Natural Killer cells (NK) and Cytotoxic T Lymphocytes (CTL) are classes of effector lymphocytes able to kill infected cells. Although NK and CTL use different receptor to recognize their target cell, they all contribute through similar mechanisms to destroy the target cell that has been recognized.

NK cells comprise 5-15% of human peripheral blood lymphocytes and are characterized by the expression of the CD56 surface antigen and the lack of CD3. They also express CD16 (FcgRII) a receptor molecule that specifically binds the Fc part of an antibody, by which they mediate antibody-dependent cellular cytoxicity. Indeed NK cells have both cytotoxicity and cytokine-producing effector functions and they are involved in early defenses against both allogeneic (nonself) cells and autologous cells undergoing various forms of stress, such as infection with viruses, bacteria, or parasites and malignant transformation. The cytockines production contributes to initiation of the antigen-specific immune response, making NK cells an important link between innate and adaptive immunity.
NK cell function is regulated by a balance between activating and inhibiting receptor signals. Several types of inhibitory NK cell receptors recognize MHC class I molecules on target cells and prevent NK cell cytotoxicity toward normal cells. Downregulation of MHC class I molecules on target cells may lead to NK cell Vmediated lysis. The process of cell target recognition is due to specific steps consisting in cell-cell contact, adhesion, formation of an immunological synapse involving specific activation receptors, granule polarization and exocytosis and target cell detachment.
In particular the mechanism of NK cells killing is the same as that used by the CTL generated in the adaptive immune response: they release cytotoxic granules on the surface of the bound target cell and the effector proteins they contain penetrate the cell membrane and induce programmed cell death. The major constituent of these granules is perforin, a cytotoxic protein able to polimerize and form transmembrane pores in target cell membrane allowing water and salts to pass into the cell that rapidly die. The other class of cytotoxic proteins are granzimes, serine proteases which activate caspase cascade and apoptosis once in the cytoplasm of the target cells. The core of lytic granules is surrounded by a lipid bilayer that contains Fas ligand and lysosomal-associated membrane glycoproteins (LAMPs). Degranulation by cytotoxic cells results in depletion of intracellular perforin and LAMP-1(CD107a) appearance at the cell surface.

NK cell deficiency states are associated with a wide range of diseases and infection susceptibility. The deficiency can be due to absence of NK cells or absence of NK cells activity.
Analysis of NK cell population by flow cytometry will overlook the majority of NK cell deficiency states. Assessment of NK cell cytotoxicity, antibody-dependent cellular cytoxicity (ADCC), cytokine responsiveness, NK cell subject phenotype and cytokine production will provide a more complete understanding of a patients NK cell status.

A number of gene defects are associated with an impairment of NK cells cytotoxicity, and are responsible of familiar hemophagocytc lymphohistiocytosis (FHL). Defined causes include defects in proteins linked to the pathway of granule-mediated cytotoxicity: perforin (FHL2); Munc13-4 (FHL3) involved in priming of the secretory granules and their infusion into plasma cell membrane; syntaxin-11 (FHL4) involved in regulating intracellular protein transport between donor and target cell; Munc18-2 (FHL5) involved in the regulation of vescicle transport to the plasma membrane by the interaction with syntaxin 11 .
Sensitive flow-based screening assays have been developed for all subtypes. Intracellular staining for perforin focused on cytotoxic lymphocytes (CD8 T cells and
NK) can accurately identify patients with PRF1 (perforin) mutations as well as carriers.
NK cell cytoxicity is a functional immunologic parameter that has been commonly evaluated, however the assay has several limitation. The classical protocol to study NK cytotoxicity is labour intensive, usually involves radioactivity and is not widely available. It is based on testing polyclonal NK-cell populations in a 51Cr-release assay for cytolytic activity against the human myeloid leukemia cell line K562, characterized by the absence of MHC expression. Briefly, K562 are labeled with 51Cr- and plated with various concentration of patient PBMCs, the co-colture are incubated for 4hr at 37 aC and the supernatants are collected to evaluate 51Cr release by gamma scintillation The percentage of lysed target cells is proportional to 51Cr release by target cells.
Recently a new assay was set up to evaluated NK and CTL function. It is based on measurement of cell degranulation through quantification CD107a surface expression by NK and CTL using flow cytometry. Diminished expression of CD107a by NK cells following stimulation and degranulation has been shown to predict defects in the genes encoding Munc 13-4 or syntaxin 11 and Munc18-2, providing a useful and rapid screening tool for FHL3 and FHL4 and FHL5. Two variants of this method can be identified, one to them is based on co-colture on cytotoxic cells with cell line target cell and subsequently evaluation of CD107a expression, the second assesses CD107a expression on IL2-activated PBMcs after stimulation with PHA or anti-CD3. The latter protocol has the advantage that can be easily performed in most routine immunology and hematology laboratory as no cell lines and radioactivity are required.

Moreover other genetic conditions may cause a clinical syndrome that largely overlaps with hemophagocytc lymphohistiocytosis, although they have some additional and specific features.
Griscelli Sindrome Type 2 (GS2), Chediak-Higashi Sydrome (CHS), and Hermansky-Pudlak syndrome type II, caused by muntation in RAB27A, LYST and AP3B1 respectively, display impaired cytotoxic activity.
X linked lymphoproliferative disease (XLP) it is caused by mutation in SH2D1A encoding the protein signaling lymphocyte activation molecule-associated protein (SAP) and BIRC4, the gene encoding for X-linked inhibitor of apoptosis (XIAP). In this case an overt defect in lymphocyte cytotoxicity, however intracytoplasmatic staining for SAP and XIAP can provide a rapid diagnosis.

Sara Ciullini Mannurita

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REFERENCES

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Kogawa K, Lee SM, Villanueva J, et al. Perforin expression in cytotoxic lymphocytes from patients with hemophagocytic lymphohistiocytosis and their family members. Blood (2002) 99:61-66.

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Wheeler RD, Cale CM, Cetica V, Arico' M, Gilmour KC. A novel assay for investigation of suspected familiar hemophagocytic lymphohisticytosis. Br J Haematol. (2010) 150:700-730.

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