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 Signaling
A structurally unique KIR, named KIR2DL4, activates potent cytokine and chemokine production, but only weak cytotoxicity responses in human NK cells. We have discovered two distinct activation signaling cascades that are initiated from KIR2DL4: 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 (Kikuchi-Maki et al., J. Immunol., 174:3859, 2005; Miah et al., J. Immunol. 180: 2922, 2008). Association with FcεRI-γ distinguishes KIR2DL4 from the DAP12-associated activating KIRs with short cytoplasmic domains (KIR2DS1-5 and KIR3DS1). Our studies defining the unique signaling pathways and surface regulation of KIR2DL4 should establish molecular mechanisms by which the receptor may play important roles in regulating NK cell cytokine responses.
We also identified the E3 ubiquitin ligase, Triad3A, as a negative regulator of 2DL4 function (Miah et al., J. Immunol., 186:2959, 2011). Triad3A was found to interact with the cytoplasmic domain of KIR2DL4 and promote polyubiquitylation and degradation of the receptor. Over-expression of Triad3A in NK cells significantly reduced total levels of KIR2DL4 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 KIR2DL4 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 KIR2DL4-mediated NF-κB signaling from internalized receptor, which functions by promoting ubiquitylation and degradation of endocytosed receptor
The only ligand previously described for KIR2DL4 is the non-classical MHC class I molecule, HLA-G, but we have been unable to verify this ligand interaction and its validity has recently been challenged by another group (Le Page et al., J. Immunol. 192:732, 2014). In collaboration with Dr. Angel Porgador (Ben Gurion University, Beer Sheva, Israel), we recently found that a recombinant soluble form of KIR2DL4 can interact with several human epithelial cancer cell lines. We utilized soluble KIR2DL4 in a whole-genomic siRNA screen to identify alternative ligand(s) for KIR2DL4 expressed on the prostate cancer cell line, PC3. Our siRNA screen revealed that KIR2DL4 interacts directly with heparan sulfate (HS) with high affinity (Brusilovsky et al., J. Immunol., 191:5256, 2013). Further studies demonstrated that the interaction with HS can modulate cytokine production responses through KIR2DL4 and can alter intracellular localization of the receptor to distinct endosomal compartments of cells. We conclude that HS represents a novel binding partner for KIR2DL4 that can regulate its function and intracellular trafficking.
Recent Departure: Jun HasegawaTop
3. Profiling NK Cell Phenotype and Function in Cancer PatientsAlexander Macfarlane, Tatiana Pazina & Poliana Patah
We are using multiparametric flow cytometry to analyze biomarkers on innate and adaptive immune cells from the peripheral blood of cancer patients. Patient samples from patients before and after treatments are being compared with those from healthy control donors to establish significant cancer-associated alterations in proportions of cell subtypes, expression of surface biomarkers, or immune functions with a heavy emphasis on the features of NK cells. The goals are to identify biomarkers that predict disease status or progression and identify potential therapeutic targets to treat cancer patients. Immune parameters are quantified by staining peripheral blood mononuclear cells for cell surface and cytosolic markers with up to 12 simultaneous fluorophore-conjugated antibodies per sample and analyzing by flow cytometry. Data is processed to quantify the percentages of individual innate and adaptive immune cell subsets in peripheral blood and the expression levels of a wide array of distinct biomarkers on these subsets.
We recently analyzed expression of immune biomarkers on fresh peripheral blood mononuclear cells (PBMC) from 90 renal cell carcinoma (RCC) patients before and after nephrectomy and 25 age-matched healthy control donors. The most striking phenotypic immune biomarker in RCC patients was a significant increase in PD-1 expression on certain PBMC in a subset of patients (MacFarlane et al., Cancer Immunol. Res., in press, 2014). Increased PD-1 expression on CD14bright myelomonocytic cells, effector T cells, and NK cells correlated to disease stage. Interestingly, this elevated PD-1 expression was dramatically reduced within weeks after surgical resection of the primary tumor. PD-1 is an inhibitory receptor on leukocytes that can suppress immune responses, particularly toward tumors, which often up-regulate PD-1 ligands. Antibodies that block the interactions between PD-1 and its ligands can reverse this inhibition and have shown significant promise in phase I trials in RCC, lung cancer, and melanoma. Based upon our data, we hypothesize that PD-1 blocking therapies: 1) will be more effective in the setting of up-regulated PD-1, 2) will enhance the functions of PD-1+ leukocytes in this subset of patients, and 3) will be most effective when administered before surgical removal of primary tumor, when PD-1 expression is highest.
