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Predominant Intracellular Retention Functional Folded State As Well As Conformation-Specific Antibody Reveal a KIR3DL1*004 With Interactions of NK Cell Receptor KIR3DL1*004 with Chaperones and Conformation-Specific Antibody Reveal a Functional Folded State As Well As This information is current as Predominant Intracellular Retention of September 23, 2021. Sabrina B. Taner, Marcelo J. Pando, Allison Roberts, Jennifer Schellekens, Steven G. E. Marsh, Karl-Johan Malmberg, Peter Parham and Frances M. Brodsky J Immunol 2011; 186:62-72; Prepublished online 29 Downloaded from November 2010; doi: 10.4049/jimmunol.0903657 http://www.jimmunol.org/content/186/1/62 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2010/11/29/jimmunol.090365 Material 7.DC1 References This article cites 75 articles, 35 of which you can access for free at: http://www.jimmunol.org/content/186/1/62.full#ref-list-1 by guest on September 23, 2021 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Interactions of NK Cell Receptor KIR3DL1*004 with Chaperones and Conformation-Specific Antibody Reveal a Functional Folded State As Well As Predominant Intracellular Retention Sabrina B. Taner,*,†,‡,1 Marcelo J. Pando,*,†,‡,1,2 Allison Roberts,* Jennifer Schellekens,x Steven G. E. Marsh,x,{ Karl-Johan Malmberg,‖ Peter Parham,# and Frances M. Brodsky*,†,‡ Variable interaction between the Bw4 epitope of HLA-B and the polymorphic KIR3DL1/S1 system of inhibitory and activating NK cell receptors diversifies the development, repertoire formation, and response of human NK cells. KIR3DL1*004, a common + KIR3DL1 allotype, in combination with Bw4 HLA-B, slows progression of HIV infection to AIDS. Analysis in this study of Downloaded from KIR3DL1*004 membrane traffic in NK cells shows this allotype is largely misfolded but stably retained in the endoplasmic reticulum, where it binds to the chaperone calreticulin and does not induce the unfolded protein response. A small fraction of KIR3DL1*004 folds correctly and leaves the endoplasmic reticulum to be expressed on the surface of primary NK and transfected NKL cells, in a form that can be triggered to inhibit NK cell activation and secretion of IFN-g. Consistent with this small proportion of correctly folded molecules, trace amounts of MHC class I coimmunoprecipitated with KIR3DL1*004. There was + no indication of any extensive intracellular interaction between unfolded KIR3DL1*004 and cognate Bw4 HLA-B. A similarly http://www.jimmunol.org/ limited interaction of Bw4 with KIR3DL1*002, when both were expressed by the same cell, was observed despite the efficient folding of KIR3DL1*002 and its abundance on the NK cell surface. Several positions of polymorphism modulate KIR3DL1 abundance at the cell surface, differences that do not necessarily correlate with the potency of allotype function. In this context, our results suggest the possibility that the effect of Bw4+ HLA-B and KIR3DL1*004 in slowing progression to AIDS is mediated by interaction of Bw4+ HLA-B with the small fraction of cell surface KIR3DL1*004. The Journal of Immunology, 2011, 186: 62–72. atural killer cells are important mediators of cytotoxicity receptor (KIR) family of receptors can both inhibit and activate and cytokine secretion in innate immunity. They also the NK cell response through recognition of HLA class I proteins N influence the adaptive immune response by secreting of the MHC (3, 4). MHC class I interaction with inhibitory KIR is by guest on September 23, 2021 immunoregulatory cytokines that stimulate T cells and dendritic also critical for NK cell education and development of the NK cell cells (1, 2). NK cells can recognize and lyse pathogen-infected, repertoire (5, 6). KIRs evolve rapidly and can exhibit a high de- tumorigenic, or allogeneic cells. Regulation of these effector gree of allelic polymorphism, approaching that of classical MHC functions stems from a balance of signaling through activating and class I genes (7). Genetic studies link specific KIR-HLA allele inhibitory receptors. On human NK cells, the killer cell Ig-like combinations with the clinical outcome of a diverse range of diseases, including viral and protozoan infection, autoimmune and inflammatory disease, tumor development, pregnancy-related dis- *Department of Bioengineering and Therapeutic Sciences, †Department of Pharma- orders, and the success of bone marrow transplantation (8–11). ‡ ceutical