Diabetes Volume 69, August 2020 1749 NKG2D Signaling Within the Pancreatic Islets Reduces NOD Diabetes and Increases Protective Central Memory CD81 T-Cell Numbers Andrew P. Trembath,1 Kelsey L. Krausz,1 Neekun Sharma,1 Ivan C. Gerling,2 Clayton E. Mathews,3 and Mary A. Markiewicz1 Diabetes 2020;69:1749–1762 | https://doi.org/10.2337/db19-0979 NKG2D is implicated in autoimmune diabetes. How- The immune receptor NKG2D, expressed by natural killer ever, the role of this receptor in diabetes pathogenesis (NK) cells and subsets of T cells (1–5), is implicated in type IMMUNOLOGY AND TRANSPLANTATION is unclear owing to conflicting results with studies in- 1 diabetes development. However, its role in disease pro- volving global inhibition of NKG2D signaling. We found gression remains unclear, with conflicting reports describ- that NKG2D and its ligands are present in human pan- ing pathogenic (6,7), nonpathogenic (8), and protective (9) creata, with expression of NKG2D and its ligands in- effects of NKG2D signaling. creased in the islets of patients with type 1 diabetes. NKG2D recognizes multiple NKG2D ligands in both To directly assess the role of NKG2D in the pancreas, we humans and mice. These are endogenous ligands, which generated NOD mice that express an NKG2D ligand in are all distantly related to MHC class I in sequence, and are b-islet cells. Diabetes was reduced in these mice. The generally believed to be functionally redundant (10,11). reduction corresponded with a decrease in the effector 1 NKG2D ligands are considered stress ligands, with their to central memory CD8 T-cell ratio. Further, NKG2D expression induced by cellular stressors, including viral signaling during in vitro activation of both mouse and 1 infection or DNA damage (10,11). Engagement of these human CD8 T cells resulted in an increased number of fi central memory CD81 T cells and diabetes protection by ligands by NKG2D on NK cells is suf cient to activate NK central memory CD81 T cells in vivo. Taken together, cell killing of NKG2D ligand-bearing target cells (1). On these studies demonstrate that there is a protective role T cells, NKG2D engagement generally costimulates T-cell 1 – for central memory CD8 T cells in autoimmune diabetes receptor driven activation, enhancing T-cell responses and that this protection is enhanced with NKG2D signal- (1,7,9,12–14). In addition, NKG2D signaling has been ing. These findings stress the importance of anatomical shown to be important in the development of mouse 1 location when determining the role NKG2D signaling plays, memory CD8 Tcells(15–17). as well as when developing therapeutic strategies targeting Early studies with the NOD mouse model led to the this pathway, in type 1 diabetes development. hypothesis that NKG2D signaling, induced by NKG2D ligands expressed on b-islet cells, enhances diabetes development (6,7). However, conflicting results were later reported that Type 1 diabetes is a T cell–mediated autoimmune disorder brought into question the importance of NKG2D to sponta- resulting in the destruction of insulin-producing pancre- neous autoimmune diabetes in this preclinical model (8,9). Our atic b-cells. b-Cell loss results in an inability of the body previous data implicated differential effects of NKG2D signal- to produce insulin. Despite significant progress in type ing induced by microbiota or islet antigen as a source of this 1 diabetes research, the immune defects that lead to the controversy (9). Therefore, new experimental approaches are development of this disease remain poorly understood. required to determine the various roles of NKG2D in diabetes. 1Department of Microbiology, Molecular Genetics and Immunology, University of This article contains supplementary material online at https://doi.org/10.2337/ Kansas Medical Center, Kansas City, KS figshare.12311675. 2 Department of Medicine, University of Tennessee, Memphis, TN A.P.T. and K.L.K. contributed to this work equally. 3Department of Pathology, Immunology and Laboratory Medicine, University of © 2020 by the American Diabetes Association. Readers may use this article as Florida College of Medicine, Gainesville, FL long as the work is properly cited, the use is educational and not for profit, and the Corresponding author: Mary A. Markiewicz, [email protected] work is not altered. More information is available at https://www.diabetesjournals Received 3 October 2019 and accepted 13 May 2020 .org/content/license. 