Impact of Kindlin-3 in NK Cell-Mediated Anti-Tumor Cytotoxicity and Inflammatory Cytokine Production

Natural Killer (NK) cells are innate lymphocytes that play a central role in anti-viral and anti-tumor responses through direct cytotoxicity and production of inflammatory cytokines.  Tumors can evade T-cell mediated immune-surveillance by down-regulation of the Class I Major Histocompatibility Complex (MHC-I) (Haworth et al, Ped Blood and Cancer, 2015).  However, this lack of ‘self' MHC-I serves as an activation stimulus for NK cells to recognize tumor cells.  The molecular mechanism for ‘self' recognition and destruction of ‘missing self' are poorly understood.  Integrins facilitate cell-to-cell interactions and are hypothesized to play a role in the ‘self' versus ‘missing self' recognition (Crozat et al, Blood, 2011). One of the critical regulators of integrin activation is Kindlin-3, which helps in their ‘inside-out' signaling. Kindlin-3 binds to the cytoplasmic tail of β2-integrin and induces a conformational change increasing ligand affinity (Ye et al, Curr Biol, 2013).  Consequently, Kindlin-3 is localized at the immunologic synapse and has been shown to interact with Adhesion and Degranulation Adaptor Protein (ADAP) (Kasirer-Friede et al, Blood 2014). As our group has shown, ADAP plays a central role in the signaling transduction for inflammatory cytokine production in NK cells (Rajasekaran et al, Nat Immunol, 2013). Clinically, defects in Kindlin-3 functions in humans are manifested by severe immune deficiency and bleeding disorder defined as Leukocyte Adhesion Deficiency-III (LAD-III).  Based on these observations, we hypothesized Kindlin-3-dependent integrin function is critical for NK cell-mediated anti-tumor cytotoxicity and production of inflammatory cytokines.

To define the role of Kindlin-3 in NK cell effector functions, we utilized a murine model. Kindlin-3 knock-in (K3KI) mice carry a double substitution mutation disrupting the binding of Kindlin-3 to β2-integrin (Xu et al, Arterioscler Thromb Vac Biol, 2014). NK cells from K3KI mice were evaluated for development and effector functions.  Flow cytometry was utilized to identify maturation and developmental populations. Inflammatory cytokine production was assessed by Interferon-γ release following NK cell and tumor co-culture as well as plate-bound antibody activation. Cytotoxicity was assessed by ⁵¹Cr-release assay and the following tumor cells were used: cells representing, 1) ‘self' (RMA and EL4 thymomas with autologous MHC-I); 2) ‘missing-self' (RMA/S with decreased MHC expression relative to RMA); 3) ‘induced self' (EL4 thymomas stably expressing H60, an activating ligand for NKG2D; 4) ‘non-self' (YAC1 lymphoma with allogeneic MHC).

Our results show an overall increase in the peripheral NK populations collected from spleens of K3KI mice, as seen in patients with LAD-III. However, no significant maturational defects were noted in the bone marrow of the K3KI mice. In vitro analyses reveal that K3KI NK cells have significantly impaired anti-tumor cytotoxicity relative to wild type controls (Figure 1).  There was a significant reduction in the cytotoxic ability of K3KI NK cells towards ‘induced self' or ‘missing self' recognition (p<0.05).   In contrast, K3KI NK cells have augmented inflammatory interferon-γ cytokine production compared to wild type controls when co cultured with the same tumor models in which cytotoxicity was significantly impaired (Figure 2).

These data reveal the essential role of Kindlin-3 interaction with integrin for the effector functions of the NK cell.  Currently, we are delineating the signaling mechanisms which mediate this divergence in NK cell functions dependent on Kindlin-3. Our studies reveal an undefined role for Kindlin-3 in NK cells and may help to identify novel therapeutic targets to modulate NK cell effector functions.