Lew are supported by a Juvenile Diabetes Foundation Program grant. Notes The online version of this article contains supplemental material.. abundant at the tumor site, and transplanted tumors were strongly rejected by either, or both, of these cell types. Blockade of a number of different known costimulatory pathways failed to prevent tumor rejection. These results reflect a critical role for NK cells and TCR+ T cells in innate immune surveillance of B cell lymphomas, mediated by as yet undetermined pathway(s) of tumor acknowledgement. strong class=”kwd-title” Keywords: immunosurveillance, effector, NK cell, tumor, perforin Introduction Immune surveillance against tumors has been debated for decades, although it has been well established using experimental tumor cell lines in mAb-treated and gene-targeted mice that this immune system recognizes and inhibits tumor growth (1C8). More recently, interest has shifted to determine whether the immune system can recognize precancerous cells, thus preventing tumor development. Mice deficient in important adaptive and innate immune effector molecules such as perforin (pfp) and IFN- have illustrated the importance of these molecules in tumor prevention in aging mice or when predisposing factors such as chemical carcinogens or loss of tumor suppressors drive carcinogenesis (5, 7, 9C11). Fiacitabine The lymphomas arising in pfp-deficient mice were of B cell origin, extremely immunogenic, and all Fiacitabine susceptible to CD8+ T cellCmediated attack when transplanted into syngeneic WT recipients (5). Fiacitabine These data as well as others (12) supported the important role adaptive immune responses play in spontaneous tumor control. The role of innate immune cells such as NK cells and TCR+ T cells in immune surveillance of tumors remains controversial. Both NK cells and TCR+ T cells express perforin (13, 14), mediate spontaneous cytotoxicity, and produce many antitumor cytokines such as IFN-, when they identify target cells via one or more of several cell surface receptors (15, 16). NK cells can spontaneously kill MHC class ICdeficient tumor cell lines in vivo (1, 6) and suppress experimental and spontaneous metastasis in mice, but you will find few models where NK cells or TCR+ T cells prevent main tumor formation (3, 17C19). Mice gene targeted for 2-microglobulin (2m) express little or no cell surface MHC class I, CD1d, or CD16 (Fc receptor III; reference 20), have greatly diminished CD8+ T cell figures, and lack CD1d-restricted T cells. We investigated spontaneous tumor development in aging 2m-deficient mice compared with mice doubly deficient in pfp and 2m, to determine whether the latter mice would develop lymphomas and additional tumors, and whether innate effector cells, such as NK cells and TCR+ T cells, could identify and eliminate such tumors given their lack of MHC class I molecules. Materials and Methods Mice. Inbred C57BL/6 WT mice were purchased from your Walter and Eliza Hall Zfp622 Institute of Medical Research (Melbourne, Australia). The following gene-targeted mice were bred at the Austin Research Institute Biological Research Laboratories (ARI-BRL; Heidelberg, Australia) and at the Peter Mac (East Melbourne, Australia): C57BL/6 perforin deficient (B6 pfp?/?); C57BL/6 -2-microglobulin deficient (B6 2m?/?); and C57BL/6 pfp?/? 2m?/? (21). All aging mice were bred, maintained, and monitored as explained previously (5). Mean lifespan (age of onset of lymphoma detected) standard error of the mean was calculated and probability of significance decided using a Mann-Whitney Rank Sum U test. C57BL/6 RAG-1?/? (Animal Resources Centre, Canning Vale, Western Australia) and C57BL/6 J18?/? (backcrossed to C57BL/6 for 12 generations and provided by Dr. M. Taniguchi, Chiba University or college, Chiba, Japan) mice were bred and managed at the Peter Mac. Congenic Ly-5.1+ B6 mice were purchased from the Animal Resources Centre and bred with B6 pfp?/? mice to generate a B6 Ly-5.1+pfp?/? collection. Mice 6C15 wk of age were used in transplantation studies in accordance with the Peter Mac animal experimental ethics committee. Circulation Cytometry. The following reagents utilized for circulation cytometry were purchased from BD Biosciences: antiCTCR-FITC or APC (H57C597); antiCNK1.1-PE (PK136); antiCLy5.2-FITC (104); and antiCTCR-biotin or FITC (clone GL3). Anti-Fc receptor (2.4G2) was used to prevent nonspecific binding by mAb. Intracellular staining was performed using the Cytoperm Kit (BD Biosciences) as per their instructions. Analysis was performed on a FACScalibur? using CellQuest software or LSR II using FACsDIVA? software (Becton Dickinson). Tumor Transplantation Experiments. Three representative (of many) B cell lymphomas from B6 pfp?/? 2m?/? mice, 2mNPN-2, 2mNPN-8, and 2mNPN-10 (B220+Ig+ TCR?) were transferred into WT, gene-targeted, and antibody-treated mice. Groups of three to five WT or gene-targeted mice were injected i.p. with increasing numbers of lymphoma cells and observed daily for tumor growth for over 150 d. Some groups of WT and gene-targeted mice were depleted of NK1.1+, asialo-GM1+, or TCR+ T cells in vivo by treatment.