1750 Role of NKG2D Signaling in Pancreatic Islets Diabetes Volume 69, August 2020 Here, we show that mRNAs encoding both NKG2D and and laser capture of islets was conducted as previously NKG2D ligands are expressed in human islets, demon- described (21). All islets in two to five sections of tissue strating that NKG2D signaling in the pancreas is likely from each donor (a minimum of 30 islets each) were relevant to type 1 diabetes pathogenesis. To directly test captured and pooled, and RNA was extracted using the the effect of NKG2D signaling in the pancreas on auto- Arcturus PicoPure RNA Isolation Kit (Applied Biosystems, immune diabetes, we generated NOD mice with constitu- Grand Island, NY). An Agilent Bioanalyzer 2100 (Agilent tive expression of the mouse NKG2D ligand RAE1e on Technologies, Santa Clara, CA) was used to assess quality b-islet cells (i.e., NOD.RIP-RAE1e mice). We found that and quantity of RNA. Samples with sufficient RNA quan- despite earlier infiltration of immune cells into the pan- tity and quality were then subjected to gene expression creas, these mice developed significantly less diabetes analysis using Affymetrix expression arrays (GeneChip compared with littermate NOD mice. This diabetes re- Human Gene 2.0 ST), and scaled normalized gene expres- 1 duction corresponded with a reduced ratio of CD8 ef- sion values were produced as previously described (22). fector T (Teff) and effector memory T (Tem) cells (Teff 1 em) 1 to CD8 central memory T cells (Tcm). Correlating with Insulitis Scoring these in vivo data, we found that stimulation of NKG2D on Insulitis was scored using standard methods (23). The 1 NOD and human CD8 T cells during in vitro activation pancreata were fixed in formalin, and 5 mmol/L sections 1 resulted in a reduced CD8 Teff 1 em:Tcm ratio. Finally, we were generated from the whole tissue and stained with 1 found that CD8 Tcm cells actively suppress NOD diabetes hematoxylin-eosin by the Washington University School of development in vivo. Taken together, these results indi- Medicine Histology Core or the Kansas Intellectual and cate that NKG2D ligand expression in pancreatic islets Developmental Disabilities Research Center Histology protects against autoimmune diabetes development by Core. Thirty islets per pancreas in nonoverlapping sections 1 enhancing the generation or survival of a protective CD8 were scored. Tcm population. Diabetes Determination RESEARCH DESIGN AND METHODS Diabetes was determined by blood glucose measurements Mice taken weekly through tail vein nick. A mouse was defined scid NOD/ShiLtJ (NOD), NOD.CB17-Prkdc /J (NOD.Scid), as diabetic on the date when the first of two consecutive and C57BL/6 (B6) mice were purchased from The Jackson blood glucose measurements $250 mg/dL was obtained, Laboratory (stock numbers 001976, 001303, and 000664, as previously described (24). respectively). B6.Cg-Tg(Ins2-cre)25Mgn/J (B6.RIP-cre) mice were purchased from The Jackson Laboratory (stock number Flow Cytometry 003573). B6.PCCALL-RAE1e and ubiquitous B6.RAE1e mice Spleens were dissociated by pressing through a 40-mm cell were generated previously (7,18) and provided by Dr. Andrey strainer in isolation buffer containing PBS, 2% FCS, and Shaw (Washington University School of Medicine, St. Louis, 2mmol/LEDTA.Pancreatawerechoppedintosmall MO). The PCCALL-RAE1e (PCCALL) allele and rat insulin sections and digested with 1 mg/mL collagenase IV (Life promoter (RIP)-cre transgene were transferred to the NOD Technologies) in Iscove’s modified Dulbecco’s medium for genetic background using speed congenic methods (19) by 10 min at 37° with shaking, and a single-cell suspension the Washington University School of Medicine Mouse Ge- was generated by pressing through a 40-mm cell strainer. netics Core. Both NOD.PCCALL and NOD.RIP-cre strains The cells were then washed at least five times, followed maintain .98% of the NOD genome, with no alterations by antibody staining at 4°C for at least 15 min. After in known Idd loci. Experimental NOD.PCCALL-RAE1e, staining, cells were washed and analyzed. Cultured cells NOD.RIP-cre, and NOD.RIP-RAE1e littermates of both were washed once in isolation buffer and stained before sexes were generated by interbreeding NOD.PCCALL and similar staining and analysis. 2 2 NOD.RIP-cre mice. NOD.B6-Klrk1tm1.1Bpol (NOD.Klrk1 / ) mice were previously generated in our laboratory (9). All NOD Diabetes Transfer mice were housed under specific pathogen–free conditions Male and female mice were used, with all transfers con- at either the Washington University School of Medicine or taining male cells going only into male NOD.Scid recipients. the University of Kansas Medical Center animal facility in Spleens and lymph nodes from nondiabetic 6- to 12- accordance with institutional guidelines and with approval week-old NOD mice were harvested and dissociated by from the institutional animal care and use committee. passing through a 40-mmcellstrainerintoisolation buffer containing PBS, 2% FCS, and 2 mmol/L EDTA. 1 Human Pancreatic Islet Microarray CD8 T cells were then enriched by negative selection using Frozen tissue from cadaveric donors was provided by the magnetic bead separation (cat. no. 558471; BD Biosciences), 1 Network for Pancreatic Organ Donors (nPOD) (20) with according to the manufacturer’s instructions. CD8 Tcells approval from the University of Florida Health Center were then stained with anti-mCD3-PE-Cy7, anti-mCD8- Review Board. The characteristics of the donors used are APC, anti-mCD44-BV650, and anti-mCD62L-BV510 anti- 1 1 1 2 shown in Supplementary Table 1.