Cytolytic immunological synapse (SMAC)
To deliver a targeted strike, an NK cell forms a cytotoxic immunological synapse with the target. This is a tight contact that forms if the target lacks inhibitory molecules (MHC-I) and the killer cell recognizes a distress signal.
- Adhesion: the contact is stabilized by $\beta_2$-integrins (LFA-1, Mac-1) and the CD2 molecule on the killer cell, which bind to target receptors (ICAM-1, ICAM-2, CD58).
- Polarization: with the help of the protein talin and F-actin filaments, the killer cell's cytoskeleton rearranges. Microtubules direct towards the synapse so that lethal granules can move along them like tracks toward the contact zone.
- Rafts: lipid rafts (membrane microdomains rich in cholesterol) bearing signaling enzymes and adaptor proteins (PLC$\gamma$, LAT, Lck) cluster into the synapse zone.
Mechanisms of target destruction: how granules work
The primary killing mechanism is perforin-dependent. A microcavity forms in the center of the synapse, where the killer cell dumps the contents of its granules. This ensures that only the target dies while neighboring healthy tissues remain unharmed.
- Membrane disruption: in the presence of calcium ions, perforin polymerizes in the target membrane, forming a wide pore.
- Apoptosis induction: granzyme B enters the cell through the pore. This serine protease directly activates executioner caspase-3 and targets mitochondria via the Bid factor.
- Mitochondrial catastrophe: the enzyme granulysin cleaves sphingomyelin into ceramide. Ceramide disrupts the target's mitochondria, releasing cytochrome c into the cytosol.
Result: the cell rapidly "shrivels", its DNA fragments, and it dies via apoptosis without inflammation. The remains are phagocytosed.
Decision making: activation and inhibition
An NK cell's behavior depends on the balance of signals:
- Activation (e.g., via NKG2D): the receptor binds stress ligands, recruits DAP-10 proteins, and triggers the PI3K kinase cascade. Calcium levels rise inside the cell, turning on transcription factors that lead to degranulation or cytokine release.
- Inhibition: if the killer cell detects normal MHC-I molecules on the target, its inhibitory receptors (KIR) phosphorylate the ITIM motif. This recruits SHP1/SHP2 phosphatases, which dephosphorylate the Vav-1 protein. The synapse disassembles within minutes, and the target survives.
Antibody-dependent cellular cytotoxicity (ADCC)
NK cells can cooperate with humoral immunity. If a target cell is coated with antibodies (opsonized), the killer cell recognizes them via its Fc receptors (Fc$\gamma$RIII or CD16).
Binding to IgG1 or IgG3 immunoglobulin fragments triggers a strong activating signal via ITAM motifs and Src kinases. This mechanism is utilized by mature killers with the CD56$^{\text{dim}}$ CD16$^{++}$ phenotype, and it drastically increases tissue clearance efficiency.
Briefly: answers to topical questions
What are the structural and functional differences between CD56$^{\text{bright}}$ and CD56$^{\text{dim}}$ NK cell subpopulations?
- CD56$^{\text{bright}}$ is an NK cell subpopulation that synthesizes cytokines upon activation. These cells express CD94/NKG2 group receptors.
- CD56$^{\text{dim}}$ is an NK cell subpopulation characterized by the development of a cytotoxic response. Mature NK cells of this subpopulation express KIR receptors and the CD56$^{\text{dim}}$ CD16$^{++}$ (or CD56$^{\text{dim}}$ CD16$^{\text{lo}}$) phenotype, demonstrating high readiness for cytolysis.
Which cytokines activate NK cells and induce the formation of LAK cells?
- IL-12 is a cytokine produced in response to viral infection, which enhances the readiness of NK cells for a cytotoxic response even before contact with targets.
- IL-2 is a cytokine whose prolonged cultivation (for 5–6 days) induces the formation of LAK cells (lymphokine-activated killers), which are IL-2-stimulated natural killers.
What is the role of DAP-10 adaptor proteins and PI3K signaling pathways in NKG2D activation?
- DAP-10 adaptor protein is non-covalently linked to the NKG2D receptor (2 DAP-10 proteins per 1 receptor) and contains an YXXM activation motif in its cytoplasmic portion, which is phosphorylated by Src family tyrosine kinases.
- PI3K signaling pathway transmits signals from the NKG2D receptor via a multimolecular complex (including 2 NKG2D molecules, 4 DAP-10 molecules, the p85 subunit of PI3K, the Grb2 adaptor protein, and Vav1), activating PI3K, PLC$\gamma$, and Rho family GTPases.
Remember the three "P"s of granule contents: Perforin Pokes the membrane, Granzyme Grants death (activates caspases), and Granulysin Grounds (destroys) mitochondria.
Class and exam questions
Why are freshly isolated tumors often not recognized by NK cells in vitro?
They often retain MHC-I molecules on their membrane (inhibitory signal) and fail to accumulate enough stress molecules to activate the killer cell.
Do natural killers destroy their targets via necrosis?
No, contact-mediated cytolysis by NK cells (stage 4) is always carried out via apoptosis. The cell is neatly fragmented without releasing toxic contents outside.
What is the alternative pathway of contact-mediated cytolysis?
This is the induction of apoptosis via membrane death receptors (FasL, TRAIL on the killer cell and Fas, DR4/5 on the target). Unlike in cytotoxic T lymphocytes, this pathway is secondary for NK cells.
What else to study on this topic
- Stages of multimolecular complex formation with the DAP-10 protein
- The role of kinases (Fyn, Lck, Syk, ZAP-70) in lipid rafts of the immunological synapse
- Regulation and return of the cell to a resting state via the SHIP1 phosphatase
- Differences in responses between CD56$^{\text{dim}}$ and CD56$^{\text{bright}}$ subpopulations
- Mechanism of LAK cell activation by interleukin-2
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