In addition, we are studying immune cell biomarkers in fresh peripheral blood and bone marrow samples from cohorts of patients with indolent B cell lymphoma (chronic lymphocytic leukemia, follicular B cell lymphoma, and marginal zone lymphoma), multiple myeloma, melanoma, and inflammatory breast cancer.Top
4. Regulation of Cell Surface Trafficking of Inhibitory KIRAmanda Purdy
We previously characterized serine and threonine phosphorylation sites on the cytoplasmic domain of KIR3DL1 (S364, S367, S394, and T399), three of which are conserved in all human inhibitory KIR (Alvarez-Arias and Campbell, J. Immunol. 179: 5281, 2007). Those studies concluded that casein kinase I (CKI) phosphorylates S364, casein kinase II (CKII) phosphorylates S367, and PKC phosphorylates S394, while the kinase phosphorylating T399 has not been identified. We further 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. The non-phosphorylatable PKC site mutant (S394A) also showed enhanced surface turnover, while a phosphomimetic mutant (S394D) demonstrated reduced turnover capacity. 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.
To extend our studies of mechanisms regulating the surface expression of KIR, we performed a yeast two-hybrid screen to identify proteins that interact with the cytoplasmic domain of KIR3DL1. This screen revealed a direct interaction of the µ2 subunit of the AP2 clathrin adaptor complex with the ITIM tyrosine residues on KIR3DL1. Studies in primary human NK cells revealed that the rate of endocytosis of KIR3DL1 was significantly diminished if the KIR/µ2 interaction was disrupted through mutation of the ITIM tyrosine residues on KIR3DL1 or expression of a dominant negative form of µ2 by lentiviral transduction. The reduced rate of endocytosis under these conditions was also associated with increased surface expression levels of KIR3DL1. We conclude that AP2 can interact with the unphosphorylated ITIM tyrosines of KIR to mediate receptor endocytosis and recycling, whereas the phosphorylated ITIMs alternatively recruit SHP-1 and SHP-2 protein tyrosine phosphatases to mediate inhibitory signaling. Based upon our evidence, we propose a model in which KIR engaged with MHC class I ligand undergo phosphorylation of ITIM tyrosines to recruit SHP-1/SHP-2, thereby preventing interactions with the endocytic pathway and retaining KIR at the cell surface to mediate effective inhibitory signaling. In this way, we have identified a mechanism by which the retention of ligand-engaged KIR at the cell surface assures immune tolerance of NK cells toward normal cells in the body.Top
5. Elotuzumab Targets NK Cell-mediated ADCC of Multiple MyelomaAshley Mentlik-James & Tatiana Pazina
Multiple myeloma (MM) is a deadly hematologic cancer characterized by clonal expansion of malignant plasma cells that reside in the bone marrow and thrive on interactions with the bone marrow microenvironment. Despite advances in treatment strategies, MM remains an incurable disease, with about 20,000 newly diagnosed cases and over 10,000 deaths per year in the U.S. Novel therapies have improved survival over the last decade, including autologous hematopoietic stem cell transplantation and the use of new drugs. Allogeneic stem cell transplantation can be curative, but is often associated with high transplantation-related mortality. Despite these advanced therapeutic options, median survival remains around 4-5 years in adults and the development of better treatments is essential.
Elotuzumab is a promising new monoclonal antibody candidate for treatment of MM. It is a fully humanized antibody that recognizes the SLAM family member, SLAMF7 (CRACC, CS1, CD319), a surface glycoprotein normally expressed on NK cells, monocytes, mature dendritic cells, a subset of T cells, and stimulated B cells. Normal plasma cells express high levels of SLAMF7, which correlates with high expression on MM cells. We are currently studying the mechanism by which elotuzumab promotes NK cell-mediated cytotoxicity of MM cells. The goals are to distinguish whether elotuzumab promotes NK cell cytotoxicity exclusively through initiating antibody-dependent cellular cytotoxicity (ADCC) or also through directly stimulating NK cell function upon binding to SLAMF7 on the NK cell surface. In addition, we hope to understand the relative importance between these and other mechanisms of action to the overall activities of elotuzumab. Studies are utilizing engineered NK-92 cell lines expressing or lacking CD16 or SLAMF7, as well as primary NK cells from healthy donors.Top