene encoding perforin (PRF1), the combination of measuring intracellular perforin as well as intracellular granzyme B has provided additional profiles that appear characteristic of HLH scenarios. Thus, reduced presence of perforin in NKcells and T-cell subsets is characteristic of perforin mutations, while increased expression of perforin and granzyme B is observed in other forms of HLH including primary inherited forms due to mutations in the gene encoding MUNC13-4, as well as secondary causes. To distinguish between these different scenarios, in the next phase of the cytotoxicity assay makeover, the screening assay will be expanded to link perforin/granzyme B profiles more directly to MUNC13-4 mutations. It is anticipated that after a period of testing and evaluation, phenotypic and functional assays of cytotoxicity will be consolidated into a single platform (FCM) method.
Mutations in MUNC13-4 account for approximately 25% of familial forms of HLH (compare with ~35% perforin for mutations). MUNC13-4 is involved in priming of the secretory lysosomes (referred to as lytic granules in cytotoxic lymphocytes, such as NK cells). Specifically, it facilitates exocytosis of these granules (containing perforin and granzyme B), such that they can engage and interact with the target cells. During this process of exocytosis, the granules fuse with the membrane of the cytotoxic cell. This feature has been exploited in an assay that measures mobilization (incorporation) of the lysosome-associated membrane-protein 1 (LAMP-1; also known as CD107a) into the cell membrane.
The basic principle of the assay involves incubating NK cells with the appropriate target cells (e.g. K562 cells) for a defined period of time and measuring CD107a expression on the surface of NK cells. Upregulation (mobilization) of CD107a is then compared to a baseline (un-stimulated) status. Due to the fact that the process of granule exocytosis does not occur in the context of defective MUNC13-4, measuring CD107a mobilization would be a useful assay to screen for this familial form of HLH.
Figure 3 shows four histograms to illustrate the assay. The histograms reflect CD107a expression on NKcells, as determined by CD56 expression and lack of CD3 expression. The perforin/granzyme B assay in this patient revealed up-regulated expression of perforin and granzyme B in both NK cells, as well as CD8-positive T cells, while NK-cell function was decreased. Genetic analysis showed biallelic mutations in MUNC13-4.
Due to the fact that this assay is in essence a NK-cell function test, measuring CD107a mobilization, can be combined with the granzyme B transfer assay. Similar to other screening assays, it is anticipated that interesting patterns of CD107a expression will become apparent in a variety of clinical scenarios beyond its original intent as a screening tool for MUNC13-4 mutations. Once combined with the Granzyme B transfer assay, these profiles may point to novel defects in cytotoxicity, and perhaps clear a pathway to the discovery of other genetic defects in HLH (keeping in mind that a large proportion of cases of familial HLH currently lack a genetic diagnosis).
These observations may invite new questions and perhaps another round of Cytotoxicity Makeover in the future.
