One focus of our research is the receptors that dictate natural killer (NK)-cell function. NK cells are an integral component of the innate immune response against infectious diseases and malignant transformation. In addition to their ability to lyse infected and transformed cells, they serve as a potent source of cytokines for potentiating both the innate and adaptive immune responses. NK cells express a large variety of "activation" receptors for recognizing potential target cells; many of these receptors recognize ligands on normal cells. "Inhibitory receptors" that also recognize ligands expressed on normal cells but are down-regulated on aberrant cells modulate potentially self-destructive activation signals.
NK cells are increasingly being cultured for immunotherapeutic infusion into patients with cancer or viral infections such as HIV. Thus, understanding how to regulate expression of key activating receptors (CD16 and NKG2D) and to sustain their expression on NK cells has obvious clinical relevance for improving the efficacy of NK cells. Likewise, identifying components critical for NK cell release of lytic and cytokine-bearing granules (like LAMP1 and -2) and the mechanism by which they function could lead to an augmentation of NK cell immunotherapeutic value.
NKG2D must associate with the DAP10 adaptor molecule to gain signaling capabilities and to facilitate cell surface expression. Because of NKG2D's importance in fighting certain viral diseases and tumorigenesis, and its demonstrated involvement in autoimmune diseases such as celiac disease and diabetes, we have an intense interest in factors that regulate the expression of NKG2D. Because DAP10 is required for NKG2D surface expression and signal transmission, our current studies are focused on understanding what regulates DAP10 gene and protein expression.
CD16, the low affinity receptor for IgG1 (FcγRIIIa), is expressed by the majority of human NK cells and is a potent activating receptor that mediates Ab-dependent cell-mediated cytotoxicity (ADCC). Down-regulation of CD16 has been linked to poor prognosis and progression of cancer and chronic infections. In HIV infections, ADCC responses to gp140 are strongly associated with a slower CD4 T-cell loss. The fact that CD16-bound IgG1 can be readily exchanged allows the target-cell specificity of NK cells to be adjusted to suit the needs of individual patients for immunotherapeutic purpose. Thus, understanding how CD16 expression is regulated has significant clinical relevance. We discovered that ex vivo NK cells cultured and expanded in the presence of IL-2 down-modulate CD16 surface expression. The changes only affected protein levels, as CD16 mRNA was stable during the first three days of culture and started to increase after four days, indicating that the CD16 down-modulation is an active post-translational process. Evidence indicates that matrix metalloproteinases (MMPs) down-modulate CD16 cell-surface expression. We are determining which MMPs are involved, how they are regulated, the cell biology of the process, and how the process can be inhibited. The goal is to enhance the immunotherapeutic value of NK cells.
Ligation of NK-cell activation receptors triggers a complex and highly regulated response leading to target-cell death. The actual killing of target cells is due to the secretion of lytic granules (also known as “secretory lysosomes”), containing perforin and granzymes, at the immunological synapse. Defects in lytic granule secretion lead to serious and often fatal diseases, such as familial hemophagocytic lymphohistiocytosis, Chediak–Higashi syndrome, and Griscelli syndrome. The granule-associated protein machinery involved in granule exocytosis in NK cells is ill defined. There are many lysosomal membrane proteins that play a variety of roles in the function of lysosomes in humans; two of them, LAMP1 and -2, compose almost 50 percent of proteins on the surface of the lysosome, yet their function is poorly understood. Given the fact that LAMP1 and -2 are the most abundant proteins in the lysosomal membrane, and NK cells utilize their secretory lysosomes to lyse target cells, we are determining the importance of these two proteins in NK-cell activity.
We are excited about our studies on the CD300 receptors; the mouse orthologues are termed CLMs, but for discussion purposes herein we refer to receptors in both species as CD300s. These receptors are a family of leukocyte regulatory molecules, mainly expressed by myeloid cells that appear to consist of paired activating and inhibitory receptors. The activating members contain a charged transmembrane residue and pair with adaptors like DAP12, whereas the inhibitory receptors contain ITIM motifs for interacting with phosphatases, as well as other motifs with either activating or inhibitory potential.
Research on CD300 molecules is in its infancy, but preliminary studies suggest that targeting CD300 receptors is likely to have significant value for modulating immune dysfunctions in disorders such as asthma and allergy and chronic inflammatory diseases such as psoriasis and Crohn’s disease. The latter are diseases known to be associated with CD300a expression. The ligands for CD300 receptors were unknown until we recently defined the primary ligands for both the mouse CD300f and human CD300a ITIM-bearing receptors as phosphatidylserine (PS) and phosphatidylethanolamine (PE), respectively. Interestingly, these phospholipids are briefly exposed on activated cells and more robustly exposed as cells become apoptotic and die. In this light, we found that both bone marrow-derived and peritoneal macrophages from CD300f -/- mice have enhanced phagocytic capacity, implying, as expected, that in this case CD300f generates an inhibitory signal. Similar results were obtained for CD300a in experiments where CD300a expression was inhibited with siRNA.
