Kerry S Campbell, PhD
Office Phone: 215-728-7761
Lab Phone: 215-728-7762
1. Role of BCAP in Regulating NK Cell Development and FunctionAlexander Macfarlane
B cell adaptor for PI3-K (BCAP) was identified as a cytosolic adaptor protein that can recruit phosphatidylinositol 3-kinase (PI3-K). B-cells from mice deficient in BCAP have an impaired ability to reach full maturity, produce less immunoglobulin, have decreased proliferative capability, are more susceptible to apoptosis, and exhibit reduced calcium mobilization in response to antigen receptor crosslinking. We recently discovered that NK cells express BCAP and studied NK cells in BCAP-deficient mice. In sharp contrast to the studies of B cells, NK cells in mice lacking BCAP are more mature, more long-lived, more resistant to apoptosis, and exhibit enhanced functional activity compared to NK cells from normal mice. The paradoxical phenotype reveals inherent differences in the signals controlling the final maturation of B cells and NK cells, which depend on positive and negative signals, respectively. Since NK cells require inhibitory receptor signaling to become functional effector cells, we hypothesize that the loss of BCAP blunts activation in a manner that is similar to inhibitory signaling, thereby augmenting NK cell terminal maturation and function. NK cells that develop under conditions lacking inhibitory receptor signaling have been shown to become hypo-responsive, due to chronic activation signaling that is not adequately suppressed. Therefore, we propose that the increased function, accumulation, and survival of mature NK cells in BCAP-deficient mice is due to better damping of a desensitizing signal that is at least partially BCAP-mediated.Top
2. Activation of Human NK Cells Through KIR2DL4 SignalingAmanda Purdy, Michael Brusilovsky & Nicholas Rodin
A structurally unique KIR, named 2DL4, activates potent cytokine and chemokine production, but only weak cytotoxicity responses in human NK cells. We have discovered two distinct activation signaling cassettes that are initiated from 2DL4: 1) physical linkage to the transmembrane adaptor protein, FcεRI--γ, which triggers calcium mobilization and weak cytotoxicity responses, and 2) FcεRI-γ-independent signaling that is likely mediated directly through the receptor’s cytoplasmic domain and is capable of stimulating production of the chemokine, MIP1α. Association with FcεRI--γ distinguishes 2DL4 from the DAP12-associated activating KIRs with short cytoplasmic domains (KIR2DS1-5 and KIR3DS1).
The only well-characterized ligand for 2DL4 is a soluble form of the non-classical MHC class I molecule, HLA-G, which is mainly produced by fetal-derived trophoblast cells in pregnant women. This has led to a hypothesis that 2DL4 may play an important role in stimulating NK cells to secrete cytokines that support placental development. Importantly, HLA-G is reportedly also expressed by numerous tumor cells, indicating that this receptor may also play important roles in tumor surveillance. We further hypothesize that additional ligand(s) may exist. Our studies defining the unique signaling pathways and surface regulation of 2DL4 should establish molecular mechanisms by which the receptor may play important roles in regulating pregnancy and preventing cancer.
Our recent studies have identified the E3 ubiquitin ligase, Triad3A, as a negative regulator of 2DL4 function. Triad3A was found to bind to the cytoplasmic domain of 2DL4 and promotes polyubiquitylation and degradation of the receptor. Over-expression of Triad3A in NK cells significantly reduced total levels of 2DL4 protein and abrogated cytokine production through the receptor, without impacting receptor surface expression or early receptor signaling responses. Our data indicated that cytokine production in response to 2DL4 engagement requires receptor internalization, and interestingly, Triad3A targeted only the internalized receptor for degradation and disrupted sustained NF-κB stimulation. Therefore, we conclude that Triad3A is a key negative regulator of sustained 2DL4-mediated NF-κB signaling from internalized 2DL4, which functions by promoting ubiquitylation and degradation of endocytosed receptorTop
3. SHP-2 Expression Negatively Regulates NK Cell FunctionAmanda Purdy
We recently showed that shRNA-mediated knockdown of the SHP-2 protein tyrosine phosphatase in NK cell lines resulted in enhanced cytotoxicity responses. In contrast, over-expression of wild type SHP-2 suppressed cytolytic function and expression of two different gain-of-function Noonan Syndrome mutant forms of SHP-2 (E76D and N308D) suppressed cytotoxicity even more profoundly. Identical impacts were observed on IFN-g production in response to target cells. Further analysis revealed that the increased cytotoxicity in SHP-2-silenced NK cells was due to enhanced polarization of the microtubule organizing cener (MTOC) toward a conjugated tumor target cell and enhanced granzyme B release, while over-expression of WT or constitutively active (gain-of-function) SHP-2 suppressed both of