NKG2D−NKG2D Ligand Interaction Inhibits the Outgrowth of Naturally Arising Low-Grade B Cell Lymphoma In Vivo This information is current as Saravanan Raju, Lena Z. Kretzmer, Olivia I. Koues, of September 29, 2021. Jacqueline E. Payton, Eugene M. Oltz, Amanda Cashen, Bojan Polic, Robert D. Schreiber, Andrey S. Shaw and Mary A. Markiewicz J Immunol published online 2 May 2016 http://www.jimmunol.org/content/early/2016/04/30/jimmun Downloaded from ol.1501982 http://www.jimmunol.org/ 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 by guest on September 29, 2021 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 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 2, 2016, doi:10.4049/jimmunol.1501982 The Journal of Immunology NKG2D–NKG2D Ligand Interaction Inhibits the Outgrowth of Naturally Arising Low-Grade B Cell Lymphoma In Vivo Saravanan Raju,* Lena Z. Kretzmer,* Olivia I. Koues,* Jacqueline E. Payton,* Eugene M. Oltz,* Amanda Cashen,† Bojan Polic,‡ Robert D. Schreiber,* Andrey S. Shaw,*,x and Mary A. Markiewicz*,{ It is now clear that recognition of nascent tumors by the immune system is critical for survival of the host against cancer. During cancer immunoediting, the ability of the tumor to escape immune recognition is important for tumor development. The immune system recognizes tumors via the presence of classical Ags and also by conserved innate mechanisms. One of these mechanisms is the NKG2D receptor that recognizes ligands whose expression is induced by cell transformation. In this study, we show that in NKG2D receptor–deficient mice, increasing numbers of B cells begin to express NKG2D ligands as they age. Their absence in wild-type mice suggests that these cells are normally cleared by NKG2D-expressing cells. NKG2D-deficient mice and mice constitutively Downloaded from expressing NKG2D ligands had increased incidence of B cell tumors, confirming that the inability to clear NKG2D ligand– expressing cells was important in tumor suppression and that NKG2D ligand expression is a marker of nascent tumors. Support- ing a role for NKG2D ligand expression in controlling the progression of early-stage B cell lymphomas in humans, we found higher expression of a microRNA that inhibits human NKG2D ligand expression in tumor cells from high-grade compared with low- grade follicular lymphoma patients. The Journal of Immunology, 2016, 196: 000–000. http://www.jimmunol.org/ he NK cell activating receptor NKG2D is expressed on all accelerated in the absence of NKG2D (18). In contrast, there was NK cells and subsets of T cells in both mouse and human no effect on the occurrence of carcinogen-induced sarcomas (18). T (1–7). NKG2D binds to a wide variety of self-ligands, all However, an experimental murine sarcoma line induced in an of which are related to MHC class I in sequence. In human, the immune-deficient mouse expressed variable levels of NKG2D li- ligands are MICA and MICB and the RAET1 family member gands and the high-ligand expressing cells were eliminated when molecules (ULBP1–6) (1, 8–10). In mouse, the ligands are the they were transferred into a wild-type host (19). In addition, treat- RAE1 (RAE1a2ε), H60 (H60a-c), and MULT-1 proteins (11–16). ment of mice with a neutralizing NKG2D Ab enhanced the sensi- These ligands are generally absent from most normal tissue; tivity of mice to carcinogen-induced fibrosarcomas (20). by guest on September 29, 2021 rather, their expression is induced under conditions of cellular Evidence from both mouse and human studies suggest tumors stress, such as viral infection, transformation, or DNA damage actively evade NKG2D recognition. Experimental murine tumors (17). Tumors of various origin express NKG2D ligands, which often lose NKG2D ligand expression as they progress (18, 19, 21). facilitates NK-mediated lysis of the tumor cells in vitro and allows Patients with certain types of cancer exhibit high serum levels of for rejection of transplantable tumors in vivo (1, 12, 14). circulating NKG2D ligands (22–24), presumably released from The role of NKG2D in tumor immunosurveillance was previ- the tumors, resulting in a downregulation of NKG2D expression on ously tested using a prostate cancer model, a B cell lymphoma NK cells and T cells, which ultimately blocks NKG2D-mediated model, and a carcinogen-induced sarcoma model. In the transgenic recognition of tumors (24). adenocarcinoma of the mouse prostate cancer model, the absence The cancer immunoediting hypothesis posits that a tumor is either of NKG2D resulted in enhanced tumor growth and more aggressive eliminated by the immune system, exists in a state of equilibrium tumors (18). Similarly, the occurrence of B cell lymphomas was with the immune system, or acquires properties that allow the tumor to grow and escape recognition by the immune system (25). Given *Department of Pathology and Immunology, Washington University School of Med- that NKG2D ligands are induced by cellular transformation (26), its icine, St. Louis, MO 63110; †Department of Medicine, Washington University role in cancer immunoediting is likely to be in the first two phases, School of Medicine, St. Louis, MO 63105; ‡Department of Histology and Embryol- ogy, Medical Faculty University of Rijeka, 51000 Rijeka, Croatia; xHoward Hughes elimination and equilibrium. Because the transgenic models of Medical Institute, Washington University School of Medicine, St Louis, MO 63110; cancer used in earlier studies enforce the development of tumors, { and Department of Microbiology, Molecular Genetics, and Immunology, University it is difficult to parse the natural role of NKG2D using these models. of Kansas Medical Center, Kansas City, KS 66160 Mice and humans are naturally prone to the development of B cell ORCIDs: 0000-0001-8904-8497 (S.R.); 0000-0001-8832-3661 (J.E.P.); 0000-0001- 6809-4566 (A.C.); 0000-0003-3930-9630 (B.P.); 0000-0001-5685-8573 (M.A.M.). lymphoma as they age (27). In this paper, we studied the natural Received for publication September 8, 2015. Accepted for publication April 4, 2016. history of spontaneously arising, non-transgene-driven B cell lym- phomas in the context of NKG2D. In mice lacking NKG2D ex- This work was supported by American Cancer Society Grant IRG-58-010-55 (to M.A.M.), the Howard Hughes Medical Institute (A.S.S.), and National Institutes of pression, NKG2D ligand expression was clearly evident on splenic Health Grant CA156690 (to J.E.P. and E.M.O.). B cells as mice aged with increasing levels correlated with aging. Address correspondence and reprint requests to Dr. Andrey S. Shaw at the current Increasing expression of NKG2D ligands also correlated with address: Genentech, One DNA Way, South San Francisco, CA 94080. E-mail address: development of B cell lymphoma. Even in mice lacking overt [email protected] tumor masses, we could easily detect multiple lymphoid aggre- Abbreviation used in this article: miRNA, microRNA. gates in multiple organs in mice lacking NKG2D expression, Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 suggesting nascent B cell lymphoma. These findings suggest that www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501982 2 NKG2D INHIBITS THE OUTGROWTH OF LOW-GRADE B CELL LYMPHOMA NKG2D ligand expression is an early marker of B cell transfor- and Klrk12/2 littermates on the same genetic background. The OT-I 2/2 mation, allowing for recognition and elimination by NKG2D- transgenic and the gC mice were purchased from The Jackson Lab- oratory. Rag-12/2 mice were purchased from Taconic. The Rag-12/2 and expressing NK and T cells. The absence of NKG2D ligands on 2 2 gC / mice were bred together in the Washington University School of the B cell lymphomas that finally arise in wild-type mice suggests Medicine animal facilities. that loss of NKG2D ligands is a requirement for tumor escape from the immune system. The human relevance of the NKG2D T cell adoptive transfer receptor–ligand axis was supported by our findings suggesting that NKG2D-expressing CTL were generated in vitro by culturing splenocytes NKG2D ligands are expressed at a higher level on low-grade and lymph node cells from OT-I TCR transgenic mice in 6-well plates 3 7 compared with high-grade human follicular lymphomas. (2.5 10 cells/well) with 1 mM OVA peptide (SIINFEKL) (provided by P. Allen, Washington University School of Medicine) for 5 d. Live cells were harvested with Ficoll–Hypaque (GE Healthcare) and labeled with 1 mM Materials and Methods CFSE (Invitrogen) and injected i.v. (107 cells/mouse). The cells were analyzed Mice 24 h later for the expression of NKG2D by staining with an NKG2D-specific All mice were housed under specific-pathogen-free conditions in the Ab (BD Biosciences), followed by flow cytometry. Washington University School of Medicine or University of Kansas Medical Tissue sectioning and staining Center animal facilities in accordance with institutional guidelines. The generation of the RAE1ε transgenic (C57BL/6/129) and Klrk12/2 Tissues were fixed in 10% formalin, and 5-mm sections were cut and (C57BL/6) mice were described previously (28, 29). The Klrk12/2 mice stained with H&E by the Developmental Biology Histology and Micros- were bred to RAE1ε transgenic heterozygous mice to generate wild-type copy Core at the Washington University School of Medicine.