In contrast, exogenous expression of CD300f in phagocytic cell lines enhanced phagocytosis of apoptotic cells. This dichotomy in function likely can be explained by the fact that both CD300a and CD300f have cytoplasmic domains with a variety of signaling motifs, some with activating and some with inhibitory potential, which suggests that these receptors can generate either activating or inhibitory signals depending on specific circumstances. We are determining exactly what signals are important for positively or negatively regulating phagocytosis.
In addition to this work on phagocytosis, studies on the other potential biological functions of the CD300 receptors will be a major part of our future research. We are particularly interested how PS and PE expression on activated/apoptotic cells regulates the interactions between stimulated T and B cells and myeloid cells (dendritic cells and macrophages), which is key to regulation of the immune response. Integral to this, we will identify the ligands for the other mouse CD300 family members. Of particular interest is whether mouse CD300a (CLM-8), like its human counterpart, and mouse CD300d (CLM-4) and -c (CLM-6) inhibitory receptors, closely related to mouse CD300a, preferentially recognize PE and/or PS or other phospholipids. Also, mouse CLM-3 and CLM-5 are putative activation receptors closely related to mouse CD300f, and it will be interesting to determine if they preferentially bind PS.
Our interest in enhancing the immunotherapeutic value of NK cells led us to study the function of Toso in NK cells. Toso (FAIM-3) was originally defined as an inhibitor of Fas-mediated apoptosis. We observed that it is dramatically down-regulated (more than 30-fold) as NK cells are activated and become susceptible to activation-induced cell death (AICD); a similar process happens in T cells. In accordance with its reported anti-apoptotic function on T cells, we hypothesized that by over-expressing Toso in NK cells we could protect them from AICD and thereby extend their immunotherapeutic usefulness. Despite a recent report to the contrary, we now have extensive data that the anti-apoptotic function ascribed to Toso is erroneous and that instead, as was recently reported, it is a specific receptor for the Fc portion of IgM and should be more appropriately named FcμR.
We continued to explore if Toso/FcμR might be an additional NK-cell activation receptor and function like the IgG1 receptor, CD16. Considering the array of IgM antibodies, including “natural” antibodies to a variety of pathogens, Toso has the possibility to vastly broaden the panel of “targets” that activate NK cells. We have found that Toso-bound IgM specific for target cells, or antibodies to IgM in a re-directed lysis assay, do not convey cytotoxic capability to NK cells, but such binding does convey activation signals as detected by phosphorylation of signaling kinases. We are determining how this signaling affects NK-cell function. As part of these studies, we acquired Toso-deficient mice. As Toso is expressed at significantly higher levels on B cells than other cell types, we have been studying the role of Toso in B-cell development, homeostasis, and function. We now think that FcμR may play a key role in regulating immune responses.
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Dr. Coligan received his Ph.D. from Indiana University and did postdoctoral research at the City of Hope Research Institute. After two years as an assistant professor at Rockefeller University, he was a founding member of the Laboratory of Immunogenetics. He has served as head of the Biological Resources Branch and Laboratory of Molecular Structure. In 1998, he joined the Laboratory of Allergic Diseases and became chief of the Receptor Cell Biology Section (RCBS). In 2007, this section moved to the Laboratory of Immunogenetics.
Konrad Krzewski Ph.D. (Staff Scientist); Jennifer Weck, Ph. D. (Lab Manager); Seung-Chul Choi, Ph.D.; Aleksandra Gil-Krzewska, Ph.D.; Miri Gitik, Ph.D.; Yousuke Murakami, Ph.D.; Giovanna Peruzzi, Ph.D.; Linjie Tian, Ph.D.; Mirna Pena (Animal Technician).
Tang X, Tian L, Esteso G, Choi SC, Barrow AD, Colonna M, Borrego F, Coligan JE. Leukocyte-associated Ig-like receptor-1-deficient mice have an altered immune cell phenotype. J Immunol. 2012 Jan 15;188(2):548-58.
Choi SC, Simhadri VR, Tian L, Gil-Krzewska A, Krzewski K, Borrego F, Coligan JE. Cutting edge: mouse CD300f (CMRF-35-like molecule-1) recognizes outer membrane-exposed phosphatidylserine and can promote phagocytosis. J Immunol. 2011 Oct 1;187(7):3483-7.
Park YP, Choi SC, Kiesler P, Gil-Krzewska A, Borrego F, Weck J, Krzewski K, Coligan JE. Complex regulation of human NKG2D-DAP10 cell surface expression: opposing roles of the γc cytokines and TGF-β1. Blood. 2011 Sep 15;118(11):3019-27.
Masilamani M, Peruzzi G, Borrego F, Coligan JE. Endocytosis and intracellular trafficking of human natural killer cell receptors. Traffic. 2009 Dec;10(12):1735-44.
Peruzzi G, Masilamani M, Borrego F, Coligan JE. Endocytosis as a mechanism of regulating natural killer cell function: unique endocytic and trafficking pathway for CD94/NKG2A. Immunol Res. 2009;43(1-3):210-22.
Fattakhova GV, Masilamani M, Narayanan S, Borrego F, Gilfillan AM, Metcalfe DD, Coligan JE. Endosomal trafficking of the ligated FcvarepsilonRI receptor. Mol Immunol. 2009 Feb;46(5):793-802.
Last Updated October 26, 2012