Chemistry, and Department of Microbiology and Immunology, University This broad influence on disease is consistent with a role for of California, San Francisco, San Francisco, CA 94143; #Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305; xAnthony pathogen-driven, rapid KIR gene evolution and diversity balanced { Nolan Research Institute, Royal Free Hospital; Cancer Institute, University College by the distinctive demands of reproductive selection (12, 13). London, Royal Free Campus, London, United Kingdom; and ‖Department of Med- icine, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Several studies link disease progression from HIV to AIDS to the Hospital, Stockholm, Sweden KIR3DL1 gene (reviewed in Refs. 8, 14). The KIR3DL1/KIR3DS1 1S.B.T. and M.J.P. contributed equally to this work. locus is highly polymorphic, with .60 different alleles currently 2 ∼ Current address: Department of Pathology, Stanford University School of Medicine, described [http://www.ebi.ac.uk/ipd/kir (15, 16)]. Of these, 80% Stanford, CA. encode inhibitory (KIR3DL1) receptors, which recognize HLA-A Received for publication November 12, 2009. Accepted for publication October 23, and HLA-B containing the Bw4 serological epitope (17, 18) and 2010. the remaining alleles encode activating (KIR3DS1) receptors. This work was supported by the National Institutes of Health (P01 AI064520). Inhibitory allotypes differ in their level of expression at the cell Address correspondence and reprint requests to Dr. Frances M. Brodsky, The G.W. surface (19, 20) and in their functional capacity to inhibit NK cell Hooper Foundation, 513 Parnassus Avenue, HSW 1529, University of California, San activation (21–24). Genetic studies indicate that the combination Francisco, CA 94143. E-mail address: [email protected] of KIR3DS1 and HLA-Bw4 is protective against progression to The online version of this article contains supplemental material. AIDS in HIV-infected individuals, but a ligand–receptor re- Abbreviations used in this paper: Chap., chaperone protein; CNX, calnexin; CRT, calreticulin; Endo H, endoglycosidase H; ER, endoplasmic reticulum; Grp78, glucose lationship between HLA-Bw4 and KIR3DS1 has not been dem- regulated protein 78; KIR, killer cell Ig-like receptor; MFI, mean fluorescence in- onstrated (discussed in Refs. 14, 25). Similarly, the combination of tensity; RT, reverse transcriptase; Tunic, tunicamycin; UPR, unfolded protein re- HLA-Bw4 and inhibitory KIR3DL1*004 has been associated with sponse; UT, untransfected; XBP-1, X-box binding protein-1. slowed progression to AIDS (26), but neither a physical nor func- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 tional interaction between KIR3DL1*004 and HLA-Bw4 has been www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903657 The Journal of Immunology 63 defined. That KIR3DL1*004 is largely retained inside the cell and (41), with additional changes to improve sequence coverage and quality is barely detectable at the cell surface (27) raises questions about (J. Schellekens, unpublished observations). Sequences were analyzed (Seq- the mechanism of action of this receptor that are investigated in Scape version 2.5,Applied Biosystems, Foster City, CA), and KIR3DL1/ KIR3DS1 alleles were identified after comparison with the KIR Immuno this study. Polymorphism Database [http://www.ebi.ac.uk/ipd/kir (15)]. The intracellular retention of KIR3DL1*004 depends on two amino acid substitutions, Leu86 and Ser182, respectively, in the D0 Flow cytometry and D1 Ig-like extracellular domains (27). These two substitutions For NKL cell transfectants, 5 3 105 cells/ml were incubated in culture are predicted to influence protein folding, which could affect both medium at various temperatures between 16˚C and 37˚C as indicated for receptor transport and ligand binding (27, 28). The selective ad- 16 h. Cells were stained with PE-conjugated anti-KIR3DL1 mAb (177407, R&D Systems, Minneapolis, MN) or isotype-matched control (20102, vantage of KIR3DL1*004 and HLA-Bw4 in responding to HIV R&D Systems) in PBS/1% BSA/0.01% sodium azide for 1 h at 4˚C. The infection (26) suggested the possibility that KIR3DL1*004 func- 177407 mAb was made against KIR3DL1, and its specificity has been tions by binding HLA-Bw4 ligand inside the cell, as has been characterized as binding both KIR3DL1 and KIR3DL2, but only in the observed for endosome-localized KIR2DL4
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