these responses. KIR-dependent inhibition of cytotoxicity was unaffected by significant reductions in SHP-2 levels, presumably because KIR were still capable of recruiting the phosphatase under these limiting conditions. In contrast, the general suppressive effect of SHP-2 on cytotoxicity and cytokine release was much more sensitive to changes in cellular SHP-2 levels. Our results establish a KIR-independent role for SHP-2 in damping NK cell cytolytic responses and suggest that SHP-2 inhibition may be a therapeutic strategy to promote tumor cell killing by NK cells. Also, the more profound suppression of cytotoxicity (>60%) by expressing the gain-of-function variants of SHP-2 (E76D and N308D), which correspond to germline mutations commonly found in Noonan Syndrome patients, suggests that such patients may have NK cell functional defects.Top
4. Regulation of Cell Surface Trafficking of Inhibitory KIR by PhosphorylationAmanda Purdy & Jennifer Oshinsky
Recently, we have characterized serine and threonine phosphorylation sites on the cytoplasmic domain of inhibitory KIR, three of which are conserved in all human inhibitory KIR. We identified four serine/threonine (S/T) phosphorylation sites on the KIR3DL1 cytoplasmic domain (S364, S367, S394, and T399). Our studies concluded that casein kinase I (CKI) phosphorylates S364, casein kinase II (CKII) phosphorylates S367, and PKC phosphorylates S394. The kinase phosphorylating T399 has not yet been identified. Pharmacological inhibitors indicated that most constitutive KIR phosphorylation is mediated by CKI, and primary phosphorylation of either S364 or S367 by CKI or CKII, respectively, promotes secondary phosphorylation of the other site.
The functional impacts of the CK and PKC phosphorylation sites were tested by mutation of the individual serines to non-phosphorylatable alanine (A) or to the phosphomimetic aspartic acid (D), which mimics the charge introduced by a phosphate at that serine within the protein sequence. We found that non-phosphorylatable mutation of the PKC phosphorylation site (S394A) or both CK phosphorylation sites (S364/367A) increases the amount of receptor expressed on the cell surface. Turnover or internalization rates of the S364/367A mutant were unchanged, but a non-phosphorylatable PKC site mutant (S394A) showed enhanced surface turnover, while a phosphomimetic mutant (S394D) demonstrated reduced turnover capacity. Finally, the phosphomimetic mutant was internalized at a slower rate and exhibited slightly reduced inhibitory function, as compared to the wild type receptor. Together, these results indicate that serine phosphorylation of KIR by PKC stabilizes the receptor on the NK cell surface. Importantly, stable surface expression of inhibitory KIR is essential to maintain self tolerance by NK cells, implying that normal phosphorylation of the serine by PKC is necessary to prevent autoimmune responses by human NK cells.Top
5. Type-2 Cytokine-producing NK CellsMargaret Joyce
Prostaglandin D2 (PGD2) has been shown to be increased in bronchiolar lavage fluid after allergen challenge and has been implicated as an important mediator of asthma, since it can cause bronchioconstriction and chemotaxis of Th2 cells. This prostaglandin is produced by mast cells and alveolar macrophages. We tested the hypothesis that PGD2 can influence the function of NK cells at sites of allergic responses. Treatment of NK cells with PGD2 was found to inhibit cytotoxicity, cytokine production (IFN-γ, TNF-α, and GM-CSF), and chemotaxis, but did not affect proliferation or differentiation. Pharmacologic studies indicated that the PGD2 effect was mediated through binding to the D-prostanoid (DP) receptor on the surface of NK cells. PGD2 binding to DP stimulated the production of cAMP, which was responsible for the inhibitory effects. Therefore, we hypothesize that PGD2 plays a critical role in suppressing inflammatory responses by NK cells at sites of allergic insult, such as at mucosal surfaces.
We have recently identified new surface markers of a small subset of human natural killer (NK) cells that produce the type-2 cytokines, IL-4 and IL-13. Our data suggest that these cells may contribute to the inflammatory response at sites of allergenic insult, such as mucosal surfaces of the lung and gastrointestinal tract. We are currently studying these human type-2 NK cells by cataloging the receptors that they express and testing their impacts on biological responses. Additional studies are attempting to characterize the type-2 NK cells in mice and test whether they can also respond to allergenic challenge in a mouse model of asthma. The findings will fill significant gaps in our understanding of environmental signals that regulate the functions of type-2 NK cells, and establish a mouse model for studying their in vivo functions. Improved understanding of these biological parameters will allow us to establish the roles of type-2 NK cells in the pathogenesis of localized allergic responses and infections, particularly in the respiratory and intestinal tracts.Top